CN111154777B - Recombinant human papilloma virus protein expression - Google Patents
Recombinant human papilloma virus protein expression Download PDFInfo
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Abstract
The application discloses a codon optimized human papillomavirus main capsid protein L1 coding gene, which can efficiently express recombinant human papillomavirus main capsid protein L1 after being transferred into yeast cells. The application also discloses a macromolecule with immunogenicity, which is mainly produced by expressing the codon-optimized human papillomavirus main capsid protein L1 coding gene in yeast cells. The application also discloses application and composition of the macromolecule with immunogenicity.
Description
The application is a divisional application of an application patent with the application date of 2014, 2-month and 18-date, the application number of 201410054725.9 and the name of recombinant human papillomavirus protein expression.
Technical Field
The present application relates to the field of molecular biology, and in particular, to genes of various human papillomavirus major capsid proteins L1 suitable for expression in Pichia pastoris by codon optimization, and vectors, strains, methods of expressing the genes containing the human papillomavirus major capsid protein L1 genes, and uses thereof.
Background
Human papillomavirus (human papilloma virus, HPV) is a non-enveloped small double-stranded circular DNA virus of the polyomaviridae subfamily of papovaviridae. HPV is transmitted by close contact between human bodies, and causes lesions such as verruca vulgaris and condyloma acuminatum of anogenital organs on the skin of infected persons, and is classified as sexually transmitted disease. In 1995, the results of the research published by the international cancer research center demonstrated that HPV has a close causal relationship with cervical cancer. It follows that HPV infection has become a pathogen that severely jeopardizes human health. Therefore, the development of the high-efficiency and low-cost HPV vaccine has very important significance for preventing female cervical cancer and sexually transmitted diseases caused by HPV infection.
More than 100 HPV types have been identified. By using a sensitive detection method, high-risk HPV-DNA can be detected from almost 100% of the diseased tissues of cervical cancer patients. HPV is classified into low-risk and high-risk types according to the relationship between HPV subtypes and female genital tract malignant tumors. Types of HPV6, 11, 34, 40, 42 are commonly found in benign cervical lesions such as cervical condyloma and cervical epithelium mild atypical hyperplasia lesions, and are HPV low-risk types; however, infections with HPV types 16, 18, 31, 33, 35, 39, 45, 52, 58 are more common in cervical dysplasia and cervical cancer, and these subtypes are at high risk. A series of studies in different populations have demonstrated that HPV type 16, 18 infection of the genital tract is highly correlated with the occurrence of cervical cancer, being more closely related to other risk factors. About 50-60% of cervical cancer patients are caused by HPV16 infection, HPV18 is about 14%, HPV45 is about 8%, HPV31 is about 5% and the remaining types are 23% (Walboomers JM. Et al J Pathol 1999;189: 12-19).
Cervical cancer is the second largest gynaecological malignancy next to breast cancer, more than 50 ten thousand women are diagnosed with cervical cancer each year worldwide, 27 ten thousand women die of this disease, and the age-standardized infection rate reaches 10.5%. As early as the 80 s, harald zur Hausen found that infection with human papillomavirus (human papilloma virus, HPV) was associated with cervical cancer onset, and subsequent extensive studies have also demonstrated that HPV is intimately associated with cervical cancer and its precancerous lesions. To date, hundreds of HPV genotypes have been found, of which about 40 can infect the genital mucosa. The cumulative lifetime probability of cervical infection with at least one HPV in a normal woman's lifetime is about 40%.
There is no systematic condyloma acuminatum and HPV related investigation at home, but the individual investigation of each region can basically explain the important role of HPV6, 11 in condyloma acuminatum. Recent epidemiological investigation results in Shenzhen region show that HPV6 and 11 type infectors account for about 80% of patients with condyloma acuminatum (data sources: distribution and significance of genital tract human papillomavirus gene subtypes of patients with condyloma acuminatum in Shenzhen city, 352 cases, tangmin; dai Yong; lv Xiaoping; li Tiyuan; third army university of medical science, 2007, 21); the investigation result of the Yiyi region shows that HPV6 and 11 type infectives account for 85.19% of condyloma acuminatum patients (data sources: genotyping and clinical analysis of 108 cases of HPV of condyloma acuminatum patients, xiong Ying; journal of Community medicine, 17 of 2007).
