CN104962567B - 6 type recombinant human papilloma virus-like particle and preparation method thereof - Google Patents

Abstract

The invention relates to a 6-type recombinant human papilloma virus-like particle and a preparation method thereof, and particularly discloses a novel polynucleotide gene segment for coding recombinant HPV 6L 1 protein, a vector containing the gene segment, a host cell comprising the vector, HPV 6L 1 fusion protein expressed by the translation of the gene segment, a pentamer and VLP (VLP) consisting of the pentamer, and application of the pentamer, the VLP protein and a vaccine composition consisting of the pentamer and the VLP protein in preparation of a medicament for preventing HPV6 infection.

Description

Type 6 recombinant human papillomavirus virus-like particle and preparation method thereof

technical field

The invention relates to a virus-like particle of human papilloma virus and a preparation method thereof. More specifically, the present invention relates to a pentamer of recombinant human papillomavirus L1 protein and a virus-like particle (Virus-like Particle, VLP) and a preparation method thereof, and a vaccine composition containing the virus-like particle in Use in the prevention of human papillomavirus infection.

Background technique

Human papillomavirus (HPV) mainly through close contact with human body, such as sexually transmitted virus, can cause a variety of human proliferative epithelial lesions, including papilloma (wart) and tumor-like lesions. Specifically, HPV-induced diseases mainly include three categories, the first category: cancers of the cervix, vagina, female vulva, penis and anus, and certain types of malignant lesions such as head and neck tumors. 100% of cervical cancer patients are caused by HPV infection, 90% of anal cancer, 40% of vulva, vagina and penis, 12% of oropharyngeal and 3% of oral cancer are attributed to HPV infection. Type 2: Benign lesions such as flat warts, condyloma acuminatum and other genital warts, which are sexually transmitted diseases and are very common among sexually active people. Although genital warts will not cause serious consequences like cancer, the lesions usually cause patients with more painful clinical symptoms such as burning pain, bleeding and pain, and at the same time produce negative psychological reactions such as embarrassment, anxiety and low self-esteem, and repeated treatment The process wastes a lot of medical resources. It is estimated that there are 30 million genital warts caused by non-carcinogenic HPV (mainly types 6 and 11) worldwide, and 20-50% of the lesions also contain mixed infection of high-risk HPV. Category 3: HPV infection can also cause recurrent respiratory papilloma (RRP), a rare, potentially fatal condition that occurs mainly in adolescence, and sometimes, a large number of papillomas can cause breathing difficulties and lead to Younger children died. Therefore, the prevention or treatment of HPV infection is of great significance to human health.

HPV is a non-enveloped double-stranded DNA virus, mainly composed of viral coat and genomic DNA (Bernard, Burket al. 2011). The HPV viral coat is an icosahedral structure composed of 360 L1 proteins (forming 72 pentamers) and up to 72 L2 proteins, with a diameter of 55–60 nm (Howley and Lowy 2007). Viral coat proteins have self-assembly properties. In vitro, L1 proteins can self-assemble alone or together with L2 proteins to form virus-like particles (Virus-likeParticle, VLP) (Chen, Garcea et al. 2000, Finnen, Erickson et al. 2003, Buck , Cheng et al. 2008, Wang and Roden 2013).

Since HPV cannot be cultured in vitro, the only way to obtain the specific antigen of the virus is to use recombinant DNA technology to prepare genetically engineered vaccines. Virus-like particles (VLPs) formed by recombinant L1 or L1/L2 assembly have no viral DNA, are safe, have antigenic epitopes similar to natural virus particles, and can produce neutralizing antibodies IgG and IgA after stimulating the body. Therefore, HPV VLPs can be used as Preventive vaccines, thereby greatly reducing the possibility of HPV-related tumors (Howley and Lowy 2007).

The key to the development of HPV vaccines is the ability to produce high-purity, stable HPV antigens in large quantities. In terms of preparation technology of HPV vaccine antigens, the commonly used expression systems for producing HPV antigens can be divided into eukaryotic expression systems and prokaryotic expression systems. Commonly used eukaryotic expression systems include poxvirus expression systems, insect baculovirus expression systems, and yeast expression systems. The HPV L1 expressed in the eukaryotic expression system can spontaneously form VLP, and often only need simple purification to obtain VLP. However, due to the low expression level of the eukaryotic expression system and the high cost of cultivation, it has brought great difficulties to large-scale industrial production. In the prokaryotic expression system, it has been reported that the expression system of Escherichia coli expresses HPV L1 protein. However, due to the low solubility of the HPV L1 protein expressed by Escherichia coli, most of the currently known purification methods obtain HPV VLP from the cell fluid with various protein types through steps such as salt-free precipitation or denaturation and renaturation. For example: in the patent CN02129070.9, the method for expressing and preparing HPV L1 multimers by prokaryotic cells is disclosed, wherein the purification process includes 3.3M urea treatment and dialysis refolding process; in the WO-0204007 patent, the L1-GST fusion protein The purification method is also through urea denaturation treatment and dialysis refolding; in the prior art, the purification method of L1 protein is also disclosed, which includes the steps of phosphate buffer ultrafiltration, dialysis and centrifugation, so that the target protein is precipitated and then redissolved. However, in these purification processes, the loss of protein is large and the yield is low, which makes it difficult to apply in large-scale production.

In terms of the homogeneity of the HPV vaccine antigen protein VLP, the particle size dispersion of the HPV L1 VLP assembled in the prior art is represented by the polyd value, and the polyd value <15% indicates that the particles have good uniformity, 15% to 30 % means that the particles have greater heterogeneity, and greater than 30% means that the particles are not uniform at all. Most of the HPV L1 VLPs prepared in the prior art are greater than 15%. Another indicator of uniform particle size is the PdI value, the PdI value is the particle size distribution coefficient, less than 0.05 is a highly uniform sample; for non-uniform samples. In the US7205125B2 patent, it is disclosed that the PdI of the mixed protein solution of two types of HPV L1 VLP is 0.07.

Therefore, there is still a need in the art for a low-cost, high-purity, high-yield, stable-quality HPV L1 protein production technology and a new method for large-scale industrial production of recombinant HPV L1 VLP.

Contents of the invention

The object of the present invention is to disclose an optimized nucleotide sequence encoding HPV6 L1 protein, a vector comprising the nucleotide sequence, a host cell comprising the vector, and an HPV L1 protein expressed by translation of the polynucleotide sequence, Tag - HPV-L1 recombinant protein, pentamers and VLPs formed by the L1 protein, and a vaccine for preventing HPV infection composed of the pentamers and VLPs as antigens.

The first aspect of the present invention provides a codon-optimized HPV6 L1 gene, the nucleotide sequence of which is SEQ NO:2.

The second aspect of the present invention provides a constructed expression vector comprising the codon-optimized HPV6 L1 gene of the first aspect of the present invention. The vector is suitable for driving heterologous DNA to translate and express HPV L1 protein in bacteria. In one embodiment, the expression vector is preferably pGEX-6p-1, pGEX-4T-2, pMAL or pET28a.

The third aspect of the present invention provides a constructed engineered bacterial cell comprising the gene of the first aspect of the present invention, or the expression vector of the second aspect. The host cell of the engineering bacteria is Escherichia coli, and in one embodiment, the host cell is preferably BL21 cell strain.

The fourth aspect of the present invention provides a Tag-HPV6 L1 fusion protein, wherein the tag Tag is 6*His.Tag, GST.Tag, SUMO.Tag, MBP.Tag, 6*His-SUMO.Tag or GST-SUMO.Tag ; L1 is HPV6 L1 full-length protein and/or C-terminal truncated by 5, 10, 15 or no more than 30 amino acids and/or N-terminal truncated by 2, 4, 6 or no more than 10 amino acid L1 protein.

The nucleotide sequence encoding Tag-HPVL1 fusion protein GST-HPV6 L1 is SEQ NO: 3, SEQ NO: 11, the nucleotide sequence of GST-SUMO-HPV6 L1 is SEQ NO: 4, SEQ NO: 12, MBP The nucleotide sequence of SEQ NO: 5, SEQ NO: 13, the nucleotide sequence of 6*His-HPV6 L1 is SEQ NO: 6, the nucleotide sequence of 6*His-SUMO-HPV6 L1 is SEQ NO: 7.

The amino acid sequence encoding Tag-HPVL1 fusion protein GST-HPV6 L1 is SEQ NO: 8, the amino acid sequence of GST-SUMO-HPV6L1 is SEQ NO: 9, and the amino acid sequence of MBP is SEQ NO: 10.

The fifth aspect of the present invention provides the HPV L1 pentamer obtained after the Tag-HPVL1 fusion protein is purified, and the VLP assembled from the pentamer. In a preferred embodiment, the average particle size of the HPV6 L1 pentameric protein is 10-15 nm PdI<0.1. In a preferred embodiment, the average particle size of HPV6 L1 VLP is 45-65nm PdI<0.1.

The sixth aspect of the present invention provides a vaccine composition, which comprises the HPV L1 pentamer or HPV L1 VLP of the present invention, and the composition further comprises pharmaceutically acceptable excipients and pharmaceutically acceptable adjuvants.

In one embodiment, the stock solution containing HPV6 L1 pentamer or VLP protein (prepared according to the above method) and aluminum hydroxide adjuvant physiological saline solution are prepared by adsorption according to the ratio of protein to aluminum content of 1:10 to obtain recombinant HPV L1 protein pentamer or VLP vaccine, stored at 4°C until use.

On the other hand, the present invention also provides a method for obtaining Tag-HPVL1 fusion protein, comprising the steps of:

A. by substituting the codons of the translation homologous protein of the HPV6 L1 gene sequence with the codons favored by Escherichia coli, the gene of the codon-optimized HPV6 L1 favored by the Escherichia coli expression system is obtained;

B. construct the Escherichia coli expression vector of HPV6 L1 gene;

C. construct the Escherichia coli expression engineering strain of Tag-HPV6 L1;

D. Induced expression and purification of the fusion protein Tag-HPV6 L1.

The prokaryotic host cell in the above method for preparing fusion protein Tag-HPV6 L1 is selected from but not limited to GI698, ER2566, BL21 (DE3), XA90, B834 (DE3), BLR (DE3).

The expression condition in the above method for preparing the fusion protein Tag-HPV6 L1 is: under the temperature condition of 20-37° C., the expression is induced for 3-20 hours. In a specific embodiment, expression is preferably induced for 16 hours at a temperature of 28°C.

The present invention also provides a method for obtaining HPV6 L1 pentamers, comprising the steps of:

a) Adsorb the fusion protein Tag-HPV6 L1 by affinity chromatography;

b) adding proteolytic enzymes to remove the Tag tag to obtain HPV6 L1 pentameric protein;

c) Purifying the HPV L1 pentameric protein to obtain L1 pentameric protein with a purity >98% and an average particle size of 10-15 nm PdI<0.1.

The protease used in the method for preparing the HPV6 L1 pentamer described above is a site-specific proteolytic enzyme that cuts off the Tag tag: recombinant 3C protease, thrombin, SUMO protease, SENP1 or TEV protease.

