CN101245099A - Amino acid sequence of recombined human papilloma virus L1 capsid protein and uses thereof - Google Patents

Amino acid sequence of recombined human papilloma virus L1 capsid protein and uses thereof Download PDF

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CN101245099A
CN101245099A CNA2007100050994A CN200710005099A CN101245099A CN 101245099 A CN101245099 A CN 101245099A CN A2007100050994 A CNA2007100050994 A CN A2007100050994A CN 200710005099 A CN200710005099 A CN 200710005099A CN 101245099 A CN101245099 A CN 101245099A
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马润林
陈小江
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Abstract

The invention relates to an amino acid sequence of a recombinant human papillomavirus L1 capsid protein, a nucleotide sequence which encodes the amino acid sequence and a recombinant vector and an expression host which contain the nucleotide sequence. The invention further relates to an application of a HPV L1 protein which is composed of the amino acid sequence in the preparation of vaccines, drug combination and diagnostic antigens or antibodies. The invention allows the recombinant HPV L1 capsid protein which is expressed in a prokaryotic system to be dissolvable in water by the modification of the HPV L1 wild-type sequence, and an L1 pentamer which has the same immunogenicity and antigenicity with the VLP of HPV L1 is obtained by expression. The invention allows the industrial production of the HPV L1 capsid protein by utilizing the prokaryotic expression system to become a reality, compared with the currently used eukaryotic expression system, the invention has the advantages of more stable quality of the products, higher yield, low cost and convenient quality control, which has great economic benefits and social effects.

Description

The aminoacid sequence of recombinant human mammilla tumor virus L 1 capsid protein and application thereof
Technical field
The present invention relates to the prevention and the treatment field of human papilloma virus infection.Particularly, the present invention relates to the aminoacid sequence of recombinant human mammilla tumor virus L 1 capsid protein, the encode nucleotide sequence of this aminoacid sequence, and the carrier and the host cell that comprise these nucleotide sequences, the invention still further relates to the application of HPV L1 albumen pentamer in preparation vaccine, pharmaceutical composition, diagnostic antigen or antibody of forming by this aminoacid sequence.
Background technology
Papilloma virus (Papilloma virus) is the dna virus that a big class infects human and other animals.Human papillomavirus (Humanpapilloma virus wherein; HPV) with human multiple disease-related, these diseases comprise optimum wart and cancer.
Papilloma virus contains two structural protein (or capsid protein) that are used to protect viral chromosome, is respectively L1 and L2.The shell of 360 L1 protein moleculars or 72 L1 pentamer formation virions; L2 is positioned at the inside of L1 shell, claims inner capsid albumen again, and each L2 molecule and a L1 pentamer link usually.Therefore, L1 and L2 are important candidate's immunogens.
Because papilloma virus formed host's specialization in the long-term evolution process, this virus are difficult to breed amplification by the cell cultures of routine, must produce this viral capsid proteins with the method for gene recombination.Up to the present, express target gene by the gene recombination scale two basic selections are arranged: prokaryotic expression and eukaryotic expression.Wherein, prokaryotic expression has productive rate height, low, the simple relatively relative sophisticated advantage with technology of system of cost for eukaryotic expression.
But the prokaryotic expression technology also exists defective: the activity expression of the biomacromolecule that molecular weight is big is difficulty relatively, and especially the biological activity of a lot of genes of the higher animal and the mankind is expressed more difficult.The basic reason of difficulty is that the host of prokaryotic expression is the low prokaryotic organism of waiting such as intestinal bacteria, processing, modified elements and modification and higher organism to the expression product polypeptide during genetic expression have important difference, express when the mankind are contour to wait biological gene, cause the secondary or the higher structure of target protein molecule to make a mistake folding easily, cause incorrect molecular conformation, thus the biological activity of influence or forfeiture expression product.
Human papillomavirus is the obligatory parasitism virus of human and other higher mammal, and self does not have gene expression system, and the expression of its full gene relies on its host fully---protein expression, translation, processing and the modification system of higher eucaryote.Therefore, in prokaryotic organism, express when resembling the such gene of L1 capsid protein, often because molecular folding and conformation make a mistake and loss of biological activity.For example, ungroomed L1 albumen is when expression in escherichia coli, and expression product usually forms water-fast inclusion body and precipitates.Sedimentary L1 albumen can't make it reply biological activity at present, promptly can't revert to the correct soluble proteins of molecule conformation.
These defectives of prokaryotic system expression higher organism gene have stoped the performance of its huge advantage to a certain extent, are the one of the main reasons that present fermentation industry does not also have all to use the protokaryon fermentation.Have active polymorphic class medicine of important biomolecule or biotechnological formulation in order to produce some, people have to adopt cost higher, also complicated eucaryon fermentation process.
One of result of the above problems is on the basis of further investigation, sequence to the target gene expression product anatomizes, do not influencing and changing on the basis of protein molecular three-dimensional structure, target gene is carried out certain modification, make it in prokaryotic cell prokaryocytes such as intestinal bacteria, realize solubility expression.
On the basis of realizing papilloma virus L1 albumen pronucleus activity expression, the L1 protein molecular of expression can form correct molecular conformation and water soluble.Soluble L1 molecule forms the polymer of L1 protein molecular because the cause of three-dimensional conformation can be carried out molecule aggregation automatically.Wherein a kind of polymerization methods is the pentamer that forms the L1 protein molecular.
Under field conditions (factors), it is old and feeble and be about to breed in a large number before dead at host cell to parasitize human body or the epithelial papilloma virus of other Mammals.During breeding, virus starts the expression of L1 capsid protein gene in host cell when starting autogene group dna replication dna, L1 albumen after the expression forms the pentamer structure automatically, the further autopolymerization of these pentamers self then, form a wild virus particle by 72 pentamers at last, the dna molecular of the inner storage one cover virus of each virion.
Human body mainly is the capsid protein molecule of identification virus surface to the identification of any intrusive viruses, not exception.The pentamer molecular surface of human mammilla tumor virus L 1 capsid protein has constituted the antigen recognition epi-position (being epitope) of human body.After virus was identified, human body produced the specific antibody virus that neutralizes, the infection and the harm of antagonism virus.In order to induce the create antagonism specific antibody of papilloma virus of human body, we express by bionic method and the capsid protein of purified virus, and produce vaccine with the L1 albumen of purifying as antigen.
Reorganization L1 albumen pentamer itself just possesses complete epitope, and therefore just can be used as antigen is used for preparing vaccine.But in the process of producing, partial L 1 pentamer can form and the duplicate viruslike particle of wild virus granular size (VLP, virus like particle) by autopolymerization.The immunogenicity of VLP and antigenicity and L1 pentamer do not have substantive difference.
In order to reduce production costs, realize the antigenic prokaryotic expression of papilloma virus, be necessary formation by genetic modification control L1 albumen VLP, help making the big or small uniformity of antigen molecule like this, be convenient to quality control, also help improving productive rate.
Summary of the invention
(1) technical problem that will solve
The aminoacid sequence that the purpose of this invention is to provide a kind of recombinant human mammilla tumor virus L 1 capsid protein makes that the human mammilla tumor virus L 1 capsid protein of expressing can be water-soluble in prokaryotic system, control the further polymerization of L1 pentamer simultaneously; Another purpose of the present invention provides the application of above-mentioned recombinant human mammilla tumor virus L 1 capsid protein aminoacid sequence.
(2) technical scheme
The reorganization aminoacid sequence of human papillomavirus capsid protein L 1 of the present invention, be that the conserved sequence VYLPP of the proteic aminoacid sequence N-of wild-type HPV L1 end or the aminoacid sequence of VYVPP upstream are replaced to GSGGG, pass through at the 5th amino acid place, conserved sequence LGRKFL downstream of its C-end simultaneously to introduce the truncation type sequence of the terminator codon formation of protein translation with respect to wild-type, wherein said HPV type is selected from HPV6, HPV11, HPV16, HPV18, HPV 26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV45, HPV 51, HPV52, HPV 53, HPV 56, HPV58, HPV 59, HPV 66, HPV 73, among the HPV 82 one or more.
The Genebank number of landing of the proteic aminoacid sequence correspondence of the wild-type L1 of above-mentioned different shaped HPV is respectively: HPV6-NC_000904, HPV11-NC_001525, HPV16-AF402678, HPV18-AY262282, HPV26-NC_001583, HPV31-NC_001527, HPV33-NC_001528, HPV35-NC_001529, HPV39-NC_001535, HPV42-NC_00153, HPV45-NC_001590, HPV51-NC_001533, HPV52-NC_001592, HPV53-NC_001593, HPV56-NC_001594, HPV58-NC_001443, HPV59-NC_001635, HPV66-NC_001695, HPV73-X94165, HPV82-AF293961.
After adopting above-mentioned strategy to transform, the reorganization aminoacid sequence of different shaped HPV correspondence is respectively: the HPV6 corresponding amino acid sequence is shown in SEQ ID NO:1, the HPV11 corresponding amino acid sequence is shown in SEQ ID NO:2, the HPV16 corresponding amino acid sequence is shown in SEQ ID NO:3, the HPV18 corresponding amino acid sequence is shown in SEQ ID NO:4, HPV 26 corresponding amino acid sequence are shown in SEQ ID NO:5, the HPV31 corresponding amino acid sequence is shown in SEQID NO:6, the HPV33 corresponding amino acid sequence is shown in SEQ ID NO:7, the HPV35 corresponding amino acid sequence is shown in SEQ ID NO:8, the HPV39 corresponding amino acid sequence is shown in SEQ ID NO:9, the HPV42 corresponding amino acid sequence is shown in SEQ ID NO:10, the HPV45 corresponding amino acid sequence is shown in SEQ ID NO:11, the HPV51 corresponding amino acid sequence is shown in SEQ ID NO:12, HPV 52 corresponding amino acid sequence are shown in SEQ ID NO:13, HPV 53 corresponding amino acid sequence are shown in SEQ ID NO:14, HPV 56 corresponding amino acid sequence are shown in SEQ ID NO:15, the HPV58 corresponding amino acid sequence is shown in SEQ ID NO:16, HPV 59 corresponding amino acid sequence are shown in SEQ ID NO:17, HPV 66 corresponding amino acid sequence are shown in SEQ ID NO:18, the HPV73 corresponding amino acid sequence is shown in SEQ IDNO:19, and HPV 82 corresponding amino acid sequence are shown in SEQ ID NO:20.
After adopting above-mentioned strategy to transform, what obtain when expressing in prokaryotic system is the proteic pentamer of HPV L1, and this pentamer can be water-soluble, and promptly HPV L1 albumen has realized that correct space is folding, has possessed biological activity.
Because the proteic dna sequence dna of coding different shaped HPV L1 all has higher conservative property, so above-mentioned transformation strategy is applicable to all HPV types, the expression product that obtains is possesses bioactive HPV L1 albumen pentamer.