HPV is a non-enveloped icosahedral symmetric virus, the viral genome DNA of which is in a closed loop shape and has a length of about 7200-8000bp, and consists of an early coding region (early region), a late coding region (late region) and a long regulatory region (long control region) located therebetween. Wherein the late coding region comprises two Open Reading Frames (ORFs) encoding viral capsid proteins L1 and L2. The L1 protein has a molecular weight of about 55kDa and is a main capsid protein, the whole virus capsid structure is supported in the form of 72 pentamers, the amino acid sequences are highly conserved in different types, and the L1 protein can stimulate the organism to generate protective antibodies. The L2 protein has smaller molecular weight and is positioned in the L1 protein.
A variety of expression systems, such as insect expression systems, yeast expression systems, prokaryotic expression systems, and mammalian cells, can be used to obtain virus-like particles (VLPs) by expressing the major capsid protein L1 alone or in combination with the expression of L1+L2. VLPs obtained by expression of L1 alone are similar in structure to the native viral capsid and can be used to induce high titer virus neutralizing antibody responses associated with protection from virus attack.
Therefore, given that the L1 protein is highly conserved inside different genotypes and can be expressed alone to form VLPs, the L1 protein has high feasibility as a target protein for HPV vaccine development. However, the commercial development and production of VLPs obtained by expressing recombinant viral proteins as HPV vaccines requires a number of technical problems to be solved, of which the first technical problem to be solved is how to increase the expression level of recombinant viral proteins. In expression systems such as escherichia coli, pichia pastoris and baculovirus, the L1 protein is often limited by the use frequency of amino acid codons in organisms, so that the expression level is low or even no expression is caused. As described in U.S. Pat. No.7,498,036 to Merck, the expression level of wild-type VLP protein in Saccharomyces cerevisiae is about 35. Mu.g/mg (VLP in the supernatant of the strain/total protein of the supernatant of the strain).
Thus, there is a need in the art for a method of expressing genes at high levels, which should be capable of expressing HPV genes at high levels, easy and convenient to handle, and at low cost.
Disclosure of Invention
In order to solve the above technical problem, according to a first aspect of the present application, there is provided an IIPV gene capable of being expressed in pichia pastoris, the gene having the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or seq id NO:10, and a nucleotide sequence shown in seq id no.
The pichia pastoris is used as an expression system for expressing recombinant proteins, has the characteristics of high expression quantity, simple and convenient operation, low cost and the like, and is more beneficial to the large-scale industrial production of human beings compared with higher insect cells and mammalian cells. Because the use frequency of amino acid codons among different species is different, when pichia pastoris is utilized to express recombinant protein, a DNA sequence which is more beneficial to translation is often obtained after codon optimization and adjustment according to the amino acid sequence of target protein. Therefore, the HPV gene subjected to codon optimization can obtain higher expression level in Pichia pastoris, and is more beneficial to research and development and production of preventive vaccines against HPV. As shown in the examples of the present application, the expression level of the codon optimized HPV6, 11, 16, 18, 58 genes of the present application in Pichia pastoris can be up to about 134. Mu.g/mg, 123. Mu.g/mg, 135. Mu.g/mg, 125. Mu.g/mg and 132. Mu.g/mg, respectively (VLP in the bacterial supernatant/total protein of the bacterial supernatant).
