METHODS AND COMPOSITIONS FOR EMERGENCY POST-INFECTION TREATMENT OF ANTHRAX

Abstract

Compositions effective for treating or preventing an anthrax infection in a subject in need thereof and recombinant proteins included in the compositions are provided. Methods for producing recombinant proteins in plants are described. Transgenic plants engineered to produce recombinant proteins as well as genetic constructs comprising nucleic acids encoding recombinant proteins thereof are also described. Methods of protecting subjects against anthrax infection with plant-derived compositions are provided.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent application Ser. No. 13/922,719, filed on Jun. 20, 2013, which claims the benefit of U.S. provisional application No. 61/663,271, filed Jun. 22, 2012, all of which is incorporated by reference as if fully set forth.

[0002] The sequence listing electronically filed with this application titled "Sequence Listing," created on Oct. 19, 2017, and having a file size of 92,962 bytes is incorporated herein by reference as if fully set forth.

FIELD OF INVENTION

[0003] The invention relates to methods and compositions to produce recombinant proteins in plants for the post-infection treatment or prophylaxis of the anthrax disease to neutralize the action of the anthrax toxin in the infected subjects. In particular the invention provides recombinant proteins, genetic constructs comprising polynucleotides encoding the proteins thereof, vectors and transgenic plants that include the genetic constructs. Methods for producing recombinant proteins, preparing compositions that include recombinant proteins and methods of protecting subjects by administering the compositions are also provided.

BACKGROUND

[0004] Despite the progress in studying mechanisms of anthrax, a zoonotic disease caused by the Gram-positive bacterium Bacillus anthracis, a problem remains concerning the prevention and/or post-exposure treatment of the infection, especially with bio-terrorism threats. Although conventional vaccines against anthrax exist, induction and maintenance of adequate protection requires multiple immunizations followed by yearly boosters which frequently cause dangerous side effects (Rainy, Young, 2004). These side effects often inhibit the use of preventive vaccines among the human population.

SUMMARY

[0005] An aspect of the invention relates to a recombinant protein. The recombinant protein includes a first protein fused to a second protein. The first protein is a toxin binding ligand. The second protein is a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein.

[0006] An aspect of the invention relates to a genetic construct. The genetic construct includes a first polynucleotide and a second polynucleotide. The first polynucleotide encodes a toxin binding ligand. The second polynucleotide encodes a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein.

[0007] An aspect of the invention relates to a genetic construct. The genetic construct includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5).

[0008] An aspect of the invention relates to a transgenic plant. The transgenic plant comprises a genetic construct. The genetic construct includes a first polynucleotide and a second polynucleotide. The first polynucleotide encodes a toxin binding ligand. The second polynucleotide encodes a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein.

[0009] An aspect of the invention relates to a method for producing a recombinant protein in a plant. The method includes contacting a plant with a genetic construct. The genetic construct includes a nucleic acid encoding the recombinant protein. The recombinant protein includes a toxin binding ligand fused to a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. The method also includes obtaining a plant including the genetic construct and expressing the recombinant protein.

[0010] An aspect of the invention relates to a method for preparing a composition effective for treating or preventing an anthrax infection in a subject. The method includes providing a recombinant protein produced by any method described herein.

[0011] An aspect of the invention relates to a method of protecting a subject against anthrax infection. The method includes providing a composition that includes a recombinant protein. The recombinant protein includes a toxin binding ligand fused to a carrier-protein. The carrier-protein is a protein selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. The composition is effective in preventing or reducing at least one symptom of an anthrax infection in a subject. The method also includes administering the composition to the subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following detailed description of the preferred embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustration, there are shown in the drawings embodiments which are presently preferred. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0013] FIG. 1A is a schematic drawing illustrating the formation of anthrax toxin complexes and their entry into cells.

[0014] FIG. 1B illustrates an infusion of a recombinant protein that includes an anthrax toxin binding ligand and a carrier protein (component A-B) into the bloodstream of the infected human or animal.

[0015] FIG. 2A illustrates a comparison of a native antibody with a recombinant protein that includes a toxin binding receptor (TBL) fused to the Fc-region of the immunoglobulin. FIG. 2B illustrates possible configurations (quaternary structures) of self-assembling A1-B recombinant proteins.

[0016] FIG. 3A illustrates a map of an intermediate plasmid that contains a nucleotide sequence encoding a synthetic gene PgA1B1.

[0017] FIG. 3B illustrates a schematic drawing of an expression cassette included in the pBIN vector.

[0018] FIGS. 4A-4B illustrate a pBI binary vector that including the TBL-Fc expression cassette (FIG. 4A) and steps for production of the recombinant TBL protein in plants (FIG. 4B).

[0019] FIGS. 5A-5F illustrate in vitro selection of transgenic tobacco plants engineered to express a recombinant TBL protein.

[0020] FIG. 5A illustrates formation of transgenic tobacco shoots on the medium supplemented with to 50 mg/L of kanamycin.

[0021] FIG. 5B illustrates development of the transgenic tobacco shoots on selection medium.

[0022] FIG. 5C illustrates the rooted transgenic tobacco plant grown in a Magenta box.

[0023] FIG. 5D illustrates a transgenic tobacco plant grown on in soil.

[0024] FIG. 5E illustrates PCR analysis of the transgenic tobacco plants using nptII-specific primers.

[0025] FIG. 5F illustrates Western blot analysis of the transgenic tobacco lines using c-myc tag-specific antibodies.

[0026] FIGS. 6A-6E illustrate in vitro selection of transgenic Echinaceia angustifolia plants engineered to express a recombinant TBL protein.

[0027] FIG. 6A illustrates formation of the transgenic Echinaceia angustifolia shoot on the medium supplemented with to 50 mg/L of kanamycin.

[0028] FIG. 6B illustrates mass-propagation of the transgenic Echinaceia angustifolia plants.

[0029] FIG. 6C illustrates the rooted transgenic Echinaceia angustifolia plant grown in a Magenta box.

[0030] FIG. 6D illustrates a transgenic Echinacea angustifolia plant growing on the selection medium supplemented with kanamycin.

[0031] FIG. 6E illustrates Western blot analysis of the transgenic Echinacea angustifolia using c-myc tag-specific antibodies.

[0032] FIGS. 7A-7C illustrate in vitro selection of transgenic Kalanchoe pinnata plants engineered to express a recombinant TBL protein

[0033] FIG. 7A illustrates formation of the transgenic Kalanchoe pinnata shoot on the medium supplemented with to 50 mg/L of kanamycin.

[0034] FIG. 7B illustrates propagation of the transgenic transgenic Kalanchoe pinnata shoots on the kanamycin selection medium.

[0035] FIG. 7C shows rooting of Kalanchoe pinnata shoots on the kanamycin selection medium.

[0036] FIG. 8 illustrates steps of the method for producing and administering plant-derived compositions effective to prevent anthrax infection in a subject.

[0037] FIGS. 9A-9C illustrates analysis of the PgA1-B1 proteins produced in plants.

[0038] FIG. 9A illustrates Western blot analysis of transgenic plants expressing PgA1-B1 proteins.

[0039] FIG. 9B illustrates Western blot analysis of a total and soluble protein extracted from transgenic tobacco plants.

[0040] FIG. 9C illustrates extraction and purification of the plant PgA1-B1 recombinant protein from tobacco leaf tissue.

[0041] FIGS. 10A-10B illustrates the ability of the recombinant protein PgA1-B1 to protect cells against the anthrax toxin.

[0042] FIG. 10A illustrates an analysis of the PA binding by the recombinant protein PgA1-B1.

[0043] FIG. 10B illustrates neutralization of the lethal toxin activity by the recombinant protein PgA1-B1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Certain terminology is used in the following description for convenience only and is not limiting. The words "right," "left," "top," and "bottom" designate directions in the drawings to which reference is made. The words "a" and "one," as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise. This terminology includes the words above specifically mentioned, derivatives thereof, and words of similar import. The phrase "at least one" followed by a list of two or more items, such as "A, B, or C," means any individual one of A, B or C as well as any combination thereof.

[0045] The onset of acute symptoms of the anthrax disease occurs due to the effects of bacterial toxin consisting of non-lethal components: protective antigen (PA) combined with lethal factor (LF) or edema factor (EF) (FIG. 1A; see Young, Collier, 2007). PA.sub.83 binds to cellular receptors where it is cleaved by a furin protease to PA.sub.63, which assembles into a heptameric pore. The pore can binds up to three units of LF, EF or both. Endocytosys of this structure leads to the entry of LF and/or EF into cytosol, where each factor causes its toxic effects. Neutralizing anthrax toxin activity could provide time for antibacterial agents or the immune system to clear up infection. Therefore, early post-infection treatment of anthrax infection with effective antitoxins that can block the action of toxin in vivo is important (Rainy, Young, 2004).

[0046] Embodiments herein provide technologies to express the chemically active recombinant anti-anthrax antitoxin in plants. FIG. 1B illustrates an infusion of recombinant proteins (Component A-B) into the bloodstream of the infected human or animal. Component A-B may be capable of binding PA.sub.83 protein and preventing it from binding to the anthrax toxin receptor on the cell surface, thus protecting cells from translocation of other toxin component, EF or LF, into the cells. Production of active recombinant antitoxin protein in plants may be easily scaled up. The potential to produce large quantities of antitoxin protein may be useful for preindustrial and industrial scale production, during threats of bioterrorism and continuous outbreaks of anthrax infections. The plant-derived compositions may also be produced at a lower cost compared to traditional antibodies. An advantage of plant-derived antitoxin compositions is that these compositions are free of mammalian pathogens.

[0047] In an embodiment, a recombinant protein is provided. The recombinant protein may include a first protein fused to a second protein. The first protein may be a toxin binding ligand (referred to herein as "component A"). The toxin binding ligand may be capable of binding anthrax toxin with high affinity.

[0048] The toxin binding ligand may be a human or animal anthrax receptor (ATR) protein. The toxin binding ligand may be a Capillary Morphogenesis Protein 2 (CMG-2; component A1). The toxin binding ligand may be a soluble domain of the CMG-2. The toxin binding ligand may be a soluble domain of another ATR protein. The toxin binding ligand may be a soluble domain of another anthrax toxin-binding polypeptide.

[0049] The toxin binding ligand may be a polypeptide capable of high affinity binding to a protective antigen (PA) region necessary for PA interaction with a lethal factor (LF) or an edema factor (EF) components of the anthrax toxin. The toxin binding ligand may be a protective antigen binding domain of a lethal factor (PA-LF; component A2).

[0050] In an embodiment, the human capillary morphogenesis protein 2 may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of: SEQ ID NO: 24 (A1-CMG2) or SEQ ID NO: 60 (PgA1-5).

[0051] In an embodiment, the PA-LFn protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of: SEQ ID NO: 25 (A2/PA-LF).

[0052] In an embodiment, the toxin binding ligand alone may be capable of protecting a subject against anthrax toxin.

[0053] The second protein may be a carrier protein (referred to herein as "component B"). The carrier-protein may be capable of improving production, stability, solubility, extraction, secondary structure or other characteristics of the recombinant protein. The carrier-protein may be capable of improving translocation of the recombinant protein through biological membranes and its delivery into the bloodstream of an infected subject. The carrier protein may be capable of simplifying purification of the recombinant protein.

[0054] The carrier-protein may be an Fc (fragment crystallizable) region of an antibody capable of interacting with cell surface receptors called Fc receptors. FIG. 2A illustrates a comparison of a native antibody (left) with a recombinant protein that includes the toxin binding receptor fused to Fc-region of an immunoglobulin. The Fc-region may be but is not limited to an IgG isotype (Component B1), an IgA isotype (Component B2), or an IgM isotype (component B3). The Fc region of an antibody may be a single isotype. The Fc region may be a combination of any of IgA, IgG, or IgM isotypes. The Fc-region of an antibody may be combined with elements of several immunoglobulin isotypes that possess properties or have the natural ability of transport across the intestinal epithelium or penetrate higher cell- and mucose surfaces. The Fc-regions of recombinant proteins may be capable of assembling into quaternary structures consisting of several similar units. For example, the toxin binding ligand fused to an Fc-region of a human or an animal antibody under native conditions may be capable of forming dimers with another Fc molecule through the formation of disulfide bonds (FIG. 2B). The assembly of the individual Fc-molecules (monomers) into quaternary structures consisting of several similar units (dimers or multimers) may be facilitated by a helper element (referred to herein as "component C"). The helper element may facilitate self-assembly of the Fc-molecules into quaternary structures after bringing them into close proximity with each other, particularly after delivery into a human or a non-human animal organism. The helper element may provide a higher stability in a bloodstream, binding avidity for the anthrax toxin due to its multivalency and ability to induce host immune response to the bound anthrax toxin. The helper element may be an IgJ antibody. The helper element may be a VP1 coat protein of the JC virus. FIG. 2B illustrates possible configurations of self-assembling A1-B recombinant proteins, where the Fc fragment (component B) originates from an antibody (Ab) of different isotypes. This figure shows that the isotype IgG (B1) may form a bivalent structure (left). The isotype IgA (B2; middle) and the isotype IgM (B3; right) may be capable of binding multiple units together in the presence of "J," a helper element, assisting in self-assembly of separate antibody molecules into dimmer or multimer structures.

[0055] The carrier-protein may be of virus origin. The carrier-protein may originate from a polyoma or a papiloma virus proteins capable of self-assembly into quaternary structures resembling virus like particles. The carrier protein may be a virus structural protein. The carrier protein may be a virus coat protein. The carrier protein may be a VP2 coat protein of the JC virus.

[0056] The carrier-protein may be capable of targeting itself as well as another covalently fused protein to plant cell oil bodies thus providing accumulation of the target protein in the plant lipid fraction, where it can be easily extracted with cheap available technologies. Particularly, the carrier-protein may be, but is not limited to, a plant oleosin. The oleosin may be capable of targeting the recombinant protein into a lipid fraction of plant cells and accumulating the recombinant protein in an outer surface of plant cell oil bodies, thus providing easy and cheap extraction of the protein from plant biomass (McLean et al., 2012, Transgenic Res, Mar. 2, Epub).

[0057] The carrier-protein may be a protein capable of changing solubility under specific temperature condition, thus providing cheap and easy extraction using recently developed technologies available on market. The carrier-protein may be a thermo-stable protein or a thermo-labile protein. The carrier-protein may be a protein from thermophilic bacteria. The carrier-protein from the termophilic bacteria may be glucuronidase or lichenase B (U.S. Pat. No. 8,173,408, incorporated herein by reference as if fully set forth). The carrier-protein may be an Elastin-Like Polypeptides capable of undergoing a reversible, inverse phase transition and providing technical simplicity, low cost, ease of scale-up of extraction using "inverse transition cycling" technique (Hassouneh et al., 2010, Curr Protocol Protein Sci 6:6.11; Meyer, Chilkoti, 1999, Nat Rev. Immunol 5:905-916, all of which are incorporated by reference as if fully set forth). The carrier protein may be any other protein.

[0058] In an embodiment, the carrier-protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 26 (B1), SEQ ID NO: 27 (B2), SEQ ID NO: 28 (B3), SEQ ID NO: 29 (B4) and SEQ ID NO: 30 (B5).

[0059] In an embodiment, the helper element may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ NO 35 (C1-protein) and SEQ ID NO: 36 (C-protein).

[0060] The first protein or the second protein may be linked to a targeting peptide. The first protein or the second protein may be linked to a peptide tag for detection or purification. The detection or purification tag may be chosen from, but is not limited to, Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, S-, HAT-, 3xFLAG, Calmodulin-binding peptide, Cellulose-binding domain, SBP, Chitin-binding domain, Glutation S-transferase, Maltose-binding protein, and Elastin-like peptide.

[0061] In an embodiment, the first protein may be fused to the second protein, or the purification tag using a flexible linker. The flexible linker may be a self-cleavable peptide. The flexible linker may be a peptide that is a site for cleaving with a specific protease, available in a composition or naturally present in mammal blood, providing, when necessary, release of the soluble toxin binding ligand from the carrier-protein or the tag during process of extraction or upon delivery of the recombinant protein into a bloodstream of a subject.

[0062] In an embodiment, the recombinant protein may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 31 (PgA1-1:B1-1).

[0063] In an embodiment, a genetic construct comprising a first polynucleotide and a second polynucleotide is provided. The first polynucleotide may encode a toxin binding ligand. The first polynucleotide may encode a CMG-2 protein. The first polynucleotide may encode a PA-LF protein.

[0064] The first polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4) and SEQ ID NO: 58 (PgA1-5). The first polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3/PA-LF) and SEQ ID NO: 57 (PgA2-4).

[0065] The first polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4) and SEQ ID NO: 58 (PgA1-5). The first polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3/PA-LF) and SEQ ID NO: 57 (PgA2-4).

[0066] The second polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2).

[0067] The second polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2).

[0068] In an embodiment, the genetic construct may further include a third polynucleotide encoding a helper element. The third polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2). The third polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2).

[0069] In an embodiment, a genetic construct may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5). The genetic construct may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5).

[0070] Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity may be measured by the Basic Local Alignment Search Tool (BLAST; Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J, 1990 "Basic local alignment search tool." J. Mol. Biol. 215:403-410, which is incorporated herein by reference as if fully set forth).

[0071] In an embodiment, the toxin binding ligand may be a derivative of a human or non-human anthrax toxin receptor protein. The toxin binding ligand may be a derivative of a bacterial protein specifically binding PA protein. The toxin binding ligand may be a variant ATR from a human or a non-human animal. The toxin binding ligand may be a variant of B. anthracis LF and EF proteins. A variant may include an amino acid sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to an amino acid sequence of native toxin-binding proteins.

[0072] Variants may include conservative amino acid substitutions, i.e., substitutions with amino acids having similar properties. Conservative substitutions may be a polar for polar amino acid (Glycine (G), Serine (S), Threonine (T), Tyrosine (Y), Cysteine (C), Asparagine (N) and Glutamine (Q)); a non-polar for non-polar amino acid (Alanine (A), Valine (V), Thyptophan W), Leucine (L), Proline (P), Methionine (M), Phenilalanine (F)); acidic for acidic amino acid Aspartic acid (D), Glutamic acid (E)); basic for basic amino acid (Arginine (R), Histidine (H), Lysine (K)); charged for charged amino acids (Aspartic acid (D), Glutamic acid (E), Histidine (H), Lysine (K) and Arginine (R)); and a hydrophobic for hydrophobic amino acid (Alanine (A), Leucine (L), Isoleucine (I), Valine (V), Proline (P), Phenilalanine (F), Tryptophan (W) and Methionone (M)). Conservative nucleotide substitutions may be made in a nucleic acid sequence by substituting a codon for an amino acid with a different codon for the same amino acid. Variants may include non-conservative substitutions.

[0073] In an embodiment, fragments of a toxin binding ligand, a carrier protein or a helper element are provided. Fragments may include 100, 150, 200, 300, 400, 600, contiguous amino acids or more, such as 700.

[0074] In an embodiment, fragments of CMG2 or PA-LF proteins are provided. Fragments may include 100, 150, 200, 300, 400, 500 contiguous amino acids or more, such as 580.

[0075] The functionality of a recombinant protein, variants or fragments thereof, may be determined using any methods. The functionality may include conferring ability to bind the components of the anthrax toxin in a solution as determined by immunodetection methods. The functionality may be assessed using protection of cells growing in vitro. The functionality of a protein, or variants, or fragments thereof, may be assessed based on an ability to protect animals after administering of a recombinant antitoxin following of the infection of animals with the causative agent of anthrax.

[0076] In an embodiment, polynucleotides are provided having a sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. In an embodiment, polynucleotides having a sequence that hybridizes to a nucleic acid having the sequence of any nucleic acid listed herein or the complement thereof are provided. In an embodiment, the hybridization conditions are low stringency conditions. In an embodiment, the hybridization conditions are moderate stringency conditions. In an embodiment, the hybridization conditions are high stringency conditions. Examples of hybridization protocols and methods for optimization of hybridization protocols are described in the following book: in Green and Sambrook. Molecular Cloning: a Laboratory Manual. 4th ed. Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press, which is incorporated by reference in as if fully set forth.

[0077] Moderate conditions may be as follows: filters loaded with DNA samples are pretreated for 2-4 hours at 68.degree. C. in a solution containing 6.times. citrate buffered saline (SSC; Amresco, Inc., Solon, Ohio), 0.5% sodium dodecyl sulfate (SDS; Amresco, Inc., Solon, Ohio), 5.times. Denhardt's solution (Amresco, Inc., Solon, Ohio), and denatured salmon sperm (Invitrogen Life Technologies, Inc. Carlsbad, Calif.). Hybridization is carried in the same solution with the following modifications: 0.01 M EDTA (Amresco, Inc., Solon, Ohio), 100 .mu.g/ml salmon sperm DNA, and 5-20.times.10.sup.6 cpm .sup.32P-labeled or fluorescently labeled probes. Filters are incubated in hybridization mixture for 16-20 hours and then washed for 15 minutes in a solution containing 2.times.SSC and 0.1% SDS. The wash solution is replaced for a second wash with a solution containing 0.1.times.SSC and 0.5% SDS and incubated an additional 2 hours at 20.degree. C. to 29.degree. C. below Tm (melting temperature in .degree. C.), where:

Tm=81.5+16.61 Log.sub.10([Na.sup.+](1.0+0.7[Na.sup.+]))+0.41(%[G+C])-(500/n)-P-F;

[0078] [Na+]=Molar concentration of sodium ions;

[0079] %[G+C]=percent of G+C bases in DNA sequence;

[0080] N=length of DNA sequence in bases;

[0081] P=a temperature correction for % mismatched base pairs (.about.1.degree. C. per 1% mismatch);

[0082] F=correction for formamide concentration (=0.63.degree. C. per 1% formamide). Filters are exposed for development in an imager or by autoradiography.

[0083] Low stringency conditions refers to hybridization conditions at low temperatures, for example, between 37.degree. C. and 60.degree. C., and the second wash with higher [Na.sup.+] (up to 0.825M) and at a temperature 40.degree. C. to 48.degree. C. below Tm. High stringency refers to hybridization conditions at high temperatures, for example, over 68.degree. C., and the second wash with [Na+]=0.0165 to 0.0330M at a temperature 5.degree. C. to 10.degree. C. below Tm.

[0084] In an embodiment, polynucleotides having a sequence that has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity along its length to a contiguous portion of a nucleic acid having any one of the sequences set forth herein or the complements thereof are provided. The contiguous portion may be the entire length of a sequence set forth herein or the complement thereof.

[0085] In an embodiment, isolated nucleic acids, polynucleotides, or oligonucleotides are provided having a portion of the sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. These isolated nucleic acids, polynucleotides, or oligonucleotides are not limited to but may have a length in the range from 10 to full length, 10 to 1000, 10 to 900, 10 to 800, 10 to 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, or 20 to 30 nucleotides or 10, 15, 20 or 25 nucleotides. An isolated nucleic acid, polynucleotide, or oligonucleotide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive. The recited length of nucleotides may start at any single position within a reference sequence (i.e., any one of the nucleic acids herein) where enough nucleotides follow the single position to accommodate the recited length. In an embodiment, a hybridization probe or primer is 85 to 100%, 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% complementary to a nucleic acid with the same length as the probe or primer and having a sequence chosen from a length of nucleotides corresponding to the probe or primer length within a portion of a sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, a hybridization probe or primer hybridizes along its length to a corresponding length of a nucleic acid having the sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, the hybridization conditions are low stringency. In an embodiment, the hybridization conditions are moderate stringency. In an embodiment, the hybridization conditions are high stringency.

[0086] The variants or fragments of the polynucleotides encoding the recombinant proteins may be identified, isolated or synthesized by any known methods. For optimal expression in a host cell, a polynucleotide sequence encoding a first and a second protein may be codon-optimized by adapting the codon usage to that most preferred in host genes. In case the host is a plant, codon usage may be optimized to native plant genes (Itakura et al. 1977 Science 198:1056; Bennetzen et al. 1982 J Mol Chem 257: 3026) using codon usage tables. Codon usage table are publicly available for various plant species (Nakamura et al. 2000 Nucl Acid Res 28: 292).

[0087] In an embodiment, a genetic construct having a nucleic acid encoding a recombinant protein may be provided in an expression cassette suitable for expression in plant cell, tissues, organs, and/or whole organism.

[0088] The expression of any one of the first and the second polynucleotide sequences or the third polynucleotide sequence of the genetic construct included the expression cassette may be under control of a promoter, which provides for transcription of the polynucleotide in a plant. The promoter may be a constitutive promoter, or tissue specific, or an inducible promoter. A constitutive promoter may provide transcription of the polynucleotides throughout most cells and tissues of the plant and during many stages of development but not necessarily all stages. An inducible promoter may initiate transcription of the polynucleotide sequences only when exposed to a particular chemical or environmental stimulus. A tissue specific promoter may be capable of initiating transcription in a particular plant tissue. Plant tissue may be, but is not limited to, a stem, leaves, trichomes, anthers, or seeds. Constitutive promoter may be, but is not limited to, the Cauliflower Mosaic Virus (CaMV) 35S promoter, the Cestrum Yellow Leaf Curling Virus promoter (CMP), or the CMP short version (CMPS), the Rubisco small subunit promoter, or the maize ubiquitin promoter.

[0089] An expression cassette may further include a terminator sequence, which terminates transcription the first and the second polynucleotide sequences or the third polynucleotide sequence and may be included at the 3' end of a transcriptional unit of the expression cassette. The terminator may be derived from a variety of plant genes. The terminator may be derived from the nopaline synthase or octopine synthase genes of Agrobacterium tumefaciens.