The invention still further relates to the nucleic acid molecule of the nucleotide sequence that comprises the above-mentioned aminoacid sequence of encoding, the common trait of the nucleotide sequence of the above-mentioned aminoacid sequence of encoding is to express the product that obtains to have and the identical immunological characteristic of wild-type HPV L1 albumen.Described nucleic acid molecule includes but not limited to following form: the nucleotide sequence of the above-mentioned aminoacid sequence of encoding and the HPV fusion sequence of the nucleotide sequence composition of albumen (as: E6/E7) in earlier stage of encoding.
The present invention also relates to comprise the expression vector of above-mentioned nucleotide sequence, these carriers are to obtain by above-mentioned nucleotide sequence being cloned in the connection carrier that comprises suitable promotor and other suitable transcriptional expression regulation and control original paper, and the carrier that wherein is used to connect comprises commercially available plasmid, bacteriophage and clay.Preferably, the carrier that is used to connect is selected from pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pET-28a, pcDNA3.1 or any other and can be used for the expression plasmid of prokaryotic expression.More preferably, the carrier that is used to connect is selected from pGEX-4T-2.The molecular cloning method concrete operations of using here are referring to " molecular cloning " (publish in August, 2002 for the third edition, Science Press).
The invention still further relates to the host cell that comprises above-mentioned expression vector, optional in intestinal bacteria, insect cell, yeast cell one or more of these host cells.Preferably, host cell is selected from intestinal bacteria.
The invention further relates to HPV L1 albumen pentamer, the proteic monomeric primary structure of this pentamer is made up of above-mentioned reorganization aminoacid sequence, and its secondary structure and higher structure are by its primary structure determined.
The present invention also provides the method for producing above-mentioned HPV L1 albumen pentamer, and it comprises the steps:
(1) will the encode nucleotide sequence of above-mentioned reorganization aminoacid sequence is cloned on the connection carrier transformed into escherichia coli;
(2) the above-mentioned intestinal bacteria of fermentation culture, and express HPV L1 albumen;
(3) separation, purifying expression product obtain HPV L1 albumen pentamer.
The invention further relates to the vaccine that comprises one or more above-mentioned reorganization HPV L1 albumen pentamers, this vaccine can be applicable to prepare the vaccine that prevention HPV infects.
Preferably, vaccine of the present invention comprises the reorganization HPV L1 albumen pentamer of HPV16, and the proteic monomeric primary structure of this pentamer is made up of the aminoacid sequence shown in the SEQ ID NO:3.
More preferably, vaccine of the present invention comprises five kinds of reorganization HPV L1 albumen pentamers, corresponding HPV type is respectively HPV6, HPV11, HPV16, HPV18 and HPV33, and the monomeric primary structure of these five kinds of HPV L1 albumen pentamers is made up of the aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, the SEQ ID NO:7 respectively.
The form of vaccine of the present invention also can be to comprise the proteic combined protein of HPV L1 albumen pentamer+L2.The immune component of above-mentioned vaccine also comprises a kind of physiologically acceptable carrier, includes but not limited to keep the simple low concentration salt solution of HPV L1 albumen pentamer integrity, 10mM NaCl for example, 0.1mM EDTA; Say on perhaps wider that carrier comprises metabolism macromole such as protein, polysaccharide, poly lactose acid, polyglyceric acid, aminoacids complex and inactivation virus particle etc. slowly.The salt that is suitable on the pharmacology also can use in the mixture of HPV L1 albumen pentamer.For example, mineral salt resembles hydrochloride, Bromide, phosphoric acid salt, vitriol etc.; Organic salt such as acetate, seminose, phenylformic acid etc.Immune component also comprises liquid, as water, physiological saline, glycerine, alcohol, but and some other material as humectant, emulsifying agent and pH buffer reagent.
Above-mentioned reorganization HPV L1 albumen pentamer or the proteic combined protein of HPV L1 albumen pentamer+L2 are inoculated to behind the human body with immune effective dose, can be lured the immune response of human body generation these recombinant proteins.This specific immune reaction of human body can help the invasion of human body prevention of human papillomavirus or neutralization to remove the human papillomavirus of having invaded.Vaccine by reorganization HPV L1 albumen pentamer or the proteic combined protein preparation of HPV L1 albumen pentamer+L2 can adopt several different methods to act on human body, comprises vein, muscle and subcutaneous injection.Vaccine of the present invention is in the scope use that can cause the effective dose of L1 protein immunization reaction.The given dose that is used for the mixture of enhancing immunity reaction changes according to L1 mixture difference.Generally speaking, the consumption of L1 pentamer is greatly between 1-500 μ g/Kg body weight.Above-mentioned dosage range is not got rid of the possibility of higher or lower dosage.For example, whether concrete dosage meeting basis is encircled with other drug dosage and is decided, or depends on individual's pharmacokinetics, drug accumulation and metabolic rate.
The invention still further relates to the pharmaceutical composition that comprises above-mentioned reorganization HPV L1 albumen pentamer.This pharmaceutical composition includes but not limited to following form: the pharmaceutical composition that for example comprises reorganization HPV L1 albumen pentamer, HPV E6/E7 albumen and other pharmaceutically acceptable auxiliary material.
The present invention also further discloses the application of above-mentioned reorganization HPV L1 albumen pentamer in preparation HPV virus immunity diagnostic antigen or antibody.Above-mentioned reorganization HPV L1 albumen pentamer can be used for producing the antibody that HPV L1 albumen is had special avidity.The specific HPV type whether these antibody can be used to exist in the direct detection of biological sample or interrelate with the particular pathologies stage.HPV L1 albumen pentamer can also be used for whether existing in the detection of biological sample antibody or the antigen of HPV virus in immunoassay.Comprise that specific combination can be produced with proven technique by those skilled in the art to the polyclone or the monoclonal antibody of the proteic single-chain antibody of L1.These by specific antigen for example the HPV different subtype lure the antibody of generation to have the specificity of the specific HPV hypotype of identification, for example HPV16 and HPV18 type.
Utilize the immunoassay of antibody response to detect L1 albumen and comprise ELISAs, Westernblot, radioimmunoassay, immunohistochemical methods mensuration, immunoprecipitation or additive method.Antibody and L1 pentamer all can be used as certification mark, for example enzyme linked immunological, radio-labeled, fluorescence or chemiluminescence.Antibody or L1 pentamer can be fixed on certain solid support, for example glass or plastics wave plate, and tissue culturing plate, porous plate, test tube, exchange are leant on, exchange column weighting material, albumen; Perhaps on the particle as microsphere, include but not limited to the uncommon or glass sphere of latex, polyphenyl second; Perhaps on the film as cellulose acetate or nylon membrane.The method of coupling protein component is well known to those skilled in the art.Only otherwise the ability of overslaugh antibody original antibody specific combination, any coupling mode all can be used.Biological sample can be the sample of any HPV of containing virus under a cloud, and for example biopsy, smear, tissue are cut sample such as skin, uterus, germinal epithelium cell, larynx, the upper respiratory tract, conjunctiva or oral cavity organization.
(3) beneficial effect
The present invention is by the transformation to HPV L1 wild-type sequence, make the reorganization HPV L1 capsid protein of in prokaryotic system, expressing realize that correct space is folding, can be water-soluble, having solved and having utilized the HPV L1 capsid protein of expressing in the prokaryotic system at present all is the difficult problem of water-fast inclusion body (not having biological activity), has broken the bottleneck of producing the HPVL1 capsid protein with prokaryotic expression system.Moreover, the present invention has also effectively controlled the proteic extent of polymerization of HPV L1, what expression obtained is to have identical immunogenicity and antigenic L1 pentamer with the VLP of HPV L1, and the L1 pentamer has for the VLP of L1 that structure is more stable, the advantage of the easier realization of escherichia coli expression.
The invention enables and utilize prokaryotic expression system industrial production HPV L1 capsid protein to become a reality, with respect to the eukaryotic expression system of present employing have that quality product is more stable, productive rate is higher, cost is low, quality control advantage easily, have great economic benefit and social effect.
Description of drawings
Fig. 1 is the SDS-polyacrylamide gel electrophoresis of the HPV16 L1 capsid protein that obtains behind the modified recombinant, swimming lane 1 expression marker among the figure, swimming lane 2 and swimming lane 3 expression albumen clear liquids, the target protein of swimming lane 4 and swimming lane 5 expression resin absorption, resin after swimming lane 6, swimming lane 7 and swimming lane 8 expression enzymes are cut, swimming lane 9 expression target protein elutriants;
Fig. 2 is the liquid chromatogram of the HPV16 L1 capsid protein that obtains behind the modified recombinant;
Fig. 3 is that the proteic electromicroscopic photograph of HPV16 L1 compares, and wherein Fig. 3 (a) represents the VLP electromicroscopic photograph of HPV16 L1, the HPV16 L1 pentamer electromicroscopic photograph that obtains behind Fig. 3 (b) expression modified recombinant.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 is the modified recombinant of example explanation to HPV L1 capsid protein aminoacid sequence with the HPV16 type
1, the detection of HPV 16 (HPV16) DNA with separate: the clinical cell sample waste (remainder that is used for the cervical exfoliated cell that conventional morphocytology checks) that may contain wild-type HPV16 virus is pacified loyal hospital-based outpatient obstetrical clinic available from Beijing.Be suspended in (about 1 * 10 in 1.0 ml waters 5Individual) cast-off cells handled 300 minutes at 55 degree Celsius through the Proteinase K of 1000 units (available from magnificent biological products company limited), and through 10000g high speed centrifugation 30 minutes, the gained supernatant liquor can be used for detecting whether have HPV virus.Infected HPV16 positive cell supernatant liquor can be directly used in the target gene fragment of pcr amplification HPV16 viral DNA sequence.
2, the molecular cloning of HPV16 type L1 protein D NA: HPV16 type L1 capsid protein gene by the special Oligonucleolide primers of target gene to obtaining through archaeal dna polymerase chain reaction (PCR).Classical PCR reaction system comprises the TAQ archaeal dna polymerase of 20ug dna profiling, 1x PCR damping fluid, forward and reverse special primer (concentration is 0.2 μ M), 1.5mM magnesium ion, 1.0 units.Reaction conditions is: 95 degree sex change 5 minutes Celsius, amplify (each circulation is 94 degree 30 seconds, 55 degree 30 seconds, 72 degree 2 minutes) through 36 PCR circulations, reaction product incubation 10 minutes under 72 degree, stopped reaction then.The forward primer sequence and the reverse primer sequence of PCR reaction see Table 1.(concrete operations are referring to " molecular cloning " third edition through the TA clone for the HPV16 L1 cDNA fragment that PCR reaction amplification is amplified, Science Press, publish in August, 2002) be cloned on the T-Easy vector plasmid (available from U.S. Promega biological reagent company).
