Patent application title: RECOMBINANT VIRUS-LIKE PARTICLES ENCODED BY MULTI-GENE VECTOR
Inventors:
Corinne John (Horgen, CH)
Christian Schaub (Wadenswil, CH)
Sabine Wellnitz (Urdorf, CH)
IPC8 Class: AA61K39295FI
USPC Class:
4242021
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) combination of antigens from multiple viral species (e.g., multivalent viral vaccine, etc.)
Publication date: 2012-03-15
Patent application number: 20120064115
Abstract:
The invention describes novel virus-like particles for use as vaccines,
diagnostic tools and R&D tools based on recombinant DNA and cell
cultivation techniques for production. The recombinant virus-like
particles of the invention are assembled by polypeptide chains that
incorporate several, in particular two or more, different epitopes which
are selected either (a) from different viral strains of the same virus
and/or (b) from different serotypes of the same virus and/or (c) from
different viral strains specific for different hosts. These epitopes are
then displayed on the particle surface.Claims:
1. A recombinant virus-like particle comprising two or more different
epitopes or different proteins comprising epitopes which are selected
either (a) from different viral strains of the same virus and/or (b) from
different serotypes of the same virus and/or (c) from different viral
strains specific for different hosts.
2. The recombinant virus-like particle according to claim 1 comprising three or more different epitopes or different proteins comprising epitopes.
3. The recombinant virus-like particle according to claim 1 comprising four or more different epitopes or different proteins comprising epitopes.
4. The recombinant virus-like particle according to claim 1 comprising six, nine or twelve epitopes or different proteins comprising epitopes.
5. The recombinant virus-like particle according to claim 1 wherein the epitopes are from three or more different virus strains or serotypes.
6. The recombinant virus-like particle according to claim 1 further comprising B- and/or T-cell epitopes.
7. The recombinant virus-like particle according to claim 1 further comprising proteins forming a complete virus-like surface and optionally capsid and/or nucleopore proteins.
8. The recombinant virus-like particle according to claim 1 further comprising fluorescent proteins, proteins useful for purification purposes of the particles or for attaching a label, and/or proteinaceous structures required for transport processes.
9. The recombinant virus-like particle according to claim 1 wherein the virus is influenza virus.
10. The recombinant virus-like particle according to claim 1 wherein the virus is human papilloma virus.
11. A vector comprising two or more polynucleotides coding for different epitopes or for different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts.
12. The vector according to claim 11 encoding a recombinant virus-like particle comprising two or more different epitopes or different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts.
13. The vector according to claim 11 which is a baculoviral vector.
14. The vector according to claim 11 comprising a polynucleotide sequence selected from SEQ ID NO: 1 to 5.
15. A host cell comprising a vector according to claim 11.
16. A vaccine comprising a recombinant virus-like particle according claim 1.
Description:
FIELD OF THE INVENTION
[0001] The invention relates to recombinant virus-like particles comprising epitopes from different virus strains, and vectors encoding these.
BACKGROUND OF THE INVENTION
[0002] There is an increasing interest in small natural biomolecules using them in different aspects in biomedicine, nanotechnology and material science. Virus simulators, virus capsids or virus-like particles are very attractive because of their regular structure, their homogenous particle size, their stability, the ease of production and the potential for manipulation. Virus-like particle possess dynamic structures, their interior is accessible and furthermore the coat is modifiable. Dependent on the application the virus-like particles could have an envelope or not and could be chosen as virus simulators. These embodiments could be used as new biological entities or targets, as vaccines, as antigens for antibody production, as research tools, as diagnostic tool, for drug delivery and bioconjunctions. These virus simulators are formed by self-assembly of envelope and or capsid proteins of many viruses. The size varies between 22-150 nm dependent on the morphology of the particular virus. The virus simulators are non-infectious because they assemble without incorporating genetic material. Dependent on the application foreign genetic material could be included in the herein described virus simulator.
[0003] A promising application of these virus simulators is the production of vaccines against various diseases because their repetitive, high density display of epitopes elicit often a strong immune response. The small size of particles is an advantage for uptake by dendritic cells. Chimeric virus simulators offer an enormous potential in selective, multi-epitope, multi-protein, multi-serotype, multi-strain, or multi-species presentation.
[0004] There exist many expression systems for the production of virus simulators which include the baculovirus/insect cell system, various mammalian cell lines, either stably or transiently transfected or transduced with viral expression vectors, furthermore various species of yeast including Saccharomyces cerevisiae and Pichia pastoris, and Escherichia coli and other bacteria.
[0005] Vaccination is dependent on the generation of a suffient immunity to protect from infectious diseases. The mostly used attenuated virus vaccines rely on limited replication of the virus in the host following immunization. But this kind of vaccination may cause severe reactions in some patients. Therefore the development of virus-like particles (VLP) as subunit vaccines is an advantage because the particles lack in general DNA or RNA genome but have the authentic conformation of the natural virus.
[0006] Vaccination is one of the most potent and cost-effective counter-measures to the threat of e.g. seasonal or pandemic influenza outbreaks. The ease of spread as an aerosol and the cause for a severe illness especially to susceptible humans are the major reasons why influenza is one of the most devastating viral diseases. Currently licensed seasonal vaccines are only partially protective, and the egg-based production is very time-consuming and cost-intensive. This strategy is vulnerable to the unanticipated emergence of epidemic strains that are poorly matched or not matched at all by the vaccine. Due to the danger of emerging strains of avian influenza or influenza of other origin novel vaccine approaches are necessary.
[0007] In another aspect the research in the field of several important viruses like HCV, HIV, Ebola etc. is very difficult because of biosafety issues. Until now there exist only a few models for investigation of viral entry and viral trafficking. Diagnostic tools are based on the genome of these viruses because of the lack of appropriate non-infectious virus models.
[0008] Presently commercial human influenza vaccines contain hemagglutinin as their only or main viral antigen. Their production starts from viruses grown on embryonated chicken eggs or, more recently, in mammalian cells in tissue culture. The production in eggs requires selection of high yield, reassorted virus strains, is limited in capacity, time-consuming (6-8 months), and expensive. Beyond that it can cause problems in vaccinated persons allergic to egg protein. The production is only possible with non-lethal bird strains. One of the most important disadvantages of the egg-based production is the limited capacity. In case of a pandemic the production of the seasonal influenza vaccine has to be stopped in favour of a pandemic influenza vaccine production which could result in even more lethal events in the long run.
[0009] Vaccines against viral diseases rely traditionally on attenuated virus strains or inactivation of infectious virus. An appropriate environment is necessary for highly pathogenic or haemorraghic viruses which constrains the production possibilities because of the biosafety level (e.g. BL3/BL4). For some viruses like human papilloma virus the attenuation will not be sufficient because the virus cannot be propagated in vitro. The ability to generate human papilloma virus (HPV)--like particles based vaccines (Gardasil, Cervarix) has changed the prospects for preventing cervical cancer in woman.
[0010] Due to the danger of emerging strains of avian influenza or other origin, novel vaccine approaches are necessary which result in an enhanced protection.
SUMMARY OF THE INVENTION
[0011] The invention relates to a recombinant virus-like particle comprising two or more different epitopes or different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts. These recombinant virus-like particles are useful as vaccines, and the invention also relates to these vaccines.
[0012] Furthermore the invention relates to a vector comprising two or more polynucleotides coding for different epitopes or for different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts, and to host cells comprising these vectors.
BRIEF DESCRIPTION OF THE FIGURE
[0013] FIG. 1: Schematic representations of vector constructs expressing multiple-epitope virus-like particles.
[0014] (A) (SEQ ID NO:1) Multivalent influenza A virus simulator containing different epitopes (M1, M2) from H1N1 viral strains as well as H3N2 (HA, NA) viral strains
[0015] (B) (SEQ ID NO:2) Chimeric human papilloma virus-like particle containing an epitope (L1) from serotypes HPV16 and HPV18
[0016] (C) (SEQ ID NO:3) Expression vector construct with embedded epitopes (HA, NA, M1, M2) from influenza B/Florida isolates
[0017] (D) (SEQ ID NO:4) Vector construct for expression of an epitope (L1) of HPV16 and HPV18 serotypes where both genes are under the control of different promoters
[0018] (E) (SEQ ID NO:5) Same Vector construct as (B) with deletion of promoter p10
[0019] The vectors contain two promoters P1 and P2 (<,>) selected from polh, p10 and pXIV very late baculoviral promoters, vp39 baculoviral late promoter, vp39polh baculoviral late/very late hybrid promoter, pca/polh, pcna, etl, p35, egt, da26 baculoviral early promoters; CMV, SV40, UBc. EF-1, RSVLTR, MT, P.sub.DS47, Ac5, P.sub.GAL and PADH. The terminator sequences T1 and T2 (T) are selected from SV40, HSVtk or BGH (bovine growth hormone). Furthermore the vector contains the transposon sites TnL and TnR (L,R) for generation of MultiBacbacmid, a loxP site (LP) for site specific homologous recombination (plasmid fusion), an origin of replication (O), ampicillin (A) and gentamycine (G) resistance genes and defined restriction sites.
[0020] FIG. 2: Analysis of expressed chimeric influenza virus-like particles.
[0021] (A) The conformation of secreted (A, lane 2) as well as intracellular VLPs (prepared from SEQ ID NO:1, A, lane 1) is verified by immunoblotting using specific antibodies against the proteins HA (H3), NA (H3) and M2 (H1). Lane 3=ladder, protein sizes in kDa. The epitopes are co-localized, which means that they are assembled in one particle.
[0022] (B) Visualization of the chimeric Influenza virus-like particle (prepared from SEQ ID NO:3) by electron microscopy using negative staining with uranyl acetate. The spikes representing epitope HA are visible. The size of the particle is in the range of 50-80 nm.
[0023] FIG. 3: Chromatographic purification and analysis of secreted multi-epitope influenza virus-like particles prepared from SEQ ID NO:1.
[0024] (A) Chromatogram of gel filtration purification. The first peak (1) contains the virus-like particle (VLP). The other peaks (2-6) represent contaminant proteins.
[0025] (B) Coomassie-stained SDS-PAGE. The multiple epitopes of the virus-like particles are verified by analyzing different fractions from the 1st peak (1) representing the start (lane 1), middle (lane 2) and end (lane 3) part of the VLP containing peak. The ladder [kDa] is represented left of the first lane. Detection of epitopes is indicated by arrows.
[0026] (C) Western blot analysis according to Coomassie-stained gel using an anti-HA antibody.
[0027] FIG. 4: Functionality of nature-like influenza virus-like particles (VLP).
[0028] Twofold serial dilution series (1:2 to 1:2048) of the purified VLPs (prepared from SEQ ID NO:3) are analyzed by standard hemagglutination assay. 50 μL purified particle solution was coated onto 96-well plate incubated with red blood cells (erythrocytes). The influenza VLP (1, upper part) are able to agglutinate red blood cells in a dose dependent manner.
[0029] Highest dilution is 1:1024. In contrast PBS, used as control (C), leads only to precipitation of erythrocytes, visible as a "dot" in the middle of the well.
[0030] FIG. 5: In vivo evaluation of multi-epitope influenza virus-like particles.
[0031] Mice are immunized either with 50 ng (mice 1-5) or 100 ng (mice 6-10) purified VLP prepared from SEQ ID NO:3, and as control with PBS (mice 11-12). Antibody titers after prime injection (3 weeks post injection) are indicated as white boxes, titers after a boost injection are indicated as black boxes (6 weeks post injection). The titers are presented as dilution of mice sera (y-axis). VLPs effectively stimulate an antibody immune response. The best results are obtained when immunization is performed with 100 μl (mice 6-10), indicating a dose dependent immune response. A clear increase of the amount of anti-VLP antibodies is observed after boost. As expected, control animals (mice 11-12) showed no immune response.