[0090] In an embodiment, an expression cassette is provided in a vector. For stable plant transformation, an expression cassette may be included in a T-DNA binary vector or a co-integrate vector. A vector may include multiple cloning sites to facilitate molecular cloning and selection markers to facilitate selection. A selection marker may be, but is not limited to, a neomycin phosphotransferase (npt) gene conferring resistance to kanamycin, a hygromycin phosphotransferase (hpt) gene conferring resistance to hygromycin, and a bar gene conferring resistance to phosphinothricin.

[0091] For transient expression of the toxin binding ligands, carrier proteins, or helper peptides in a plant, an expression cassette may be included in a viral-based vector. A viral-based vector may be obtained from a virus, which is not infectious for a mammalian object and therefore not requiring elimination of the vector components from the compositions herein. A viral-based vector may be, but is not limited to, a tobacco mosaic virus (TMV)-based vector or a potato virus X (PVX)-based vector.

[0092] In an embodiment, a transgenic plant is provided. The transgenic plant may include a genetic construct. The genetic construct may include a first polynucleotide and a second polynucleotide. The first polynucleotide may encode a toxin binding ligand. The second polynucleotide may encode a carrier-protein. The carrier-protein may be but is not limited to Fc-IgA, Fc-IgG, Fc-IgM, oleosin, or VP2 coat protein. The toxin binding ligand may be a capillary morphogenesis protein 2. The toxin binding ligand may be a PA-LF protein. The first polynucleotide may encode the capillary morphogenesis protein 2 or a PA-LF protein. The first polynucleotide may include, consist essentially of, or consisting of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4 (PgA2-2), SEQ ID NO: 5 (A2/PA-LF), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4), SEQ ID NO: 57 (PgA2-4), and SEQ ID NO: 58 (PgA1-5). The second polynucleotide may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2).

[0093] The transgenic plant may further include a third polynucleotide. The third polynucleotide may encode a helper element. The third polynucleotide may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2).

[0094] The transgenic plant may include a genetic construct that includes, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20

[0095] (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5) and SEQ ID NO: 38 (PgA1-5:B 1-5).

[0096] The transgenic plant may be any plant, or a part of a plant. The part of a plant may be a stem, a leaf, a flower, a seed, or a callus. The transgenic plant may be a progeny, or descendant of a transgenic plant. The transgenic plant may be obtained through crossing of a transgenic plant and non-transgenic plant as long as it retains the genetic construct as described above. The transgenic plant may be a crop cultivated for purposes of obtaining food, feed or plant derived products including carbohydrates, oil, and medicinal ingredients. A crop plant may be selected from group consisting of: tomato, tobacco, pepper, eggplant, lettuce, sunflower, oilseed rape, broccoli, cauliflower and cabbage crops, cucumber, carrot, melon, watermelon, pumpkin, squash, sugar beet, peanut, chard, Swiss chard, soybean, cotton, beans, cassava, potatoes, sweet potato, okra, barley, pearl millet, wheat, rye, buckwheat, sorghum, rice. The transgenic plant may include forage grasses. The transgenic plant may include tree species and fleshy fruit species. The transgenic plants may include grapes, peaches, plums, cherries, strawberries, cranberries, mangos, and bananas.

[0097] The transgenic plant may be a medicinal plant. A medicinal plant may be a plant thought to have medicinal property and used in herbalism. A medicinal plant may be selected from a group consisting of, but not limited to: Arthemis nobilis, Calendula officinalis, Caragana sinica, Codonopsis pilosulae, Echinacea angustifolia, Hedyotis diffusa, Houttuynia cordata, Hydrastis canadensis, Kalanchoe pinnata, Lonicera japonica, Morinda offcinalis, and Oenothera odorata.

[0098] The transgenic plant may be edible or medicinal plants. Edible or medicinal plants may not contain health-threatening components. Edible or medicinal plants may not require any special purification and may be used similar to conventional biologically active dietary supplements produced from plants.

[0099] In an embodiment, a method for producing a recombinant protein in a plant is provided. The method may include steps of contacting a plant with a genetic construct. The genetic construct may include a nucleic acid encoding the recombinant protein. The recombinant protein may include a toxin binding ligand fused to a carrier protein. The carrier protein may be but is not limited to Fc-IgA, Fc-IgG, Fc-IgM, oleosin, or VP2 coat protein. The method may also include obtaining a plant that includes the genetic construct and expressing the recombinant protein.

[0100] In an embodiment of the method, the toxin binding ligand may be a capillary morphogenesis protein 2. The toxin binding ligand may be a PA-LF protein.

[0101] The step of contacting may include contacting with a vectors providing for stable transformation of a plant. The step of contacting may include contacting with a vector providing for transient expression in a plant. The vector may include a first polynucleotide encoding a toxin binding ligand and a second polynucleotide sequence encoding a carrier protein. A method may further include contacting a plant with another vector that includes a third polynucleotide encoding a helper element.

[0102] In an embodiment, the step of contacting may include contacting with the nucleic acid that may include a first polynucleotide encoding a toxin binding ligand. The nucleic acid may also include a second polynucleotide encoding a carrier protein. The first polynucleotide may include, consists essentially of, consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4) and SEQ ID NO: 58 (PgA1-5), SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3 /PA-LF), and SEQ ID NO: 57 (PgA2-4). The second polynucleotide may include, consists essentially of, consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2).

[0103] In an embodiment of the method, the nucleic acid may further include a third polynucleotide encoding a helper element. The third polynucleotide may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2).

[0104] In an embodiment of the method, the step of contacting may include contacting with a genetic construct that may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5).

[0105] In an embodiment of the method, the recombinant protein including the toxin binding ligand and a carrier-protein that may be capable of protecting a subject against anthrax toxin. The helper element may help to establish secondary structures from the molecules of the recombinant protein consisting of the toxin binding ligand and the carrier protein.

[0106] The plant may be created by Agrobacterium-mediated transformation using a vector that includes a nucleic acid encoding the recombinant protein herein. The transgenic plant may be created by other methods for modifying plants. The transgenic plant may be created by direct uptake of plasmid DNA. The transformed plant may be stably transformed. The stably transformed plant may incorporate the genetic construct into the genome of the plant.

[0107] The plant may be transformed with a viral vector for transient expression of a recombinant protein in a plant. The viral vector may be delivered to a plant by any method. For reference, see U.S. patents (U.S. Pat. Nos. 5,889,190; 5,889,191; 5,316,931; 5,589,367; 7,667,092; 7,670,801; 7,763,458; 8,093,458; and 8,003,381), all of which are incorporated by reference herein as if fully set forth. Viral vectors may be T-DNA vectors. Plants may be infiltrated with a diluted Agrobacterium suspension carrying T-DNAs encoding viral replicons. The resulting plants may have a high copy number of RNA molecules that encode a recombinant protein. A recombinant protein may be produced in a transgenic plant in a short period of time. A recombinant protein may be produced in the transgenic plant in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after transformation. The transgenic plants may have a high copy number of RNA molecules that encode recombinant proteins. Recombinant proteins may be produced in plants rapidly and in increasing volumes of biomass containing recombinant proteins that may not require changes in growing conditions. Features of transient expression system may include non-integration of external genes into plant genome, and thus reducing or eliminating the risk of releasing transgene into environment through pollen, seeds, or other routes. Further, no intact and replication-competent virus may be produced, thus, reducing or eliminating the risk of virus mediated spreading of the recombinant genes. Protein production may be performed in closed indoor settings.

[0108] In an embodiment, the method may include obtaining a plant expressing the recombinant antitoxin binding protein. The method may include also obtaining a plant expressing the helper peptide. The method may further include crossing the plant expressing the recombinant protein with the plant expressing the helper element.

[0109] An embodiment of any of the method may further include breeding the transgenic plant and obtaining its progeny, or its descendant. The progeny or the descendant may include the genetic construct.

[0110] In an embodiment, any of the method further may include obtaining a seed of the transgenic plant. The seed may include the genetic construct that includes the recombinant protein.

[0111] The method may further include isolating and purifying the recombinant protein from the plant.

[0112] In an embodiment a method for preparing a composition effective for treating or preventing an anthrax infection in a subject is also provided. The method may include providing a recombinant protein produced by any methods described herein. The therapeutic composition may include a toxin binding ligand (component A) alone. The therapeutic composition may include a toxin binding ligand fused to a carrier-protein (components A and B). The therapeutic composition may include a mixture of two or more recombinant antitoxins of different types related to different component B parts. The therapeutic composition may include any composition described herein and a helper element (component C). The plant-derived therapeutic compositions may neutralize, delay, or attenuate the fatal action of the anthrax toxin in a subject.

[0113] In an embodiment, a plant-derived therapeutic composition may include active agents. Active agents may include at least one of recombinant antitoxin proteins produced in plants. The therapeutic composition may be therapeutically effective. Therapeutic efficacy may depend on effective amounts of active agents and time of administering necessary to achieve the desired result.

[0114] In an embodiment, the therapeutic compositions in a "therapeutically effective amount", i.e., the amount sufficient to protect against accumulation of active deadly toxin in serum, or disappearance of disease symptoms in a subject. Disappearance of disease symptoms may be assessed by decrease of internalization of active LF or EF components of the anthrax toxin by living cells in the subject's body or by increase of a surviving time of the subject after contact with pathogen. The plant-derived compositions may be administered using any amount and any route of administration effective for a protective action.

[0115] The exact dosage may be chosen by the physician based on a variety of factors and in view of individual patients. Dosage and administration may be adjusted to provide sufficient levels of the active agent or agents or to maintain the desired effect. For example, factors which may be taken into account include time of potential infection, the type and amount of infection agent and severity of a disease; weight of the patient; availability of other means for treatment or prophylaxis.

[0116] Therapeutic efficacy and toxicity of active agents in a composition may be determined by standard pharmaceutical procedures, for example, by determining the therapeutically effective dose of the agent in 50% of the population (ED.sub.50) and the lethal dose to 50% of the population (LD.sub.50) with or without therapeutic agent in cells cultured in vitro or experimental animals. Plant-derived therapeutic compositions may be evaluated based on the dose ratio of toxic to therapeutic effects (LD.sub.50/ED.sub.50), called the therapeutic index, the largest value of which may be used for assessment. The data obtained from and animal studies may be used in formulating a dosage for human use.

[0117] The therapeutic dose according to currently accepted norm in animal models of anthrax infection may be at least 50 microgram (50 .mu.g) of antitoxin/dose/animal. As plant-based recombinant antitoxin may be readily produced and inexpensively engineered and designed and stored, lesser or greater doses for large animals may be economically feasible.

[0118] In an embodiment, the method may also include providing the therapeutic composition that includes a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" refers to solvents, diluents, preservatives, dispersion or suspension aids, isotonic agents, thickening or emulsifying agents, solid binders, and lubricants, appropriate for the particular dosage form. The pharmaceutically acceptable carrier may be any known carrier that may be used in formulating pharmaceutical compositions and knows techniques for the preparation thereof. See Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995. The pharmaceutically acceptable carriers may include, but are not limited to Ringer's solution, isotonic saline, starches, potato starch, sugars, glucose, powdered tragacant, malt, gelatin, talc, cellulose and its derivatives, ethyl cellulose, sodium carboxymethyl cellulose, cellulose acetate excipients, cocoa butter, suppository waxes, agar, alginic acid, oils, cottonseed oil, peanut oil, safflower oil, sesame oil, olive oil, soybean oil, corn oil, glycols, propylene glycol, esters, ethyl laurate, ethyl oleate, buffering agents, aluminum hydroxide, magnesium hydroxide, phosphate buffer solutions, pyrogen-free water, ethyl alcohol, other non-toxic compatible lubricants, sodium lauryl sulfate, magnesium stearate, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents. Pharmaceutically acceptable carriers may also include preservatives and antioxidants.

[0119] In an embodiment, the method includes providing an adjuvant. The adjuvant may be any adjuvant. As used herein, the term "adjuvant" refers to a pharmacological or immunological agent which when administered with an antigen nonspecifically enhances the recipient's response to that antigen. The adjuvant may be but is not limited to Alum, oil-in-water nannoemulsion (MF59.TM.), the glycolipid monophosphoryl lipid A (MPL.RTM.), virus-like particles (VLP), the cholera toxin B subunit (CTB), montanides ISA51 and ISA720, saponines Quil-A, ISCOM and QS-21, syntax adjuvant formulation (SAF), muramyl dipeptides (MDP), immunostimulatory oligonucleotides, TLR ligands, Escherichia coli heat-labile exotoxin, or lipid-based adjuvants (Vajdy et al., 2004 Imm and Cell Biol 82:617; Schroder et al., 1999 Vaccine 17:2096, all of which are incorporated by reference herein as if fully set forth).

[0120] In an embodiment, a method of protecting a subject against anthrax infection is provided. The method may include providing a composition that includes a recombinant protein. The recombinant protein may include a toxin binding ligand fused to a carrier-protein. The carrier protein may be a protein selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. The composition may effective in preventing or reducing at least one symptom of an anthrax infection in a subject. The method may also include administering the composition to the subject in need thereof.

[0121] In an embodiment of the method, the toxin binding ligand may be a capillary morphogenesis protein 2. The toxin binding ligand may be a PA-LF protein. The toxin binding ligand may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 24 (A1-CMG2), SEQ ID NO: 25 (A2/PA-LF) and SEQ ID NO: 60 (PgA1-5).

[0122] In an embodiment of the method, the carrier-protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 26 (B1), SEQ ID NO: 27 (B2), SEQ ID NO: 28 (B3), SEQ ID NO: 29 (B4) and SEQ ID NO: 30 (B5).

[0123] In an embodiment of the method, the recombinant protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 31 (PgA1-1:B1-1).

[0124] In an embodiment, the subject may be a mammal. The mammal may be but is not limited to an agricultural animal, an equine, a high value zoo animal, or a research animal. The mammal may be a human.

[0125] In an embodiment, the step of administering the composition may include a route selected from the group consisting of: intravenous, intramuscular, intraperitoneal, intradermal, mucosal, cutaneous, and subcutaneous. The step of administering may be achieved through intranasal administration. The intranasal administration may include inhalation or nasal drops. A therapeutic composition may be administered to a recipient by any routes. A therapeutic composition may be introduced by injection, inhalation, oral, or intranasal route of administration. A therapeutic composition may be introduced by a parenteral or mucosal route of administration. Routes may include administering a composition orally, intrapulmonaryly, transdermally, rectally, intravaginally, intraperitoneally, intracisternally, and or ectopically. A mucosal route may include administering a therapeutic composition to any mucosal surface of the body of the recipient. Mucosal administration differs from "systemic" or "parenteral" administration. Systemic administration may include administering compositions to a non-mucosal surface, e.g., intraperitoneal, intramuscular, sub-, or transcutaneous, intra- or transdermal, or intravenous administration.

[0126] In an embodiment, the step of administering may be achieved by using a formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage. A therapeutic composition may be administered in liquid dosage forms. Liquid dosage forms may be prepared for oral, nasal, inhalation, or transdermal administration. Liquid dosage forms may include, but not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, and suspensions. Liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters or sorbitan, and mixtures of thereof. Besides inert diluents, the compositions may also include ingredients stimulating protein translocation via mucosal tissues and/or absorption/permeability enhancers including but not limited to bile salts, surfactants, fusidic acid derivates, phosphatidylcholines, cyclodextrines, alcohols, low molecular weight polyethylene glycol etc. Liquid dosage forms may be available in forms optimal for use with inhalator devices.

[0127] Dosage forms for topical or transdermal administration of a therapeutical composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalant, or patches. Powders and sprays may content therapeutic proteins admixed with excipients such as talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder, or mixture of these substances. Sprays may additionally contain customary propellants, for example, chlorofluorohydrocarbons. The therapeutic proteins may be admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be appropriate.

[0128] In an embodiment, administering the compositions may include a direct needle-free infusion of the raw, concentrated or partially purified extract of plants containing recombinant protein into human or non-human animal bloodstream. The step of administering may be achieved with the help of existing conventional devices and techniques enhancing absorption/permeability of biological surfaces. These devices or techniques may include but not limited to transdermal patches adapted to the purposes of this invention, or pulmonary delivery through an immunoglobulin transport pathway using conventional inhalators, intranasal spraying, or feeding the subject plant extract.

[0129] In an embodiment, administering of a plant-derived therapeutic composition may be a preventive treatment of subjects to promote emergency post-infection prophylaxis of a contact with the infectious agent. Administering of a plant-derived composition may be a therapeutic measure for neutralization anthrax toxin produced by bacterial pathogen Bacillus anthracis and minimizing complications associated with accumulation of deadly toxin in patients infected with the pathogen bacteria. Administering the plant-derived composition may be used for treatment of a variety of, symptoms and consequences of various forms of anthrax disease arising from infection with pathogenic bacteria Bacillus anthracis. Plant-derived therapeutic compositions may be useful to treat patients being in contact with anthrax toxin, pathogen, infected animal or human, belonging to a group of risk of biological weapon attack.

[0130] In an embodiment, the method may further include measuring cell viability in the presence of different concentrations of the anthrax toxin, wherein cell viability is a percentage of surviving cells protected by the antitoxin in comparison to the complete lysis in the control.

[0131] In an embodiment, the method may further include measuring survival of animals after challenging with lethal concentration of the anthrax toxin or B. anthracis spores, followed by administration of protective amounts of the recombinant antitoxin. The survival may be a percentage of live animals protected by the antitoxin in comparison to unprotected objects in the control.

[0132] A skilled person will recognize that many suitable variations of the methods may be substituted for or used in addition to those described above and in the claims It should be understood that the implementation of other variations and modifications of the embodiments of the invention and its various aspects will be apparent to one skilled in the art, and that the invention is not limited by the specific embodiments described herein and in the claims. The present application mentions various patents, scientific articles, and other publications, each of which is hereby incorporated in its entirety by reference.

[0133] Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein. Further embodiments herein may be described by reference to any one of the appended claims following claim 1 and reading the chosen claim to depend from any one or more preceding claim.

EXAMPLES

[0134] The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below.

Example 1

Recombinant Antitoxin Proteins Optimized for Expression in Plants

[0135] A soluble extracellular domain (aa 35-220) of the human CMG2 protein, sCMG2 (component A1), capable of binding PA protein and neutralization of the anthrax toxin (Scobie et al., 2005) or a fusion of the component A1 with the human IgG1 Fc-fragment (component B1), resulting in a component A1-B1 recombinant antitoxin, were chosen to first to test in a transient plant expression system. Expression cassettes were designed with or without ER targeting and retention signals and two commercial affinity tags (c-myc and 6.times.His). All cassettes were sub-cloned into a plant transformation vector suitable for transient expression together with commercially available helper plasmids.

[0136] Further optimization of the human CMG2 extracellular domain (amino acids 35-220) included the removal of a native signal peptide, the transmembrane domain and the cytoplasmic tail and resulted in Component A1 (PgA1). Fusion of a human CMG2 soluble extracellular domain (aa 35-220) with a human IgG1 Fc-fragment (Component B1, PgB1) resulted in Component A1-B1 (PgA1-B1) constructs. All sequences were optimized in a stepwise fashion as described in Table 1 using ELISA and Western blots for experimental quantitative confirmation.

TABLE-US-00001 TABLE 1 Stepwise optimization of therapeutic proteins for expression in plants Variable parameter Outcome 1. Sequence analysis and Analysis of codon usage, mRNA genus/species-specific thermostability, cryptic intron splice optimization to improve sites, polyA signals, overall yields of evaluation and correction of sequences recombinant protein 2. Optimal days post-infiltration Expression at different time points post (transient transformation only) infiltration 3.ER localization signal choice Expression with and without targeting signal sequences 4.ER retention signal presence Expression with and without C- terminal HDEL (SEQ ID NO: 64)/ KDEL (SEQ ID NO: 65) peptide 5. Affinity tags selection Expression and recovery with commercially available tags (c-Myc and 6xHis) 6. N. benthamiana glycosylation Expression and functionality in a model in knock-out mutants (transient wild type plant and glycosylation transformation only) knockout mutants that confer mammalian type glycans

[0137] Using the strategies outlined in Table 1 the nucleic acid sequences encoding the sCMG2-based recombinant subunit antitoxin and the recombinant Fc-fusion antitoxin were optimized for a better plant-specific production.

[0138] Each of the expression cassettes encoding the recombinant polypeptides also contained specific restriction/ligation sites required for direct subcloning into the plasmid carrier. The 5' terminal region positioned at the NcoI site CCATGG was introduced in frame with the Kawasaki motif or Kozak-like sequence immediately before the initiation translation ATG codon 5'-gacaccATGG (SEQ ID NO: 39). The respective BglII and SacI sites were identified before and after the stop codon at the 3' terminal region AGATCTccaataaGAGCTC-3' (SEQ ID NO: 40) in both constructs. Additionally, the NotI site as follows: 5'-tcttGCGGCCGCagga-3' (SEQ ID NO: 41), was identified between Components A1 and B1 in the PgA1-B1 construct. These cloning sites were eliminated from the rest of the sequence body during the process of optimization.

[0139] PgA1 and PgA1-B1 expression cassettes were synthesized. These cassettes contained the synthetic anthrax toxin receptor sCMG2 or synthetic sCMG2 fused with the Fc fragment inserted into expression cassettes. The expression cassettes also contained C-terminus-specific tags, such as c-Myc and/or 6.times.His tags, and N-terminus plant-specific intracellular targeting signals. Two targeting signal sequences included the plant BAA gene encoding the amino acid sequence MANKHLSLSLFLVLLGLSASLASG (SEQ ID NO: 42) and the plant APBP1 gene, encoding the amino acid sequence MIVLSVGSASSSPIVVVFSVALLLFYFSETSLG (SEQ ID NO: 43). A flexible linker was introduced into the PgA1-B1 construct between the synthetic sCMG2 and the Fc fragment GGGSGNS (SEQ ID NO: 44). A short peptide was also introduced in front of the flexible linker to facilitate cleavage by a mammal serum protease, such as Thrombin LVPRGS (SEQ ID NO: 45), or Factor Xa protease IEGR (SEQ ID NO: 46).

[0140] The synthetic expression cassettes were introduced into plant transformation vectors harboring compatible cloning sites for NcoI (harbors ATG, first codon and without targeting signal peptide) and SacI. At the C-terminus of the expression cassettes, affinity tags such as c-Myc and/or 6.times.His were linked to endoplasmic reticulum (ER) retention signal HDEL (SEQ ID NO: 64) and inserted into the expression cassette immediately prior the stop codon. An Invitrogene/Geneart optimization with Gene with GeneOptimizer.RTM. sequence processing included the following parameters:

[0141] (i) Identification of the optimal sequence elements, such as restriction/ligation sites.

[0142] (ii) Elimination of cryptic splice sites and RNA destabilizing sequence elements for increased RNA stability.

[0143] (iii) Addition of RNA stabilizing sequence elements.

[0144] (iv) Codon optimization and G/C content adaptation for plant expression system.

[0145] (v) Intron removal.

[0146] (vi) Avoidance of templates compromising RNA secondary structures.

[0147] Additionally, regions with very high (>80%) or very low (<30%) GC content were avoided. For expression in plants, an average GC content of 58% was desirable. During the optimization process the following cis-acting sequence motifs were avoided: internal TATA-boxes, chi-sites and ribosomal entry sites. At-rich or GC-rich sequence stretches in codons, other RNA instability motifs, repeat sequences and RNAs secondary structures, cryptic splice donor and acceptor sites in eukaryotes. In some cases, negative cis-acting motifs were not removed.

[0148] The optimization considered successful if negative cis-acting sites which may negatively influence expression were eliminated, and GC content was adjusted to prolong mRNA half-life.

[0149] The expression cassettes included nucleic acid sequences encoding the soluble form of extracellular domain (aa 35-220) of human CMG2 protein (sCMG2, component A1) capable of binding PA protein and neutralization of the anthrax toxin; a fusion of the component A1 with human IgG1 Fc-fragment (component B1), resulting in a recombinant antitoxin, a component A1-B1. Expression cassettes were designed and tested in commercial plasmids for stable transformation/expression

[0150] PgA1 expression cassettes. PgA1 included a soluble extracellular domain of human CMG2 protein, sCMG2 (component A1), capable of binding PA protein and neutralization of the anthrax toxin that has no native signal peptide (amino acids 35-220), transmembrane domain and cytoplasmic tail. These elements were optimized for expression in plants and named sCMG2. PgA1 was designed for transient and stable plant transformation/expression. A 581 bp nucleic acid sequence of PgA1-1(SEQ ID NO: 1) encoding the PgA protein (190 aa) designed for cloning in the MagnICON expression vectors is as follows:

TABLE-US-00002 (SEQ ID NO: 1) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGAAGATCTCCAATAAGAGCTC

[0151] A 575 bp nucleic acid sequence of PgA1-2 (SEQ ID NO: 2) encoding the PgA1 protein (190 aa) designed for stable expression is as follows:

TABLE-US-00003 (SEQ ID NO: 2) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGAAGATCTCCAATAA

[0152] PgA1-B1 expression cassettes. A soluble extracellular domain of human CMG2 protein, component A1 as described above, fused with human IgG1 Fc-fragment (component B1), resulting in a component A1-B1 recombinant antitoxin optimized for expression in plants. PgA1-B1 expression cassettes were designed for transient and stable plant transformation.