According to a second aspect of the present invention there is provided a method of expressing the HPV L1 gene in pichia pastoris, comprising the steps of:
(1) Cloning the HPV L1 genes of the invention into expression vectors respectively;
(2) Transforming the expression vector obtained in the step (1) into a pichia pastoris strain;
(3) Screening the transformed strains obtained in the step (2) by using antibiotics to obtain one or more strains with the best growth condition;
(4) Further screening the strain obtained in the step (3) by testing the expression quantity of each HPV L1 gene to obtain one or more strains with the highest expression quantity;
(5) And (3) performing expression by using the strain obtained in the step (4) to obtain HPV L1 proteins respectively.
According to a specific embodiment of the present invention, the expression vector in step (1) is a ppiczαb vector and the antibiotic used in step (3) is Zeocin. .
According to a specific embodiment of the present invention, the Pichia pastoris strain used in the step (2) is Pichia pastoris X-33 strain.
According to a specific embodiment of the present invention, the operation of testing the expression level of the HPV L1 gene in the step (4) is performed by a Western blot method.
According to a specific embodiment of the present invention, the expression step in step (5) is a fermentation step performed in a fermenter.
According to a third aspect of the present invention there is provided an expression vector comprising each HPV L1 gene of the present invention.
According to a specific embodiment of the present invention, the expression vector containing each HPV L1 gene of the present invention is derived from a ppiczαb vector.
According to a fourth aspect of the present invention, there is provided a Pichia pastoris strain containing the HPV L1 gene or expression vector of the present invention, which is capable of high level expression production of each HPV L1 protein, more advantageous for the development and production of prophylactic vaccines against HPV.
Drawings
FIG. 1 shows agarose electrophoresis patterns of HPV6L1 gene after double digestion. 1: DL5000DNA Marker (Takara Co.); 2: bstBI+KpnI double cleavage pPICZαB-6L1 sample 1;3: bstBI+KpnI double-digested pPICZαB-6L1 sample 2.
FIG. 2 shows agarose electrophoresis patterns of HPV11L1 gene after double digestion. 1: DL5000DNA Marker (Takara Co.); 2: bstBI+KpnI double-digested pPICZαB-11L1 sample 1.
FIG. 3 shows agarose electrophoresis patterns of HPV16L1 gene after double digestion. 1: DL5000DNA Marker (Takara Co.); 2: pPICZαB-16L1 sample 1;3: pPICZαB-16L1 sample 2;4: bstBI+KpnI double cleavage pPICZαB-16L1 sample 1; bstBI+KpnI double-digested pPICZαB-16L1 sample 2.
FIG. 4 shows agarose electrophoresis patterns of HPV18L1 gene after double digestion. 1: DL5000DNA Marker (Takara Co.); 2: bstBI+KpnI double digested pPICZαB-18L1 samples.
FIG. 5 shows agarose electrophoresis patterns of HPV58L1 gene after double digestion. 1: bstBI+KpnI double-digested pPICZαB-58L1 sample; 2: DL5000DNA Marker (Takara Co.).
FIG. 6 shows Western-blot identification of HPV6L1 supernatant after disruption. 1-8: recombinant expression strains; 9: pageRuler Prestained Protein Ladder;10: and (5) empty host bacteria.
FIG. 7 shows Western-blot identification of HPV11L1 supernatant after disruption. 1-8: recombinant expression strains; 9: pageRuler Prestained Protein Ladder;10: and (5) empty host bacteria.
FIG. 8 shows Western-blot identification of HPvl6L1 supernatant after disruption. 1-8: recombinant expression strains; 9: pageRuler Prestained Protein Ladder;10: and (5) empty host bacteria.
FIG. 9 shows Western-blot identification of HPV18L1 supernatants after disruption. 1: pageRuler Prestained Protein Ladder;2-10: recombinant expression strains.
FIG. 10 shows Western-blot identification of HPV58L1h supernatant after disruption. 1: pageRuler Prestained Protein Ladder;2-5: recombinant expression strains.
FIG. 11 shows SDS-PAGE electrophoresis of HPV6L1 protein samples. 1-9: recombinant purified protein samples; 10: pageRuler Prestained Protein Ladder.