[0032] FIG. 6: Confirmation of specific immune response to multi-epitope influenza VLP by hemagglutination inhibition assay.
[0033] The ELISA test was performed with sera taken at week 6 post injection to analyze the presence of specific anti-HA antibodies. Multi-epitope virus-like particles prepared from SEQ ID NO:3 were coated onto a 96-well plate, mixed with the sera and incubated for 30 min. The sera were tested in a series of twofold dilutions (1:2 to 1:1024). After incubation erythrocytes were added and incubated for further 30 min. Specific anti-Influenza-HA antibodies from different mice binding to multi-epitope virus-like particles result in inhibition of erythrocyte agglutination up to a dilution 1:128 (1) and dilution 1:256 (2), visible as erythrocyte precipitation ("dot") in the middle of the well. No hemagglutination inhibition is observed with sera sample of the control mouse (C).
[0034] FIG. 7: Screening of best expression conditions using 50 mL bioreactors.
[0035] The initial cell amount in the range of 1-2×106 cells/mL (TOI, 1 or 2), virus inoculum (MOI, 0.01-2) and time of harvest (days post infection, d2-d6) were determined by dot blot analysis.
[0036] (A) Determination of best expression parameters of an expression construct carrying only one epitope L1 which is used as control. Detection by a specific anti-HPV18 antibody (Abcam).
[0037] (B) Determination of best expression parameters of a multi-epitope expression construct carrying two epitopes from different serotypes (SEQ ID NO:2). Detection by specific anti-HPV16-(Camvir, Santa Cruz) and anti-HPV18-(Abcam) antibodies against the two epitopes HPV16 L1 and HPV18 L1.
[0038] FIG. 8: Chromatographic purification and analysis of multi-epitope human papilloma virus-like particles prepared from SEQ ID NO:2.
[0039] (A) Chromatogram of anion exchange chromtography using DEAE-sepharose column. Flow through (1), wash (2) and elution peaks (3-5) are indicated by numbers (1-5). The increased ionic strength of elution buffer is shown by a line [%]. 3=elution with 300 mM NaCl, 4=elution with 420 mM NaCl, 5=elution with 680 mM NaCl.
[0040] (B) Coomassie-stained SDS-PAGE. The presence of multiple epitopes of the virus-like particles were verified by analyzing different parts of the chromatogramm. Lane 1=ladder [kDa], lane 2=VLP before purification, lane 3=wash step, lane 4=elution with 300 mM NaCl, lane 5=elution with 420 mM NaCl, lane 6=elution with 500 mM NaCl, lane 7=elution with 680 mM NaCl. Epitopes are indicated by arrows (L1).
[0041] (C) Western blot analysis with coomassie-stained gel using specific antibodies against two epitopes which are indicated by arrows (L1).
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention aims at producing novel virus-like particles for use as vaccines, diagnostic tools and R&D tools based on recombinant DNA and cell cultivation techniques for production. Particles of the invention meet the demand for vaccines suitable to combat a potential pandemic influenza outbreak. The recombinant virus-like particles of the invention are assembled by polypeptide chains that incorporate several, in particular two or more, such as two, three, four or five, or also multiples of three, such as six, nine or twelve, different epitopes or different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts. These epitopes are then displayed on the particle surface. Selection of epitopes from different strains, serotypes and/or viruses specific for different hosts results in a multifunctional virus-like particle mimicking natural changes of viruses as they occur in nature, e.g. as observed in April 2009 during the outbreak of swine influenza. State of the art virus-like particles are either composed of a single protein or comprise up to three different epitopes derived from the same viral strain. The particle of the invention is encoded by a single DNA vector, either viral or plasmid based, which is used for the production in a host cell such as insect cells, bacterial cells and mammalian cells. In a preferred embodiment, the DNA vector is a baculovirus vector and the host cell an insect cell.
[0043] Epitopes of the invention are immunogenic peptides consisting of between 4 and 1000 amino acids, preferably between 6 and 100 amino acids, and are preferably neutralization epitopes. Neutralization epitopes are epitopes which, when bound by antibodies as the results of an immunogenic response, lead to neutralization of the virus carrying such a neutralizing epitope. Epitopes as understood herein may be repetitive, and may be part of a larger protein, in particular part of an antigen, part of a viral surface protein or part of a viral membrane protein. Such epitopes incorporated in viral surface proteins or viral membrane proteins are preferred. If the intended use of the virus-like particles according to the invention is as a R&D tool, diagnostics tool or a virus simulator it is important that the epitopes are part of complete viral proteins providing a complete virus-type surface.
[0044] Different viral strains of the invention are, for example, different strains of influenza virus, for example influenza virus A strains H1N1, H5N1, H9N1, H1N2, H2N2, H3N2 or H9N2, or also influenza virus B or influenza virus C.
[0045] Different serotypes of the invention are, for example, different serotypes of human papilloma virus (HPV), for example serotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 48, 52, 58 62, 66, 68, 70, 73 and 82, but also from the proto-oncogenic types HPV 5, 8, 14, 17, 20 and 47 or from papilloma relevant types HPV 6, 11, 13, 26, 28, 32 and 60.
[0046] Virus strains specific for different hosts means particularly adapted to the corresponding host, and are, for example, human influenza virus strains, swine influenza virus strains and avian influenza virus strains. In this context, specific for a host means that the virus is easily transmitted from one host to another host of the same type, but not to a different type of host. For example, an avian virus strain is easily transmitted from birds to other birds, but not to other animals or to humans.
[0047] In a preferred embodiment the particle comprising epitopes from different strains, serotypes and/or viruses specific for different hosts are combined with B- and/or T-cell epitopes in order to induce a broader immune response.
[0048] In another preferred embodiment the virus-like particle consists of proteins forming a complete virus-like surface, optionally further comprising capsid and nucleopore proteins. The virus-like particle of the invention may further comprise fluorescent proteins, proteins useful for purification purposes of the particles or for attaching a label, and proteinaceous structures required for transport processes and stability.
[0049] The herein described polypeptides and virus like particles are generated in a shorter time and in unlimited amounts compared to actual vaccine manufacturing processes, due to the use of specific genetic and process engineering tools. The capability to assemble the required viral genes by modern molecular biology methods, such as the MultiBac technology (WO 2005/085456; I. Berger et al., Nature Biotechnology 22, 1583, 2004), Polybac technology (WO 2007/054250), or gene synthesis, for instance, allows for fast assembly of the coding DNA vector. The use of these technologies does not require any physical transfer of original, potentially dangerous viruses during the development, manufacturing or administration of virus-like particles and vaccines of the invention. For the construction of particles of the invention it is sufficient to use nucleotide sequences from an infected individual. This stands in major contrast to classical egg-based methods for generating vaccines, which require genetically modified virus as a seed-strain virus. Particles of the invention are manufactured using modern disposable tissue culture techniques which allow for high production capacity. In the preferred embodiment of baculoviral vector and insect cells as host cells the manufacturing process can be quickly set-up, and production times are short, i.e. in the range of weeks rather than months compared to egg-based methods. Additionally, the construction of disposable tissue culture facilities is less time-consuming and costly compared to setting up an egg-based facility. As a consequence large amounts of vaccine for a full population can be produced and re-produced within short time frames, and several different types of vaccines, e.g. seasonal influenza vaccines and pandemic influenza vaccines, can easily be produced in parallel. Difficult decisions by health authorities for the one or the other vaccine due to capacity limits of egg-based vaccine manufacturing plants will not be required.
[0050] The invention relates to a recombinant virus-like particle comprising two or more, such as two, three, four or five, or also multiples of three, such as six, nine or twelve, different epitopes or different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts. Preferred are recombinant virus-like particle comprising three or more, preferably four or more different epitopes or different proteins comprising epitopes. Likewise preferred are recombinant virus-like particle comprising multiples of three, such as six, nine or twelve different epitopes or different proteins comprising epitopes. The epitopes are selected from two different strains, serotypes or virus strains specific for different hosts, or from three different strains, serotypes or virus strains specific for different hosts, or from four different strains or serotypes. Preferred are virus-like particles comprising several epitopes from three different strains or serotypes. Likewise preferred are virus-like particles comprising several epitopes from virus strains specific for two or three different hosts.
[0051] Furthermore the invention relates to a vector comprising two or more, such as two, three, four or five, or also multiples of three, such as six, nine or twelve, different polynucleotides coding for epitopes or for different proteins comprising epitopes which are selected either (a) from different viral strains of the same virus and/or (b) from different serotypes of the same virus and/or (c) from different viral strains specific for different hosts. "Polynucleotides" as used herein may represent a chain of between 12 and 3,000 nucleotides, includes oligonucleotides as commonly designated, and may be a viral gene or open reading frame from the mentioned different viral sources, in particular genes or open reading frames encoding viral surface proteins or viral membrane proteins.
[0052] Preferred are vectors coding for preferred virus-like particles mentioned hereinbefore.
[0053] Most preferred is a vector comprising a polynucleotide sequence selected from SEQ ID NO: 1 to 5.
[0054] In preferred embodiments a virus-like particle of the invention comprises
[0055] (1) the same type of a surface protein from two or three different strains or serotypes of the same virus;
[0056] (2) a mixture of more than two different surface proteins from different viral strains, e.g. from influenza virus strains H5N1 and H1N1;
[0057] (3) a mixture of different surface proteins combined from viruses specific for different hosts, e.g. from influenza virus specific for swine, human and/or avian hosts.
[0058] Viruses considered as source for epitopes to be comprised in virus-like particles of the invention are, for example, influenza virus, HPV, HIV, CMV, Dengue, HCV and Newcastle Disease Virus. Epitopes may be derived from other viruses and from bacteria. Particularly preferred is influenza virus. Equally preferred is human papilloma virus (HPV).
[0059] Vectors considered are DNA vectors, and can either be a plasmid vector or a viral vector. Methods to assemble such vectors are standard methods of state of the art molecular biology. Preferred methods are MultiBac such as described in WO 2005/085456 and in I. Berger et al., Nature Biotechnology 22, 1583, 2004, or Polybac methods such as described in WO 2007/054250, combined with CAP® technology and state of the art gene synthesis technologies. These technologies allow assembling a multi-gene co-expression DNA vector which is suitable for expression in different host cells. The preferred DNA vector of the invention is a baculoviral vector.
[0060] The host cell used for the expression of vectors of the invention can be any prokaryotic (e.g. E. coli) or eukaryotic expression cell line. For expression of a preferred baculoviral vector an insect cell line is preferred. Examples of insect cell lines are, e.g., SF9, SF21, Hi-5, Express Sf+, and S2 Schneider cells. For expression in a eukaryotic system, mammalian cells are preferred, in particular human cells, e.g. HeLa, Huh7, HEK293, HepG2, BHK, CHO, MT-2, bone-marrow fibroblasts, primary neural cells, or embryonic cells. For expression in yeast S. cerevisiae, S. pombe, C. albicans, or P. pastoris cells may be used.
[0061] Cultivation and propagation of host cells according to the invention can be done in any vessel, bioreactor or disposable unit providing the appropriate conditions for the particular host cell.
[0062] The virus-like particles of the invention can be used as vaccines. Furthermore they may be used as antigens in diagnostic tools, antigens for antibody generation, and as virus simulators for research and development tools, e.g. viral entry studies and virus-host interaction studies.
[0063] Vaccines according to the invention contain the recombinant virus-like particle in aqueous solution optionally further containing viscosity-regulating compounds, stabilizing compounds and/or adjuvants increasing the immunogenicity, as is known in the state of the art.