3, proteic immunogenicity of the modification of HPV16 L1 protein gene: HPV 16 L1 and antigenicity depend on its correct correct three-dimensional conformation that molecular folding produced.The purpose that HPV16 L1 protein gene sequence is modified is the solubility when increasing the target recombinant gene prokaryotic, thereby utilizes E.coli to produce the L1 albumen of biologically active.It is the design special primer that the basic step of genetic modification plays, and utilizes then with the special primer and amplifies the L1 target gene fragment for the template pcr amplification.Improved reorganization HPV16 L1 protein gene dna sequence dna includes BamH I restriction enzyme site at its 5 '-end, and the DNA codon that contains the GSGGG that encodes simultaneously is to replace the amino acid whose codon in conserved sequence VYLPP upstream of encoding amino acid sequence N-end.The forward primer sequence and the reverse primer sequence that are used to transform see Table 1.Downstream primer is introduced terminator codon UAA, causes L1 cDNA to reduce about 25 amino acid at 3 '-end.The PCR reaction system comprises the TAQ archaeal dna polymerase of 20ug dna profiling, 1x PCR damping fluid, forward and reverse special primer (concentration is 0.2 μ M), 1.5mM magnesium ion, 1.0 units.Reaction conditions is: 95 degree sex change 5 minutes Celsius, amplify (each circulation is 94 degree 30 seconds, 55 degree 30 seconds, 72 degree 2 minutes) through 36 PCR circulations, reaction product incubation 10 minutes under 72 degree, stopped reaction then.
The HPV16 L1 cDNA product that PCR reaction amplification is amplified is cut the formation cohesive terminus through BamHI and XhoI Restriction Enzyme enzyme, simultaneously expression plasmid pGEX-4T-2 (available from magnificent biological products company limited) DNA cuts digestion with BamHI and XhoI enzyme, uses T4 ligase enzyme (available from U.S. Promega biological reagent company) connection PCR product and expression plasmid again.Connect product (concrete operations of electricity conversion referring to " molecular cloning " third edition, Science Press, in August, 2002 publication) in electricity is transferred to e. coli bl21 (available from magnificent biological products company limited) host cell.
4, proteic prokaryotic expression of L1 and detection: be loaded with HPV16 L1 gene and insert after the segmental expression plasmid importing e. coli bl21 cell, (prescription is seen " molecular cloning " third edition to use the LB substratum, Science Press, in August, 2002 publication) carrying out 37 degree cultivates, bacterial classification inserts the back growth to begin to induce with induced expression agent IPTG (available from U.S. Promega company) in 12 hours, harvested cell after 9 hours, cell repeats smudge cells 2 times with Niro Soavi NS2006 type high pressure homogenization machine under 800bar, the microscopy cell crashing ratio reaches more than 90%.L1 albumen in the supernatant solution is used through the affinity chromatography purifying protein: prepackage gsh-agarose resin (Amersham company produce Glutathione Sepharose 4 B) chromatographic column, and get concentration and be 50% Glutathione Sepharose 4 B homogenate and put into chromatographic column (every 200ml albumen clear liquid needs 5-10ml homogenate).(component is: 50mmol/L Tric-HCl with the buffer A of 5-10 column volume doubly, 200mmol/LNaCl, 1mmol/L EDTA, pH8.0) washing resin, the albumen clear liquid is added in the chromatographic column, evenly and after at room temperature acting on 20 minutes emit filtered solution with mixed with resin, with the buffer A washing resin post of 10 times of column volumes.
Detect: (" molecular cloning " third edition is seen in concrete operations through density scan with the SDS-polyacrylamide gel electrophoresis, Science Press, publish in August, 2002) the proteic purity of testing goal (gel electrophoresis the results are shown in accompanying drawing 1), the HPV16 L1 purity of protein that results expression obtains is up to 95%.
Use the liquid phase size exclusive chromatography [to carry out gel filtration chromatography again with the AKTA FPLC filtering system that Amersham Biosciences produces, gel filter medium is the Hiload Superdex 200 that Amersham produces, moving phase is that (composition is seen " molecular cloning " third edition for the phosphate buffered saline of pH7.4, Science Press, publish in August, 2002)] detect, the results are shown in accompanying drawing 2, this figure shows that it is water-soluble expressing the HPV16 L1 albumen that obtains, and has promptly possessed biological activity.
Use the HPV16 L1 albumen of electron microscopic examination purifying then, the results are shown in accompanying drawing 3 (b), contrast accompanying drawing 3 (a) [the VLP electromicroscopic photograph of HPV16 L1], what as seen obtain is HPV16 L1 pentamer.
The modified recombinant of the L1 capsid protein aminoacid sequence of embodiment 2 all the other HPV types
The operation steps of the modified recombinant of the L1 capsid protein aminoacid sequence of all the other HPV types (comprising HPV6, HPV11, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV45, HPV 51, HPV 52, HPV 53, HPV 56, HPV58, HPV 59, HPV 66, HPV 73, HPV 82) is all identical with embodiment 1 with detection method, and difference sees Table 1.
Table 1
The HPV type It is right to clone the used primer of L1 protein gene The used primer of the modification of L1 protein gene is not to (forward primer comprises the amino acid whose Linker dna sequence dna of coding GSGGG)
HPV6 Forward: 5 '-GCATTGCCTGACTCGTCTCT-3 ' is reverse: 5 '-CATGTTGGTACTGCGTGTGG-3 ' Forward: 5 '-TCCTCCTAACCCTGTATCCAAAGTTG-3 ' is reverse: 5 '-ATAGGTATCTAATGTACCATTTGG-3 '
HPV11 Forward: 5 '-TGTATCCAAGGTTGTTGCCA-3 ' is reverse: 5 '-ACAGGTCATCAGGCACAGGT-3 ' Forward: 5 '-GATGCGTATGTTAAACGCACCAACAT-3 ' is reverse: 5 '-AACCAAAGTTCCAGTCCTCCAAAAC-3 '
HPV16 Forward: 5 '-CCTCCTGTCCCAGTATCTAAGG-3 ' is reverse: 5 '-CTTTAATCCTGCTTGTAGTAA-3 ' Forward: 5 '-GTCCCAGTATCTAAGGTTGTAAGC-3 ' is reverse: 5 '-TAGTAAAAATTTGCGTCCTAAAGG-3 '
HPV18 Forward: 5 '-CCTCCTTCTGTGGCAAGAGT-3 ' is reverse: 5 '-AAGTCCATGGCACCATATCC-3 ' Forward: 5 '-TGGCCCATTGTATCACCCACGGCCC-3 ' is reverse: 5 '-CTTATTTTCAGCCGGTGCAGCATCC-3 '
HPV26 Forward: 5 '-GGTAGAGGACAGCCATTAGGC-3 ' is reverse: 5 '-TGAAATATAAATTGTAATTC-3 ' Forward: 5 '-ATCTTCCTCCCACCCCTGTGTCTCGG-3 ' is reverse: 5 '-CAGGTAGTAGCAGAGTTTTTAATAAAC-3 '
HPV31 Forward: 5 '-TGCTTACAGTAGGCCATCCA-3 ' is reverse: 5 '-CCAACCGTGCCTGATCTATT-3 ' Forward: 5 '-CACCTGTCCCAGTGTCTAAAGTTGTA-3 ' is reverse: 5 '-AATTAACCTACCCAAAATACATAATCT-3 '
HPV33 Forward: 5 '-CCACAGTGTACCTGCCTCCT-3 ' is reverse: 5 '-GACCTTGTGCACGTTGTAGC-3 ' Forward: 5 '-CTGCCTCCTGTACCTGTATCTAAAG-3 ' is reverse: 5 '-AAACTGATCTAAATCTGCTGAAAATT-3 '
HPV35 Forward: 5 '-CATGCAGGCAGTTCTAGGCT-3 ' is reverse: 5 '-GTGCATCGGAGGTTACCATA-3 ' Forward: 5 '-CCTGCCTCCAGTGTCAGTGTCTAAG-3 ' is reverse: 5 '-TTTACGGCCCAACGGAAACTGATCT-3 '
HPV39 Forward: 5 '-TTATGTTACACGCACAGGCA-3 ' is reverse: 5 '-CACCATACCACCACGATTCC-3 ' Forward: 5 '-GCCTCCACCTTCTGTGGCGAAGG-3 ' is reverse: 5 '-TTAAGTCAACATTCCAAAACTGAC-3 '
HPV42 Forward: 5 '-ATGTGCAACGCACCAACTAC-3 ' is reverse: 5 '-CCATAGGCCTCAGCAGACAT-3 ' Forward: 5 '-ATCCTAGTTATTTTTGGCGTAGGCGC-3 ' is reverse: 5 '-GAAATTGATCTAAATCAGTAGAAAAC-3 '
HPV45 Forward: 5 '-CACCTTCTGTGGCCAGAGTT-3 ' is reverse: 5 '-AACAGTTGTTCACGGCGTAG-3 ' Forward: 5 '-TGGCTTTGTGGCGGCCTAGTGAC-3 ' is reverse: 5 '-TTTATCATATGGATCCTGCTTTTC-3 '
HPV51 Forward: 5 '-GAATATATCACACGCACCGGCA-3 ' is reverse: 5 '-TTGTGACCCTGCGCACGGTGG-3 ' Forward: 5 '-ACCTGTGTCTCGAATTGTGAATACA-3 ' is reverse: 5 '-GAGGTAATGTTAATCCAAAATTCCAC-3 '
HPV52 Forward: 5 '-ATGCAGGCAGTTCTCGATTA-3 ' is reverse: 5 '-CTCGCCATGACGAAGGTATT-3 ' Forward: 5 '-ACGTCGCAGGCGTAAACGTTTTCC-3 ' is reverse: 5 '-CACCTCCCAAAAACATATAGTCCT-3 '
HPV53 Forward: 5 '-ATAGGGTGTTTAGAGTACGCC-3 ' is reverse: 5 '-GGGGCGGTCCCTGCCATTAC-3 ' Forward: 5 '-CATTACAACGGATGCCTATGTA-3 ' is reverse: 5 '-ATTCATATTCCTCTGCATGTC-3 '
HPV56 Forward: 5 '-TATCATGCAGGCAGTTCACG-3 ' is reverse: 5 '-TCAGCCTTAGATTCCTGCAA-3 ' Forward: 5 '-CTCCTTTGCATTATGGCCTGTGTA-3 ' is reverse: 5 '-GACATGTTATAGCTGTGCTTCTAAC-3 '
HPV58 Forward: 5 '-CACTGTGTACCTGCCTCCTG-3 ' is reverse: 5 '-TATGACCTTGTGCACGCTGT-3 ' Forward: 5 '-AGATGTCCGTGTGGCGGCCTAG-3 ' is reverse: 5 '-ACCTCCCAAAAAGTATATTTATTT-3 '
HPV59 Forward: 5 '-GGTAGCTAAGGTTGTCAGCAC-3 ' is reverse: 5 '-CCTGATCTATTCCTTTTTAGT-3 ' Forward: 5 '-AGCTAAGGTTGTCAGCACTGATG-3 ' is reverse: 5 '-CCAAAACTTTAGTTTGTCATAA-3 '
HPV66 Forward: 5 '-GATGACACTGAGGTCTCTAA-3 ' is reverse: 5 '-TACAAATCTGTAGGAAGGGC-3 ' Forward: 5 '-ATGTAAAACGTACCAGTATATT-3 ' is reverse: 5 '-TCCCAAAACTTATATTTAGCCA-3 '
HPV73 Forward: 5 '-AGGAGCGCCTAGTATGGGCC-3 ' is reverse: 5 '-GATGGTGTTGCAGTATTGCC-3 ' Forward: 5 '-GTAGCACACGTTTGTTGGCTG-3 ' is reverse: 5 '-TTAAGATCTACATCCCAAAAGG-3 '
HPV82 Forward: 5 '-CCTTTTGGGATGTAGATCTTAA-3 ' is reverse: 5 '-TTTCAGTATCATCATACTTATTA-3 ' Forward: 5 '-ATAACCCGCACCGGCATATATTA-3 ' is reverse: 5 '-ACGGTCCAAAACTTATATTTTG-3 '
The L1 capsid protein aminoacid sequence of above-mentioned HPV type is behind modified recombinant, and the result who obtains is all identical with the HPV16 L1 capsid protein that obtains among the embodiment 1, has promptly all obtained water-soluble L1 pentamer.
