CN105039359B - 16 type recombinant human papilloma virus-like particle and preparation method thereof - Google Patents

Abstract

The invention relates to a 16-type recombinant human papilloma virus-like particle and a preparation method thereof, and particularly provides a novel polynucleotide gene segment for coding recombinant HPV16L1 protein, a vector containing the gene segment, a host cell comprising the vector, HPV16L1 fusion protein and a pentamer which are translated and expressed by the gene segment, and VLP consisting of the pentamer, and also discloses 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 HPV16 infection.

Description

16 type recombinant human papilloma virus-like particle and preparation method thereof

Technical Field

The present invention relates to virus-like particles of human papilloma virus and a method for preparing the same. More particularly, the invention relates to a pentamer of recombinant human papilloma Virus L1 protein, a Virus-like Particle (VLP), a preparation method thereof and application of a vaccine composition containing the VLP in preventing human papilloma Virus infection.

Background

Human Papillomaviruses (HPV) are known to cause numerous proliferative epithelial lesions in humans, including papillomas (warts) and neoplasias, mainly by close contact with the Human body, such as sexually transmitted viruses. Specifically, HPV-induced diseases mainly include 3 major types, 1 st, cancer of cervix, vagina, female vulva, penis and anus, and some 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 oropharynx and 3% of oral cancer are due to HPV infection. Group 2 benign lesions, such as genital warts, including flat warts and condyloma acuminata, are sexually transmitted diseases and are common in people with active sexual activity. Although genital warts do not have as serious consequences as cancer, the lesions often cause painful clinical symptoms such as burning, bleeding and pain for the patient, while creating negative psychological reactions such as embarrassment, anxiety and self-mutism, and the process of repeated treatment wastes a great deal of medical resources. It is estimated that there are 3000 tens of thousands of genital warts worldwide caused by non-oncogenic HPV (mainly types 6 and 11), with 20-50% of lesions also containing mixed infections with high-risk HPV types. Group 3 HPV infection can also cause Recurrent Respiratory Papillomas (RRP), a rare, potentially fatal disease that occurs primarily in adolescents, and sometimes, a large number of papillomas can cause dyspnea and death in younger children.

HPV is a membrane-free double-stranded DNA virus, consisting mainly of viral coat and genomic DNA (Bernard, Burket al.2011). The HPV virus coat is an icosahedral structure consisting of 360L 1 proteins (forming 72 pentamers) and at most 72L 2 proteins, and has a diameter of 55-60 nm (Howley and Lowy 2007). The viral coat protein has self-assembly properties, and L1 protein alone or together with L2 protein self-assembles in vitro to form Virus-like particles (VLPs) (Chen, Garce 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 specific antigen of the virus can be obtained only by using the method of recombinant DNA technology to prepare the genetic engineering vaccine. The virus-like particles VLPs formed by assembling the recombinant Ll or L1/L2 have no virus DNA, good safety and antigen epitope similar to natural virus particles, and can generate neutralizing antibodies IgG and IgA after stimulating an organism, so the HPV VLPs can be used as a preventive vaccine, thereby greatly reducing the possibility of generating related tumors caused by infecting HPV (Howley and Lowy 2007).

The key to developing HPV vaccine is the ability to prepare high purity, stable HPV antigens in large quantities. In the aspect of HPV vaccine antigen preparation technology, the expression systems for producing HPV antigens that are currently used can be classified into eukaryotic expression systems and prokaryotic expression systems. Examples of eukaryotic expression systems that are commonly used include poxvirus expression systems, insect baculovirus expression systems, and yeast expression systems. HPV L1 expressed in eukaryotic expression systems spontaneously forms VLPs, which are often obtained by simple purification. However, the expression level of the eukaryotic expression system is low, the culture cost is high, and great difficulty is brought to large-scale industrial production. The expression of HPV L1 protein in prokaryotic expression system by means of E.coli expression system has been reported. However, since the HPV L1 protein expressed by escherichia coli is low in solubility, the HPV VLPs are finally purified from the cell sap with the complicated protein types by the currently known purification methods, such as salt-free precipitation or denaturation and renaturation. For example: in patent CN02129070.9, a method for expressing and preparing HPV L1 multimer by prokaryotic cell is disclosed, wherein the purification process comprises treatment by 3.3M urea and dialysis renaturation process; the purification of the L1-GST fusion protein in WO-0204007 patent was also carried out by urea denaturation and dialytic renaturation; it is also disclosed in the prior art that the purification method of L1 protein comprises the steps of ultrafiltration dialysis with phosphate buffer and centrifugation to precipitate the target protein for reconstitution. However, in these purification processes, the amount of protein lost is large, the yield is low, and it is difficult to apply the method to mass production.

In the aspect of uniformity of HPV vaccine antigen protein VLP, the particle size dispersion of HPV L1VLP assembled in the prior art is expressed by using poly d value, wherein the poly d value is less than 15% to indicate that the particles have good uniformity, between 15% and 30% to indicate that the particles have large heterogeneity, and more than 30% to indicate that the particles are not uniform enough. The HPV L1VLP prepared in the prior art is more than 15%. Another indication that the particle size is uniform is the PdI value, which is the particle size distribution coefficient, less than 0.05 being a highly uniform sample; 0.05 to 0.1, 0.1 to 0.3, and more than 0.3. The PdI of the mixed protein solution of HPV16-HPV 18L 1 VLPs disclosed in the US7205125B2 patent is 0.07.

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

Disclosure of Invention

The invention aims to disclose an optimized nucleotide sequence for coding HPV16L1 protein, a vector comprising the nucleotide sequence, a host cell comprising the vector, HPV L1 protein which is translated and expressed by the polynucleotide sequence, Tag-HPV-L1 recombinant protein, pentamer and VLP formed by the L1 protein, and a vaccine for preventing HPV infection by taking the pentamer and VLP as antigens.

The invention provides a codon-optimized HPV16L1 gene, the nucleotide sequence of which is SEQ NO: 2.

in a second aspect, the present invention provides a constructed expression vector comprising the gene of codon-optimized HPV16L1 of the first aspect of the invention. The vector is suitable for driving heterologous DNA to express HPV L1 protein translationally in bacteria. In one embodiment, the expression vector is preferably pGEX-6p-1, pGEX-4T-2, pMAL, or pET28 a.

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

The fourth aspect of the invention provides a Tag-HPV16L1 fusion protein, wherein the Tag is 6 His Tag, GST Tag, sumo Tag, mbp Tag, 6 His-sumo Tag or GST-sumo Tag; l1 is HPV16L1 full length protein and/or L1 protein truncated 5, 10, 15 or no more than 30 amino acids at the C-terminus and/or 2, 4, 6 or no more than 10 amino acids at the N-terminus.

The nucleotide sequence of the fusion protein GST-HPV 16L1 of the coding Tag-HPVL1 is SEQ NO: 3. the amino acid sequence of SEQ NO: 11, the nucleotide sequence of GST-SUMO-HPV 16L1 is SEQ NO: 4. the amino acid sequence of SEQ NO: 12, nucleotide sequence of MBP SEQ NO: 5. the amino acid sequence of SEQ NO: 13, 6 His-HPV 16L1 has the nucleotide sequence of SEQ NO: 6, 6 His-SUMO-HPV 16L1 nucleotide sequence of SEQ NO: 7.

the amino acid sequence of the fusion protein GST-HPV 16L1 of the coding Tag-HPVL1 is SEQ NO: 8, the amino acid sequence of GST-SUMO-HPV 16L1 is SEQ NO: 9, amino acid sequence of MBP SEQ NO: 10.

the fifth aspect of the present invention provides a pentamer of HPV L1 obtained by purifying a Tag-HPVL1 fusion protein, and a VLP assembled from the pentamer. In a preferred embodiment, the average particle size of the HPV16L1 pentamer protein is 10-15 nm PdI < 0.1. In a preferred embodiment, the HPV16L 1VLP has an average particle size of 45-65 nm PdI < 0.1.

In a sixth aspect, the present invention provides a vaccine composition comprising a pentamer of HPV L1 or a VLP of HPV L1 of the invention, said composition further comprising a pharmaceutically acceptable excipient and a pharmaceutically acceptable adjuvant.

In one embodiment, recombinant HPV L1 protein pentamer or VLP vaccine is prepared by separately adsorbing a protein stock solution (prepared according to the above method) containing HPV16L1 pentamer or VLP with an aluminum hydroxide adjuvant physiological saline solution according to a ratio of 1:10 protein to aluminum content, and is stored at 4 ℃ for later use.

In another aspect, the present invention also provides a method for obtaining a Tag-HPVL1 fusion protein, comprising the steps of:

A. replacing codons of the translation homologous protein of the HPV16L1 gene sequence by codons preferred by escherichia coli to obtain a codon-optimized HPV16L1 gene preferred by an escherichia coli expression system;

B. constructing an Escherichia coli expression vector of the HPV16L1 gene;

C. constructing an escherichia coli expression engineering strain of Tag-HPV16L 1;

D. inducing expression and purifying to obtain fusion protein Tag-HPV16L 1.

The prokaryotic host cell in the above method for preparing the fusion protein Tag-HPV16L1 is selected from but not limited to GI698, ER2566, BL21(DE3), XA90, B834(DE3), BLR (DE 3).

The expression conditions in the method for preparing the fusion protein Tag-HPV16L1 are as follows: under the temperature condition of 20-37 ℃, the induction expression is carried out for 3-20 hours. In one embodiment, preferably at 28 ℃ temperature, induced expression for 16 hours.

The invention also provides a method for obtaining the HPV16L1 pentamer, which comprises the following steps:

a) adsorbing the fusion protein Tag-HPV16L1 by an affinity chromatography method;

b) adding a proteolytic enzyme to excise the Tag label to obtain HPV16L1 pentamer protein;

c) purifying the HPVL1 pentamer protein to obtain the L1 pentamer protein with the purity of more than 98% and the average particle size of 10-15 nm PdI of less than 0.1.

The protease used in the above method for preparing HPV16L1 pentamer is a site-specific proteolytic enzyme that cleaves the Tag: recombinant 3C protease, thrombin, SUMO protease, SENP1 or TEV protease.

The purification method in the method for preparing HPV16L1 pentamer is selected from, but not limited to, ion exchange chromatography, hydrophobic chromatography, molecular sieve (or gel filtration or molecular exclusion) chromatography; preferably the purification comprises ion exchange chromatography and molecular sieve chromatography.

The purification method in the method for preparing HPV16L1 pentamer further comprises using a reducing agent, such as DTT.