[0064] In a particular embodiment H3N2 Influenza virus-like particles are constructed using the methods of Berger et al., Nature Biotechnology, 2004, WO2005/085456 and WO 2007/054250 and CAP® technology. At least one M1 and M2 gene of the H1N1 Influenza strain A/Puerto Rico/834 is cloned by PCR amplification into the transfer vector pFL (WO 2007/054250, FIG. 1) together with the HA gene of influenza A/Brisbane10/2007 and the NA gene of influenza A/Brisbane10/2007. The construct is confirmed by DNA sequencing.
[0065] In another particular embodiment the same cloning techniques are used to introduce at least one L1 gene of both serotypes HPV16 and HPV18 or further serotypes from the group 2, 4, 6, 11, 31, 33-35, 39, 40-45, 51-53, 55-59, 62, 66, 68, 70, 73, and 77, cloned into the transfer vector pFL (FIG. 1).
Experimental Part
[0066] The baculoviral vectors termed MultiBac or YFPMultiBac (WO 2005/085456), which allow propagation in E.coli cells, and CAP® technology are used for generation of recombinant AcNPVs (Autographa californica nuclear polyhedrosis virus, a baculovirus) using a conventional system (Fitzgerald et al., Nature Methods, 3, 1021, 2006). 10 ng of the multi-gene vector is transformed in DH10MultiBac and/or DH10YFPMuItiBac competent cells. Positive clones are selected by blue/white screening and PCR. The corresponding MultiBac bacmid DNA is isolated using the Birnboim & Doly method. Recombinant AcNPVs are generated by transfection of 1 μg of multi-gene MultiBac bacmid in 0.9×106 Sf21 (Invitrogen) cells using transfection reagent Fugene (Roche) according to the manufacturers protocol. Virus amplification is done as described previously (Fitzgerald et al., Nature Methods, 3, 1021, 2006). The titer of all recombinant AcNPVs is determined by plaque assay described in the Bac-to-Bac-Manual (Invitrogen). Protein production parameters like multiplicity of infection (MOI), cell number (TOI) and time of harvest (TOH) are analyzed with small scale expression studies.
EXAMPLE 1
Generation of Expression Vector Construct
[0067] To manufacture various constructs the multiplication module M present in the transfer vector pFL (WO 2005/085456 and CAP® technology) is used according to the described method in WO 2005/085456. DNA of epitopes are either obtained by isolation of viral RNA from original virus followed by reverse transcription combined with PCR (for the influenza virus epitopes) or by gene synthesis (provided by the company Geneart). The reverse transcription is performed using the RevertAid® H Minus First strand cDNA Synthesis kit (Fermentas) according to the manufactures protocol. The cDNA (2 μL) is used as template for PCR reaction. The following conditions are used based on the manufacturer's protocol. For a 50 μL total volume reaction 0.2 mM dNTP (NEB), 1.2% DMSO, 0.5 μM reverse and forward primer (Microsynth), 10 μl 5×Phusion GC reaction buffer and 2U Phusion Hot Start Polymerase (Finnzyme) are used. For multigene assembly the appropriate restriction sites (BstZ17I, Spel, Pmel, AvrII) are introduced using PCR. The PCR fragments are cut with the restriction enzymes followed by ligation and transformation processes to integrate the multiplication module into the transfer vector. The ligation is done over night at 4° C. using 500 ng linearized transfer vector (pFL), 4 μL PCR product and 1U T4-DNA-ligase (Fermentas). To generate the plasmid 4 μL ligation solution are added to 50 μl competent DH5α cells and incubated for 30 min on ice. After a heat shock at 42° C. for 30 sec and a 2 min cold shock at 4° C., 200 μl LB medium is added and incubated for 1 h at 37° C. and 220 rpm. Afterwards 80 μl of the cell suspension is plated on LB agar plates containing the appropriate antibiotics, in this case 100 μg ampicillin and 100 μg gentamycin. The whole procedure is repeated until all epitopes are introduced into the transfer vector.
[0068] Influenza epitopes are selected from the genes HA and NA, both chosen from a H3N2/Brisbane10/2007 strain whereas epitopes from M1 and M2 are chosen from H1N1/Puerto Rico/834 strain. The M1 epitope is controlled by the promoter p10, all other epitopes are controlled by the polyhedrin promoter polh. All epitopes are present on the same vector construct (FIG. 1A, SEQ ID NO:1). Influenza B/Florida/2006 isolates are chosen to generate a construct with multiple epitopes from the genes HA, NA, M1 and M2 (FIG. 10, SEQ ID NO:3). Human papillomavirus epitopes are selected from the gene L1 from the cancer relevant serotypes HPV16 and HPV18 and are unified in one vector construct. Both epitopes are controlled by the polyhedrin promoter polh (FIG. 1B, SEQ ID NO:2). To improve the expression yield the p10 promoter is deleted in a further construct (FIG. 1E, SEC ID NO:5). In another construct HPV16 epitope is controlled by p10 promoter whereas the polyhedrin promoter polh is chosen for the HPV18 epitope (FIG. 1D, SEQ ID NO:4).
EXAMPLE 2
Generation of Recombinant Baculovirus
[0069] This virus contains multiple epitopes to generate virus-like particles or virus simulators which present these epitopes on their surface. The virus-like particles can be used for different applications, e.g. as vaccines in the influenza field. The AcNPV-derived baculovirus contains multiple different epitopes from the viral strains recommended by WHO for the 2008/2009-VLP vaccination campaign. All genes of the transfer vector are transposed by site specific homologous recombination into MultiBac cells according to the protocol of WO 2005/085456.
[0070] 10 ng transfer vector are added to 100 μl MultiBac competent cells and incubated for 30 min at 4° C. After a heat shock at 42° C. for 45 sec and a 2 min cold shock at 4° C. 400 μl LB medium is added and the cell solution is incubated for 4 h at 37° C. and 220 rpm. Different dilutions are plated on appropriate LB agar plates containing various antibiotic resistances. Based on blue/white and PCR screening several correct MultiBac clones are selected. The corresponding MultiBac bacmid DNA is isolated using the Birnboim & Doly method. At least four MultiBac bacmid clones are selected for initial transfection of insect cells like Sf9 or Sf21 to generate the recombinant AcNPV-derived baculovirus. This is generated by transfection of 1 μg of multi-gene MultiBac bacmid in 0.9×106 Sf21 (Invitrogen) cells using transfection reagent Fugene (Roche) according to the manufacturer's protocol. Virus amplification is done as described previously (Fitzgerald et al., Nature Methods, 3, 1021, 2006; Bac-to-Bac-Manual, Invitrogen). The virus is amplified to expand the volume and increase the infectious titer which is determined by plaque assay according to Bac-to-Bac-Manual (Invitrogen). The best expression construct is determined by 50 mL small scale expression experiments followed by determination of protein yield by Bradford Assay (ADV, Cytosceleton). Expression of best expressor is further verified by Western blot analysis with antibodies against the multiple different epitopes (FIG. 2A).
EXAMPLE 3
Production and Purification of Multi-Epitope Influenza Virus-Like Particles in Insect Cells
[0071] After determination of the best expression construct the biotechnological production parameters like cell line, cell amount (TOI), amount of recombinant virus inoculum (multiplicity of infection, MOI) and time of harvest (TOH) are determined in 50 mL bioreactors. A matrix of different TOI, MOI and TOH are designed according to Eibl, Riesen and John (Bioforum 03/2009) and Friesen J. (Bachelor thesis, University of Applied Science, Esslingen, Germany). The expression of secreted or intracellular multi-epitope virus-like particles is observed for six to eight days with daily sample taking. For intracellular particles (e.g. HPV) the cell pellets are lysed with 50 mM TrisCl, pH 7.6, 100 mM NaCl, 0.1%TritonX100 and centrifuged for 10 min, at 4° C. and 8000×g. The epitopes of the virus-like particles present in the supernatant are further verified using a Dot-blot apparatus (Biometra) followed by Western blotting with specific antibodies. Conditions resulting in the highest yield are defined as expression parameters preferring a harvest time between three and four days. According to these defined parameters the virus-like particles are produced either in shaker flasks or wave cultibags in fall armyworm Spodoptera frugiperda cells Sf9 and Sf21. For multi-epitope influenza virus-like particles expression Sf21 cells are chosen with the following conditions: 1.5×106cells/mL, MOI 0.05 and harvest time at day four post infection. Cells are propagated at 27° C. without carbon dioxide and fetal calf serum supplementation. According to defined time of harvest the secreted virus-like particles are collected by centrifugation at 500-1000×g for 20 min at 4° C. The supernatant volume containing the particles is reduced for purification by tangential flow filtration using cassettes (Sartocon-Slice 200, Sartorius and CentramateOS, PALL) with a cut-off of 100 kDa. The purification of virus-like particles is performed with scalable chromatographic methods and sucrose gradient ultracentrifugation.
[0072] The chromatographic purification is a multi-step purification using cation exchange, anion exchange and gel filtration chromatography. The supernatant is loaded onto a CaptoQ column connected to an FPLC-system (AEKTA purifier, GE Healthcare) in 50 mM phosphate buffer, pH 7.4. The particles are eluted with increasing salt concentrations in a linear gradient using 50 mM phosphate, 1 M NaCl, pH 7.4. The particle containing fractions are pooled and further purified by gel filtration chromatography (VLP from SEQ ID NO:1, FIG. 3). The purification is performed in 50 mM phosphate, 150 mM NaCl, pH 7.4 buffer using a HighLoad Superdex 200 pg column. All chromatography steps are analyzed by SDS-PAGE followed by coomassie staining and immunoblotting.
EXAMPLE 4
Analysis of Purified Influenza Virus-Like Particles
[0073] To confirm the presence of the different epitopes purified material is analyzed by SDS-PAGE followed by coomassie staining or Western blot. 150 μl of different chromatography fractions are loaded on a 4-12% Bis-Tris NuPAGE gel (Invitrogen), run for 15 min at 150 V and for 45 min at 175 V and coomassie stained using SimplyBlueSafestain (Invitrogen). For immunoblotting the proteins are transferred onto a nitrocellulose membrane (BioRAD) at 19 V for 40 min using a semi-dry apparatus (BioRAD). After blocking unspecific binding sites for 30 min with 5% non-fat-dry-milk-TrisCl-Tween20 (0.1%) solution, the membrane is incubated over night at 4° C. with antibodies against HA, NA and matrix proteins. The membrane is washed several times with TrisCl-Tween20 (0.1%) buffer. Dependent on the source of primary antibody the second antibody is either an anti-mouse or anti-rabbit connected with alkaline phosphatase or horse-radish-peroxidase for detection. A co-localization of these proteins show the assembly and the production derived from one expression vector and one baculovirus (FIG. 3B, from SEQ ID NO:1). This co-localization can also be shown for the expression constructs containing the genes HA, NA and both matrix proteins M1 and M2 including their membrane anchors.
EXAMPLE 5
Functionality of Influenza Virus-Like Particles (VLP)
[0074] To analyze if the VLPs correctly integrate the hemagglutinin protein (HA) in their surface, a standard hemagglutination assay using red blood cells (RBC) from chicken is performed (FIG. 4, VLP from SEQ ID NO:3). Twofold serial dilutions of the purified VLPs are carried out with PBS (1×) in V-formed 96 well plates. An equal amount of erythrocytes (1% solution) is added and incubated for 1 h at 4° C. The appearance of RBC aggregates on the bottom of the well indicates lack of hemagglutination. Titers are expressed as the inverse of the highest dilution of the purified VLP solution to agglutinate RBCs. The results obtained show that VLPs are able to agglutinate chicken erythrocytes and demonstrate indirectly the presence of HA on the VLP surface. The negative control (PBS) shows no agglutination.