[0153] A 1286 bp nucleic acid sequence of PgA1-3:B1-3 (SEQ ID NO: 16) encoding the PgA1-B1 protein (425 aa) designed for transient expression vectors is as follows:

TABLE-US-00004 (SEQ ID NO: 16) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACAAGACCC ACACCTGCCCTCCTTGCCCTGCTCCTGAGCTCCTCGGTGGTCCTTCTGTC TTCCTCTTCCCTCCTAAGCCTAAGGACACCCTCATGATCTCTCGTACCCC TGAGGTCACCTGCGTCGTCGTCGACGTCTCTCACGAGGACCCTGAGGTCA AGTTCAACTGGTACGTCGACGGTGTCGAGGTCCACAACGCTAAGACCAAG CCTCGTGAGGAGCAGTACAACTCTACCTACCGTGTCGTCTCTGTCCTCAC CGTCCTCCACCAGGACTGGCTCAACGGTAAGGAGTACAAGTGCAAGGTCT CTAACAAGGCTCTCCCTGCTCCTATCGAGAAGACCATCTCTAAGGCTAAG GGTCAGCCTCGTGAGCCTCAGGTCTACACCCTCCCTCCTTCTCGTGAGGA GATGACCAAGAACCAGGTCTCTCTCACCTGCCTCGTCAAGGGTTTCTACC CTTCTGACATCGCTGTCGAGTGGGAGTCTAACGGTCAGCCTGAGAACAAC TACAAGACCACCCCTCCTGTCCTCGACTCTGACGGTTCTTTCTTCCTCTA CTCTAAGCTCACCGTCGACAAGTCTCGTTGGCAGCAGGGTAACGTCTTCT CTTGCTCTGTCATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCT CTCTCTCTCTCTCCTGGTAAGGACCTCTAAGAGCTC

[0154] A 1283 bp nucleic acid sequence of PgA1-4:B1-4 (SEQ ID NO: 17) encoding the PgA1-B1 protein (442 aa) designed for stable transformation is as follows:

TABLE-US-00005 (SEQ ID NO: 17) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACAAGACCC ACACCTGCCCTCCTTGCCCTGCTCCTGAGCTCCTCGGTGGTCCTTCTGTC TTCCTCTTCCCTCCTAAGCCTAAGGACACCCTCATGATCTCTCGTACCCC TGAGGTCACCTGCGTCGTCGTCGACGTCTCTCACGAGGACCCTGAGGTCA AGTTCAACTGGTACGTCGACGGTGTCGAGGTCCACAACGCTAAGACCAAG CCTCGTGAGGAGCAGTACAACTCTACCTACCGTGTCGTCTCTGTCCTCAC CGTCCTCCACCAGGACTGGCTCAACGGTAAGGAGTACAAGTGCAAGGTCT CTAACAAGGCTCTCCCTGCTCCTATCGAGAAGACCATCTCTAAGGCTAAG GGTCAGCCTCGTGAGCCTCAGGTCTACACCCTCCCTCCTTCTCGTGAGGA GATGACCAAGAACCAGGTCTCTCTCACCTGCCTCGTCAAGGGTTTCTACC CTTCTGACATCGCTGTCGAGTGGGAGTCTAACGGTCAGCCTGAGAACAAC TACAAGACCACCCCTCCTGTCCTCGACTCTGACGGTTCTTTCTTCCTCTA CTCTAAGCTCACCGTCGACAAGTCTCGTTGGCAGCAGGGTAACGTCTTCT CTTGCTCTGTCATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCT CTCTCTCTCTCTCCTGGTAAGGACCTCGATCTCCAAAAGCTTATTAGCGA GGAGGATCTTCATCACCATCACCATCACTAAGAGCTC

[0155] PgA1-B2 expression cassettes. A soluble extracellular domain of human CMG2 protein, component A1 as described above, fused with human IgA1 Fc-fragment (component B2), resulting in a component A1-B2 recombinant antitoxin optimized for expression in plants. PgA1-B2 expression cassettes were designed for transient and stable plant transformation/expression.

[0156] A 1377 bp nucleic acid sequence of PgA1-3:B2-3(SEQ ID NO: 18) encoding the PgA1-B2.sub.Nt protein (458 aa) designed for transient expression vectors is as follows:

TABLE-US-00006 (SEQ ID NO: 18) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACGTCACCG TCCCTTGCCCTGTCCCTTCTACCCCTCCTACCCCTTCTCCTTCTACCCCT CCTACCCCTTCTCCTTCTTGCTGCCACCCTCGTCTCTCTCTCCACCGTCC TGCTCTCGAGGACCTCCTCCTCGGTTCTGAGGCTAACCTCACCTGCACCC TCACCGGTCTCCGTGACGCTTCTGGTGTCACCTTCACCTGGACCCCTTCT TCTGGTAAGTCTGCTGTCCAGGGTCCTCCTGAGCGTGACCTCTGCGGTTG CTACTCTGTCTCTTCTGTCCTCCCTGGTTGCGCTGAGCCTTGGAACCACG GTAAGACCTTCACCTGCACCGCTGCTTACCCTGAGTCTAAGACCCCTCTC ACCGCTACCCTCTCTAAGTCTGGTAACACCTTCCGTCCTGAGGTCCACCT CCTCCCTCCTCCTTCTGAGGAGCTCGCTCTCAACGAGCTCGTCACCCTCA CCTGCCTCGCTCGTGGTTTCTCTCCTAAGGACGTCCTCGTCCGTTGGCTC CAGGGTTCTCAGGAGCTCCCTCGTGAGAAGTACCTCACCTGGGCTTCTCG TCAGGAGCCTTCTCAGGGTACCACCACCTTCGCTGTCACCTCTATCCTCC GTGTCGCTGCTGAGGACTGGAAGAAGGGTGACACCTTCTCTTGCATGGTC GGTCACGAGGCTCTCCCTCTCGCTTTCACCCAGAAGACCATCGACCGTCT CGCTGGTAAGCCTACCCACGTCAACGTCTCTGTCGTCATGGCTGAGGTCG ACGGTACCTGCTACCCAATAAGAGCTC

[0157] A 1273 bp nucleic acid sequence of PgA1-4:B2-4(SEQ ID NO: 19) encoding the PgA1-B2 protein (454 aa) designed for stable transformation vectors is as follows:

TABLE-US-00007 (SEQ ID NO: 19) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACGTCACCG TCCCTTGCCCTGTCCCTTCTACCCCTCCTACCCCTTCTCCTTCTACCCCT CCTACCCCTTCTCCTTCTTGCTGCCACCCTCGTCTCTCTCTCCACCGTCC TGCTCTCGAGGACCTCCTCCTCGGTTCTGAGGCTAACCTCACCTGCACCC TCACCGGTCTCCGTGACGCTTCTGGTGTCACCTTCACCTGGACCCCTTCT TCTGGTAAGTCTGCTGTCCAGGGTCCTCCTGAGCGTGACCTCTGCGGTTG CTACTCTGTCTCTTCTGTCCTCCCTGGTTGCGCTGAGCCTTGGAACCACG GTAAGACCTTCACCTGCACCGCTGCTTACCCTGAGTCTAAGACCCCTCTC ACCGCTACCCTCTCTAAGTCTGGTAACACCTTCCGTCCTGAGGTCCACCT CCTCCCTCCTCCTTCTGAGGAGCTCGCTCTCAACGAGCTCGTCACCCTCA CCTGCCTCGCTCGTGGTTTCTCTCCTAAGGACGTCCTCGTCCGTTGGCTC CAGGGTTCTCAGGAGCTCCCTCGTGAGAAGTACCTCACCTGGGCTTCTCG TCAGGAGCCTTCTCAGGGTACCACCACCTTCGCTGTCACCTCTATCCTCC GTGTCGCTGCTGAGGACTGGAAGAAGGGTGACACCTTCTCTTGCATGGTC GGTCACGAGGCTCTCCCTCTCGCTTTCACCCAGAAGACCATCGACCGTCT CGCTGGTAAGCCTACCCACGTCAACGTCTCTGTCGTCATGGCTGAGGTCG ACGGTACCTGCTACTAAAGATCT

[0158] PgA1-B3 expression cassettes. A soluble extracellular domain of human CMG2 protein, component A1 as above, fused with human IgM Fc-fragment (component B3), resulting in a component A1-B3 recombinant antitoxin optimized for expression in plants. PgA1-B3 expression cassettes were designed for transient and stable plant transformation and expression.

[0159] A 1662 bp nucleic acid sequence of PgA1-3:B3-3 (SEQ ID NO: 20) encoding the PgA1-B3 protein (553 aa) for transient expression vectors is as follows:

TABLE-US-00008 (SEQ ID NO: 20) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGTCCCTCTCC CTGTCATCGCTGAGCTCCCTCCTAAGGTCTCTGTCTTCGTCCCTCCTCGT GACGGTTTCTTCGGTAACCCTCGTAAGTCTAAGCTCATCTGCCAGGCTAC CGGTTTCTCTCCTCGTCAGATCCAGGTCTCTTGGCTCCGTGAGGGTAAGC AGGTCGGTTCTGGTGTCACCACCGACCAGGTCCAGGCTGAGGCTAAGGAG TCTGGTCCTACCACCTACAAGGTCACCTCTACCCTCACCATCAAGGAGTC TGACTGGCTCTCTCAGTCTATGTTCACCTGCCGTGTCGACCACCGTGGTC TCACCTTCCAGCAGAACGCTTCTTCTATGTGCGTCCCTGACCAGGACACC GCTATCCGTGTCTTCGCTATCCCTCCTTCTTTCGCTTCTATCTTCCTCAC CAAGTCTACCAAGCTCACCTGCCTCGTCACCGACCTCACCACCTACGACT CTGTCACCATCTCTTGGACCCGTCAGAACGGTGAGGCTGTCAAGACCCAC ACCAACATCTCTGAGTCTCACCCTAACGCTACCTTCTCTGCTGTCGGTGA GGCTTCTATCTGCGAGGACGACTGGAACTCTGGTGAGCGTTTCACCTGCA CCGTCACCCACACCGACCTCCCTTCTCCTCTCAAGCAGACCATCTCTCGT CCTAAGGGTGTCGCTCTCCACCGTCCTGACGTCTACCTCCTCCCTCCTGC TCGTGAGCAGCTCAACCTCCGTGAGTCTGCTACCATCACCTGCCTCGTCA CCGGTTTCTCTCCTGCTGACGTCTTCGTCCAGTGGATGCAGCGTGGTCAG CCTCTCTCTCCTGAGAAGTACGTCACCTCTGCTCCTATGCCTGAGCCTCA GGCTCCTGGTCGTTACTTCGCTCACTCTATCCTCACCGTCTCTGAGGAGG AGTGGAACACCGGTGAGACCTACACCTGCGTCGTCGCTCACGAGGCTCTC CCTAACCGTGTCACCGAGCGTACCGTCGACAAGTCTACCGGTAAGCCTAC CCTCTACAACGTCTCTCTCGTCATGTCTGACACCGCTGGTACCTGCTACC CAATAAGAGCTC

[0160] A 1658 bp nucleic acid sequence of PgA-4:B3-4 (SEQ ID NO: 21) encoding the PgA1-B3 protein (549 aa) for stable transformation vectors is as follows:

TABLE-US-00009 (SEQ ID NO: 21) CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGTCCCTCTCC CTGTCATCGCTGAGCTCCCTCCTAAGGTCTCTGTCTTCGTCCCTCCTCGT GACGGTTTCTTCGGTAACCCTCGTAAGTCTAAGCTCATCTGCCAGGCTAC CGGTTTCTCTCCTCGTCAGATCCAGGTCTCTTGGCTCCGTGAGGGTAAGC AGGTCGGTTCTGGTGTCACCACCGACCAGGTCCAGGCTGAGGCTAAGGAG TCTGGTCCTACCACCTACAAGGTCACCTCTACCCTCACCATCAAGGAGTC TGACTGGCTCTCTCAGTCTATGTTCACCTGCCGTGTCGACCACCGTGGTC TCACCTTCCAGCAGAACGCTTCTTCTATGTGCGTCCCTGACCAGGACACC GCTATCCGTGTCTTCGCTATCCCTCCTTCTTTCGCTTCTATCTTCCTCAC CAAGTCTACCAAGCTCACCTGCCTCGTCACCGACCTCACCACCTACGACT CTGTCACCATCTCTTGGACCCGTCAGAACGGTGAGGCTGTCAAGACCCAC ACCAACATCTCTGAGTCTCACCCTAACGCTACCTTCTCTGCTGTCGGTGA GGCTTCTATCTGCGAGGACGACTGGAACTCTGGTGAGCGTTTCACCTGCA CCGTCACCCACACCGACCTCCCTTCTCCTCTCAAGCAGACCATCTCTCGT CCTAAGGGTGTCGCTCTCCACCGTCCTGACGTCTACCTCCTCCCTCCTGC TCGTGAGCAGCTCAACCTCCGTGAGTCTGCTACCATCACCTGCCTCGTCA CCGGTTTCTCTCCTGCTGACGTCTTCGTCCAGTGGATGCAGCGTGGTCAG CCTCTCTCTCCTGAGAAGTACGTCACCTCTGCTCCTATGCCTGAGCCTCA GGCTCCTGGTCGTTACTTCGCTCACTCTATCCTCACCGTCTCTGAGGAGG AGTGGAACACCGGTGAGACCTACACCTGCGTCGTCGCTCACGAGGCTCTC CCTAACCGTGTCACCGAGCGTACCGTCGACAAGTCTACCGGTAAGCCTAC CCTCTACAACGTCTCTCTCGTCATGTCTGACACCGCTGGTACCTGCTACT AAAGATCT

[0161] PgB4-A1 expression cassettes. A coat protein VP2 of the human polyoma virus (JC virus) capable of self-assembly into VLPs (component B4) was fused with the soluble extracellular domain of human CMG2 protein, (component A1), resulting in a component B4-A1 recombinant antitoxin. PgB4-A1 expression cassettes were optimized for transient and stable plant transformation and expression.

[0162] A 1064 bp nucleic acid sequence of PgB4-2: A1-3 (SEQ ID NO: 22) encoding the PgB4-A1 protein (354 aa) for transient or stable plant transformation/expression vectors is as follows:

TABLE-US-00010 (SEQ ID NO: 22) CCATGGGTGCTGCTCTCGCTCTCCTCGGTGACCTCGTCGCTACCGTCTCT GAGGCTGCTGCTGCTACCGGTTTCTCTGTCGCTGAGATCGCTGCTGGTGA GGCTGCTGCTACCATCGAGGTCGAGATCGCTTCTCTCGCTACCGTCGAGG GTATCACCTCTACCTCTGAGGCTATCGCTGCTATCGGTCTCACCCCTGAG ACCTACGCTGTCATCACCGGTGCTCCTGGTGCTGTCGCTGGTTTCGCTGC TCTCGTCCAGACCGTCACCGGTGGTTCTGCTATCGCTCAGCTCGGTTACC GTTTCTTCGCTGACTGGGACCACAAGGTCTCTACCGTCGGTCTCTTCCAG CAGCCTGCTATGGCTCTCCAGCTCTTCAACCCTGAGGACTACTACGACAT CCTCTTCCCTGGTGTCAACGCTTTCGTCAACAACATCCACTACCTCGACC CTCGTCACTGGGGTCCTTCTCTCTTCTCTACCATCTCTCAGGCTTTCTGG AACCTCGTCCGTGACGACCTCCCTGCTCTCACCTCTCAGGAGATCCAGCG TCGTACCCAGAAGCTCTTCGTCGAGTCTCTCGCTCGTTTCCTCGAGGAGA CCACCTGGGCTATCGTCAACTCTCCTGCTAACCTCTACAACTACATCTCT GACTACTACTCTCGTCTCTCTCCTGTCCGTCCTTCTATGGTCCGTCAGGT CGCTCAGCGTGAGGGTACCTACATCTCTTTCGGTCACTCTTACACCCAGT CTATCGACGACGCTGACTCTATCCAGGAGGTCACCCAGCGTCTCGACCTC AAGACCCCTAACGTCCAGTCTGGTGAGTTCATCGAGCGTTCTATCGCTCC TGGTGGTGCTAACCAGCGTTCTGCTCCTCAGTGGATGCTCCCTCTCCTCC TCGGTCTCTACGGTACCGTCACCCCTGCTCTCGAGGCTTACGAGGACGGT CCTAACAAGAAGAAGCGTCGTAAGGAGGGTCCTCGTGCTTCTTCTAAGAC CTCTTACAAGCGTCGTTCTCGTTCTTCTCGTTCTGGAGGTGGATCTGGAA ATTCTGCGGCCGCA

[0163] PgB5-A1 expression cassettes. Arabidopsis thaliana oleosin (OLE) protein is capable of targeting itself, as well as, another covalently fused protein to plant cell oil bodies, thus providing accumulation of the target protein in plant lipid fraction making it easily extractable ("component B5"). OLE protein was used for cloning as a fusion with CMG2 ("component A1"), resulting in "component B5-A1" recombinant antitoxin, and named PgB5-A1. PgB5-A1 constructs were optimized for transient and stable plant transformation/expression.

[0164] A 530 bp nucleic acid sequence PgB5-2: A1-3 (SEQ ID NO: 22) encoding the PgB5 protein (176 aa) for cloning as a fusion with the ATR-encoding DNA fragment in transient or stable plant transformation/expression vectors is as follows:

TABLE-US-00011 (SEQ ID NO: 22) CCATGGCCGACACGGCCAGGGGCACGCACCACGACATCATCGGCAGGGAC CAGTACCCGATGATGGGCAGGGACAGGGACCAGTACCAGATGTCCGGCAG GGGCTCCGACTACTCCAAGTCCAGGCAGATCGCCAAGGCCGCCACGGCCG TGACGGCCGGCGGCTCCCTCCTCGTGCTCTCCTCCCTCACGCTCGTGGGC ACGGTGATCGCCCTCACGGTGGCCACGCCGCTCCTCGTGATCTTCTCCCC GATCCTCGTGCCGGCCCTCATCACGGTGGCCCTCCTCATCACGGGCTTCC TCTCCTCCGGCGGCTTCGGCATCGCCGCCATCACGGTGTTCTCCTGGATC TACAAGTACGCCACGGGCGAGCACCCGCAGGGCTCCGACAAGCTCGACTC CGCCAGGATGAAGCTCGGCTCCAAGGCCCAGGACCTCAAGGACAGGGCCC AGTACTACGGCCAGCAGCACACGGGCGGCGAGCACGACAGGGACAGGACG AGGGGCGGCCAGCACACGACGGCGGCCGCA

[0165] PgA2 expression cassettes. PA-binding domain of LF (LFn, amino acids 28-263) of the B. anthracis three-component anthrax toxin (component A2), capable of binding the PA region was optimized for expression in plants. PgA2 designs for transient and stable plant transformation/expression are shown as follows.

[0166] A 722 bp nucleic acid sequence of PgA2-1(SEQ ID NO: 3) encoding the PgA2 protein (237 aa) for cloning in transient expression vector is as follows:

TABLE-US-00012 (SEQ ID NO: 3) CCATGGGTGACGTCGGTATGCACGTCAAGGAGAAGGAGAAGAACAAGGAC GAGAACAAGCGTAAGGACGAGGAGCGTAACAAGACCCAGGAGGAGCACCT CAAGGAGATCATGAAGCACATCGTCAAGATCGAGGTCAAGGGTGAGGAGG CTGTCAAGAAGGAGGCTGCTGAGAAGCTCCTCGAGAAGGTCCCTTCTGAC GTCCTCGAGATGTACAAGGCTATCGGTGGTAAGATCTACATCGTCGACGG TGACATCACCAAGCACATCTCTCTCGAGGCTCTCTCTGAGGACAAGAAGA AGATCAAGGACATCTACGGTAAGGACGCTCTCCTCCACGAGCACTACGTC TACGCTAAGGAGGGTTACGAGCCTGTCCTCGTCATCCAGTCTTCTGAGGA CTACGTCGAGAACACCGAGAAGGCTCTCAACGTCTACTACGAGATCGGTA AGATCCTCTCTCGTGACATCCTCTCTAAGATCAACCAGCCTTACCAGAAG TTCCTCGACGTCCTCAACACCATCAAGAACGCTTCTGACTCTGACGGTCA GGACCTCCTCTTCACCAACCAGCTCAAGGAGCACCCTACCGACTTCTCTG TCGAGTTCCTCGAGCAGAACTCTAACGAGGTCCAGGAGGTCTTCGCTAAG GCTTTCGCTTACTACATCGAGCCTCAGCACCGTGACGTCCTCCAGCTCTA CGCTCCTGAGGCTTAAGAGCTC

[0167] A 728 bp nucleic acid sequence of PgA2-2(SEQ ID NO: 4) encoding the PgA2 protein (241 aa) for stable plant transformation vectors is as follows:

TABLE-US-00013 SEQ ID NO: 4) CCATGGGTGACGTCGGTATGCACGTCAAGGAGAAGGAGAAGAACAAGGAC GAGAACAAGCGTAAGGACGAGGAGCGTAACAAGACCCAGGAGGAGCACCT CAAGGAGATCATGAAGCACATCGTCAAGATCGAGGTCAAGGGTGAGGAGG CTGTCAAGAAGGAGGCTGCTGAGAAGCTCCTCGAGAAGGTCCCTTCTGAC GTCCTCGAGATGTACAAGGCTATCGGTGGTAAGATCTACATCGTCGACGG TGACATCACCAAGCACATCTCTCTCGAGGCTCTCTCTGAGGACAAGAAGA AGATCAAGGACATCTACGGTAAGGACGCTCTCCTCCACGAGCACTACGTC TACGCTAAGGAGGGTTACGAGCCTGTCCTCGTCATCCAGTCTTCTGAGGA CTACGTCGAGAACACCGAGAAGGCTCTCAACGTCTACTACGAGATCGGTA AGATCCTCTCTCGTGACATCCTCTCTAAGATCAACCAGCCTTACCAGAAG TTCCTCGACGTCCTCAACACCATCAAGAACGCTTCTGACTCTGACGGTCA GGACCTCCTCTTCACCAACCAGCTCAAGGAGCACCCTACCGACTTCTCTG TCGAGTTCCTCGAGCAGAACTCTAACGAGGTCCAGGAGGTCTTCGCTAAG GCTTTCGCTTACTACATCGAGCCTCAGCACCGTGACGTCCTCCAGCTCTA CGCTCCTGAGGCTGGAGATCTCCAATAA

[0168] PgA2-B1 expression cassettes. PA-binding domain of LF (LF, amino acids 28-263) of the B. anthracis three-component anthrax toxin (component A2), capable of binding the PA region was optimized for expression in plants as fusion with human IgG1 Fc-fragment (component B1), resulting in a component A2-B1 recombinant antitoxin. PgA2-B1 designs for transient and stable plant transformation/expression are shown as follows.

[0169] A 721 bp nucleic acid sequence encoding PgA2-4: B1-3 (SEQ ID NO: 37) the PgA2 protein (239 aa) for cloning as a fusion with the Fc-encoding DNA fragment in the transient (as in SEQ ID NO: 16) or stable transformation/expression vectors (as in SEQ ID NO: 17):

TABLE-US-00014 (SEQ ID NO: 37) CCATGGGTGACGTCGGTATGCACGTCAAGGAGAAGGAGAAGAACAAGGAC GAGAACAAGCGTAAGGACGAGGAGCGTAACAAGACCCAGGAGGAGCACCT CAAGGAGATCATGAAGCACATCGTCAAGATCGAGGTCAAGGGTGAGGAGG CTGTCAAGAAGGAGGCTGCTGAGAAGCTCCTCGAGAAGGTCCCTTCTGAC GTCCTCGAGATGTACAAGGCTATCGGTGGTAAGATCTACATCGTCGACGG TGACATCACCAAGCACATCTCTCTCGAGGCTCTCTCTGAGGACAAGAAGA AGATCAAGGACATCTACGGTAAGGACGCTCTCCTCCACGAGCACTACGTC TACGCTAAGGAGGGTTACGAGCCTGTCCTCGTCATCCAGTCTTCTGAGGA CTACGTCGAGAACACCGAGAAGGCTCTCAACGTCTACTACGAGATCGGTA AGATCCTCTCTCGTGACATCCTCTCTAAGATCAACCAGCCTTACCAGAAG TTCCTCGACGTCCTCAACACCATCAAGAACGCTTCTGACTCTGACGGTCA GGACCTCCTCTTCACCAACCAGCTCAAGGAGCACCCTACCGACTTCTCTG TCGAGTTCCTCGAGCAGAACTCTAACGAGGTCCAGGAGGTCTTCGCTAAG GCTTTCGCTTACTACATCGAGCCTCAGCACCGTGACGTCCTCCAGCTCTA CGCTCCTGAGGCTGCGGCCGC

[0170] Helper element (component C)/PgC1 expression cassettes. Human immunoglobulin J polypeptide, linker protein for immunoglobulin Alpha and Mu polypeptides (IgJ, amino acids 34-159) (component C1), that has no native signal peptide (amino acids 1-33), was optimized for expression in plants. IgJ is capable of helping IgA and/or IgM subunits to self-assemble into quaternary structure. PgC1 cassettes were designed for transient and stable plant transformation/expression.