Sequence table
＜110〉Ma Runlin
Chen Xiaojiang
＜120〉aminoacid sequence of recombinant human mammilla tumor virus L 1 capsid protein and application thereof
<130>MA0702
<160>20
<170>PatentIn version 3.3
<210>1
<211>465
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV6 L1
<400>1
Gly Ser Gly Gly Gly Val Tyr Val Pro Pro Pro Asn Pro Val Ser Lys
1 5 10 15
Val Val Ala Thr Asp Ala Tyr Val Thr Arg Thr Asn Ile Phe Tyr His
20 25 30
Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Ser Ile
35 40 45
Lys Arg Ala Asn Lys Thr Val Val Pro Lys Val Ser Gly Tyr Gln Tyr
50 55 60
Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe Ala Leu Pro
65 70 75 80
Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu Val Trp Ala Cys
85 90 95
Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Val Ser
100 105 110
Gly His Pro Phe Leu Asn Lys Tyr Asp Asp Val Glu Asn Ser Gly Ser
115 120 125
Gly Gly Asn Pro Gly Gln Asp Asn Arg Val Asn Val Gly Met Asp Tyr
130 135 140
Lys Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro Pro Leu Gly Glu
145 150 155 160
His Trp Gly Lys Gly Lys Gln Cys Thr Asn Thr Pro Val Gln Ala Gly
165 170 175
Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile Gln Asp Gly Asp
180 185 190
Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala Asp Leu Gln Thr
195 200 205
Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Gly Thr Thr Cys Lys Tyr
210 215 220
Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro Tyr Gly Asp Arg Leu Phe
225 230 235 240
Phe Phe Leu Arg Lys Glu Gln Met Phe Ala Arg His Phe Phe Asn Arg
245 250 255
Ala Gly Glu Val Gly Glu Pro Val Pro Asp Thr Leu Ile Ile Lys Gly
260 265 270
Ser Gly Asn Arg Thr Ser Val Gly Ser Ser Val Tyr Val Asn Thr Pro
275 280 285
Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe Asn Lys Pro Tyr
290 295 300
Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn
305 310 315 320
Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Thr
325 330 335
Leu Cys Ala Ser Val Thr Thr Ser Ser Thr Tyr Thr Asn Ser Asp Tyr
340 345 350
Lys Glu Tyr Met Arg His Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe
355 360 365
Gln Leu Cys Ser Ile Thr Leu Ser Ala Glu Val Met Ala Tyr Ile His
370 375 380
Thr Met Asn Pro Ser Gly Leu Glu Asp Trp Asn Phe Gly Leu Ser Pro
385 390 395 400
Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr Val Gln Ser Gln
405 410 415
Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu Lys Pro Asp Pro
420 425 430
Tyr Lys Asn Leu Ser Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser
435 440 445
Ser Glu Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser
450 455 460
Gly
465
<210>2
<211>465
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV11 L1
<400>2
Gly Ser Gly Gly Gly Val Tyr Val Pro Pro Pro Asn Pro Val Ser Lys
1 5 10 15
Val Val Ala Thr Asp Ala Tyr Val Lys Arg Thr Asn Ile Phe Tyr His
20 25 30
Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Tyr Ser Ile
35 40 45
Lys Lys Val Asn Lys Thr Val Val Pro Lys Val Ser Gly Tyr Gln Tyr
50 55 60
Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe Ala Leu Pro
65 70 75 80
Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu Val Trp Ala Cys
85 90 95
Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Val Ser
100 105 110
Gly His Pro Leu Leu Asn Lys Tyr Asp Asp Val Glu Asn Ser Gly Gly
115 120 125
Tyr Gly Gly Asn Pro Gly Gln Asp Asn Arg Val Asn Val Gly Met Asp
130 135 140
Tyr Lys Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro Pro Leu Gly
145 150 155 160
Glu His Trp Gly Lys Gly Thr Gln Cys Ser Asn Thr Ser Val Gln Asn
165 170 175
Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile Gln Asp Gly
180 185 190
Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala Asp Leu Gln
195 200 205
Thr Asn Lys Ser Asp Val Pro Leu Asp Ile Cys Gly Thr Val Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro Tyr Gly Asp Arg Leu
225 230 235 240
Phe Phe Tyr Leu Arg Lys Glu Gln Met Phe AlaArg His Phe Phe Asn
245 250 255
Arg Ala Gly Thr Val Gly Glu Pro Val Pro Asp Asp Leu Leu Val Lys
260 265 270
Gly Gly Asn Asn Arg Ser Ser Val Ala Ser Ser Ile Tyr Val His Thr
275 280 285
Pro Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe Asn Lys Pro
290 295 300
Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly
305 310 315 320
Asn His Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met
325 330 335
Thr Leu Cys Ala Ser Val Ser Lys Ser Ala Thr Tyr Thr Asn Ser Asp
340 345 350
Tyr Lys Glu Tyr Met Arg His Val Glu Glu Phe Asp Leu Gln Phe Ile
355 360 365
Phe Gln Leu Cys Ser Ile Thr Leu Ser Ala Glu Val Met Ala Tyr Ile
370 375 380
His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe Gly Leu Ser
385 390 395 400
Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr Arg Tyr Val Gln Ser Gln
405 410 415
Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu Lys Gln Asp Pro
420 425 430
Tyr Lys Asp Met Ser Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser
435 440 445
Ser Glu Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser
450 455 460
Gly
465
<210>3
<211>467
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV16 L1
<400>3
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala
20 25 30
Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys
35 40 45
Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu Gln
50 55 60
Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe
65 70 75 80
Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala
85 90 95
Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile
100 105 110
Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser
115 120 125
Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met
130 135 140
Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile
145 150 155 160
Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn
165 170 175
Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp
180 185 190
Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu
195 200 205
Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys
210 215 220
Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser
225 230 235 240
Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe
245 250 255
Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile
260 265 270
Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro
275 280 285
Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn Lys
290 295 300
Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn
340 345 350
Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln
355 360 365
Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr
370 375 380
Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly
385 390 395 400
Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val
405 410 415
Thr Gln Ala Ile Ala Cys Gln Lys His Thr Pro Pro Ala Pro Lys Glu
420 425 430
Asp Ser Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
435 440 445
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
450 455 460
Gln Ala Gly
465
<210>4
<211>469
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV18 L1
<400>4
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Pro Ser Val Ala Arg Val
1 5 10 15
Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser Ile Phe Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg Val Pro
35 40 45
Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys Val Ser Ala Tyr Gln
50 55 60
Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe Gly Leu
65 70 75 80
Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp Ala
85 90 95
Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Leu
100 105 110
Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser Ser His
115 120 125
Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val
130 135 140
Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile
145 150 155 160
Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser
165 170 175
Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp
180 185 190
Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu
195 200 205
Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys
210 215 220
Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser
225 230 235 240
Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp
245 250 255
Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile
260 265 270
Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro
275 280 285
Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp
305 310 315 320
His Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp
340 345 350
Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met
370 375 380
Ser Tyr Ile Gln Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe
385 390 395 400
Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe
405 410 415
Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu
420 425 430
Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys
435 440 445
Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe
450 455 460
Leu Val Gln Ala Gly
465
<210>5
<211>469
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV26 L1
<400>5
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Thr Pro Val Ser Arg Val
1 5 10 15
Val Asn Thr Asp Glu Tyr Val Thr Arg Thr Gly Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Leu Gly His Pro Tyr Phe Ser Ile Pro
35 40 45
Lys Thr Gly Gln Lys Ala Glu Ile Pro Lys Val Ser Ala Tyr Gln Tyr
50 55 60
Arg Val Phe Arg Val His Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro
65 70 75 80
Asp Pro Gln Leu Tyr Asn Pro Asp Thr Glu Arg Leu Val Trp Ala Cys
85 90 95
Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Ile Gly Leu Ser
100 105 110
Gly His Pro Leu Phe Asn Lys Leu Asp Asp Thr Glu Asn Ser His Leu
115 120 125
Ala Thr Val Asn Ala Asp Thr Asp Asn Arg Asp Asn Val Ser Val Asp
130 135 140
Asn Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Thr Pro Pro Leu Gly
145 150 155 160
Glu His Trp Gly Ile Gly Thr Ile Cys Lys Asn Thr Gln Thr Gln Arg
165 170 175