The HPV16L1 pentamer protein obtained after final purification in the method for preparing the HPV16L1 pentamer has an average particle size of 10-15 nm PdI < 0.1.

The invention also provides a method for assembling the HPV16L1 pentamer into VLPs, which comprises the following steps:

mixing the L1 pentamer protein solution with the average particle size of 10-15 nm PdI <0.1 with an assembly buffer solution to finally obtain the HPV16L 1VLP protein solution with the pH value of 5.0-5.9, the salt concentration of 500-2000 mM and the average particle size of 45-65 nm PdI <0.1, preferably obtain the HPV16L 1VLP protein solution with the pH value of 5.7 and the salt concentration of 1300 mM.

The assembly buffer includes, but is not limited to, Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citric acid buffer, histidine buffer, boric acid buffer, and the like.

In the method for assembling the HPV16L1 pentamer into VLPs, protective agents can be added into the protein liquid of the HPV16L 1-VLP, such as: 0.01-0.1 polysorbate 80.

The invention also provides another method for assembling VLP, namely a low-temperature freezing treatment assembling method, which comprises the following steps:

putting the HPV L1 pentamer protein solution into a buffer solution with the pH value of 5.5-8.0 and the salt concentration of 150-1000 mM, completely freezing at the temperature of-20-80 ℃, preferably freezing for 24 hours, and then placing at room temperature until the protein stock solution is dissolved to obtain the HPV16L 1VLP protein solution with the average particle size of 45-65 nm and PdI < 0.1.

In another aspect, the invention also provides the use of pentamers of HPV L1, VLPs and vaccine compositions comprising the pentamers or VLPs in the preparation of a medicament for the prevention of HPV infection.

According to the present invention, the vaccine of the present invention may take a form acceptable to patients, including but not limited to injection or nasal or buccal inhalation or vaginal administration, preferably injection and intramuscular injection.

Description and explanation of related terms in the present invention

According to the present invention, the term "E.coli expression system" means a system consisting of E.coli (strain) derived from commercially available sources, exemplified herein but not limited thereto: GI698, ER2566, BL21(DE3), XA90, DH (5a), B834(DE3), BLR (DE 3).

According to the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to obtain expression of the genetic material element carried by the vector in the host cell. By way of example, the carrier includes: a plasmid; bacteriophage; cosmids, and the like.

According to the present invention, the term "vaccine excipient or carrier" refers to a substance selected from one or more of, including but not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents such as, but not limited to, phosphate buffers, surfactants include cationic, anionic or nonionic surfactants. By way of example but not limitation: polysorbate 80. Adjuvants are exemplified by, but not limited to, aluminum hydroxide, aluminum phosphate, freund's complete adjuvant, freund's incomplete adjuvant, and the like. Ionic strength enhancers are exemplified by, 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 obtaining recombinant HPV L1 protein according to the present invention, the buffer refers to a solution capable of greatly reducing the pH fluctuation range upon addition of a small amount of acid or alkali and water, and includes, but is not limited to, Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, boric acid buffer, and the like.

According to the present invention, the cell disruption includes, but is not limited to, one or more of disruption by a homogenizer, sonication, milling, high pressure extrusion, lysozyme treatment;

according to the present invention, in the method of obtaining recombinant HPV L1 protein according to the present invention, the salt used includes, but is not limited to, neutral salt, especially one or more of alkali metal salt, ammonium salt, hydrochloride, sulfate, bicarbonate, phosphate or hydrogen phosphate, especially NaCI, KCl, CaCl2, NH4Cl, KCI, NH4CI, MgSO4, (NH4)2SO 4. NaCI is preferred. Reducing agents used include, but are not limited to, DTT, 2-mercaptoethanol. The amount used includes, but is not limited to, 2 mM-lO 0mM, preferably 10-15 mM.

Advantageous effects

The invention provides a synthetic gene, the gene sequence is a nucleotide sequence which is subjected to codon optimization according to the codon preference of escherichia coli, and the sequence encodes an HPV L1 protein amino acid sequence. The research finds that the expression amount of the L1 protein of the nucleic acid sequence subjected to codon optimization is obviously improved compared with that of the nucleic acid sequence not subjected to codon optimization.

The escherichia coli expression system disclosed by the invention has the advantages of high expression level, easiness in culture and operation, low production cost and the like. However, it is still difficult to directly obtain a large amount of soluble HPV L1 protein using only this expression system because L1 protein is very easy to form inclusion bodies, i.e., insoluble polymers having no biological activity. In addition, even if a large amount of inclusion bodies are obtained, in order to obtain biologically active proteins, the inclusion bodies must be denatured and renatured, and a large amount of protein is often lost in this process. In order to solve the problem, the invention adopts a fusion technology to perform fusion expression on the L1 gene and a protein which assists the correct folding of the protein, such as glutathione-S-transferase (GST), SUMO, MBP, 6 His-SUMO or GST-SUMO, and the like, so that the solubility and the yield of the protein are improved, and GST-SUMO-HPVL1, 6 His-SUMO-HPVL1 enable no residue of exogenous amino acid at the N end of HPV L1 protein, and the GST-SUMO is found as a fusion tag and a molecular chaperone expressed by recombinant protein HPVL1 and has the functions of resisting proteolysis, obviously increasing the expression amount of the recombinant protein, promoting the correct folding of the target protein, improving the solubility and the like. Therefore, the technical route adopted by the invention is to adopt a tag protein fusion technology when constructing an HPV L1 protein expression vector, on one hand, the solubility of the target protein is improved and the yield is improved through the fusion protein formed by the tag protein and the L1 protein, on the other hand, the purification characteristic of the target protein can be carried out through the GST fusion tag by utilizing the methods of affinity chromatography and proteolytic enzyme excision fusion substance tag, thereby realizing the one-step purification of the various cell lysates to obtain the HPV L1 protein with the purity of more than 70 percent, greatly improving the purification efficiency and further improving the yield of the final product HPV L1 protein.

The technical route of the invention, which is firstly used for obtaining the high-purity HPV L1 pentamer protein through expression, separation and purification and then manually controlling and assembling to form the VLP, can solve the problems of low purity, high degradation ratio and low yield of VLP directly purified from cell disruption solutions with various proteins in the prior art, and obtains the high-purity pentamer in-vitro assembled VLP and the VLP storage condition.

In addition, the inventors have surprisingly found a new assembly condition and method: namely, a low-temperature freezing process assembly method. The VLP obtained by the method can change the protein (PdI is more than 0.1) with non-uniform particle size assembled before freeze thawing into the VLP with particle size which is expected by theory and is uniform, and the PdI is less than 0.1, is more stable compared with the VLP obtained by the prior art, can be stored in buffers with different salt concentrations and wider pH value range, and is more convenient for the dilution and preparation of final vaccine preparations.

The HPV L1VLP protein obtained by recombination has good immunogenicity, can induce high-titer neutralizing antibodies aiming at homotype HPV, prevents HPV infection to 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 attached drawings. All references disclosed herein are incorporated by reference in their entirety.

Drawings

FIG. l: and (3) an SDS-PAGE gel electrophoresis picture of GST-HPV 16L1 protein affinity and enzymolysis. The M lane is a protein mass standard lane and comprises the following components from top to bottom: 94kDa, 66kDa,45kDa,33kDa,26kDa and 20kdat, the left lane is a resin for affinity adsorption of GST-L1, and the right lane is a resin for affinity adsorption of GST and L1 after enzymatic hydrolysis.

FIG. 2: SDS-PAGE gel electrophoresis picture of GST-SUMO-HPV 16L1 protein after affinity and enzymolysis. The M lane is a protein mass standard (94 kDa, 66kDa,45kDa,33kDa,26kDa,20kDa from top to bottom), the left lane is a resin for affinity adsorption of GST-SUMO-L1, and the right lane is a resin for enzymatic hydrolysis of GST-SUMO and L1.

FIG. 3: SDS-PAGE gel electrophoresis picture of MBP-HPV 16L1 protein after affinity and enzymolysis. The M lane is a protein mass standard (94 kDa, 66kDa,45kDa,33kDa,26kDa,20kDa from top to bottom), the left lane is a resin for affinity adsorption of MBP-L1, and the right lane is a resin for adsorption of MBP and L1 after enzymolysis.

FIG. 4: 6H HIS-SUMO-HPV 16L1 protein after affinity and enzymolysis, SDS-PAGE gel electrophoresis picture. The M lane is the protein mass standard (94 kDa, 66kDa,45kDa,33kDa,26kDa,20kDa from top to bottom), the left lane is the resin for affinity adsorption of 6 HIS-SUMO-L1, and the right lane is the resin for 6 HIS-SUMO and L1 after enzymolysis. The lysated protein with 6 × HIS-SUMO tag was not cleaved by the protease as shown by gel electrophoresis.

FIG. 5: the SDS-PAGE gel electrophoresis picture of the recombinant HPV16L1 pentamer protein purified by molecular sieve chromatography is shown. The M lane is the protein mass standard (94 kDa, 66kDa,45kDa,33kDa,26kDa,20kDa from top to bottom), and the other lane is the HPV L1 protein.

FIG. 6: dynamic light scattering observations of HPV16L1 pentamer. The result showed that the particle diameter of the pentamer was 12.62nM and the particle size distribution PdI was 0.030.

FIG. 7: dynamic light scattering observations of HPV16L1 VLPs. The results showed that the diameter of the particle size of the VLPs was 47.79nM and the particle size distribution PdI was 0.019.

FIG. 8: transmission electron micrograph of HPV16L1 pentameric protein.

FIG. 9: transmission electron micrograph of HPV16L 1VLP protein.

FIG. 10: high pressure liquid phase molecular sieve chromatogram of HPV16L1 pentamer protein showing that highly purified L1 pentamer protein is more than 98% pure.

FIG. 11: high pressure liquid phase molecular sieve chromatograms of HPV16L 1VLP proteins showing that highly purified VLP proteins are greater than 98% pure.

FIG. 12: the mean titer level of neutralizing antibodies was measured 4 weeks after the second booster immunization of mice vaccinated with each experimental group of HPV16L1 pentamer and HPV 6L1 VLPs.

The invention is further illustrated by the following examples. These examples are not limiting.

Example i: design and synthesis of codon-optimized HPV L1 gene

The gene sequence is derived from various HPV sequences disclosed on PUBMED. All HPV DNA sequences were synthesized after codon optimization of selected HPV DNA sequences with reference to E.coli bias towards gene transcription codons. Primers were designed based on the synthetic DNA sequence and PCR amplification was performed using the synthetic gene as a template. The resulting codon optimized sequence was verified by DNA sequencing.

