Expression Vector

Abstract

The present invention provides an expression vector for cell-surface expression of proteins.

Description

[0001] Since the discovery of monoclonal antibodies (mAbs) in 1975 by Kohler and Milstein, they have become molecular tools of inestimable value. Due to their high specificity, monoclonal antibodies (mAbs) are used for standard techniques throughout biology, being the key to the characterisation of protein function and distribution. Besides their usage in basic research, mAbs are also widely utilised as diagnostic and therapeutic agents. Due to this wide range of applications the generation of mAbs became a standard procedure. However, it can still be problematic, since for studies in physiological settings, it is important that the mAbs recognise the antigen in its native conformation.

[0002] Most commonly mAbs are raised against synthetic peptides derived from the predicted sequence of the target protein. Unfortunately, these Abs, though strongly reactive with peptide, frequently fail to recognise the native protein. Another standard procedure to generate mAbs uses recombinantly expressed protein. Prokaryotic expression systems are the most widely used expression hosts. But when studying mammalian surface proteins it is often necessary to use mammalian expression systems, as they are more likely to produce functional proteins with the appropriate disulfide-bonds, posttranslational glycosylations or proteolytic modifications. Purification of recombinant proteins is often a tedious undertaking, frequently representing a limiting step towards obtaining antibodies. Although introduction of affinity tags simplify purification, it often remains difficult to obtain recombinant protein in native conformation and in sufficient yield and purity. This applies most notably to membrane-associated proteins, as they are likely to lose their native structure during purification processes.

[0003] When attempting to generate mAbs capable of recognising the protein in its native context it is also critical to use protein in native conformation not only in the immunisation step but also for the screening procedure. Many standard hybridoma-screening protocols, such as the immobilisation of recombinant proteins on solid supports, may significantly alter protein conformation. For these reasons, mAbs selected on the basis of binding to a recombinant protein may not bind the same protein when it is in its native context.

[0004] Therefore, there is a need for an antigen expression system allowing the expression of antigens in native confirmation on the cell surface of cells.

[0005] In a first object the present invention provides a nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence encoding a secretion signal peptide, a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence encoding a transmembrane domain of glycophorin.

[0006] In a preferred embodiment of the nucleic acid expression vector, the transmembrane domain of glycophorin is the transmembrane domain of glycophorin A.

[0007] In a further preferred embodiment of the nucleic acid expression vector, the transmembrane domain of glycophorin A is the mouse glycophorin A transmembrane domain or the Armenian hamster glycophorin A domain.

[0008] In a further preferred embodiment of the nucleic acid expression vector, the mouse glycophorin A transmembrane domain comprises the amino acid sequence disclosed in Seq. Id. No. 1 and the Armenian hamster glycophorin A domain comprises the amino acid sequence disclosed in Seq. Id. No. 12.

[0009] In a further preferred embodiment of the nucleic acid expression vector, the secretion signal peptide is the secretion signal peptide of bee-venom melittin.

[0010] In a further preferred embodiment of the nucleic acid expression vector, the secretion signal peptide of bee-venom melittin comprises the amino acid sequence disclosed in Seq. Id. No. 2.

[0011] In a further preferred embodiment, the nucleic acid expression vector further comprises downstream (3') of the cloning site for inserting a polynucleotide sequence encoding a protein to be expressed a polynucleotide sequence encoding a FLAG tag comprising the amino acid sequence of Seq. Id. No. 3.

[0012] In a further preferred embodiment, the nucleic acid expression vector further comprises downstream (3') of the polynucleotide sequence encoding the transmembrane domain of glycophorin a polynucleotide sequence encoding a His tag, preferably a His tag comprising the amino acid sequence disclosed in Seq. Id. No. 4.

[0013] In a further preferred embodiment of the nucleic acid expression vector, the cloning site comprises the restriction enzyme cleavage sites of NheI, KpnI, BamHI, EcoRI, EcoRV and NotI.

[0014] In a further preferred embodiment, the nucleic acid expression vector comprises a polynucleotide sequence selected from the group consisting of Seq. Id. No. 5, Seq. Id. No. 13, Seq. Id. No. 14 and Seq. Id. No. 15.

[0015] In a further preferred embodiment of the nucleic acid expression vector, the protein to be expressed is a membrane associated protein.

[0016] a second object, the present invention provides a cell comprising the vector of the present invention, preferably a mammalian cell, more preferably a HEK cell.

[0017] In a third object, the present invention provides a method for the generation of monoclonal antibodies against a specific protein comprising the steps:

[0018] a) immunisation of a non-human animal with cells expressing on its cell surfaces the specific protein using the vector of the present invention,

[0019] b) isolating spleen cells of the non-human animals of step a),

[0020] c) fusing the spleen cells of step b) with myeloma cells to generate B cell hybridomas and

[0021] d) identification of B cell hybridomas expressing antibodies directed against the specific protein.

[0022] In a preferred embodiment of the method of the present invention, the non-human animal is a mouse or Armenian hamster.

[0023] "Nucleic acid expression vector" refers to an assembly which is capable of directing the expression of a sequence or gene of interest. The nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. In addition, the vector may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the vector to exist as single-stranded DNA (e.g., a M 13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication). A "vector" is capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated.

[0024] The term "protein" as used herein, refers to a polymer of amino acids, and not to a specific length. Thus, peptides, oligopeptides and protein fragments are included within the definition of polypeptide.

[0025] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g. U.S. Pat. No. 4,816,567 (Cabilly et al.) and Mage and Lamoyi (1987) in Monoclonal Antibody Production Techniques and Applications, pp. 79-97, Marcel Dekker, Inc., New York). The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al. (1990) Nature 348:552-554, for example.

SHORT DESCRIPTION OF FIGURES

[0026] FIG. 1A shows the primary structure of human ABCA1 (Seq. Id. No. 7), rat TMEM27 (Seq. Id. No. 9) and P. falciparum PFF0620c (Seq. Id. No. 11) proteins used in the examples. The domains used for the constructs described are marked with the diagonal lines with the amino acids at the N and C termini indicated;

[0027] FIG. 1B shows schematic diagrams of the expressed protein constructs derived from the vectors described in the examples. The extracellular domains are equivalent to the ones shown in FIG. 1A;

[0028] FIG. 2 shows a Westerm blot using anti-FLAG M2-HRP conjugated antibody (Sigma) of total cell lysates from HEK293 cells transfected with pANITA2-ABCA1 and pANITA2-TMEM27. Strong expression with bands at appropriate molecular weights is seen;

[0029] FIG. 3A shows cell-surface expression of PI-10620C on stably transfected HEK cells. Fluorescence (column 2 & 3) and differential interference contrast micrographs (column 1) of non-transfected HEK cells (line 1) and HEK cells displaying PFF0620C (line 2). Cells were grown on chamber-slides and stained without fixation with anti-FLAG antibody and FITC-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI;

[0030] FIG. 3B shows extracellular localisation of PFF0620C on stably transfected HEK cells. Fluorescence (line 1 & 3) and differential interference contrast micrograph (line 2 & 4) of PFF0620C-HEK cells after staining with anti-FLAG (left column) or anti-6× His antibodies (right column) and FITC-labelled anti-mouse IgG antibodies. With the anti-FLAG antibody living cells and methanol-fixed cells were stained, whereas the anti-His antibody only stained methanol-fixed cells, indicating intracellular localisation of the His-tag and extracellular localisation of the FLAG-tag together with the P. falciparum derived protein domain;

[0031] FIG. 4 shows the results of a screening of antibodies for binding to transfected cells. In a second step, all wells positive for IgG production were screened for antibody binding to transfected cells by IFA (immuno fluorescence assay). Transfected and non-transfected HEK cells spotted onto multiwell glass-slides were stained with individual hybridoma supernatants and analysed by fluorescence microscopy;

[0032] FIG. 5 shows a western blot analysis of the reactivity of generated monoclonal antibodies with the recombinant P. falciparum proteins. Specificity of representative monoclonal antibodies for the corresponding recombinant proteins is demonstrated by Western-blot analysis. Lysates of PFF0620c- (line 1), control pANITA2 constructs containing unrelated proteins (lines 2 & 3) and non-transfected HEK cells (line 4) were probed with anti-6× His mAb and an anti PFF0620cmAb generated as described, respectively.

[0033] FIG. 6 shows that PFD 1130w-specific monoclonal antibodies inhibit parasite growth in vivo.

EXAMPLES

[0034] Expression Proteins on the Cell Surface of Mammalian Cells.

[0035] The P. falciparum ORF PFF0620c, human ABCA1 extracellular domain and rat TMEM27 extracellular domain were expressed on the cell surface of HEK cells using the expression plasmids pANITA2-PFF0620C; pANITA2-ABCA1 or pANITA2-TMEM27 respectively. To ensure high levels of expression on the cell surface, the genes were modified in several ways (FIG. 1): i. the endogenous sequences were codon-optimised for expression in mammalian cells and only predicted extracelluar domains were used; ii. the endogenous secretion signal sequences were replaced by the secretion signal sequence of bee-venom melittin; iii. for membrane anchoring the transmembrane domain encoding sequence of mouse glycophorin A was used instead of the predicted GPI-attachment signal sequence or predicted transmembrane domains; iv. to allow expression analysis, a FLAG tag was inserted N-terminally of the transmembrane domain and a 6× His tag was placed at the C-terminus. The two tags were positioned just before and after the transmembrane domain to facilitate verification of the extracellular localisation of the recombinantly expressed antigens.

[0036] HEK-derived cell lines expressing P. falciparum PFF0620c, human ABCA1 extracellular domain and rat TMEM27 extracellular domain were established by stable transfection.

[0037] To obtain highly expressing cell lines, transfectants were separated into high-expressing cell-pools by fluorescent-activated-cell-sorting after surface staining with anti-FLAG antibodies. The mean fluorescence intensity of the cells gated for sorting into the high-expressing cell pool was 2.1-4.3 times higher than that of all transfectants.

[0038] Human ABCA1 and rat TMEM27-expressing cell lines were tested for expression by Western blot analysis, showing a high level of expression of a protein with the expected molecular weight. (FIG. 2) Cell surface expression of the P. falciparum PFF0620c protein was shown by immunofluoresence analysis with anti-FLAG antibody yielding strong signals on living cells. (FIG. 3) In contrast, staining with anti-6× His antibody gave strong signals only on methanol fixed cells but not on living cells (FIG. 3B). These results verified that PFF0620c is expressed and anchored in the cell wall with the FLAG-tag lying extracellularly and the His-tag lying intracellularly.

[0039] Development of malaria antigen specific antibodies in mice immunised with transfected HEK cells

[0040] The high-expressing cell pool of PFF0620c-HEK was used to immunise NMRI mice. Mice received intravenous injections of 10⁶ cells on three consecutive days and another suite of three daily injections two weeks later. Development of serum antibody titres was analysed by flow cytometry comparing immune-staining of the transfectant with that of non-transfected HEK cells. The fluorescence intensity observed with the transfectant was fourfold higher than that of non-transfected control HEK cells. This indicated that the mice had mounted an antibody response against the malaria antigen expressed on the surface of the transfected HEK cells.