Gly Asp Cys Pro Pro Leu Glu Leu Ile Ser Ser Ile Ile Glu Asp Gly
180 185 190
Asp Met Ile Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Ala Leu Gln
195 200 205
Ala Thr Lys Ser Asp Val Pro Ile Asp Ile Ser Gln Ser Thr Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Thr Tyr Gly Asn Ser Met
225 230 235 240
Phe Phe Phe Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Tyr Asn
245 250 255
Lys Ala Gly Ala Val Gly Asp Ala Ile Pro Thr Thr Leu Tyr Ile Lys
260 265 270
Gly Ala Glu Ser Gly Arg Glu Pro Pro Thr Ser Ser Ile Tyr Ser Ala
275 280 285
Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Gly Asn Gln Leu Phe Val Thr Cys Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Leu Thr Ile Ser Thr Leu Ser Ala Ala Ser Ala Ser Thr Pro Phe Lys
340 345 350
Pro Ser Asp Tyr Lys Gln Phe Ile Arg His Gly Glu Glu Tyr Glu Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Thr Asp Val Met
370 375 380
Ala Tyr Ile His Leu Met Asn Ala Ser Ile Leu Glu Asp Trp Asn Phe
385 390 395 400
Gly Leu Thr Leu Pro Pro Thr Ala Ser Leu Glu Asp Ala Tyr Arg Phe
405 410 415
Ile Lys Asn Ser Ala Thr Thr Cys Gln Arg Asn Ala Pro Pro Val Pro
420 425 430
Lys Glu Asp Pro Phe Gln Lys Phe Lys Phe Trp Asp Val Asp Leu Lys
435 440 445
Glu Lys Phe Ser Ile Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
450 455 460
Met Leu Gln Ala Gly
465
<210>6
<211>469
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV31 L1
<400>6
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn Ile Tyr Tyr His Ala
20 25 30
Gly Ser Ala Arg Leu Leu Thr Val Gly His Pro Tyr Tyr Ser Ile Pro
35 40 45
Lys Ser Asp Asn Pro Lys Lys Ile Val Val Pro Lys Val Ser Gly Leu
50 55 60
Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly
65 70 75 80
Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp
85 90 95
Ala Cys Val Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly
100 105 110
Ile Ser Gly His Pro Leu Leu Asn Lys Phe Asp Asp Thr Glu Asn Ser
115 120 125
Asn Arg Tyr Ala Gly Gly Pro Gly Thr Asp Asn Arg Glu Cys Ile Ser
130 135 140
Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Leu Gly Cys Lys Pro Pro
145 150 155 160
Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Ser Asn Asn Ala Ile
165 170 175
Thr Pro Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Ser Val Ile Gln
180 185 190
Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Ala
195 200 205
Leu Gln Asp Thr Lys Ser Asn Val Pro Leu Asp Ile Cys Asn Ser Ile
210 215 220
Cys Lys Tyr Pro Asp Tyr Leu Lys Met Val Ala Glu Pro Tyr Gly Asp
225 230 235 240
Thr Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Phe
245 250 255
Phe Asn Arg Ser Gly Thr Val Gly Glu Ser Val Pro Thr Asp Leu Tyr
260 265 270
Ile Lys Gly Ser Gly Ser Thr Ala Thr Leu Ala Asn Ser Thr Tyr Phe
275 280 285
Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn
290 295 300
Lys Pro Tyr Trp Met Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys
305 310 315 320
Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr
325 330 335
Asn Met Ser Val Cys Ala Ala Ile Ala Asn Ser Asp Thr Thr Phe Lys
340 345 350
Ser Ser Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Phe Asp Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Ser Ala Asp Ile Met
370 375 380
Thr Tyr Ile His Ser Met Asn Pro Ala Ile Leu Glu Asp Trp Asn Phe
385 390 395 400
Gly Leu Thr Thr Pro Pro Ser Gly Ser Leu Glu Asp Thr Tyr Arg Phe
405 410 415
Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Thr Ala Pro Gln Lys Pro
420 425 430
Lys Glu Asp Pro Phe Lys Asp Tyr Val Phe Trp Glu Val Asn Leu Lys
435 440 445
Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
450 455 460
Leu Leu Gln Ala Gly
465
<210>7
<211>467
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV33 L1
<400>7
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Ser Ile Lys
35 40 45
Asn Pro Thr Asn Ala Lys Lys Leu Leu Val Pro Lys Val Ser Gly Leu
50 55 60
Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly
65 70 75 80
Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp
85 90 95
Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly
100 105 110
Ile Ser Gly His Pro Leu Leu Asn Lys Phe Asp Asp Thr Glu Thr Gly
115 120 125
Asn Lys Tyr Pro Gly Gln Pro Gly Ala Asp Asn Arg Glu Cys Leu Ser
130 135 140
Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Leu Gly Cys Lys Pro Pro
145 150 155 160
Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Thr Asn Ala Ala Pro
165 170 175
Ala Asn Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Ile Ile Glu Asp
180 185 190
Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Lys Thr Leu
195 200 205
Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Gly Ser Thr Cys
210 215 220
Lys Tyr Pro Asp Tyr Leu Lys Met Thr Ser Glu Pro Tyr Gly Asp Ser
225 230 235 240
Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe
245 250 255
Asn Arg Ala Gly Thr Leu Gly Glu Ala Val Pro Asp Asp Leu Tyr Ile
260 265 270
Lys Gly Ser Gly Thr Thr Ala Ser Ile Gln Ser Ser Ala Phe Phe Pro
275 280 285
Thr Pro Ser Gly Ser Met Val Thr Ser Glu Ser Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Gly Asn Gln Val Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Met Thr Leu Cys Thr Gln Val Thr Ser Asp Ser Thr Tyr Lys Asn Glu
340 345 350
Asn Phe Lys Glu Tyr Ile Arg His Val Glu Glu Tyr Asp Leu Gln Phe
355 360 365
Val Phe Gln Leu Cys Lys Val Thr Leu Thr Ala Glu Val Met Thr Tyr
370 375 380
Ile His Ala Met Asn Pro Asp Ile Leu Glu Asp Trp Gln Phe Gly Leu
385 390 395 400
Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe Val Thr
405 410 415
Ser Gln Ala Ile Thr Cys Gln Lys Thr Val Pro Pro Lys Glu Lys Glu
420 425 430
Asp Pro Leu Gly Lys Tyr Thr Phe Trp Glu Val Asp Leu Lys Glu Lys
435 440 445
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
450 455 460
Gln Ala Gly
465
<210>8
<211>469
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV35 L1
<400>8
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Ser Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn Ile Tyr Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Tyr Ala Ile Lys
35 40 45
Lys Gln Asp Ser Asn Lys Ile Ala Val Pro Lys Val Ser Gly Leu Gln
50 55 60
Tyr Arg Val Phe Arg Val Lys Leu Pro Asp Pro Asn Lys Phe Gly Phe
65 70 75 80
Pro Asp Thr Ser Phe Tyr Asp Pro Cys Leu Gln Arg Leu Val Trp Ala
85 90 95
Cys Thr Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile
100 105 110
Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Leu Asn
115 120 125
Lys Tyr Val Gly Asn Ser Gly Asn Ser Gly Thr Asp Asn Arg Glu Cys
130 135 140
Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Arg
145 150 155 160
Pro Pro Ile Gly Glu His Trp Gly Lys Gly Thr Pro Cys Asn AlaAsn
165 170 175
Gln Val Lys Ala Gly Glu Cys Pro Pro Leu Glu Leu Leu Asn Thr Val
180 185 190
Leu Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe
195 200 205
Thr Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Leu Asp Ile Cys Ser
210 215 220
Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Lys Met Val Ser Glu Pro Tyr
225 230 235 240
Gly Asp Met Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg
245 250 255
His Leu Phe Asn Arg Ala Gly Thr Val Gly Glu Thr Val Pro Ala Asp
260 265 270
Leu Tyr Ile Lys Gly Thr Thr Gly Thr Leu Pro Ser Thr Ser Tyr Phe
275 280 285
Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn
290 295 300
Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys
305 310 315 320
Trp Ser Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr
325 330 335
Asn Met Ser Val Cys Ser Ala Val Ser Ser Ser Asp Ser Thr Tyr Lys
340 345 350
Asn Asp Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met
370 375 380
Thr Tyr Ile His Ser Met Asn Pro Ser Ile Leu Glu Asp Trp Asn Phe
385 390 395 400
Gly Leu Thr Pro Pro Pro Ser Gly Thr Leu Glu Asp Thr Tyr Arg Tyr
405 410 415
Val Thr Ser Gln Ala Val Thr Cys Gln Lys Pro Ser Ala Pro Lys Pro
420 425 430
Lys Asp Asp Pro Leu Lys Asn Tyr Thr Phe Trp Glu Val Asp Leu Lys
435 440 445
Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
450 455 460
Leu Leu Gln Ala Gly
465
<210>9
<211>468
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV39 L1
<400>9
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Pro Ser Val Ala Lys Val
1 5 10 15
Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Gly Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Val Gly His Pro Tyr Phe Lys Val Gly
35 40 45
Met Asn Gly Gly Arg Lys Gln Asp Ile Pro Lys Val Ser Ala Tyr Gln
50 55 60
Tyr Arg Val Phe Arg Val Thr Leu Pro Asp Pro Asn Lys Phe Ser Ile
65 70 75 80
Pro Asp Ala Ser Leu Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp Ala
85 90 95
Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile
100 105 110
Ser Gly His Pro Leu Tyr Asn Arg Gln Asp Asp Thr Glu Asn Ser Pro
115 120 125
Phe Ser Ser Thr Thr Asn Lys Asp Ser Arg Asp Asn Val Ser Val Asp
130 135 140
Tyr Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Val Pro Ala Ile Gly
145 150 155 160
Glu His Trp Gly Lys Gly Lys Ala Cys Lys Pro Asn Asn Val Ser Thr
165 170 175
Gly Asp Cys Pro Pro Leu Glu Leu Val Asn Thr Pro Ile Glu Asp Gly
180 185 190
Asp Met Ile Asp Thr Gly Tyr Gly Ala Met Asp Phe Gly Ala Leu Gln
195 200 205
Glu Thr Lys Ser Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Val Tyr Gly Asp Ser Met
225 230 235 240
Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn
245 250 255
Arg Gly Gly Met Val Gly Asp Ala Ile Pro Ala Gln Leu Tyr Ile Lys
260 265 270
Gly Thr Asp Ile Arg Ala Asn Pro Gly Ser Ser Val Tyr Cys Pro Ser
275 280 285
Pro Ser Gly Ser Met Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro
290 295 300
Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Ile Cys Trp His
305 310 315 320
Asn Gln Leu Phe Leu Thr Val Val Asp Thr Thr Arg Ser Thr Asn Phe
325 330 335
Thr Leu Ser Thr Ser Ile Glu Ser Ser Ile Pro Ser Thr Tyr Asp Pro
340 345 350
Ser Lys Phe Lys Glu Tyr Thr Arg His Val Glu Glu Tyr Asp Leu Gln
355 360 365
Phe Ile Phe Gln Leu Cys Thr Val Thr Leu Thr Thr Asp Val Met Ser
370 375 380
Tyr Ile His Thr Met Asn Ser Ser Ile Leu Asp Asn Trp Asn Phe Ala