The purification method in the above method for preparing HPV6 L1 pentamer is selected from but not limited to ion exchange chromatography, hydrophobic chromatography, molecular sieve (or gel filtration or molecular exclusion) chromatography; preferably purification includes ion exchange chromatography and Molecular sieve chromatography.

The purification method in the above method for preparing the HPV6 L1 pentamer also includes the use of a reducing agent, such as adding DTT.

The average particle size of the HPV6 L1 pentamer protein obtained after the final purification in the above method for preparing the HPV6 L1 pentamer is 10-15 nm PdI<0.1.

The present invention also provides a method for assembling HPV6 L1 pentamers into VLPs, comprising the steps of:

Mix the L1 pentamer protein solution with an average particle size of 10-15nm PdI<0.1 with the assembly buffer, and finally obtain a pH value of 5.0-5.9, a salt concentration of 500-2000 mM, and an average particle size of 45-65nm PdI<0.1 For the HPV6 L1 VLP protein liquid, it is preferable to obtain the HPV6 L1 VLP protein liquid with a pH value of 5.7 and a salt concentration of 1300 mM.

Assembly buffers include but are not limited to Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, borate buffer, etc.

In the above method of assembling VLPs from HPV6 L1 pentamers, a protective agent, such as 0.01-0.1 polysorbate 80, may also be added to the HPV6 L1-VLP protein solution.

The present invention also provides another method for assembling VLPs—low temperature freezing treatment assembly method, comprising the following steps:

Put the HPV L1 pentamer protein solution in buffer solution with a pH value of 5.5 to 8.0 and a salt concentration of 150 to 1000 mM, and completely freeze it at -20 to -80°C, preferably for 24 hours, and then place it at room temperature to The protein stock solution was melted to obtain the HPV6 L1VLP protein solution with an average particle size of 45-65nm PdI<0.1.

On the other hand, the present invention also provides the application of the HPV L1 pentamer, VLP and the vaccine composition comprising the pentamer or VLP in the preparation of a medicament for preventing HPV infection.

According to the present invention, the vaccine of the present invention may be in a form acceptable to the patient, including but not limited to injection or nasal or oral inhalation or vaginal administration, preferably injection and intramuscular injection.

Explanation and interpretation of relevant terms in the present invention

According to the present invention, the term "Escherichia coli expression system" refers to the composition of Escherichia coli (bacterial strains) and vectors, wherein Escherichia coli (bacterial strains) are derived from commercially available, examples but not limited to: GI698, ER2566, BL21 ( DE3), XA90, DH(5a), B834 (DE3), BLR (DE3).

According to the present invention, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect the host cell, so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors include: plasmids; bacteriophages; cosmids, and the like.

According to the present invention, the term "vaccine excipient or carrier" refers to one or more of them, including but not limited to: pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators are exemplified but not limited to phosphate buffer, and surfactants include cationic, anionic or nonionic surfactants. For example but not limited to: Polysorbate 80. Adjuvants include but are not limited to aluminum hydroxide, aluminum phosphate, complete Freund's adjuvant, incomplete Freund's adjuvant and the like. Ionic strength enhancers are exemplified but not limited to sodium chloride.

According to the present invention, the term "chromatography" includes, but is not limited to: ion exchange chromatography (e.g. cation exchange chromatography, anion exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (e.g. hydroxyapatite chromatography), molecular sieve chromatography (gel filtration or size exclusion chromatography), affinity chromatography.

According to the present invention, in the method for recombinant HPV L1 protein obtained in the present invention, the buffer refers to a solution that can greatly reduce the range of pH fluctuations when adding a small amount of acid or alkali and water, including but not limited to Tris buffer, phosphoric acid Salt buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, boric acid buffer, etc.

According to the present invention, the cell disruption includes, but is not limited to, one or more methods of homogenizer disruption, homogenizer disruption, ultrasonic treatment, grinding, high-pressure extrusion, and lysozyme treatment;

According to the present invention, in the method for the recombinant HPV L1 protein that the present invention obtains, used salt includes but not limited to neutral salt, particularly alkali metal salt, ammonium salt, hydrochloride, sulfate, bicarbonate, phosphoric acid Salt or hydrogen phosphate, especially one or more of NaCl, KCl, CaCl2, NH4Cl, KCl, NH4Cl, MgSO4, (NH4)2SO4. NaCI is preferred. The reducing agents used include, but are not limited to, DTT, 2-mercaptoethanol. The amount used includes, but is not limited to, 2 mM to 100 mM, preferably 10 to 15 mM.

Beneficial effect

The invention provides a synthetic gene, the gene sequence is a codon-optimized nucleotide sequence according to the codon preference of Escherichia coli, and the sequence encodes the amino acid sequence of HPV L1 protein. The study found that the expression level of L1 protein in the codon-optimized nucleic acid sequence was significantly increased compared with that in the non-codon-optimized nucleic acid sequence.

The Escherichia coli expression system disclosed in the present invention has the advantages of high expression level, easy cultivation and operation, and low production cost. However, it is still difficult to directly obtain a large amount of soluble HPV L1 protein only by using this expression system, because the L1 protein is very easy to form inclusion bodies, that is, insoluble polymers without biological activity. In addition, even if a large number of inclusion bodies are obtained, in order to obtain biologically active proteins, the inclusion bodies must be denatured and refolded, and a large amount of protein is often lost during this process. In order to solve this problem, the present invention adopts fusion technology to combine L1 gene with proteins that assist in correct protein folding, such as glutathione sulfur transferase (GST), SUMO, MBP, 6*His-SUMO or GST-SUMO, etc. Fusion expression not only improves the solubility and yield of the protein, but also GST-SUMO-HPVL1, 6*His-SUMO-HPVL1 makes there is no residue of exogenous amino acids at the N-terminus of the HPV L1 protein, and GST-SUMO is found as The fusion tag and molecular chaperone expressed by the recombinant protein HPVL1 have the functions of resisting protease hydrolysis, significantly increasing the expression of recombinant protein, promoting the correct folding of the target protein, and improving solubility. Therefore, the technical route adopted in the present invention is to use the tag protein fusion technology when constructing the HPV L1 protein expression vector. On the one hand, the fusion protein formed by the tag protein and the L1 protein is used to improve the solubility and yield of the target protein; The fusion tag can use affinity chromatography and proteolytic enzymes to remove the fusion tag method to purify the target protein, so that the HPV L1 protein with a purity of more than 70% can be obtained in one step from a variety of cell lysates , greatly improving the purification efficiency, thereby increasing the yield of the final product HPV L1 protein.

The technical route provided by the present invention to obtain high-purity HPV L1 pentameric protein by first expressing, separating and purifying, and then manually controlling the assembly to form VLP can solve the problem of directly purifying VLP from cell disruption liquid with various miscellaneous proteins in the current known technology The problems of low purity, high degradation ratio, and low yield were obtained by in vitro assembly of high-purity pentamer VLP and storage conditions of VLP.

In addition, the present inventors unexpectedly discovered a new assembly condition and method: that is, low-temperature freezing treatment assembly method. The VLP obtained by this method can transform the protein with uneven particle size (PdI greater than 0.1) assembled before freezing and thawing into a VLP with a uniform particle size in line with theoretical expectations and a PdI less than 0.1, which is more efficient than the VLP obtained by the prior art. It is stable and can be stored in buffers with different salt concentrations and a wider pH range, which is more convenient for dilution and preparation of the final vaccine preparation.

The recombined HPV L1 VLP protein of the invention has good immunogenicity, can induce high-titer neutralizing antibodies against the same type of HPV, prevents HPV from infecting human body, and is a good vaccine form.

These and other aspects of the invention will be apparent upon reference to the following detailed description and accompanying drawings. All references disclosed herein are hereby incorporated by reference in their entirety.

Description of drawings

Figure 1: GST-HPV6 L1 protein affinity and SDS-PAGE gel electrophoresis after enzymatic hydrolysis. Lane M is the protein quality standard lane from top to bottom: 94kDa, 66kDa, 45kDa, 33kDa, 26kDa, 20kdat, the left lane is the resin for affinity adsorption of GST-L1, and the right lane is the resin for GST and L1 after enzymatic hydrolysis.

Figure 2: SDS-PAGE gel electrophoresis of GST-SUMO-HPV6 L1 protein after affinity and enzymatic digestion. Lane M is the protein standard (from top to bottom: 94kDa, 66kDa, 45kDa, 33kDa, 26kDa, 20kda), the left lane is the resin for affinity adsorption of GST-SUMO-L1, and the right lane is GST-SUMO after enzymatic digestion Resin with L1.

Figure 3: SDS-PAGE gel electrophoresis of MBP-HPV6 L1 protein after affinity and enzymatic digestion. Lane M is the protein standard (from top to bottom: 94kDa, 66kDa, 45kDa, 33kDa, 26kDa, 20kda), the left lane is the resin for affinity adsorption of MBP-L1, and the right lane is the resin for MBP and L1 after enzymatic hydrolysis .

Figure 4: SDS-PAGE gel electrophoresis of 6*HIS-SUMO-HPV6 L1 protein after affinity and enzymatic digestion. Lane M is the protein standard (from top to bottom: 94kDa, 66kDa, 45kDa, 33kDa, 26kDa, 20kda), the left lane is the resin for affinity adsorption of 6*HIS-SUMO-L1, and the right lane is the 6*HIS-SUMO-L1 after enzymatic digestion. *HIS-SUMO and L1 resins. Gel electrophoresis showed that the protease did not cut the fusion protein with 6*HIS-SUMO tag.

Fig. 5: SDS-PAGE gel electrophoresis picture of recombinant HPV6 L1 pentameric protein purified by molecular sieve chromatography of the present invention. Lane M is the protein standard (from top to bottom: 94kDa, 66kDa, 45kDa, 33kDa, 26kDa, 20kda), and the other lane is HPV L1 protein.

Figure 6: Dynamic light scattering observations of the HPV6 L1 pentamer. The results show that the particle diameter of the pentamer is 12.89nM, and the particle size distribution PdI is 0.058.

Figure 7: Dynamic light scattering observations of HPV6 L1 VLPs. The results showed that the particle size diameter of VLP was 50.29 nM, and the particle size distribution PdI was 0.040.

Figure 8: Transmission electron micrographs of HPV6 L1 pentameric protein.

Figure 9: Transmission electron micrographs of HPV6 L1 VLP protein.

Figure 10: High pressure liquid phase molecular sieve chromatogram of HPV6 L1 pentamer protein, which shows that the purity of the highly purified L1 pentamer protein is greater than 98%.

Figure 11: High pressure liquid phase molecular sieve chromatogram of HPV6 L1 VLP protein, which shows that the purity of the highly purified VLP protein is greater than 98%.

Figure 12: After the mice were vaccinated with the HPV6 L1 pentamer vaccine in each experimental group, the average titer levels of neutralizing antibodies were detected 4 weeks after the second booster immunization.

Figure 13: The average titer levels of neutralizing antibodies were detected 4 weeks after the second booster immunization after mice were inoculated with HPV6 L1 VLP vaccine in each experimental group.