[0041] Spleen cells of mice immunised with the transfected HEK cells were fused with PAI myeloma cells to generate B cell hybridoma. Fused cells were distributed in microtitre culture plate wells. To identify hybridoma cells that produce PFF0620c-specific antibodies a two-step screening procedure was used that completely obviates the requirement for purified recombinant proteins. First all culture wells were tested for IgG production by ELISA. Between 18 and 29%, of the tested wells were positive. In a second step all wells positive for IgG production were screened for antibody binding to transfected cells by IFA. Transfected and non-transfected HEK cells spotted onto multiwell glass-slides were stained with individual hybridoma supernatants and analysed by fluorescence microscopy (FIG. 4). Non-transfected HEK cells served as a negative control for each sample. Numerous clones positive on the transfected cells were also positive on non-transfected cells. However, the fusion yielded also numerous wells containing antibodies strongly reactive with the transfectant but not reactive with untransfected HEK cells. All other antibodies were specific for the transfected cells used for immunisation and did not stain control transfectants. From wells of this category, 17 hybridoma clones were derived by recloning from the PFF0620c-fusion.

[0042] The specificity of the monoclonal antibodies was further confirmed by Western blot analysis (FIG. 5). 16 of the mAbs stained the corresponding recombinant protein in the lysate of the transfectant used for immunisation, but not in lysates of control transfected or untransfected HEK cells.

[0043] PFD1130w-Specific Monoclonal Antibodies Inhibit Parasite Growth In Vivo

[0044] We evaluated the in vivo parasite inhibitory activity of anti-PFD1130w mAbs in a P. falciparum SCID mouse model. The anti-PFD1130w mAbs were produced using the same methods and vectors that were used for the generation of the mAbs against P. falciparum PFF0620c (see methods section below). This model uses non-myelodepleted NOD-scid IL2Rnull mice engrafted with human erythrocytes in order to allow the growth of P. falciparum. Groups of three mice with a parasitemia of 0.58±0.14% were injected once with 2.5 mg anti-PFD1130w c12 mAb, 0.5 mg anti-PFD 1130w c12 mAb or 2.5 mg isotype/subclass control mAb per mouse, respectively. Parasitemia of all mice was monitored for the next six days. While the parasitemia in mice that had received PBS only or the control mAb increased continuously, reaching 11.3±0.8% after six days, parasitemia of mice that received 0.5 mg anti-PFD1130w c12 mAb increased to a much lower extent, reaching 5.6±1.3% after six days. Parasitemia of mice receiving 2.5 mg anti-144)1130w c12 mAb stayed low till the end of the experiment (1.4±0.3% on day 6). The difference in parasitemia after 6 days compared to the negative control group was highly significant (two-sided t-test; P<0.0001) (FIG. 6).

[0045] The fact that anti-PFD1130w mAbs inhibit parasite growth in vivo indicates the power of the described entirely cell-based technology to generate mAbs that bind the endogenous protein in its native context.

[0046] Methods

[0047] Construction of Plasmids and Transformation

[0048] A double-stranded oligonucleotide encoding the secretion signal sequence of bee-venom melittin was ligated to NheI digested pcDNA3.1(+) (Invitrogen) resulting in plasmid pcDNA3.1_BVM, with a single NheI site retained 3' of the signal sequence. A mouse glycophorin cytoplasmic and transmembrane domain cDNA was obtained by rtPCR (Invitrogen SuperScript III First Strand Synthesis kit and Roche Expand High Fidelity PCR System) using RNA extracted from bone marrow as a template. The resulting PCR amplicon being cloned into a pCR2.1 cloning vector. Primers to mouse glycophorin contained a 5' NotI site and 3' histidine tag followed by a stop codon and EagI site. The glycophorin-6His fragment was excised with EagI and ligated to NotI-digested pcDNA3.1_BVM resulting in plasmid pcDNA3.1_BVM_GP with the pcDNA3.1 NotI site preserved at the 5' end of the glycophorin sequence. To create the finished expression vector (pANITA2) a double-stranded oligonucleotide was ligated into NotI-digested pcDNA3.1_BVM_GP encoding a Flag-tag flanked by short linker sequences and resulting in a unique NotI site to the 5' side of the Flag-tag.

[0049] Rat TMEM27 extracellular domain (aa 15-130 of Seq. Id. No. 9); a predicted extracellular domain of P. falciparum gene PFF0620C (aa 21-353 of Seq. Id. No. 11) and human ABCA1 N-terminal extracellular domain (aa 43-640 of Seq. Id. No. 7) cDNA sequences were synthesised with optimisation of codon usage to give high expression in mammalian cell culture. The genes were ligated into the unique NheI and NotI sites of the pANITA2 vector and the sequence of the vectors confirmed by DNA sequencing. The resulting plasmids are hereafter referred to as pA-NITA2-TMEM27; pANITA2-PFF0620C or pANITA2-ABCA1 respectively.

[0050] In pANITA3.1 and pANITA3.3, the native pcDNA3.1 XbaI and XhoI sites were also removed by site-directed mutagenesis. The features of the multiple cloning sites and fusion-protein-coding sequences are shown in the table 1 below, with numbering from the insert start.

[0051] Armenian hamster glycophorin sequence was determined by PCR-cloning and nucleotide sequencing using the Chinese hamster glycophorin sequence as a guide for primer design and cDNA generated from Armenian hamster bone-marrow RNA preparations. The following sequences are depicted in table 1: pANITA2 with Kozak sequence=Seq. Id. No. 15, pANITA3.1=Seq. Id. No. 13 and pANITA3.3=Seq. Id. No. 14.

TABLE-US-00001 TABLE 1 Comparison of expression vectors Vector element pANITA2 pANITA3.1 pANITA3.3 Kozak sequence 1-12 1-12 1-12 Bee venom melittin signal 9-72 9-72 9-72 sequence Unique NheI restriction site 70-75 70-75 70-75 Unique KpnI restriction site 82-87 82-87 82-87 Unique BamHI restriction site 94-99 94-99 94-99 Unique EcoRI restriction site 106-111 106-111 106-111 Unique EcoRV restriction site 112-117 112-117 112-117 Unique XbaI restriction site -- 118-123 118-123 Unique NotI restriction site 124-131 124-131 124-131 Flag tag/Enterokinase cleavage 133-156 133-156 133-156 site Unique HindIII restriction site -- 154-159 154-159 Mouse glycophorin membrane 172-369 163-369 -- anchor Armenian hamster glycophorin -- -- 178-375 membrane anchor 6-His tag 382-399 382-399 388-405 Stop codons 400-405 400-405 406-411

[0052] Establishment of HEK 293 Cell Lines Stably Expressing PFF0620C, TMEM27 or ABCA1 Domains.

[0053] 293 HEK cells were transfected with pANITA2-TMEM27; pANITA2-PFF0620C or pA-NITA2-ABCA1 using JetPEI® (PolyPlus) transfection reagent following the manufacturer's protocol. Antibiotic selection was started 48 h after transfection. The selection medium containing 500 ug/ml of Geneticin (Gibco) was exchanged every 3-4 days. After non-antibiotic resistant cells had died off and resistant cells started growing normally, a high-expressing pool was generated by FACS. Cells were dissociated with enzyme-free dissociation buffer (Cell dissociation buffer enzyme-free Hanks'-based, Gibco), washed with blocking buffer (PBS containing 3% BSA). The cells were then incubated with 200 μl of 100 μg/ml anti-FLAG mAb=FLAG-27 diluted in blocking buffer for 15 min on ice. The cells were then washed with blocking buffer and incubated with 200 μl of 100 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in blocking buffer for 15 min on ice. After a final wash the labelled cells were analysed and sorted using a BD FACSAria running FACSDiva software. All analyses were performed using appropriate scatter gates to exclude cellular debris and aggregates. Gating settings were set to collect highly labelled cells. Post-sorting, the cells were collected in culture medium with 20% FCS and plated in 35 mm wells

[0054] Immunofluorescence Staining of Living HEK Cells

[0055] For immunofluorescence staining of live HEK cells chamber slides (4-well chamber-slide, Lab-Tek®, Nunc®) were used. Wells were coated with 100 mg/l poly-D-lysine in H₂O in a humid box at room temperature over night. After washing the wells three times with sterile H₂O, 40'000 cells were seeded per well. Three days later the immunostaining was performed by incubating the wells with 500 μl of an appropriate mAb diluted in serum-free culture medium for 30 min on ice. After washing two times with serum-free culture medium 500 μl of 100 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in serum-free culture medium were added to the wells and incubated for 30 min on ice. Finally, the wells were rinsed twice with serum-free culture medium and once with DPBS (Dulbecco's Phosphate-Buffered Saline containing calcium, Gibco). The slides were mounted with mounting solution containing DAPI (ProLong® Gold antifade reagent with DAPI, Invitrogen) and covered with a coverslip. Stainings were assessed as described above.

[0056] Immunisation of Mice

[0057] NMRI mice were immunised by intravenous injections of 10⁶ stably transfected HEK cells. Cells were thawed, washed and resuspended in 0.9% NaCl. Injections were accomplished on three consecutive days and after two weeks again on three consecutive days. After the boost, blood was collected and the serum was tested for the presence of anti-PFF0620C antibodies by IFA using stably transfected 293 HEK cells.

[0058] Animals with serum strongly reactive with expressing cells were selected for fusion. These received a final injection of 10⁶ cells two and one day before the fusion. Mice were sacrificed and the spleen was removed. Spleen cells were harvested by trituration under sterile conditions and fused with the myeloma cell partner (PAI mouse myeloma cells, derived from P-3X63-Ag8) using polyethylene glycol 1500 (Roche Diagnostics). The fusion mix was plated into multiwell plates and hybridomas were selected by growing in HAT medium supplemented with culture supernatant of mouse macrophages P388. Wells were screened for specific IgG production between 2-3 weeks post-fusion by ELISA and IFA as described below. Cells from wells positive in initial screens were cloned by limiting dilution to obtain monoclonal populations.

[0059] IgG ELISA Screen

[0060] Maxisorp® plates (Nunc) were coated overnight at 4° C. in a humid box with 100 μl of 5 μg/ml goat anti-mouse IgG (y-chain specific) mAb (Sigma) diluted in PBS. After two washings with PBS containing 0.05% Tween-20, wells were blocked with blocking buffer (50 mM Tris, 140 mM NaCl, 5 mM EDTA, 0.05% NONidet P40, 0.25% gelatine, 1% BSA) for 1 h at 37° C. and afterwards washed two times. 50 μl hybridoma supernatants were added to the wells and incubated for 1 h at 37° C. After washing 4 times, plates were incubated with 50 μl horseradish peroxidase-conjugated goat anti-mouse IgG (y-chain specific) (Sigma) diluted 1:1000 in blocking buffer for 1 h at room-temperature in a humid box in the dark. After washing 4 times, TMB peroxidase substrate solution was added and the colour change monitored.

[0061] Antibody Production and Characterisation

[0062] Identification of antibody isotypes was performed using a Mouse Monoclonal Antibody Isotyping Kit (ISO2, Sigma). For large-scale mAb production hybridoma cell lines were cultured in 500 ml roller-bottles (Corning). MAbs were purified by affinity chromatography using protein A or protein G Sepharose.