385 390 395 400
Val Ala Pro Pro Pro Ser Ala Ser Leu Val Asp Thr Tyr Arg Tyr Leu
405 410 415
Gln Ser Ala Ala Ile Thr Cys Gln Lys Asp Ala Pro Ala Pro Glu Lys
420 425 430
Lys Asp Pro Tyr Asp Gly Leu Lys Phe Trp Asn Val Asp Leu Arg Glu
435 440 445
Lys Phe Ser Leu Glu Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu
450 455 460
Leu Gln Ala Arg
465
<210>10
<211>467
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV42L1
<400>10
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Pro Pro Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Gln Arg Thr Asn Tyr Phe Tyr His Ala
20 25 30
Ser Ser Ser Arg Leu Leu Val Val Gly His Pro Tyr Tyr Ser Ile Thr
35 40 45
Lys Arg Pro Asn Lys Thr Ser Ile Pro Lys Val Ser Gly Leu Gln Tyr
50 55 60
Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Thr Leu Pro
65 70 75 80
Glu Thr Asn Leu Tyr Asn Pro Glu Thr Gln Arg Met Val Trp Ala Cys
85 90 95
Val Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser
100 105 110
Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Pro Thr
115 120 125
Tyr Gly Gly Gly Pro Gly Thr Asp Asn Arg Glu Asn Val Ser Met Asp
130 135 140
Tyr Lys Gln Thr Gln Leu Cys Leu Val Gly Cys Lys Pro Ala Ile Gly
145 150 155 160
Glu His Trp Gly Lys Gly Thr Ala Cys Thr Pro Gln Ser Asn Gly Asp
165 170 175
Cys Pro Pro Leu Glu Leu Lys Asn Ser Phe Ile Gln Asp Gly Asp Met
180 185 190
Val Asp Val Gly Phe Gly Ala Leu Asp Phe Gly Ala Leu Gln Ser Ser
195 200 205
Lys Ala Glu Val Pro Leu Asp Ile Val Asn Ser Ile Thr Lys Tyr Pro
210 215 220
Asp Tyr Leu Lys Met Ser Ala Glu Ala Tyr Gly Asp Ser Met Phe Phe
225 230 235 240
Phe Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe Asn Arg Ala
245 250 255
Gly Ala Ile Gly Glu Pro Val Pro Asp Glu Leu Tyr Thr Lys Ala Ala
260 265 270
Asn Asn Ala Ser Gly Arg His Asn Leu Gly Ser Ser Ile Tyr Tyr Pro
275 280 285
Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu Gln Gln Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Gly Asn Gln Leu Phe Leu Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Met Thr Leu Cys Ala Thr Ala Thr Ser Gly Asp Thr Tyr Thr Ala Ala
340 345 350
Asn Phe Lys Glu Tyr Leu Arg His Ala Glu Glu Tyr Asp Val Gln Phe
355 360 365
Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Val Glu Val Met Ser Tyr
370 375 380
Ile His Asn Met Asn Pro Asn Ile Leu Glu Glu Trp Asn Val Gly Val
385 390 395 400
Ala Pro Pro Pro Ser Gly Thr Leu Glu Asp Ser Tyr Arg Tyr Val Gln
405 410 415
Ser Glu Ala Ile Arg Cys Gln Ala Lys Val Thr Thr Pro Glu Lys Lys
420 425 430
Asp Pro Tyr Ser Asp Phe Trp Phe Trp Glu Val Asn Leu Ser Glu Lys
435 440 445
Phe Ser Thr Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
450 455 460
Gln Ala Gly
465
<210>11
<211>472
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV45 L1
<400>11
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Pro Ser Val Ala Arg Val
1 5 10 15
Val Ser Thr Asp Asp Tyr Val Ser Arg Thr Ser Ile Phe Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg Val Val
35 40 45
Pro Asn Gly Ala Gly Asn Lys Gln Ala Val Pro Lys Val Ser Ala Tyr
50 55 60
Gln Tyr Arg Val Phe Arg Val Ala Leu Pro Asp Pro Asn Lys Phe Gly
65 70 75 80
Leu Pro Asp Ser Thr Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp
85 90 95
Ala Cys Val Gly Met Glu Ile Gly Arg Gly Gln Pro Leu Gly Ile Gly
100 105 110
Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser Ala
115 120 125
His Ala Ala Thr Ala Val Ile Thr Gln Asp Val Arg Asp Asn Val Ser
130 135 140
Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Val Pro Ala
145 150 155 160
Ile Gly Glu His Trp Ala Lys Gly Thr Leu Cys Lys Pro Ala Gln Leu
165 170 175
Gln Pro Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Ile Ile Glu
180 185 190
Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr
195 200 205
Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile
210 215 220
Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp
225 230 235 240
Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe
245 250 255
Trp Asn Arg Ala Gly Val Met Gly Asp Thr Val Pro Thr Asp Leu Tyr
260 265 270
Ile Lys Gly Thr Ser Ala Asn Met Arg Glu Thr Pro Gly Ser Cys Val
275 280 285
Tyr Ser Pro Ser Pro Ser Gly Ser Ile Ile Thr Ser Asp Ser Gln Leu
290 295 300
Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly
305 310 315 320
Ile Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg
325 330 335
Ser Thr Asn Leu Thr Leu Cys Ala Ser Thr Gln Asn Pro Val Pro Ser
340 345 350
Thr Tyr Asp Pro Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu
355 360 365
Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala
370 375 380
Glu Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu Glu Asn
385 390 395 400
Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr
405 410 415
Tyr Arg Phe Val Gln Ser Val Ala Val Thr Cys Gln Lys Asp Thr Thr
420 425 430
Pro Pro Glu Lys Gln Asp Pro Tyr Asp Lys Leu Lys Phe Trp Thr Val
435 440 445
Asp Leu Lys Glu Lys Phe Ser Ser Asp Leu Asp Gln Tyr Pro Leu Gly
450 455 460
Arg Lys Phe Leu Val Gln Ala Gly
465 470
<210>12
<211>468
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV51 L1
<400>12
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Ala Pro Val Ser Arg Ile
1 5 10 15
Val Asn Thr Glu Glu Tyr Ile Thr Arg Thr Gly Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Ile Thr Leu Gly His Pro Tyr Phe Pro Ile Pro
35 40 45
Lys Thr Ser Thr Arg Ala Ala Ile Pro Lys Val Ser Ala Phe Gln Tyr
50 55 60
Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro
65 70 75 80
Asp Pro Asn Leu Tyr Asn Pro Asp Thr Asp Arg Leu Val Trp Gly Cys
85 90 95
Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Leu Ser
100 105 110
Gly His Pro Leu Phe Asn Lys Tyr Asp Asp Thr Glu Asn Ser Arg Ile
115 120 125
Ala Asn Gly Asn Ala Gln Gln Asp Val Arg Asp Asn Thr Ser Val Asp
130 135 140
Asn Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Ala Pro Pro Ile Gly
145 150 155 160
Glu His Trp Gly Ile Gly Thr Thr Cys Lys Asn Thr Pro Val Pro Pro
165 170 175
Gly Asp Cys Pro Pro Leu Glu Leu Val Ser Ser Val Ile Gln Asp Gly
180 185 190
Asp Met Ile Asp Thr Gly Phe Gly Ala Met Asp Phe Ala Ala Leu Gln
195 200 205
Ala Thr Lys Ser Asp Val Pro Leu Asp Ile Ser Gln Ser Val Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Thr Tyr Gly Asn Ser Met
225 230 235 240
Phe Phe His Leu Arg Arg Glu Gln Ile Phe Ala Arg His Tyr Tyr Asn
245 250 255
Lys Leu Val Gly Val Gly Glu Asp Ile Pro Asn Asp Tyr Tyr Ile Lys
260 265 270
Gly Ser Gly Asn Gly Arg Asp Pro Ile Glu Ser Tyr Ile Tyr Ser Ala
275 280 285
Thr Pro Ser Gly Ser Met Ile Thr Ser Asp Ser Gln Ile Phe Asn Lys
290 295 300
Pro Tyr Trp Leu His Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Asn Asn Gln Leu Phe Ile Thr Cys Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Leu Thr Ile Ser Thr Ala Thr Ala Ala Val Ser Pro Thr Phe Thr Pro
340 345 350
Ser Asn Phe Lys Gln Tyr Ile Arg His Gly Glu Glu Tyr Glu Leu Gln
355 360 365
Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Thr Glu Val Met Ala
370 375 380
Tyr Leu His Thr Met Asp Pro Thr Ile Leu Glu Gln Trp Asn Phe Gly
385 390 395 400
Leu Thr Leu Pro Pro Ser Ala Ser Leu Glu Asp Ala Tyr Arg Phe Val
405 410 415
Arg Asn Ala Ala Thr Ser Cys Gln Lys Asp Thr Pro Pro Gln Ala Lys
420 425 430
Pro Asp Pro Leu Ala Lys Tyr Lys Phe Trp Asp Val Asp Leu Lys Glu
435 440 445
Arg Phe Ser Leu Asp Leu Asp Gln Phe Ala Leu Gly Arg Lys Phe Leu
450 455 460
Leu Gln Val Gly
465
<210>13
<211>472
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV52 L1
<400>13
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Val Gly His Pro Tyr Phe Ser Ile Lys
35 40 45
Asn Thr Ser Ser Gly Asn Gly Lys Lys Val Leu Val Pro Lys Val Ser
50 55 60
Gly Leu Gln Tyr Arg Val Phe Arg Ile Lys Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu Thr Gln Arg Leu
85 90 95
Val Trp Ala Cys Thr Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Phe Asp Asp Thr Glu
115 120 125
Thr Ser Asn Lys Tyr Ala Gly Lys Pro Gly Ile Asp Asn Arg Glu Cys
130 135 140
Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Lys
145 150 155 160
Pro Pro Ile Gly Glu His Trp Gly Lys Gly Thr Pro Cys Asn Asn Asn
165 170 175
Ser Gly Asn Pro Gly Asp Cys Pro Pro Leu Gln Leu Ile Asn Ser Val
180 185 190
Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe
195 200 205
Asn Thr Leu Gln Ala Ser Lys Ser Asp Val Pro Ile Asp Ile Cys Ser
210 215 220
Ser Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ser Glu Pro Tyr
225 230 235 240
Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg
245 250 255
His Phe Phe Asn Arg Ala Gly Thr Leu Gly Asp Pro Val Pro Gly Asp
260 265 270
Leu Tyr Ile Gln Gly Ser Asn Ser Gly Aan Thr Ala Thr Val Gln Ser
275 280 285
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Glu Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Glu Val Lys Lys Glu Ser
340 345 350
Thr Tyr Lys Asn Glu Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu
355 360 365
Phe Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
370 375 380
Asp Val Met Thr Tyr Ile His Lys Met Asp Ala Thr Ile Leu Glu Asp
385 390 395 400
Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Glu Asp Thr
405 410 415
Tyr Arg Phe Val Thr Ser Thr Ala Ile Thr Cys Gln Lys Asn Thr Pro
420 425 430
Pro Lys Gly Lys Glu Asp Pro Leu Lys Asp Tyr Met Phe Trp Glu Val
435 440 445
Asp Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
450 455 460
Arg Lys Phe Leu Leu Gln Ala Gly
465 470
<210>14
<211>465
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV53 L1
<400>14
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Thr Pro Val Ser Lys Val
1 5 10 15
Ile Thr Thr Asp Ala Tyr Val Lys Arg Thr Thr Ile Phe Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Val Gly His Pro Tyr Tyr Pro Ile Ser
35 40 45
Lys Ser Gly Lys Ala Asp Ile Pro Lys Val Ser Ala Phe Gln Tyr Arg
50 55 60
Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro Asp
65 70 75 80
Thr Asn Ile Phe Asn Pro Asp Gln Glu Arg Leu Val Trp Ala Cys Val
85 90 95
Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Val Ser Gly
100 105 110
His Pro Leu Phe Asn Arg Leu Asp Asp Thr Glu Ser Ser Ser Ile Ala
115 120 125
Ile Gln Asp Thr Ala Pro Asp Ser Arg Asp Asn Val Ser Val Asp Pro
130 135 140
Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Ala Pro Ala Ile Gly Glu
145 150 155 160
His Trp Thr Lys Gly Thr Ala Cys Arg Ser Thr Pro Thr Thr Ala Gly
165 170 175
Asp Cys Pro Pro Leu Glu Leu Ile Asn Ser Pro Ile Glu Asp Gly Asp
180 185 190
Met Val Asp Thr Gly Phe Gly Ala Leu Asn Phe Lys Ala Leu Gln Glu
195 200 205
Ser Lys Ser Asp Val Pro Leu Asp Ile Val Gln Ser Thr Cys Lys Tyr
210 215 220
Pro Asp Tyr Leu Lys Met Ser Ala Asp Ala Tyr Gly Asp Ser Met Trp
225 230 235 240
Phe Tyr Leu Arg Arg Glu Gln Leu Phe Thr Arg His Phe Phe Asn Arg
245 250 255
Ala Gly Val Ile Gly Glu Glu Ile Pro Asn Asp Leu Tyr Ile Lys Gly
260 265 270
Ser Asn Gly Arg Asp Pro Pro Pro Ser Ser Val Tyr Val Ala Thr Pro
275 280 285
Ser Gly Ser Met Ile Thr Ser Glu Ala Gln Leu Phe Asn Lys Pro Tyr
290 295 300
Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Asn Asn
305 310 315 320
Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Asn Thr Asn Met Thr
325 330 335
Leu Ser Ala Thr Thr Gln Ser Met Ser Thr Tyr Asn Ser Lys Gln Ile
340 345 350
Lys Gln Tyr Val Arg His Ala Glu Glu Tyr Glu Leu Gln Phe Val Phe
355 360 365
Gln Leu Cys Lys Ile Ser Leu Ser Ala Glu Val Met Ala Tyr Leu His
370 375 380
Thr Met Asn Ser Thr Leu Leu Glu Asp Trp Asn Ile Gly Leu Ser Pro
385 390 395 400
Pro Val Ala Thr Ser Leu Glu Asp Lys Tyr Arg Tyr Val Lys Ser Ala
405 410 415
Ala Ile Thr Cys Gln Lys Asp Gln Pro Pro Pro Glu Lys Gln Asp Pro
420 425 430
Leu Ser Lys Tyr Lys Phe Trp Glu Val Asn Leu Gln Asn Ser Phe Ser
435 440 445
Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Met Gln Val
450 455 460
Gly
465
<210>15
<211>466
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV56 L1
<400>15
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Thr Pro Val Ser Lys Val
1 5 10 15
Val Ala Thr Asp Ser Tyr Val Lys Arg Thr Ser Ile Phe Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Tyr Ser Val Thr
35 40 45
Lys Asp Asn Thr Lys Thr Asn Ile Pro Lys Val Ser Ala Tyr Gln Tyr
50 55 60
Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro
65 70 75 80
Asp Thr Asn Ile Tyr Asn Pro Asp Gln Glu Arg Leu Val Trp Ala Cys
85 90 95
Val Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Ala Gly Leu Ser
100 105 110
Gly His Pro Leu Phe Asn Arg Leu Asp Asp Thr Glu Ser Ser Asn Leu
115 120 125
Ala Asn Asn Asn Val Ile Glu Asp Ser Arg Asp Asn Ile Ser Val Asp
130 135 140
Gly Lys Gln Thr Gln Leu Cys Ile Val Gly Cys Thr Pro Ala Met Gly
145 150 155 160
Glu His Trp Thr Lys Gly Ala Val Cys Lys Ser Thr Gln Val Thr Thr
165 170 175
Gly Asp Cys Pro Pro Leu Ala Leu Ile Asn Thr Pro Ile Glu Asp Gly
180 185 190
Asp Met Ile Asp Thr Gly Phe Gly Ala Met Asp Phe Lys Val Leu Gln
195 200 205
Glu Ser Lys Ala Glu Val Pro Leu Asp Ile Val Gln Ser Thr Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Ala Tyr Gly Asp Ser Met
225 230 235 240
Trp Phe Tyr Leu Arg Arg Glu Gln Leu Phe Ala Arg His Tyr Phe Asn
245 250 255
Arg Ala Gly Lys Val Gly Glu Thr Ile Pro Ala Glu Leu Tyr Leu Lys
260 265 270
Gly Ser Asn Gly Arg Glu Pro Pro Pro Ser Ser Val Tyr Val Ala Thr
275 280 285
Pro Ser Gly Ser Met Ile Thr Ser Glu Ala Gln Leu Phe Asn Lys Pro
290 295 300
Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly
305 310 315 320
Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met
325 330 335
Thr Ile Ser Thr Ala Thr Glu Gln Leu Ser Lys Tyr Asp Ala Arg Lys
340 345 350
Ile Asn Gln Tyr Leu Arg His Val Glu Glu Tyr Glu Leu Gln Phe Val
355 360 365
Phe Gln Leu Cys Lys Ile Thr Leu Ser Ala Glu Val Met Ala Tyr Leu
370 375 380
His Asn Met Asn Ala Asn Leu Leu Glu Asp Trp Asn Ile Gly Leu Ser
385 390 395 400
Pro Pro Val Ala Thr Ser Leu Glu Asp Lys Tyr Arg Tyr Val Arg Ser
405 410 415
Thr Ala Ile Thr Cys Gln Arg Glu Gln Pro Pro Thr Glu Lys Gln Asp
420 425 430
Pro Leu Ala Lys Tyr Lys Phe Trp Asp Val Asn Leu Gln Asp Ser Phe
435 440 445
Ser Thr Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Met Gln
450 455 460
Leu Gly
465
<210>16
<211>467
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV58 L1
<400>16
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys
35 40 45
Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser Gly Leu
50 55 60
Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly
65 70 75 80
Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp
85 90 95
Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly
100 105 110
Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu Thr Ser
115 120 125
Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys Leu Ser
130 135 140
Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro
145 150 155 160
Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn Ala Ala
165 170 175
Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile Glu Asp
180 185 190
Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly Thr Leu
195 200 205
Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys
210 215 220
Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly Asp Ser
225 230 235 240
Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe
245 250 255
Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu Tyr Ile
260 265 270
Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe Phe Pro
275 280 285
Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys Asn Asp
340 345 350
Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu Gln Phe
355 360 365
Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met Thr Tyr
370 375 380
Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe Gly Leu
385 390 395 400
Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe Val Thr
405 410 415
Ser Gln Ala Ile Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu Lys Glu
420 425 430
Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
435 440 445
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
450 455 460
Gln Ser Gly
465
<210>17
<211>469
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV59 L1
<400>17
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Pro Ser Val Ala Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Ser Ile Phe Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Thr Val Gly His Pro Tyr Phe Lys Val Pro
35 40 45
Lys Gly Gly Asn Gly Arg Gln Asp Val Pro Lys Val Ser Ala Tyr Gln
50 55 60
Tyr Arg Val Phe Arg Val Lys Leu Pro Asp Pro Asn Lys Phe Gly Leu
65 70 75 80
Pro Asp Asn Thr Val Tyr Asp Pro Asn Ser Gln Arg Leu Val Trp Ala
85 90 95
Cys Val Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Leu
100 105 110
Ser Gly His Pro Leu Tyr Asn Lys Leu Asp Asp Thr Glu Asn Ser His
115 120 125
Val Ala Ser Ala Val Asp Thr Lys Asp Thr Arg Asp Asn Val Ser Val
130 135 140
Asp Tyr Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Val Pro Ala Ile
145 150 155 160
Gly Glu His Trp Thr Lys Gly Thr Ala Cys Lys Pro Thr Thr Val Val
165 170 175
Gln G1y Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Pro Ile Glu Asp
180 185 190
Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Lys Leu Leu
195 200 205
Gln Asp Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys
210 215 220
Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Ala Tyr Gly Asp Ser
225 230 235 240
Met Phe Phe Cys Leu Arg Arg Glu Gln Val Phe Ala Arg His Phe Trp
245 250 255
Asn Arg Ser Gly Thr Met Gly Asp Gln Leu Pro Glu Ser Leu Tyr Ile
260 265 270
Lys Gly Thr Asp Ile Arg Ala Asn Pro Gly Ser Tyr Leu Tyr Ser Pro
275 280 285
Ser Pro Ser Gly Ser Val Val Thr Ser Asp Ser Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu His Lys Ala Gln Gly Leu Asn Asn Gly Ile Cys Trp
305 310 315 320
His Asn Gln Leu Phe Leu Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Leu Ser Val Cys Ala Ser Thr Thr Ser Ser Ile Pro Asn Val Tyr Thr
340 345 350
Pro Thr Ser Phe Lys Glu Tyr Ala Arg His Val Glu Glu Phe Asp Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Thr Glu Val Met
370 375 380
Ser Tyr Ile His Asn Met Asn Thr Thr Ile Leu Glu Asp Trp Asn Phe
385 390 395 400
Gly Val Thr Pro Pro Pro Thr Ala Ser Leu Val Asp Thr Tyr Arg Phe
405 410 415
Val Gln Ser Ala Ala Val Thr Cys Gln Lys Asp Thr Ala Pro Pro Val
420 425 430
Lys Gln Asp Pro Tyr Asp Lys Leu Lys Phe Trp Pro Val Asp Leu Lys
435 440 445
Glu Arg Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
450 455 460
Leu Leu Gln Leu Gly
465
<210>18
<211>466
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV66 L1
<400>18
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Thr Pro Val Ser Lys Val
1 5 10 15
Val Ala Thr Asp Thr Tyr Val Lys Arg Thr Ser Ile Phe Tyr His Ala
20 25 30
Gly Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr Tyr Ser Val Ser
35 40 45
Lys Ser Gly Thr Lys Thr Asn Ile Pro Lys Val Ser Ala Tyr Gln Tyr
50 55 60
Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro
65 70 75 80
Asp Pro Ser Phe Tyr Asn Pro Asp Gln Glu Arg Leu Val Trp Ala Cys
85 90 95
Val Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Ala Gly Leu Ser
100 105 110
Gly His Pro Leu Phe Asn Arg Leu Asp Asp Thr Glu Val Ser Asn Leu
115 120 125
Ala Gly Asn Asn Val Ile Glu Asp Ser Arg Asp Asn Ile Ser Val Asp
130 135 140
Cys Lys Gln Thr Gln Leu Cys Ile Val Gly Cys Ala Pro Ala Leu Gly
145 150 155 160
Glu His Trp Thr Lys Gly Ala Val Cys Lys Ser Thr Pro Gly Asn Thr
165 170 175
Gly Asp Cys Pro Pro Leu Ala Leu Val Asn Thr Pro Ile Glu Asp Gly
180 185 190
Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Lys Leu Leu Gln
195 200 205
Glu Ser Lys Ala Glu Val Pro Leu Asp Ile Val Gln Ser Thr Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Ala Tyr Gly Asp Ser Met
225 230 235 240
Trp Phe Tyr Leu Arg Arg Glu Gln Leu Phe Ala Arg His Tyr Phe Asn
245 250 255
Arg Ala Gly Asn Val Gly Glu Ala Ile Pro Thr Asp Leu Tyr Trp Lys
260 265 270
Gly Gly Asn Gly Arg Asp Pro Pro Pro Ser Ser Val Tyr Val Ala Thr
275 280 285