Below in conjunction with embodiment the present invention is further described by way of example. These examples are non-limiting.

Embodiment 1: the design and synthesis of the HPV L1 gene of codon optimization

The gene sequences were derived from the various types of HPV sequences published on PUBMED. All HPV DNA sequences were synthesized after codon optimization of the selected HPV DNA sequences according to the codon preference of Escherichia coli for gene transcription. Primers were designed according to the synthetic DNA sequence, and the synthetic gene was used as a template for PCR amplification. The resulting codon-optimized sequences were verified by DNA sequencing.

DNA sequences of various types of HPV before and after optimization:

SEQ NO.1: DNA sequence of HPV6 type L1 before optimization

SEQ NO.2: DNA sequence of optimized HPV6 type L1

Example 2: Construction and identification of recombinant vector pGEX-6P-1-GST-HPV6 L1:

Primers for amplifying the DNA fragment of HPV6 L1: (the restriction sites are BamHI and XhoI, respectively)

Forward-HPV6 L1-ApaI:5'ACTTCAGGATCC ATGTGGCGTCCGTCTGACTCTA

Reverse-HPV6 L1-XhoI: 5'ATCTCACTCGAGCTA ACGTTTGGTT TTAGCACGTTT

PCR amplification reaction system: 10 x Pfu buffer 20 μL, Pfu enzyme 4 μL, 10 mM dNTP 2.5 μL, 5’ Primer (5 μM) 10 μL, 3’ Primer (5 μM) 10 μL, template DNA 50 ng, add d2H2O to 200 μL.

Gene PCR amplification conditions: 95°C for 3 min; 95°C for 30 sec, 58°C for 30 sec, 72°C for 4 min; cycle 32 times; 72°C for 10 min.

The L1 gene fragment containing BamH I and XhoI restriction sites and the vector pGEX-6P-1 were subjected to BamH I/XhoI double digestion treatment, and then the recovered gene fragment was combined with pGEX-6P-1 containing the corresponding cohesive end using T4 DNA ligase. 6P-1 was subjected to ligation reaction at 16°C for 10-15 h.

After the ligation reaction, the ligation product was transformed into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Shanghai Sangon Biotechnology Co., Ltd., the nucleotide sequence of the fusion recombinant GST-HPV6-L1 protein was obtained as SEQ NO.3, and the amino acid sequence was SEQ NO. .8.

The fusion recombinant vector GST-HPV-L1 with GST tag was prepared according to the method in this example, and its gene sequence was SEQ NO.11.

Example 3: Construction of recombinant vector pGEX-6P-1m-GST-SUMO-HPV6 L1 vector

Construction of pGEX-6p-1m vector: In order to make the ApaI restriction site (GGGCCC) near the multi-restriction site the only ApaI restriction site of the vector, point mutation was carried out without changing the protein expression sequence of the lacI gene ApaI (3890) can be eliminated by changing the Gly codon GGC in another ApaI recognition sequence GGGCCC of the commercially available pGEX-6p-1 vector to its synonymous codon GGT. Through such modification, ApaI can be used to insert and express genes.

Primers for amplifying the DNA fragment of SUMO: (restriction sites are ApaI and BamHI respectively)

Forward -SUMO-ApaI: ACTTCAGGGCCCTCTGACCAGGAAGCTAAACCGTC

Reverse-SUMO-BamHI: CGCGGATCCACCGGTCTGTTCCTGGTAAAC

Primers for amplifying the DNA fragment of HPV6 L1: (the restriction sites are BamHI and XhoI, respectively)

Forward-HPV6 L1-ApaI:5'ACTTCAGGATCC ATGTGGCGTCCGTCTGACTCTA

Reverse-HPV6 L1-XhoI: 5'ATCTCACTCGAGCTA ACGTTTGGTT TTAGCACGTTT

PCR amplification reaction system: 10 x Pfu buffer 20 μL, Pfu enzyme 4 μL, 10 mM dNTP 2.5 μL, 5’ Primer (5 μM) 10 μL, 3’ Primer (5 μM) 10 μL, template DNA 50 ng, add d2H2O to 200 μL.

Gene PCR amplification conditions: 1.5 min at 95°C; 30 sec at 95°C, 30 sec at 58°C, 1 min at 72°C; 32 cycles; 10 min at 72°C.

The conditions for gene PCR amplification are the same as those in the above-mentioned examples.

Enzyme digestion ligation: The SUMO gene fragment containing ApaI and BamHI restriction sites and the vector pGEX-6P-1m were subjected to ApaI/BamHI double digestion treatment, and then the recovered gene fragment was combined with the corresponding cohesive end using T4 DNA ligase The pGEX-6P-1m was used for ligation reaction at 16°C for 10-15h.

Transformation identification: After the ligation reaction, transform the ligated product into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the fusion recombinant vector pGSTSUMO-6p-1m.

Reenzyme digestion and ligation: The L1 gene fragment containing BamHI and Xho1 restriction sites and the recombinant vector pGSTSUMO-6p-1m were subjected to BamHI/Xho1 double digestion treatment, and then the recovered gene fragment was combined with the corresponding cohesive DNA using T4 DNA ligase The terminal pGSTSUMO-6p1m was ligated at 16°C for 10-15 h.

Re-transformation identification: after the ligation reaction, transform the ligation product into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the fusion recombinant vector GST-SUMO-L1 with the GST-SUMO tag. Its gene sequence is SEQ NO. 4. The amino acid sequence is SEQ NO.9.

The fusion recombinant vector GST-SUMO-L1 with the GST-SUMO tag was prepared according to the method in this example, and its gene sequence is SEQ NO.12.

Embodiment 4: Construction of recombinant vector pMAL-MBP-HPV6 L1

Primers for amplifying the DNA fragment of HPV6 L1: (the enzyme cutting sites are EcoRI and HindIII respectively)

Forward-HPV6 L1-EcoRI:5’ ACTTCA GAATTC ATGTGGCGTCCGTCTGACTCTA

Reverse-HPV6 L1-HindIII:5’ ATCTCA AAGCTTCTA ACGTTTGGTT TTAGCACGTTT

The L1 gene fragment containing EcoRI and HindIII restriction sites and the carrier pMAL were subjected to EcoRI/HindIII double digestion treatment, and then the recovered gene fragment was ligated with the pMAL containing the corresponding cohesive end using T4 DNA ligase, 16 ℃ 10 ~15 hours.

After the ligation reaction, the ligation product was transformed into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Shanghai Sangon Biotechnology Co., Ltd. to obtain the gene sequence SEQ NO.5 of the fusion recombinant MBP-HPV6-L1 protein, and the amino acid sequence was SEQ NO.10.

The fusion recombinant vector MBP-HPV-L1 with the MBP tag was prepared according to the method in this example, and its gene sequence was SEQ NO.13.

Example 5: Construction of recombinant vector pET28a-6*His-HPV6 L1

Primers for amplifying the DNA fragment of HPV6 L1: (The enzyme cutting sites are NdeI and XhoI, respectively, pET28a)

Forward-HPV6 L1-NdeI:5’ GACTTCA CATATGATGTGGCGTCCGTCTGACTCTA

Reverse-HPV6 L1-XhoI:5' CATCTCACTCGAGCTA ACGTTTGGTT TTAGCACGTTT

The L1 gene fragment containing NdeI and XhoI restriction sites and the carrier pMAL were subjected to NdeI/XhoI double digestion treatment, and then the recovered gene fragment was ligated with pET28a containing the corresponding cohesive end using T4 DNA ligase, at 16°C for 10 ~15 hours.

After the ligation reaction, the ligation product was transformed into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Shanghai Sangon Biotechnology Co., Ltd. to obtain the gene sequence SEQ NO.6 of the fusion recombinant MBP-HPV6-L1 protein.

Example 6: Construction of recombinant vector 6*His-SUMO-HPV6 L1 vector

Primers for amplifying the DNA fragment of SUMO: (the enzyme cutting sites are NdeI and BamHI respectively)

Forward -SUMO-NdeI: GGAATTCCATATGTCTGACCAGGAAGCTAAACCGTC

Reverse-SUMO-BamHI: CGC GGATCCACCGGTCTGTTCCTGGTAAAC

Primers for amplifying the DNA fragment of HPV6 L1: (the restriction sites are BamHI and XhoI, respectively)

Forward-HPV6 L1-ApaI:5'ACTTCAGGATCC ATGTGGCGTCCGTCTGACTCTA

Reverse-HPV6 L1-XhoI: 5'ATCTCACTCGAGCTA ACGTTTGGTT TTAGCACGTTT

The PCR amplification conditions and reaction system of SUMO gene and L1 gene are the same as those described in the above examples.

Enzyme digestion ligation: The SUMO gene fragment containing NdeI and BamHI restriction sites and the vector pET-28a were subjected to NdeI/BamHI double digestion treatment, and then the recovered gene fragment was combined with pET28a containing the corresponding sticky end using T4 DNA ligase Ligation reaction, 10-15 h at 16 °C.

Transformation identification: After the ligation reaction, transform the ligated product into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the fusion recombinant vector pETSUMO-28a.

Reenzyme digestion and ligation: The L1 gene fragment containing BamHI and Xho1 restriction sites and the recombinant vector pETSUMO-28a were subjected to BamHI/Xho1 double digestion treatment, and then the recovered gene fragment was combined with the corresponding cohesive end using T4 DNA ligase. pETSUMO-28a was subjected to ligation reaction at 16°C for 10-15 h.

Re-transformation identification: after the ligation reaction, transform the ligation product into the host strain DH5α for recombinant screening. The screened monoclonal colony was expanded and cultured and the plasmid was extracted, and then sequenced by Shanghai Sangon Biotechnology Co., Ltd. to obtain the gene sequence SEQ NO.7 of the fusion recombinant MBP-HPV6-L1 protein.

Embodiment 7: Expression of recombinant HPV L1 pentameric protein

The recombinant vectors of Examples 2, 3, 4, 5 and 6 with correct sequencing results were transformed into Escherichia coli BL21 host cells, and used as engineering bacteria expressing recombinant proteins to express HPV L1 protein. The engineering culture medium was 2YT medium (10 g/L tryptone; 5 g/L yeast powder; 10 g/L NaCl). Pick a single spot of bacteria containing the recombinant plasmid and place it in 10ml 2YT medium (containing 100μg/ml ampicillin) at 230 revolutions per minute (rpm), shaking at 37°C overnight. Transfer 5 ml of overnight bacteria to 500 ml (containing 100 μg/ml ampicillin) 2YT liquid medium, shake culture at 37°C until the recombinant engineering bacteria grow to OD600nm≈0.4~1, add IPTG at a final concentration of 0.2mM to induce, Induce the expression of the recombinant protein for more than 6 hours under the condition of 28°C.