DNA sequences of HPV types before and after optimization:

1, SEQ No. 1: DNA sequence of HPV16 type L1 before optimization

SEQ NO. 2: optimized HPV16 type L1 DNA sequence

Example 2: construction and identification of recombinant vector pGEX-6P-1-GST-HPV 16L 1:

DNA fragment primers for amplification of HPV16L 1: (cleavage sites are BamHI and XhoI, respectively)

Forward-HPV16 L1-ApaI:5’ACTTCAGGATCC ATGTCTCTGTGGCTGCCGTCTG

Reverse-HPV16 L1-XhoI:5’ATCTCACTCGAG CTA CAGTTTACGC TGTTTACGTTT

PCR amplification reaction System: 10 Xpfu buffer 20. mu.L, Pfu enzyme 4. mu.L, 10 mM dNTP 2.5. mu.L, 5 'Primer (5. mu.M) 10. mu.L, 3' Primer (5. mu.M) 10. mu.L, template DNA 50 ng, plus d2H2O to 200. mu.L.

The gene PCR amplification conditions are as follows: 3 min at 95 ℃; 30 sec at 95 ℃, 30 sec at 58 ℃ and 4 min at 72 ℃; circulating for 32 times; 10 min at 72 ℃.

Carrying out BamH I/XhoI double enzyme digestion treatment on an L1 gene fragment containing BamH I and XhoI enzyme digestion sites and a vector pGEX-6P-1, and then carrying out ligation reaction on the recovered gene fragment and pGEX-6P-1 containing a corresponding cohesive end by using T4 DNA ligase at 16 ℃ for 10-15 h.

And carrying out amplification culture on the screened monoclonal colony, extracting plasmids, and sequencing by Shanghai bio-engineering company to obtain the fusion recombinant GST-HPV16-L1 protein, wherein the nucleotide sequence is SEQ NO.3, and the amino acid sequence is SEQ NO. 8.

A fusion recombinant vector GST-HPV-L1 with GST tag is prepared according to the method of the embodiment, and the gene sequence is SEQ NO. 11.

Example 3: construction of recombinant vector pGEX-6P-1 m-GST-SUMO-HPV 16L1 vector

Construction of pGEX-6p-1m vector: in order to ensure that ApaI enzyme cutting sites (GGGCCC) near the multi-enzyme cutting sites are the only ApaI enzyme cutting sites of the vector, the Gly codon GGC in another ApaI recognition sequence GGGCCC of a commercial pGEX-6p-1 vector is changed into the same sense codon GGT by a point mutation technology on the premise of not changing the protein expression sequence of the lacI gene, and the ApaI can be eliminated (3890). ApaI is made available to insert a site for expression of a gene by such a modification.

DNA fragment primers for amplification of SUMO: (cleavage sites were ApaI and BamHI, respectively)

Forward -SUMO-ApaI: ACTTCAGGGCCCTCTGACCAGGAAGCTAAACCGTC

Reverse-SUMO-BamHI: CGCGGATCCACCGGTCTGTTCCTGGTAAAC

DNA fragment primers for amplification of HPV16L 1: (cleavage sites are BamHI and XhoI, respectively)

Forward-HPV16 L1-ApaI:5’ACTTCAGGATCC ATGTCTCTGTGGCTGCCGTCTG

Reverse-HPV16 L1-XhoI:5’ATCTCACTCGAG CTACAGTTTACGC TGTTTACGTTT

PCR amplification reaction System: 10 Xpfu buffer 20. mu.L, Pfu enzyme 4. mu.L, 10 mM dNTP 2.5. mu.L, 5 'Primer (5. mu.M) 10. mu.L, 3' Primer (5. mu.M) 10. mu.L, template DNA 50 ng, plus d2H2O to 200. mu.L.

The gene PCR amplification conditions are as follows: 1.5 min at 95 ℃; 30 sec at 95 ℃, 30 sec at 58 ℃ and 1 min at 72 ℃; circulating for 32 times; 10 min at 72 ℃.

The gene PCR amplification conditions were the same as in the above examples.

Enzyme digestion connection: carrying out Apa I/BamHI double enzyme digestion treatment on the SUMO gene fragment containing ApaI and BamHI enzyme digestion sites and the vector pGEX-6P-1m, and then carrying out a ligation reaction on the recovered gene fragment and pGEX-6P-1m containing the corresponding cohesive end by using T4 DNA ligase at 16 ℃ for 10-15 h.

Transformation and identification, namely transforming a ligation product into host bacteria DH5 α after ligation reaction to screen recombinants, carrying out amplification culture on screened monoclonal colonies, extracting plasmids, and sequencing by Jinzhi Biotech, Suzhou to obtain a fusion recombinant vector pGSTSMO-6 p-1 m.

And (3) secondary enzyme digestion and connection: the L1 gene fragment containing BamHI and Xho1 enzyme cutting sites and the recombinant vector pGSTSMO-6 p-1m are subjected to BamHI/Xho1 double enzyme cutting treatment, and then the recovered gene fragment is subjected to ligation reaction with pGSTSMMO-6 p1m containing corresponding cohesive ends by using T4 DNA ligase at 16 ℃ for 10-15 h.

And performing secondary transformation and identification, namely transforming a connection product after connection reaction into host bacteria DH5 α to screen a recombinant, performing amplification culture on the screened monoclonal colony, extracting a plasmid, and sequencing by Soviken Jinzhi Biotech Co., Ltd to obtain a fusion recombinant vector GST-SUMO-L1 with a GST-SUMO label, wherein the gene sequence is SEQ NO.4, and the amino acid sequence is SEQ NO. 9.

A fusion recombinant vector GST-SUMO-L1 with a GST tag, whose gene sequence is SEQ NO.12, was prepared by reference to the procedure of this example.

Example 4: construction of recombinant vector pMAL-MBP-HPV 16L1

DNA fragment primers for amplification of HPV16L 1: (restriction sites were EcoRI and HindIII, respectively)

Forward-HPV16 L1-EcoRI:5’ ACTTCA GAATTC ATGTCTCTGTGGCTGCCGTCTG

Reverse-HPV16 L1-HindIII:5’ ATCTCA AAGCTT CTACAGTTTACGCTGTTTACGTTT

Carrying out EcoRI/HindIII double enzyme digestion treatment on an L1 gene fragment containing EcoRI and HindIII enzyme digestion sites and a vector pMAL, then carrying out ligation reaction on the recovered gene fragment and pMAL containing a corresponding cohesive end by using T4 DNA ligase, and carrying out 10-15 h at 16 ℃.

And after the ligation reaction, transforming the ligation product into host bacteria DH5 α to screen recombinants, carrying out amplification culture on the screened monoclonal colony, extracting plasmids, and sequencing by Shanghai bio-engineering company to obtain a gene sequence SEQ NO.5 of the fusion recombinant MBP-HPV16-L1 protein, wherein the amino acid sequence is SEQ NO. 10.

A fusion recombinant vector MBP-HPV-L1 with MBP label is prepared according to the method of the embodiment, and the gene sequence is SEQ NO. 13.

Example 5: construction of recombinant vector pET28a-6 His-HPV 16L1

DNA fragment primers for amplification of HPV16L 1: (the cleavage sites were NdeI and XhoI, respectively, pET28 a)

Forward-HPV16 L1-NdeI:5’ GACTTCA CATATGATGTCTCTGTGGCTGCCGTCTG

Reverse-HPV16 L1-XhoI:5’ CATCTCACTCGAGCTA CAGTTTACGCTGTTTACGTTT

The L1 gene fragment containing NdeI and XhoI enzyme cutting sites and the vector pMAL are subjected to NdeI/XhoI double enzyme cutting treatment, and then the recovered gene fragment and pET28a containing the corresponding cohesive end are subjected to ligation reaction by utilizing T4 DNA ligase at 16 ℃ for 10-15 h.

And transforming a connecting product into host bacteria DH5 α to screen a recombinant after a connecting reaction, carrying out amplification culture on the screened monoclonal colony, extracting a plasmid, and sequencing by Shanghai bio-engineering company to obtain a gene sequence SEQ NO.6 of the fusion recombinant MBP-HPV16-L1 protein.

Example 6: recombinant vector 6 His-SUMO-HPV 16L1 vector construction

DNA fragment primers for amplification of SUMO: (the cleavage sites were NdeI and BamHI, respectively)

Forward -SUMO-NdeI: GGAATTCCATATGTCTGACCAGGAAGCTAAACCGTC

Reverse-SUMO-BamHI: CGC GGATCCACCGGTCTGTTCCTGGTAAAC

DNA fragment primers for amplification of HPV16L 1: (cleavage sites are BamHI and XhoI, respectively)

Forward-HPV16 L1-ApaI:5’ACTTCAGGATCC ATGTCTCTGTGGCTGCCGTCTG

Reverse-HPV16 L1-XhoI:5’ATCTCACTCGAG CTA CAGTTTACGC TGTTTACGTTT

The conditions and reaction system for PCR amplification of the SUMO gene and the L1 gene were as described in the above examples.

Enzyme digestion connection: the SUMO gene fragment containing NdeI and BamHI enzyme cutting sites and a vector pET-28a are subjected to NdeI/BamHI double enzyme cutting treatment, and then the recovered gene fragment and pET28a containing corresponding cohesive ends are subjected to ligation reaction by using T4 DNA ligase at 16 ℃ for 10-15 h.

Transformation and identification, namely transforming a ligation product into a host bacterium DH5 α after ligation reaction to screen recombinants, carrying out amplification culture on the screened monoclonal colony, extracting a plasmid, and then sequencing by Soviken Zhi Biotech Co., Ltd to obtain a fusion recombinant vector pETSUMO-28 a.

And (3) secondary enzyme digestion and connection: carrying out BamHI/Xho1 double enzyme digestion treatment on an L1 gene fragment containing BamHI and Xho1 enzyme digestion sites and a recombinant vector pETSUMO-28a, and then carrying out a ligation reaction on the recovered gene fragment and pETSUMO-28a containing a corresponding cohesive end by using T4 DNA ligase at 16 ℃ for 10-15 h.

And performing secondary transformation and identification, namely transforming a connecting product into host bacteria DH5 α after the connection reaction to screen a recon, performing amplification culture on the screened monoclonal colony, extracting a plasmid, and sequencing by Shanghai bio-organisms to obtain a gene sequence SEQ NO.7 of the fusion recombinant MBP-HPV16-L1 protein.