EXAMPLE 6
In vivo Evaluation of Influenza VLP
[0075] The immunogenicity (stimulation of immune system) of VLPs (prepared from SEQ ID NO:3) is tested in vivo using two groups of mice which are subcutaneously immunized in a prime boost schedule at week 0 and week 3 respectively. Immunization is performed using 50 or 100 μl (50 or 100 ng) of VLPs in suspension. To determine the quality of the immune response of the VLPs alone, no adjuvant is used. Mice are bled at week 3 and 6 and sera are analyzed to look for antibody responses against the immunized VLPs. Results obtained show that VLPs are effective at stimulating an antibody immune response. The best results are obtained when immunization is performed with 100 μl, indicating a dose dependent immune response. A clear increase of the amount of anti-VLP antibodies is observed after boost. As expected, naive animals show no immune response.
[0076] To reconfirm the specificity of the immune response, a hemagglutination inhibition test is performed at week 6 to analyze the presence of specific anti-HA antibodies. The results show that specific anti-Influenza-HA antibodies are able to inhibit erythrocyte agglutination up to a dilution 128 (mouse 6) and a dilution 256 (mouse 8). No hemagglutination inhibition is observed with sera sample of the naive mouse. Newly generated multi-epitope influenza-VLPs are able to stimulate the immune system in a dose dependent manner. When the immune system is re-stimulated with a boost, the immune response increases at least 15 times. The specificity of the elicited immune response is analyzed by ELISA and a hemagglutination test.
EXAMPLE 7
Production and Purification of Multi-Epitope Virus-Like Particles Carrying Human Papillomavirus Epitopes in Various Cell Lines
[0077] The biotechnological production parameters like cell line, cell amount (TOI), amount of recombinant virus inoculum (multiplicity of infection, MOD and time of harvest (TOH) are determined in 50 mL bioreactors (FIG. 7) according to Eibl et al. (Bioforum 3/2009). According to these defined parameters the virus-like particles are produced either in shaker flasks or wave cultibags in fall armyworm Spodoptera frugiperda cells Sf9 and Sf21. Multiple-epitope papilloma virus-like particles are expressed from SEQ ID NO:2 in Sf21cells using 2×106cells/mL, MOI 0.5 and harvest time at day three post infection. Cells are propagated at 27° C. without carbon dioxide and fetal calf serum supplementation. At defined harvest time (three days post infection) intracellular virus-like particles are collected by centrifugation at 500-1000×g for 20 min at 4° C. The cells are lysed using hypotonic phosphate buffers followed by ultrasonication. After a centrifugation step at 4° C. and 2000×g the supernatant was collected. The purification of virus-like particles is performed with scalable chromatographic methods and sucrose gradient ultra-centrifugation. The chromatographic purification is a multi-step purification using cation exchange, anion exchange and gel filtration chromatography. The supernatant is loaded onto a CaptoDEAE column connected to an FPLC-system (AEKTA purifier, GE Healthcare) in 50 mM phosphate buffer, pH 7.4. The particles are eluted with increasing salt concentrations in a linear gradient using 50 mM phosphate, 1 M NaCl, pH 7.4 (FIG. 8A). The particle containing fractions are pooled and further purified using a hydroxyapatite column. Binding is performed in 20 mM phosphate buffer, pH 7.0 followed by linear gradient elution with 500 mM phosphate, 150 mM NaCl, pH 7.0. To polish the multi-epitope particles a gel filtration chromatography is done. The purification is performed in 50 mM phosphate, 150 mM NaCl, pH 7.4 buffer using a HighLoad Superdex 200 pg column. All chromatography steps are analyzed by SDS-PAGE followed by coomassie staining and immunoblotting.
EXAMPLE 8
Analysis of Purified Chimeric Human Papilloma Virus-Like Particles
[0078] To confirm the presence of the different epitopes in purified material, the VLP prepared from SEQ ID NO:2 is analyzed by SDS-PAGE followed by coomassie staining (FIG. 8B) and Western blot (FIG. 8C). For immunoblotting antibodies against L1 protein of different serotypes are used. 150 pl of different chromatography fractions are loaded on a 4-12% Bis-Tris NuPAGE gel (Invitrogen), run for 15 min at 150 V and a for 45 min at 175 V and coomassie stained using SimplyBlueSafestain (Invitrogen). For immunoblotting the proteins are transferred onto a nitrocellulose membrane (BioRAD) at 19 V for 40 min using a semi-dry apparatus (BioRAD). After blocking unspecific binding sites for 30 min with 5% non-fat-dry-milk-TrisCl-Tween20 (0.1%) solution, the membrane is incubated over night at 4° C. with antibodies against L1 epitopes. Camvir antibody (SantaCruz) is used for HPV16 and anti-HPV18ab (Abcam) for HPV18 detection. The membrane is washed several times with TrisCl-Tween20 (0.1%) buffer. The membrane is incubated for 1 h with an anti-mouse antibody connected with alkaline phosphatase for detection. The epitopes are detected by BCIP/NPT solution. A co-localization of these proteins show the assembly and production derived from one expression vector and one baculovirus.
EXAMPLE 9
Functionality of Chimeric Human Papilloma Virus-Like Particles (VLP)
[0079] To analyze if the human papilloma virus-like particles prepared from SEQ ID NO:2 correctly integrate the L1 protein in their surface, a standard ELISA assay is performed. Twofold serial dilutions of the purified VLPs are carried out with PBS (1×) in V-formed 96 well plates. An equal amount of a serotype specific antibody (concentration 1:1000) is added and incubated for 1 h at 37° C. Appropriate binding of the antibody to the L1 protein is detected using a second antibody with a horse-radish peroxidase and a chemoluminescent detection system. The results obtained show binding of antibodies to the recombinant expressed epitopes in a dose dependent manner. The negative control (PBS) showed no binding.
Sequence CWU
1
5110482DNAArtificialmulti-epitope influenza A virus-like particle
1ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
360ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga
420acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
480gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
540ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
600tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
660ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
720ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
780aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
840gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
900ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
960gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
1020cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
1080cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
1140acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
1200caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
1260taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
1320ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
1380aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
1440agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
1500atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
1560tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
1620tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
1680gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
1740taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
1800aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
1860ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
1920catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
1980ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
2040ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
2100agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
2160taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
2220atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
2280cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
2340ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
2400accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
2460gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
2520tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg
2580agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
2640caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
2700aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
2760cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg
2820gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc
2880gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac
2940ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc
3000ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag
3060cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat
3120ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc
3180ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta
3240atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag
3300atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
3360gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
3420gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg
3480cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga
3540aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
3600agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc
3660gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg
3720aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg
3780cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc
3840aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag
3900tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct
3960atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca
4020acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta
4080tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg
4140agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg
4200tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc
4260ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt
4320catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct
4380cccggtaccg catgctatgc atgcggccgc tcacttgaac cgttgcatct gcacccccat
4440tcgtttctga taggcctgca aattttcaag aagatcattt ttcagaccag cactggagct
4500aggatgagtc ccaatggttc tcatcgcttg caccatttgc ctagcctgac tagcaacctc
4560catggcctct gctgcttgct cactcgatcc agccatttgc tccatagcct tagctgtagt
4620gctggctaaa accattctgt tctcatgtct gattagtgga ttggttgttg tcaccatttg
4680cctatgagac cgatgctggg agtcagcaat ctgttcacag gttgcacata ccaggccaaa
4740tgccacttca gtggtcacag cccccatcct gttgtatatg aggcccatac aactggcaag
4800tgcaccagca gaataactga gtgagatttc tttggcccca tggaatgtta tctccctctt
4860gagcttccta tacagtttaa ctgctttgtc catgttattt ggatccccgt tcccattaag
4920ggcagtttgg acaaagcgtc tacgctgcag tcctcgctca ctgggcacgg tgagcgtgaa
4980cacaaatcct aaaatcccct tagtcagagg tgacaggatt ggtcttgtct ttagccattc
5040catgagaacc tcaagatcgg tgttcttccc tgcaaagaca tcttcaagtc tctgtgcgat
5100ctcggctttg agggggcctg acgggatgat agagagaacg tacgtttcga cctcggttag
5160aagactcatg gtggatccat cccgggtgat caagtcttcg tcgagtgatt gtaaataaaa
5220tgtaatttac agtatagtat tttaattaat atacaaatga tttgataata attcttattt
5280aactataata tattgtgttg ggttgaatta aaggtccgta tactagtatc gattcgcgac
5340ctactccgga atattaatag atcatggaga taattaaaat gataaccatc tcgcaaataa
5400ataagtattt tactgttttc gtaacagttt tgtaataaaa aaacctataa atattccgga
5460ttattcatac cgtcccacca tcgggcgcgg atccaccatg aatccaaatc aaaagataat
5520aacgattggc tctgtttctc tcaccatttc cacaatatgc ttcttcatgc aaattgccat
5580cttgataact actgtaacat tgcatttcaa gcaatatgaa ttcaactccc ccccaaacaa
5640ccaagtgatg ctgtgtgaac caacaataat agaaagaaac ataacagaga tagtgtatct
5700gaccaacacc accatagaga aggaaatatg ccccaaacta gcagaataca gaaattggtc
5760aaagccgcaa tgtgacatta caggatttgc acctttttct aaggacaatt cgattaggct
5820ttccgctggt ggggacatct gggtgacaag agaaccttat gtgtcatgcg atcctgacaa
5880gtgttatcaa tttgcccttg gacagggaac aacactaaac aacgtgcatt caaatgacac