[0063] DNA and Protein Sequences

TABLE-US-00002 Gene/Protein name Species Description Seq. Id. No. Glycophorin A Mouse Transmembrane + cyto- 1 plasmic domain of glycophorin A Melittin Bee Secretion signal of bee 2 venom melittin Flag tag -- Flag tag 3 His tag -- His tag 4 Expression vector -- Expression vector sequence 5 pANITA2 comprising secretion (without Kozak sequence) signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A ABCA1 Human DNA encoding human 6 ABCA1 protein ABCA1 Human ABCA1 protein 7 TMEM27 Rat DNA encoding rat 8 TMEM27 TMEM27 Rat TMEM27 protein 9 PFF0620C Plasmodium falciparum DNA encoding 3D7 protein 10 PFF0620C Plasmodium falciparum 3D7 protein 11 Glycophorin A Armenian hamster Transmembrane + cyto- 12 plasmic domain of glycophorin A Expression vector -- Expression vector sequence 13 pANITA3.1 comprising secretion signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A Expression vector -- Expression vector sequence 14 pANITA3.3 comprising secretion signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of Armenian hamster glycophorin A Expression vector -- Expression vector sequence 15 pANITA2 with comprising secretion Kozak sequence (nt signal of bee venom 1-12) melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A

[0064] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Sequence CWU 1

1

15166PRTMus musculus 1His Asp Phe Pro Ala Leu Val Met Ile Leu Ile Ile Leu Gly Val Met 1 5 10 15 Ala Gly Ile Ile Gly Thr Ile Leu Leu Ile Ser Tyr Cys Ile Ser Arg 20 25 30 Met Thr Lys Lys Ser Ser Val Asp Ile Gln Ser Pro Glu Gly Gly Asp 35 40 45 Asn Ser Val Pro Leu Ser Ser Ile Glu Gln Thr Pro Asn Glu Glu Ser 50 55 60 Ser Asn 65 221PRTApis mellifera 2Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile 1 5 10 15 Ser Phe Ile Tyr Ala 20 38PRTArtificial SequenceFLAG tag 3Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 410PRTArtificial SequenceHis Tag 4Val Ser Gly Gly His His His His His His 1 5 10 5390DNAArtificial SequenceExpression construct 5atgaagttcc tggtgaatgt ggccttggtg ttcatggtgg tgtacatcag cttcatctac 60gctagcctta agggtaccga gctcggatcc actagtgaat tcgatatcga cgtcgcggcc 120gctgactaca aagacgatga cgacaagagc cctagggccg cacacgattt tcctgcacta 180gtgatgatac tcataatttt gggcgtgatg gcagggatta tcggaactat ccttcttatc 240tcttactgta tcagccgaat gacaaagaaa agttcagttg acatccaatc tcctgagggt 300ggtgacaaca gtgtgccttt gagttctatt gagcagactc ctaatgaaga gtcctccaat 360gttagcggcg gccatcacca tcaccatcac 39067140DNAHomo sapiensCDS(314)..(7099) 6gtaattgcga gcgagagtga gtggggccgg gacccgcaga gccgagccga cccttctctc 60ccgggctgcg gcagggcagg gcggggagct ccgcgcacca acagagccgg ttctcagggc 120gctttgctcc ttgttttttc cccggttctg ttttctcccc ttctccggaa ggcttgtcaa 180ggggtaggag aaagagacgc aaacacaaaa gtggaaaaca gttaatgacc agccacggcg 240tccctgctgt gagctctggc cgctgccttc cagggctccc gagccacacg ctgggggtgc 300tggctgaggg aac atg gct tgt tgg cct cag ctg agg ttg ctg ctg tgg 349 Met Ala Cys Trp Pro Gln Leu Arg Leu Leu Leu Trp 1 5 10 aag aac ctc act ttc aga aga aga caa aca tgt cag ctg ctg ctg gaa 397Lys Asn Leu Thr Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu Leu Glu 15 20 25 gtg gcc tgg cct cta ttt atc ttc ctg atc ctg atc tct gtt cgg ctg 445Val Ala Trp Pro Leu Phe Ile Phe Leu Ile Leu Ile Ser Val Arg Leu 30 35 40 agc tac cca ccc tat gaa caa cat gaa tgc cat ttt cca aat aaa gcc 493Ser Tyr Pro Pro Tyr Glu Gln His Glu Cys His Phe Pro Asn Lys Ala 45 50 55 60 atg ccc tct gca gga aca ctt cct tgg gtt cag ggg att atc tgt aat 541Met Pro Ser Ala Gly Thr Leu Pro Trp Val Gln Gly Ile Ile Cys Asn 65 70 75 gcc aac aac ccc tgt ttc cgt tac ccg act cct ggg gag gct ccc gga 589Ala Asn Asn Pro Cys Phe Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly 80 85 90 gtt gtt gga aac ttt aac aaa tcc att gtg gct cgc ctg ttc tca gat 637Val Val Gly Asn Phe Asn Lys Ser Ile Val Ala Arg Leu Phe Ser Asp 95 100 105 gct cgg agg ctt ctt tta tac agc cag aaa gac acc agc atg aag gac 685Ala Arg Arg Leu Leu Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp 110 115 120 atg cgc aaa gtt ctg aga aca tta cag cag atc aag aaa tcc agc tca 733Met Arg Lys Val Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser Ser 125 130 135 140 aac ttg aag ctt caa gat ttc ctg gtg gac aat gaa acc ttc tct ggg 781Asn Leu Lys Leu Gln Asp Phe Leu Val Asp Asn Glu Thr Phe Ser Gly 145 150 155 ttc ctg tat cac aac ctc tct ctc cca aag tct act gtg gac aag atg 829Phe Leu Tyr His Asn Leu Ser Leu Pro Lys Ser Thr Val Asp Lys Met 160 165 170 ctg agg gct gat gtc att ctc cac aag gta ttt ttg caa ggc tac cag 877Leu Arg Ala Asp Val Ile Leu His Lys Val Phe Leu Gln Gly Tyr Gln 175 180 185 tta cat ttg aca agt ctg tgc aat gga tca aaa tca gaa gag atg att 925Leu His Leu Thr Ser Leu Cys Asn Gly Ser Lys Ser Glu Glu Met Ile 190 195 200 caa ctt ggt gac caa gaa gtt tct gag ctt tgt ggc cta cca agg gag 973Gln Leu Gly Asp Gln Glu Val Ser Glu Leu Cys Gly Leu Pro Arg Glu 205 210 215 220 aaa ctg gct gca gca gag cga gta ctt cgt tcc aac atg gac atc ctg 1021Lys Leu Ala Ala Ala Glu Arg Val Leu Arg Ser Asn Met Asp Ile Leu 225 230 235 aag cca atc ctg aga aca cta aac tct aca tct ccc ttc ccg agc aag 1069Lys Pro Ile Leu Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser Lys 240 245 250 gag ctg gct gaa gcc aca aaa aca ttg ctg cat agt ctt ggg act ctg 1117Glu Leu Ala Glu Ala Thr Lys Thr Leu Leu His Ser Leu Gly Thr Leu 255 260 265 gcc cag gag ctg ttc agc atg aga agc tgg agt gac atg cga cag gag 1165Ala Gln Glu Leu Phe Ser Met Arg Ser Trp Ser Asp Met Arg Gln Glu 270 275 280 gtg atg ttt ctg acc aat gtg aac agc tcc agc tcc tcc acc caa atc 1213Val Met Phe Leu Thr Asn Val Asn Ser Ser Ser Ser Ser Thr Gln Ile 285 290 295 300 tac cag gct gtg tct cgt att gtc tgc ggg cat ccc gag gga ggg ggg 1261Tyr Gln Ala Val Ser Arg Ile Val Cys Gly His Pro Glu Gly Gly Gly 305 310 315 ctg aag atc aag tct ctc aac tgg tat gag gac aac aac tac aaa gcc 1309Leu Lys Ile Lys Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala 320 325 330 ctc ttt gga ggc aat ggc act gag gaa gat gct gaa acc ttc tat gac 1357Leu Phe Gly Gly Asn Gly Thr Glu Glu Asp Ala Glu Thr Phe Tyr Asp 335 340 345 aac tct aca act cct tac tgc aat gat ttg atg aag aat ttg gag tct 1405Asn Ser Thr Thr Pro Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser 350 355 360 agt cct ctt tcc cgc att atc tgg aaa gct ctg aag ccg ctg ctc gtt 1453Ser Pro Leu Ser Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu Leu Val 365 370 375 380 ggg aag atc ctg tat aca cct gac act cca gcc aca agg cag gtc atg 1501Gly Lys Ile Leu Tyr Thr Pro Asp Thr Pro Ala Thr Arg Gln Val Met 385 390 395 gct gag gtg aac aag acc ttc cag gaa ctg gct gtg ttc cat gat ctg 1549Ala Glu Val Asn Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp Leu 400 405 410 gaa ggc atg tgg gag gaa ctc agc ccc aag atc tgg acc ttc atg gag 1597Glu Gly Met Trp Glu Glu Leu Ser Pro Lys Ile Trp Thr Phe Met Glu 415 420 425 aac agc caa gaa atg gac ctt gtc cgg atg ctg ttg gac agc agg gac 1645Asn Ser Gln Glu Met Asp Leu Val Arg Met Leu Leu Asp Ser Arg Asp 