Example 7: expression of recombinant HPV L1 pentamer protein

The recombinant vectors of the correct sequencing results of the examples 2, 3, 4, 5 and 6 are transformed into an Escherichia coli BL21 host cell, and used as an engineering bacterium for expressing recombinant proteins to express HPV L1 proteins. The engineering bacteria culture medium is 2YT culture medium (10 g/L tryptone, 5 g/L yeast powder, 10 g/L NaCl). Single spots of the cells containing the recombinant plasmid were picked up in 10ml of 2YT medium (containing 100. mu.g/ml ampicillin), and cultured at 230 revolutions per minute (rpm) overnight at 37 ℃ with shaking. Transferring 5ml of overnight bacteria into 500 ml (containing 100 mu g/ml ampicillin) 2YT liquid culture medium, shaking and culturing at 37 ℃ until the recombinant engineering bacteria grow to OD600nm which is approximately equal to 0.4-1, adding IPTG with the final concentration of 0.2mM for induction and expression of recombinant protein for more than 6h at 28 ℃.

Cell collection and disruption: centrifuging the fermentation culture, discarding the supernatant, harvesting thallus precipitate, and weighing; the pellet was washed with buffer L (pH 8.0, 50mM Tris, 200 mM NaCl, 5mM DTT), then resuspended in buffer L for ultrasonication, followed by centrifugation of the lysate by a high speed centrifuge (16000 rpm, 30 min, 4 ℃) and the supernatant was collected.

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

Detecting the expression quantity of the Tag-HPV L1 pentamer protein in escherichia coli before loading by adopting an ELISA sandwich method, and detecting a sample and a test sample:

coating antibody: self-made anti-HPV 16L1 mouse monoclonal antibody.

Comparison products: high-purity HPV16L1 protein is prepared.

And (3) testing the sample: the test product Tag-HPV16L1 was diluted with the sample diluent to a concentration within the control gradient dilution range.

Enzyme-labeled antibody: the self-made horseradish peroxidase-labeled rabbit anti-HPV 16L1 protein polyclonal antibody.

Results calculation the mean of the parallel wells was calculated to control line concentration OD₄₅₀Making a linear equation for the absorption values of the corresponding L1 protein antigens, wherein the variation coefficient between parallel sample wells is not more than 10%, and performing a linear regression equation R²Not less than 0.980, OD of the sample₄₅₀The absorption value is substituted into an equation to calculate the content of the L1 protein antigen in the test sample after dilution, and the content of the L1 protein antigen in the test sample is obtained by multiplying the content by the corresponding dilution times, which is shown in Table 1.

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

Example 9: recombinant HPV L1 pentamer protein affinity chromatography

Affinity chromatography of recombinant proteins with GST tag: the affinity column was filled with 5ml of GST agarose affinity chromatography medium, equilibrated with Buffer L (pH 8.0, 50mM Tris, 200 mM NaCl, 5mM DTT), and loaded with the protein solution of example 8 with GST or GST-SUMO tag, and washed with Buffer L until no protein was eluted, and the affinity was complete. The affinity medium was suspended at 5mLBuffer L and samples were taken for detection and calculation of the total amount of bound L1 protein in the medium.

Affinity chromatography of MBP-tagged recombinant proteins: the affinity column was loaded with 5ml of an Amylose-Resin affinity chromatography medium, equilibrated with Buffer L (pH 8.0, 50mM Tris, 200 mM NaCl, 5mM DTT), and loaded with the protein solution of example 8 with GST or GST-SUMO tag, and washed with Buffer L until no protein eluted, and the affinity was complete. The affinity medium was suspended at 5mLBuffer L and samples were taken for detection and calculation of the total amount of bound L1 protein in the medium.

Affinity chromatography of recombinant proteins with 6 × HIS tag: 5ml of Ni-NTA gel was loaded on a column, and 10 column volumes of an equilibration solution (50 mmol/L NaH2PO4, 300mmol/L NaCl, 20mmol/L imidizole, pH adjusted to 8 with NaOH) were slowly added to the column to fully equilibrate the Ni-NTA gel at a flow rate of 1 ml/min. The supernatant from example 8, after filtration and with 6 × His tag, was taken and after complete gel entry, the gel was washed further with 10 column volumes of equilibration solution, maintaining the flow rate at 1 ml/min. Eluting with the balance solution until no protein flows out, and finishing the affinity. Sampling was performed to detect and calculate the total amount of bound L1 protein in the medium.

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

According to the mass ratio of the target protein to the protease of 100: 1, adding enzyme amount, wherein the protein with GST-HPV-L1 is cut by 3C protein, the protein with GST-SUMO-HPV-L1 and 6 His-SUMO-HPV-L1 is cut by SENP1 protein, the protein with Mbp-HPV-L1 is cut by Factor Xa protein, the protein with 6 His-HPV-L1 is cut by Thrombin protein, and after 2h of mixed digestion, respectively eluting and collecting HPV16L1 pentamer protein solution obtained after each protease digestion.

The L1 protein solution after the 3C enzyme cuts the GST tag is detected by SDS-PAGE gel electrophoresis, the result is shown in figure 1 affinity chromatography electrophoresis result, and the experiment shows that 90% of the target protein can 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. FIG. 3 shows that Factor Xa protease cleaves proteins with Mbp-HPV-L1, as detected by SDS-PAGE gel electrophoresis. FIGS. 1-3 illustrate that the HPV16L1 protein of 55kDa is obtained.

The thrombobin protease did not cleave the 6 His-HPV-L1 protein; the protein solution from 6 His-SUMO-L1 digested with SENP1 was examined by SDS-PAGE gel electrophoresis, and the results are shown in FIG. 4, which shows that SENP1 protease failed to cleave 6 His-SUMO-tagged fusion proteins.

Example 11: purification of recombinant HPV L1 pentamer protein

And (3) purifying by molecular sieve chromatography: the HPV16L1 pentamer protein collected in the previous example after enzyme digestion and purification is respectively purified, the HPV16L1 pentamer protein collected by ion exchange chromatography can be firstly subjected to further molecular sieve chromatography by using gel filtration medium of Superdex200 (manufactured by GE company) without ion exchange step, the mobile phase of the molecular sieve is pH8.0, 10 mM Tris and 100 mM NaCl, and the fraction of the ultraviolet absorption peak of the HPV16L1 pentamer protein is collected.

Determination of sample purity after purification: and (3) sampling the collected protein solution, and detecting by SDS-PAGE gel electrophoresis, wherein the final purities of the pentamer of the target protein HPV16L1 are all more than 98% after molecular sieve chromatography, and the detailed picture is shown in FIG. 5, and the SDS-PAGE gel electrophoresis picture of the protein of the pentamer of the recombinant HPV16L1 after molecular sieve chromatography.

Determination of the protein concentration of the sample: protein concentration was measured by the Bradford method using a standard sample of 2mg/ml BAS prepared from 100ug/ul to 500ug/ul, and 10ul diluted BSA +200ul Bradford working solution was taken from the sample reaction: standard curve y = 0.0013 x-0.0294, R = 0.9986, OD of determination sample₅₉₅Substituting the standard curve to calculate the protein concentration of the sample, and the result is shown in Table 2.

TABLE 2 Bradford method for detecting recombinant HPV16L1 pentamer protein concentration

Note: the sample group 1 is an HPV L1 pentamer protein solution obtained by purifying GST-HPV L1 by a molecular sieve; the sample group 2 is an HPV L1 pentameric protein solution obtained by purifying GST-SUMO-HPV L1 through a molecular sieve; and the sample group 3 is an HPV L1 pentamer protein solution obtained by purifying the Mbp-HPV L1 by a molecular sieve.

Example 12: assembly of recombinant HPV16L1 pentamer proteins into VLPs

After being placed and stabilized in the following salt concentration (NaCl) and pH value conditions, HPV L1 pentamer solution sample groups 1, 2 and 3 are subjected to particle size and particle size distribution measurement (the particle size distribution coefficient PdI is an index of particle size dispersion degree, less than 0.05 is a highly uniform sample, 0.05-0.1 is a quasi-uniform sample, 0.1-0.3 is a sample with poor uniformity, and more than 0.3 is a non-uniform sample) by using a dynamic light scattering particle sizer of Malvern Zetasizer NanoZS, and the HPV16L1 pentamer protein is assembled to obtain VLPs with uniform particle sizes (PdI is less than 0.05).

Table 3 particle size detection of assembled HPV16L1 VLPs at different pH and salt concentrations

Note: the sample group 1 is a HPV L1VLP protein solution obtained by purifying GST-HPV L1 by a molecular sieve; the sample group 2 is an HPV L1VLP protein solution obtained by purifying GST-SUMO-HPV L1 through a molecular sieve; sample group 3 is an HPV L1VLP protein solution obtained by purifying Mbp-HPV L1 with a molecular sieve.

Example 13: dynamic Light Scattering (DLS) particle size determination of L1 pentamer and VLP proteins

The instrument is a dynamic light scattering particle size analyzer of a Malvern Zetasizer NanoZS, HPV16L1 pentamer and HPV16L 1VLP protein finally prepared by each sample group are taken for detection, and the average particle size and the dispersity index PdI (indicating the uniformity of the protein) are measured, so that the uniformity of the L1 pentamer and the VLP protein finally prepared by each group of samples is indicated. The particle size distribution of the pentameric protein finally prepared in sample set 2 and the HPV16L 1VLP protein obtained by the assembly thereof is shown in FIGS. 6 and 7.

Example 14: preparation of HPV16L1 pentamer and VLP

According to the technology adopted in the above embodiments 1-13, the HPV16L1 protein with the sequences 11, 12 and 13 is prepared, and the protein can be purified to obtain the protein with the purity of more than 98%, and the HPV16L1 pentamer protein with the average particle size of 10-15 nm and the PdI of less than 0.1 is obtained. Further assembling to obtain HPV16L 1VLP protein with the average particle size of 45-65 nm and PdI < 0.1.

Example 15: experiment for influence of low-temperature freezing treatment on L1 pentamer protein in-vitro assembly VLP

VLPs were assembled by placing the L1 pentamer protein at room temperature under the salt concentration and PH conditions of the table below, and their particle size and PdI values were measured. Then, the mixture is frozen at-80 ℃ for 24 hours, then is placed at room temperature until the protein solution is melted, and the particle size and the PdI value of the mixture are detected again. The protein (PdI is more than 0.1) with nonuniform particle size and assembled under the condition of room temperature (before freeze thawing) can be changed into VLP protein with particle size which is consistent with theoretical expectation and is uniform, and the PdI is less than 0.1 through freezing treatment.