FIG. 12 shows SDS-PAGE electrophoresis of HPV11L1 protein samples. 1- -4: recombinant purified protein samples; 5: pageRuler Prestained Protein Ladder;6-8: recombinant purified protein samples.
FIG. 13 shows SDS-PAGE electrophoresis of HPV16L1 protein samples. 1-7: recombinant purified protein samples; 8: pageRuler Prestained Protein Ladder.
FIG. 14 shows SDS-PAGE electrophoresis of HPV18L1 protein samples. 1-4: recombinant purified protein samples; 5: pageRuler Prestained Protein Ladder;6-7: recombinant purified protein samples.
FIG. 15 shows SDS-PAGE electrophoresis of IIPV58L1 protein samples. 1-5: recombinant purified protein samples; 6: pageRuler Prestained Protein Ladder;7-8: recombinant purified protein samples.
FIG. 16 shows a transmission electron micrograph of virus-like particles after HPV6L1 purification.
FIG. 17 shows a transmission electron micrograph of virus-like particles after HPV11L1 purification.
FIG. 18 shows transmission electron micrographs of virus-like particles after HPV16L1 purification.
FIG. 19 shows transmission electron micrographs of virus-like particles after HPV18L1 purification.
FIG. 20 shows a transmission electron micrograph of virus-like particles after HPV58L1 purification.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 is the amino acid sequence of wild type HPV6L 1.
SEQ ID NO:2 is the amino acid sequence of wild type HPV11L 1.
SEQ ID NO:3 is the amino acid sequence of wild type HPV16L 1.
SEQ ID NO:4 is the wild type HPV18LI amino acid sequence.
SEQ ID NO:5 is the wild type HPV58L1 amino acid sequence.
SEQ ID NO:6 is the nucleotide sequence of the IIPV6L1 gene of the present invention.
SEQ ID NO:7 is the nucleotide sequence of the HPV11L1 gene of the invention.
SEQ ID NO:8 is the nucleotide sequence of the HPV16L1 gene of the invention.
SEQ ID NO:9 is the nucleotide sequence of the HPvl8L1 gene of the invention.
SEQ ID NO:10 is the nucleotide sequence of the HPV58L1 gene of the invention.
Detailed Description
The present invention is described in detail by the following examples so that those skilled in the art can better understand the present invention. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless otherwise indicated, temperatures are in units of degrees celsius or are ambient temperatures, and pressures are near or equal to atmospheric pressure. Restriction enzymes used in the examples below were purchased from New England Biolab, unless otherwise indicated. It should be understood that the instrumentation used in the examples below is conventional in the art unless otherwise indicated. Unless otherwise indicated, the media used are conventional media available on the market, the ingredients and amounts of which are well known to those skilled in the art. For simplicity, various common abbreviations are possible to use herein, the meaning of which is fully understood by those skilled in the art.
Examples
Example 1: HPVL1 codon optimized design
There are 64 genetic codes, but most organisms tend to utilize some of these codons. The genes of pichia and humans have respective preferences for codon usage. Since the genetic code is degenerate, each amino acid is encoded by more than one codon, and the frequency of use of codons for the same amino acid in a wild-type gene is different. Codon preference of Pichia pastoris may lead to low translation efficiency and expression level of recombinant proteins, the inventors have genetically engineered according to wild type HPV6L1 amino acid sequence (Genebank CBY 85547.1), HPV11L1 amino acid sequence (Genebank CCE 60515.1), HPV16L1 amino acid sequence (Genebank AAC 09292.1), HPV18L1 amino acid sequence (Genebank AAP 20601.1) and HPV58L1 amino acid sequence (Genebank BAA 31851.1): the most frequently used and higher codons are used for all of its amino acid genes. The codon usage frequency of Pichia yeast is shown in Table 1 (see http:// www.kazusa.or.jp/codon /). Then, on the basis of this, in order to avoid that the GC proportion of the translated mRNA is too high, the secondary structure of the mRNA influences the translation efficiency and some commonly used cleavage sites, the inventors have modified the highest frequency codons, such as AAT for some asparagine (Asn) highest frequency codons AAC, AAA for lysine (Lys) highest frequency codons AAG, GAC for aspartic acid (Asp) highest frequency codons GAT, TTT for phenylalanine (Phe) highest frequency codons TTT for TTC, TAT for tyrosine (Tyr) highest frequency codons TAC and GGA for glycine (Gly) highest frequency codons GGT. The modified HPVL1 gene sequence does not contain the following intron recognition sequences and transcription factor binding sites: ATGACTCAT and TGACTA (transcription factor GCN4 binding site); ataaa (binding site for GAL 4); TATTTAA (TBP binding site); TTAGTAA and TTACTAA (YAP 1 binding site); ATGACTAAT; ACTAATTAGG.