430 435 440 aat gac cac ttt tgg gaa cag cag ttg gat ggc tta gat tgg aca gcc 1693Asn Asp His Phe Trp Glu Gln Gln Leu Asp Gly Leu Asp Trp Thr Ala 445 450 455 460 caa gac atc gtg gcg ttt ttg gcc aag cac cca gag gat gtc cag tcc 1741Gln Asp Ile Val Ala Phe Leu Ala Lys His Pro Glu Asp Val Gln Ser 465 470 475 agt aat ggt tct gtg tac acc tgg aga gaa gct ttc aac gag act aac 1789Ser Asn Gly Ser Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr Asn 480 485 490 cag gca atc cgg acc ata tct cgc ttc atg gag tgt gtc aac ctg aac 1837Gln Ala Ile Arg Thr Ile Ser Arg Phe Met Glu Cys Val Asn Leu Asn 495 500 505 aag cta gaa ccc ata gca aca gaa gtc tgg ctc atc aac aag tcc atg 1885Lys Leu Glu Pro Ile Ala Thr Glu Val Trp Leu Ile Asn Lys Ser Met 510 515 520 gag ctg ctg gat gag agg aag ttc tgg gct ggt att gtg ttc act gga 1933Glu Leu Leu Asp Glu Arg Lys Phe Trp Ala Gly Ile Val Phe Thr Gly 525 530 535 540 att act cca ggc agc att gag ctg ccc cat cat gtc aag tac aag atc 1981Ile Thr Pro Gly Ser Ile Glu Leu Pro His His Val Lys Tyr Lys Ile 545 550 555 cga atg gac att gac aat gtg gag agg aca aat aaa atc aag gat ggg 2029Arg Met Asp Ile Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp Gly 560 565 570 tac tgg gac cct ggt cct cga gct gac ccc ttt gag gac atg cgg tac 2077Tyr Trp Asp Pro Gly Pro Arg Ala Asp Pro Phe Glu Asp Met Arg Tyr 575 580 585 gtc tgg ggg ggc ttc gcc tac ttg cag gat gtg gtg gag cag gca atc 2125Val Trp Gly Gly Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile 590 595 600 atc agg gtg ctg acg ggc acc gag aag aaa act ggt gtc tat atg caa 2173Ile Arg Val Leu Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr Met Gln 605 610 615 620 cag atg ccc tat ccc tgt tac gtt gat gac atc ttt ctg cgg gtg atg 2221Gln Met Pro Tyr Pro Cys Tyr Val Asp Asp Ile Phe Leu Arg Val Met 625 630 635 agc cgg tca atg ccc ctc ttc atg acg ctg gcc tgg att tac tca gtg 2269Ser Arg Ser Met Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser Val 640 645 650 gct gtg atc atc aag ggc atc gtg tat gag aag gag gca cgg ctg aaa 2317Ala Val Ile Ile Lys Gly Ile Val Tyr Glu Lys Glu Ala Arg Leu Lys 655 660 665 gag acc atg cgg atc atg ggc ctg gac aac agc atc ctc tgg ttt agc 2365Glu Thr Met Arg Ile Met Gly Leu Asp Asn Ser Ile Leu Trp Phe Ser 670 675 680 tgg ttc att agt agc ctc att cct ctt ctt gtg agc gct ggc ctg cta 2413Trp Phe Ile Ser Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu 685 690 695 700 gtg gtc atc ctg aag tta gga aac ctg ctg ccc tac agt gat ccc agc 2461Val Val Ile Leu Lys Leu Gly Asn Leu Leu Pro Tyr Ser Asp Pro Ser 705 710 715 gtg gtg ttt gtc ttc ctg tcc gtg ttt gct gtg gtg aca atc ctg cag 2509Val Val Phe Val Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu Gln 720 725 730 tgc ttc ctg att agc aca ctc ttc tcc aga gcc aac ctg gca gca gcc 2557Cys Phe Leu Ile Ser Thr Leu Phe Ser Arg Ala Asn Leu Ala Ala Ala 735 740 745 tgt ggg ggc atc atc tac ttc acg ctg tac ctg ccc tac gtc ctg tgt 2605Cys Gly Gly Ile Ile Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys 750 755 760 gtg gca tgg cag gac tac gtg ggc ttc aca ctc aag atc ttc gct agc 2653Val Ala Trp Gln Asp Tyr Val Gly Phe Thr Leu Lys Ile Phe Ala Ser 765 770 775 780 ctg ctg tct cct gtg gct ttt ggg ttt ggc tgt gag tac ttt gcc ctt 2701Leu Leu Ser Pro Val Ala Phe Gly Phe Gly Cys Glu Tyr Phe Ala Leu 785 790 795 ttt gag gag cag ggc att gga gtg cag tgg gac aac ctg ttt gag agt 2749Phe Glu Glu Gln Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu Ser 800 805 810 cct gtg gag gaa gat ggc ttc aat ctc acc act tcg gtc tcc atg atg 2797Pro Val Glu Glu Asp Gly Phe Asn Leu Thr Thr Ser Val Ser Met Met 815 820 825 ctg ttt gac acc ttc ctc tat ggg gtg atg acc tgg tac att gag gct 2845Leu Phe Asp Thr Phe Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala 830 835 840 gtc ttt cca ggc cag tac gga att ccc agg ccc tgg tat ttt cct tgc 2893Val Phe Pro Gly Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe Pro Cys 845 850 855 860 acc aag tcc tac tgg ttt ggc gag gaa agt gat gag aag agc cac cct 2941Thr Lys Ser Tyr Trp Phe Gly Glu Glu Ser Asp Glu Lys Ser His Pro 865 870 875 ggt tcc aac cag aag aga ata tca gaa atc tgc atg gag gag gaa ccc 2989Gly Ser Asn Gln Lys Arg Ile Ser Glu Ile Cys Met Glu Glu Glu Pro 880 885 890 acc cac ttg aag ctg ggc gtg tcc att cag aac ctg gta aaa gtc tac 3037Thr His Leu Lys Leu Gly Val Ser Ile Gln Asn Leu Val Lys Val Tyr 895 900 905 cga gat ggg atg aag gtg gct gtc gat ggc ctg gca ctg aat ttt tat 3085Arg Asp Gly Met Lys Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr 910 915 920 gag ggc cag atc acc tcc ttc ctg ggc cac aat gga gcg ggg aag acg 3133Glu Gly Gln Ile Thr Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr 925 930 935 940 acc acc atg tca atc ctg acc ggg ttg ttc ccc ccg acc tcg ggc acc 3181Thr Thr Met Ser Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Thr 945 950 955 gcc tac atc ctg gga aaa gac att cgc tct gag atg agc acc atc cgg 3229Ala Tyr Ile Leu Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile Arg 960 965 970 cag aac ctg ggg gtc tgt ccc cag cat aac gtg ctg ttt gac atg ctg 3277Gln Asn Leu Gly Val Cys Pro Gln His Asn Val Leu Phe Asp Met Leu 975 980 985 act gtc gaa gaa cac atc tgg ttc tat gcc cgc ttg aaa ggg ctc tct 3325Thr Val Glu Glu His Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser 990 995 1000 gag aag cac gtg aag gcg gag atg gag cag atg gcc ctg gat gtt 3370Glu Lys His Val Lys Ala Glu Met Glu Gln Met Ala Leu Asp Val 1005 1010 1015 ggt ttg cca tca agc aag ctg aaa agc aaa aca agc cag ctg tca 3415Gly Leu Pro Ser Ser Lys Leu Lys Ser Lys Thr Ser Gln Leu Ser 1020 1025 1030 ggt gga atg cag aga aag cta tct gtg gcc ttg gcc ttt gtc ggg 3460Gly Gly Met Gln Arg Lys Leu Ser Val Ala Leu Ala Phe Val Gly 1035 1040 1045 gga tct aag gtt gtc att ctg gat gaa ccc aca gct ggt gtg gac 3505Gly Ser Lys Val Val Ile Leu Asp Glu Pro Thr Ala Gly Val Asp 1050 1055 1060 cct tac tcc cgc agg gga ata tgg gag ctg ctg ctg aaa tac cga 3550Pro Tyr Ser Arg Arg Gly Ile Trp Glu Leu Leu Leu Lys Tyr Arg 1065 1070 1075 caa ggc cgc acc att att ctc tct aca cac cac atg gat gaa gcg 3595Gln Gly Arg Thr Ile Ile Leu Ser Thr His His Met Asp Glu Ala 1080 1085 1090 gac gtc ctg ggg gac agg att gcc atc atc tcc cat ggg aag ctg 3640Asp Val Leu Gly Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu 1095 1100 1105 tgc tgt gtg ggc tcc tcc ctg ttt ctg aag aac cag ctg gga aca 3685Cys Cys Val Gly Ser Ser Leu Phe Leu Lys Asn Gln Leu Gly Thr 1110 1115 1120 ggc tac tac ctg acc ttg gtc aag aaa gat gtg gaa tcc tcc ctc 3730Gly Tyr Tyr Leu Thr Leu Val Lys Lys Asp Val Glu Ser Ser Leu 1125 1130 1135 agt tcc tgc aga aac agt agt agc act gtg tca tac ctg aaa aag 3775Ser Ser Cys Arg Asn Ser Ser Ser Thr Val Ser Tyr Leu Lys Lys 1140 1145 1150 gag gac agt gtt tct cag agc agt tct gat gct ggc ctg ggc agc 3820Glu Asp Ser Val Ser Gln Ser Ser Ser Asp Ala Gly Leu Gly Ser 1155 1160 1165 gac cat gag agt gac acg ctg acc atc gat gtc tct gct atc tcc 3865Asp His Glu Ser Asp Thr Leu Thr Ile Asp Val Ser Ala Ile Ser 1170 1175 1180