Table 4 detection of particle size of HPV16L pentamer protein assembled VLP at room temperature

TABLE 5 particle size test results of assembled VLPs after freezing treatment of HPV16L pentamer protein at-80 deg.C

Example 16: morphological examination of HPV16L1 pentamer and VLP

And (3) observing by a transmission electron microscope: the HPV16L1 pentamer protein obtained by purification and the HPV16L 1-VLP protein obtained by assembly in each example were observed by a transmission electron microscope platform used in the biophysics of the Chinese academy of sciences. Preparation of frozen sample and photographing process:

1) the liquid nitrogen box is filled with liquid nitrogen, and when the liquid level is not boiling, ethane is slowly injected into the cooled copper bowl to be cooled into liquid.

2) The copper mesh was hydrophilically treated in a PDC-32 type plasma cleaner.

3) In a Vitrobot TM Mark IV frozen sample preparation apparatus, 3.5. mu.L of a pentamer and VLP sample was adsorbed on a 300 mesh QUANTIFOIL copper mesh, and after absorbing water for 4s, the sample was frozen by liquid ethane.

4) The samples were quickly transferred to liquid nitrogen for storage.

5) When the frozen photographs were collected, the electron dose was 20 e-/Å 2 data were recorded by a 300 kV Titan Krios transmission electron microscope Gatan UltraScan 4000 CCD with an acceleration voltage of 300 kV.

The results show that in the HPV16L1 pentameric protein sample group, a large number of pentameric proteins with the diameter of about 10nm, which is consistent with the theoretical size, are visible in the visual field; in the HPV16L 1-VLP protein sample group, a large number of virus-like particles (VLPs) with a diameter of around 50nm, which are consistent in particle size with theory, were found, and were uniform. Wherein the transmission electron micrograph of the sample obtained by enzyme-cutting purified HPV16L1 pentamer of GST-SUMO label group (sample group 2) is shown in figure 8, and the transmission electron micrograph of the VLP protein assembled after enzyme-cutting purification of Mbp label group (sample group 3) is shown in figure 9.

Example 17: purity detection of HPV16L1 protein stock solution

The molecular exclusion high performance liquid chromatography determination comprises the steps of performing molecular exclusion high performance liquid chromatography determination on a chromatographic column Agilent Bio SEC-5um, 2000 Å, 7.8 multiplied by 300mm, the column volume is about 15m 1, the molecular weight range is larger than or equal to lO, OOOkDa, taking 0.1mol/L phosphate buffer solution (25.8 g of disodium hydrogen phosphate and 4.37g of sodium dihydrogen phosphate are weighed and added with ultrapure water to be dissolved, adjusting the pH to 6.8 by using phosphoric acid, and fixing the volume to 1000 ml) with pH6.8 as a mobile phase, the flow rate is 1ml/min, the detection wavelength is 280nm, the column temperature is 25 ℃, the sample loading amount is not less than 20ug, the theoretical number of sample column plates at the main peak is not less than 1000, the tailing factor is less than 2.0, 5 needles are continuously injected, and the relative standard deviation of.

The protein stock solutions of the HPV16L1 pentamer finally prepared and the assembled VLP of the purified sample 2 group are respectively diluted to the concentration of 1mg/ml, the sample loading amount is 20ul, the protein stock solutions are injected into a high pressure liquid chromatograph, the purity is calculated according to the area normalization method according to the detection method, the purity of all processed proteins is more than 98%, and the results are shown in an attached figure 10, a table 6, an attached figure 11 and a table 7.

TABLE 6 HPLC PROTEIN PURITY TESTING OF HPV16L1 PENTAMER

TABLE 7 HPLC PROTEIN PURITY TESTING OF HPV16L1 ASSEMBLED VLPs

Peak(s)	Retention time	Area of	Area%
				1	13.854	2673380	100.00
Total of		2673380	100.00

Example 18: HPV VLP stability assay

The HPV16 VLP protein finally prepared from each sample group is placed at 25 ℃ for 14 days to 28 days under the buffer conditions of the following table for particle size detection, and the results are shown in the following table, which proves that the HPV16 VLP is stable in storage at pH 5.0 to 5.9 and salt concentration of 500-2000 mM. The detection results of HPV16 VLPs obtained in sample group 3 after 14-28 days at pH 5.0 to 5.9 and at a salt concentration of 500-2000 mM are shown in the following table.

TABLE 8 detection results of particle size of HPV16L 1VLP after standing at 25 ℃ for 14-28 days

Example 19: preparation of monovalent vaccines comprising HPV L1 pentamer or VLP

And adsorbing and preparing the HPV16L1 pentamer or VLP protein stock solution containing each sample group and an aluminum hydroxide adjuvant physiological saline solution according to the ratio of the protein to the aluminum content of 1:10 respectively to prepare the recombinant HPV L1 protein pentamer or VLP vaccine, and storing at 4 ℃ for later use.

Example 20: immunogenicity assays for HPV L1 pentamers and VLPs

The L1 pentamer or VLP vaccine was taken, and sterilized saline was added to dilute the vaccine into a 20. mu.g/ml concentration of the pentamer or VLP protein vaccine, respectively, and BALB/c mice were injected intramuscularly at 0.1ml per one mouse, 10 per group. Mice were boosted every 4 weeks for 2 total immunizations. After 4 weeks of booster immunization, the neutralizing antibody titer against homotype HPV in the serum of mice after each immunization was determined by a pseudovirus cell neutralization assay, and the results are shown in fig. 12.

The results show that the neutralizing antibody can reach a high level 4 weeks after the secondary immunization when the HPV L1 pentamer and VLP protein vaccine is used for inoculating the mice. The experimental result proves that the HPV L1 pentamer and the assembled VLP vaccine can generate neutralizing antibodies in animals, which indicates that the HPV L1 pentamer and the VLP protein vaccine have immunogenicity in human clinical tests, namely, the diseases caused by HPV homotype viruses can be prevented.

SEQUENCE LISTING

<110> Beijing Kangle guard Biotechnology Ltd

<120>16 type recombinant human papilloma virus-like particle and preparation method thereof

<130>2014

<160>13

<170>PatentIn version 3.3

<210>1

<211>1518

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<213> Artificial sequence

<400>1

atgtctcttt ggctgcctag tgaggccact gtctacttgc ctcctgtccc agtatctaag 60

gttgtaagca cggatgaata tgttgcacgc acaaacatat attatcatgc aggaacatcc 120

agactacttg cagttggaca tccctatttt cctattaaaa aacctaacaa taacaaaata 180

ttagttccta aagtatcagg attacaatac agggtattta gaatacattt acctgacccc 240

aataagtttg gttttcctga cacctcattt tataatccag atacacagcg gctggtttgg 300

gcctgtgtag gtgttgaggt aggtcgtggt cagccattag gtgtgggcat tagtggccat 360

cctttattaa ataaattgga tgacacagaa aatgctagtg cttatgcagc aaatgcaggt 420

gtggataata gagaatgtat atctatggat tacaaacaaa cacaattgtg tttaattggt 480

tgcaaaccac ctatagggga acactggggc aaaggatccc catgtaccaa tgttgcagta 540

aatccaggtg attgtccacc attagagtta ataaacacag ttattcagga tggtgatatg 600

gttgatactg gctttggtgc tatggacttt actacattac aggctaacaa aagtgaagtt 660

ccactggata tttgtacatc tatttgcaaa tatccagatt atattaaaat ggtgtcagaa 720

ccatatggcg acagcttatt tttttattta cgaagggaac aaatgtttgt tagacattta 780

tttaataggg ctggtgctgt tggtgaaaat gtaccagacg atttatacat taaaggctct 840

gggtctactg caaatttagc cagttcaaat tattttccta cacctagtgg ttctatggtt 900

acctctgatg cccaaatatt caataaacct tattggttac aacgagcaca gggccacaat 960

aatggcattt gttggggtaa ccaactattt gttactgttg ttgatactac acgcagtaca 1020

aatatgtctt tatgtgctgc catatctact tcagaaacta catataaaaa tactaacttt 1080

aaggagtacc tacgacatgg ggaggaatat gatttacagt ttatttttca actgtgcaaa 1140

ataaccttaa ctgcagacgt tatgacatac atacattcta tgaattctac tattttggag 1200

gactggaatt ttggtctaca acctccccca ggaggcacac tagaagatac ttataggttt 1260

gtaacatccc aggcaattgc ttgtcaaaaa catacacctc cagcacctaa agaagatccc 1320

cttaaaaaat acactttttg ggaagtaaat ttaaaggaaa agttttctgc agacctagat 1380

cagtttcctt taggacgcaa atttttacta caagcaggat tgaaggccaa accaaaattt 1440

acattaggaa aacgaaaagc tacacccacc acctcatcta cctctacaac tgctaaacgc 1500

aaacaacgta agctgtaa 1518

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cgtctgctgg ctgttggtca cccgtacttc ccgatcaaaa aaccgaacaa caacaaaatc 180

ctggttccga aagtttctgg tctgcagtac cgtgttttcc gtatccacct gccggacccg 240

aacaaattcg gtttcccgga cacctctttc tacaacccgg acacccagcg tctggtttgg 300

gcttgcgttg gtgttgaagt tggtcgtggt cagccgctgg gtgttggtat ctctggtcac 360

ccgctgctga acaaactgga cgacaccgaa aacgcttctg cttacgctgc taacgctggt 420

gttgacaacc gtgaatgcat ctctatggac tacaaacaga cccagctgtg cctgatcggt 480

tgcaaaccgc cgatcggtga acactggggt aaaggttctc cgtgcaccaa cgttgctgtt 540

aacccgggtg actgcccgcc gctggaactg atcaacaccg ttatccagga cggtgacatg 600

gttgacaccg gtttcggtgc tatggacttc accaccctgc aggctaacaa atctgaagtt 660

ccgctggaca tctgcacctc tatctgcaaa tacccggact acatcaaaat ggtttctgaa 720

ccgtacggtg actctctgtt cttctacctg cgtcgtgaac agatgttcgt tcgtcacctg 780

ttcaaccgtg ctggtgctgt tggtgaaaac gttccggacg acctgtacat caaaggttct 840

ggttctaccg ctaacctggc ttcttctaac tacttcccga ccccgtctgg ttctatggtt 900

acctctgacg ctcagatctt caacaaaccg tactggctgc agcgtgctca gggtcacaac 960

aacggtatct gctggggtaa ccagctgttc gttaccgttg ttgacaccac ccgttctacc 1020

aacatgtctc tgtgcgctgc tatctctacc tctgaaacca cctacaaaaa caccaacttc 1080

aaagaatacc tgcgtcacgg tgaagaatac gacctgcagt tcatcttcca gctgtgcaaa 1140

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gactggaact tcggtctgca gccgccgccg ggtggtaccc tggaagacac ctaccgtttc 1260