5940agtacgtgat aggacccctt atcggaccct attgatgaat gagttaggtg ttccttttca
6000tctggggacc aagcaagtgt gcatagcatg gtccagctca agttgtcacg atggaaaagc
6060atggctgcat gtttgtataa cgggggatga taaaaatgca actgctagct tcatttacaa
6120tgggaggctt gtagatagta ttgtttcatg gtccaaagaa atcctcagga cccaggagtc
6180agaatgcgtt tgtatcaatg gaacttgtac agtagtaatg actgatggga gtgcttcagg
6240aaaagctgat actaaaatac tattcattga ggaggggaaa atcgttcata ctagcacatt
6300gtcaggaagt gctcagcatg tcgaggagtg ctcctgctat cctcgatatc ctggtgtcag
6360atgtgtctgc agagacaact ggaaaggctc caataggccc atcgtagata taaacataaa
6420ggatcatagc actgtttcca gttatgtgtg ttcaggactt gttggagaca cacccagaaa
6480aaacgacagc tccagcagta gccattgttt ggatcctaac aatgaagaag gtggtcatgg
6540agtgaaaggc tgggcctttg atgatggaaa tgacgtgtgg atgggaagaa cgatcagcga
6600gaagtcgcgc ttagggtatg aaaccttcaa agtcattgaa ggctggtcca accctaagtc
6660caaattgcag ataaataggc aagtcatagt tgacagaggt aataggtccg gttattctgg
6720tattttctct gttgaaggca aaagctgcat caatcggtgc ttttatgtgg agttgataag
6780gggaagaaaa gaggaaactg aagtcttgtg gacctcaaac agtattgttg tgttttgtgg
6840cacctcaggt acatatggaa caggctcatg gcctgatggg gcggacatca atctcatgcc
6900tatataagta ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
6960taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
7020ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
7080caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
7140cttatcatgt ctggatctga tcactgcttg agcctagaag atccggctgc taacaaagcc
7200cgaaaggaag ctgagttggc tgctgtggct agcttatcta gaaatattaa tagatcatgg
7260agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt ttcgtaacag
7320ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca ccatcgggcg
7380caccatgagt cttctaaccg aggtcgaaac gcctatcaga aacgaatggg ggtgcagatg
7440caacggttca agtgatcctc tcactattgc cgcaaatatc attgggatct tgcacttgac
7500attgtggatt cttgatcgtc tttttttcaa atgcatttac cgtcgcttta aatacggact
7560gaaaggaggg ccttctacgg aaggagtgcc aaagtctatg agggaagaat atcgaaagga
7620acagcagagt gctgtggatg ctgacgatgg tcattttgtc agcatagagc tggagtaagt
7680actagaggat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
7740tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
7800ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
7860catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
7920tctggatctg atcactgctt gagcctagaa gatccggctg ctaacaaagc ccgaaaggaa
7980gctgagttgg ctgctgccac cgctgagcaa taactatcat aaccggaata ttaatagatc
8040atggagataa ttaaaatgat aaccatctcg caaataaata agtattttac tgttttcgta
8100acagttttgt aataaaaaaa cctataaata ttccggatta ttcataccgt cccaccatcg
8160ggcgcggatc caccatgaag actatcattg ctttgagcta cattctatgt ctggttttca
8220ctcaaaaact tcccggaaat gacaacagca cggcaacgct gtgccttggg caccatgcag
8280taccaaacgg aacgatagtg aaaacaatca cgaatgacca aattgaagtt actaatgcta
8340ctgagctggt tcagagttcc tcaacaggtg aaatatgcga cagtcctcat cagatccttg
8400atggagaaaa ctgcacacta atagatgctc tattgggaga ccctcagtgt gatggcttcc
8460aaaataagaa atgggacctt tttgttgaac gcagcaaagc ctacagcaac tgttaccctt
8520atgatgtgcc ggattatgcc tcccttaggt cactagttgc ctcatccggc acactggagt
8580ttaacaatga aagcttcaat tggactggag tcactcaaaa cggaacaagc tctgcttgca
8640taaggagatc taataacagt ttctttagta gattgaattg gttgacccac ttaaaattca
8700aatacccagc attgaacgtg actatgccaa acaatgaaaa atttgacaaa ttgtacattt
8760ggggggttca ccacccgggt acggacaatg accaaatctt cccgtatgct caagcatcag
8820gaagaatcac agtctctacc aaaagaagcc aacaaactgt aatcccgaat atcggatcta
8880gacccagagt aaggaatatc cccagcagaa taagcatcta ttggacaata gtaaaaccgg
8940gagacatact tttgattaac agcacaggga atctaattgc tcctaggggt tacttcaaaa
9000tacgaagtgg gaaaagctca ataatgagat cagatgcacc cattggcaaa tgcaattctg
9060aatgcatcac tccaaacgga agcattccca atgacaaacc attccaaaat gtaaacagga
9120tcacatacgg ggcctgtccc agatatgtta agcaaaacac tctgaaattg gcaacaggga
9180tgcgaaatgt accagagaaa caaactagag gcatatttgg cgcaatcgcg ggtttcatag
9240aaaatggttg ggagggaatg gtggatggtt ggtacggttt caggcatcaa aattctgagg
9300gaataggaca agcagcagat ctcaaaagca ctcaagcagc aatcgatcaa atcaatggga
9360agctgaatag gttgatcggg aaaaccaacg agaaattcca tcagattgaa aaagaattct
9420cagaagtcga agggagaatt caggaccttg agaaatatgt tgaggacacc aaaatagatc
9480tctggtcata caacgcggag cttcttgttg ccctggagaa ccaacataca attgatctaa
9540ctgactcaga aatgaacaaa ctgtttgaaa aaacaaagaa gcaactgagg gaaaatgctg
9600aggatatggg caatggttgt ttcaaaatat accacaaatg tgacaatgcc tgcataggat
9660caatcagaaa tggaacttat gaccacgatg tatacagaga tgaagcatta aacaaccggt
9720tccagatcaa gggcgttgag ctgaagtcag gatacaaaga ttggatccta tggatttcct
9780ttgccatatc atgttttttg ctttgtgttg ctttgttggg gttcatcatg tgggcctgcc
9840aaaaaggcaa cattaggtgc aacatttgca tttgagtact agaggatcat aatcagccat
9900accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
9960aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
10020aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
10080tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgatc actgcttgag
10140cctagaagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
10200tgagcaataa ctatcataac ccctaggaga tccgaaccag ataagtgaaa tctagttcca
10260aactattttg tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct
10320attttgtcac tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc
10380aactattttg tccgcccaca gcggggcatt tttcttcctg ttatgttttt aatcaaacat
10440cctgccaact ccatgtgaca aaccgtcatc ttcggctact tt
1048228917DNAArtificialmulti-epitope human papilloma virus-like
particle 2ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt
ttgtaattga 60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct
gtagcggcgc 120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
ccagcgccct 180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
gctttccccg 240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
ggcacctcga 300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
gatagacggt 360ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt
ccaaactgga 420acaacactca accctatctc ggtctattct tttgatttat aagggatttt
gccgatttcg 480gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
taacaaaata 540ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
cctatttgtt 600tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
tgataaatgc 660ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
gcccttattc 720ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa 780aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
ctcaacagcg 840gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
acttttaaag 900ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
ctcggtcgcc 960gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
aagcatctta 1020cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg 1080cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
tttttgcaca 1140acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
gaagccatac 1200caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
cgcaaactat 1260taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
atggaggcgg 1320ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
attgctgata 1380aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
ccagatggta 1440agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
gatgaacgaa 1500atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
tcagaccaag 1560tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
aggatctagg 1620tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
tcgttccact 1680gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
tttctgcgcg 1740taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
ttgccggatc 1800aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata 1860ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
gcaccgccta 1920catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc 1980ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
ggctgaacgg 2040ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
agatacctac 2100agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg 2160taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
aacgcctggt 2220atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
ttgtgatgct 2280cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
cggttcctgg 2340ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
tctgtggata 2400accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
accgagcgca 2460gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
cttacgcatc 2520tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg
gttacggttg 2580agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc
ttaaactgaa 2640caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt
tgtaaactga 2700aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct
aaagcaaact 2760cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc
caagggcatg 2820gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg
ccgggaagcc 2880gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
agtgcatcac 2940ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
cgtaatctgc 3000ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
gattgatgag 3060cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
agatatagat 3120ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
caacaaccgc 3180ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
tcccgaggta 3240atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
gaccgaaaag 3300atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
tgcgaatgat 3360gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
taacatcgtt 3420gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg
gcgtaacgcg 3480cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
caagccatga 3540aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
cagttgcgtg 3600agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
aactgggttc 3660gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc
agcgaagtcg 3720aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg
catcgtcagg 3780cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg
cccttgcttc 3840aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc
ccggatgaag 3900tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag
gactctagct 3960atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa
ctgctgatca 4020acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta
tacgaagtta 4080tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac
gttggctgcg 4140agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga
aacgcgggcg 4200tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg
ggaccgaacc 4260ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt
ttattgccgt 4320catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc
tcccccatct 4380cccggtaccg catgctatgc atatcccggg tgatcaagtc ttcgtcgagt
gattgtaaat 4440aaaatgtaat ttacagtata gtattttaat taatatacaa atgatttgat
aataattctt 4500atttaactat aatatattgt gttgggttga attaaaggtc cgtatactag
tatcgattcg 4560cgacctactc cggaatatta atagatcatg gagataatta aaatgataac
catctcgcaa 4620ataaataagt attttactgt tttcgtaaca gttttgtaat aaaaaaacct
ataaatattc 4680cggattattc ataccgtccc accatcgggc gcggatccgc caccatgtcc
ctgtggctgc 4740cctccgaggc taccgtgtac ctgccccccg tgcccgtgtc caaggtggtg
tccaccgacg 4800agtacgtggc tcgtaccaac atctactacc acgctggcac ctcccgtctg
ctggctgtgg 4860gtcaccccta cttccccatc aagaagccca acaacaacaa gatcctggtg
cccaaggtgt 4920ccggcctgca gtaccgtgtg ttccgtatcc acctgcccga ccccaacaag
ttcggtttcc 4980ccgacacctc cttctacaac cctgacaccc agcgcctcgt gtgggcttgc
gtgggcgtgg 5040aggtcggccg tggccagccc ctgggtgtcg gtatctccgg tcaccccctg
ctgaacaagc 5100tggacgacac cgagaacgct tccgcttacg ctgctaacgc tggtgtcgac
aaccgcgagt 5160gcatctccat ggactacaag cagacccagc tgtgcctgat cggttgcaag
ccccccatcg 5220gcgagcactg gggcaagggt tccccctgca ccaacgtggc tgtgaacccc
ggcgactgcc 5280cccctctcga gctgatcaac accgtgatcc aggacggcga catggtggac
accggtttcg 5340gtgctatgga cttcaccacc ctgcaggcta acaagtccga ggtgcccctg
gacatctgca 5400cctccatctg caagtacccc gactacatca agatggtgtc cgagccctac
ggcgactccc 5460tgttcttcta cctgcgtcgt gagcagatgt tcgtgcgtca cctgttcaac
cgtgctggtg 5520ctgtgggcga gaacgtgccc gacgacctgt acatcaaggg ttccggttcc
accgctaacc 5580tggcttccag caactacttc cctaccccct ccggttccat ggtcacctcc
gacgctcaga 5640tcttcaacaa gccctactgg ctgcagcgtg ctcagggtca caacaacggt
atctgctggg 5700gcaaccagct gttcgtgacc gtggtcgaca ccacccgttc caccaacatg