aac ctc atc agg aag cat gtg tct gaa gcc cgg ctg gtg gaa gac 3910Asn Leu Ile Arg Lys His Val Ser Glu Ala Arg Leu Val Glu Asp 1185 1190 1195 ata ggg cat gag ctg acc tat gtg ctg cca tat gaa gct gct aag 3955Ile Gly His Glu Leu Thr Tyr Val Leu Pro Tyr Glu Ala Ala Lys 1200 1205 1210 gag gga gcc ttt gtg gaa ctc ttt cat gag att gat gac cgg ctc 4000Glu Gly Ala Phe Val Glu Leu Phe His Glu Ile Asp Asp Arg Leu 1215 1220 1225 tca gac ctg ggc att tct agt tat ggc atc tca gag acg acc ctg 4045Ser Asp Leu Gly Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu 1230 1235 1240 gaa gaa ata ttc ctc aag gtg gcc gaa gag agt ggg gtg gat gct 4090Glu Glu Ile Phe Leu Lys Val Ala Glu Glu Ser Gly Val Asp Ala 1245 1250 1255 gag acc tca gat ggt acc ttg cca gca aga cga aac agg cgg gcc 4135Glu Thr Ser Asp Gly Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala 1260 1265 1270 ttc ggg gac aag cag agc tgt ctt cgc ccg ttc act gaa gat gat 4180Phe Gly Asp Lys Gln Ser Cys Leu Arg Pro Phe Thr Glu Asp Asp 1275 1280 1285 gct gct gat cca aat gat tct gac ata gac cca gaa tcc aga gag 4225Ala Ala Asp Pro Asn Asp Ser Asp Ile Asp Pro Glu Ser Arg Glu 1290 1295 1300 aca gac ttg ctc agt ggg atg gat ggc aaa ggg tcc tac cag gtg 4270Thr Asp Leu Leu Ser Gly Met Asp Gly Lys Gly Ser Tyr Gln Val 1305 1310 1315 aaa ggc tgg aaa ctt aca cag caa cag ttt gtg gcc ctt ttg tgg 4315Lys Gly Trp Lys Leu Thr Gln Gln Gln Phe Val Ala Leu Leu Trp 1320 1325 1330 aag aga ctg cta att gcc aga cgg agt cgg aaa gga ttt ttt gct 4360Lys Arg Leu Leu Ile Ala Arg Arg Ser Arg Lys Gly Phe Phe Ala 1335 1340 1345 cag att gtc ttg cca gct gtg ttt gtc tgc att gcc ctt gtg ttc 4405Gln Ile Val Leu Pro Ala Val Phe Val Cys Ile Ala Leu Val Phe 1350 1355 1360 agc ctg atc gtg cca ccc ttt ggc aag tac ccc agc ctg gaa ctt 4450Ser Leu Ile Val Pro Pro Phe Gly Lys Tyr Pro Ser Leu Glu Leu 1365 1370 1375 cag ccc tgg atg tac aac gaa cag tac aca ttt gtc agc aat gat 4495Gln Pro Trp Met Tyr Asn Glu Gln Tyr Thr Phe Val Ser Asn Asp 1380 1385 1390 gct cct gag gac acg gga acc ctg gaa ctc tta aac gcc ctc acc 4540Ala Pro Glu Asp Thr Gly Thr Leu Glu Leu Leu Asn Ala Leu Thr 1395 1400 1405 aaa gac cct ggc ttc ggg acc cgc tgt atg gaa gga aac cca atc 4585Lys Asp Pro Gly Phe Gly Thr Arg Cys Met Glu Gly Asn Pro Ile 1410 1415 1420 cca gac acg ccc tgc cag gca ggg gag gaa gag tgg acc act gcc 4630Pro Asp Thr Pro Cys Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala 1425 1430 1435 cca gtt ccc cag acc atc atg gac ctc ttc cag aat ggg aac tgg 4675Pro Val Pro Gln Thr Ile Met Asp Leu Phe Gln Asn Gly Asn Trp 1440 1445 1450 aca atg cag aac cct tca cct gca tgc cag tgt agc agc gac aaa 4720Thr Met Gln Asn Pro Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys 1455 1460 1465 atc aag aag atg ctg cct gtg tgt ccc cca ggg gca ggg ggg ctg 4765Ile Lys Lys Met Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu 1470 1475 1480 cct cct cca caa aga aaa caa aac act gca gat atc ctt cag gac 4810Pro Pro Pro Gln Arg Lys Gln Asn Thr Ala Asp Ile Leu Gln Asp 1485 1490 1495 ctg aca gga aga aac att tcg gat tat ctg gtg aag acg tat gtg 4855Leu Thr Gly Arg Asn Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val 1500 1505 1510 cag atc ata gcc aaa agc tta aag aac aag atc tgg gtg aat gag 4900Gln Ile Ile Ala Lys Ser Leu Lys Asn Lys Ile Trp Val Asn Glu 1515 1520 1525 ttt agg tat ggc ggc ttt tcc ctg ggt gtc agt aat act caa gca 4945Phe Arg Tyr Gly Gly Phe Ser Leu Gly Val Ser Asn Thr Gln Ala 1530 1535 1540 ctt cct ccg agt caa gaa gtt aat gat gcc atc aaa caa atg aag 4990Leu Pro Pro Ser Gln Glu Val Asn Asp Ala Ile Lys Gln Met Lys 1545 1550 1555 aaa cac cta aag ctg gcc aag gac agt tct gca gat cga ttt ctc 5035Lys His Leu Lys Leu Ala Lys Asp Ser Ser Ala Asp Arg Phe Leu 1560 1565 1570 aac agc ttg gga aga ttt atg aca gga ctg gac acc aaa aat aat 5080Asn Ser Leu Gly Arg Phe Met Thr Gly Leu Asp Thr Lys Asn Asn 1575 1580 1585 gtc aag gtg tgg ttc aat aac aag ggc tgg cat gca atc agc tct 5125Val Lys Val Trp Phe Asn Asn Lys Gly Trp His Ala Ile Ser Ser 1590 1595 1600 ttc ctg aat gtc atc aac aat gcc att ctc cgg gcc aac ctg caa 5170Phe Leu Asn Val Ile Asn Asn Ala Ile Leu Arg Ala Asn Leu Gln 1605 1610 1615 aag gga gag aac cct agc cat tat gga att act gct ttc aat cat 5215Lys Gly Glu Asn Pro Ser His Tyr Gly Ile Thr Ala Phe Asn His 1620 1625 1630 ccc ctg aat ctc acc aag cag cag ctc tca gag gtg gct ctg atg 5260Pro Leu Asn Leu Thr Lys Gln Gln Leu Ser Glu Val Ala Leu Met 1635 1640 1645 acc aca tca gtg gat gtc ctt gtg tcc atc tgt gtc atc ttt gca 5305Thr Thr Ser Val Asp Val Leu Val Ser Ile Cys Val Ile Phe Ala 1650 1655 1660 atg tcc ttc gtc cca gcc agc ttt gtc gta ttc ctg atc cag gag 5350Met Ser Phe Val Pro Ala Ser Phe Val Val Phe Leu Ile Gln Glu 1665 1670 1675 cgg gtc agc aaa gca aaa cac ctg cag ttc atc agt gga gtg aag 5395Arg Val Ser Lys Ala Lys His Leu Gln Phe Ile Ser Gly Val Lys 1680 1685 1690 cct gtc atc tac tgg ctc tct aat ttt gtc tgg gat atg tgc aat 5440Pro Val Ile Tyr Trp Leu Ser Asn Phe Val Trp Asp Met Cys Asn 1695 1700 1705 tac gtt gtc cct gcc aca ctg gtc att atc atc ttc atc tgc ttc 5485Tyr Val Val Pro Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe 1710 1715 1720 cag cag aag tcc tat gtg tcc tcc acc aat ctg cct gtg cta gcc 5530Gln Gln Lys Ser Tyr Val Ser Ser Thr Asn Leu Pro Val Leu Ala 1725 1730 1735 ctt cta ctt ttg ctg tat ggg tgg tca atc aca cct ctc atg tac 5575Leu Leu Leu Leu Leu Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr 1740 1745 1750 cca gcc tcc ttt gtg ttc aag atc ccc agc aca gcc tat gtg gtg 5620Pro Ala Ser Phe Val Phe Lys Ile Pro Ser Thr Ala Tyr Val Val 1755 1760 1765 ctc acc agc gtg aac ctc ttc att ggc att aat ggc agc gtg gcc 5665Leu Thr Ser Val Asn Leu Phe Ile Gly Ile Asn Gly Ser Val Ala 1770 1775 1780 acc ttt gtg ctg gag ctg ttc acc gac aat aag ctg aat aat atc 5710Thr Phe Val Leu Glu Leu Phe Thr Asp Asn Lys Leu Asn Asn Ile 1785 1790 1795 aat gat atc ctg aag tcc gtg ttc ttg atc ttc cca cat ttt tgc 5755Asn Asp Ile Leu Lys Ser Val Phe Leu Ile Phe Pro His Phe Cys 1800 1805 1810 ctg gga cga ggg ctc atc gac atg gtg aaa aac cag gca atg gct 5800Leu Gly Arg Gly Leu Ile Asp Met Val Lys Asn Gln Ala Met Ala 1815 1820 1825 gat gcc ctg gaa agg ttt ggg gag aat cgc ttt gtg tca cca tta 5845Asp Ala Leu Glu Arg Phe Gly Glu Asn Arg Phe Val Ser Pro Leu 1830 1835 1840 tct tgg gac ttg gtg gga cga aac ctc ttc gcc atg gcc gtg gaa 5890Ser Trp Asp Leu Val Gly Arg Asn Leu Phe Ala Met Ala Val Glu 1845 1850 1855 ggg gtg gtg ttc ttc ctc att act gtt ctg atc cag tac aga ttc 5935Gly Val Val Phe Phe Leu Ile Thr Val Leu Ile Gln Tyr Arg Phe 1860 1865 1870 ttc atc agg ccc aga cct gta aat gca aag cta tct cct ctg aat 5980Phe Ile Arg Pro Arg Pro Val Asn Ala Lys Leu Ser Pro Leu Asn 1875 1880 1885 gat gaa gat gaa gat gtg agg cgg gaa aga cag aga att ctt gat 6025Asp Glu Asp Glu Asp Val Arg Arg Glu Arg Gln Arg Ile Leu Asp 1890 1895 1900 ggt gga ggc cag aat gac atc tta gaa atc aag gag ttg acg aag 6070Gly Gly Gly Gln Asn Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys 1905 1910 1915 ata tat aga agg aag cgg aag cct gct gtt gac agg att tgc gtg 6115Ile Tyr Arg Arg Lys Arg Lys Pro Ala Val Asp Arg Ile Cys Val 1920 1925 1930 ggc att cct cct ggt gag tgc ttt ggg ctc ctg gga gtt aat ggg 6160Gly Ile Pro Pro Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly 1935 1940 1945 gct gga aaa tca tca act ttc aag atg tta aca gga gat acc act 6205Ala Gly Lys Ser Ser Thr Phe Lys Met Leu Thr Gly Asp Thr Thr 1950 1955 1960 gtt acc aga gga gat gct ttc ctt aac aaa aat agt atc tta tca 6250Val Thr Arg Gly Asp Ala Phe Leu Asn Lys Asn Ser Ile Leu Ser 1965 1970 1975 aac atc cat gaa gta cat cag aac atg ggc tac tgc cct cag ttt 6295Asn Ile His Glu Val His Gln Asn Met Gly Tyr Cys Pro Gln Phe 1980 1985 1990 gat gcc atc aca gag ctg ttg act ggg aga gaa cac gtg gag ttc 6340Asp Ala Ile Thr Glu Leu Leu Thr Gly Arg Glu His Val Glu Phe 1995 2000 2005 ttt gcc ctt ttg aga gga gtc cca gag aaa gaa gtt ggc aag gtt 6385Phe Ala Leu Leu Arg Gly Val Pro Glu Lys Glu Val Gly Lys Val 2010 2015 2020 ggt gag tgg gcg att cgg aaa ctg ggc ctc gtg aag tat gga gaa 6430Gly Glu Trp Ala Ile Arg Lys Leu Gly Leu Val Lys Tyr Gly Glu 2025 2030 2035 aaa tat gct ggt aac tat agt gga ggc aac aaa cgc aag ctc tct 6475Lys Tyr Ala Gly Asn Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser 2040 2045 2050 aca gcc atg gct ttg atc ggc ggg cct cct gtg gtg ttt ctg gat 6520Thr Ala Met Ala Leu Ile Gly Gly Pro Pro Val Val Phe Leu Asp 2055 2060 2065 gaa ccc acc aca ggc atg gat ccc aaa gcc cgg cgg ttc ttg tgg 6565Glu Pro Thr Thr Gly Met Asp Pro Lys Ala Arg Arg Phe Leu Trp 2070 2075 2080 aat tgt gcc cta agt gtt gtc aag gag ggg aga tca gta gtg ctt 6610Asn Cys Ala Leu Ser Val Val Lys Glu Gly Arg Ser Val Val Leu 2085 2090 2095 aca tct cat agt atg gaa gaa tgt gaa gct ctt tgc act agg atg 6655Thr Ser His Ser Met Glu Glu Cys Glu Ala Leu Cys Thr Arg Met 2100 2105 2110 gca atc atg gtc aat gga agg ttc agg tgc ctt ggc agt gtc cag 6700Ala Ile Met Val Asn Gly Arg Phe Arg Cys Leu Gly Ser Val Gln 2115 2120 2125 cat cta aaa aat agg ttt gga gat ggt tat aca ata gtt gta cga 6745His Leu Lys Asn Arg Phe Gly Asp Gly Tyr Thr Ile Val Val Arg 2130 2135 2140 ata gca ggg tcc aac ccg gac ctg aag cct gtc cag gat ttc ttt 6790Ile Ala Gly Ser Asn Pro Asp Leu Lys Pro Val Gln Asp Phe Phe 2145 2150 2155 gga ctt gca ttt cct gga agt gtt cta aaa gag aaa cac cgg aac 6835Gly Leu Ala Phe Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn 2160 2165 2170 atg cta caa tac cag ctt cca tct tca tta tct tct ctg gcc agg 6880Met Leu Gln Tyr Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg 2175 2180 2185 ata ttc agc atc ctc tcc cag agc aaa aag cga ctc cac ata gaa 6925Ile Phe Ser Ile Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu 2190 2195 2200 gac tac tct gtt tct cag aca aca ctt gac caa gta ttt gtg aac 6970Asp Tyr Ser Val Ser Gln Thr Thr Leu Asp Gln Val Phe Val Asn 2205 2210 2215 ttt gcc aag gac caa agt gat gat gac cac tta aaa gac ctc tca 7015Phe Ala Lys Asp Gln Ser Asp Asp Asp His Leu Lys Asp Leu Ser 2220 2225 2230 tta cac aaa aac cag aca gta gtg gac gtt gca gtt ctc aca tct 7060Leu His Lys Asn Gln Thr Val Val Asp Val Ala Val Leu Thr Ser 2235 2240 2245 ttt cta cag gat gag aaa gtg aaa gaa agc tat gta tga agaatcctgt 7109Phe Leu Gln Asp Glu Lys Val Lys Glu Ser Tyr Val 2250 2255 2260 tcatacgggg tggctgaaag taaagaggaa c 714072261PRTHomo sapiens 7Met Ala Cys Trp Pro Gln Leu Arg Leu Leu Leu Trp Lys Asn Leu Thr 1 5 10 15 Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu Leu Glu Val Ala Trp Pro 20 25 30 Leu Phe Ile Phe Leu Ile Leu Ile Ser Val Arg Leu Ser Tyr Pro Pro 35 40 45 Tyr Glu Gln His Glu Cys His Phe Pro Asn Lys Ala Met Pro Ser Ala 50 55 60 Gly Thr Leu Pro Trp Val Gln Gly Ile Ile Cys Asn Ala Asn Asn Pro 65 70 75 80 Cys Phe Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn 85 90 95 Phe Asn Lys Ser Ile Val Ala Arg Leu Phe Ser Asp Ala Arg Arg Leu 100 105 110 Leu Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp Met Arg Lys Val 115 120 125 Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser Ser Asn Leu Lys Leu 130 135 140 Gln Asp Phe Leu Val Asp Asn Glu Thr Phe Ser Gly Phe Leu Tyr His 145 150 155 160 Asn Leu Ser Leu Pro Lys Ser Thr Val Asp Lys Met Leu Arg Ala Asp 165 170 175 Val Ile Leu His Lys Val Phe Leu Gln Gly Tyr Gln Leu His Leu Thr 180 185 190 Ser Leu Cys Asn Gly Ser Lys Ser Glu Glu Met Ile Gln Leu Gly Asp 195 200 205 Gln Glu Val Ser Glu Leu Cys Gly Leu Pro Arg Glu Lys Leu Ala Ala 210 215 220 Ala Glu Arg Val Leu Arg Ser Asn Met Asp Ile Leu Lys Pro Ile Leu 225 230 235 240 Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser Lys Glu Leu Ala Glu 245 250 255 Ala Thr Lys Thr Leu Leu His Ser Leu Gly Thr Leu Ala Gln Glu Leu 260 265 270 Phe Ser Met Arg Ser Trp Ser Asp Met Arg Gln Glu Val Met Phe Leu 275 280 285 Thr Asn Val Asn Ser Ser Ser Ser Ser Thr Gln Ile Tyr Gln Ala Val 290 295 300 Ser Arg Ile Val Cys Gly His Pro Glu Gly Gly Gly