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cagttcccgc tgggtcgtaa attcctgctg caggctggtc tgaaagctaa accgaaattc 1440

accctgggta aacgtaaagc taccccgacc acctcttcta cctctaccac cgctaaacgt 1500

aaacagcgta aactg 1515

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gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggaagttc tgttccaggg gcccctggga tccatgtctc tgtggctgcc gtctgaagct 720

accgtttacc tgccgccggt tccggtttct aaagttgttt ctaccgacga atacgttgct 780

cgtaccaaca tctactacca cgctggtacc tctcgtctgc tggctgttgg tcacccgtac 840

ttcccgatca aaaaaccgaa caacaacaaa atcctggttc cgaaagtttc tggtctgcag 900

taccgtgttt tccgtatcca cctgccggac ccgaacaaat tcggtttccc ggacacctct 960

ttctacaacc cggacaccca gcgtctggtt tgggcttgcg ttggtgttga agttggtcgt 1020

ggtcagccgc tgggtgttgg tatctctggt cacccgctgc tgaacaaact ggacgacacc 1080

gaaaacgctt ctgcttacgc tgctaacgct ggtgttgaca accgtgaatg catctctatg 1140

gactacaaac agacccagct gtgcctgatc ggttgcaaac cgccgatcgg tgaacactgg 1200

ggtaaaggtt ctccgtgcac caacgttgct gttaacccgg gtgactgccc gccgctggaa 1260

ctgatcaaca ccgttatcca ggacggtgac atggttgaca ccggtttcgg tgctatggac 1320

ttcaccaccc tgcaggctaa caaatctgaa gttccgctgg acatctgcac ctctatctgc 1380

aaatacccgg actacatcaa aatggtttct gaaccgtacg gtgactctct gttcttctac 1440

ctgcgtcgtg aacagatgtt cgttcgtcac ctgttcaacc gtgctggtgc tgttggtgaa 1500

aacgttccgg acgacctgta catcaaaggt tctggttcta ccgctaacct ggcttcttct 1560

aactacttcc cgaccccgtc tggttctatg gttacctctg acgctcagat cttcaacaaa 1620

ccgtactggc tgcagcgtgc tcagggtcac aacaacggta tctgctgggg taaccagctg 1680

ttcgttaccg ttgttgacac cacccgttct accaacatgt ctctgtgcgc tgctatctct 1740

acctctgaaa ccacctacaa aaacaccaac ttcaaagaat acctgcgtca cggtgaagaa 1800

tacgacctgc agttcatctt ccagctgtgc aaaatcaccc tgaccgctga cgttatgacc 1860

tacatccact ctatgaactc taccatcctg gaagactgga acttcggtct gcagccgccg 1920

ccgggtggta ccctggaaga cacctaccgt ttcgttacct ctcaggctat cgcttgccag 1980

aaacacaccc cgccggctcc gaaagaagac ccgctgaaaa aatacacctt ctgggaagtt 2040

aacctgaaag aaaaattctc tgctgacctg gaccagttcc cgctgggtcg taaattcctg 2100

ctgcaggctg gtctgaaagc taaaccgaaa ttcaccctgg gtaaacgtaa agctaccccg 2160

accacctctt ctacctctac caccgctaaa cgtaaacagc gtaaactg 2208

<210>4

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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 gccctctgac caggaagcta aaccgtctac cgaagacctg 720

ggtgacaaaa aagaaggtga atacatcaaa ctgaaagtta tcggtcagga ctcttctgaa 780

atccacttca aagttaaaat gaccacccac ctgaaaaaac tgaaagaatc ttactgccag 840

cgtcagggtg ttccgatgaa ctctctgcgt ttcctgttcg aaggtcagcg tatcgctgac 900

aaccacaccc cgaaagaact gggtatggaa gaagaagacg ttatcgaagt ttaccaggaa 960

cagaccggtg gatccatgtc tctgtggctg ccgtctgaag ctaccgttta cctgccgccg 1020

gttccggttt ctaaagttgt ttctaccgac gaatacgttg ctcgtaccaa catctactac 1080

cacgctggta cctctcgtct gctggctgtt ggtcacccgt acttcccgat caaaaaaccg 1140

aacaacaaca aaatcctggt tccgaaagtt tctggtctgc agtaccgtgt tttccgtatc 1200

cacctgccgg acccgaacaa attcggtttc ccggacacct ctttctacaa cccggacacc 1260

cagcgtctgg tttgggcttg cgttggtgtt gaagttggtc gtggtcagcc gctgggtgtt 1320

ggtatctctg gtcacccgct gctgaacaaa ctggacgaca ccgaaaacgc ttctgcttac 1380

gctgctaacg ctggtgttga caaccgtgaa tgcatctcta tggactacaa acagacccag 1440

ctgtgcctga tcggttgcaa accgccgatc ggtgaacact ggggtaaagg ttctccgtgc 1500

accaacgttg ctgttaaccc gggtgactgc ccgccgctgg aactgatcaa caccgttatc 1560

caggacggtg acatggttga caccggtttc ggtgctatgg acttcaccac cctgcaggct 1620

aacaaatctg aagttccgct ggacatctgc acctctatct gcaaataccc ggactacatc 1680

aaaatggttt ctgaaccgta cggtgactct ctgttcttct acctgcgtcg tgaacagatg 1740

ttcgttcgtc acctgttcaa ccgtgctggt gctgttggtg aaaacgttcc ggacgacctg 1800

tacatcaaag gttctggttc taccgctaac ctggcttctt ctaactactt cccgaccccg 1860

tctggttcta tggttacctc tgacgctcag atcttcaaca aaccgtactg gctgcagcgt 1920

gctcagggtc acaacaacgg tatctgctgg ggtaaccagc tgttcgttac cgttgttgac 1980

accacccgtt ctaccaacat gtctctgtgc gctgctatct ctacctctga aaccacctac 2040

aaaaacacca acttcaaaga atacctgcgt cacggtgaag aatacgacct gcagttcatc 2100

ttccagctgt gcaaaatcac cctgaccgct gacgttatga cctacatcca ctctatgaac 2160

tctaccatcc tggaagactg gaacttcggt ctgcagccgc cgccgggtgg taccctggaa 2220

gacacctacc gtttcgttac ctctcaggct atcgcttgcc agaaacacac cccgccggct 2280

ccgaaagaag acccgctgaa aaaatacacc ttctgggaag ttaacctgaa agaaaaattc2340

tctgctgacc tggaccagtt cccgctgggt cgtaaattcc tgctgcaggc tggtctgaaa 2400

gctaaaccga aattcaccct gggtaaacgt aaagctaccc cgaccacctc ttctacctct 2460

accaccgcta aacgtaaaca gcgtaaactg 2490

<210>5

<211>2688

<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 ctgacggggg 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 ttcatgtctc tgtggctgcc gtctgaagct 1200

accgtttacc tgccgccggt tccggtttct aaagttgttt ctaccgacga atacgttgct 1260

cgtaccaaca tctactacca cgctggtacc tctcgtctgc tggctgttgg tcacccgtac 1320

ttcccgatca aaaaaccgaa caacaacaaa atcctggttc cgaaagtttc tggtctgcag 1380

taccgtgttt tccgtatcca cctgccggac ccgaacaaat tcggtttccc ggacacctct 1440

ttctacaacc cggacaccca gcgtctggtt tgggcttgcg ttggtgttga agttggtcgt 1500

ggtcagccgc tgggtgttgg tatctctggt cacccgctgc tgaacaaact ggacgacacc 1560

gaaaacgctt ctgcttacgc tgctaacgct ggtgttgaca accgtgaatg catctctatg 1620

gactacaaac agacccagct gtgcctgatc ggttgcaaac cgccgatcgg tgaacactgg 1680

ggtaaaggtt ctccgtgcac caacgttgct gttaacccgg gtgactgccc gccgctggaa 1740

ctgatcaaca ccgttatcca ggacggtgac atggttgaca ccggtttcgg tgctatggac 1800

ttcaccaccc tgcaggctaa caaatctgaa gttccgctgg acatctgcac ctctatctgc 1860

aaatacccgg actacatcaa aatggtttct gaaccgtacg gtgactctct gttcttctac 1920

ctgcgtcgtg aacagatgtt cgttcgtcac ctgttcaacc gtgctggtgc tgttggtgaa 1980

aacgttccgg acgacctgta catcaaaggt tctggttcta ccgctaacct ggcttcttct 2040

aactacttcc cgaccccgtc tggttctatg gttacctctg acgctcagat cttcaacaaa 2100

ccgtactggc tgcagcgtgc tcagggtcac aacaacggta tctgctgggg taaccagctg 2160

ttcgttaccg ttgttgacac cacccgttct accaacatgt ctctgtgcgc tgctatctct 2220

acctctgaaa ccacctacaa aaacaccaac ttcaaagaat acctgcgtca cggtgaagaa 2280

tacgacctgc agttcatctt ccagctgtgc aaaatcaccc tgaccgctga cgttatgacc 2340

tacatccact ctatgaactc taccatcctg gaagactgga acttcggtct gcagccgccg 2400

ccgggtggta ccctggaaga cacctaccgt ttcgttacct ctcaggctat cgcttgccag 2460

aaacacaccc cgccggctcc gaaagaagac ccgctgaaaa aatacacctt ctgggaagtt 2520

aacctgaaag aaaaattctc tgctgacctg gaccagttcc cgctgggtcg taaattcctg 2580

ctgcaggctg gtctgaaagc taaaccgaaa ttcaccctgg gtaaacgtaa agctaccccg 2640

accacctctt ctacctctac caccgctaaa cgtaaacagc gtaaactg 2688

<210>6

<211>1578

<212>DNA

<213> Artificial sequence

<400>6

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgatgtctc tgtggctgcc gtctgaagct accgtttacc tgccgccggt tccggtttct 120