[0171] A 395 bp nucleic acid sequence of PgC1-2 (SEQ ID NO: 32) encoding the PgC1 protein (128 aa) for transient expression vectors is as follows:

TABLE-US-00015 (SEQ ID NO: 32) CCATGGGAAAGTGCAAGTGCGCTCGTATCACCTCTCGTATCATCCGTTCT TCTGAGGACCCTAACGAGGACATCGTCGAGCGTAACATCCGTATCATCGT CCCTCTCAACAACCGTGAGAACATCTCTGACCCTACCTCTCCTCTCCGTA CCCGTTTCGTCTACCACCTCTCTGACCTCTGCAAGAAGTGCGACCCTACC GAGGTCGAGCTCGACAACCAGATCGTCACCGCTACCCAGTCTAACATCTG CGACGAGGACTCTGCTACCGAGACCTGCTACACCTACGACCGTAACAAGT GCTACACCGCTGTCGTCCCTCTCGTCTACGGTGGTGAGACCAAGATGGTC GAGACCGCTCTCACCCCTGACGCTTGCTACCCTGACTAAGAGCTC

[0172] A 401 bp nucleic acid sequence of PgC1-3 (SEQ ID NO: 33) encoding the PgC1.sub.Nt protein (132 aa) for stable transformation vectors is as follows:

TABLE-US-00016 (SEQ ID NO: 33) CCATGGGAAAGTGCAAGTGCGCTCGTATCACCTCTCGTATCATCCGTTCT TCTGAGGACCCTAACGAGGACATCGTCGAGCGTAACATCCGTATCATCGT CCCTCTCAACAACCGTGAGAACATCTCTGACCCTACCTCTCCTCTCCGTA CCCGTTTCGTCTACCACCTCTCTGACCTCTGCAAGAAGTGCGACCCTACC GAGGTCGAGCTCGACAACCAGATCGTCACCGCTACCCAGTCTAACATCTG CGACGAGGACTCTGCTACCGAGACCTGCTACACCTACGACCGTAACAAGT GCTACACCGCTGTCGTCCCTCTCGTCTACGGTGGTGAGACCAAGATGGTC GAGACCGCTCTCACCCCTGACGCTTGCTACCCTGACGCAGATCTCCAATA A

[0173] VLP helper element/PgC2 expression cassettes. A coat protein VP1 of human polyoma virus capable of self-assembling into VLPs and necessary for the inclusion of JC virus VP2 protein into the same VLP assembly ("component C2"), resulting in a component C2, was optimized for expression in plants. PgC2 design for Magnifection and stable plant transformation is shown as follows.

[0174] A 1133 bp nucleic acid PgC2-2 (SEQ ID NO: 34) encoding the PgC2 protein (374 aa) for cloning in MagnICON expression vector or in pIV1.2/pIV1.3 Impact Vectors (Plant Research International, Wageningen, The Netherlands):

TABLE-US-00017 (SEQ ID NO: 34) CCATGGCCCCAACAAAGAGAAAAGGAGAAAGGAAGGACCCAGTGCAAGTT CCAAAACTTCTCATAAGAGGAGGAGTAGAAGTTCTTGAAGTTAAAACTGG AGTTGACTCAATTACAGAGGTAGAATGCTTCTTAACTCCAGAAATGGGTG ACCCAGATGAGCATCTTAGGGGTTTTAGTAAGTCAATATCTATATCAGAT ACATTTGAAAGTGACTCCCCAAATAGGGACATGCTTCCTTGTTACAGTGT GGCCAGGATTCCACTACCTAATCTAAATGAGGATCTAACTTGTGGAAATA TACTCATGTGGGAGGCTGTGACATTAAAGACTGAGGTTATAGGAGTGACA AGTTTGATGAATGTGCATTCTAATGGTCAAGCAACTCATGACAATGGTGC AGGTAAGCCAGTGCAGGGTACAAGTTTTCATTTCTTTTCTGTTGGAGGTG AGGCTTTAGAATTACAGGGAGTGCTTTTTAATTACAGAACAAAGTACCCA GATGGAACAATTTTTCCAAAGAATGCCACAGTGCAATCTCAAGTCATGAA CACAGAGCATAAGGCGTACCTAGATAAGAACAAAGCATATCCTGTTGAAT GTTGGGTTCCTGATCCAACTAGAAATGAAAACACAAGATATTTTGGTACA CTAACAGGAGGAGAAAATGTTCCTCCAGTTCTTCATATAACAAACACTGC CACAACAGTGTTGCTTGATGAATTTGGTGTTGGACCACTTTGTAAAGGTG ACAACTTATACTTGTCAGCTGTTGATGTCTGTGGTATGTTTACAAACAGG TCTGGTTCCCAGCAGTGGAGAGGACTCTCCAGATATTTTAAGGTGCAGCT AAGGAAGAGGAGGGTTAAGAACCCATACCCAATTTCTTTCCTTCTTACTG ATTTGATTAACAGAAGGACTCCTAGAGTTGATGGACAGCCTATGTATGGT ATGGATGCTCAAGTAGAGGAGGTTAGAGTTTTTGAGGGAACAGAGGAGCT TCCAGGAGACCCAGACATGATGAGATACGTTGACAAATATGGACAGTTGC AGACAAAGATGCTGGCGGCCGCAGATCTCCAAAAGCTTATTAGCGAGGAG GATCTTCATCACCATCACCATCACTAAGAGCTC

[0175] PgB1, PgB2, and PgB2 expression cassettes. Human IgG1, IgA1, and IgM Fc-fragment (components B1, B2, and B3) polypeptides were used as carrier proteins. B1, B2 and B3 constructs were optimized for transient and stable plant transformation/expression.

[0176] A 687 bp nucleic acid sequence of PgB1-1(SEQ ID NO: 6) encoding the PgB1 polypeptide (229 aa) for cloning as a fusion with A1 or A2 Component in transient or stable plant transformation/expression vectors is as follows:

TABLE-US-00018 (SEQ ID NO: 6) GATAAAACTCATACTTGTCCTCCTTGTCCTGCTCCTGAACTTCTTGGTGG TCCTTCTGTTTTTCTTTTTCCTCCTAAACCTAAAGATACTCTTATGATTT CTCGTACTCCTGAAGTTACTTGTGTTGTTGTTGATGTTTCTCATGAAGAT CCTGAAGTTAAATTTAATTGGTATGTTGATGGTGTTGAAGTTCATAATGC TAAAACTAAACCTCGTGAAGAACAATATAATTCTACTTATCGTGTTGTTT CTGTTCTTACTGTTCTTCATCAAGATTGGCTTAATGGTAAAGAATATAAA TGTAAAGTTTCTAATAAAGCTCTTCCTGCTCCTATTGAAAAAACTATTTC TAAAGCTAAAGGTCAACCTCGTGAACCTCAAGTTTATACTCTTCCTCCTT CTCGTGAAGAAATGACTAAAAATCAAGTTTCTCTTACTTGTCTTGTTAAA GGTTTTTATCCTTCTGATATTGCTGTTGAATGGGAATCTAATGGTCAACC TGAAAATAATTATAAAACTACTCCTCCTGTTCTTGATTCTGATGGTTCTT TTTTTCTTTATTCTAAACTTACTGTTGATAAATCTCGTTGGCAACAAGGT AATGTTTTTTCTTGTTCTGTTATGCATGAAGCTCTTCATAATCATTATAC TCAAAAATCTCTTTCTCTTTCTCCTGGTAAAGATCTT

[0177] A 774 bp nucleic acid sequence of PgB2-1 (SEQ ID NO: 7) encoding the PgB2 polypeptide (258 aa) for cloning as a fusion with A1 or A2 Component in transient or stable plant transformation/expression vectors is as follows:

TABLE-US-00019 (SEQ ID NO: 7) GATGTTACTGTTCCTTGTCCTGTTCCTTCTACTCCTCCTACTCCTTCTCC TTCTACTCCTCCTACTCCTTCTCCTTCTTGTTGTCATCCTCGTCTTTCTC TTCATCGTCCTGCTCTTGAAGATCTTCTTCTTGGTTCTGAAGCTAATCTT ACTTGTACTCTTACTGGTCTTCGTGATGCTTCTGGTGTTACTTTTACTTG GACTCCTTCTTCTGGTAAATCTGCTGTTCAAGGTCCTCCTGAACGTGATC TTTGTGGTTGTTATTCTGTTTCTTCTGTTCTTCCTGGTTGTGCTGAACCT TGGAATCATGGTAAAACTTTTACTTGTACTGCTGCTTATCCTGAATCTAA AACTCCTCTTACTGCTACTCTTTCTAAATCTGGTAATACTTTTCGTCCTG AAGTTCATCTTCTTCCTCCTCCTTCTGAAGAACTTGCTCTTAATGAACTT GTTACTCTTACTTGTCTTGCTCGTGGTTTTTCTCCTAAAGATGTTCTTGT TCGTTGGCTTCAAGGTTCTCAAGAACTTCCTCGTGAAAAATATCTTACTT GGGCTTCTCGTCAAGAACCTTCTCAAGGTACTACTACTTTTGCTGTTACT TCTATTCTTCGTGTTGCTGCTGAAGATTGGAAAAAAGGTGATACTTTTTC TTGTATGGTTGGTCATGAAGCTCTTCCTCTTGCTTTTACTCAAAAAACTA TTGATCGTCTTGCTGGTAAACCTACTCATGTTAATGTTTCTGTTGTTATG GCTGAAGTTGATGGTACTTGTTAT

[0178] A 1059 bp nucleic acid sequence of PgB3-1 (SEQ ID NO: 8) encoding the PgB3 polypeptide (353 aa) for cloning as a fusion with A1 or A2 Component in transient or stable plant transformation/expression vectors is as follows:

TABLE-US-00020 (SEQ ID NO: 8) GTTCCTCTTCCTGTTATTGCTGAACTTCCTCCTAAAGTTTCTGTTTTTGT TCCTCCTCGTGATGGTTTTTTTGGTAATCCTCGTAAATCTAAACTTATTT GTCAAGCTACTGGTTTTTCTCCTCGTCAAATTCAAGTTTCTTGGCTTCGT GAAGGTAAACAAGTTGGTTCTGGTGTTACTACTGATCAAGTTCAAGCTGA AGCTAAAGAATCTGGTCCTACTACTTATAAAGTTACTTCTACTCTTACTA TTAAAGAATCTGATTGGCTTTCTCAATCTATGTTTACTTGTCGTGTTGAT CATCGTGGTCTTACTTTTCAACAAAATGCTTCTTCTATGTGTGTTCCTGA TCAAGATACTGCTATTCGTGTTTTTGCTATTCCTCCTTCTTTTGCTTCTA TTTTTCTTACTAAATCTACTAAACTTACTTGTCTTGTTACTGATCTTACT ACTTATGATTCTGTTACTATTTCTTGGACTCGTCAAAATGGTGAAGCTGT TAAAACTCATACTAATATTTCTGGATGATTGGAATTCTGGTGAACGTTTT ACTTGTACTGTTACTCATACTGATCTTCCTTCTCCTCTTAAACAAACTAT TTCTCGTCCTAAAGGTGTTGCTCTTCATCGTCCTGATGTTTATCTTCTTC CTCCTGCTCGTGAACAACTTAATCTTCGTGAATCTGCTACTATTACTTGT CTTGTTACTGGTTTTTCTCCTGCTGATGTTTTTGTTCAATGGATGCAACG TGGTCAACCTCTTTCTCCTGAAAAATATGTTACTTCTGCTCCTATGCCTG AACCTCAAGCTCCTGGTCGTTATTTTGCTCATTCTATTCTTACTGTTTCT GAAGAAGAATGGAATACTGGTGAAACTTATACTTGTGTTGTTGCTCATGA AGCTCTTCCTAATCGTGTTACTGAACGTACTGTTGATAAATCTACTGGTA AACCTACTCTTTATAATGTTTCTCTTGTTATGTCTGATACTGCTGGTACT TGTTAT

[0179] Table 2 lists and describes all sequences included herein.

TABLE-US-00021 SEQ SEQ NAME DESCRIPTION ID NO PgA1-1 construct Extracellular domain 1 of human CMG2 for transient expression (DNA) PgA1-2 construct Extracellular domain 2 of human CMG2 for stable transformation (DNA) PgA2-1 construct PA-binding domain of 3 Lethal factor LF for transient expression (DNA) PgA2-2 construct PA-binding domain of 4 Lethal factor LF for stable transformation(DNA) A2-3-PA-LF construct Native PA-binding 5 domain LF of B. anthracis lethal factor LF (DNA) Accession No: M29081.1 PgB1-1 construct Human Fc-IgG1 6 fragment optimized for medicinal plant (DNA) PgB2-1 construct Human Fc-IgA1 7 fragment optimized for medicinal plant (DNA) PgB3-1 construct Human Fc-IgM 8 fragment optimized for medicinal plant (DNA) PgB1-2 construct Native Fc-IgG 9 fragment (DNA) PgB2-2 construct Native Fc-IgA 10 fragment (DNA) PgB3-2 construct Native Fc-IgM 11 fragment (DNA) PgB4-1 construct Native VP2 coat 12 protein of JC virus (DNA) PgB5 construct Native A. thaliana 13 oleosin (DNA) PgC1-1 construct Native IgJ gene 14 C2-1 construct Native VP1 coat 15 protein of JC virus (DNA) PgA1-3:B1-3 construct sCMG2-IgG1(Fc) for 16 transient expression (DNA) PgA1-4:B1-4 construct CMG2-IgG1(Fc) for 17 stable transformation (DNA) PgA1-3:B2-4 construct sCMG2-IgA1 (Fc) 18 for transient expression (DNA) PgA1-4:B2-4 construct sCMG2-IgA1 (Fc) 19 for stable transformation (DNA) PgA1-3:B3-3 construct CMG2-IgM1 (Fc) 20 for transient expression (DNA) PgA1-4:B3-4 construct sCMG2-IgM1 (Fc) 21 for stable transformation (DNA) PgA1-3:B4-2 construct VP2 polyoma 22 (JCvirus)-sCMG2 (DNA) PgA1-3:B5-2 A. thaliana oleosin 23 (OLE)-sCMG2 (DNA) A1-CMG2 protein Native example of 24 human CMG2 protein A2-protein-PA-binding LF Native example of PA 25 protein binding domain of B. anthracis lethal factor LF protein B1-protein Native Fc-IgG protein 26 Accession No. AY172957 B2-protein Native Fc-IgA protein 27 Accession No. S71043 B3-protein Native Fc-IgM 28 protein Accession No. X67301 S50847 B4-protein Native VP2 coat 29 protein of JC virus Accession No. NC_001699 B5-protein Native A. thaliana 30 oleosin Accession No. X62353 S38026 PgA1-1:B1-1 protein Amino acid sequence 31 PgA1B1 C1-2 construct Human IgJ 32 polypeptide (linker for IGgs Alpha and Mu) for transient expression C1-3 construct Human IgJ 33 polypeptide (linker for Igs Alpha and Mu) for stable transformation (DNA) C2-2 construct A coat protein of 34 human polyoma virus C1-protein IgJ protein 35 Accession No. NM_144646 C2-protein VP1 coat protein of 36 JC virus Accession No. NC_001699 PgA2-4:B1-3 construct PA-binding domain of 37 lethal factor-Fc-IgG1 (DNA) PgA1-5:B1-5 construct sCMG2-hIgG1(Fc) 38 gene optimized for expression in plants (DNA) Kozak-like sequence Regulatory element 39 (DNA) BGLII and SACI sites Restriction sites 40 NOTI site Restriction site 41 BAA targeting peptide Targeting peptide 42 ABP1 targeting peptide Targeting peptide 43 Flexible linker Linker 44 Trombin cleavage peptide Cleavage peptide 45 Factor Faa protease Cleavage peptide 46 cleavage peptide PgA1-3 construct Extracellular domain 47 of human CMG2 (DNA) PgB1-3 construct Human Fc-IgG1 48 fragment (DNA) PgA1-4 construct Extracellular domain 49 of human CMG2 (DNA) PgB1-4 construct Human Fc-IgG1 50 fragment (DNA) PgB2-3 construct Human Fc-IgA1 51 fragment (DNA) PgB2-4 construct Human Fc-IgA1 52 fragment (DNA) PgB3-3 construct Fc-IgM fragment 53 (DNA) PgB3-4 construct Fc-IgM fragment 54 (DNA) PgB4-2 construct VP2 coat protein of 55 JC virus (DNA PgB5-2 construct A. thaliana oleosin 56 (DNA) PgA2-4 construct Human Fc-IgA1 57 fragment (DNA) PgA1-5 construct Extracellular domain 58 of human CMG2 (DNA) PgB1-5 construct Human Fc-IgG1 59 fragment DNA PgA1-5 protein Extracellular domain 60 of human CMG2 protein PgB1-5 protein Human Fc-IgG1 61 fragment protein NPTII forward primer PCR primer 62 NPTII reverse primer PCR primer 63

Example 2

Construction of the Expression Cassette for Production of Recombinant ATR-FC Fusion Proteins in Plants

[0180] A synthetic gene encoding PgA1B1 (SEQ ID NO: 38) was assembled from synthetic nucleotides and/or PCR products. The fragment was cloned into the pMK-RQ (KanR) plasmid using SfiI and SfiI cloning sites and resulted in a plasmid 12AA3X5C_PgA1B1_pMK-RQ (FIG. 3A). The plasmid includes an origin of replication Col E1, the restriction sites NarI, DraII, NcoI, Eco57I, and SfiI in the body of vector pMK-RQ, and the restriction sites NcoI, XhoI, HindIII, and BamHI in the 1297 bp synthetic PgA1B1 gene (SEQ ID NO: 39). The plasmid DNA was purified from transformed bacteria and concentration determined with UV spectroscopy. The final construct was verified by sequencing. The sequence congruence within the used restriction sites was 100%.

[0181] The sequence encoding PgA1B1 (SEQ ID NO: 38) was then cloned into an expression cassette using NcoI and BglII cloning sites. The expression cassette also included nucleic acid sequence encoding the human IgG1 Fc fragment, P.sub.rbcS promoter and T.sub.rbcS termination signals (FIG. 3B). The expression cassette also included the restriction sites XbaI, NcoI, BglII, and SacI for cloning of the expression cassette into the plant vector. The expression cassette includes PgA1B1, a synthetic nucleotide sequence encoding a soluble recombinant protein (component A1-B1) capable of binding an anthrax toxin PA protein. The recombinant protein includes ATR, which is covalently linked to the Fc fragment of an immunoglobulin G (Fc-IgG) using a short flexible polypeptide linker or hinge. The expression cassette also includes c-myc and His, peptide tags for easy purification of the recombinant protein. The cassette includes the KDEL (SEQ ID NO: 65) short signal peptide directing the expressed recombinant protein to a specific compartment of a plant cell. The expression cassette was than cloned into the pBI121 binary vector (based on the pBIN19 vector) using XbaI and SacI cloning sites (Bevan, M, 1984, Nucl Acids Res:12, 8711-8721).

[0182] FIG. 4A schematically shows a plant expression vector. As shown, the vector includes a backbone derived from a conventional pBI binary vector covalently linked to the T-DNA surrounded by the right border, RB, and the left border, LB. The T-DNA includes the nptll gene for kanamycin selection. The nptII gene is linked to the expression cassette that includes the Rubisco promoter, P.sub.rbcS; the recombinant protein PgA1B1; the purification tag, Tag; the endoplasmic reticulum compartment sorting signal, KDEL (SEQ ID NO: 65); and the translation termination signal of the Rubisco gene, RbcT. FIG. 4B is a diagram illustrating ways of using vectors for production of PgA1B1 recombinant proteins in plant. DNA of the expression vectors can be introduced into plant cells either by using Agrobacterium, or by direct DNA transfer. This figure shows that the plant expression vector can be used for stable or transient transformation of plants. In the transient system, plant expression vectors are not integrated into the genomes of the transformed plants. In this system, the recombinant proteins can be produced after a short period of tissue incubation with the vectors. In the stable transformation system, only plant cells with the T-DNAs of the expression vectors integrated into the plant genomes are selected. Such transgenic plants can transfer the T-DNAs to the progeny which may also produce the recombinant proteins.

Example 3

Plants Transformation

[0183] Stable Transformation of Tobacco Plants

[0184] Stable transformation of tobacco was performed as described in Golovkin et al., 2007, which is incorporated herein by reference in entirety. Tobacco (Nicotiana tabacum cv Wisconsin 38) were used for all experiments. Tobacco seeds were surface sterilized and incubated on the solidified with 0.7% agar Murashige-Skoog medium (Physiol. Plant, 1962, 15:473) supplemented with 1% sucrose, pH 5.8 at 24.degree. C., with 16/8 h light/dark photoperiod. 2-3 weeks old seedlings were excised and transferred to flasks containing the MS medium supplemented with 0.7% agar and 3% sucrose. Sterile leaf explants were transformed by using the described above kanamycin-resistant construct encoding recombinant ATR-Fc protein in the Agrobacterium tumefaciens strain LB4404. Leaves of 5-6 week-old aseptically grown plants were cut into segments 0.5 to 0.7 cm in size and inoculated for 10 minutes with the Agrobacterium cell suspension diluted to OD.sub.600nm 0.5. Inoculated leaf explants were blotted dry and plated onto Petri dishes with solidified MS medium supplemented with 3% sucrose and 0.7% agar. After 2 days of co-cultivation at 24.degree. C. in the dark, plant explants were transferred to the selection/regeneration MS medium supplemented with 3% sucrose, 1 mg/l BAP, 0.1 mg/l NAA, 100 mg/l kanamycin, 300 mg/l timentin, 0.7% agar and incubated at 24.degree. C. and 16/8 h light/dark photoperiod. After 5-6 weeks of selection, the putative transgenic green shoots were formed (FIGS. 5A-5B). These shoots were excised and transferred to Magenta boxes containing MS medium supplemented with 100 mg/l kanamycin, 3% sucrose, 0.7% agar and 200 mg/l timentin for rooting, FIG. 5C, where transgenic shoots were developing on the medium supplemented with kanamycin. Control wild type tobacco plants failed to grow under similar conditions (FIG. 5A). Plants that rooted in the presence of 100 mg/l kanamycin were tested for expression of recombinant products. The best transgenic lines were chosen for root induction and then were transferred to soil to mature and set seeds (FIG. 5D). Plants were grown in 10-15 cm pot in the greenhouse at 25.degree. C. and 16/8 day/night period.

[0185] Selected transgenic lines were screened by a PCR reaction for the T-DNA integration based on the detection of the nptII marker encoding resistance to kanamycin (FIG. 4A) linked to recombinant PgA1B1 gene. The nptII-specific primers were as follows: 5-'TGAATGAACTGCAGGACGA-3' (forward; SEQ ID NO: 62) and 5'-AGCCAACGTATGTCCTGAT-3' (reverse; SEQ ID NO: 63). The PCR reaction condition included 30 cycles of polymerization at the annealing temperature 56.degree. C. using Taq polymerase protocol from Promega. As shown on FIG. 5E, over 50% tested plants were positive as they produced PCR products of expected molecular size of approximately 500 bp.

[0186] Transgenic plants were further screened with Western blotting for the presence of the ATR-Fc protein fused with the c-myc tag (FIG. 3B). A total crude protein extracts of selected tobacco lines were separated on SDS-PACE and tested by Western blot analysis using the c-myc monoclonal antibody mAb (ATCC, Manassas) and the goat anti-mouse horse radish conjugated antibody (Ab) (Upstate NY) (FIG. 5F). As shown on FIG. 5F, the recombinant product of the expected size of approximately 50 kDa was detected as a single band in all but one tested samples containing a total soluble protein (TSP). The correct molecular weights were verified by pre-stained Universal Kaleidoscope protein marker (BioRad).

[0187] Transformation of Echinacea plants

[0188] Plant material. Seeds of Echinacea plants were obtained from Horizon Herbs Co. (Williams, Oreg.). Seeds were sterilized with 70% ethanol for 1 min and 25% commercial bleach solution for 10 min. After washing with sterile distilled water, seeds were placed in the germination MS medium containing 10 g/L sucrose and 8 g/L agar. Efficient germination and in vitro culturing was carried out at 24.degree. C. at 16 h-light/8 h-dark photoperiods and 40 .mu.E/m2/S1 light intensity. Cotyledons and young leaves were used for transformation experiments.

[0189] Transformation. Agrobacterium tumefaciens strain LBA4404 carrying the pBI binary vector for PgA1B1 protein expression was used for transformation. Cotyledons and young leaves were cut into 3-5 mm explants and incubated in Agrobacterium suspension (OD.sub.600nm 0.5) for 10 min. After blotting dry with sterile filter paper, explants were transferred to solid MS co-cultivation medium supplemented with 100 .mu.M acetosyringone and incubated in the dark for 2 days at 24.degree. C. After co-cultivation, explants were transferred to the first selection/regeneration MS medium supplemented with 30 g/L sucrose, 1 mg/L BAP, 1 mg/L thidiazuron, 0.3 mg/L NAA, 30 mg/L kanamycin, 300 mg/L timentin and 8 g/L agar. After 10 days, explants were transferred to the second selection/regeneration MS medium supplemented with 30 g/L sucrose, 1 mg/L BAP, 1 mg/L thidiazuron, 0.1 mg/L NAA, 50 mg/L kanamycin, 300 mg/L timentin and 8 g/L agar. After 4-5 weeks of culturing on the second selection medium, putatively transformed green shoots were formed (FIGS. 6A-6B and 6D). It was noticed that the cotyledon explants produced 2.5 times more putative transgenic shoots than the leaf explants. Healthy green shoots that reached 1-2 cm in height were excised and transferred to the MS medium supplemented with 30 g/L sucrose, 8 g/L agar, 300 mg/L timentin and 50 mg/L kanamycin for rooting. Putative transgenic shoots produced roots after 1-2 weeks of cultivation and showed good growth on the selection medium (FIG. 6C).

[0190] Transgenic plants were further screened with Western blotting for the presence of the PgA1B1 -c-myc fusions using the c-myc monoclonal antibody mAb (ATCC, Manassas) and the goat anti-mouse horse radish conjugated antibody (Ab) (Upstate NY) (FIG. 6E). As shown on FIG. 6E, the recombinant product of the expected size of approximately 50 kDa was detected in many samples.

[0191] Transformation of Kalanchoe Plants.