Therefore, the inventor optimally designs a plurality of nucleotide sequences of HPV L1 genes suitable for pichia pastoris expression, fully synthesizes the HPV L1 genes according to the sequences, clones the HPV L1 genes into the existing pichia pastoris expression vector, and constructs a recombinant pichia pastoris expression strain through homologous recombination and screening of high-concentration antibiotics; fermenting and culturing recombinant Pichia pastoris and inducing intracellular expression of HPV L1 protein with methanol. The nucleotide sequences of the brand-new HPV L1 genes are respectively screened out, HPV L1 proteins can be respectively expressed in pichia pastoris, virus-like particles (VLPs) are simultaneously formed in cells, the purity of the purified virus-like particles is more than 90% after the bacterial supernatant is purified by a chromatography method, and the purified virus-like particles have extremely high immunogenicity after adsorbing an aluminum adjuvant and can be used as a vaccine for preventing cervical cancer of people. The nucleotide sequence of the HPV L1 gene optimally designed is shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or seq id NO: shown at 10.
TABLE 1Pichia Yeast codon table
Example 2: construction of HPV L1 recombinant expression vector
The HPV L1 gene sequence thus synthesized was cloned into the pPICZalphaB vector (Invitrogen) by the following method. The HPV L1DNA fragments with BstBI and KpnI at both ends are amplified by PCR, and PCR primers. HPV6L1, forward primer: 5'-CGATGGAACTTCGAAACGATGTGGAGACC-3' (BstBI) (SEQ ID NO: 11); reverse primer: 5'-GACCTGGGTACCCTATTATCTCTTGGTTTTAG-3' (KpnI) (SEQ ID NO: 12). HPV11L1, forward primer: 5'-CGATGGAACTTCGAAACGATGTGGAGACC-3' (BstBI) (SEQ ID NO: 13); reverse primer: 5'-GACCTGGGTACCCTATTATTTCTTGG-3' (KpnT) (SEQ ID NO: 14). HPV16L1, forward primer: 5'-CGATGGAACTTCGAAACGATGATGTCCTTG-3' (BstBI) (SEQ ID NO: 15); reverse primer: 5'-GACCTGGGTACCCTATTAAAGCTTTC-3' (KpnI) (SEQ ID NO: 16). HPV18L1, forward primer: 5'-CGATGGAACTTCGAAACGATGGCTTTGTGG-3' (BstBI) (SEQ ID NO: 17); reverse primer: 5'-GACCTGGGTACCCTATTATTTTCTGGC-3' (KpnI) (SEQ ID NO: 18). HPV58LI, forward primer: 5'-CGATGGAACTTCGAAACGATGTCC TTTGG-3' (BstBI) (SEQ ID NO: 19); reverse primer: 5'-GACCTGGGTACCCTATTATTTCTTAAC-3' (KpnI) (SEQ ID NO: 20). PCR procedure: the operation was completed by cycling 30 times at 94℃for 5 minutes, 94℃for 30 seconds, 55℃for 30 seconds, and 72℃for 1 minute for 50 seconds, 72℃for 10 minutes, and 10℃for 10 minutes. The PCR products were identified by agarose gel electrophoresis and the 1500bp band was recovered (Qiagen gel extraction kit). The recovered fragment was digested with pPICZalphaB in combination with BstBI and KpnI (New England Biolab), and agarose gel electrophoresis was performed to identify and recover fragments of about 1500bp and 3600bp, respectively. After recovery, HPVL1 and pPICZalphaB were ligated in a 5:1 molar ratio overnight with T4 ligase (Takara) at 16℃and the ligation product was transformed into E.coli DH 5. Alpha. The next day, spread on low-salt LB plates (containing 25ug/mL Zeocin) and incubated overnight at 37 ℃. The plasmid was extracted by cloning after partial transformation, and was identified by double digestion (BstBI+KpnI) and detected by agarose electrophoresis (FIG. 1). The positive recombinant clone obtained by identification is stored after being verified to be correct by DNA sequencing, and the recombinant vectors are named pPICZ6L1, pPICZ11L1pPICZ16L1, pPICZ18L1 and pPICZ58L1 respectively.