Leu Lys Ile Lys 305 310 315 320 Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala Leu Phe Gly Gly 325 330 335 Asn Gly Thr Glu Glu Asp Ala Glu Thr Phe Tyr Asp Asn Ser Thr Thr 340 345 350 Pro Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser Ser Pro Leu Ser 355 360 365 Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu Leu Val Gly Lys Ile Leu 370 375 380 Tyr Thr Pro Asp Thr Pro Ala Thr Arg Gln Val Met Ala Glu Val Asn 385 390 395 400 Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp Leu Glu Gly Met Trp 405 410 415 Glu Glu Leu Ser Pro Lys Ile Trp Thr Phe Met Glu Asn Ser Gln Glu 420 425 430 Met Asp Leu Val Arg Met Leu Leu Asp Ser Arg Asp Asn Asp His Phe 435 440 445 Trp Glu Gln Gln Leu Asp Gly Leu Asp Trp Thr Ala Gln Asp Ile Val 450 455 460 Ala Phe Leu Ala Lys His Pro Glu Asp Val Gln Ser Ser Asn Gly Ser 465 470 475 480 Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr Asn Gln Ala Ile Arg 485 490 495 Thr Ile Ser Arg Phe Met Glu Cys Val Asn Leu Asn Lys Leu Glu Pro 500 505 510 Ile Ala Thr Glu Val Trp Leu Ile Asn Lys Ser Met Glu Leu Leu Asp 515 520 525 Glu Arg Lys Phe Trp Ala Gly Ile Val Phe Thr Gly Ile Thr Pro Gly 530 535 540 Ser Ile Glu Leu Pro His His Val Lys Tyr Lys Ile Arg Met Asp Ile 545 550 555 560 Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp Gly Tyr Trp Asp Pro 565 570 575 Gly Pro Arg Ala Asp Pro Phe Glu Asp Met Arg Tyr Val Trp Gly Gly 580 585 590 Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile Ile Arg Val Leu 595 600 605 Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr Met Gln Gln Met Pro Tyr 610 615 620 Pro Cys Tyr Val Asp Asp Ile Phe Leu Arg Val Met Ser Arg Ser Met 625 630 635 640 Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser Val Ala Val Ile Ile 645 650 655 Lys Gly Ile Val Tyr Glu Lys Glu Ala Arg Leu Lys Glu Thr Met Arg 660 665 670 Ile Met Gly Leu Asp Asn Ser Ile Leu Trp Phe Ser Trp Phe Ile Ser 675 680 685 Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val Ile Leu 690 695 700 Lys Leu Gly Asn Leu Leu Pro Tyr Ser Asp Pro Ser Val Val Phe Val 705 710 715 720 Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu Gln Cys Phe Leu Ile 725 730 735 Ser Thr Leu Phe Ser Arg Ala Asn Leu Ala Ala Ala Cys Gly Gly Ile 740 745 750 Ile Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys Val Ala Trp Gln 755 760 765 Asp Tyr Val Gly Phe Thr Leu Lys Ile Phe Ala Ser Leu Leu Ser Pro 770 775 780 Val Ala Phe Gly Phe Gly Cys Glu Tyr Phe Ala Leu Phe Glu Glu Gln 785 790 795 800 Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu Ser Pro Val Glu Glu 805 810 815 Asp Gly Phe Asn Leu Thr Thr Ser Val Ser Met Met Leu Phe Asp Thr 820 825 830 Phe Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala Val Phe Pro Gly 835 840 845 Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe Pro Cys Thr Lys Ser Tyr 850 855 860 Trp Phe Gly Glu Glu Ser Asp Glu Lys Ser His Pro Gly Ser Asn Gln 865 870 875 880 Lys Arg Ile Ser Glu Ile Cys Met Glu Glu Glu Pro Thr His Leu Lys 885 890 895 Leu Gly Val Ser Ile Gln Asn Leu Val Lys Val Tyr Arg Asp Gly Met 900 905 910 Lys Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr Glu Gly Gln Ile 915 920 925 Thr Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser 930 935 940 Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Thr Ala Tyr Ile Leu 945 950 955 960 Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile Arg Gln Asn Leu Gly 965 970 975 Val Cys Pro Gln His Asn Val Leu Phe Asp Met Leu Thr Val Glu Glu 980 985 990 His Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser Glu Lys His Val 995 1000 1005 Lys Ala Glu Met Glu Gln Met Ala Leu Asp Val Gly Leu Pro Ser 1010 1015 1020 Ser Lys Leu Lys Ser Lys Thr Ser Gln Leu Ser Gly Gly Met Gln 1025 1030 1035 Arg Lys Leu Ser Val Ala Leu Ala Phe Val Gly Gly Ser Lys Val 1040 1045 1050 Val Ile Leu Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr Ser Arg 1055 1060 1065 Arg Gly Ile Trp Glu Leu Leu Leu Lys Tyr Arg Gln Gly Arg Thr 1070 1075 1080 Ile Ile Leu Ser Thr His His Met Asp Glu Ala Asp Val Leu Gly 1085 1090 1095 Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu Cys Cys Val Gly 1100 1105 1110 Ser Ser Leu Phe Leu Lys Asn Gln Leu Gly Thr Gly Tyr Tyr Leu 1115 1120 1125 Thr Leu Val Lys Lys Asp Val Glu Ser Ser Leu Ser Ser Cys Arg 1130 1135 1140 Asn Ser Ser Ser Thr Val Ser Tyr Leu Lys Lys Glu Asp Ser Val 1145 1150 1155 Ser Gln Ser Ser Ser Asp Ala Gly Leu Gly Ser Asp His Glu Ser 1160 1165 1170 Asp Thr Leu Thr Ile Asp Val Ser Ala Ile Ser Asn Leu Ile Arg 1175 1180 1185 Lys His Val Ser Glu Ala Arg Leu Val Glu Asp Ile Gly His Glu 1190 1195 1200 Leu Thr Tyr Val Leu Pro Tyr Glu Ala Ala Lys Glu Gly Ala Phe 1205 1210 1215 Val Glu Leu Phe His Glu Ile Asp Asp Arg Leu Ser Asp Leu Gly 1220 1225 1230 Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu Glu Glu Ile Phe 1235 1240 1245 Leu Lys Val Ala Glu Glu Ser Gly Val Asp Ala Glu Thr Ser Asp 1250 1255 1260 Gly Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala Phe Gly Asp Lys 1265 1270 1275 Gln Ser Cys Leu Arg Pro Phe Thr Glu Asp Asp Ala Ala Asp Pro 1280 1285 1290 Asn Asp Ser Asp Ile Asp Pro Glu Ser Arg Glu Thr Asp Leu Leu 1295 1300 1305 Ser Gly Met Asp Gly Lys Gly Ser Tyr Gln Val Lys Gly Trp Lys 1310 1315 1320 Leu Thr Gln Gln Gln Phe Val Ala Leu Leu Trp Lys Arg Leu Leu 1325 1330 1335 Ile Ala Arg Arg Ser Arg Lys Gly Phe Phe Ala Gln Ile Val Leu 1340 1345 1350 Pro Ala Val Phe Val Cys Ile Ala Leu Val Phe Ser Leu Ile Val 1355 1360 1365 Pro Pro Phe Gly Lys Tyr Pro Ser Leu Glu Leu Gln Pro Trp Met 1370 1375 1380 Tyr Asn Glu Gln Tyr Thr Phe Val Ser Asn Asp Ala Pro Glu Asp 1385 1390 1395 Thr Gly Thr Leu Glu Leu Leu Asn Ala Leu Thr Lys Asp Pro Gly 1400 1405 1410 Phe Gly Thr Arg Cys Met Glu Gly Asn Pro Ile Pro Asp Thr Pro 1415 1420 1425 Cys Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala Pro Val Pro Gln 1430 1435 1440 Thr Ile Met Asp Leu Phe Gln Asn Gly Asn Trp Thr Met Gln Asn 1445 1450 1455 Pro Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys Ile Lys Lys Met 1460 1465 1470 Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu Pro Pro Pro Gln 1475 1480 1485 Arg Lys Gln Asn Thr Ala Asp Ile Leu Gln Asp Leu Thr Gly Arg 1490 1495 1500 Asn Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val Gln Ile Ile Ala 1505 1510 1515 Lys Ser Leu Lys Asn Lys Ile Trp Val Asn Glu Phe Arg Tyr Gly 1520 1525 1530 Gly Phe Ser Leu Gly Val Ser Asn Thr Gln Ala Leu Pro Pro Ser 1535 1540 1545 Gln Glu Val Asn Asp Ala Ile Lys Gln Met Lys Lys His Leu Lys 1550 1555 1560 Leu Ala Lys Asp Ser Ser Ala Asp Arg Phe Leu Asn Ser Leu Gly 1565 1570 1575 Arg Phe Met Thr Gly Leu Asp Thr Lys Asn Asn Val Lys Val Trp 1580 1585 1590 Phe Asn Asn Lys Gly Trp His Ala Ile Ser Ser Phe Leu Asn Val 1595 1600 1605 Ile Asn Asn Ala Ile Leu Arg Ala Asn Leu Gln Lys Gly Glu Asn 1610 1615 1620 Pro Ser His Tyr Gly Ile Thr Ala Phe Asn His Pro Leu Asn Leu 1625 1630 1635 Thr Lys Gln Gln Leu Ser Glu Val Ala Leu Met Thr Thr Ser Val 1640 1645 1650 Asp Val Leu Val Ser Ile Cys Val Ile Phe Ala Met Ser Phe Val 1655 1660 1665 Pro Ala Ser Phe Val Val Phe Leu Ile Gln Glu Arg Val Ser Lys 1670 1675 1680 Ala Lys His Leu Gln Phe Ile Ser Gly Val Lys Pro Val Ile Tyr 1685 1690 1695 Trp Leu Ser Asn Phe Val Trp Asp Met Cys Asn Tyr Val Val Pro 1700 1705 1710 Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe Gln Gln Lys Ser 1715 1720 1725 Tyr Val Ser Ser Thr Asn Leu Pro Val Leu Ala Leu Leu Leu Leu 1730 1735 1740 Leu Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr Pro Ala Ser Phe 1745 1750 1755 Val Phe Lys Ile Pro Ser Thr Ala Tyr Val Val Leu Thr Ser Val 1760 1765 1770 Asn Leu Phe Ile Gly Ile Asn Gly Ser Val Ala Thr Phe Val Leu 1775 1780 1785 Glu Leu Phe Thr Asp Asn Lys Leu Asn Asn Ile Asn Asp Ile Leu 1790 1795 1800 Lys Ser Val Phe Leu Ile Phe Pro His Phe Cys Leu Gly Arg Gly 1805 1810 1815 Leu Ile Asp Met Val Lys Asn Gln Ala Met Ala Asp Ala Leu Glu 1820 1825 1830 Arg Phe Gly Glu Asn Arg Phe Val Ser Pro Leu Ser Trp Asp Leu 1835 1840 1845 Val Gly Arg Asn Leu Phe Ala Met Ala Val Glu Gly Val Val Phe 1850 1855 1860 Phe Leu Ile Thr Val Leu Ile Gln Tyr Arg Phe Phe Ile Arg Pro 1865 1870 1875 Arg Pro Val Asn Ala Lys Leu Ser Pro Leu Asn Asp Glu Asp Glu 1880 1885 1890 Asp Val Arg Arg Glu Arg Gln Arg Ile Leu Asp Gly Gly Gly Gln 1895 1900 1905 Asn Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys Ile Tyr Arg Arg 1910 1915 1920 Lys Arg Lys Pro Ala Val Asp Arg Ile Cys Val Gly Ile Pro Pro 1925 1930 1935 Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala Gly Lys Ser 1940 1945 1950 Ser Thr Phe Lys Met Leu Thr Gly Asp Thr Thr Val Thr Arg Gly 1955 1960 1965 Asp Ala Phe Leu Asn Lys Asn Ser Ile Leu Ser Asn Ile His Glu 1970 1975 1980 Val His Gln Asn Met Gly Tyr Cys Pro Gln Phe Asp Ala Ile Thr 1985 1990 1995 Glu Leu Leu Thr Gly Arg Glu His Val Glu Phe Phe Ala Leu Leu 2000 2005 2010 Arg Gly Val Pro Glu Lys Glu Val Gly Lys Val Gly Glu Trp Ala 2015 2020 2025 Ile Arg Lys Leu Gly Leu Val Lys Tyr Gly Glu Lys Tyr Ala Gly 2030 2035 2040 Asn Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser Thr Ala Met Ala 2045 2050 2055 Leu Ile Gly Gly Pro Pro Val Val Phe Leu Asp Glu Pro Thr Thr 2060 2065 2070 Gly Met Asp Pro Lys Ala Arg Arg Phe Leu Trp Asn Cys Ala Leu 2075 2080 2085 Ser Val Val Lys Glu Gly Arg Ser Val Val Leu Thr Ser His Ser 2090 2095 2100 Met Glu Glu Cys Glu Ala Leu Cys Thr Arg Met Ala Ile Met Val 2105 2110 2115 Asn Gly Arg Phe Arg Cys Leu Gly Ser Val Gln His Leu Lys Asn 2120 2125 2130 Arg Phe Gly Asp Gly Tyr Thr Ile Val Val Arg Ile Ala Gly Ser 2135 2140 2145 Asn Pro Asp Leu Lys Pro Val Gln Asp Phe Phe Gly Leu Ala Phe 2150 2155 2160 Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn Met Leu Gln Tyr 2165 2170 2175 Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg Ile Phe Ser Ile 2180 2185 2190 Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu Asp Tyr Ser Val 2195 2200 2205 Ser Gln Thr Thr Leu Asp Gln Val Phe Val Asn Phe Ala Lys Asp 2210 2215 2220 Gln Ser Asp Asp Asp His Leu Lys Asp Leu Ser Leu His Lys Asn 2225 2230 2235 Gln Thr Val Val Asp Val Ala Val Leu Thr Ser Phe Leu Gln Asp 2240 2245 2250 Glu Lys Val Lys Glu Ser Tyr Val 2255 2260 8780DNARattus norvegicusCDS(63)..(731) 8gcagctttaa gtagagagtg gatttttgtc tcagtttgtc ttctgtttgc gactctgaaa 60ga atg ctg tgg gca ctc ttt ttc ctg gtg act act att cac gct gaa 107 Met Leu Trp Ala Leu Phe Phe Leu Val Thr Thr Ile His Ala Glu 1 5 10 15 ctc tgc cgt cca gat gca gaa aat gcc ttt aaa gta aga ctt agc atc 155Leu Cys Arg Pro Asp Ala Glu Asn Ala Phe Lys Val Arg Leu Ser Ile 20 25 30 aaa gca gct ctt gga gat aaa gcg tat gtc tgg gac aca gat gaa gaa 203Lys Ala Ala Leu Gly Asp Lys Ala Tyr Val Trp Asp Thr Asp Glu Glu 35 40 45 tat ctc ttc aga gca atg gtg gca ttc tcc atg aga aaa gtt ccc aac 251Tyr Leu Phe Arg Ala Met Val Ala Phe Ser Met Arg Lys Val Pro Asn 50 55 60 aga gaa gga aca gaa att tcc cac gtc ctg ctt tgc aat gta acc cag 299Arg Glu Gly Thr Glu Ile Ser His Val Leu Leu Cys Asn Val Thr Gln 65 70 75 aga gtg tca ttc tgg ttt gtg gtc aca gac cct ttg aaa aac cat act 347Arg Val Ser Phe Trp Phe Val Val Thr Asp Pro Leu Lys Asn His Thr 80 85 90 95 ctt cct gca gct gaa gta cag tca gcc ata aga atg aac agg aac cgg 395Leu Pro Ala Ala Glu Val Gln Ser Ala Ile Arg Met Asn Arg Asn Arg 100 105 110 atc aac agt gca ttc ttt ttg gat gat cat act ctg gaa ttt tta aaa 443Ile Asn Ser Ala Phe Phe Leu Asp Asp His Thr Leu Glu Phe Leu Lys 115 120 125 att cct tcc act ctt gct ccc ccg atg gat cca tct gtg ccc gtc tgg 491Ile Pro Ser Thr Leu Ala Pro Pro Met Asp Pro Ser Val Pro Val Trp 130 135 140 att att gta ttt ggt gtg ata ttt tgc att gtt aca gtt gca att gca 539Ile Ile Val Phe Gly Val Ile Phe Cys Ile Val Thr Val Ala Ile Ala 145 150 155 cta ctg gtt tta tcc gga atc cgg caa cga