aaagttgttt ctaccgacga atacgttgct cgtaccaaca tctactacca cgctggtacc 180

tctcgtctgc tggctgttgg tcacccgtac ttcccgatca aaaaaccgaa caacaacaaa 240

atcctggttc cgaaagtttc tggtctgcag taccgtgttt tccgtatcca cctgccggac 300

ccgaacaaat tcggtttccc ggacacctct ttctacaacc cggacaccca gcgtctggtt 360

tgggcttgcg ttggtgttga agttggtcgt ggtcagccgc tgggtgttgg tatctctggt 420

cacccgctgc tgaacaaact ggacgacacc gaaaacgctt ctgcttacgc tgctaacgct 480

ggtgttgaca accgtgaatg catctctatg gactacaaac agacccagct gtgcctgatc 540

ggttgcaaac cgccgatcgg tgaacactgg ggtaaaggtt ctccgtgcac caacgttgct 600

gttaacccgg gtgactgccc gccgctggaa ctgatcaaca ccgttatcca ggacggtgac 660

atggttgaca ccggtttcgg tgctatggac ttcaccaccc tgcaggctaa caaatctgaa 720

gttccgctgg acatctgcac ctctatctgc aaatacccgg actacatcaa aatggtttct 780

gaaccgtacg gtgactctct gttcttctac ctgcgtcgtg aacagatgtt cgttcgtcac 840

ctgttcaacc gtgctggtgc tgttggtgaa aacgttccgg acgacctgta catcaaaggt 900

tctggttcta ccgctaacct ggcttcttct aactacttcc cgaccccgtc tggttctatg 960

gttacctctg acgctcagat cttcaacaaa ccgtactggc tgcagcgtgc tcagggtcac 1020

aacaacggta tctgctgggg taaccagctg ttcgttaccg ttgttgacac cacccgttct 1080

accaacatgt ctctgtgcgc tgctatctct acctctgaaa ccacctacaa aaacaccaac 1140

ttcaaagaat acctgcgtca cggtgaagaa tacgacctgc agttcatctt ccagctgtgc 1200

aaaatcaccc tgaccgctga cgttatgacc tacatccact ctatgaactc taccatcctg 1260

gaagactgga acttcggtct gcagccgccg ccgggtggta ccctggaaga cacctaccgt 1320

ttcgttacct ctcaggctat cgcttgccag aaacacaccc cgccggctcc gaaagaagac 1380

ccgctgaaaa aatacacctt ctgggaagtt aacctgaaag aaaaattctc tgctgacctg 1440

gaccagttcc cgctgggtcg taaattcctg ctgcaggctg gtctgaaagc taaaccgaaa 1500

ttcaccctgg gtaaacgtaa agctaccccg accacctctt ctacctctac caccgctaaa 1560

cgtaaacagc gtaaactg 1578

<210>7

<211>1869

<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 gtcagggtgt tccgatgaac 240

tctctgcgtt tcctgttcga aggtcagcgt atcgctgaca accacacccc gaaagaactg 300

ggtatggaag aagaagacgt tatcgaagtt taccaggaac agaccggtgg atccatgtct 360

ctgtggctgc cgtctgaagc taccgtttac ctgccgccgg ttccggtttc taaagttgtt 420

tctaccgacg aatacgttgc tcgtaccaac atctactacc acgctggtac ctctcgtctg 480

ctggctgttg gtcacccgta cttcccgatc aaaaaaccga acaacaacaa aatcctggtt 540

ccgaaagttt ctggtctgca gtaccgtgtt ttccgtatcc acctgccgga cccgaacaaa 600

ttcggtttcc cggacacctc tttctacaac ccggacaccc agcgtctggt ttgggcttgc 660

gttggtgttg aagttggtcg tggtcagccg ctgggtgttg gtatctctgg tcacccgctg 720

ctgaacaaac tggacgacac cgaaaacgct tctgcttacg ctgctaacgc tggtgttgac 780

aaccgtgaat gcatctctat ggactacaaa cagacccagc tgtgcctgat cggttgcaaa 840

ccgccgatcg gtgaacactg gggtaaaggt tctccgtgca ccaacgttgc tgttaacccg 900

ggtgactgcc cgccgctgga actgatcaac accgttatcc aggacggtga catggttgac 960

accggtttcg gtgctatgga cttcaccacc ctgcaggcta acaaatctga agttccgctg 1020

gacatctgca cctctatctg caaatacccg gactacatca aaatggtttc tgaaccgtac 1080

ggtgactctc tgttcttcta cctgcgtcgt gaacagatgt tcgttcgtca cctgttcaac 1140

cgtgctggtg ctgttggtga aaacgttccg gacgacctgt acatcaaagg ttctggttct 1200

accgctaacc tggcttcttc taactacttc ccgaccccgt ctggttctat ggttacctct 1260

gacgctcaga tcttcaacaa accgtactgg ctgcagcgtg ctcagggtca caacaacggt 1320

atctgctggg gtaaccagct gttcgttacc gttgttgaca ccacccgttc taccaacatg 1380

tctctgtgcg ctgctatctc tacctctgaa accacctaca aaaacaccaa cttcaaagaa 1440

tacctgcgtc acggtgaaga atacgacctg cagttcatct tccagctgtg caaaatcacc 1500

ctgaccgctg acgttatgac ctacatccac tctatgaact ctaccatcct ggaagactgg 1560

aacttcggtc tgcagccgcc gccgggtggt accctggaag acacctaccg tttcgttacc 1620

tctcaggcta tcgcttgcca gaaacacacc ccgccggctc cgaaagaaga cccgctgaaa 1680

aaatacacct tctgggaagt taacctgaaa gaaaaattct ctgctgacct ggaccagttc 1740

ccgctgggtc gtaaattcct gctgcaggct ggtctgaaag ctaaaccgaa attcaccctg 1800

ggtaaacgta aagctacccc gaccacctct tctacctcta ccaccgctaa acgtaaacag 1860

cgtaaactg 1869

<210>8

<211>736

<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 Ser Leu Trp Leu Pro Ser Glu Ala

225 230 235 240

Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp

245 250 255

Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala Gly Thr Ser Arg

260 265 270

Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn

275 280 285

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

290 295 300

Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser

305 310 315 320

Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly Val

325 330 335

Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly His Pro

340 345 350

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

355 360 365

Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln

370 375 380

Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile Gly Glu His Trp

385 390395 400

Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro Gly Asp Cys

405 410 415

Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val

420 425 430

Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln Ala Asn Lys

435 440 445

Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp

450 455 460

Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Tyr

465 470 475 480

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

485 490 495

Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys Gly Ser Gly

500 505 510

Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr Pro Ser Gly

515 520 525

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

530 535 540

Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu

545550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His Ser

610 615 620

Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu Gln Pro Pro

625 630 635 640

Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val Thr Ser Gln Ala

645 650 655

Ile Ala Cys Gln Lys His Thr Pro Pro Ala Pro Lys Glu Asp Pro Leu

660 665 670

Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala

675 680 685

Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly

690 695 700

Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys Ala Thr Pro

705 710 715 720

Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Gln Arg Lys Leu

725 730 735

<210>9

<211>830

<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 105110

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

260265 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 Ser Leu Trp Leu Pro Ser Glu Ala Thr Val

325 330 335

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

340 345 350

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

355 360 365

Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys

370 375 380

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

385 390 395 400

His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr

405 410 415

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

420 425 430

Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu

435 440 445

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

450 455 460

Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln

465 470 475 480

Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys

485 490 495

Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro

500 505 510

Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val Asp Thr

515 520 525

Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu

530 535 540

Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile

545 550 555 560

Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg

565 570 575

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

580 585 590

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

595 600 605

Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met

610 615 620

Val Thr Ser Asp Ala Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg

625 630 635 640

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

645 650 655

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

660 665 670

Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr

675 680 685

Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys

690 695 700

Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His Ser Met Asn

705 710 715 720

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

725 730 735

Gly Thr Leu Glu Asp Thr Tyr ArgPhe Val Thr Ser Gln Ala Ile Ala

740 745 750

Cys Gln Lys His Thr Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys

755 760 765

Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu

770 775 780

Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys

785 790 795 800

Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr

805 810 815

Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Gln Arg Lys Leu

820 825 830

<210>10

<211>896

<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

3540 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 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 ThrAsn

355 360 365

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

370 375 380

Glu Gly Arg Ile Ser Glu Phe Met Ser Leu Trp Leu Pro Ser Glu Ala

385 390 395 400

Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp

405 410 415

Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala Gly Thr Ser Arg

420 425 430

Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn

435 440 445

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

450 455 460

Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser

465 470 475 480

Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly Val

485 490 495

Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly His Pro

500 505 510

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

515 520 525

Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln

530 535 540

Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile Gly Glu His Trp

545 550 555 560

Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro Gly Asp Cys

565 570 575

Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val

580 585 590

Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln Ala Asn Lys

595 600 605

Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp

610 615 620

Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Tyr

625 630 635 640

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

645 650 655

Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys Gly Ser Gly

660 665 670

Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr Pro Ser Gly

675 680 685

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

690 695 700

Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His Ser

770 775 780

Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu Gln Pro Pro

785 790 795 800

Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val Thr Ser Gln Ala

805 810 815

Ile Ala Cys Gln Lys His Thr Pro Pro Ala Pro Lys Glu Asp Pro Leu

820 825 830

Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala

835 840 845

Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly

850 855 860

Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys Ala Thr Pro

865 870 875 880

Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Gln Arg Lys Leu

885 890 895

<210>11

<211>2097

<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 gccctctgaa gctaccgttt acctgccgcc ggttccggtt 720

tctaaagttg tttctaccga cgaatacgtt gctcgtacca acatctacta ccacgctggt 780

acctctcgtc tgctggctgt tggtcacccg tacttcccga tcaaaaaacc gaacaacaac 840

aaaatcctgg ttccgaaagt ttctggtctg cagtaccgtg ttttccgtat ccacctgccg 900

gacccgaaca aattcggttt cccggacacc tctttctaca acccggacac ccagcgtctg 960

gtttgggctt gcgttggtgt tgaagttggt cgtggtcagc cgctgggtgt tggtatctct 1020

ggtcacccgc tgctgaacaa actggacgac accgaaaacg cttctgctta cgctgctaac 1080

gctggtgttg acaaccgtga atgcatctct atggactaca aacagaccca gctgtgcctg 1140

atcggttgca aaccgccgat cggtgaacac tggggtaaag gttctccgtg caccaacgtt 1200

gctgttaacc cgggtgactg cccgccgctg gaactgatca acaccgttat ccaggacggt 1260

gacatggttg acaccggttt cggtgctatg gacttcacca ccctgcaggc taacaaatct 1320

gaagttccgc tggacatctg cacctctatc tgcaaatacc cggactacat caaaatggtt 1380

tctgaaccgt acggtgactc tctgttcttc tacctgcgtc gtgaacagat gttcgttcgt 1440

cacctgttca accgtgctgg tgctgttggt gaaaacgttc cggacgacct gtacatcaaa 1500

ggttctggtt ctaccgctaa cctggcttct tctaactact tcccgacccc gtctggttct 1560

atggttacctctgacgctca gatcttcaac aaaccgtact ggctgcagcg tgctcagggt 1620

cacaacaacg gtatctgctg gggtaaccag ctgttcgtta ccgttgttga caccacccgt 1680

tctaccaaca tgtctctgtg cgctgctatc tctacctctg aaaccaccta caaaaacacc 1740

aacttcaaag aatacctgcg tcacggtgaa gaatacgacc tgcagttcat cttccagctg 1800

tgcaaaatca ccctgaccgc tgacgttatg acctacatcc actctatgaa ctctaccatc 1860

ctggaagact ggaacttcgg tctgcagccg ccgccgggtg gtaccctgga agacacctac 1920

cgtttcgtta cctctcaggc tatcgcttgc cagaaacaca ccccgccggc tccgaaagaa 1980

gacccgctga aaaaatacac cttctgggaa gttaacctga aagaaaaatt ctctgctgac 2040

ctggaccagt tcccgctggg tcgtaaattc ctgctgcagg ctggtctgaa agcttag 2097

<210>12

<211>2472

<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 gaccacccac ctgaaaaaac tgaaagaatc ttactgccag 840