Example 3: construction and expression of HPV L1 recombinant expression strain
Linearizing pPICZ6L1 with SacI, phenol after the cleavage reaction is finished: the protein was removed with chloroform, and then 2.5 volumes of absolute ethanol was added, 1/10 volume of 3M NaAc (pH5.2) was used to precipitate the DNA, and the resulting precipitate was washed with 75%2 alcohol, dried, and then dissolved in a small amount of sterile ddH20, and the Pichia pastoris host bacteria (Invitrogen) were electrotransferred, plated on YPDS plates (containing 180. Mu.g/mL Zeocin), and cultured at 30℃for 3 days to obtain hundreds of clones. From this, several ten clones were picked and inoculated on YPD plates (containing 1500. Mu.g/mL Zeocin), and plasmid high copy strains were selected and cultured at 30℃for 2 days. And (3) partial clones grow faster, a plurality of clones with the best growth condition are selected and inoculated in 5mL YPD liquid culture medium, the BMMY culture medium is replaced after 24 hours, and bacterial cells are collected after 0.5% methanol induction for 48 hours. After the thalli are broken by glass beads, supernatant obtained by centrifugation is identified by Western-blot (figure 6), and the primary antibody is self-made rabbit polyclonal antibody. And freezing the strain with the highest expression level at-80 ℃ to be used as a working seed for fermentation tank culture.
Linearizing pPICZ11L1 with SacI, phenol after the cleavage reaction: the protein was removed with chloroform, then 2.5 volumes of absolute ethanol was added, 1/10 volumes of 3M NaAc (pH5.2) was used to precipitate the DNA, the resulting precipitate was washed with 75% ethanol, dried, and then dissolved in a small amount of sterile ddH20 to electrotransfer Pichia pastoris host bacteria (Invitrogen), spread on YPDS plates (containing 180. Mu.g/mL Zeocin), and incubated at 30℃for 3 days to obtain hundreds of clones. From this, several ten clones were picked and inoculated on YPD plates (containing 1500. Mu.g/mL Zeocin), and plasmid high copy strains were selected and cultured at 30℃for 2 days. And (3) partial clones grow faster, a plurality of clones with the best growth condition are selected and inoculated in 5mL YPD liquid culture medium, the BMMY culture medium is replaced after 24 hours, and bacterial cells are collected after 0.5% methanol induction for 48 hours. After the thalli are broken by glass beads, supernatant obtained by centrifugation is identified by Western-blot (figure 7), and the primary antibody is self-made rabbit polyclonal antibody. And freezing the strain with the highest expression level at-80 ℃ to be used as a working seed for fermentation tank culture.