tctctgtgcg 5760ctgctatctc cacctccgag actacctaca agaacaccaa cttcaaggag
tacctgcgtc 5820acggcgagga gtacgacctg cagttcatct tccagctgtg caagatcacc
ctgaccgctg 5880acgtgatgac ctacatccac tccatgaact ccactatcct cgaagattgg
aacttcggtc 5940tgcagccccc tcccggtggc accctcgagg acacctaccg tttcgtcacc
tcccaggcta 6000tcgcttgcca gaagcacacc ccccctgctc ccaaggagga ccccctgaag
aagtacacct 6060tctgggaggt caacctgaag gagaagttct ccgctgacct ggaccagttc
cccctgggtc 6120gcaagttcct gctgcaggcc ggactgaagg ccaagcccaa gttcaccctg
ggcaagcgca 6180aggctacccc caccacctcc tccacctcca ccaccgctaa gaggaagaag
cgcaagctgt 6240aaaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt
gtagaggttt 6300tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
atgaatgcaa 6360ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
aatagcatca 6420caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
tccaaactca 6480tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctagaaga
tccggctgct 6540aacaaagccc gaaaggaagc tgagttggct gctgtggcta gctttgttta
actttaagaa 6600ggagatacat ctagaaatat taatagatca tggagataat taaaatgata
accatctcgc 6660aaataaataa gtattttact gttttcgtaa cagttttgta ataaaaaaac
ctataaatat 6720tccggattat tcataccgtc ccaccatcgg gcgcggatcc gccaccatgg
ctctgtggcg 6780tccctccgac aacaccgtgt acctgccccc tccctccgtg gctcgtgtgg
tcaacaccga 6840cgactacgtg acccgtacct ccatcttcta ccacgctggt tcctcccgtc
tgctgaccgt 6900gggcaacccc tacttccgtg tgcccgctgg cggtggcaac aagcaggaca
tccccaaggt 6960gtccgcttac cagtaccgtg tgttccgtgt gcagctgccc gaccccaaca
agttcggtct 7020gcccgacacc tccatctaca accccgagac tcagcgcctc gtgtgggctt
gcgctggtgt 7080cgagatcggt cgtggccagc ccctgggtgt cggcctgtcc ggtcacccct
tctacaacaa 7140gctggacgac accgagtcct cccacgctgc tacctccaac gtgtccgagg
acgtgcgcga 7200caacgtgtct gtggactaca agcagaccca gctgtgcatc ctgggttgcg
ctcccgctat 7260cggcgagcac tgggctaagg gcaccgcttg caagtcccgt cctctgtccc
agggcgactg 7320cccccctctc gagctgaaga acaccgtgct cgaggacggc gacatggtgg
acaccggtta 7380cggtgctatg gacttcagca ccctgcagga caccaagtgc gaggtgcccc
tggacatctg 7440ccagtccatc tgcaagtacc ccgactacct gcagatgtcc gctgacccct
acggcgactc 7500tatgttcttc tgcctgcgtc gtgagcagct gttcgctcgt cacttctgga
accgtgctgg 7560caccatgggt gacaccgtgc cccagtccct gtacatcaag ggcaccggca
tgcgtgcttc 7620ccccggttcc tgcgtgtact ccccttcccc ctccggttcc atcgtgacct
ccgactccca 7680gctgttcaac aagccctact ggctgcacaa ggctcagggt cacaacaacg
gtgtctgctg 7740gcacaaccag ctgttcgtga ccgtggtcga caccacccgt tccaccaacc
tgaccatctg 7800cgcttccacc cagtcccccg tgcccggcca gtacgacgct accaagttca
agcagtactc 7860ccgtcacgtg gaggagtacg acctgcagtt catcttccag ctctgcacta
tcaccctgac 7920cgctgacgtg atgtcctaca tccactccat gaactcctct atcctcgaag
attggaactt 7980cggtgtcccc cctcccccca ctacctccct ggtggacact taccgtttcg
tgcagtccgt 8040ggctatcacc tgccagaagg acgctgctcc cgctgagaac aaggacccct
acgacaagct 8100gaagttctgg aacgtggacc tgaaggagaa gttctccctg gacctggacc
agtaccccct 8160gggtcgcaag ttcctggtgc aggctggcct gaggcgcaag cccaccatcg
gtccccgcaa 8220gcgttccgct ccctccgcta ccacctcctc caagcccgcc aagcgtgtgc
gtgtgcgcgc 8280tcgcaagtaa gctagcaagc ttgtcgagaa gtactagagg atcataatca
gccataccac 8340atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
acctgaaaca 8400taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
gttacaaata 8460aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
ctagttgtgg 8520tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatcactgc
ttgagcctag 8580aagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
accgctgagc 8640aataactatc ataaccccta ggagatccga accagataag tgaaatctag
ttccaaacta 8700ttttgtcatt tttaattttc gtattagctt acgacgctac acccagttcc
catctatttt 8760gtcactcttc cctaaataat ccttaaaaac tccatttcca cccctcccag
ttcccaacta 8820ttttgtccgc ccacagcggg gcatttttct tcctgttatg tttttaatca
aacatcctgc 8880caactccatg tgacaaaccg tcatcttcgg ctacttt
8917310595DNAArtificialmulti-epitope influenza B virus-like
particle 3ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt
ttgtaattga 60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct
gtagcggcgc 120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
ccagcgccct 180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
gctttccccg 240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
ggcacctcga 300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
gatagacggt 360ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt
ccaaactgga 420acaacactca accctatctc ggtctattct tttgatttat aagggatttt
gccgatttcg 480gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
taacaaaata 540ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
cctatttgtt 600tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
tgataaatgc 660ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
gcccttattc 720ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa 780aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
ctcaacagcg 840gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
acttttaaag 900ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
ctcggtcgcc 960gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
aagcatctta 1020cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg 1080cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
tttttgcaca 1140acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
gaagccatac 1200caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
cgcaaactat 1260taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
atggaggcgg 1320ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
attgctgata 1380aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
ccagatggta 1440agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
gatgaacgaa 1500atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
tcagaccaag 1560tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
aggatctagg 1620tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
tcgttccact 1680gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
tttctgcgcg 1740taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
ttgccggatc 1800aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata 1860ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
gcaccgccta 1920catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc 1980ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
ggctgaacgg 2040ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
agatacctac 2100agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg 2160taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
aacgcctggt 2220atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
ttgtgatgct 2280cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
cggttcctgg 2340ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
tctgtggata 2400accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
accgagcgca 2460gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
cttacgcatc 2520tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg
gttacggttg 2580agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc
ttaaactgaa 2640caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt
tgtaaactga 2700aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct
aaagcaaact 2760cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc
caagggcatg 2820gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg
ccgggaagcc 2880gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
agtgcatcac 2940ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
cgtaatctgc 3000ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
gattgatgag 3060cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
agatatagat 3120ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
caacaaccgc 3180ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
tcccgaggta 3240atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
gaccgaaaag 3300atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
tgcgaatgat 3360gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
taacatcgtt 3420gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg
gcgtaacgcg 3480cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
caagccatga 3540aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
cagttgcgtg 3600agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
aactgggttc 3660gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc
agcgaagtcg 3720aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg
catcgtcagg 3780cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg
cccttgcttc 3840aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc
ccggatgaag 3900tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag
gactctagct 3960atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa
ctgctgatca 4020acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta
tacgaagtta 4080tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac
gttggctgcg 4140agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga
aacgcgggcg 4200tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg
ggaccgaacc 4260ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt
ttattgccgt 4320catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc
tcccccatct 4380cccggtacct tataagtatt tcctcacaag agctgaattt cccatagagc
tctgttttag 4440cacttccatt acatctttgg caattccttc tccattcttt tgacttgctc
ctagagatct 4500caacactcca atgttgtttt gcagctcttc tgctagtttt tggacgtctt
ctccctttcc 4560cattccattc attgtctttg ctgtgttcat agctgaaacc atctgcattt
ctcgtcttac 4620tccaggtacc gaagaccttg ctgctctgct atgggctcta tgcgagtgcg
atgcttgttt 4680ctcgcataaa gcacagagcg ttcctagttt tacttgcatt gaatagtttt
cagggtttag 4740gtacatgacc ataagacaat atagtaatgc tgagctttcg tggccttctg
ctatttcaaa 4800tgcttcatga aagcttacac atcttctcat ttttctctca gctagaatta
ggcctttctt 4860ctttgttgct gttgttccca ttcctgacag gggctctgtg atgaatctcc
tttttctttc 4920ttggtctttg ggttttaaaa agcatataga ggcaccaatt agtgcttttt
gtatatcagt 4980taggcacctt ttgtttttta tccattccaa agcagaatct aggtcaaatt
ctttcccacc 5040gaaccaacag tgtaattttt cagctagttc tgctttgcct tctccatctt
ctattagtga 5100aagcaggtag gcaattgtgt ctccaaacag cgacatggtg gcatcccggg
tgatcaagtc 5160ttcgtcgagt gattgtaaat aaaatgtaat ttacagtata gtattttaat
taatatacaa 5220atgatttgat aataattctt atttaactat aatatattgt gttgggttga
attaaaggtc 5280cgtatactag tatcgattcg cgacctactc cggaatatta atagatcatg
gagataatta 5340aaatgataac catctcgcaa ataaataagt attttactgt tttcgtaaca
gttttgtaat 5400aaaaaaacct ataaatattc cggattattc ataccgtccc accatcgggc
gcggatccac 5460catgaaggca ataattgtac tactcatggt agtaacatcc aatgcagatc
gaatctgcac 5520tggaataaca tcttcaaact cacctcatgt ggtcaaaaca gccactcaag
gggaggtcaa 5580tgtgactggt gtgataccac taacaacaac accaacaaaa tcttattttg
caaatctcaa 5640aggaacaagg accagaggga aactatgccc agactgtctc aactgcacag
atctggatgt 5700ggctttgggc agaccaatgt gtgtggggac cacaccttcg gcgaaagctt
caatactcca 5760cgaagtcaaa cctgttacat ccgggtgctt tcctataatg cacgacagaa
caaaaatcag 5820gcaactaccc aatcttctca gaggatatga aaatatcagg ctatcaaccc
aaaacgtcat 5880cgatgcggaa aaggcaccag gaggacccta cagacttgga acctcaggat
cttgccctaa 5940cgctaccagt aagagcggat ttttcgcaac aatggcttgg gctgtcccaa
aggacaacaa 6000caaaaatgca acgaacccac taacagtaga agtaccatac atttgtacag
aaggggaaga 6060ccaaatcact gtttgggggt tccattcaga tgacaaaacc caaatgaaga
acctctatgg 6120agactcaaat cctcaaaagt tcacctcatc tgctaatgga gtaaccacac
actatgtttc 6180tcagattggc agcttcccag atcaaacaga agacggagga ctaccacaaa
gcggcaggat 6240tgttgttgat tacatgatgc aaaaacctgg gaaaacagga acaattgtct
accaaagagg 6300tgttttgttg cctcaaaagg tgtggtgcgc gagtggcagg agcaaagtaa
taaaagggtc 6360cttgccttta attggtgaag cagattgcct tcatgaaaaa tacggtggat
taaacaaaag 6420caagccttac tacacaggag aacatgcaaa agccatagga aattgcccaa
tatgggtgaa 6480aacacctttg aagcttgcca atggaaccaa atatagacct cctgcaaaac
tattaaagga 6540aaggggtttc ttcggagcta ttgctggttt cctagaagga ggatgggaag
gaatgattgc 6600aggctggcac ggatacacat ctcacggagc acatggagtg gcagtggcgg
cggaccttaa 6660gagtacgcaa gaagctataa acaagataac aaaaaatctc aattctttga
gtgagctaga 6720agtaaagaat cttcaaagac taagtggtgc catggatgaa ctccacaacg
aaatactcga 6780gctggatgag aaagtggatg atctcagagc tgacactata agctcgcaaa
tagaacttgc 6840agtcttgctt tccaacgaag gaataataaa cagtgaagat gagcatctat
tggcacttga 6900gagaaaacta aagaaaatgc tgggtccctc tgctgtagag ataggaaatg
gatgcttcga 6960aaccaaacac aagtgcaacc agacctgctt agacaggata gctgctggca
cctttaatgc 7020aggagaattt tctctcccca cttttgattc actgaacatt actgctgcat