[0192] Plant material. Fresh leaves of Kalanchoe pinnata were obtained from Tropilab Inc. (St. Petersburg, Fla.) and surface sterilized by immersion in 70% ethanol for 1 min, followed by soaking in 25% of bleach solution for 8 min. After rinsing 3 times in sterile distilled water, and blotting dry with sterile filter paper, leaf segments (1.times.1 cm) were cultured on MS basal medium supplemented with 30 g/l sucrose, 1 mg/l BAP, 0.1 mg/l NAA and 7 g/l agar. Explants were cultivated at 24.degree. C. at 16 h-light /8 h-dark photoperiods and 40 .mu.E/m2/S1 light intensity. Explants developed shoots after 5-6 weeks in culture. Shoots were excised and transferred to the Magenta boxes containing basal MS medium supplemented with 30 g/L sucrose and 7 g/L agar. Shoots formed roots and produced whole plants within 3-5 weeks. For propagation of plant material stem segments with axillary buds were transferred to the fresh MS medium supplemented with 30 g/L sucrose and 7 g/L agar.

[0193] Transformation. Agrobacterium tumefaciens strain LBA4404 containing the pBI-PgA1B1 construct was used for transformation experiments. Agrobacteria were grown at 28.degree. C. on solid LB plates supplemented with 50 mg/L kanamycin and 20 mg/L rifampicin. A single colony was used to inoculate 20 mL of LB liquid medium with the same antibiotics. Agrobacterium culture was incubated 1-2 days at 150 rpm on a shaker. The suspension of Agrobacterium was diluted with a liquid MS medium to obtain OD.sub.600 0.5.

[0194] For transformation experiments leaves of aseptically propagated 2 moths-old plants were cut into 0.6 to 0.7 cm pieces and inoculated with Agrobacterium suspensions for 10 min. After blotting dry with sterile filter paper, explants were transferred to the co-cultivation MS medium supplemented with 100 .mu.M acetosyringone and incubated in the dark for 2 days at 24.degree. C. After co-cultivation, explants were transferred to the selection regeneration MS basal medium supplemented with 2 mg/L BAP, 0.1 mg/L NAA, 50 mg/L kanamycin and 300 mg/L timentin. All explants were sub-cultured every 2-3 weeks onto the fresh medium with the same combination of plant hormones and antibiotics. After 5-6 weeks, explants produced shoots on the selection medium (FIG. 7A). FIG. 7A shows the transformed explant developing shoots (left) and the non-transformed explant bleaching on the selection medium containing 50 mg/L of kanamycin. Putative transgenic green regenerants were transferred to MS medium supplemented with 50 mg/L kanamycin and 300 mg/L timentin for rooting (FIG. 7B). In the presence of 50 mg/L kanamycin transgenic kalanchoe shoots showed good growth and development of roots compare to the non-transgenic shoots that did not produce roots and eventually died. FIG. 7C shows the transgenic kalanchoe plant rooted on the selection medium. Transgenic kalanchoe plants had showed no morphological abnormalities and resembled non-transgenic plants of the same age.

[0195] FIG. 8 is a schematic drawing explaining the process of production of plant-derived composition. As shown in this figure, vectors that include the expression constructs encoding recombinant ATR proteins are introduced into either crop or medicinal plants. The recombinant ATR proteins are extracted and may be used in plant-derived therapeutic compositions either alone or in combination with enhancers and adjuvants.

Example 4

Analysis of Activity of Synthetic Recombinant Proteins

[0196] Extraction of Soluble Protein from Transgenic Tobacco Plants.

[0197] Total and soluble plant proteins were extracted from transgenic tobacco and medicinal plants as described by Golovkin et al., 2007. Plant tissue sample were collected, immediately frozen in liquid nitrogen and stored at -80.degree. C. until extraction. Recombinant product was extracted from frozen plant tissues directly using equal amount (V/W) of Laemmli loading buffer for the total/insoluble extract or soluble buffer containing 0.1M Na phosphate pH7.4, 0.3M NaCl, 3% Glycerol, 0.1 mM EDTA, 2 mM .beta.-ME and 0.05% of plant protease inhibitors cocktail (Sigma) for a total soluble protein, concentrated and brought into an equal volume of loading buffer. Protein was extracted from the transgenic tobacco line shown on was used for further analysis (FIG. 9A).

[0198] Extraction of Protein from Transgenic Echinacea Plants.

[0199] Total plant protein was extracted from transgenic Echinacea plants using similar protocol as in Golovkin et al., 2007. Plant tissue sample were collected, immediately frozen in liquid nitrogen and store at -80.degree. C. until extraction. Recombinant product was extracted from frozen plant tissues directly with equal amount (V/W) of Laemmli loading buffer and further used for Western blot analysis (FIG. 6E).

[0200] Purification of Soluble Recombinant Proteins

[0201] About 200 g of frozen leaf material was grounded in 5 volumes of the extraction buffer containing 50-100 mM Na Phosphate, pH7.4, 0.3M NaCl, 0.2% Tween-20, 1.5mM .beta.-Mercaptoethanol, 0.05% Plant Protein Inhibitors Cocktail (Sigma) using Brinkman Polytron Homogenizer at 27,000 rpm. Insoluble parts were pelleted (Beckman) at 16,000 rpm for 20 min at 4.degree. C. Following the flow-filtering through Miracloth (Calbiochem), the PgA1B1 protein was purified in a single-step protocol, using protein A agarose as described earlier (Spitsin et al., 2009; Andrianov et al., 2010). FIG. 9C illustrates extraction of the plant PgA1B1 recombinant protein from tobacco leaf tissue and purification of the recombinant protein on the protein-A agarose column. In this figure, "M" is a protein molecular weight marker, "IgG" is a purified conventional IgG antibody with the heavy ("H") and light ("L") chains eluted from the protein-A agarose column and used as a standard. "Total" stands for a total plant protein extract before purification. "FT" indicates flow through the fraction from the column. "Eluate" marks the final purified plant-derived PgA1B1 product identified as the "PgA1B1" 48 kDa band monomer of the PgA1B1 protein and the upper "dimer" band.

[0202] In vitro characterization and quantification of protein expression was performed with ELISA and Western blot analysis (FIG. 9A) essentially as described by Golovkin et al., 2007. FIG. 9A shows a total protein extracted from the transgenic tobacco plant on PAAG gel (left panel) and immunodetection of PgA1B1 protein expressed in plants using Western blotting (right panel). In this figure, "M" is a protein molecular weight marker, "neg" is an extract of untransformed plant, "Tr" marks total proteins from PgA1B1-expressing transgenic plants, "+" is a positive control from bacteria, "-" is a total protein extract from a wild type plant, "Transgenic" refers to protein extracts from different transgenic tobacco lines. "PgA1B1" indicates the position of the 48 kDa TBL-Fc fusion protein and upper band represents the corresponding "dimer".

[0203] FIG. 9B demonstrates comparison of a total and soluble PgA1B1 protein in transgenic tobacco plants using Western blot analysis. In this figure, "+" is a positive control, "-" is a negative control from wild type plants, "Sol" and "Tot" stands for soluble and total protein fractions, respectively. Analysis as shown in FIG. 9B confirmed that almost all fusion proteins are expressed as soluble proteins in plants. Highly pure preparations of plant-derived PgA1B1 protein were extracted from plant tissue minimal concentration of 1.5 mg/ml and yield of at least 3 mg per Kg of raw plant tissue weight.

Example 5

Affinity Binding of the Anthrax Toxin by Plant-Derived Toxin Binding Ligand

[0204] As shown earlier by VWA/I domain of the native CMG2 protein may bind PA in a divalent cation-dependent manner (Bradley et al., 2001; Lacy et al., 2004; Scobie et al. 2003). The ability of plant-derived PgAB fusion protein to bind PA was confirmed by two kinds of experiments.

[0205] Affinity Pulls Down Assay of PA Protein from Solution

[0206] Affinity of the PA protein to the plant-derived PgA1B1 protein was demonstrated by using protein A agarose beads. Specific binding of 0.5 .mu.g of commercial anthrax PA protein (List Laboratories, Campbell, Calif.) was mixed with 5 .mu.(=7.25 .mu.g) of purified PgA1B1 protein was done in 100 .mu.l of TBS buffer containing 0.05 Tween-20 (TBSTS), 3% BSA, and 1 mM MgCl.sub.2 by incubating it at 4.degree. C. overnight with gentle shaking. A positive control, 2 .mu.l (aprox. 8-10 .mu.g) of commercial anti-PA goat antiserum (List Laboratories, Campbell, Calif.) was used instead of PgA1B1 protein. Bound protein complexes were rescued from the solution using MagnaBind Protein A Beads (Pierce, Rockford, Ill.). The beads were eluted and analyzed by Western immunoassay using anti-PA mAbs (Biodesign, Saco, Me.). Both anti-PA mAbs and plant-derived PgA1B1 were shown to efficiently bind PA protein. Under experimental conditions, a plant-derived PgA1B1 recombinant protein was more efficient in binding anthrax toxin PA component then the control commercial antibody.

[0207] Detection of Binding PA Protein to Plant-Derived TBL by ELISA

[0208] FIG. 10A illustrates an analysis of the PA binding by the recombinant PgA1B1 protein. Concentration dependency of the plant-derived PgA1B1 binding to the surface-immobilized of PA protein was detected by sandwich ELISA. ELISA was performed in a 96-well plate coated with 0.3 .mu.g/well of the PA protein (List Laboratories, Campbell, Calif.) preparation in 50 .mu.l TBS per well at 4.degree. C. overnight followed by blocking with TBS containing 0.025% Tween (TBST2.5) and 3% BSA. The PA protein preparation was incubated with increasing concentrations of plant PgA1B1 in the presence (dark bars) of or without magnesium ions (Mg.sup.2+) (light bars). Bound PgA1B1 was detected using c-myc-specific mAbs. Dilutions of PgA1B1 protein starting at 300 ng/well in 50 .mu.l TBST2.5 with 1 mM MgCl.sub.2 were added and incubated 1 h to set up binding, then washed 2.times.5' in TBST2.5. Primary c-myc specific antibody (Invitrogen, Carlsbad, Calif.) were applied at 1/1000-1/2000 dilution for an hour washed vigorously' in TBST2.5. After incubation with secondary AP-anti-mouse conjugate (Sigma, Saint Louis, Mo.) at 1/2000 dilution, plates were developed by using pNPP Substrate (Sigma, Saint Louis, Mo.) as recommended by manufacturer and OD.sub.405 nm determined. Strong affinity of PgA1B1 to PA was demonstrated in the presence of Mg.sup.2+ ions, which is characteristic to a native ATR/sCMG2 protein (Scobie et al. 2003).

[0209] In vitro Protection of Macrophage Cells Against Anthrax Toxin with Plant ATR.

[0210] Referring to FIG. 10B neutralizing-antibody activity was determined in host monocyte-macrophages cells J774A.1 (American Type Culture Collection, Manassas, VA) essentially as described by Little et al., 1990. Various concentrations of purified plant PgA1B1 (diamonds) or an irrelevant serum from mice immunized with PBS buffer (Negative serum, squares) were premixed with the anthrax lethal toxin before addition to the monocyte-macrophage J774A.1 cells. Cells were incubated with lethal concentrations of the PA-LF toxin in the presence of different concentrations of either the recombinant PgA1B1 protein or sera from mice immunized with PBS buffer (Negative serum). Lactate dehydrogenase activity was measured by a Cytotoxicity Detection Kit (Roche, Indianapolis, Ind.). Cell viability was calculated as a percentage of surviving cells to the complete lysis achieved with 1% Triton X-100. Data points represent the deviation of mean values in triplicate samples. PA and LF components of the anthrax toxin (at 0.1 .mu.g/ml) were combined with plant purified PgA1B1 and negative serum. The antiserum-toxin mixtures were added to J774A.1 at 4.times.10.sup.4 cells per well. Lactate dehydrogenase activity was measured by the Cytotoxicity Detection kit (Roche, Indianapolis, Ind.). The percentage of cell lysis was calculated as the mean of "A.sub.490 of serum/PA toxin/target cell mix" minus "A490 of target cell control" divided by "A490 of cells treated with 2% Triton X-100" minus "A490 of cells incubated with medium." Neutralization of anthrax toxin was observed for purified plant-derived PgA1B1 at working concentration of 0.6 .mu.g/ml where 50% cells survived confirming its capability to protect cells against the anthrax toxin. That is in a good agreement with previously described result for native sCMG2 protein (Scobie et al., 2005; Vuyisich et al., 2008; Wycoff et al., 2011; Thomas et al., 2012). No neutralization activity was detected in negative control serum.

Example 6

Administration of Antitoxin Composition to a Subject

[0211] Administration of a therapeutic antitoxin composition into bloodstream of animal subjects could be done with the help of commercial needle-free technique developed by Apogee Technologies (Norwood, Mass.), based on using micro needle patches for transdermal administration of protein-based therapeuticals. Up to 1.5 .mu.g/cm.sup.2 of c-myc tagged PgA1B1 plant-derived protein, produced as described in Example 1, was loaded onto the micro-needle patch in a formulation recommended by manufacturer. The patches containing micro-needle array are then manually applied on the skin and released upon a pressure applied on the center of the patch for 1 min to facilitate micro-needle insertion. After 30 min the amount of intramuscular recombinant protein could be estimated histologically using c-myc-specific antibody demonstrating complete dissolution of the formulation in the animal.

REFERENCES

[0212] Andrianov V., Brodzik R., Spitsin S., Bandurska K., McManus H., Koprowski H., Golovkin M. Production of recombinant anthrax toxin receptor (ATR/CMG2) fused with human Fc in planta. Protein Expression and Purification 70:158-162 (2010).

[0213] Bradley K. A., Mogridge J., Mourez M., Collier R. J., Young J. A. Identification of the cellular receptor for anthrax toxin. Nature 414:225-229 (2001).

[0214] Golovkin M., Spitsin S., Andrianov V., et al., Smallpox subunit vaccine produced in planta confers protection in mice. Proc. Natl. Acad. Sci., 104:6864-6869 (2007).

[0215] Golovkin M. Production of recombinant pharmaceuticals using plant biotechnology. In: Bioprocess Science and Technology, Series Biochemistry Research Trends Ed. Min-Tze Liong. Nova Sci.Publ., Inc., USA. ISBN:978-1-61122-950-9 (2011).

[0216] Golovkin M. Plant-Derived vaccines: Plant biotechnology for production of recombinant pharmaceuticals, Human Vaccines 7(3), Landes Bioscience (2011).

[0217] Lacy D. B., Wigelsworth D. J., Scobie H. M., Young J. A. and Collier R. J. Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: an anthrax toxin receptor. Proc Natl Acad Sci USA, 101: 6367-6372 (2004).

[0218] Manayani D. J., Thomas D., Dryden K. A., Reddy V., Silath M. E., Marlett J. M., Rainey G. J. A., Pique M. E., Scobie H. M., Yeager M., Young J. A. T. A viral nanoparticle with dual function as an anthrax antitoxin and vaccine. PLoS Pathog 3:e142 (2007).

[0219] Rainey G. J. A., Young J. A. T. Antitoxins: novel strategies to target agents of bioterrorism. Nat. Rev. Microbiol., 2: 721-726 (2004).

[0220] Scobie H. M., et al. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc Natl Acad Sci USA, 100: 5170-74 (2003).

[0221] Scobie H. M., et al. A soluble receptor decoy protects rats against anthrax lethal toxin challenge. The Journal of Infectious Diseases, 192: 1047-51 (2005).

[0222] Spitsin S., Andrianov V., Pogrebnyak N. et al., Immunological assessment of plant-derived avian flu H5/HA1 variants. Vaccine, 27:1289-1292 (2009).

[0223] Thomas D., Naughton J., Cote C., Welkos S., Manchester M., Young J. A. T. Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment. PLoS ONE, 7:e34611 (2012).

[0224] Vuyisich M., Gnanakaran S., Lovchik J. A., Lyons C. R., Gupta G. A dual-purpose protein ligand for effective therapy and sensitive diagnosis of anthrax. Protein J 27:292-302 (2008).

[0225] Wycoff K. L., Belle A., Deppe D., Schaefer L, Maclean J. M., Haase S., Trilling A. K., Liu S., Leppla S. H., Geren I. N., Pawlik J., Peterson J. W. Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax. Antimicrobial Agents and Chemotherapy 55:132-139 (2011).

[0226] Young J. A., Collier R. J. Anthrax Toxin: Receptor Binding, Internalization, Pore Formation, and Translocation. Annu. Rev. Biochem. 76:243-265 (2007).

[0227] The references cited throughout this application are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0228] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