Cell collection and crushing: Centrifuge the fermentation culture, discard the supernatant, harvest the cell pellet, and weigh it; wash the pellet with buffer L (pH 8.0, 50 mM Tris, 200 mM NaCl, 5 mM DTT), and then resuspend it Ultrasonic disruption was carried out in buffer L, and then the broken bacteria liquid was centrifuged by a high-speed centrifuge (16000 rpm, 30 min, 4°C), and the supernatant was collected.

Example 8: Detection of recombinant HPV L1 pentamer protein expression in Escherichia coli

ELISA sandwich method was used to detect the expression level of Tag-HPV L1 pentamer protein in Escherichia coli before loading on affinity chromatography, samples and test items:

Coating antibody: self-made anti-HPV6 L1 mouse monoclonal antibody.

Control substance: self-made high-purity HPV6 L1 protein.

Test product: Dilute the test product Tag-HPV6 L1 with the sample diluent until the concentration is within the range of the gradient dilution concentration of the reference product.

Enzyme-labeled antibody: homemade horseradish peroxidase-labeled rabbit anti-HPV6 L1 protein polyclonal antibody.

Calculation of results: calculate the average value of parallel wells, make a linear equation for its corresponding L1 protein antigen with the concentration _OD of the reference substance series concentration, the coefficient of variation between parallel sample wells must not be greater than 10%, and the linear regression equation R must not be less ^than 0.980, Substituting the OD ₄₅₀ absorption value of the test product into the equation to calculate the L1 protein antigen content of the test product after dilution, and then multiplying by the corresponding dilution factor is the L1 protein antigen content in the test product, see Table 1.

Table 1 Antigen content of Tag-HPV L1 protein after detection of expression

Embodiment 9: Recombinant HPV L1 pentamer protein affinity chromatography

Affinity chromatography of recombinant protein with GST tag: 5 ml of GST agarose affinity chromatography medium is loaded into the affinity column, and the column is equilibrated with buffer L (pH 8.0, 50 mM Tris, 200 mM NaCl, 5 mM DTT), Then load the protein solution with the GST or GST-SUMO label in Example 8, and wash with Buffer L until no protein flows out, and the affinity is completed. Suspend the affinity medium with 5mL Buffer L, take a sample to detect and calculate the total amount of bound L1 protein in the medium.

Affinity chromatography of recombinant protein with MBP tag: put 5ml of Amylose-Resin affinity chromatography medium into the affinity column, equilibrate the column with buffer L (pH 8.0, 50 mM Tris, 200 mM NaCl, 5mM DTT), Then load the protein solution with the GST or GST-SUMO label in Example 8, and wash with Buffer L until no protein flows out, and the affinity is completed. Suspend the affinity medium with 5mL Buffer L, take a sample to detect and calculate the total amount of bound L1 protein in the medium.

Affinity chromatography of recombinant protein with 6*HIS tag: take 5ml Ni-NTA gel column, slowly add 10 times column volume of equilibrium solution (50mmol/L NaH2PO4, 300mmol/L Nacl, 20mmol/L imidazole , adjust the pH value to 8 with NaOH) to fully balance the Ni-NTA gel, the flow rate is 1ml/min. Take the filtered supernatant with 6*His tag in Example 8, and after it completely enters the gel, continue to wash the gel with 10 times the column volume of the equilibrium solution, and keep the flow rate at 1ml/min. Elute with equilibrium solution until no protein flows out, and the affinity is completed. Take a sample to detect and calculate the total amount of bound L1 protein in the medium.

Example 10: Enzymatic digestion and purification of recombinant Tag-HPV L1 protein

Add the amount of enzyme according to the mass ratio of the target protein to protease 100:1, the protein with GST-HPV-L1 is digested with 3C protease, and the protein with GST-SUMO-HPV-L1 and 6*His-SUMO-HPV-L1 Digest with SENP1 protease, digest protein with Mbp-HPV-L1 with Factor Xa protease, digest protein with 6*His-HPV-L1 with Thrombin protease, mix and digest for 2 hours, then elute and collect each protease The resulting HPV6 L1 pentamer protein solution.

The L1 protein solution after the 3C enzyme digested the GST tag was detected by SDS-PAGE gel electrophoresis, and the results are shown in Figure 1. The results of affinity chromatography electrophoresis showed that 90% of the target protein could be cut off. Fig. 2 shows that SENP1 protease cleaves the protein with GST-SUMO-HPV-L1, which is detected by SDS-PAGE gel electrophoresis. Figure 3 shows Factor Xa protease digestion of protein with Mbp-HPV-L1, detected by SDS-PAGE gel electrophoresis. Figures 1 to 3 illustrate that HPV6 L1 protein of 55 kDa was obtained.

Thrombin protease did not cut 6*His-HPV-L1 protein; the protein solution of 6*His-SUMO-L1 digested with SENP1 was detected by SDS-PAGE gel electrophoresis, the results are shown in Figure 4, showing that SENP1 protease failed to cut Fusion protein with 6*His-SUMO tag.

Embodiment 11: Purification of recombinant HPV L1 pentameric protein

Molecular sieve chromatographic purification: Purify the HPV6 L1 pentamer protein collected by enzyme digestion and purification in the previous example, first through the HPV6 L1 pentamer protein collected by ion exchange chromatography, or directly use Superdex200 without ion exchange step The gel filtration medium (produced by GE Company) was further subjected to molecular sieve chromatography, and the molecular sieve mobile phase was pH8.0, 10 mM Tris, 100 mM NaCl, and the fractions of the UV absorption peak of the HPV6 L1 pentameric protein were collected.

Determination of sample purity after purification: The collected protein solution was sampled and detected by SDS-PAGE gel electrophoresis. The final purity of the target protein HPV6 L1 pentamer was greater than 98% after purification by molecular sieve chromatography. See Figure 5 for details. After purification by molecular sieve chromatography SDS-PAGE gel electrophoresis picture of recombinant HPV6 L1 pentameric protein.

Determination of sample protein concentration: use the Bradford method for protein concentration detection, use the standard sample 2mg/ml BAS to prepare and dilute from 100ug/ul to 500ug/ul, take 10ul diluted BSA+200ul Bradford working solution for the sample reaction system: the standard curve is y = 0.0013 x - 0.0294, R² = 0.9986, measure the OD ₅₉₅ of the sample, substitute it into the standard curve, and calculate the protein concentration of the sample. The results are shown in Table 2.

Table 2 Bradford method to detect recombinant HPV6 L1 pentamer protein concentration

Note: Sample group 1 is the HPV L1 pentamer protein solution obtained from GST-HPV L1 purified by molecular sieves; sample group 2 is the HPV L1 pentamer protein solution obtained from GST-SUMO-HPV L1 purified by molecular sieves; sample group 3 is Mbp-HPV L1 is purified by molecular sieve to obtain HPV L1 pentameric protein solution.

Embodiment 12: Recombinant HPV6 L1 pentamer protein is assembled into VLP

Under the conditions of the following salt concentration (NaCl) and pH value, HPV L1 pentamer solution sample groups 1, 2 and 3 were placed stably, and the particle size and Particle size distribution measurement (the particle size distribution coefficient PdI value is an index of particle size dispersion, less than 0.05 is a highly uniform sample; Homogeneous samples), HPV6 L1 pentamer protein assembly to obtain VLPs with uniform particle size (PdI<0.05).

Table 3 The particle size detection of assembled HPV6 L1 VLP under different pH and salt concentration conditions

Note: Sample group 1 is HPV L1 VLP protein solution obtained after GST-HPV L1 purified by molecular sieve; sample group 2 is HPV L1 VLP protein solution obtained by GST-SUMO-HPV L1 purified by molecular sieve; sample group 3 is Mbp-HPV L1 purified HPV L1 VLP protein solution was obtained after molecular sieve purification.

Example 13: Particle Size Determination of L1 Pentamer and VLP Proteins by Dynamic Light Scattering (DLS)

The instrument is the dynamic light scattering particle size analyzer of Malvern Zetasizer NanoZS, and the HPV6 L1 pentamer and HPV6 L1 VLP protein finally prepared in each sample group are taken for detection, and the average particle size and dispersibility index PdI (indicating the homogeneity of the protein ), indicating that the final preparation of L1 pentamer and VLP protein in each group of samples was uniform. The particle size distribution of the pentameric protein finally produced in sample group 2 and the HPV6 L1 VLP protein assembled therefrom are shown in Figures 6 and 7 for details.

Example 14: Preparation of HPV6 L1 Pentamer and VLP

According to the technology adopted in the above-mentioned Examples 1-13 of the present invention, HPV6 L1 proteins with sequences 11, 12, and 13 are prepared, and the above proteins can be purified to obtain proteins with a purity of more than 98%, and an average particle size of 10-15nm PdI< 0.1 HPV6 L1 pentameric protein. The HPV6 L1 VLP protein with an average particle size of 45-65nm PdI<0.1 was further assembled.

Example 15: Morphological detection of HPV6 L1 pentamers and VLPs

Transmission electron microscope observation: The purified HPV6 L1 pentamer protein and the assembled HPV6 L1-VLP protein obtained in the examples were observed by the Institute of Biophysics, Chinese Academy of Sciences using a transmission electron microscope platform. Frozen sample preparation and photographing process:

1) Fill the liquid nitrogen box with liquid nitrogen, and when the liquid level is not boiling, slowly inject ethane into the cooling copper bowl to cool it into a liquid state.

2) Make the copper mesh hydrophilic in the PDC-32 plasma cleaner.

3) In the Vitrobot TM Mark IV frozen sample preparation equipment, 3.5 μL of pentamer and VLP samples were adsorbed on a 300-mesh QUANTIFOIL copper grid, and after absorbing water for 4 seconds, the samples were frozen by liquid ethane.

4) Quickly transfer the sample to liquid nitrogen for storage.

5) When collecting cryographs, the electron dose is 20 e-/Å2. Data were recorded with a Gatan UltraScan 4000 CCD on a 300 kV TitanKrios transmission electron microscope at 300 kV. The accelerating voltage is 300 kV.

The results show that in the HPV6 L1 pentamer protein sample group, a large number of pentameric proteins with a diameter consistent with the theoretical size can be seen in the field of vision; in the HPV6 L1-VLP protein sample group, a large number of pentameric proteins with a particle size consistent with the theoretical size can be seen Virus-like particles (VLP) with a diameter of about 50nm, uniform and consistent. Among them, the transmission electron micrograph of the sample obtained by digesting and purifying the HPV6 L1 pentamer of the GST-SUMO tag group (sample group 2) is shown in Figure 8. The electron microscope photo is shown in attached drawing 9.

Example 16: Purity detection of HPV6 L1 protein stock solution

Molecular exclusion high-performance liquid chromatography determination: chromatographic column Agilent Bio SEC-5um, 2000Å, 7.8 × 300mm, column volume about 15 ml, molecular weight range ≥ 10,000kDa; 0.1mol/L phosphate buffer with pH 6.8 ( Weigh 25.8g of disodium hydrogen phosphate and 4.37g of sodium dihydrogen phosphate, add ultrapure water to dissolve, adjust the pH to 6.8 with phosphoric acid, and adjust the volume to 1000ml with ultrapure water) as the mobile phase; the flow rate is 1ml/min; the detection wavelength 280nm; column temperature 25°C, sample volume not less than 20ug, theoretical plate number of the main peak of the sample not less than 1000, tailing factor less than 2.0, continuous injection of 5 needles, relative standard deviation of peak area not greater than 3%.