aga agg aac aag aaa gga 587Leu Leu Val Leu Ser Gly Ile Arg Gln Arg Arg Arg Asn Lys Lys Gly 160 165 170 175 cca cct gga gtg gag gat gca gaa gac aag tgt gaa aac atc atc aca 635Pro Pro Gly Val Glu Asp Ala Glu Asp Lys Cys Glu Asn Ile Ile Thr 180 185 190 att gaa aat ggc atc cct tgt gat ccc ttg gac atg aag gga ggg cac 683Ile Glu Asn Gly Ile Pro Cys Asp Pro Leu Asp Met Lys Gly Gly His 195 200 205 att aat gat ggc ttc ttg aca gag gat gag cgt ctc acc cct ctc tga 731Ile Asn Asp Gly Phe Leu Thr Glu Asp Glu Arg Leu Thr Pro Leu 210 215 220 gagttacagt cttgtaagaa aatttcaaga tgcttgaatg tgatagaca 7809222PRTRattus norvegicus 9Met Leu Trp Ala Leu Phe Phe Leu Val Thr Thr Ile His Ala Glu Leu 1 5 10 15 Cys Arg Pro Asp Ala Glu Asn Ala Phe Lys Val Arg Leu Ser Ile Lys 20 25 30 Ala Ala Leu Gly Asp Lys Ala Tyr Val Trp Asp Thr Asp Glu Glu Tyr 35 40 45 Leu Phe Arg Ala Met Val Ala Phe Ser Met Arg Lys Val Pro Asn Arg 50 55 60 Glu Gly Thr Glu Ile Ser His Val Leu Leu Cys Asn Val Thr Gln Arg 65 70 75 80 Val Ser Phe Trp Phe Val Val Thr Asp Pro Leu Lys Asn His Thr Leu 85 90 95 Pro Ala Ala Glu Val Gln Ser Ala Ile Arg Met Asn Arg Asn Arg Ile 100 105 110 Asn Ser Ala Phe Phe Leu Asp Asp His Thr Leu Glu Phe Leu Lys Ile 115 120 125 Pro Ser Thr Leu Ala Pro Pro Met Asp Pro Ser Val Pro Val Trp Ile 130 135 140 Ile Val Phe Gly Val Ile Phe Cys Ile Val Thr Val Ala Ile Ala Leu 145 150 155 160 Leu Val Leu Ser Gly Ile Arg Gln Arg Arg Arg Asn Lys Lys Gly Pro 165 170 175 Pro Gly Val Glu Asp Ala Glu Asp Lys Cys Glu Asn Ile Ile Thr Ile 180 185 190 Glu Asn Gly Ile Pro Cys Asp Pro Leu Asp Met Lys Gly Gly His Ile 195 200 205 Asn Asp Gly Phe Leu Thr Glu Asp Glu Arg Leu Thr Pro Leu 210 215 220 101116DNAPlasmodium falciparumCDS(1)..(1116) 10atg cat ata gtg agc ttt att att ttt ttc ttt gca tta ttt ttt cca 48Met His Ile Val Ser Phe Ile Ile Phe Phe Phe Ala Leu Phe Phe Pro 1 5 10 15 att tcc atc tgt tat aaa ata aat ggg gta tgt gat ttt tcg agc gaa 96Ile Ser Ile Cys Tyr Lys Ile Asn Gly Val Cys Asp Phe Ser Ser Glu 20 25 30 ggg cta agt ttg ttg cca gaa gaa aag tta gat ttt tct gta tca agg 144Gly Leu Ser Leu Leu Pro Glu Glu Lys Leu Asp Phe Ser Val Ser Arg 35 40 45 aat gta gat aaa tta tct gat gaa aac aat gta aga cat tgt gta cat 192Asn Val Asp Lys Leu Ser Asp Glu Asn Asn Val Arg His Cys Val His 50 55 60 ttt agt aag ggt ttt gaa tat tta cgt ttt ata tgt cca atg aga aaa 240Phe Ser Lys Gly Phe Glu Tyr Leu Arg Phe Ile Cys Pro Met Arg Lys 65 70 75 80 gat aat tat gaa gga att gaa att cgt cct gtt gaa tgt ttt gaa tat 288Asp Asn Tyr Glu Gly Ile Glu Ile Arg Pro Val Glu Cys Phe Glu Tyr 85 90 95 att cat att gaa gga aga gaa cac aaa tta agc gag ata tta aaa ggt 336Ile His Ile Glu Gly Arg Glu His Lys Leu Ser Glu Ile Leu Lys Gly 100 105 110 agt tta tat gaa aaa agt ata aat gat aat ata atg acg aga gat gtt 384Ser Leu Tyr Glu Lys Ser Ile Asn Asp Asn Ile Met Thr Arg Asp Val 115 120 125 ttt att cct cca act att tat gaa gat atg ttt ttt gaa tgt aca tgt 432Phe Ile Pro Pro Thr Ile Tyr Glu Asp Met Phe Phe Glu Cys Thr Cys 130 135 140 gat aat agt tta acc ttt aaa aat aat atg att ggt ata aga ggt ata 480Asp Asn Ser Leu Thr Phe Lys Asn Asn Met Ile Gly Ile Arg Gly Ile 145 150 155 160 atg aaa atc cat tta aaa aaa aat att tta tat gga tgt gat ttt gat 528Met Lys Ile His Leu Lys Lys Asn Ile Leu Tyr Gly Cys Asp Phe Asp 165 170 175 cat gat gaa aaa tta atg aaa aat aaa aca gca ttt aca aat ttt tat 576His Asp Glu Lys Leu Met Lys Asn Lys Thr Ala Phe Thr Asn Phe Tyr 180 185 190 gat aaa caa aaa att tta cca tta ata ggt aat aat aat aat gat gat 624Asp Lys Gln Lys Ile Leu Pro Leu Ile Gly Asn Asn Asn Asn Asp Asp 195 200 205 gat aat aat gat gat gat aat aat aat gat aat aat aat aat gat aat 672Asp Asn Asn Asp Asp Asp Asn Asn Asn Asp Asn Asn Asn Asn Asp Asn 210 215 220 aat aat aat aat aat aat aat aat aat aat aat aat aat aat aat aat 720Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn 225 230 235 240 aat aat att act tgt aat gtt act att aaa aaa tct caa gtt tat tta 768Asn Asn Ile Thr Cys Asn Val Thr Ile Lys Lys Ser Gln Val Tyr Leu 245 250 255 gga att ata tgc cca gat gga tat act tta tat cca aat gat tgt ttt 816Gly Ile Ile Cys Pro Asp Gly Tyr Thr Leu Tyr Pro Asn Asp Cys Phe 260 265 270 aaa aat gtt ata tat gat aat aat att att ata cca tta aaa aaa att 864Lys Asn Val Ile Tyr Asp Asn Asn Ile Ile Ile Pro Leu Lys Lys Ile 275 280 285 ata cca cat gat att tta tat cat caa gac aaa aac aaa aga att act 912Ile Pro His Asp Ile Leu Tyr His Gln Asp Lys Asn Lys Arg Ile Thr 290 295 300 ttt gct tca ttt aca tta aat ata aat gaa aat cca cca gga ttc aca 960Phe Ala Ser Phe Thr Leu Asn Ile Asn Glu Asn Pro Pro Gly Phe Thr 305 310 315 320 tgt tat tgt att aaa gat caa aca aat att aat aac cca ctt atc gta 1008Cys Tyr Cys Ile Lys Asp Gln Thr Asn Ile Asn Asn Pro Leu Ile Val 325 330 335 aac ttc cat ttt tca aat caa gaa aca tca tat gca aca aaa aat aaa 1056Asn Phe His Phe Ser Asn Gln Glu Thr Ser Tyr Ala Thr Lys Asn Lys 340 345 350 aat ctc ttc ttt tat ttt att ttc atc ttc cct ttt ctt tat gtt att 1104Asn Leu Phe Phe Tyr Phe Ile Phe Ile Phe Pro Phe Leu Tyr Val Ile 355 360 365 ttg tta tta taa 1116Leu Leu Leu 370 11371PRTPlasmodium falciparum 11Met His Ile Val Ser Phe Ile Ile Phe Phe Phe Ala Leu Phe Phe Pro 1 5 10 15 Ile Ser Ile Cys Tyr Lys Ile Asn Gly Val Cys Asp Phe Ser Ser Glu 20 25 30 Gly Leu Ser Leu Leu Pro Glu Glu Lys Leu Asp Phe Ser Val Ser Arg 35 40 45 Asn Val Asp Lys Leu Ser Asp Glu Asn Asn Val Arg His Cys Val His 50 55 60 Phe Ser Lys Gly Phe Glu Tyr Leu Arg Phe Ile Cys Pro Met Arg Lys 65 70 75 80 Asp Asn Tyr Glu Gly Ile Glu Ile Arg Pro Val Glu Cys Phe Glu Tyr 85 90 95 Ile His Ile Glu Gly Arg Glu His Lys Leu Ser Glu Ile Leu Lys Gly 100 105 110 Ser Leu Tyr Glu Lys Ser Ile Asn Asp Asn Ile Met Thr Arg Asp Val 115 120 125 Phe Ile Pro Pro Thr Ile Tyr Glu Asp Met Phe Phe Glu Cys Thr Cys 130 135 140 Asp Asn Ser Leu Thr Phe Lys Asn Asn Met Ile Gly Ile Arg Gly Ile 145 150 155 160 Met Lys Ile His Leu Lys Lys Asn Ile Leu Tyr Gly Cys Asp Phe Asp 165 170 175 His Asp Glu Lys Leu Met Lys Asn Lys Thr Ala Phe Thr Asn Phe Tyr 180 185 190 Asp Lys Gln Lys Ile Leu Pro Leu Ile Gly Asn Asn Asn Asn Asp Asp 195 200 205 Asp Asn Asn Asp Asp Asp Asn Asn Asn Asp Asn Asn Asn Asn Asp Asn 210 215 220 Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn 225 230 235 240 Asn Asn Ile Thr Cys Asn Val Thr Ile Lys Lys Ser Gln Val Tyr Leu 245 250 255 Gly Ile Ile Cys Pro Asp Gly Tyr Thr Leu Tyr Pro Asn Asp Cys Phe 260 265 270 Lys Asn Val Ile Tyr Asp Asn Asn Ile Ile Ile Pro Leu Lys Lys Ile 275 280 285 Ile Pro His Asp Ile Leu Tyr His Gln Asp Lys Asn Lys Arg Ile Thr 290 295 300 Phe Ala Ser Phe Thr Leu Asn Ile Asn Glu Asn Pro Pro Gly Phe Thr 305 310 315 320 Cys Tyr Cys Ile Lys Asp Gln Thr Asn Ile Asn Asn Pro Leu Ile Val 325 330 335 Asn Phe His Phe Ser Asn Gln Glu Thr Ser Tyr Ala Thr Lys Asn Lys 340 345 350 Asn Leu Phe Phe Tyr Phe Ile Phe Ile Phe Pro Phe Leu Tyr Val Ile 355 360 365 Leu Leu Leu 370 1273PRTCricetulus migratorius 12Gln Arg Val Asp His His Phe Asn Glu Pro Val Thr Ile Ala Ile Ile 1 5 10 15 Leu Gly Met Ile Ala Gly Ile Val Gly Thr Ile Leu Leu Ile Tyr Tyr 20 25 30 Leu Ile Ser Leu Ile Thr Lys Lys Ile Ser Ala Asp Lys Gln Pro Pro 35 40 45 Lys Ser Glu Asn Thr Asp Glu Pro Pro Ser Pro Ile Glu Gln Ile Ile 50 55 60 Val Gln Glu Glu His Asp Ser Ile Val 65 70 13405DNAArtificial SequenceExpression vector 13gccgccacta tgaagttcct ggtgaatgtg gccttggtgt tcatggtggt gtacatcagc 60ttcatctacg ctagccttaa gggtaccgag ctcggatcca ctagtgaatt cgatatctct 120agagcggccg ctgactacaa agacgatgac gacaagcttt caccaattca acacgatttt 180cctgcactag tgatgatact cataattttg ggcgtgatgg cagggattat cggaactatc 240cttcttatct cttactgtat cagccgaatg acaaagaaaa gttcagttga catccaatct 300cctgagggtg gtgacaacag tgtgcctttg agttctattg agcagactcc taatgaagag 360tcctccaatg ttagcggcgg ccatcaccat caccatcact gataa 40514411DNAArtificial SequenceExpression vector 14gccgccacta tgaagttcct ggtgaatgtg gccttggtgt tcatggtggt gtacatcagc 60ttcatctacg ctagccttaa gggtaccgag ctcggatcca ctagtgaatt cgatatctct 120agagcggccg ctgactacaa agacgatgac gacaagcttc aaagagttga tcaccatttt 180aatgagccag tgactatagc cattattttg ggcatgatcg ctggtatcgt tggaactatc 240cttctcattt attacttaat cagcctaata acaaagaaaa tttcagctga caaacaacct 300cccaagagtg aaaacacgga tgagccacca agtcctattg aacagattat tgttcaagaa 360gagcatgaca gcattgttag cggcggccat caccatcacc atcactgata a 41115405DNAArtificial SequenceExpression vector 15gccgccacta tgaagttcct ggtgaatgtg gccttggtgt tcatggtggt gtacatcagc 60ttcatctacg ctagccttaa gggtaccgag ctcggatcca ctagtgaatt cgatatcgac 120gtcgcggccg ctgactacaa agacgatgac gacaagagcc ctagggccgc acacgatttt 180cctgcactag tgatgatact cataattttg ggcgtgatgg cagggattat cggaactatc 240cttcttatct cttactgtat cagccgaatg acaaagaaaa gttcagttga catccaatct 300cctgagggtg gtgacaacag tgtgcctttg agttctattg agcagactcc taatgaagag 360tcctccaatg ttagcggcgg ccatcaccat caccatcact gataa 405