ctttaaatga 7080tgatggattg gataaccata ctatactgct ctattactca actgctgctt
ctagtttggc 7140tgtaacattg atgctagcta tttttattgt ttatatggtc tccagagaca
acgtttcatg 7200ctccatctgt ctataagtcg acgtactaga ggatcataat cagccatacc
acatttgtag 7260aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
cataaaatga 7320atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
taaagcaata 7380gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
ggtttgtcca 7440aactcatcaa tgtatcttat catgtctgga tctgatcact gcttgagcct
agaagatccg 7500gctgctaaca aagcccgaaa ggaagctgag ttggctgctg tggctagctt
tgtttaactt 7560taagaaggag atacatctag aaatattaat agatcatgga gataattaaa
atgataacca 7620tctcgcaaat aaataagtat tttactgttt tcgtaacagt tttgtaataa
aaaaacctat 7680aaatattccg gattattcat accgtcccac catcgggcgc ggatccacca
tgctaccttc 7740aactatacaa acgttaaccc tatttctcac atcaggggga gtgttattat
cactatatgt 7800gtcagcttca ttgtcatact tactatattc ggatatattg ctaaaatttt
cacaaacaga 7860aataactgca ccaataatgc cattggattg tgcaaacgca tcaaatgttc
aggctgtgaa 7920ccgttctgca gcaaaagggg tgacacttct tctcccagaa ccggagtgga
cataccctcg 7980tttatcttgc ccgggctcaa cctttcagaa agcactccta attagccccc
atagattcgg 8040agaaaccaaa ggaaactcag ctcccttgat aataagggaa ccttttattg
cttgtggacc 8100aacggaatgc aaacactttg ctctaaccca ttatgcagct caaccagggg
gatactacaa 8160tggaacaaga gaagacagaa acaagctgag gcatctaatt tcagtcaaat
tgggcaaaat 8220cccaacagta gaaaactcca ttttccatat ggcagcttgg agcgggtccg
catgccatga 8280tggtaaagaa tggacatata tcggagttga tggccccgac agtaatgcat
tactcaaaat 8340aaaatatgga gaagcatata ctgacacata ccattcctat gcaaaaaaca
tcctaaggac 8400acaagaaagt gcctgcaatt gcatcggggg agattgttat cttatgataa
ctgatggccc 8460agcttcaggg attagtgaat gcagattcct taagattcga gagggccgaa
taataaaaga 8520aatatttcca acaggaagag taaaacatac tgaggaatgc acatgcggat
ttgccagcaa 8580caaaaccata gaatgtgctt gtagagataa cagttacaca gcaaaaagac
cctttgtcaa 8640attaaatgtg gagactgata cagcggaaat aagattgatg tgcacagaga
cttatttgga 8700cacccccaga ccaaatgatg gaagcataac agggccttgc gaatctgatg
gggacaaagg 8760gagtggaggc atcaagggag gatttgttca tcaaagaatg gcatccaaga
ttggaaggtg 8820gtactctcga acgatgtcta aaactaaaag aatggggatg ggactgtatg
taaagtatga 8880tggagaccca tggactgaca gtgaagccct tgctcttagt ggagtaatgg
tttcgatgga 8940agaacctggt tggtattcct ttggcttcga aataaaagat aagaaatgtg
atgtcccctg 9000tattgggata gaaatggtac atgatggtgg gaaaacgact tggcactcag
cagcaacagc 9060catttactgt ttaatgggct caggacaact gctgtgggac actgtcacag
gtgttgatat 9120ggctctgtaa atcgatgaca agcttgtcga gaagtactag aggatcataa
tcagccatac 9180cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
tgaacctgaa 9240acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
atggttacaa 9300ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
attctagttg 9360tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgatcac
tgcttgagcc 9420tagaagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
gggtgcctaa 9480tgagtgagct aactcacatt aattgcccgg aatattaata gatcatggag
ataattaaaa 9540tgataaccat ctcgcaaata aataagtatt ttactgtttt cgtaacagtt
ttgtaataaa 9600aaaacctata aatattccgg attattcata ccgtcccacc atcgggcgcg
gatccaccat 9660gctcgaacca tttcagattc tttcaatttg ttcttttatc ttatcagctc
tccatttcat 9720ggcttggaca atagggcatt tgaatcaaat aaaaagagga ataaacatga
aaatacgaat 9780aaaaggtcca aacaaagaga caataaacag agaggtatca attttgagac
acagttacca 9840aaaagaaatc caggccaaag aaacaatgaa ggaagtactc tctgacaaca
tggaggtgtt 9900gagtgaccac ataataattg aggggctttc tgccgaagag ataataaaaa
tgggtgaaac 9960agttttggag atagaagaat tgcattaagt actagaggat cataatcagc
cataccacat 10020ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
ctgaaacata 10080aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
tacaaataaa 10140gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
agttgtggtt 10200tgtccaaact catcaatgta tcttatcatg tctggatctg atcactgctt
gagcctagaa 10260gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
cgctgagcaa 10320taactatcat aacccctagg agatccgaac cagataagtg aaatctagtt
ccaaactatt 10380ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca
tctattttgt 10440cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt
cccaactatt 10500ttgtccgccc acagcggggc atttttcttc ctgttatgtt tttaatcaaa
catcctgcca 10560actccatgtg acaaaccgtc atcttcggct acttt
1059548411DNAArtificialmulti-epitope papilloma virus-like
particle 4ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt
ttgtaattga 60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct
gtagcggcgc 120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
ccagcgccct 180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
gctttccccg 240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
ggcacctcga 300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
gatagacggt 360ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt
ccaaactgga 420acaacactca accctatctc ggtctattct tttgatttat aagggatttt
gccgatttcg 480gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
taacaaaata 540ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
cctatttgtt 600tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
tgataaatgc 660ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
gcccttattc 720ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa 780aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
ctcaacagcg 840gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
acttttaaag 900ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
ctcggtcgcc 960gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
aagcatctta 1020cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg 1080cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
tttttgcaca 1140acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
gaagccatac 1200caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
cgcaaactat 1260taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
atggaggcgg 1320ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
attgctgata 1380aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
ccagatggta 1440agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
gatgaacgaa 1500atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
tcagaccaag 1560tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
aggatctagg 1620tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
tcgttccact 1680gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
tttctgcgcg 1740taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
ttgccggatc 1800aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata 1860ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
gcaccgccta 1920catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc 1980ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
ggctgaacgg 2040ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
agatacctac 2100agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg 2160taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
aacgcctggt 2220atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
ttgtgatgct 2280cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
cggttcctgg 2340ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
tctgtggata 2400accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
accgagcgca 2460gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
cttacgcatc 2520tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg
gttacggttg 2580agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc
ttaaactgaa 2640caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt
tgtaaactga 2700aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct
aaagcaaact 2760cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc
caagggcatg 2820gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg
ccgggaagcc 2880gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
agtgcatcac 2940ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
cgtaatctgc 3000ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
gattgatgag 3060cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
agatatagat 3120ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
caacaaccgc 3180ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
tcccgaggta 3240atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
gaccgaaaag 3300atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
tgcgaatgat 3360gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
taacatcgtt 3420gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg
gcgtaacgcg 3480cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
caagccatga 3540aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
cagttgcgtg 3600agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
aactgggttc 3660gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc
agcgaagtcg 3720aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg
catcgtcagg 3780cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg
cccttgcttc 3840aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc
ccggatgaag 3900tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag
gactctagct 3960atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa
ctgctgatca 4020acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta
tacgaagtta 4080tctggtttaa acgtacccgt agtggctatg gcagggcttg ccgccccgac
gttggctgcg 4140agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga
aacgcgggcg 4200tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg
ggaccgaacc 4260ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt
ttattgccgt 4320catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc
tcccccatct 4380cccggtaccg catgctatgc ataagctttt acagcttgcg cttcttcctc
ttagcggtgg 4440tggaggtgga ggaggtggtg ggggtagcct tgcgcttgcc cagggtgaac
ttgggcttgg 4500ccttcagtcc ggcctgcagc aggaacttgc gacccagggg gaactggtcc
aggtcagcgg 4560agaacttctc cttcaggttg acctcccaga aggtgtactt cttcaggggg
tcctccttgg 4620gagcaggggg ggtgtgcttc tggcaagcga tagcctggga ggtgacgaaa
cggtaggtgt 4680cctcgagggt gccaccggga gggggctgca gaccgaagtt ccaatcttcg
aggatagtgg 4740agttcatgga gtggatgtag gtcatcacgt cagcggtcag ggtgatcttg
cacagctgga 4800agatgaactg caggtcgtac tcctcgccgt gacgcaggta ctccttgaag
ttggtgttct 4860tgtaggtagt ctcggaggtg gagatagcag cgcacagaga catgttggtg
gaacgggtgg 4920tgtcgaccac ggtcacgaac agctggttgc cccagcagat accgttgttg
tgaccctgag 4980cacgctgcag ccagtagggc ttgttgaaga tctgagcgtc ggaggtgacc
atggaaccgg 5040agggggtagg gaagtagttg ctggaagcca ggttagcggt ggaaccggaa
cccttgatgt 5100acaggtcgtc gggcacgttc tcgcccacag caccagcacg gttgaacagg
tgacgcacga 5160acatctgctc acgacgcagg tagaagaaca gggagtcgcc gtagggctcg
gacaccatct 5220tgatgtagtc ggggtacttg cagatggagg tgcagatgtc caggggcacc
tcggacttgt 5280tagcctgcag ggtggtgaag tccatagcac cgaaaccggt gtccaccatg
tcgccgtcct 5340ggatcacggt gttgatcagc tcgagagggg ggcagtcgcc ggggttcaca
gccacgttgg 5400tgcaggggga acccttgccc cagtgctcgc cgatgggggg cttgcaaccg
atcaggcaca 5460gctgggtctg cttgtagtcc atggagatgc actcgcggtt gtcgacacca
gcgttagcag 5520cgtaagcgga agcgttctcg gtgtcgtcca gcttgttcag cagggggtga
ccggagatac 5580cgacacccag gggctggcca cggccgacct ccacgcccac gcaagcccac
acgaggcgct 5640gggtgtcagg gttgtagaag gaggtgtcgg ggaaaccgaa cttgttgggg
tcgggcaggt 5700ggatacggaa cacacggtac tgcaggccgg acaccttggg caccaggatc
ttgttgttgt 5760tgggcttctt gatggggaag taggggtgac ccacagccag cagacgggag
gtgccagcgt 5820ggtagtagat gttggtacga gccacgtact cgtcggtgga caccaccttg
gacacgggca 5880cggggggcag gtacacggta gcctcggagg gcagccacag ggacatggtg
gcggatccat 5940cccgggtgat caagtcttcg tcgagtgatt gtaaataaaa tgtaatttac
agtatagtat 6000tttaattaat atacaaatga tttgataata attcttattt aactataata
tattgtgttg 6060ggttgaatta aaggtccgta tactagtatc gattcgcgac ctactccgga
atattaatag 6120atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt
tactgttttc 6180gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac
cgtcccacca 6240tcgggcgcgg atccgccacc atggctctgt ggcgtccctc cgacaacacc
gtgtacctgc 6300cccctccctc cgtggctcgt gtggtcaaca ccgacgacta cgtgacccgt
acctccatct 6360tctaccacgc tggttcctcc cgtctgctga ccgtgggcaa cccctacttc
cgtgtgcccg 6420ctggcggtgg caacaagcag gacatcccca aggtgtccgc ttaccagtac
cgtgtgttcc 6480gtgtgcagct gcccgacccc aacaagttcg gtctgcccga cacctccatc
tacaaccccg 6540agactcagcg cctcgtgtgg gcttgcgctg gtgtcgagat cggtcgtggc
cagcccctgg 6600gtgtcggcct gtccggtcac cccttctaca acaagctgga cgacaccgag