cgtcagggtg ttccgatgaa ctctctgcgt ttcctgttcg aaggtcagcg tatcgctgac 900

aaccacaccc cgaaagaact gggtatggaa gaagaagacg ttatcgaagt ttaccaggaa 960

cagaccggtg gatcctctga agctaccgtt tacctgccgc cggttccggt ttctaaagtt 1020

gtttctaccg acgaatacgt tgctcgtacc aacatctact accacgctgg tacctctcgt 1080

ctgctggctg ttggtcaccc gtacttcccg atcaaaaaac cgaacaacaa caaaatcctg 1140

gttccgaaag tttctggtct gcagtaccgt gttttccgta tccacctgcc ggacccgaac 1200

aaattcggtt tcccggacac ctctttctac aacccggaca cccagcgtct ggtttgggct 1260

tgcgttggtg ttgaagttgg tcgtggtcag ccgctgggtg ttggtatctc tggtcacccg 1320

ctgctgaaca aactggacga caccgaaaac gcttctgctt acgctgctaa cgctggtgtt 1380

gacaaccgtg aatgcatctc tatggactac aaacagaccc agctgtgcct gatcggttgc 1440

aaaccgccga tcggtgaaca ctggggtaaa ggttctccgt gcaccaacgt tgctgttaac 1500

ccgggtgact gcccgccgct ggaactgatc aacaccgtta tccaggacgg tgacatggtt 1560

gacaccggtt tcggtgctat ggacttcacc accctgcagg ctaacaaatc tgaagttccg 1620

ctggacatct gcacctctat ctgcaaatac ccggactaca tcaaaatggt ttctgaaccg 1680

tacggtgact ctctgttctt ctacctgcgt cgtgaacaga tgttcgttcg tcacctgttc 1740

aaccgtgctg gtgctgttgg tgaaaacgtt ccggacgacc tgtacatcaa aggttctggt 1800

tctaccgcta acctggcttc ttctaactac ttcccgaccc cgtctggttc tatggttacc 1860

tctgacgctc agatcttcaa caaaccgtac tggctgcagc gtgctcaggg tcacaacaac 1920

ggtatctgct ggggtaacca gctgttcgtt accgttgttg acaccacccg ttctaccaac 1980

atgtctctgt gcgctgctat ctctacctct gaaaccacct acaaaaacac caacttcaaa 2040

gaatacctgc gtcacggtga agaatacgac ctgcagttca tcttccagct gtgcaaaatc 2100

accctgaccg ctgacgttat gacctacatc cactctatga actctaccat cctggaagac 2160

tggaacttcg gtctgcagcc gccgccgggt ggtaccctgg aagacaccta ccgtttcgtt 2220

acctctcagg ctatcgcttg ccagaaacac accccgccgg ctccgaaaga agacccgctg 2280

aaaaaataca ccttctggga agttaacctg aaagaaaaat tctctgctga cctggaccag 2340

ttcccgctgg gtcgtaaatt cctgctgcag gctggtctga aagctaaacc gaaattcacc 2400

ctgggtaaac gtaaagctac cccgaccacc tcttctacct ctaccaccgc taaacgtaaa 2460

cagcgtaaac tg 2472

<210>13

<211>2643

<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 ctgacggggg 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 ttcatgtctc tgtggctgcc gtctgaagct 1200

accgtttacc tgccgccggt tccggtttct aaagttgttt ctaccgacga atacgttgct 1260

cgtaccaaca tctactacca cgctggtacc tctcgtctgc tggctgttgg tcacccgtac 1320

ttcccgatca aaaaaccgaa caacaacaaa atcctggttc cgaaagtttc tggtctgcag 1380

taccgtgttt tccgtatcca cctgccggac ccgaacaaat tcggtttccc ggacacctct 1440

ttctacaacc cggacaccca gcgtctggtt tgggcttgcg ttggtgttga agttggtcgt 1500

ggtcagccgc tgggtgttgg tatctctggt cacccgctgc tgaacaaact ggacgacacc 1560

gaaaacgctt ctgcttacgc tgctaacgct ggtgttgaca accgtgaatg catctctatg 1620

gactacaaac agacccagct gtgcctgatc ggttgcaaac cgccgatcgg tgaacactgg 1680

ggtaaaggtt ctccgtgcac caacgttgct gttaacccgg gtgactgccc gccgctggaa 1740

ctgatcaaca ccgttatcca ggacggtgac atggttgaca ccggtttcgg tgctatggac 1800

ttcaccaccc tgcaggctaa caaatctgaa gttccgctgg acatctgcac ctctatctgc 1860

aaatacccgg actacatcaa aatggtttct gaaccgtacg gtgactctct gttcttctac 1920

ctgcgtcgtg aacagatgtt cgttcgtcac ctgttcaacc gtgctggtgc tgttggtgaa 1980

aacgttccgg acgacctgta catcaaaggt tctggttcta ccgctaacct ggcttcttct 2040

aactacttcc cgaccccgtc tggttctatg gttacctctg acgctcagat cttcaacaaa 2100

ccgtactggc tgcagcgtgc tcagggtcac aacaacggta tctgctgggg taaccagctg 2160

ttcgttaccg ttgttgacac cacccgttct accaacatgt ctctgtgcgc tgctatctct 2220

acctctgaaa ccacctacaa aaacaccaac ttcaaagaat acctgcgtca cggtgaagaa 2280

tacgacctgc agttcatctt ccagctgtgc aaaatcaccc tgaccgctga cgttatgacc 2340

tacatccact ctatgaactc taccatcctg gaagactgga acttcggtct gcagccgccg 2400

ccgggtggta ccctggaaga cacctaccgt ttcgttacct ctcaggctat cgcttgccag 2460

aaacacaccc cgccggctcc gaaagaagac ccgctgaaaa aatacacctt ctgggaagtt 2520

aacctgaaag aaaaattctc tgctgacctg gaccagttcc cgctgggtcg taaattcctg 2580

ctgcaggctg gtctgaaagc taaaccgaaa ttcaccctgg gtaaacgtaa agctaccccg 2640

acc 2643

Claims (7)

1. A method of making HPV16L1 VLPs, comprising: the method is assembled by low-temperature freezing treatment and comprises the following steps:

putting an HPV16L1 pentamer protein liquid with the average particle size of 10-15 nm PdI <0.1 into an assembly buffer solution with the pH value of 5.5-8.0 and the salt concentration of 150-1000 mM, completely freezing at the temperature of-20 to-80 ℃, and then putting at room temperature until a protein stock solution is melted to obtain an HPV16L 1VLP protein liquid with the average particle size of 45-65 nm PdI < 0.1;

the HPV16L 1VLP protein is assembled by HPV16L1 pentamer protein, and the HPV16L1 pentamer protein is obtained by purifying Tag-HPV16L1 fusion protein;

the fusion protein of Tag-HPV16L1 comprises: the amino acid sequence coded by the human papillomavirus HPV16L1 gene optimized by the escherichia coli preference codon is used as HPV16L1 protein, and the nucleotide sequence of the human papillomavirus HPV16L1 optimized by the escherichia coli preference codon is shown in SEQ NO. 2; and a Tag, wherein the Tag is GST.tag, MBP.tag or GST-SUMO.tag;

the nucleotide sequence of the fusion protein of Tag-HPV16L1 is SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.11, SEQ NO.12 or SEQ NO. 13.

2. A method of making HPV16L1 VLPs of claim 1, wherein: the HPV16L1 protein is a full-length protein, or an L1 protein truncated by no more than 30 amino acids at the C-terminus and/or no more than 10 amino acids at the N-terminus.

3. A method of making HPV16L1 VLPs of claim 1, wherein: the amino acid sequence of the fusion protein of the Tag-HPV16L1 is SEQ NO.8, SEQ NO.9 or SEQ NO. 10.

4. A method of making HPV16L1 VLPs of claim 1, 2 or 3, wherein: the preparation method of the Tag-HPV16L1 fusion protein comprises the following steps:

obtaining a codon-optimized gene of HPV16L1 by substituting codons of the HPV16L1 gene sequence with codons preferred by Escherichia coli;

constructing an Escherichia coli expression vector of the HPV16L1 gene;

constructing an escherichia coli expression engineering strain of Tag-HPV16L 1;

and (4) performing induced expression and purifying to obtain the fusion protein of Tag-HPV16L 1.

5. A method of making HPV16L1 VLPs according to claim 4 wherein: the prokaryotic host cell Escherichia coli is GI698, ER2566, BL21(DE3), XA90, B834(DE3) or BLR (DE 3).

6. A method of making HPV16L1 VLPs of claim 1, wherein: the preparation method of the HPV16L1 pentamer protein comprises the following steps:

adsorbing the fusion protein Tag-HPV16L1 by an affinity chromatography method;

adding a proteolytic enzyme to remove the Tag label to obtain HPV16L1 pentamer protein;

purifying the HPV16L1 pentamer protein to obtain the HPV16L1 pentamer protein with the purity of more than 98 percent and the average particle size of 10-15 nm and PdI of less than 0.1.

7. The method of making an HPV16L 1VLP of claim 6, wherein: the proteolytic enzyme is a site-specific protease for excision of the Tag, recombinant 3C protease, thrombin, SUMO protease, SENP1, or TEV protease.

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