Linearizing pPICZ16L1 with SacI, phenol after the cleavage reaction: the protein was removed with chloroform, then 2.5 volumes of absolute ethanol was added, 1/10 volumes of 3M NaAc (pH5.2) was used to precipitate the DNA, the resulting precipitate was washed with 75% ethanol, dried, and then dissolved in a small amount of sterile ddH20 to electrotransfer Pichia pastoris host bacteria (Invitrogen), spread on YPDS plates (containing 180. Mu.g/mL Zeocin), and incubated at 30℃for 3 days to obtain hundreds of clones. From this, several ten clones were picked and inoculated on YPD plates (containing 1500. Mu.g/mL Zeocin), and plasmid high copy strains were selected and cultured at 30℃for 2 days. And (3) partial clones grow faster, a plurality of clones with the best growth condition are selected and inoculated in 5mL YPD liquid culture medium, the BMMY culture medium is replaced after 24 hours, and bacterial cells are collected after 0.5% methanol induction for 48 hours. After the thalli are broken by glass beads, supernatant obtained by centrifugation is identified by Western-blot (figure 8), and the primary antibody is self-made rabbit polyclonal antibody. And freezing the strain with the highest expression level at-80 ℃ to be used as a working seed for fermentation tank culture.
Linearizing pPICZ18L1 with SacI, phenol after the cleavage reaction: the protein was removed with chloroform, and then 2.5 volumes of absolute ethanol was added, 1/10 volumes of 3M NaAc (pH5.2) was used to precipitate the DNA, and the resulting precipitate was washed with 75%7 alcohol, dried, and then dissolved in a small amount of sterile ddH2O to obtain a pellet, which was used to electrophoresed a Pichia pastoris host cell (Invitrogen), plated on YPDS plates (containing 180. Mu.g/mL Zeocin), and incubated at 30℃for 3 days to obtain hundreds of clones. From this, several ten clones were picked and inoculated on YPD plates (containing 1500. Mu.g/mL Zeocin), and plasmid high copy strains were selected and cultured at 30℃for 2 days. And (3) partial clones grow faster, a plurality of clones with the best K generation condition are selected and inoculated in 5mL YPD liquid culture medium, BMMY culture medium is replaced after 24 hours, and bacterial cells are collected after 0.5% methanol induction for 48 hours. After the thalli are broken by glass beads, supernatant obtained by centrifugation is identified by Western-blot (figure 9), and the primary antibody is self-made rabbit polyclonal antibody. And freezing the strain with the highest expression level at-80 ℃ to be used as a working seed for fermentation tank culture.
Linearizing pPICZ58L1 with SacI, phenol after the cleavage reaction: the protein was removed with chloroform, then 2.5 volumes of absolute ethanol was added, 1/10 volumes of 3M NaAc (pH5.2) was used to precipitate the DNA, the resulting precipitate was washed with 75% ethanol, dried, and then dissolved in a small amount of sterile ddH20 to electrotransfer Pichia pastoris host bacteria (Invitrogen), spread on YPDS plates (containing 180. Mu.g/mL Zeocin), and incubated at 30℃for 3 days to obtain hundreds of clones. From this, several ten clones were picked and inoculated on YPD plates (containing 1500. Mu.g/mL Zeocin), and plasmid high copy strains were selected and cultured at 30℃for 2 days. And (3) partial clones grow faster, a plurality of clones with the best growth condition are selected and inoculated in 5mL YPD liquid culture medium, the BMMY culture medium is replaced after 24 hours, and bacterial cells are collected after 0.5% methanol induction for 48 hours. After the thalli are broken by glass beads, supernatant obtained by centrifugation is identified by Western-blot (figure 10), and the primary antibody is self-made rabbit polyclonal antibody. And freezing the strain with the highest expression level at-80 ℃ to be used as a working seed for fermentation tank culture.