Take 2 groups of purified samples and finally prepare the HPV6 L1 pentamer and the protein stock solution of the assembled VLP, respectively dilute the concentration to 1mg/ml, inject 20ul of the sample into the high-pressure liquid chromatograph, detect according to the above method, press The purity was calculated by the area normalization method, and the purity of all treated proteins was greater than 98%. The results are shown in Figure 10 and Table 4, Figure 11 and Table 5.

Table 4 HPLC protein purity detection of HPV6 L1 pentamer

Table 5 HPLC protein purity detection of VLP after HPV6 L1 assembly peak keep time area area% 1 13.995 2615610 100.00 total 2615610 100.00

Embodiment 17: HPV VLP stability experiment

The final HPV6 VLP protein prepared by each sample group was placed at 25°C for 14 to 28 days under the buffer conditions in the following table, and the particle size was detected. Stable storage at 500-2000mM. The test results of the HPV6 VLP obtained in sample group 3 after being stored for 14-28 days at a pH of 5.0 to 5.9 and a salt concentration of 500 to 2000 mM are shown in the table below.

Table 6 The particle size detection results of HPV6 L1 VLP stored at 25°C for 14-28 days

实施例18：制备含有HPV L1 五聚体或VLP的单价疫苗

Example 18: Preparation of monovalent vaccine containing HPV L1 pentamer or VLP

Recombinant HPV L1 protein pentamer or VLP can be prepared by adsorbing the HPV6 L1 pentamer or VLP protein stock solution containing each sample group with aluminum hydroxide adjuvant saline solution according to the ratio of protein to aluminum content of 1:10 Vaccines are stored at 4°C until use.

Example 19: Immunogenicity Assay of HPV L1 Pentamers and VLPs

Take the above-mentioned L1 pentamer or VLP vaccine respectively, add sterilized normal saline and dilute to 20 μg/ml concentration of the pentamer or VLP protein vaccine, inject BALB/c mice intramuscularly with 0.1ml each, 10 in each group Only. Mice were boosted every 4 weeks for a total of 2 immunizations. Four weeks after the booster immunization, the neutralizing antibody titer against the same type of HPV in the mouse serum after each immunization was measured by the pseudovirus cell neutralization assay, and the results are shown in Figures 12 and 13 .

The results showed that HPV L1 pentamer and VLP protein vaccinated mice, and neutralizing antibodies could reach a very high level 4 weeks after the second immunization. The experimental results prove that both the HPV L1 pentamer and the assembled VLP vaccine can produce neutralizing antibodies in animals, indicating that both the HPV L1 pentamer and the VLP protein vaccine have immunogenicity in human clinical trials, that is, they can prevent HPV isotypes Diseases caused by viruses.

SEQUENCE LISTING

<110> Beijing Kangleguard Biotechnology Co., Ltd.

<120> Type 6 recombinant human papillomavirus virus-like particle and preparation method thereof

<130> 2014

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 1503

<212>DNA

<213> Artificial sequence

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atgtggcggc ctagcgacag cacagtatat gtgcctcctc ctaaccctgt atccaaagtt 60

gttgccacgg atgcttatgt tactcgcacc aacatatttt atcatgccag cagttctaga 120

cttcttgcag tgggacatcc ttattttcc ataaaacggg ctaacaaaac tgttgtgcca 180

aaggtgtcag gatatcaata cagggtattt aaggtggtgt taccagatcc taacaaattt 240

gcattgcctg actcgtctct tttcgatccc acaacacaac gtttagtatg ggcatgcaca 300

ggcctagagg tgggcagggg acagccatta ggtgtgggtg taagtggaca tcctttccta 360

aataaatatg atgatgttga aaattcaggg agtggtggta accctggaca ggataacagg 420

gttaatgtag gtatggatta taaacaaaca caattatgca tggttggatg tgccccccct 480

ttgggcgagc attggggtaa aggtaaacag tgtactaata cacctgtaca ggctggtgac 540

tgcccgccct tagaacttat taccagtgtt atacagatg gcgatatggt tgacacaggc 600

tttggtgcta tgaattttgc tgatttgcag accaataaat cagatgttcc tattgacata 660

tgtggcacta catgtaaata tccagattat ttacaaatgg ctgcagaccc atatggtgat 720

agattatttt tttttctacg gaaggaacaa atgtttgcca gacatttttt taacagggct 780

ggcgaggtgg gggaacctgt gcctgataca cttataatta agggtagtgg aaatcgcacg 840

tctgtaggga gtagtatata tgttaacacc ccgagcggct ctttggtgtc ctctgaggca 900

caattgttta ataagccata ttggctacaa aaagcccagg gacataacaa tggtatttgt 960

tggggtaatc aactgtttgt tactgtggta gataccacac gcagtaccaa catgacatta 1020

tgtgcatccg taactacatc ttccacatac accaattctg attataaaga gtacatgcgt 1080

catgtggaag agtatgattt acaatttatt tttcaattat gtagcattac attgtctgct 1140

gaagtaatgg cctatattca cacaatgaat ccctctgttt tggaagactg gaactttggg 1200

ttatcgcctc ccccaaatgg tacattagaa gatacctata ggtatgtgca gtcacaggcc 1260

attacctgtc aaaagcccac tcctgaaaag gaaaagccag atccctataa gaaccttagt 1320

ttttgggagg ttaatttaaa agaaaagttt tctagtgaat tggatcagta tcctttggga 1380

cgcaagtttt tgttacaaag tggatatagg ggacggtcct ctattcgtac aggtgttaag 1440

cgccctgctg tttccaaagc ctctgctgcc cctaaacgta agcgcgccaa aactaaaagg 1500

taa 1503

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atgtggcgtc cgtctgactc taccgtttac gttccgccgc cgaacccggt ttctaaagtt 60

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ctgctggctg ttggtcaccc gtacttctct atcaaacgtg ctaacaaaac cgttgttccg 180

aaagtttctg gttaccagta ccgtgttttc aaagttgttc tgccggaccc gaacaaattc 240

gctctgccgg actcttctct gttcgacccg accacccagc gtctggtttg ggcttgcacc 300

ggtctggaag ttggtcgtgg tcagccgctg ggtgttggtg tttctggtca cccgttcctg 360

aacaaatacg acgacgttga aaactctggt tctggtggta acccgggtca ggacaaccgt 420

gttaacgttg gtatggacta caaacagacc cagctgtgca tggttggttg cgctccgccg 480

ctgggtgaac actggggtaa aggtaaacag tgcaccaaca ccccggttca ggctggtgac 540

tgcccgccgc tggaactgat cacctctgtt atccaggacg gtgacatggt tgacaccggt 600

ttcggtgcta tgaacttcgc tgacctgcag accaacaaat ctgacgttcc gatcgacatc 660

tgcggtacca cctgcaaata cccggactac ctgcagatgg ctgctgaccc gtacggtgac 720

cgtctgttct tcttcctgcg taaagaacag atgttcgctc gtcacttctt caaccgtgct 780

ggtgaagttg gtgaaccggt tccggacacc ctgatcatca aaggttctgg taaccgtacc 840

tctgttggtt cttctatcta cgttaacacc ccgtctggtt ctctggtttc ttctgaagct 900

cagctgttca acaaaccgta ctggctgcag aaagctcagg gtcacaacaa cggtatctgc 960

tggggtaacc agctgttcgt taccgttgtt gacaccaccc gttctaccaa catgaccctg 1020

tgcgcttctg ttaccacctc ttctacctac accaactctg actacaaaga atacatgcgt 1080

cacgttgaag aatacgacct gcagttcatc ttccagctgt gctctatcac cctgtctgct 1140

gaagttatgg cttacatcca caccatgaac ccgtctgttc tggaagactg gaacttcggt 1200

ctgtctccgc cgccgaacgg taccctggaa gacacctacc gttacgttca gtctcaggct 1260

atcacctgcc agaaaccgac cccggaaaaa gaaaaaccgg acccgtacaa aaacctgtct 1320

ttctgggaag ttaacctgaa agaaaaattc tcttctgaac tggaccagta cccgctgggt 1380

cgtaaattcc tgctgcagtc tggttaccgt ggtcgttctt ctatccgtac cggtgttaaa 1440

cgtccggctg tttctaaagc ttctgctgct ccgaaacgta aacgtgctaa aaccaaacgt 1500

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<213> Artificial sequence

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atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

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tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggaagttc tgttccaggg gcccctggga tccatgtggc gtccgtctga ctctaccgtt 720

tacgttccgc cgccgaaccc ggtttctaaa gttgttgcta ccgacgctta cgttacccgt 780

accaacatct tctaccacgc ttcttcttct cgtctgctgg ctgttggtca cccgtacttc 840

tctatcaaac gtgctaacaa aaccgttgtt ccgaaagttt ctggttacca gtaccgtgtt 900

ttcaaagttg ttctgccgga cccgaacaaa ttcgctctgc cggactcttc tctgttcgac 960

ccgaccaccc agcgtctggt ttgggcttgc accggtctgg aagttggtcg tggtcagccg 1020

ctgggtgttg gtgtttctgg tcacccgttc ctgaacaaat acgacgacgt tgaaaactct 1080

ggttctggtg gtaacccggg tcaggacaac cgtgttaacg ttggtatgga ctacaaacag 1140

accccagctgt gcatggttgg ttgcgctccg ccgctgggtg aacactgggg taaaggtaaa 1200

cagtgcacca acaccccggt tcaggctggt gactgcccgc cgctggaact gatcacctct 1260

gttatccagg acggtgacat ggttgacacc ggtttcggtg ctatgaactt cgctgacctg 1320

cagaccaaca aatctgacgt tccgatcgac atctgcggta ccacctgcaa atacccggac 1380

tacctgcaga tggctgctga cccgtacggt gaccgtctgt tcttcttcct gcgtaaagaa 1440

cagatgttcg ctcgtcactt cttcaaccgt gctggtgaag ttggtgaacc ggttccggac 1500

accctgatca tcaaaggttc tggtaaccgt acctctgttg gttcttctat ctacgttaac 1560

accccgtctg gttctctggt ttcttctgaa gctcagctgt tcaacaaacc gtactggctg 1620

cagaaagctc agggtcacaa caacggtatc tgctggggta accagctgtt cgttaccgtt 1680

gttgacacca cccgttctac caacatgacc ctgtgcgctt ctgttaccac ctcttctacc 1740

tacaccaact ctgactacaa agaatacatg cgtcacgttg aagaatacga cctgcagttc 1800

atcttccagc tgtgctctat caccctgtct gctgaagtta tggcttacat ccacaccatg 1860

aacccgtctg ttctggaaga ctggaacttc ggtctgtctc cgccgccgaa cggtaccctg 1920

gaagacacct accgttacgt tcagtctcag gctatcacct gccagaaacc gaccccggaa 1980

aaagaaaaac cggacccgta caaaaacctg tctttctggg aagttaacct gaaagaaaaa 2040

ttctcttctg aactggacca gtacccgctg ggtcgtaaat tcctgctgca gtctggttac 2100

cgtggtcgtt cttctatccg taccggtgtt aaacgtccgg ctgtttctaa agcttctgct 2160

gctccgaaac gtaaacgtgc taaaaccaaa cgt 2193

<210> 4

<211> 2475

<212>DNA

<213> Artificial sequence

<400> 4

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggaagttc tgttccaggg gccctctgac caggaagcta aaccgtctac cgaagacctg 720