Sequence CWU 1

1

651581DNAArtificial SequenceSynthetic construct, PgA1-1 construct 1ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gaagatctcc aataagagct c 5812575DNAArtificial SequenceSynthetic construct, PgA1-2 construct 2ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gaagatctcc aataa 5753722DNAArtificial SequenceSynthetic construct, PgA2-1 construct 3ccatgggtga cgtcggtatg cacgtcaagg agaaggagaa gaacaaggac gagaacaagc 60gtaaggacga ggagcgtaac aagacccagg aggagcacct caaggagatc atgaagcaca 120tcgtcaagat cgaggtcaag ggtgaggagg ctgtcaagaa ggaggctgct gagaagctcc 180tcgagaaggt cccttctgac gtcctcgaga tgtacaaggc tatcggtggt aagatctaca 240tcgtcgacgg tgacatcacc aagcacatct ctctcgaggc tctctctgag gacaagaaga 300agatcaagga catctacggt aaggacgctc tcctccacga gcactacgtc tacgctaagg 360agggttacga gcctgtcctc gtcatccagt cttctgagga ctacgtcgag aacaccgaga 420aggctctcaa cgtctactac gagatcggta agatcctctc tcgtgacatc ctctctaaga 480tcaaccagcc ttaccagaag ttcctcgacg tcctcaacac catcaagaac gcttctgact 540ctgacggtca ggacctcctc ttcaccaacc agctcaagga gcaccctacc gacttctctg 600tcgagttcct cgagcagaac tctaacgagg tccaggaggt cttcgctaag gctttcgctt 660actacatcga gcctcagcac cgtgacgtcc tccagctcta cgctcctgag gcttaagagc 720tc 7224728DNAArtificial SequenceSynthetic construct, PgA2-2 construct 4ccatgggtga cgtcggtatg cacgtcaagg agaaggagaa gaacaaggac gagaacaagc 60gtaaggacga ggagcgtaac aagacccagg aggagcacct caaggagatc atgaagcaca 120tcgtcaagat cgaggtcaag ggtgaggagg ctgtcaagaa ggaggctgct gagaagctcc 180tcgagaaggt cccttctgac gtcctcgaga tgtacaaggc tatcggtggt aagatctaca 240tcgtcgacgg tgacatcacc aagcacatct ctctcgaggc tctctctgag gacaagaaga 300agatcaagga catctacggt aaggacgctc tcctccacga gcactacgtc tacgctaagg 360agggttacga gcctgtcctc gtcatccagt cttctgagga ctacgtcgag aacaccgaga 420aggctctcaa cgtctactac gagatcggta agatcctctc tcgtgacatc ctctctaaga 480tcaaccagcc ttaccagaag ttcctcgacg tcctcaacac catcaagaac gcttctgact 540ctgacggtca ggacctcctc ttcaccaacc agctcaagga gcaccctacc gacttctctg 600tcgagttcct cgagcagaac tctaacgagg tccaggaggt cttcgctaag gctttcgctt 660actacatcga gcctcagcac cgtgacgtcc tccagctcta cgctcctgag gctggagatc 720tccaataa 7285732DNABacillus anthracismisc_feature(1)..(732)PgA2-3, DNA encoding PA-binding domain LFn 5gagaataaga gaaaagatga agaacgaaat aaaacacagg aagagcattt aaaggaaatc 60atgaaacaca ttgtaaaaat agaagtaaaa ggggaggaag ctgttaaaaa agaggcagca 120gaaaagctac ttgagaaagt accatctgat gttttagaga tgtataaagc aattggagga 180aagatatata ttgtggatgg tgatattaca aaacatatat ctttagaagc attatctgaa 240gataagaaaa aaataaaaga catttatggg aaagatgctt tattacatga acattatgta 300tatgcaaaag aaggatatga acccgtactt gtaatccaat cttcggaaga ttatgtagaa 360aatactgaaa aggcactgaa cgtttattat gaaataggta agatattatc aagggatatt 420ttaagtaaaa ttaatcaacc atatcagaaa tttttagatg tattaaatac cattaaaaat 480gcatctgatt cagatggaca agatctttta tttactaatc agcttaagga acatcccaca 540gacttttctg tagaattctt ggaacaaaat agcaatgagg tacaagaagt atttgcgaaa 600gcttttgcat attatatcga gccacagcat cgtgatgttt tacagcttta tgcaccggaa 660gcttttaatt acatggataa atttaacgaa caagaaataa atctatcctt ggaagaactt 720aaagatcaac gg 7326687DNAArtificial SequenceSynthetic construct, PgB1-1 construct 6gataaaactc atacttgtcc tccttgtcct gctcctgaac ttcttggtgg tccttctgtt 60tttctttttc ctcctaaacc taaagatact cttatgattt ctcgtactcc tgaagttact 120tgtgttgttg ttgatgtttc tcatgaagat cctgaagtta aatttaattg gtatgttgat 180ggtgttgaag ttcataatgc taaaactaaa cctcgtgaag aacaatataa ttctacttat 240cgtgttgttt ctgttcttac tgttcttcat caagattggc ttaatggtaa agaatataaa 300tgtaaagttt ctaataaagc tcttcctgct cctattgaaa aaactatttc taaagctaaa 360ggtcaacctc gtgaacctca agtttatact cttcctcctt ctcgtgaaga aatgactaaa 420aatcaagttt ctcttacttg tcttgttaaa ggtttttatc cttctgatat tgctgttgaa 480tgggaatcta atggtcaacc tgaaaataat tataaaacta ctcctcctgt tcttgattct 540gatggttctt tttttcttta ttctaaactt actgttgata aatctcgttg gcaacaaggt 600aatgtttttt cttgttctgt tatgcatgaa gctcttcata atcattatac tcaaaaatct 660ctttctcttt ctcctggtaa agatctt 6877774DNAArtificial SequenceSynthetic construct, PgB2-1 construct 7gatgttactg ttccttgtcc tgttccttct actcctccta ctccttctcc ttctactcct 60cctactcctt ctccttcttg ttgtcatcct cgtctttctc ttcatcgtcc tgctcttgaa 120gatcttcttc ttggttctga agctaatctt acttgtactc ttactggtct tcgtgatgct 180tctggtgtta cttttacttg gactccttct tctggtaaat ctgctgttca aggtcctcct 240gaacgtgatc tttgtggttg ttattctgtt tcttctgttc ttcctggttg tgctgaacct 300tggaatcatg gtaaaacttt tacttgtact gctgcttatc ctgaatctaa aactcctctt 360actgctactc tttctaaatc tggtaatact tttcgtcctg aagttcatct tcttcctcct 420ccttctgaag aacttgctct taatgaactt gttactctta cttgtcttgc tcgtggtttt 480tctcctaaag atgttcttgt tcgttggctt caaggttctc aagaacttcc tcgtgaaaaa 540tatcttactt gggcttctcg tcaagaacct tctcaaggta ctactacttt tgctgttact 600tctattcttc gtgttgctgc tgaagattgg aaaaaaggtg atactttttc ttgtatggtt 660ggtcatgaag ctcttcctct tgcttttact caaaaaacta ttgatcgtct tgctggtaaa 720cctactcatg ttaatgtttc tgttgttatg gctgaagttg atggtacttg ttat 77481006DNAArtificial SequenceSynthetic construct, PgB3-1 construct 8gttcctcttc ctgttattgc tgaacttcct cctaaagttt ctgtttttgt tcctcctcgt 60gatggttttt ttggtaatcc tcgtaaatct aaacttattt gtcaagctac tggtttttct 120cctcgtcaaa ttcaagtttc ttggcttcgt gaaggtaaac aagttggttc tggtgttact 180actgatcaag ttcaagctga agctaaagaa tctggtccta ctacttataa agttacttct 240actcttacta ttaaagaatc tgattggctt tctcaatcta tgtttacttg tcgtgttgat 300catcgtggtc ttacttttca acaaaatgct tcttctatgt gtgttcctga tcaagatact 360gctattcgtg tttttgctat tcctccttct tttgcttcta tttttcttac taaatctact 420aaacttactt gtcttgttac tgatcttact acttatgatt ctgttactat ttcttggact 480cgtcaaaatg gtgaagctgt taaaactcat actaatattt ctggatgatt ggaattctgg 540tgaacgtttt acttgtactg ttactcatac tgatcttcct tctcctctta aacaaactat 600ttctcgtcct aaaggtgttg ctcttcatcg tcctgatgtt tatcttcttc ctcctgctcg 660tgaacaactt aatcttcgtg aatctgctac tattacttgt cttgttactg gtttttctcc 720tgctgatgtt tttgttcaat ggatgcaacg tggtcaacct ctttctcctg aaaaatatgt 780tacttctgct cctatgcctg aacctcaagc tcctggtcgt tattttgctc attctattct 840tactgtttct gaagaagaat ggaatactgg tgaaacttat acttgtgttg ttgctcatga 900agctcttcct aatcgtgtta ctgaacgtac tgttgataaa tctactggta aacctactct 960ttataatgtt tctcttgtta tgtctgatac tgctggtact tgttat 100691431DNAArtificial SequenceSynthetic construct, PgB1-2 construct 9atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag 60gtgcagctgg tgcagtctgg ggctgaggtg aagaagcctg ggtcctcggt gaaggtctcc 120tgcaaggctt ctggaggcac cttcaacagg tatactgtca actgggtgcg acaggcccct 180ggacaagggc ttgagtggat gggaggcatc atccctatct ttggtacagc aaactacgca 240cagaggttcc agggcagact caccattacc gcggacgaat ccacgagcac agcctacatg 300gagctgagca gcctgagatc tgatgacacg gccgtgtatt tctgtgcgag agagaatctc 360gataattcgg ggacttatta ttatttctca ggctggttcg acccctgggg ccagggaacc 420ctggtcaccg tctcctcagc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 480tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 540gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 600gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 660agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 720gacaagagag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 780cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 840atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 900gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 960cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1020gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1080atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1140cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1200ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1260aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctatag caagctcacc 1320gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1380ctgcacaacc actacacgca gaagagcctc tccctgtccc cgggtaaatg a 1431101799DNAArtificial SequenceSynthetic construct, PgB2-2 construct 10cctctctgtg ctgggttcct ccagtgtaga ggagaggcag gtacagcctg tcctcctggg 60gacatggcat gagggccgcg tcctcacagc gcattctgtg ttccagcatc cccgaccagc 120cccaaggtct tcccgctgag cctcgacagc accccccaag atgggaacgt ggtcgtcgca 180tgcctggtcc agggcttctt cccccaggag ccactcagtg tgacctggag cgaaagcgga 240cagaacgtga ccgccagaaa cttcccacct agccaggatg cctccgggga cctgtacacc 300acgagcagcc agctgaccct gccggccaca cagtgcccag acggcaagtc cgtgacatgc 360cacgtgaagc actacacgaa ttccagccag gatgtgactg tgccctgccg aggtcagagg 420gcaggctggg gagtggggcg gggccacccc gtcctgccct gacactgcgc ctgcacccgt 480gttccccaca gggagccgcc ccttcactca caccagagtg gaccgcgggc cgagccccag 540gaggtggtgg tggacaggcc aggaggggcg aggcgggggc acggggaagg gcgttctgac 600cagctcaggc catctctcca ctccagttcc cccacctccc ccatgctgcc acccccgact 660gtcgctgcac cgaccggccc tcgaggacct gctcttaggt tcagaagcga acctcacgtg 720cacactgacc ggcctgagag atgcctctgg tgccaccttc acctggacgc cctcaagtgg 780gaagagcgct gttcaaggac cacctgagcg tgacctctgt ggctgctaca gcgtgtccag 840tgtcctgcct ggctgtgccc agccatggaa ccatggggag accttcacct gcactgctgc 900ccaccccgag ttgaagaccc cactaaccgc caacatcaca aaatccggtg ggtccagacc 960ctgctcgggg ccctgctcag tgctctggtt tgcaaagcat attcccggcc tgcctcctcc 1020ctcccaatcc tgggctccag tgctcatgcc aagtacagag ggaaactgag gcaggctgag 1080gggccaggac acagcccagg gtgcccacca gagcagaggg gctctctcat cccctgccca 1140gccccctgac ctggctctct accctccagg aaacacattc cggcccgagg tccacctgct 1200gccgccgccg tcggaggagc tggccctgaa cgagctggtg acgctgacgt gcctggcacg 1260tggcttcagc cccaaggatg tgctggttcg ctggctgcag gggtcacagg agctgccccg 1320cgagaagtac ctgacttggg catcccggca ggagcccagc cagggcacca ccaccttcgc 1380tgtgaccagc atactgcgcg tggcagccga ggactggaag aagggggaca ccttctcctg 1440catggtgggc cacgaggccc tgccgctggc cttcacacag aagaccatcg accgcttggc 1500gggtaaaccc acccatgtca atgtgtctgt tgtcatggcg gaggtggacg gcacctgcta 1560ctgagccgcc cgcctgtccc cacccctgaa taaactccat gctcccccaa gcagccccac 1620gcttccatcc ggcgcctgtc tgtccatcct cagggtctca gcacttggga aagggccagg 1680gcatggacag ggaagaatac cccctgccct gagcctcggg gggcccctgg cacccccatg 1740agactttcca ccctggtgtg agtgtgagtt gtgagtgtga gagtgtgtgg tgcaggagg 1799111453DNAArtificial SequenceSynthetic construct, PgB3-2 construct 11gggagtgcat ccgccccaac ccttttcccc ctcgtctcct gtgagaattc cccgtcggat 60acgagcagcg tggccgttgg ctgcctcgca caggacttcc ttcccgactc catcactttc 120tcctggaaat acaagaacaa ctctgacatc agcagcaccc ggggcttccc atcagtcctg 180agagggggca agtacgcagc cacctcacag gtgctgctgc cttccaagga cgtcatgcag 240ggcacagacg aacacgtggt gtgcaaagtc cagcacccca acggcaacaa agaaaagaac 300gtgcctcttc cagtgattgc cgagctgcct cccaaagtga gcgtcttcgt cccaccccgc 360gacggcttct tcggcaaccc ccgcaagtcc aagctcatct gccaggccac gggtttcagt 420ccccggcaga ttcaggtgtc ctggctgcgc gaggggaagc aggtggggtc tggcgtcacc 480acggaccagg tgcaggctga ggccaaagag tctgggccca cgacctacaa ggtgaccagc 540acactgacca tcaaagagag cgactggctc agccagagca tgttcacctg ccgcgtggat 600cacaggggcc tgaccttcca gcagaatgcg tcctccatgt gtgtccccga tcaagacaca 660gccatccggg tcttcgccat ccccccatcc tttgccagca tcttcctcac caagtccacc 720aagttgacct gcctggtcac agacctgacc acctatgaca gcgtgaccat ctcctggacc 780cgccagaatg gcgaagctgt gaaaacccac accaacatct ccgagagcca ccccaatgcc 840actttcagcg ccgtgggtga ggccagcatc tgcgaggatg actggaattc cggggagagg 900ttcacgtgca ccgtgaccca cacagacctg ccctcgccac tgaagcagac catctcccgg 960cccaaggggg tggccctgca caggcccgat gtctacttgc tgccaccagc ccgggagcag 1020ctgaacctgc gggagtcggc caccatcacg tgcctggtga cgggcttctc tcccgcggac 1080gtcttcgtgc agtggatgca gagggggcag cccttgtccc cggagaagta tgtgaccagc 1140gccccaatgc ctgagcccca ggccccaggc cggtacttcg cccacagcat cctgaccgtg 1200tccgaagagg aatggaacac gggggagacc tacacctgcg tggtggccca tgaggccctg 1260cccaacaggg tcaccgagag gaccgtggac aagtccaccg gtaaacccac cctgtacaac 1320gtgtccctgg tcatgtccga cacagctggc acctgctact gaccctgctg gcctgcccac 1380aggctcgggg cggctggccg ctctgtgtgt gcatgcaaac taacccgtgt caacggggtg 1440agatgttgca tct 1453121035DNAArtificial SequenceSynthetic construct, PgB4-1 construct 12atgggtgccg cacttgcact tttgggggac ctagttgcta ctgtttctga ggctgctgct 60gccacaggat tttcagtagc tgaaattgct gctggagagg ctgctgctac tatagaagtt 120gaaattgcat cccttgctac tgtagagggg attacaagta cctctgaggc tatagctgct 180ataggcctta ctcctgaaac atatgctgta ataactggag ctccgggggc tgtagctggg 240tttgctgcat tggttcaaac tgtaactggt ggtagtgcta ttgctcagtt gggatataga 300ttttttgctg actgggatca taaagtttca acagttgggc tttttcagca gccagctatg 360gctttacaat tatttaatcc agaagactac tatgatattt tatttcctgg agtgaatgcc 420tttgttaaca atattcacta tttagatcct agacattggg gcccgtcctt gttctccaca 480atctcccagg ctttttggaa tcttgttaga gatgatttgc cagccttaac ctctcaggaa 540attcagagaa gaacccaaaa actatttgtt gaaagtttag caaggttttt ggaagaaact 600acttgggcaa tagttaattc accagctaac ttatataatt atatttcaga ctattattct 660agattgtctc cagttaggcc ctctatggta aggcaagttg cccaaaggga gggaacctat 720atttcttttg gccactcata cacccaaagt atagatgatg cagacagcat tcaagaagtt 780acccaaaggc tagatttaaa aaccccaaat gtgcaatctg gtgaatttat agaaagaagt 840attgcaccag gaggtgcaaa tcaaagatct gctcctcaat ggatgttgcc tttactttta 900gggttgtacg ggactgtaac acctgctctt gaagcatatg aagatggccc caacaaaaag 960aaaaggagaa aggaaggacc ccgtgcaagt tccaaaactt cttataagag gaggagtaga 1020agttctagaa gttaa 1035131800DNAArabidopsis thalianamisc_feature(1)..(1800)PgB5-1 construct 13ccatggctat acccaacctc ggtcttggtc acaccaggaa ctctctggta agctagctcc 60actccccaga aacaaccggc gccaaattgc cggaattgct gacctgaaga cggaacatca 120tcgtcgggtc cttgggcgat tgcggcggaa gatgggtcag cttgggcttg aggacgagac 180ccgaatcgag tctgttgaaa ggttgttcat tgggatttgt atacggagat tggtcgtcga 240gaggtttgag ggaaaggaca aatgggtttg gctctggaga aagagagtgc ggctttagag 300agagaattga gaggtttaga gagagatgcg gcggcgatga cgggaggaga gacgacgagg 360acctgcatta tcaaagcagt gacgtggtga aatttggaac ttttaagagg cagatagatt 420tattatttgt atccattttc ttcattgttc tagaatgtcg cggaacaaat tttaaaacta 480aatcctaaat ttttctaatt ttgttgccaa tagtggatat gtgggccgta tagaaggaat 540ctattgaagg cccaaaccca tactgacgag cccaaaggtt cgttttgcgt tttatgtttc 600ggttcgatgc caacgccaca ttctgagcta ggcaaaaaac aaacgtgtct ttgaatagac 660tcctctcgtt aacacatgca gcggctgcat ggtgacgcca ttaacacgtg gcctacaatt 720gcatgatgtc tccattgaca cgtgacttct cgtctccttt cttaatatat ctaacaaaca 780ctcctacctc ttccaaaata tatacacatc tttttgatca atctctcatt caaaatctca 840ttctctctag taaacaagaa caaaaaaatg gcggatacag ctagaggaac ccatcacgat 900atcatcggca gagaccagta cccgatgatg ggccgagacc gagaccagta ccagatgtcc 960ggacgaggat ctgactactc caagtctagg cagattgcta aagctgcaac tgctgtcaca 1020gctggtggtt ccctccttgt tctctccagc cttacccttg ttggaactgt catagctttg 1080actgttgcaa cacctctgct cgttatcttc agcccaatcc ttgtcccggc tctcatcaca 1140gttgcactcc tcatcaccgg ttttctttcc tctggagggt ttggcattgc cgctataacc 1200gttttctctt ggatttacaa gtaagcacac atttatcatc ttacttcata attttgtgca 1260atatgtgcat gcatgtgttg agccagtagc tttggatcaa tttttttggt cgaataacaa 1320atgtaacaat aagaaattgc aaattctagg gaacatttgg ttaactaaat acgaaatttg 1380acctagctag cttgaatgtg tctgtgtata tcatctatat aggtaaaatg cttggtatga 1440tacctattga ttgtgaatag gtacgcaacg ggagagcacc cacagggatc agacaagttg 1500gacagtgcaa ggatgaagtt gggaagcaaa gctcaggatc tgaaagacag agctcagtac 1560tacggacagc aacatactgg tggggaacat gaccgtgacc gtactcgtgg tggccagcac 1620actacttaag ttaccccact gatgtcatcg tcatagtcca ataactccaa tgtcggggag 1680ttagtttatg aggaataaag tgtttagaat ttgatcaggg ggagataata aaagccgagt 1740ttgaatcttt ttgttataag taatgtttat gtgtgtttct atatgttgtc aaatggtacc 1800141413DNAArtificial SequenceSynthetic construct, PgC1-1 construct 14ttgtgattgt ttttagtttg ttagctgcct ggagtgttat tttaagaaag cagaagcacc 60atcatttgca cactccttat agatcacaca ccttaaccct gacttttttt gctccagttt 120ttcagaagaa gtgaagtcaa gatgaagaac catttgcttt tctggggagt cctggcggtt 180tttattaagg ctgttcatgt gaaagcccaa gaagatgaaa ggattgttct tgttgacaac 240aaatgtaagt gtgcccggat tacttccagg atcatccgtt cttccgaaga tcctaatgag 300gacattgtgg agagaaacat ccgaattatt gttcctctga acaacaggga gaatatctct 360gatcccacct caccattgag aaccagattt gtgtaccatt tgtctgacct ctgtaaaaaa 420tgtgatccta cagaagtgga gctggataat cagatagtta ctgctaccca gagcaatatc 480tgtgatgaag acagtgctac agagacctgc tacacttatg acagaaacaa

gtgctacaca 540gctgtggtcc cactcgtata tggtggtgag accaaaatgg tggaaacagc cttaacccca 600gatgcctgct atcctgacta atttaagtca ttgctgactg catagctctt tttcttgaga 660ggctctccat tttgattcag aaagttagca tatttattac caatgaattt gaaaccaggg 720cttttttttt tttttgggtg atgtaaaacc aactccctgc caccaaaata attaaaatag 780tcacattgtt atctttatta ggtaatcact tcttaattat atgttcatac tctaagtatc 840aaaatcttcc aattatcatg ctcacctgaa agaggtatgc tctcttagga atacagtttc 900tagcattaaa caaataaaca aggggagaaa ataaaactca aggactgaaa atcaggaggt 960gtaataaaat gttcctcgca ttcccccccg cttttttttt tttttttgac tttgccttgg 1020agagccagag cttccgcatt ttctttacta ttctttttaa aaaaagtttc actgtgtaga 1080gaacatatat gcataaacat aggtcaatta tatgtctcca ttagaaaaat aataattgga 1140aaacatgttc tagaactagt tacaaaaata atttaaggtg aaatctctaa tatttataaa 1200agtagcaaaa taaatgcata attaaaatat atttggacat aacagacttg gaagcagatg 1260atacagactt ctttttttca taatcaggtt agtgtaagaa attgccattt gaaacaatcc 1320attttgtaac tgaaccttat gaaatatatg tatttcatgg tacgtattct ctagcacagt 1380ctgagcaatt aaatagattc ataagcataa aaa 1413151065DNAArtificial SequenceSynthetic construct, PgC2-1 construct 15atggccccaa caaaaagaaa aggagaaagg aaggaccccg tgcaagttcc aaaacttctt 60ataagaggag gagtagaagt tctagaagtt aaaactgggg ttgactcaat tacagaggta 120gaatgctttt taactccaga aatgggtgac ccagatgagc atcttagggg ttttagtaag 180tcaatatcta tatcagatac atttgaaagt gactccccaa atagggacat gcttccttgt 240tacagtgtgg ccagaattcc actacccaat ctaaatgagg atctaacctg tggaaatata 300ctcatgtggg aggctgtgac cttaaaaact gaggttatag gggtgacaag tttgatgaat 360gtgcactcta atgggcaagc aactcatgac aatggtgcag ggaagccagt gcagggcacc 420agctttcatt ttttttctgt tgggggggag gctttagaat tacagggggt gctttttaat 480tacagaacaa agtacccaga tggaacaatt tttccaaaga atgccacagt gcaatctcaa 540gtcatgaaca cagagcacaa ggcgtaccta gataagaaca aagcatatcc tgttgaatgt 600tgggttcctg atcccaccag aaatgaaaac acaagatatt ttgggacact aacaggagga 660gaaaatgttc ctccagttct tcatataaca aacactgcca caacagtgtt gcttgatgaa 720tttggtgttg ggccactttg caaaggtgac aacttatact tgtcagctgt tgatgtctgt 780ggcatgttta caaacaggtc tggttcccag cagtggagag gactctccag atattttaag 840gtgcagctaa ggaaaaggag ggttaaaaac ccctacccaa tttctttcct tcttactgat 900ttaattaaca gaaggactcc tagagttgat gggcagccta tgtatggcat ggatgctcaa 960gtagaggagg ttagagtttt tgagggaaca gaggagcttc caggggaccc agacatgatg 1020agatacgttg acaaatatgg acagttgcag acaaaaatgc tgtaa 1065161286DNAArtificial SequenceSynthetic construct, PgA1-3B1-3 contsruct 16ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gcggccgcag gaggtggatc tggaaattct gacaagaccc 600acacctgccc tccttgccct gctcctgagc tcctcggtgg tccttctgtc ttcctcttcc 660ctcctaagcc taaggacacc ctcatgatct ctcgtacccc tgaggtcacc tgcgtcgtcg 720tcgacgtctc tcacgaggac cctgaggtca agttcaactg gtacgtcgac ggtgtcgagg 780tccacaacgc taagaccaag cctcgtgagg agcagtacaa ctctacctac cgtgtcgtct 840ctgtcctcac cgtcctccac caggactggc tcaacggtaa ggagtacaag tgcaaggtct 900ctaacaaggc tctccctgct cctatcgaga agaccatctc taaggctaag ggtcagcctc 960gtgagcctca ggtctacacc ctccctcctt ctcgtgagga gatgaccaag aaccaggtct 1020ctctcacctg cctcgtcaag ggtttctacc cttctgacat cgctgtcgag tgggagtcta 1080acggtcagcc tgagaacaac tacaagacca cccctcctgt cctcgactct gacggttctt 1140tcttcctcta ctctaagctc accgtcgaca agtctcgttg gcagcagggt aacgtcttct 1200cttgctctgt catgcacgag gctctccaca accactacac ccagaagtct ctctctctct 1260ctcctggtaa ggacctctaa gagctc 1286171337DNAArtificial SequenceSynthetic construct, PgA1-4B1-4 construct 17ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gcggccgcag gaggtggatc tggaaattct gacaagaccc 600acacctgccc tccttgccct gctcctgagc tcctcggtgg tccttctgtc ttcctcttcc 660ctcctaagcc taaggacacc ctcatgatct ctcgtacccc tgaggtcacc tgcgtcgtcg 720tcgacgtctc tcacgaggac cctgaggtca agttcaactg gtacgtcgac ggtgtcgagg 780tccacaacgc taagaccaag cctcgtgagg agcagtacaa ctctacctac cgtgtcgtct 840ctgtcctcac cgtcctccac caggactggc tcaacggtaa ggagtacaag tgcaaggtct 900ctaacaaggc tctccctgct cctatcgaga agaccatctc taaggctaag ggtcagcctc 960gtgagcctca ggtctacacc ctccctcctt ctcgtgagga gatgaccaag aaccaggtct 1020ctctcacctg cctcgtcaag ggtttctacc cttctgacat cgctgtcgag tgggagtcta 1080acggtcagcc tgagaacaac tacaagacca cccctcctgt cctcgactct gacggttctt 1140tcttcctcta ctctaagctc accgtcgaca agtctcgttg gcagcagggt aacgtcttct 1200cttgctctgt catgcacgag gctctccaca accactacac ccagaagtct ctctctctct 1260ctcctggtaa ggacctcgat ctccaaaagc ttattagcga ggaggatctt catcaccatc 1320accatcacta agagctc 1337181377DNAArtificial SequenceSynthetic construct, PgA1-3B2-3 construct 18ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gcggccgcag gaggtggatc tggaaattct gacgtcaccg 600tcccttgccc tgtcccttct acccctccta ccccttctcc ttctacccct cctacccctt 660ctccttcttg ctgccaccct cgtctctctc tccaccgtcc tgctctcgag gacctcctcc 720tcggttctga ggctaacctc acctgcaccc tcaccggtct ccgtgacgct tctggtgtca 780ccttcacctg gaccccttct tctggtaagt ctgctgtcca gggtcctcct gagcgtgacc 840tctgcggttg ctactctgtc tcttctgtcc tccctggttg cgctgagcct tggaaccacg 900gtaagacctt cacctgcacc gctgcttacc ctgagtctaa gacccctctc accgctaccc 960tctctaagtc tggtaacacc ttccgtcctg aggtccacct cctccctcct ccttctgagg 1020agctcgctct caacgagctc gtcaccctca cctgcctcgc tcgtggtttc tctcctaagg 1080acgtcctcgt ccgttggctc cagggttctc aggagctccc tcgtgagaag tacctcacct 1140gggcttctcg tcaggagcct tctcagggta ccaccacctt cgctgtcacc tctatcctcc 1200gtgtcgctgc tgaggactgg aagaagggtg acaccttctc ttgcatggtc ggtcacgagg 1260ctctccctct cgctttcacc cagaagacca tcgaccgtct cgctggtaag cctacccacg 1320tcaacgtctc tgtcgtcatg gctgaggtcg acggtacctg ctacccaata agagctc 1377191373DNAArtificial SequenceSynthetic construct, PgA1-4B2-4 construct 19ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gcggccgcag gaggtggatc tggaaattct gacgtcaccg 600tcccttgccc tgtcccttct acccctccta ccccttctcc ttctacccct cctacccctt 660ctccttcttg ctgccaccct cgtctctctc tccaccgtcc tgctctcgag gacctcctcc 720tcggttctga ggctaacctc acctgcaccc tcaccggtct ccgtgacgct tctggtgtca 780ccttcacctg gaccccttct tctggtaagt ctgctgtcca gggtcctcct gagcgtgacc 840tctgcggttg ctactctgtc tcttctgtcc tccctggttg cgctgagcct tggaaccacg 900gtaagacctt cacctgcacc gctgcttacc ctgagtctaa gacccctctc accgctaccc 960tctctaagtc tggtaacacc ttccgtcctg aggtccacct cctccctcct ccttctgagg 1020agctcgctct caacgagctc gtcaccctca cctgcctcgc tcgtggtttc tctcctaagg 1080acgtcctcgt ccgttggctc cagggttctc aggagctccc tcgtgagaag tacctcacct 1140gggcttctcg tcaggagcct tctcagggta ccaccacctt cgctgtcacc tctatcctcc 1200gtgtcgctgc tgaggactgg aagaagggtg acaccttctc ttgcatggtc ggtcacgagg 1260ctctccctct cgctttcacc cagaagacca tcgaccgtct cgctggtaag cctacccacg 1320tcaacgtctc tgtcgtcatg gctgaggtcg acggtacctg ctactaaaga tct 1373201662DNAArtificial SequenceSynthetic cosntruct, PgA1-3 B3-3 construct 20ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gcggccgcag gaggtggatc tggaaattct gtccctctcc 600ctgtcatcgc tgagctccct cctaaggtct ctgtcttcgt ccctcctcgt gacggtttct 660tcggtaaccc tcgtaagtct aagctcatct gccaggctac cggtttctct cctcgtcaga 720tccaggtctc ttggctccgt gagggtaagc aggtcggttc tggtgtcacc accgaccagg 780tccaggctga ggctaaggag tctggtccta ccacctacaa ggtcacctct accctcacca 840tcaaggagtc tgactggctc tctcagtcta tgttcacctg ccgtgtcgac caccgtggtc 900tcaccttcca gcagaacgct tcttctatgt gcgtccctga ccaggacacc gctatccgtg 960tcttcgctat ccctccttct ttcgcttcta tcttcctcac caagtctacc aagctcacct 1020gcctcgtcac cgacctcacc acctacgact ctgtcaccat ctcttggacc cgtcagaacg 1080gtgaggctgt caagacccac accaacatct ctgagtctca ccctaacgct accttctctg 1140ctgtcggtga ggcttctatc tgcgaggacg actggaactc tggtgagcgt ttcacctgca 1200ccgtcaccca caccgacctc ccttctcctc tcaagcagac catctctcgt cctaagggtg 1260tcgctctcca ccgtcctgac gtctacctcc tccctcctgc tcgtgagcag ctcaacctcc 1320gtgagtctgc taccatcacc tgcctcgtca ccggtttctc tcctgctgac gtcttcgtcc 1380agtggatgca gcgtggtcag cctctctctc ctgagaagta cgtcacctct gctcctatgc 1440ctgagcctca ggctcctggt cgttacttcg ctcactctat cctcaccgtc tctgaggagg 1500agtggaacac cggtgagacc tacacctgcg tcgtcgctca cgaggctctc cctaaccgtg 1560tcaccgagcg taccgtcgac aagtctaccg gtaagcctac cctctacaac gtctctctcg 1620tcatgtctga caccgctggt acctgctacc caataagagc tc 1662211658DNAArtificial SequenceSynthetic construct, PgA1-2B3-2 construct 21ccatggaaca accttcttgt cgtcgagctt ttgatcttta ttttgttctt gataaatctg 60gttctgttgc taataattgg attgaaattt ataattttgt tcaacaactt gctgaaagat 120ttgtttctcc tgaaatgaga ctttctttta ttgttttttc ttctcaagct actattattc 180ttcctcttac tggtgataga ggaaaaattt ctaaaggact tgaggatttg aaaagagttt 240ctcctgttgg tgaaacttat attcatgagg gacttaaact tgctaatgaa caaattcaaa 300aagctggtgg tcttaaaact tcttctatta ttattgctct tactgatgga aaacttgatg 360gtcttgttcc ttcttatgct gaaaaagaag ctaaaatttc aagatcactt ggtgcttctg 420tttattgtgt tggtgttctt gattttgaac aagctcaact tgaaagaatt gctgattcta 480aagaacaagt ttttcctgtt aagggtggat ttcaagctct taaaggaatt attaattcta 540ttcttgctca atcttgtact gcggccgcag gaggtggatc tggaaattct gtccctctcc 600ctgtcatcgc tgagctccct cctaaggtct ctgtcttcgt ccctcctcgt gacggtttct 660tcggtaaccc tcgtaagtct aagctcatct gccaggctac cggtttctct cctcgtcaga 720tccaggtctc ttggctccgt gagggtaagc aggtcggttc tggtgtcacc accgaccagg 780tccaggctga ggctaaggag tctggtccta ccacctacaa ggtcacctct accctcacca 840tcaaggagtc tgactggctc tctcagtcta tgttcacctg ccgtgtcgac caccgtggtc 900tcaccttcca gcagaacgct tcttctatgt gcgtccctga ccaggacacc gctatccgtg 960tcttcgctat ccctccttct ttcgcttcta tcttcctcac caagtctacc aagctcacct 1020gcctcgtcac cgacctcacc acctacgact ctgtcaccat ctcttggacc cgtcagaacg 1080gtgaggctgt caagacccac accaacatct ctgagtctca ccctaacgct accttctctg 1140ctgtcggtga ggcttctatc tgcgaggacg actggaactc tggtgagcgt ttcacctgca 1200ccgtcaccca caccgacctc ccttctcctc tcaagcagac catctctcgt cctaagggtg 1260tcgctctcca ccgtcctgac gtctacctcc tccctcctgc tcgtgagcag ctcaacctcc 1320gtgagtctgc taccatcacc tgcctcgtca ccggtttctc tcctgctgac gtcttcgtcc 1380agtggatgca gcgtggtcag cctctctctc ctgagaagta cgtcacctct gctcctatgc 1440ctgagcctca ggctcctggt cgttacttcg ctcactctat cctcaccgtc tctgaggagg 1500agtggaacac cggtgagacc tacacctgcg tcgtcgctca cgaggctctc cctaaccgtg 1560tcaccgagcg taccgtcgac aagtctaccg gtaagcctac cctctacaac gtctctctcg 1620tcatgtctga caccgctggt acctgctact aaagatct 1658221064DNAArtificial SequenceSynthetic construct PgB4-2 A1-3construct 22ccatgggtgc tgctctcgct ctcctcggtg acctcgtcgc taccgtctct gaggctgctg 60ctgctaccgg tttctctgtc gctgagatcg ctgctggtga ggctgctgct accatcgagg 120tcgagatcgc ttctctcgct accgtcgagg gtatcacctc tacctctgag gctatcgctg 180ctatcggtct cacccctgag acctacgctg tcatcaccgg tgctcctggt gctgtcgctg 240gtttcgctgc tctcgtccag accgtcaccg gtggttctgc tatcgctcag ctcggttacc 300gtttcttcgc tgactgggac cacaaggtct ctaccgtcgg tctcttccag cagcctgcta 360tggctctcca gctcttcaac cctgaggact actacgacat cctcttccct ggtgtcaacg 420ctttcgtcaa caacatccac tacctcgacc ctcgtcactg gggtccttct ctcttctcta 480ccatctctca ggctttctgg aacctcgtcc gtgacgacct ccctgctctc acctctcagg 540agatccagcg tcgtacccag aagctcttcg tcgagtctct cgctcgtttc ctcgaggaga 600ccacctgggc tatcgtcaac tctcctgcta acctctacaa ctacatctct gactactact 660ctcgtctctc tcctgtccgt ccttctatgg tccgtcaggt cgctcagcgt gagggtacct 720acatctcttt cggtcactct tacacccagt ctatcgacga cgctgactct atccaggagg 780tcacccagcg tctcgacctc aagaccccta acgtccagtc tggtgagttc atcgagcgtt 840ctatcgctcc tggtggtgct aaccagcgtt ctgctcctca gtggatgctc cctctcctcc 900tcggtctcta cggtaccgtc acccctgctc tcgaggctta cgaggacggt cctaacaaga 960agaagcgtcg taaggagggt cctcgtgctt cttctaagac ctcttacaag cgtcgttctc 1020gttcttctcg ttctggaggt ggatctggaa attctgcggc cgca 106423530DNAArtificial SequenceSynthetic construct; PgA1-1B5 construct 23ccatggccga cacggccagg ggcacgcacc acgacatcat cggcagggac cagtacccga 60tgatgggcag ggacagggac cagtaccaga tgtccggcag gggctccgac tactccaagt 120ccaggcagat cgccaaggcc gccacggccg tgacggccgg cggctccctc ctcgtgctct 180cctccctcac gctcgtgggc acggtgatcg ccctcacggt ggccacgccg ctcctcgtga 240tcttctcccc gatcctcgtg ccggccctca tcacggtggc cctcctcatc acgggcttcc 300tctcctccgg cggcttcggc atcgccgcca tcacggtgtt ctcctggatc tacaagtacg 360ccacgggcga gcacccgcag ggctccgaca agctcgactc cgccaggatg aagctcggct 420ccaaggccca ggacctcaag gacagggccc agtactacgg ccagcagcac acgggcggcg 480agcacgacag ggacaggacg aggggcggcc agcacacgac ggcggccgca 53024186PRTHomo sapiensMISC_FEATURE(1)..(186)A1-CMG2 protein 24Glu Gln Pro Ser Cys Arg Arg Ala Phe Asp Leu Tyr Phe Val Leu Asp 1 5 10 15 Lys Ser Gly Ser Val Ala Asn Asn Trp Ile Glu Ile Tyr Asn Phe Val 20 25 30 Gln Gln Leu Ala Glu Arg Phe Val Ser Pro Glu Met Arg Leu Ser Phe 35 40 45 Ile Val Phe Ser Ser Gln Ala Thr Ile Ile Leu Pro Leu Thr Gly Asp 50 55 60 Arg Gly Lys Ile Ser Lys Gly Leu Glu Asp Leu Lys Arg Val Ser Pro 65 70 75 80 Val Gly Glu Thr Tyr Ile His Glu Gly Leu Lys Leu Ala Asn Glu Gln 85 90 95 Ile Gln Lys Ala Gly Gly Leu Lys Thr Ser Ser Ile Ile Ile Ala Leu 100 105 110 Thr Asp Gly Lys Leu Asp Gly Leu Val Pro Ser Tyr Ala Glu Lys Glu 115 120 125 Ala Lys Ile Ser Arg Ser Leu Gly Ala Ser Val Tyr Cys Val Gly Val 130 135 140 Leu Asp Phe Glu Gln Ala Gln Leu Glu Arg Ile Ala Asp Ser Lys Glu 145 150 155 160 Gln Val Phe Pro Val Lys Gly Gly Phe Gln Ala Leu Lys Gly Ile Ile 165 170 175 Asn Ser Ile Leu Ala Gln Ser Cys Thr Glu 180 185 25244PRTBacillus anthracisMISC_FEATURE(1)..(244)A2-PA-LF protein 25Glu Asn Lys Arg Lys Asp Glu Glu Arg Asn Lys Thr Gln Glu Glu His 1 5 10 15 Leu Lys Glu Ile Met Lys His Ile Val Lys Ile Glu Val Lys Gly Glu 20 25 30 Glu Ala Val Lys Lys Glu Ala Ala Glu Lys Leu Leu Glu Lys Val Pro 35 40 45 Ser Asp Val Leu Glu Met Tyr Lys Ala Ile Gly Gly Lys Ile Tyr Ile 50 55 60 Val Asp Gly Asp Ile Thr Lys His Ile Ser Leu Glu Ala Leu Ser Glu 65 70 75 80 Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly Lys Asp Ala Leu Leu His 85 90 95 Glu His Tyr Val Tyr Ala Lys Glu Gly Tyr Glu Pro Val Leu Val Ile 100 105