Pro Ser Gly Ser Met Ile Thr Ser Glu Ala Gln Leu Phe Asn Lys Pro
290 295 300
Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly
305 310 315 320
Asn Gln Val Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met
325 330 335
Thr Ile Asn Ala Ala Lys Ser Thr Leu Thr Lys Tyr Asp Ala Arg Glu
340 345 350
Ile Asn Gln Tyr Leu Arg His Val Glu Glu Tyr Glu Leu Gln Phe Val
355 360 365
Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Val Met Ala Tyr Leu
370 375 380
His Asn Met Asn Asn Thr Leu Leu Asp Asp Trp Asn Ile Gly Leu Ser
385 390 395 400
Pro Pro Val Ala Thr Ser Leu Glu Asp Lys Tyr Arg Tyr Ile Lys Ser
405 410 415
Thr Ala Ile Thr Cys Gln Arg Glu Gln Pro Pro Ala Glu Lys Gln Asp
420 425 430
Pro Leu Ala Lys Tyr Lys Phe Trp Glu Val Asn Leu Gln Asp Ser Phe
435 440 445
Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Met Gln
450 455 460
Leu Gly
465
<210>19
<211>470
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV73 L1
<400>19
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Val Ser Val Ser Lys Val
1 5 10 15
Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Thr Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys
35 40 45
Asp Ser Gln Lys Arg Lys Thr Ile Val Pro Lys Val Ser Gly Leu Gln
50 55 60
Tyr Arg Val Phe Arg Leu Arg Leu Pro Asp Pro Asn Lys Phe Gly Phe
65 70 75 80
Pro Asp Ala Ser Phe Tyr Asn Pro Asp Lys Glu Arg Leu Val Trp Ala
85 90 95
Cys Ser Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Ile Gly Thr
100 105 110
Ser Gly Asn Pro Phe Met Asn Lys Leu Asp Asp Thr Glu Asn Ala Pro
115 120 125
Lys Tyr Ile Ala Gly Gln Asn Thr Asp Gly Arg Glu Cys Met Ser Val
130 135 140
Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Arg Pro Pro Leu
145 150 155 160
Gly Glu His Trp Gly Pro Gly Thr Pro Cys Thr Ser Gln Thr Val Asn
165 170 175
Thr Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Pro Ile Gln Asp
180 185 190
Gly Asp Met Ile Asp Val Gly Phe Gly Ala Met Asp Phe Lys Ala Leu
195 200 205
Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Ser Asn Thr Thr Cys
210 215 220
Lys Tyr Pro Asp Tyr Leu Gly Met Ala Ala Asp Pro Tyr Gly Asp Ser
225 230 235 240
Met Trp Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe
245 250 255
Asn Arg Ala Gly Asp Thr Gly Asp Lys Ile Pro Asp Asp Leu Met Ile
260 265 270
Lys Gly Thr Gly Asn Thr Ala Thr Pro Ser Ser Cys Val Phe Tyr Pro
275 280 285
Thr Pro Ser Gly Ser Met Val Ser Ser Asp Ala Gln Leu Phe Asn Lys
290 295 300
Pro Tyr Trp Leu Gln Lys Ala Gln Gly Gln Asn Asn Gly Ile Cys Trp
305 310 315 320
His Asn Gln Leu Phe Leu Thr Val Val Asp Thr Thr Arg Ser Thr Asn
325 330 335
Phe Ser Val Cys Val Gly Thr Gln Ala Ser Ser Ser Thr Thr Thr Tyr
340 345 350
Ala Asn Ser Asn Phe Lys Glu Tyr Leu Arg His Ala Glu Glu Phe Asp
355 360 365
Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Ser Leu Thr Thr Glu Val
370 375 380
Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Glu Trp Asn
385 390 395 400
Phe Gly Leu Thr Pro Pro Pro Ser Gly Thr Leu Glu Glu Thr Tyr Arg
405 410 415
Tyr Val Thr Ser Gln Ala Ile Ser Cys Gln Arg Pro Gln Pro Pro Lys
420 425 430
Glu Thr Glu Asp Pro Tyr Ala Lys Leu Ser Phe Trp Asp Val Asp Leu
435 440 445
Lys Glu Lys Phe Ser Ala Glu Leu Asp Gln Phe Pro Leu Gly Arg Lys
450 455 460
Phe Leu Leu Gln Leu Gly
465 470
<210>20
<211>469
<212>PRT
＜213〉artificial sequence
<220>
<223>HPV83 L1
<400>20
Gly Ser Gly Gly Gly Val Tyr Leu Pro Pro Ala Pro Val Ser Arg Ile
1 5 10 15
Val Asn Thr Glu Glu Tyr Ile Thr Arg Thr Gly Ile Tyr Tyr Tyr Ala
20 25 30
Gly Ser Ser Arg Leu Ile Thr Leu Gly His Pro Tyr Phe Ser Ile Pro
35 40 45
Lys Thr Asn Thr Arg Ala Glu Ile Pro Lys Val Ser Ala Phe Gln Tyr
50 55 60
Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro
65 70 75 80
Asp Pro Asn Leu Phe Asn Pro Asp Thr Asp Arg Leu Val Trp Gly Cys
85 90 95
Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Leu Ser
100 105 110
Gly His Pro Leu Phe Asn Lys Tyr Asp Asp Thr Glu Asn Ser Arg Phe
115 120 125
Ala Asn Gly Asn Asp Gln Gln Asp Val Arg Asp Asn Ile Ser Val Asp
130 135 140
Asn Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Ala Pro Pro Ile Gly
145 150 155 160
Glu His Trp Ala Thr Gly Thr Thr Cys Lys Asn Val Pro Val Pro Gln
165 170 175
Gly Asp Cys Pro Pro Leu Glu Leu Val Ser Thr Val Ile Glu Asp Gly
180 185 190
Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Ala Asn Leu Gln
195 200 205
Ala Thr Lys Ser Asp Val Pro Leu Asp Ile Ala Gln Ser Val Cys Lys
210 215 220
Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Thr Tyr Gly Asn Ser Met
225 230 235 240
Phe Phe His Leu Arg Arg Glu Gln Ile Phe Ala Arg His Tyr Tyr Asn
245 250 255
Lys Ala Gly Val Val Gly Asp Ala Ile Pro Asp Lys Ala Tyr Ile Lys
260 265 270
Gly Thr Gly Ala Gly Arg Asp Pro Ile Ser Ser Tyr Ile Tyr Ser Ala
275 280 285
Thr Pro Ser Gly Ser Met Ile Thr Ser Asp Ser Gln Ile Phe Asn Lys
290 295 300
Pro Tyr Trp Leu His Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp
305 310 315 320
Asn Asn Gln Leu Phe Ile Thr Cys Val Asp Thr Thr Lys Ser Thr Asn
325 330 335
Leu Thr Ile Ser Thr Ala Val Thr Pro Ser Val Ala Gln Thr Phe Thr
340 345 350
Pro Ala Asn Phe Lys Gln Tyr Ile Arg His Gly Glu Glu Tyr Glu Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Thr Glu Ile Met
370 375 380
Ala Tyr Leu His Thr Met Asp Ser Thr Ile Leu Glu Gln Trp Asn Phe
385 390 395 400
Gly Leu Thr Leu Pro Pro Ser Ala Ser Leu Glu Asp Ala Tyr Arg Phe
405 410 415
Val Lys Asn Ala Ala Thr Ser Cys His Lys Asp Ser Pro Pro Gln Ala
420 425 430
Lys Glu Asp Pro Leu Ala Lys Tyr Lys Phe Trp Asn Val Asp Leu Lys
435 440 445
Glu Arg Phe Ser Leu Asp Leu Asp Gln Phe Ala Leu Gly Arg Lys Phe
450 455 460
Leu Leu Gln Ile Gly
465

Claims (12)

1. the aminoacid sequence of a recombinant human mammilla tumor virus L 1 capsid protein, it is characterized in that the conserved sequence VYLPP of the proteic aminoacid sequence N-of wild-type HPV L1 end or the aminoacid sequence of VYVPP upstream are replaced to GSGGG, pass through at the 5th amino acid place, conserved sequence LGRKFL downstream of its C-end simultaneously to introduce the truncation type sequence of the terminator codon formation of protein translation with respect to wild-type, wherein said HPV type is selected from HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV73, among the HPV82 one or more.
2. aminoacid sequence according to claim 1, wherein the HPV6 corresponding amino acid sequence is shown in SEQ ID NO:1, the HPV11 corresponding amino acid sequence is shown in SEQ ID NO:2, the HPV16 corresponding amino acid sequence is shown in SEQID NO:3, the HPV18 corresponding amino acid sequence is shown in SEQ ID NO:4, the HPV26 corresponding amino acid sequence is shown in SEQ ID NO:5, the HPV31 corresponding amino acid sequence is shown in SEQ ID NO:6, the HPV33 corresponding amino acid sequence is shown in SEQ ID NO:7, the HPV35 corresponding amino acid sequence is shown in SEQ ID NO:8, the HPV39 corresponding amino acid sequence is shown in SEQ ID NO:9, the HPV42 corresponding amino acid sequence is shown in SEQ ID NO:10, the HPV45 corresponding amino acid sequence is shown in SEQ ID NO:11, the HPV51 corresponding amino acid sequence is shown in SEQ IDNO:12, HPV 52 corresponding amino acid sequence are shown in SEQ ID NO:13, the HPV53 corresponding amino acid sequence is shown in SEQ ID NO:14, the HPV56 corresponding amino acid sequence is shown in SEQ ID NO:15, the HPV58 corresponding amino acid sequence is shown in SEQ IDNO:16, the HPV59 corresponding amino acid sequence is shown in SEQ ID NO:17, the HPV66 corresponding amino acid sequence is shown in SEQ ID NO:18, the HPV73 corresponding amino acid sequence is shown in SEQID NO:19, and the HPV82 corresponding amino acid sequence is shown in SEQ ID NO:20.
3. a nucleic acid molecule is characterized in that it comprises the nucleotide sequence of the described aminoacid sequence of coding claim 1.
4. an expression vector is characterized in that it comprises the described nucleotide sequence of claim 3.
5. a host cell is characterized in that it comprises the described carrier of claim 4.
6. host cell according to claim 5 is characterized in that it is a Bacillus coli cells.
7. the proteic pentamer of HPV L1 is characterized in that its primary structure is made up of the described aminoacid sequence of claim 1, and its secondary structure and higher structure are determined by above-mentioned primary structure.
8. the application of the proteic pentamer of HPV L1 according to claim 7 in the vaccine that preparation prevention HPV infects.
9. application according to claim 8, the HPV type that it is characterized in that being used to preparing the vaccine that described prevention HPV infects comprises HPV16.
10. application according to claim 8, the HPV type that it is characterized in that being used to preparing the vaccine that described prevention HPV infects comprises HPV6, HPV11, HPV16, HPV18 and HPV33.
11. the application of the proteic pentamer of HPV L1 according to claim 7 in pharmaceutical compositions.
12. the application of the proteic pentamer of HPV L1 according to claim 7 in preparation immunodiagnosis antigen or antibody.
CNA2007100050994A 2007-02-14 2007-02-14 Amino acid sequence of recombined human papilloma virus L1 capsid protein and uses thereof Pending CN101245099A (en)

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CN101890160B (en) * 2009-04-28 2014-06-18 北京康乐卫士生物技术有限公司 Multivalent recombinant human papilloma virus vaccine and application thereof
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CN104710515B (en) * 2013-12-17 2019-11-29 北京康乐卫士生物技术股份有限公司 Human mammilla tumor virus L 1 protein mutant and preparation method thereof
CN104761623B (en) * 2013-12-17 2019-11-29 北京康乐卫士生物技术股份有限公司 A kind of inhibitor formed with the inhibition HPV L1 pentamer that alpha-helix 5 is target
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