ggtgacaaaa aagaaggtga atacatcaaa ctgaaagtta tcggtcagga ctcttctgaa 780

atccacttca aagttaaaat gaccaccac ctgaaaaaac tgaaagaatc ttactgccag 840

cgtcagggtg ttccgatgaa ctctctgcgt ttcctgttcg aaggtcagcg tatcgctgac 900

aaccaacaccc cgaaagaact gggtatggaa gaagaagacg ttatcgaagt ttaccaggaa 960

cagaccggtg gatccatgtg gcgtccgtct gactctaccg tttacgttcc gccgccgaac 1020

ccggtttcta aagttgttgc taccgacgct tacgttaccc gtaccaacat cttctaccac 1080

gcttcttctt ctcgtctgct ggctgttggt cacccgtact tctctatcaa acgtgctaac 1140

aaaaccgttg ttccgaaagt ttctggttac cagtaccgtg ttttcaaagt tgttctgccg 1200

gacccgaaca aattcgctct gccggactct tctctgttcg acccgaccac ccagcgtctg 1260

gtttgggctt gcaccggtct ggaagttggt cgtggtcagc cgctgggtgt tggtgtttct 1320

ggtcacccgt tcctgaacaa atacgacgac gttgaaaact ctggttctgg tggtaacccg 1380

ggtcaggaca accgtgttaa cgttggtatg gactacaaac agacccagct gtgcatggtt 1440

ggttgcgctc cgccgctggg tgaacactgg ggtaaaggta aacagtgcac caacacccccg 1500

gttcaggctg gtgactgccc gccgctggaa ctgatcacct ctgttatcca ggacggtgac 1560

atggttgaca ccggtttcgg tgctatgaac ttcgctgacc tgcagaccaa caaatctgac 1620

gttccgatcg acatctgcgg taccacctgc aaatacccgg actacctgca gatggctgct 1680

gacccgtacg gtgaccgtct gttcttcttc ctgcgtaaag aacagatgtt cgctcgtcac 1740

ttcttcaacc gtgctggtga agttggtgaa ccggttccgg acaccctgat catcaaaggt 1800

tctggtaacc gtacctctgt tggttcttct atctacgtta acaccccgtc tggttctctg 1860

gtttcttctg aagctcagct gttcaacaaa ccgtactggc tgcagaaagc tcagggtcac 1920

aacaacggta tctgctgggg taaccagctg ttcgttaccg ttgttgacac cacccgttct 1980

accaacatga ccctgtgcgc ttctgttacc acctcttcta cctacaccaa ctctgactac 2040

aaagaataca tgcgtcacgt tgaagaatac gacctgcagt tcatcttcca gctgtgctct 2100

atcaccctgt ctgctgaagt tatggcttac atccacacca tgaacccgtc tgttctggaa 2160

gactggaact tcggtctgtc tccgccgccg aacggtaccc tggaagacac ctaccgttac 2220

gttcagtctc aggctatcac ctgccagaaa ccgaccccgg aaaaagaaaa accggacccg 2280

tacaaaaacc tgtctttctg ggaagttaac ctgaaagaaa aattctcttc tgaactggac 2340

cagtacccgc tgggtcgtaa attcctgctg cagtctggtt accgtggtcg ttcttctatc 2400

cgtaccggtg ttaaacgtcc ggctgtttct aaagcttctg ctgctccgaa acgtaaacgt 2460

gctaaaacca aacgt 2475

<210> 5

<211> 2673

<212>DNA

<213> Artificial sequence

<400> 5

atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60

ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120

ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180

atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240

accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300

aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360

gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420

aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480

ctgattgctg ctgacgggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540

gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600

aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660

ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720

gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780

ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840

ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900

ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960

actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020

tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080

gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140

aacctcggga tcgagggaag gatttcagaa ttcatgtggc gtccgtctga ctctaccgtt 1200

tacgttccgc cgccgaaccc ggtttctaaa gttgttgcta ccgacgctta cgttacccgt 1260

accaacatct tctaccacgc ttcttcttct cgtctgctgg ctgttggtca cccgtacttc 1320

tctatcaaac gtgctaacaa aaccgttgtt ccgaaagttt ctggttacca gtaccgtgtt 1380

ttcaaagttg ttctgccgga cccgaacaaa ttcgctctgc cggactcttc tctgttcgac 1440

ccgaccacccc agcgtctggt ttgggcttgc accggtctgg aagttggtcg tggtcagccg 1500

ctgggtgttg gtgtttctgg tcacccgttc ctgaacaaat acgacgacgt tgaaaactct 1560

ggttctggtg gtaacccggg tcaggacaac cgtgttaacg ttggtatgga ctacaaacag 1620

accccagctgt gcatggttgg ttgcgctccg ccgctgggtg aacactgggg taaaggtaaa 1680

cagtgcacca acaccccggt tcaggctggt gactgcccgc cgctggaact gatcacctct 1740

gttatccagg acggtgacat ggttgacacc ggtttcggtg ctatgaactt cgctgacctg 1800

cagaccaaca aatctgacgt tccgatcgac atctgcggta ccacctgcaa atacccggac 1860

tacctgcaga tggctgctga cccgtacggt gaccgtctgt tcttcttcct gcgtaaagaa 1920

cagatgttcg ctcgtcactt cttcaaccgt gctggtgaag ttggtgaacc ggttccggac 1980

accctgatca tcaaaggttc tggtaaccgt acctctgttg gttcttctat ctacgttaac 2040

accccgtctg gttctctggt ttcttctgaa gctcagctgt tcaacaaacc gtactggctg 2100

cagaaagctc agggtcacaa caacggtatc tgctggggta accagctgtt cgttaccgtt 2160

gttgacacca cccgttctac caacatgacc ctgtgcgctt ctgttaccac ctcttctacc 2220

tacaccaact ctgactacaa agaatacatg cgtcacgttg aagaatacga cctgcagttc 2280

atcttccagc tgtgctctat caccctgtct gctgaagtta tggcttacat ccacaccatg 2340

aacccgtctg ttctggaaga ctggaacttc ggtctgtctc cgccgccgaa cggtaccctg 2400

gaagacacct accgttacgt tcagtctcag gctatcacct gccagaaacc gaccccggaa 2460

aaagaaaaac cggacccgta caaaaacctg tctttctggg aagttaacct gaaagaaaaa 2520

ttctcttctg aactggacca gtacccgctg ggtcgtaaat tcctgctgca gtctggttac 2580

cgtggtcgtt cttctatccg taccggtgtt aaacgtccgg ctgtttctaa agcttctgct 2640

gctccgaaac gtaaacgtgc taaaaccaaa cgt 2673

<210> 6

<211> 1563

<212>DNA

<213> Artificial sequence

<400> 6

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgatgtggc gtccgtctga ctctaccgtt tacgttccgc cgccgaaccc ggtttctaaa 120

gttgttgcta ccgacgctta cgttacccgt accaacatct tctaccacgc ttcttcttct 180

cgtctgctgg ctgttggtca cccgtacttc tctatcaaac gtgctaacaa aaccgttgtt 240

ccgaaagttt ctggttacca gtaccgtgtt ttcaaagttg ttctgccgga cccgaacaaa 300

ttcgctctgc cggactcttc tctgttcgac ccgaccaccc agcgtctggt ttgggcttgc 360

accggtctgg aagttggtcg tggtcagccg ctgggtgttg gtgtttctgg tcacccgttc 420

ctgaacaaat acgacgacgt tgaaaactct ggttctggtg gtaacccggg tcaggacaac 480

cgtgttaacg ttggtatgga ctacaaacag acccagctgt gcatggttgg ttgcgctccg 540

ccgctgggtg aacactgggg taaaggtaaa cagtgcacca acaccccggt tcaggctggt 600

gactgcccgc cgctggaact gatcacctct gttatccagg acggtgacat ggttgacacc 660

ggtttcggtg ctatgaactt cgctgacctg cagaccaaca aatctgacgt tccgatcgac 720

atctgcggta ccacctgcaa atacccggac tacctgcaga tggctgctga cccgtacggt 780

gaccgtctgt tcttcttcct gcgtaaagaa cagatgttcg ctcgtcactt cttcaaccgt 840

gctggtgaag ttggtgaacc ggttccggac accctgatca tcaaaggttc tggtaaccgt 900

acctctgttg gttcttctat ctacgttaac accccgtctg gttctctggt ttcttctgaa 960

gctcagctgt tcaacaaacc gtactggctg cagaaagctc agggtcacaa caacggtatc 1020

tgctggggta accagctgtt cgttaccgtt gttgacacca cccgttctac caacatgacc 1080

ctgtgcgctt ctgttaccac ctcttctacc tacaccaact ctgactacaa agaatacatg 1140

cgtcacgttg aagaatacga cctgcagttc atcttccagc tgtgctctat caccctgtct 1200

gctgaagtta tggcttacat ccacaccatg aacccgtctg ttctggaaga ctggaacttc 1260

ggtctgtctc cgccgccgaa cggtaccctg gaagaacacct accgttacgt tcagtctcag 1320

gctatcacct gccagaaacc gaccccggaa aaagaaaaac cggacccgta caaaaacctg 1380

tctttctggg aagttaacct gaaagaaaaa ttctcttctg aactggacca gtacccgctg 1440

ggtcgtaaat tcctgctgca gtctggttac cgtggtcgtt cttctatccg taccggtgtt 1500

aaacgtccgg ctgtttctaa agcttctgct gctccgaaac gtaaacgtgc taaaaccaaa 1560

cgt 1563

<210> 7

<211> 1854

<212>DNA

<213> Artificial sequence

<400> 7

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgtctgacc aggaagctaa accgtctacc gaagacctgg gtgacaaaaa agaaggtgaa 120