tcctcccacg 6660ctgctacctc caacgtgtcc gaggacgtgc gcgacaacgt gtctgtggac
tacaagcaga 6720cccagctgtg catcctgggt tgcgctcccg ctatcggcga gcactgggct
aagggcaccg 6780cttgcaagtc ccgtcctctg tcccagggcg actgcccccc tctcgagctg
aagaacaccg 6840tgctcgagga cggcgacatg gtggacaccg gttacggtgc tatggacttc
agcaccctgc 6900aggacaccaa gtgcgaggtg cccctggaca tctgccagtc catctgcaag
taccccgact 6960acctgcagat gtccgctgac ccctacggcg actctatgtt cttctgcctg
cgtcgtgagc 7020agctgttcgc tcgtcacttc tggaaccgtg ctggcaccat gggtgacacc
gtgccccagt 7080ccctgtacat caagggcacc ggcatgcgtg cttcccccgg ttcctgcgtg
tactcccctt 7140ccccctccgg ttccatcgtg acctccgact cccagctgtt caacaagccc
tactggctgc 7200acaaggctca gggtcacaac aacggtgtct gctggcacaa ccagctgttc
gtgaccgtgg 7260tcgacaccac ccgttccacc aacctgacca tctgcgcttc cacccagtcc
cccgtgcccg 7320gccagtacga cgctaccaag ttcaagcagt actcccgtca cgtggaggag
tacgacctgc 7380agttcatctt ccagctctgc actatcaccc tgaccgctga cgtgatgtcc
tacatccact 7440ccatgaactc ctctatcctc gaagattgga acttcggtgt cccccctccc
cccactacct 7500ccctggtgga cacttaccgt ttcgtgcagt ccgtggctat cacctgccag
aaggacgctg 7560ctcccgctga gaacaaggac ccctacgaca agctgaagtt ctggaacgtg
gacctgaagg 7620agaagttctc cctggacctg gaccagtacc ccctgggtcg caagttcctg
gtgcaggctg 7680gcctgaggcg caagcccacc atcggtcccc gcaagcgttc cgctccctcc
gctaccacct 7740cctccaagcc cgccaagcgt gtgcgtgtgc gcgctcgcaa gtaagctagc
aagcttgtcg 7800agaagtacta gaggatcata atcagccata ccacatttgt agaggtttta
cttgctttaa 7860aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
gttgttgtta 7920acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
aatttcacaa 7980ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
aatgtatctt 8040atcatgtctg gatctgatca ctgcttgagc ctagaagatc cggctgctaa
caaagcccga 8100aaggaagctg agttggctgc tgccaccgct gagcaataac tatcataacc
cctaggagat 8160ccgaaccaga taagtgaaat ctagttccaa actattttgt catttttaat
tttcgtatta 8220gcttacgacg ctacacccag ttcccatcta ttttgtcact cttccctaaa
taatccttaa 8280aaactccatt tccacccctc ccagttccca actattttgt ccgcccacag
cggggcattt 8340ttcttcctgt tatgttttta atcaaacatc ctgccaactc catgtgacaa
accgtcatct 8400tcggctactt t
841158445DNAArtificialmulti-epitope papilloma virus-like
particle 5ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt
ttgtaattga 60ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct
gtagcggcgc 120attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
ccagcgccct 180agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
gctttccccg 240tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
ggcacctcga 300ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
gatagacggt 360ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt
ccaaactgga 420acaacactca accctatctc ggtctattct tttgatttat aagggatttt
gccgatttcg 480gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
taacaaaata 540ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
cctatttgtt 600tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
tgataaatgc 660ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
gcccttattc 720ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa 780aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
ctcaacagcg 840gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
acttttaaag 900ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
ctcggtcgcc 960gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
aagcatctta 1020cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg 1080cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
tttttgcaca 1140acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
gaagccatac 1200caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
cgcaaactat 1260taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
atggaggcgg 1320ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
attgctgata 1380aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
ccagatggta 1440agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
gatgaacgaa 1500atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
tcagaccaag 1560tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
aggatctagg 1620tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
tcgttccact 1680gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
tttctgcgcg 1740taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
ttgccggatc 1800aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata 1860ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
gcaccgccta 1920catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc 1980ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
ggctgaacgg 2040ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
agatacctac 2100agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg 2160taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
aacgcctggt 2220atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
ttgtgatgct 2280cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
cggttcctgg 2340ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
tctgtggata 2400accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
accgagcgca 2460gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
cttacgcatc 2520tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg
gttacggttg 2580agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc
ttaaactgaa 2640caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt
tgtaaactga 2700aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct
aaagcaaact 2760cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc
caagggcatg 2820gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg
ccgggaagcc 2880gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
agtgcatcac 2940ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
cgtaatctgc 3000ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
gattgatgag 3060cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
agatatagat 3120ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
caacaaccgc 3180ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
tcccgaggta 3240atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
gaccgaaaag 3300atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
tgcgaatgat 3360gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
taacatcgtt 3420gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg
gcgtaacgcg 3480cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
caagccatga 3540aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
cagttgcgtg 3600agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
aactgggttc 3660gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc
agcgaagtcg 3720aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg
catcgtcagg 3780cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg
cccttgcttc 3840aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc
ccggatgaag 3900tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag
gactctagct 3960atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa
ctgctgatca 4020acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta
tacgaagtta 4080tctggtttcg acctactccg gaatattaat agatcatgga gataattaaa
atgataacca 4140tctcgcaaat aaataagtat tttactgttt tcgtaacagt tttgtaataa
aaaaacctat 4200aaatattccg gattattcat accgtcccac catcgggcgc ggatccgcca
ccatgtccct 4260gtggctgccc tccgaggcta ccgtgtacct gccccccgtg cccgtgtcca
aggtggtgtc 4320caccgacgag tacgtggctc gtaccaacat ctactaccac gctggcacct
cccgtctgct 4380ggctgtgggt cacccctact tccccatcaa gaagcccaac aacaacaaga
tcctggtgcc 4440caaggtgtcc ggcctgcagt accgtgtgtt ccgtatccac ctgcccgacc
ccaacaagtt 4500cggtttcccc gacacctcct tctacaaccc tgacacccag cgcctcgtgt
gggcttgcgt 4560gggcgtggag gtcggccgtg gccagcccct gggtgtcggt atctccggtc
accccctgct 4620gaacaagctg gacgacaccg agaacgcttc cgcttacgct gctaacgctg
gtgtcgacaa 4680ccgcgagtgc atctccatgg actacaagca gacccagctg tgcctgatcg
gttgcaagcc 4740ccccatcggc gagcactggg gcaagggttc cccctgcacc aacgtggctg
tgaaccccgg 4800cgactgcccc cctctcgagc tgatcaacac cgtgatccag gacggcgaca
tggtggacac 4860cggtttcggt gctatggact tcaccaccct gcaggctaac aagtccgagg
tgcccctgga 4920catctgcacc tccatctgca agtaccccga ctacatcaag atggtgtccg
agccctacgg 4980cgactccctg ttcttctacc tgcgtcgtga gcagatgttc gtgcgtcacc
tgttcaaccg 5040tgctggtgct gtgggcgaga acgtgcccga cgacctgtac atcaagggtt
ccggttccac 5100cgctaacctg gcttccagca actacttccc taccccctcc ggttccatgg
tcacctccga 5160cgctcagatc ttcaacaagc cctactggct gcagcgtgct cagggtcaca
acaacggtat 5220ctgctggggc aaccagctgt tcgtgaccgt ggtcgacacc acccgttcca
ccaacatgtc 5280tctgtgcgct gctatctcca cctccgagac tacctacaag aacaccaact
tcaaggagta 5340cctgcgtcac ggcgaggagt acgacctgca gttcatcttc cagctgtgca
agatcaccct 5400gaccgctgac gtgatgacct acatccactc catgaactcc actatcctcg
aagattggaa 5460cttcggtctg cagccccctc ccggtggcac cctcgaggac acctaccgtt
tcgtcacctc 5520ccaggctatc gcttgccaga agcacacccc ccctgctccc aaggaggacc
ccctgaagaa 5580gtacaccttc tgggaggtca acctgaagga gaagttctcc gctgacctgg
accagttccc 5640cctgggtcgc aagttcctgc tgcaggccgg actgaaggcc aagcccaagt
tcaccctggg 5700caagcgcaag gctaccccca ccacctcctc cacctccacc accgctaaga
ggaagaagcg 5760caagctgtaa aagcttgtcg agaagtacta gaggatcata atcagccata
ccacatttgt 5820agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
aacataaaat 5880gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
aataaagcaa 5940tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
gtggtttgtc 6000caaactcatc aatgtatctt atcatgtctg gatctgatca ctgcttgagc
ctagaagatc 6060cggctgctaa caaagcccga aaggaagctg agttggctgc tgtggctagc
tttgtttaac 6120tttaagaagg agatacatct agaaatatta atagatcatg gagataatta
aaatgataac 6180catctcgcaa ataaataagt attttactgt tttcgtaaca gttttgtaat
aaaaaaacct 6240ataaatattc cggattattc ataccgtccc accatcgggc gcggatccgc
caccatggct 6300ctgtggcgtc cctccgacaa caccgtgtac ctgccccctc cctccgtggc
tcgtgtggtc 6360aacaccgacg actacgtgac ccgtacctcc atcttctacc acgctggttc
ctcccgtctg 6420ctgaccgtgg gcaaccccta cttccgtgtg cccgctggcg gtggcaacaa
gcaggacatc 6480cccaaggtgt ccgcttacca gtaccgtgtg ttccgtgtgc agctgcccga
ccccaacaag 6540ttcggtctgc ccgacacctc catctacaac cccgagactc agcgcctcgt
gtgggcttgc 6600gctggtgtcg agatcggtcg tggccagccc ctgggtgtcg gcctgtccgg
tcaccccttc 6660tacaacaagc tggacgacac cgagtcctcc cacgctgcta cctccaacgt
gtccgaggac 6720gtgcgcgaca acgtgtctgt ggactacaag cagacccagc tgtgcatcct
gggttgcgct 6780cccgctatcg gcgagcactg ggctaagggc accgcttgca agtcccgtcc
tctgtcccag 6840ggcgactgcc cccctctcga gctgaagaac accgtgctcg aggacggcga
catggtggac 6900accggttacg gtgctatgga cttcagcacc ctgcaggaca ccaagtgcga
ggtgcccctg 6960gacatctgcc agtccatctg caagtacccc gactacctgc agatgtccgc
tgacccctac 7020ggcgactcta tgttcttctg cctgcgtcgt gagcagctgt tcgctcgtca
cttctggaac 7080cgtgctggca ccatgggtga caccgtgccc cagtccctgt acatcaaggg
caccggcatg 7140cgtgcttccc ccggttcctg cgtgtactcc ccttccccct ccggttccat
cgtgacctcc 7200gactcccagc tgttcaacaa gccctactgg ctgcacaagg ctcagggtca
caacaacggt 7260gtctgctggc acaaccagct gttcgtgacc gtggtcgaca ccacccgttc
caccaacctg 7320accatctgcg cttccaccca gtcccccgtg cccggccagt acgacgctac
caagttcaag 7380cagtactccc gtcacgtgga ggagtacgac ctgcagttca tcttccagct
ctgcactatc 7440accctgaccg ctgacgtgat gtcctacatc cactccatga actcctctat
cctcgaagat 7500tggaacttcg gtgtcccccc tccccccact acctccctgg tggacactta
ccgtttcgtg 7560cagtccgtgg ctatcacctg ccagaaggac gctgctcccg ctgagaacaa
ggacccctac 7620gacaagctga agttctggaa cgtggacctg aaggagaagt tctccctgga
cctggaccag 7680taccccctgg gtcgcaagtt cctggtgcag gctggcctga ggcgcaagcc
caccatcggt 7740ccccgcaagc gttccgctcc ctccgctacc acctcctcca agcccgccaa
gcgtgtgcgt 7800gtgcgcgctc gcaagtaagc tagcaagctt gtcgagaagt actagaggat
cataatcagc 7860cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
ccccctgaac 7920ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
ttataatggt 7980tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
actgcattct 8040agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg
atcactgctt 8100gagcctagaa gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
ctgctgccac 8160cgctgagcaa taactatcat aacccctagg agatccgaac cagataagtg
aaatctagtt 8220ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac
ccagttccca 8280tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc
cctcccagtt 8340cccaactatt ttgtccgccc acagcggggc atttttcttc ctgttatgtt
tttaatcaaa 8400catcctgcca actccatgtg acaaaccgtc atcttcggct acttt
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