110 Gln Ser Ser Glu Asp Tyr Val Glu Asn Thr Glu Lys Ala Leu Asn Val 115 120 125 Tyr Tyr Glu Ile Gly Lys Ile Leu Ser Arg Asp Ile Leu Ser Lys Ile 130 135 140 Asn Gln Pro Tyr Gln Lys Phe Leu Asp Val Leu Asn Thr Ile Lys Asn 145 150 155 160 Ala Ser Asp Ser Asp Gly Gln Asp Leu Leu Phe Thr Asn Gln Leu Lys 165 170 175 Glu His Pro Thr Asp Phe Ser Val Glu Phe Leu Glu Gln Asn Ser Asn 180 185 190 Glu Val Gln Glu Val Phe Ala Lys Ala Phe Ala Tyr Tyr Ile Glu Pro 195 200 205 Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu Ala Phe Asn Tyr 210 215 220 Met Asp Lys Phe Asn Glu Gln Glu Ile Asn Leu Ser Leu Glu Glu Leu 225 230 235 240 Lys Asp Gln Arg 26476PRTHomo sapiensMISC_FEATURE(1)..(476)B1 protein 26Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe 35 40 45 Asn Arg Tyr Thr Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala 65 70 75 80 Gln Arg Phe Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Asn Leu Asp Asn Ser Gly Thr Tyr Tyr Tyr 115 120 125 Phe Ser Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 27340PRTHomo sapiensMISC_FEATURE(1)..(340)B2 protein 27Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr 1 5 10 15 Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Ser Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110 Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125 Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140 Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly 145 150 155 160 Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175 Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180 185 190 Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys 195 200 205 Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser 210 215 220 Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg 225 230 235 240 Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255 Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265 270 Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285 Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His 290 295 300 Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala 305 310 315 320 Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp 325 330 335 Gly Thr Cys Tyr 340 28353PRTHomo sapiensMISC_FEATURE(1)..(353)B3 protein 28Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe 1 5 10 15 Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu 20 25 30 Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp 35 40 45 Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val 50 55 60 Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser 65 70 75 80 Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln Ser Met Phe Thr 85 90 95 Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser 100 105 110 Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro 115 120 125 Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys 130 135 140 Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr 145 150 155 160 Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser 165 170 175 His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu 180 185 190 Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr 195 200 205 Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val 210 215 220 Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln 225 230 235 240 Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe 245 250 255 Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu 260 265 270 Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala 275 280 285 Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu 290 295 300 Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu 305 310 315 320 Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro 325 330 335 Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys 340 345 350 Tyr 29344PRTJC virusMISC_FEATURE(1)..(344)B4 VP coat protein 29Met Gly Ala Ala Leu Ala Leu Leu Gly Asp Leu Val Ala Thr Val Ser 1 5 10 15 Glu Ala Ala Ala Ala Thr Gly Phe Ser Val Ala Glu Ile Ala Ala Gly 20 25 30 Glu Ala Ala Ala Thr Ile Glu Val Glu Ile Ala Ser Leu Ala Thr Val 35 40 45 Glu Gly Ile Thr Ser Thr Ser Glu Ala Ile Ala Ala Ile Gly Leu Thr 50 55 60 Pro Glu Thr Tyr Ala Val Ile Thr Gly Ala Pro Gly Ala Val Ala Gly 65 70 75 80 Phe Ala Ala Leu Val Gln Thr Val Thr Gly Gly Ser Ala Ile Ala Gln 85 90 95 Leu Gly Tyr Arg Phe Phe Ala Asp Trp Asp His Lys Val Ser Thr Val 100 105 110 Gly Leu Phe Gln Gln Pro Ala Met Ala Leu Gln Leu Phe Asn Pro Glu 115 120 125 Asp Tyr Tyr Asp Ile Leu Phe Pro Gly Val Asn Ala Phe Val Asn Asn 130 135 140 Ile His Tyr Leu Asp Pro Arg His Trp Gly Pro Ser Leu Phe Ser Thr 145 150 155 160 Ile Ser Gln Ala Phe Trp Asn Leu Val Arg Asp Asp Leu Pro Ala Leu 165 170 175 Thr Ser Gln Glu Ile Gln Arg Arg Thr Gln Lys Leu Phe Val Glu Ser 180 185 190 Leu Ala Arg Phe Leu Glu Glu Thr Thr Trp Ala Ile Val Asn Ser Pro 195 200 205 Ala Asn Leu Tyr Asn Tyr Ile Ser Asp Tyr Tyr Ser Arg Leu Ser Pro 210 215 220 Val Arg Pro Ser Met Val Arg Gln Val Ala Gln Arg Glu Gly Thr Tyr 225 230 235 240 Ile Ser Phe Gly His Ser Tyr Thr Gln Ser Ile Asp Asp Ala Asp Ser 245 250 255 Ile Gln Glu Val Thr Gln Arg Leu Asp Leu Lys Thr Pro Asn Val Gln 260 265 270 Ser Gly Glu Phe Ile Glu Arg Ser Ile Ala Pro Gly Gly Ala Asn Gln 275 280 285 Arg Ser Ala Pro Gln Trp Met Leu Pro Leu Leu Leu Gly Leu Tyr Gly 290 295 300 Thr Val Thr Pro Ala Leu Glu Ala Tyr Glu Asp Gly Pro Asn Lys Lys 305 310 315 320 Lys Arg Arg Lys Glu Gly Pro Arg Ala Ser Ser Lys Thr Ser Tyr Lys 325 330 335 Arg Arg Ser Arg Ser Ser Arg Ser 340 30173PRTArabidopsis thalianaMISC_FEATURE(1)..(173)B5 oleosin protein 30Met Ala Asp Thr Ala Arg Gly Thr His His Asp Ile Ile Gly Arg Asp 1 5 10 15 Gln Tyr Pro Met Met Gly Arg Asp Arg Asp Gln Tyr Gln Met Ser Gly 20 25 30 Arg Gly Ser Asp Tyr Ser Lys Ser Arg Gln Ile Ala Lys Ala Ala Thr 35 40 45 Ala Val Thr Ala Gly Gly Ser Leu Leu Val Leu Ser Ser Leu Thr Leu 50 55 60 Val Gly Thr Val Ile Ala Leu Thr Val Ala Thr Pro Leu Leu Val Ile 65 70 75 80 Phe Ser Pro Ile Leu Val Pro Ala Leu Ile Thr Val Ala Leu Leu Ile 85 90 95 Thr Gly Phe Leu Ser Ser Gly Gly Phe Gly Ile Ala Ala Ile Thr Val 100 105 110 Phe Ser Trp Ile Tyr Lys Tyr Ala Thr Gly Glu His Pro Gln Gly Ser 115 120 125 Asp Lys Leu Asp Ser Ala Arg Met Lys Leu Gly Ser Lys Ala Gln Asp 130 135 140 Leu Lys Asp Arg Ala Gln Tyr Tyr Gly Gln Gln His Thr Gly Gly Glu 145 150 155 160 His Asp Arg Asp Arg Thr Arg Gly Gly Gln His Thr Thr 165 170 31427PRTArtificial SequenceSynthetic construct, Pg A1-5B1-5 protein 31Met Glu Gln Pro Ser Cys Arg Arg Ala Phe Asp Leu Tyr Phe Val Leu 1 5 10 15 Asp Lys Ser Gly Ser Val Ala Asn Asn Trp Ile Glu Ile Tyr Asn Phe 20 25 30 Val Gln Gln Leu Ala Glu Arg Phe Val Ser Pro Glu Met Arg Leu Ser 35 40 45 Phe Ile Val Phe Ser Ser Gln Ala Thr Ile Ile Leu Pro Leu Thr Gly 50 55 60 Asp Arg Gly Lys Ile Ser Lys Gly Leu Glu Asp Leu Lys Arg Val Ser 65 70 75 80 Pro Val Gly Glu Thr Tyr Ile His Glu Gly Leu Lys Leu Ala Asn Glu 85 90 95 Gln Ile Gln Lys Ala Gly Gly Leu Lys Thr Ser Ser Ile Ile Ile Ala 100 105 110 Leu Thr Asp Gly Lys Leu Asp Gly Leu Val Pro Ser Tyr Ala Glu Lys 115 120 125 Glu Ala Lys Ile Ser Arg Ser Leu Gly Ala Ser Val Tyr Cys Val Gly 130 135 140 Val Leu Asp Phe Glu Gln Ala Gln Leu Glu Arg Ile Ala Asp Ser Lys 145 150 155 160 Glu Gln Val Phe Pro Val Lys Gly Gly Phe Gln Ala Leu Lys Gly Ile 165 170 175 Ile Asn Ser Ile Leu Ala Gln Ser Cys Thr Glu Gly Gly Gly Ser Gly 180 185 190 Asn Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 195 200 205 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 210 215 220 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 225 230 235 240 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 245 250 255 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 260 265 270 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 275 280 285 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 290 295 300 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 305 310 315 320 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 325 330 335 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 340 345 350 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 355 360 365 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 370 375 380 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 385 390 395 400 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 405 410 415 Leu Ser Pro Gly Lys Asp Leu Ile Glu Gly Arg 420 425 32395DNAHomo sapiensmisc_feature(1)..(395)C1-2 construct 32ccatgggaaa