tacatcaaac tgaaagttat cggtcaggac tcttctgaaa tccacttcaa agttaaaatg 180

accacccacc tgaaaaaact gaaagaatct tactgccagc gtcagggtgttccgatgaac 240

tctctgcgtt tcctgttcga aggtcagcgt atcgctgaca accacaccccc gaaagaactg 300

ggtatggaag aagaagacgt tatcgaagtt taccaggaac agaccggtgg atccatgtgg 360

cgtccgtctg actctaccgt ttacgttccg ccgccgaacc cggtttctaa agttgttgct 420

accgacgctt acgttacccg taccaacatc ttctaccacg cttcttcttc tcgtctgctg 480

gctgttggtc acccgtactt ctctatcaaa cgtgctaaca aaaccgttgt tccgaaagtt 540

tctggttacc agtaccgtgttttcaaagtt gttctgccgg acccgaacaa attcgctctg 600

ccggactctt ctctgttcga cccgaccacc cagcgtctgg tttgggcttg caccggtctg 660

gaagttggtc gtggtcagcc gctgggtgtt ggtgtttctg gtcacccgtt cctgaacaaa 720

tacgacgacg ttgaaaactc tggttctggt ggtaacccgg gtcaggaca ccgtgttaac 780

gttggtatgg actacaaaca gacccagctg tgcatggttg gttgcgctcc gccgctgggt 840

gaacactggg gtaaaggtaa acagtgcacc aacacccccgg ttcaggctgg tgactgcccg 900

ccgctggaac tgatcacctc tgttatccag gacggtgaca tggttgacac cggtttcggt 960

gctatgaact tcgctgacct gcagaccaac aaatctgacg ttccgatcga catctgcggt 1020

accacctgca aatacccgga ctacctgcag atggctgctg accccgtacgg tgaccgtctg 1080

ttcttcttcc tgcgtaaaga acagatgttc gctcgtcact tcttcaaccg tgctggtgaa 1140

gttggtgaac cggttccgga caccctgatc atcaaaggtt ctggtaaccg taccctctgtt 1200

ggttcttcta tctacgttaa caccccgtct ggttctctgg tttcttctga agctcagctg 1260

ttcaacaaac cgtactggct gcagaaagct cagggtcaca acaacggtat ctgctggggt 1320

aaccagctgt tcgttaccgt tgttgacacc acccgttcta ccaacatgac cctgtgcgct 1380

tctgttacca cctcttctac cctaccaac tctgactaca aagaatacat gcgtcacgtt 1440

gaagaatacg acctgcagtt catcttccag ctgtgctcta tcaccctgtc tgctgaagtt 1500

atggcttaca tccacaccat gaacccgtct gttctggaag actggaactt cggtctgtct 1560

ccgccgccga acggtaccct ggaagacacc taccgttacg ttcagtctca ggctatcacc 1620

tgccagaaac cgaccccgga aaaagaaaaa ccggacccgt acaaaaacct gtctttctgg 1680

gaagttaacc tgaaagaaaa attctcttct gaactggacc agtacccgct gggtcgtaaa 1740

ttcctgctgc agtctggtta ccgtggtcgt tcttctatcc gtaccggtgt taaacgtccg 1800

gctgtttcta aagcttctgc tgctccgaaa cgtaaacgtg ctaaaaccaa acgt 1854

<210> 8

<211> 731

<212> PRT

<213> Artificial sequence

<400> 8

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu

210 215 220

Phe Gln Gly Pro Leu Gly Ser Met Trp Arg Pro Ser Asp Ser Thr Val

225 230 235 240

Tyr Val Pro Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala

245 250 255

Tyr Val Thr Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu

260 265 270

Leu Ala Val Gly His Pro Tyr Phe Ser Ile Lys Arg Ala Asn Lys Thr

275 280 285

Val Val Pro Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val

290 295 300

Leu Pro Asp Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp

305 310 315 320

Pro Thr Thr Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly

325 330 335

Arg Gly Gln Pro Leu Gly Val Gly Val Ser Gly His Pro Phe Leu Asn

340 345 350

Lys Tyr Asp Asp Val Glu Asn Ser Gly Ser Gly Gly Asn Pro Gly Gln

355 360 365

Asp Asn Arg Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys

370 375 380

Met Val Gly Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Lys

385 390 395 400

Gln Cys Thr Asn Thr Pro Val Gln Ala Gly Asp Cys Pro Pro Leu Glu

405 410 415

Leu Ile Thr Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe

420 425 430

Gly Ala Met Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro

435 440 445

Ile Asp Ile Cys Gly Thr Thr Cys Lys Tyr Pro Asp Tyr Leu Gln Met

450 455 460

Ala Ala Asp Pro Tyr Gly Asp Arg Leu Phe Phe Phe Leu Arg Lys Glu

465 470 475 480

Gln Met Phe Ala Arg His Phe Phe Asn Arg Ala Gly Glu Val Gly Glu

485 490 495

Pro Val Pro Asp Thr Leu Ile Ile Lys Gly Ser Gly Asn Arg Thr Ser

500 505 510

Val Gly Ser Ser Ile Tyr Val Asn Thr Pro Ser Gly Ser Leu Val Ser

515 520 525

Ser Glu Ala Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln

530 535 540

Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val

545 550 555 560

Val Asp Thr Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Thr

565 570 575

Thr Ser Ser Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His

580 585 590

Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr

595 600 605

Leu Ser Ala Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val

610 615 620

Leu Glu Asp Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu

625 630 635 640

Glu Asp Thr Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys

645 650 655

Pro Thr Pro Glu Lys Glu Lys Pro Asp Pro Tyr Lys Asn Leu Ser Phe

660 665 670

Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Tyr

675 680 685

Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Ser

690 695 700

Ser Ile Arg Thr Gly Val Lys Arg Pro Ala Val Ser Lys Ala Ser Ala

705 710 715 720

Ala Pro Lys Arg Lys Arg Ala Lys Thr Lys Arg

725 730

<210> 9

<211> 825

<212> PRT

<213> Artificial sequence

<400> 9

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu

210 215 220

Phe Gln Gly Pro Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu

225 230 235 240

Gly Asp Lys Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln

245 250 255

Asp Ser Ser Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys

260 265 270

Lys Leu Lys Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser

275 280 285

Leu Arg Phe Leu Phe Glu Gly Gln Arg Ile Ala Asp Asn His Thr Pro

290 295 300

Lys Glu Leu Gly Met Glu Glu Glu Asp Val Ile Glu Val Tyr Gln Glu

305 310 315 320

Gln Thr Gly Gly Ser Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val

325 330 335

Pro Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val

340 345 350

Thr Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala

355 360 365

Val Gly His Pro Tyr Phe Ser Ile Lys Arg Ala Asn Lys Thr Val Val

370 375 380

Pro Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro

385 390 395 400

Asp Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr

405 410 415

Thr Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly

420 425 430

Gln Pro Leu Gly Val Gly Val Ser Gly His Pro Phe Leu Asn Lys Tyr

435 440 445

Asp Asp Val Glu Asn Ser Gly Ser Gly Gly Asn Pro Gly Gln Asp Asn

450 455 460

Arg Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val

465 470 475 480

Gly Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Lys Gln Cys

485 490 495

Thr Asn Thr Pro Val Gln Ala Gly Asp Cys Pro Pro Leu Glu Leu Ile

500 505 510

Thr Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala

515 520 525

Met Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Ile Asp

530 535 540

Ile Cys Gly Thr Thr Thr Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala

545 550 555 560

Asp Pro Tyr Gly Asp Arg Leu Phe Phe Phe Leu Arg Lys Glu Gln Met

565 570 575

Phe Ala Arg His Phe Phe Asn Arg Ala Gly Glu Val Gly Glu Pro Val

580 585 590

Pro Asp Thr Leu Ile Ile Lys Gly Ser Gly Asn Arg Thr Ser Val Gly

595 600 605

Ser Ser Ile Tyr Val Asn Thr Pro Ser Gly Ser Leu Val Ser Ser Glu

610 615 620

Ala Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His

625 630 635 640

Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp

645 650 655

Thr Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Thr Thr Ser

660 665 670

Ser Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu

675 680 685

Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser

690 695 700

Ala Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu

705 710 715 720

Asp Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp

725 730 735

Thr Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr

740 745 750

Pro Glu Lys Glu Lys Pro Asp Pro Tyr Lys Asn Leu Ser Phe Trp Glu

755 760 765

Val Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Tyr Pro Leu

770 775 780

Gly Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Ser Ser Ile

785 790 795 800

Arg Thr Gly Val Lys Arg Pro Ala Val Ser Lys Ala Ser Ala Ala Pro

805 810 815

Lys Arg Lys Arg Ala Lys Thr Lys Arg

820 825

<210> 10

<211> 891

<212> PRT

<213> Artificial sequence

<400> 10

Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys

1 5 10 15

Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr

20 25 30

Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe

35 40 45

Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala

50 55 60

His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile

65 70 75 80

Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp

85 90 95

Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu

100 105 110

Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys

115 120 125

Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly

130 135 140

Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro

145 150 155 160

Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys

165 170 175

Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly

180 185 190

Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp

195 200 205

Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala

210 215 220

Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys

225 230 235 240

Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gly Gln Pro Ser

245 250 255

Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro

260 265 270

Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp

275 280 285

Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala

290 295 300

Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala

305 310 315 320

Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln

325 330 335

Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala

340 345 350

Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn

355 360 365

Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile

370 375 380

Glu Gly Arg Ile Ser Glu Phe Met Trp Arg Pro Ser Asp Ser Thr Val

385 390 395 400

Tyr Val Pro Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala

405 410 415

Tyr Val Thr Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu

420 425 430

Leu Ala Val Gly His Pro Tyr Phe Ser Ile Lys Arg Ala Asn Lys Thr

435 440 445

Val Val Pro Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val

450 455 460

Leu Pro Asp Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp

465 470 475 480

Pro Thr Thr Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly

485 490 495

Arg Gly Gln Pro Leu Gly Val Gly Val Ser Gly His Pro Phe Leu Asn

500 505 510

Lys Tyr Asp Asp Val Glu Asn Ser Gly Ser Gly Gly Asn Pro Gly Gln

515 520 525

Asp Asn Arg Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys

530 535 540

Met Val Gly Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Lys

545 550 555 560

Gln Cys Thr Asn Thr Pro Val Gln Ala Gly Asp Cys Pro Pro Leu Glu

565 570 575

Leu Ile Thr Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe

580 585 590

Gly Ala Met Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro

595 600 605

Ile Asp Ile Cys Gly Thr Thr Cys Lys Tyr Pro Asp Tyr Leu Gln Met

610 615 620

Ala Ala Asp Pro Tyr Gly Asp Arg Leu Phe Phe Phe Leu Arg Lys Glu

625 630 635 640

Gln Met Phe Ala Arg His Phe Phe Asn Arg Ala Gly Glu Val Gly Glu

645 650 655

Pro Val Pro Asp Thr Leu Ile Ile Lys Gly Ser Gly Asn Arg Thr Ser

660 665 670

Val Gly Ser Ser Ile Tyr Val Asn Thr Pro Ser Gly Ser Leu Val Ser

675 680 685

Ser Glu Ala Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln

690 695 700

Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val

705 710 715 720

Val Asp Thr Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Thr

725 730 735

Thr Ser Ser Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His

740 745 750

Val Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr

755 760 765

Leu Ser Ala Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val

770 775 780

Leu Glu Asp Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu

785 790 795 800

Glu Asp Thr Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys

805 810 815

Pro Thr Pro Glu Lys Glu Lys Pro Asp Pro Tyr Lys Asn Leu Ser Phe

820 825 830

Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Tyr

835 840 845

Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Ser

850 855 860

Ser Ile Arg Thr Gly Val Lys Arg Pro Ala Val Ser Lys Ala Ser Ala

865 870 875 880

Ala Pro Lys Arg Lys Arg Ala Lys Thr Lys Arg

885 890

<210> 11

<211> 2088

<212>DNA

<213> Artificial sequence

<400> 11

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggaagttc tgttccaggg gccctctgac tctaccgttt acgttccgcc gccgaacccg 720