Claims

1. A nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence comprising a sequence encoding a secretion signal peptide, a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence comprising a sequence encoding a transmembrane domain of glycophorin.

2. The nucleic acid vector of claim 1, wherein the transmembrane domain of glycophorin is the transmembrane domain of glycophorin A.

3. The nucleic acid vector of claim 1 or 2, wherein the transmembrane domain of glycophorin A is the mouse glycophorin A transmembrane domain or Armenian hamster glycophorin A domain.

4. The nucleic acid vector of claim 3, wherein the mouse glycophorin A domain comprises the amino acids disclosed in Seq. Id. No. 1 and the Armenian hamster glycophorin A domain comprises the amino acid sequence disclosed in Seq. Id. No. 12.

5. The nucleic acid vector of claims 1-4, wherein the secretion signal peptide is the secretion signal peptide of bee-venom melittin.

6. The nucleic acid vector of claim 5, wherein the secretion signal peptide of bee-venom melittin comprises the amino acid sequence disclosed in Seq. Id. No. 2.

7. The nucleic acid vector of claims 1-6, further comprising downstream (3') of the cloning site for inserting a polynucleotide sequence encoding a protein to be expressed a polynucleoide sequence encoding a FLAG tag comprising the amino acid sequence of Seq. Id. No. 3.

8. The nucleic acid vector of claims 1-7, further comprising downstream (3') of the polynucleotide sequence encoding the transmembrane domain of glycophorin a polynucleotide sequence encoding a His tag, preferably a His tag comprising the amino acid sequence disclosed in Seq. Id. No. 4.

9. The nucleic acid vector of claims 1-8, wherein the cloning site comprises the restriction enzyme cleavage sites of NheI, KpnI, BamHI, EcoRI, EcoRV and NotI.

10. The nucleic acid vector of claims 1-9 comprising a polynucleotide sequence selected from the group consisting of Seq. Id. No. 5, Seq. Id. No. 13, Seq. Id. No. 14 or Seq. Id. No. 15.

11. The nucleic acid vector of claims 1-10, wherein the protein to be expressed is a membrane associated protein.

12. A cell comprising the vector of claims 1 to 11, preferably a mammalian cell, more preferably a HEK cell.

13. Use of a cell of claim 12 for the expression of proteins suitable for antibody generation.

14. Use of a cell of claim 12 for the immunisation of a non-human animal for antibody generation, preferably monoclonal antibodies.

15. A method for the generation of monoclonal antibodies against a specific protein comprising the steps: a) immunisation of a non-human animal with cells expressing on its cell surfaces the specific protein using the vector of claims 1 to 11, b) isolating spleen cells of the non-human animals of step a), c) fusing the spleen cells of step b) with myeloma cells to generate B cell hybridomas and d) identification of B cell hybridomas expressing antibodies directed against the specific protein.

16. The method of claim 15, wherein the non-human animal is a mouse or hamster.

17. The invention as hereinbefore described.

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