gtgcaagtgc gctcgtatca cctctcgtat catccgttct tctgaggacc 60ctaacgagga catcgtcgag cgtaacatcc gtatcatcgt ccctctcaac aaccgtgaga 120acatctctga ccctacctct cctctccgta cccgtttcgt ctaccacctc tctgacctct 180gcaagaagtg cgaccctacc gaggtcgagc tcgacaacca gatcgtcacc gctacccagt 240ctaacatctg cgacgaggac tctgctaccg agacctgcta cacctacgac cgtaacaagt 300gctacaccgc tgtcgtccct ctcgtctacg gtggtgagac caagatggtc gagaccgctc 360tcacccctga cgcttgctac cctgactaag agctc 39533401DNAhomo sapiensmisc_feature(1)..(401)C1-3 construct 33ccatgggaaa gtgcaagtgc gctcgtatca cctctcgtat catccgttct tctgaggacc 60ctaacgagga catcgtcgag cgtaacatcc gtatcatcgt ccctctcaac aaccgtgaga 120acatctctga ccctacctct cctctccgta cccgtttcgt ctaccacctc tctgacctct 180gcaagaagtg cgaccctacc gaggtcgagc tcgacaacca gatcgtcacc gctacccagt 240ctaacatctg cgacgaggac tctgctaccg agacctgcta cacctacgac cgtaacaagt 300gctacaccgc tgtcgtccct ctcgtctacg gtggtgagac caagatggtc gagaccgctc 360tcacccctga cgcttgctac cctgacgcag atctccaata a 401341133DNAArtificial SequenceSynthetic construct, C2-2 construct 34ccatggcccc aacaaagaga aaaggagaaa ggaaggaccc agtgcaagtt ccaaaacttc 60tcataagagg aggagtagaa gttcttgaag ttaaaactgg agttgactca attacagagg 120tagaatgctt cttaactcca gaaatgggtg acccagatga gcatcttagg ggttttagta 180agtcaatatc tatatcagat acatttgaaa gtgactcccc aaatagggac atgcttcctt 240gttacagtgt ggccaggatt ccactaccta atctaaatga ggatctaact tgtggaaata 300tactcatgtg ggaggctgtg acattaaaga ctgaggttat aggagtgaca agtttgatga 360atgtgcattc taatggtcaa gcaactcatg acaatggtgc aggtaagcca gtgcagggta 420caagttttca tttcttttct gttggaggtg aggctttaga attacaggga gtgcttttta 480attacagaac aaagtaccca gatggaacaa tttttccaaa gaatgccaca gtgcaatctc 540aagtcatgaa cacagagcat aaggcgtacc tagataagaa caaagcatat cctgttgaat 600gttgggttcc tgatccaact agaaatgaaa acacaagata ttttggtaca ctaacaggag 660gagaaaatgt tcctccagtt cttcatataa caaacactgc cacaacagtg ttgcttgatg 720aatttggtgt tggaccactt tgtaaaggtg acaacttata cttgtcagct gttgatgtct 780gtggtatgtt tacaaacagg tctggttccc agcagtggag aggactctcc agatatttta 840aggtgcagct aaggaagagg agggttaaga acccataccc aatttctttc cttcttactg 900atttgattaa cagaaggact cctagagttg atggacagcc tatgtatggt atggatgctc 960aagtagagga ggttagagtt tttgagggaa cagaggagct tccaggagac ccagacatga 1020tgagatacgt tgacaaatat ggacagttgc agacaaagat gctggcggcc gcagatctcc 1080aaaagcttat tagcgaggag gatcttcatc accatcacca tcactaagag ctc 113335159PRTHomo sapiensMISC_FEATURE(1)..(159)C-1-protein 35Met Lys Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15 Ala Val His Val Lys Ala Gln Glu Asp Glu Arg Ile Val Leu Val Asp 20 25 30 Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser 35 40 45 Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val 50 55 60 Pro Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg 65 70 75 80 Thr Arg Phe Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro 85 90 95 Thr Glu Val Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn 100 105 110 Ile Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg 115 120 125 Asn Lys Cys Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr 130 135 140 Lys Met Val Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp 145 150 155 36354PRTJC virusMISC_FEATURE(1)..(354)C2 protein 36Met Ala Pro Thr Lys Arg Lys Gly Glu Arg Lys Asp Pro Val Gln Val 1 5 10 15 Pro Lys Leu Leu Ile Arg Gly Gly Val Glu Val Leu Glu Val Lys Thr 20 25 30 Gly Val Asp Ser Ile Thr Glu Val Glu Cys Phe Leu Thr Pro Glu Met 35 40 45 Gly Asp Pro Asp Glu His Leu Arg Gly Phe Ser Lys Ser Ile Ser Ile 50 55 60 Ser Asp Thr Phe Glu Ser Asp Ser Pro Asn Arg Asp Met Leu Pro Cys 65 70 75 80 Tyr Ser Val Ala Arg Ile Pro Leu Pro Asn Leu Asn Glu Asp Leu Thr 85 90 95 Cys Gly Asn Ile Leu Met Trp Glu Ala Val Thr Leu Lys Thr Glu Val 100 105 110 Ile Gly Val Thr Ser Leu Met Asn Val His Ser Asn Gly Gln Ala Thr 115 120 125 His Asp Asn Gly Ala Gly Lys Pro Val Gln Gly Thr Ser Phe His Phe 130 135 140 Phe Ser Val Gly Gly Glu Ala Leu Glu Leu Gln Gly Val Leu Phe Asn 145 150 155 160 Tyr Arg Thr Lys Tyr Pro Asp Gly Thr Ile Phe Pro Lys Asn Ala Thr 165 170 175 Val Gln Ser Gln Val Met Asn Thr Glu His Lys Ala Tyr Leu Asp Lys 180 185 190 Asn Lys Ala Tyr Pro Val Glu Cys Trp Val Pro Asp Pro Thr Arg Asn 195 200 205 Glu Asn Thr Arg Tyr Phe Gly Thr Leu Thr Gly Gly Glu Asn Val Pro 210 215 220 Pro Val Leu His Ile Thr Asn Thr Ala Thr Thr Val Leu Leu Asp Glu 225 230 235 240 Phe Gly Val Gly Pro Leu Cys Lys Gly Asp Asn Leu Tyr Leu Ser Ala 245 250 255 Val Asp Val Cys Gly Met Phe Thr Asn Arg Ser Gly Ser Gln Gln Trp 260 265 270 Arg Gly Leu Ser Arg Tyr Phe Lys Val Gln Leu Arg Lys Arg Arg Val 275 280 285 Lys Asn Pro Tyr Pro Ile Ser Phe Leu Leu Thr Asp Leu Ile Asn Arg 290 295 300 Arg Thr Pro Arg Val Asp Gly Gln Pro Met Tyr Gly Met Asp Ala Gln 305 310 315 320 Val Glu Glu Val Arg Val Phe Glu Gly Thr Glu Glu Leu Pro Gly Asp 325 330 335 Pro Asp Met Met Arg Tyr Val Asp Lys Tyr Gly Gln Leu Gln Thr Lys 340 345 350 Met Leu 37721DNAArtificial SequenceSynthetic construct, PgA2-4B1-3 construct 37ccatgggtga cgtcggtatg cacgtcaagg agaaggagaa gaacaaggac gagaacaagc 60gtaaggacga ggagcgtaac aagacccagg aggagcacct caaggagatc atgaagcaca 120tcgtcaagat cgaggtcaag ggtgaggagg ctgtcaagaa ggaggctgct gagaagctcc 180tcgagaaggt cccttctgac gtcctcgaga tgtacaaggc tatcggtggt aagatctaca 240tcgtcgacgg tgacatcacc aagcacatct ctctcgaggc tctctctgag gacaagaaga 300agatcaagga catctacggt aaggacgctc tcctccacga gcactacgtc tacgctaagg 360agggttacga gcctgtcctc gtcatccagt cttctgagga ctacgtcgag aacaccgaga 420aggctctcaa cgtctactac gagatcggta agatcctctc tcgtgacatc ctctctaaga 480tcaaccagcc ttaccagaag ttcctcgacg tcctcaacac catcaagaac gcttctgact 540ctgacggtca ggacctcctc ttcaccaacc agctcaagga gcaccctacc gacttctctg 600tcgagttcct cgagcagaac tctaacgagg tccaggaggt cttcgctaag gctttcgctt 660actacatcga gcctcagcac cgtgacgtcc tccagctcta cgctcctgag gctgcggccg 720c 721381297DNAArtificial SequenceSynthetic construct, Pg1-1B1-1-1 construct 38agtcccatgg aacaaccatc ttgccgtagg gctttcgatc tctacttcgt gctcgataag 60tccggctctg ttgctaacaa ctggatcgaa atctacaact tcgtgcagca gctcgctgag 120agattcgttt ctccagagat gaggctctcc ttcatcgtgt tctcttcaca ggctactatc 180atcctcccac tcactggtga taggggcaag atttctaagg gactcgagga tctcaagagg 240gtgtcaccag ttggagagac ttacattcac gagggactca agcttgctaa cgagcagatt 300caaaaggctg gcggcctcaa gacttcctcc attattatcg ctctcactga tggcaagctc 360gatggacttg ttccatccta cgctgagaaa gaggctaaga tcagtcgttc ccttggcgct 420tctgtttact gcgttggagt gcttgatttc gagcaggctc agcttgagag gatcgctgat 480tccaaagagc aggttttccc agttaagggc ggattccaag ctctcaaggg catcatcaac 540tccatccttg ctcagtcttg tactgagggt ggtggatccg gaaactccga taagactcat 600acttgtccac catgcccagc tccagaactt cttggaggac catctgtgtt cttgttccca 660ccaaagccaa aggatactct catgatctcc aggactccag aggttacatg cgttgtggtt 720gatgtgtctc acgaggatcc agaggtgaag ttcaactggt atgtggatgg tgttgaggtg 780cacaacgcta agactaagcc acgtgaggaa cagtacaact ccacttacag ggtggtgtct 840gtgcttactg tgcttcacca ggattggctc aacggcaaag agtacaagtg caaggtgtcc 900aacaaggctc tcccagctcc aatcgaaaag actatctcca aggctaaggg acagccaagg 960gaaccacagg tttacactct tccaccatcc agggaagaga tgactaagaa ccaggtgtcc 1020cttacttgcc tcgtgaaggg attctaccca tccgatattg ctgttgagtg ggagtctaat 1080ggccagccag agaacaacta caagactact ccaccagtgc tcgattccga tggctcattc 1140ttcttgtact ccaagctcac tgtggataag tccaggtggc agcagggaaa cgttttctct 1200tgctctgtta tgcacgaggc tctccacaac cactacactc agaagtcctt gtccttgtcc 1260ccaggcaagg atcttattga gggaagaaga tctccaa 12973910DNAArtificial SequenceSynthetic construct, Kozak-like sequence 39gacaccatgg 104019DNAArtificial SequenceSynthetic construct, BglII and Sac sites 40agatctccaa taagagctc 194116DNAArtificial SequenceSynthetic construct, NotI site 41tcttgcggcc gcagga 164224PRTArtificial SequenceSynthetic construct, BAA targeting peptide 42Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly 1 5 10 15 Leu Ser Ala Ser Leu Ala Ser Gly 20 4333PRTArtificial SequenceSynthetic construct, APBP1 targeting peptide 43Met Ile Val Leu Ser Val Gly Ser Ala Ser Ser Ser Pro Ile Val Val 1 5 10 15 Val Phe Ser Val Ala Leu Leu Leu Phe Tyr Phe Ser Glu Thr Ser Leu 20 25 30 Gly 447PRTArtificial SequenceSynthetic construct, flexible linker 44Gly Gly Gly Ser Gly Asn Ser 1 5 456PRTArtificial SequenceSyntehtic construct, Thrombin cleavage factor 45Leu Val Pro Arg Gly Ser 1 5 464PRTArtificial SequenceSynthetic construct, factor Xa protease cleavage peptide 46Ile Glu Gly Arg 1 47558DNAArtificial SequenceSynthetic construct, PgA1-3 construct 47atggaacaac cttcttgtcg tcgagctttt gatctttatt ttgttcttga taaatctggt 60tctgttgcta ataattggat tgaaatttat aattttgttc aacaacttgc tgaaagattt 120gtttctcctg aaatgagact ttcttttatt gttttttctt ctcaagctac tattattctt 180cctcttactg gtgatagagg aaaaatttct aaaggacttg aggatttgaa aagagtttct 240cctgttggtg aaacttatat tcatgaggga cttaaacttg ctaatgaaca aattcaaaaa 300gctggtggtc ttaaaacttc ttctattatt attgctctta ctgatggaaa acttgatggt 360cttgttcctt cttatgctga aaaagaagct aaaatttcaa gatcacttgg tgcttctgtt 420tattgtgttg gtgttcttga ttttgaacaa gctcaacttg aaagaattgc tgattctaaa 480gaacaagttt ttcctgttaa gggtggattt caagctctta aaggaattat taattctatt 540cttgctcaat cttgtact 55848690DNAArtificial SequenceSynthetic construct, PgB1-3 construct 48gacaagaccc acacctgccc tccttgccct gctcctgagc tcctcggtgg tccttctgtc 60ttcctcttcc ctcctaagcc taaggacacc ctcatgatct ctcgtacccc tgaggtcacc 120tgcgtcgtcg tcgacgtctc tcacgaggac cctgaggtca agttcaactg gtacgtcgac 180ggtgtcgagg tccacaacgc taagaccaag cctcgtgagg agcagtacaa ctctacctac 240cgtgtcgtct ctgtcctcac cgtcctccac caggactggc tcaacggtaa ggagtacaag 300tgcaaggtct ctaacaaggc tctccctgct cctatcgaga agaccatctc taaggctaag 360ggtcagcctc gtgagcctca ggtctacacc ctccctcctt ctcgtgagga gatgaccaag 420aaccaggtct ctctcacctg cctcgtcaag ggtttctacc cttctgacat cgctgtcgag 480tgggagtcta acggtcagcc tgagaacaac tacaagacca cccctcctgt cctcgactct 540gacggttctt tcttcctcta ctctaagctc accgtcgaca agtctcgttg gcagcagggt 600aacgtcttct cttgctctgt catgcacgag gctctccaca accactacac ccagaagtct 660ctctctctct ctcctggtaa ggacctctaa 69049588DNAArtificial SequenceSynthetic construct, PgA1-4 construct 49atggaacaac cttcttgtcg tcgagctttt gatctttatt ttgttcttga taaatctggt 60tctgttgcta ataattggat tgaaatttat aattttgttc aacaacttgc tgaaagattt 120gtttctcctg aaatgagact ttcttttatt gttttttctt ctcaagctac tattattctt 180cctcttactg gtgatagagg aaaaatttct aaaggacttg aggatttgaa aagagtttct 240cctgttggtg aaacttatat tcatgaggga cttaaacttg ctaatgaaca aattcaaaaa 300gctggtggtc ttaaaacttc ttctattatt attgctctta ctgatggaaa acttgatggt 360cttgttcctt cttatgctga aaaagaagct aaaatttcaa gatcacttgg tgcttctgtt 420tattgtgttg gtgttcttga ttttgaacaa gctcaacttg aaagaattgc tgattctaaa 480gaacaagttt ttcctgttaa gggtggattt caagctctta aaggaattat taattctatt 540cttgctcaat cttgtactgc ggccgcagga ggtggatctg gaaattct 58850680DNAArtificial SequenceSynthetic construct, PgB1-4 construct 50gacaagaccc acacctgccc tccttgccct gctcctgagc tcctcggtgg tccttctgtc 60ttcctcttcc ctcctaagcc taaggacacc ctcatgatct ctcgtacccc tgaggtcacc 120tgcgtcgtcg tcgacgtctc tcacgaggac cctgaggtca agttcaactg gtacgtcgac 180ggtgtcgagg tccacaacgc taagaccaag cctcgtgagg agcagtacaa ctctacctac 240cgtgtcgtct ctgtcctcac cgtcctccac caggactggc tcaacggtaa ggagtacaag 300tgcaaggtct ctaacaaggc tctccctgct cctatcgaga agaccatctc taaggctaag 360ggtcagcctc gtgagcctca ggtctacacc ctccctcctt ctcgtgagga gatgaccaag 420aaccaggtct ctctcacctg cctcgtcaag ggtttctacc cttctgacat cgctgtcgag 480tgggagtcta acggtcagcc tgagaacaac tacaagacca cccctcctgt cctcgactct 540gacggttctt tcttcctcta ctctaagctc accgtcgaca agtctcgttg gcagcagggt 600aacgtcttct cttgctctgt catgcacgag gctctccaca accactacac ccagaagtct 660ctctctctct ctcctggtaa 68051790DNAArtificial SequenceSynthetic construct, PgB2-3 construct 51ggaaattctg acgtcaccgt cccttgccct gtcccttcta cccctcctac cccttctcct 60tctacccctc ctaccccttc tccttcttgc tgccaccctc gtctctctct ccaccgtcct 120gctctcgagg acctcctcct cggttctgag gctaacctca cctgcaccct caccggtctc 180cgtgacgctt ctggtgtcac cttcacctgg accccttctt ctggtaagtc tgctgtccag 240ggtcctcctg agcgtgacct ctgcggttgc tactctgtct cttctgtcct ccctggttgc 300gctgagcctt ggaaccacgg taagaccttc acctgcaccg ctgcttaccc tgagtctaag 360acccctctca ccgctaccct ctctaagtct ggtaacacct tccgtcctga ggtccacctc 420ctccctcctc cttctgagga gctcgctctc aacgagctcg tcaccctcac ctgcctcgct 480cgtggtttct ctcctaagga cgtcctcgtc cgttggctcc agggttctca ggagctccct 540cgtgagaagt acctcacctg ggcttctcgt caggagcctt ctcagggtac caccaccttc 600gctgtcacct ctatcctccg tgtcgctgct gaggactgga agaagggtga caccttctct 660tgcatggtcg gtcacgaggc tctccctctc gctttcaccc agaagaccat cgaccgtctc 720gctggtaagc ctacccacgt caacgtctct gtcgtcatgg ctgaggtcga cggtacctgc 780tacccaataa 79052777DNAArtificial SequenceSynthetic construct, PgB2-4 construct 52gacgtcaccg tcccttgccc tgtcccttct acccctccta ccccttctcc ttctacccct 60cctacccctt ctccttcttg ctgccaccct cgtctctctc tccaccgtcc tgctctcgag 120gacctcctcc tcggttctga ggctaacctc acctgcaccc tcaccggtct ccgtgacgct 180tctggtgtca ccttcacctg gaccccttct tctggtaagt ctgctgtcca gggtcctcct 240gagcgtgacc tctgcggttg ctactctgtc tcttctgtcc tccctggttg cgctgagcct 300tggaaccacg gtaagacctt cacctgcacc gctgcttacc ctgagtctaa gacccctctc 360accgctaccc tctctaagtc tggtaacacc ttccgtcctg aggtccacct cctccctcct 420ccttctgagg agctcgctct caacgagctc gtcaccctca cctgcctcgc tcgtggtttc 480tctcctaagg acgtcctcgt ccgttggctc cagggttctc aggagctccc tcgtgagaag 540tacctcacct gggcttctcg tcaggagcct tctcagggta ccaccacctt cgctgtcacc 600tctatcctcc gtgtcgctgc tgaggactgg aagaagggtg acaccttctc ttgcatggtc 660ggtcacgagg ctctccctct cgctttcacc cagaagacca tcgaccgtct cgctggtaag 720cctacccacg tcaacgtctc tgtcgtcatg gctgaggtcg acggtacctg ctactaa 777531066DNAArtificial SequenceSynthetic construct, PgB3-3 construct 53gtccctctcc ctgtcatcgc tgagctccct cctaaggtct ctgtcttcgt ccctcctcgt 60gacggtttct tcggtaaccc tcgtaagtct aagctcatct gccaggctac cggtttctct 120cctcgtcaga tccaggtctc ttggctccgt gagggtaagc aggtcggttc tggtgtcacc 180accgaccagg tccaggctga ggctaaggag tctggtccta ccacctacaa ggtcacctct 240accctcacca tcaaggagtc tgactggctc tctcagtcta tgttcacctg ccgtgtcgac 300caccgtggtc tcaccttcca gcagaacgct tcttctatgt gcgtccctga ccaggacacc 360gctatccgtg tcttcgctat ccctccttct ttcgcttcta tcttcctcac caagtctacc 420aagctcacct gcctcgtcac cgacctcacc acctacgact ctgtcaccat ctcttggacc 480cgtcagaacg gtgaggctgt caagacccac accaacatct ctgagtctca ccctaacgct 540accttctctg ctgtcggtga ggcttctatc tgcgaggacg actggaactc tggtgagcgt 600ttcacctgca ccgtcaccca caccgacctc ccttctcctc tcaagcagac catctctcgt 660cctaagggtg tcgctctcca ccgtcctgac gtctacctcc tccctcctgc tcgtgagcag 720ctcaacctcc gtgagtctgc taccatcacc tgcctcgtca ccggtttctc tcctgctgac 780gtcttcgtcc agtggatgca gcgtggtcag cctctctctc ctgagaagta cgtcacctct 840gctcctatgc ctgagcctca ggctcctggt cgttacttcg ctcactctat cctcaccgtc 900tctgaggagg agtggaacac cggtgagacc tacacctgcg tcgtcgctca cgaggctctc 960cctaaccgtg tcaccgagcg taccgtcgac aagtctaccg gtaagcctac cctctacaac 1020gtctctctcg tcatgtctga caccgctggt acctgctacc caataa 1066541062DNAArtificial SequenceSynthetic cosntruct, PgB3-4 construct 54gtccctctcc ctgtcatcgc tgagctccct cctaaggtct ctgtcttcgt ccctcctcgt 60gacggtttct tcggtaaccc tcgtaagtct aagctcatct gccaggctac cggtttctct 120cctcgtcaga tccaggtctc ttggctccgt gagggtaagc aggtcggttc tggtgtcacc 180accgaccagg tccaggctga ggctaaggag tctggtccta ccacctacaa ggtcacctct 240accctcacca tcaaggagtc tgactggctc tctcagtcta tgttcacctg ccgtgtcgac 300caccgtggtc tcaccttcca gcagaacgct tcttctatgt gcgtccctga

ccaggacacc 360gctatccgtg tcttcgctat ccctccttct ttcgcttcta tcttcctcac caagtctacc 420aagctcacct gcctcgtcac cgacctcacc acctacgact ctgtcaccat ctcttggacc 480cgtcagaacg gtgaggctgt caagacccac accaacatct ctgagtctca ccctaacgct 540accttctctg ctgtcggtga ggcttctatc tgcgaggacg actggaactc tggtgagcgt 600ttcacctgca ccgtcaccca caccgacctc ccttctcctc tcaagcagac catctctcgt 660cctaagggtg tcgctctcca ccgtcctgac gtctacctcc tccctcctgc tcgtgagcag 720ctcaacctcc gtgagtctgc taccatcacc tgcctcgtca ccggtttctc tcctgctgac 780gtcttcgtcc agtggatgca gcgtggtcag cctctctctc ctgagaagta cgtcacctct 840gctcctatgc ctgagcctca ggctcctggt cgttacttcg ctcactctat cctcaccgtc 900tctgaggagg agtggaacac cggtgagacc tacacctgcg tcgtcgctca cgaggctctc 960cctaaccgtg tcaccgagcg taccgtcgac aagtctaccg gtaagcctac cctctacaac 1020gtctctctcg tcatgtctga caccgctggt acctgctact aa 1062551032DNAArtificial SequenceSynthetic construct, PgB4-2 construct 55atgggtgctg ctctcgctct cctcggtgac ctcgtcgcta ccgtctctga ggctgctgct 60gctaccggtt tctctgtcgc tgagatcgct gctggtgagg ctgctgctac catcgaggtc 120gagatcgctt ctctcgctac cgtcgagggt atcacctcta cctctgaggc tatcgctgct 180atcggtctca cccctgagac ctacgctgtc atcaccggtg ctcctggtgc tgtcgctggt 240ttcgctgctc tcgtccagac cgtcaccggt ggttctgcta tcgctcagct cggttaccgt 300ttcttcgctg actgggacca caaggtctct accgtcggtc tcttccagca gcctgctatg 360gctctccagc tcttcaaccc tgaggactac tacgacatcc tcttccctgg tgtcaacgct 420ttcgtcaaca acatccacta cctcgaccct cgtcactggg gtccttctct cttctctacc 480atctctcagg ctttctggaa cctcgtccgt gacgacctcc ctgctctcac ctctcaggag 540atccagcgtc gtacccagaa gctcttcgtc gagtctctcg ctcgtttcct cgaggagacc 600acctgggcta tcgtcaactc tcctgctaac ctctacaact acatctctga ctactactct 660cgtctctctc ctgtccgtcc ttctatggtc cgtcaggtcg ctcagcgtga gggtacctac 720atctctttcg gtcactctta cacccagtct atcgacgacg ctgactctat ccaggaggtc 780acccagcgtc tcgacctcaa gacccctaac gtccagtctg gtgagttcat cgagcgttct 840atcgctcctg gtggtgctaa ccagcgttct gctcctcagt ggatgctccc tctcctcctc 900ggtctctacg gtaccgtcac ccctgctctc gaggcttacg aggacggtcc taacaagaag 960aagcgtcgta aggagggtcc tcgtgcttct tctaagacct cttacaagcg tcgttctcgt 1020tcttctcgtt ct 103256519DNAArtificial SequenceSynthetic construct, PgB5-2 construct 56atggccgaca cggccagggg cacgcaccac gacatcatcg gcagggacca gtacccgatg 60atgggcaggg acagggacca gtaccagatg tccggcaggg gctccgacta ctccaagtcc 120aggcagatcg ccaaggccgc cacggccgtg acggccggcg gctccctcct cgtgctctcc 180tccctcacgc tcgtgggcac ggtgatcgcc ctcacggtgg ccacgccgct cctcgtgatc 240ttctccccga tcctcgtgcc ggccctcatc acggtggccc tcctcatcac gggcttcctc 300tcctccggcg gcttcggcat cgccgccatc acggtgttct cctggatcta caagtacgcc 360acgggcgagc acccgcaggg ctccgacaag ctcgactccg ccaggatgaa gctcggctcc 420aaggcccagg acctcaagga cagggcccag tactacggcc agcagcacac gggcggcgag 480cacgacaggg acaggacgag gggcggccag cacacgacg 51957711DNAArtificial SequenceSynthetic construct, PgA2-4 construct 57atgggtgacg tcggtatgca cgtcaaggag aaggagaaga acaaggacga gaacaagcgt 60aaggacgagg agcgtaacaa gacccaggag gagcacctca aggagatcat gaagcacatc 120gtcaagatcg aggtcaaggg tgaggaggct gtcaagaagg aggctgctga gaagctcctc 180gagaaggtcc cttctgacgt cctcgagatg tacaaggcta tcggtggtaa gatctacatc 240gtcgacggtg acatcaccaa gcacatctct ctcgaggctc tctctgagga caagaagaag 300atcaaggaca tctacggtaa ggacgctctc ctccacgagc actacgtcta cgctaaggag 360ggttacgagc ctgtcctcgt catccagtct tctgaggact acgtcgagaa caccgagaag 420gctctcaacg tctactacga gatcggtaag atcctctctc gtgacatcct ctctaagatc 480aaccagcctt accagaagtt cctcgacgtc ctcaacacca tcaagaacgc ttctgactct 540gacggtcagg acctcctctt caccaaccag ctcaaggagc accctaccga cttctctgtc 600gagttcctcg agcagaactc taacgaggtc caggaggtct tcgctaaggc tttcgcttac 660tacatcgagc ctcagcaccg tgacgtcctc cagctctacg ctcctgaggc t 71158560DNAArtificial SequenceSynthetic construct, PgA1-5 construct 58atggaacaac catcttgccg tagggctttc gatctctact tcgtgctcga taagtccggc 60ctgttgctaa caactggatc gaaatctaca acttcgtgca gcagctcgct gagagattcg 120tttctccaga gatgaggctc tccttcatcg tgttctcttc acaggctact atcatcctcc 180cactcactgg tgataggggc aagatttcta agggactcga ggatctcaag agggtgtcac 240cagttggaga gacttacatt cacgagggac tcaagcttgc taacgagcag attcaaaagg 300ctggcggcct caagacttcc tccattatta tcgctctcac tgatggcaag ctcgatggac 360ttgttccatc ctacgctgag aaagaggcta agatcagtcg ttcccttggc gcttctgttt 420actgcgttgg agtgcttgat ttcgagcagg ctcagcttga gaggatcgct gattccaaag 480agcaggtttt cccagttaag ggcggattcc aagctctcaa gggcatcatc aactccatcc 540ttgctcagtc ttgtactgag 56059690DNAArtificial SequenceSynthetic construct, PgB1-5 construct 59gataagactc atacttgtcc accatgccca gctccagaac ttcttggagg accatctgtg 60ttcttgttcc caccaaagcc aaaggatact ctcatgatct ccaggactcc agaggttaca 120tgcgttgtgg ttgatgtgtc tcacgaggat ccagaggtga agttcaactg gtatgtggat 180ggtgttgagg tgcacaacgc taagactaag ccacgtgagg aacagtacaa ctccacttac 240agggtggtgt ctgtgcttac tgtgcttcac caggattggc tcaacggcaa agagtacaag 300tgcaaggtgt ccaacaaggc tctcccagct ccaatcgaaa agactatctc caaggctaag 360ggacagccaa gggaaccaca ggtttacact cttccaccat ccagggaaga gatgactaag 420aaccaggtgt cccttacttg cctcgtgaag ggattctacc catccgatat tgctgttgag 480tgggagtcta atggccagcc agagaacaac tacaagacta ctccaccagt gctcgattcc 540gatggctcat tcttcttgta ctccaagctc actgtggata agtccaggtg gcagcaggga 600aacgttttct cttgctctgt tatgcacgag gctctccaca accactacac tcagaagtcc 660ttgtccttgt ccccaggcaa ggatcttatt 69060187PRTArtificial SequenceSynthetic construct, PgA1-5 protein 60Met Glu Gln Pro Ser Cys Arg Arg Ala Phe Asp Leu Tyr Phe Val Leu 1 5 10 15 Asp Lys Ser Gly Ser Val Ala Asn Asn Trp Ile Glu Ile Tyr Asn Phe 20 25 30 Val Gln Gln Leu Ala Glu Arg Phe Val Ser Pro Glu Met Arg Leu Ser 35 40 45 Phe Ile Val Phe Ser Ser Gln Ala Thr Ile Ile Leu Pro Leu Thr Gly 50 55 60 Asp Arg Gly Lys Ile Ser Lys Gly Leu Glu Asp Leu Lys Arg Val Ser 65 70 75 80 Pro Val Gly Glu Thr Tyr Ile His Glu Gly Leu Lys Leu Ala Asn Glu 85 90 95 Gln Ile Gln Lys Ala Gly Gly Leu Lys Thr Ser Ser Ile Ile Ile Ala 100 105 110 Leu Thr Asp Gly Lys Leu Asp Gly Leu Val Pro Ser Tyr Ala Glu Lys 115 120 125 Glu Ala Lys Ile Ser Arg Ser Leu Gly Ala Ser Val Tyr Cys Val Gly 130 135 140 Val Leu Asp Phe Glu Gln Ala Gln Leu Glu Arg Ile Ala Asp Ser Lys 145 150 155 160 Glu Gln Val Phe Pro Val Lys Gly Gly Phe Gln Ala Leu Lys Gly Ile 165 170 175 Ile Asn Ser Ile Leu Ala Gln Ser Cys Thr Glu 180 185 61229PRTArtificial SequenceSynthetic construct, PgB1-5 protein 61Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Asp Leu 225 6219DNAArtificial SequenceSyntehtic construct, NPTII forward primer 62tgaatgaact gcaggacga 196319DNAArtificial SequenceSynthetic construct, NPTII reverse primer 63agccaacgta tgtcctgat 19644PRTArtificial SequenceSynthetic construct, HDEL peptide 64His Asp Glu Leu 1 654PRTArtificial SequenceSynthetic construct, KDEL peptide 65Lys Asp Glu Leu 1

Claims

1. A genetic construct comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a PA-LF protein, and the second polynucleotide encodes a carrier-protein selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein.

2. The genetic construct of claim 8, wherein the first polynucleotide includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3/PA-LF), and SEQ ID NO: 57 (PgA2-4).

3. The genetic construct of claim 8, wherein the second polynucleotide includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2).

4. The genetic construct of claim 1 further comprising a third polynucleotide encoding a helper element comprising a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2).

5. The genetic construct of claim 1 comprising a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 37 (PgA2-4:B1-5).

6. A recombinant protein produced by the genetic construct of claim 1.

7. A transgenic plant produced by the genetic construct of claim 1.

8. The transgenic plant of claim 7, wherein the plant is a crop plant selected from the group consisting of: tomato, tobacco, pepper, eggplant, lettuce, oilseed rape, broccoli, cauliflower, cabbage crops, cucumber, melon, pumpkin, squash, peanut, soybeans, corn, rice, barley, buckwheat, sugar cane, cotton, beans, cassava, potatoes, sweet potatoes, and okra.

9. The transgenic plant of claim 7, wherein the plant is a medicinal plant selected from the group consisting of: Caragana sinica, Codonopsis pilosulae, Hedyotis diffusa, Houttuynia cordata, Lonicera japonica, Morinda officinalis, Oenothera odorata, Kalanchoe pinnata, Echinacea angustifolia, Calendula officinalis, and Arthemis nobilis.

10. A method for producing a recombinant protein in a plant, wherein the method comprises: contacting a plant with a genetic construct of claim 1; obtaining a plant including the genetic construct and expressing a recombinant protein, wherein the recombinant protein is encoded by the genetic construct.

11. The method of claim 10, wherein the method further comprises isolating and purifying the recombinant protein from the plant.

12. A method for preparing a composition effective for treating or preventing an anthrax infection in a subject, wherein the method comprises providing a recombinant protein produced by the method of claim 10.

13. The method of claim 12, wherein the method further comprises providing a pharmaceutically acceptable carrier.

14. The method of claim 12, wherein the method further comprises providing an adjuvant.

15. A method of protecting a subject against anthrax infection, wherein the method comprises: providing a composition that includes a recombinant protein of claim 6, wherein the composition is effective in preventing or reducing at least one symptom of an anthrax infection in a subject; and administering the composition to the subject in need thereof.

16. The method of claim 15, wherein the subject is a mammal.

17. The method of claim 15, wherein the mammal is selected from the group consisting of: an agricultural animal, an equine, a high value zoo animal, a research animal and a human.

18. The method of claim 15, wherein administering includes a route selected from the group consisting of: intravenous, intramuscular, intraperitoneal, intradermal, mucosal, cutaneous and subcutaneous.

19. The method of claim 15, wherein administering is intranasal.

20. The method of claim 19, wherein intranasal administering is provided in the form of inhalation or nasal drops.