gtttctaaag ttgttgctac cgacgcttac gttacccgta ccaacatctt ctaccacgct 780

tcttcttctc gtctgctggc tgttggtcac ccgtacttct ctatcaaacg tgctaacaaa 840

accgttgttc cgaaagtttc tggttaccag taccgtgttt tcaaagttgt tctgccggac 900

ccgaacaaat tcgctctgcc ggactcttct ctgttcgacc cgaccaccca gcgtctggtt 960

tgggcttgca ccggtctgga agttggtcgt ggtcagccgc tgggtgttgg tgtttctggt 1020

cacccgttcc tgaacaaata cgacgacgtt gaaaactctg gttctggtgg taacccgggt 1080

caggacaacc gtgttaacgt tggtatggac tacaaacaga cccagctgtg catggttggt 1140

tgcgctccgc cgctgggtga aacactggggt aaaggtaaac agtgcaccaa caccccggtt 1200

caggctggtg actgcccgcc gctggaactg atcacctctg ttatccagga cggtgacatg 1260

gttgacaccg gtttcggtgc tatgaacttc gctgacctgc agaccaacaa atctgacgtt 1320

ccgatcgaca tctgcggtac cacctgcaaa tacccggact acctgcagat ggctgctgac 1380

ccgtacggtg accgtctgtt cttcttcctg cgtaaagaac agatgttcgc tcgtcacttc 1440

ttcaaccgtg ctggtgaagt tggtgaaccg gttccggaca ccctgatcat caaaggttct 1500

ggtaaccgta cctctgttgg ttcttctatc tacgttaaca ccccgtctgg ttctctggtt 1560

tcttctgaag ctcagctgtt caacaaaccg tactggctgc agaaagctca gggtcacaac 1620

aacggtatct gctggggtaa ccagctgttc gttaccgttg ttgacaccac ccgttctacc 1680

aacatgaccc tgtgcgcttc tgttaccacc tcttctacct acaccaactc tgactacaaa 1740

gaatacatgc gtcacgttga agaatacgac ctgcagttca tcttccagct gtgctctatc 1800

accctgtctg ctgaagttat ggcttacatc cacaccatga accccgtctgt tctggaagac 1860

tggaacttcg gtctgtctcc gccgccgaac ggtaccctgg aagacaccta ccgttacgtt 1920

cagtctcagg ctatcacctg ccagaaaccg accccggaaa aagaaaaacc ggacccgtac 1980

aaaaacctgt ctttctggga agttaacctg aaagaaaaat tctcttctga actggaccag 2040

tacccgctgg gtcgtaaatt cctgctgcag tctggttacc gtggttag 2088

<210> 12

<211> 2457

<212>DNA

<213> Artificial sequence

<400> 12

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggaagttc tgttccaggg gccctctgac caggaagcta aaccgtctac cgaagacctg 720

ggtgacaaaa aagaaggtga atacatcaaa ctgaaagtta tcggtcagga ctcttctgaa 780

atccacttca aagttaaaat gaccaccac ctgaaaaaac tgaaagaatc ttactgccag 840

cgtcagggtg ttccgatgaa ctctctgcgt ttcctgttcg aaggtcagcg tatcgctgac 900

aaccaacaccc cgaaagaact gggtatggaa gaagaagacg ttatcgaagt ttaccaggaa 960

cagaccggtg gatcctctac cgtttacgtt ccgccgccga acccggtttc taaagttgtt 1020

gctaccgacg cttacgttac ccgtaccaac atcttctacc acgcttcttc ttctcgtctg 1080

ctggctgttg gtcacccgta cttctctatc aaacgtgcta acaaaaccgt tgttccgaaa 1140

gtttctggtt accagtaccg tgttttcaaa gttgttctgc cggacccgaa caaattcgct 1200

ctgccggact cttctctgtt cgacccgacc acccagcgtc tggtttgggc ttgcaccggt 1260

ctggaagttg gtcgtggtca gccgctgggt gttggtgttt ctggtcaccc gttcctgaac 1320

aaatacgacg acgttgaaaa ctctggttct ggtggtaacc cgggtcagga caaccgtgtt 1380

aacgttggta tggactacaa acagacccag ctgtgcatgg ttggttgcgc tccgccgctg 1440

ggtgaacact ggggtaaagg taaacagtgc accaacaccc cggttcaggc tggtgactgc 1500

ccgccgctgg aactgatcac ctctgttatc caggacggtg acatggttga caccggtttc 1560

ggtgctatga acttcgctga cctgcagacc aacaaatctg acgttccgat cgacatctgc 1620

ggtaccacct gcaaataccc ggactacctg cagatggctg ctgacccgta cggtgaccgt 1680

ctgttcttct tcctgcgtaa agaacagatg ttcgctcgtc acttcttcaa ccgtgctggt 1740

gaagttggtg aaccggttcc ggacaccctg atcatcaaag gttctggtaa ccgtacctct 1800

gttggttctt ctatctacgt taacaccccg tctggttctc tggtttcttc tgaagctcag 1860

ctgttcaaca aaccgtactg gctgcagaaa gctcagggtc acaacaacgg tatctgctgg 1920

ggtaaccagc tgttcgttac cgttgttgac accacccgtt ctaccaacat gaccctgtgc 1980

gcttctgtta ccaccctcttc tacctacacc aactctgact acaaagaata catgcgtcac 2040

gttgaagaat acgacctgca gttcatcttc cagctgtgct ctatcaccct gtctgctgaa 2100

gttatggctt acatccacac catgaacccg tctgttctgg aagactggaa cttcggtctg 2160

tctccgccgc cgaacggtac cctggaagac acctaccgtt acgttcagtc tcaggctatc 2220

acctgccaga aaccgacccc ggaaaaagaa aaaccggacc cgtacaaaaa cctgtctttc 2280

tgggaagtta acctgaaaga aaaattctct tctgaactgg accagtaccc gctgggtcgt 2340

aaattcctgc tgcagtctgg ttaccgtggt cgttcttcta tccgtaccgg tgttaaacgt 2400

ccggctgttt ctaaagcttc tgctgctccg aaacgtaaac gtgctaaaac caaacgt 2457

<210> 13

<211> 2628

<212>DNA

<213> Artificial sequence

<400> 13

atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60

ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120

ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180

atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240

accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300

aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360

gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420

aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480

ctgattgctg ctgacgggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540

gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600

aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660

ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720

gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780

ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840

ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900

ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960

actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020

tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080

gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140

aacctcggga tcgagggaag gatttcagaa ttcatgtggc gtccgtctga ctctaccgtt 1200

tacgttccgc cgccgaaccc ggtttctaaa gttgttgcta ccgacgctta cgttacccgt 1260

accaacatct tctaccacgc ttcttcttct cgtctgctgg ctgttggtca cccgtacttc 1320

tctatcaaac gtgctaacaa aaccgttgtt ccgaaagttt ctggttacca gtaccgtgtt 1380

ttcaaagttg ttctgccgga cccgaacaaa ttcgctctgc cggactcttc tctgttcgac 1440

ccgaccacccc agcgtctggt ttgggcttgc accggtctgg aagttggtcg tggtcagccg 1500

ctgggtgttg gtgtttctgg tcacccgttc ctgaacaaat acgacgacgt tgaaaactct 1560

ggttctggtg gtaacccggg tcaggacaac cgtgttaacg ttggtatgga ctacaaacag 1620

accccagctgt gcatggttgg ttgcgctccg ccgctgggtg aacactgggg taaaggtaaa 1680

cagtgcacca acaccccggt tcaggctggt gactgcccgc cgctggaact gatcacctct 1740

gttatccagg acggtgacat ggttgacacc ggtttcggtg ctatgaactt cgctgacctg 1800

cagaccaaca aatctgacgt tccgatcgac atctgcggta ccacctgcaa atacccggac 1860

tacctgcaga tggctgctga cccgtacggt gaccgtctgt tcttcttcct gcgtaaagaa 1920

cagatgttcg ctcgtcactt cttcaaccgt gctggtgaag ttggtgaacc ggttccggac 1980

accctgatca tcaaaggttc tggtaaccgt acctctgttg gttcttctat ctacgttaac 2040

accccgtctg gttctctggt ttcttctgaa gctcagctgt tcaacaaacc gtactggctg 2100

cagaaagctc agggtcacaa caacggtatc tgctggggta accagctgtt cgttaccgtt 2160

gttgacacca cccgttctac caacatgacc ctgtgcgctt ctgttaccac ctcttctacc 2220

tacaccaact ctgactacaa agaatacatg cgtcacgttg aagaatacga cctgcagttc 2280

atcttccagc tgtgctctat caccctgtct gctgaagtta tggcttacat ccacaccatg 2340

aacccgtctg ttctggaaga ctggaacttc ggtctgtctc cgccgccgaa cggtaccctg 2400

gaagacacct accgttacgt tcagtctcag gctatcacct gccagaaacc gaccccggaa 2460

aaagaaaaac cggacccgta caaaaacctg tctttctggg aagttaacct gaaagaaaaa 2520

ttctcttctg aactggacca gtacccgctg ggtcgtaaat tcctgctgca gtctggttac 2580

cgtggtcgtt cttctatccg taccggtgtt aaacgtccgg ctgtttct 2628

Claims (3)

1. A method of preparing HPV 6L 1 VLPs, characterized in that the method comprises the steps of:

constructing an escherichia coli expression vector containing a coding gene of Tag-HPV 6L 1 fusion protein shown as SEQ NO. 5;

constructing an escherichia coli expression engineering strain containing the expression vector, carrying out induction expression in the escherichia coli expression engineering strain, and purifying by an affinity chromatography method to obtain the Tag-HPV 6L 1 fusion protein;

carrying out enzyme digestion on the purified Tag label of the Tag-HPV 6L 1 fusion protein by adopting Factor Xa protease to obtain HPV 6L 1 pentamer protein;

purifying the HPV 6L 1 pentamer protein to obtain an HPV 6L 1 pentamer protein solution with the purity of more than 98 percent and the average particle size of 10-15nm, pdI (positive pressure induced plasticity) < -0.1;

mixing the HPV 6L 1 pentamer protein solution with an assembly buffer solution to finally obtain an HPV 6L 1VLP protein solution with the pH value of 5.0-5.9, the salt concentration of 500-2000 mM, the average particle size of 45-65nm and PdI (Positive pressure induced plasticity) knot of 0.1.

2. A method of making HPV 6L 1 VLPs according to claim 1, wherein: the amino acid sequence of the Tag-HPV 6L 1 fusion protein is shown in SEQ NO.10.

3. A method of making HPV 6L 1 VLPs according to claim 1, wherein: wherein the E.coli host cell is GI698, ER2566, BL21 (DE 3), XA90, B834 (DE 3) or BLR (DE 3).

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