Example 4: fermenter culture of HPV L1 recombinant protein
From the working seed pool of IIPV6L 1-expressing genetically engineered bacteria obtained in example 3, 1 MycobacteriumThe glycerol stock was thawed and 100. Mu.L of the glycerol stock was inoculated into 5mL of YPD medium, and incubated at 280 revolutions per minute (rpm) at 30℃for 20 hours. The cell density reaches an OD600 of about 1-2. And the microscopic examination has no contamination of miscellaneous bacteria. 1mL of the qualified activating solution was inoculated into 500mL of YPD medium, 280rpm, and incubated at 30℃for 20 hours. The density of the thallus reaches OD 600 About 2-6. And the microscopic examination has no contamination of miscellaneous bacteria. Basic salt culture medium BSM1 (K) for fermentation 2 SO 4 273g，MgSO 4 109g，CaSO 4 ·2H 2 O17.6g，H 3 PO 4 400.5mL, KOH62g, 600g of glycerol, PTM160mL, 1mL of enemy, deionized water to 15L) without antibiotic, and after preparation, sterilization in a 30L fermenter (Bioengineering Co.). Sterilizing at 121deg.C for 30 min, and cooling to 30deg.C. Inoculating the activated seed liquid into a tank at a ratio of 1:15. The fermentation temperature is 30.0+ -0.5deg.C, initial pH is 5.00+ -0.05, initial rotation speed is 300rpm, aeration rate is 0.5vvm, DO (dissolved oxygen value) is 100%, and PTM1 (CuSO) is added 4 ·5H 2 06.0g，NaI 0.008g，MnSO 4 3.0g，NaMo0 4 0.2g，H 3 BO 3 0.02g，ZnSO 4 20.0g，CoCl 2 0.5g，FeSO 4 ·7H 2 O65.0g，biotin0.2g，H2SO 4 5.0mL deionized water was added to 1L) trace salts. The initial proliferation stage is about 24 hours, the dissolved oxygen value is maintained to be not lower than 20%, and when the carbon source is consumed, the dissolved oxygen value is rapidly increased, and the wet weight of the thallus reaches about 100g/L. The initial two hours were supplemented with a 50% volume percent glycerol solution (12 mL of PTM1 per liter added) at a rate of 200mL/h per hour. After two hours of feeding, 300mL/h was used. Through regulating stirring speed, air flow and tank pressure<0.8 bar) maintains dissolved oxygen levels above 30%. After the addition of the culture medium for about 4 hours, the addition of the culture medium was stopped and the dissolved oxygen value was increased when the wet weight of the cells was about 200 g/L. Simultaneously, the pH value is controlled to be 6.00+/-0.05, and methanol (12 mL of PTM1 is added per liter) is added for induction. The initial methanol addition was controlled at 30mL/h. The amount of methanol added was slowly increased, and after 4 hours of methanol induction, the feed rate was set at 90mL/h. The dissolved oxygen value is maintained to be higher than 20 percent by volume, the temperature is maintained to be 30 ℃, and the pH value is controlled to be 6.00+/-0.05. And discharging fermentation liquor after the fermentation is finished after the induction is carried out for 40 hours. Collecting thallus by centrifugation at 4 ℃ and wetting thallus And the weight is 400g/L.
From the working seed pool of HPV11L 1-expressing genetically engineered bacteria obtained in example 3, 1 strain glycerol cryopreservation tube was taken, 100. Mu.L was aspirated after thawing and inoculated into 5mL YPD medium at 280 revolutions per minute (rpm), and incubated at 30℃for 20 hours. The density of the thallus reaches OD 600 About 1-2. And the microscopic examination has no contamination of miscellaneous bacteria. 1mL of the qualified activating solution was inoculated into 500mL of YPD medium, 280rpm, and incubated at 30℃for 20 hours. The cell density reaches an OD600 of about 2-6. And the microscopic examination has no contamination of miscellaneous bacteria. Basic salt culture medium BSM1 (K) for fermentation 2 SO 4 273g，MgSO 4 109g，CaSO 4 ·2H 2 O17.6g，H 3 PO 4 400.5mL, KOH62g, glycerol 600g, PTML60mL, bufomide 1mL, deionized water to 15L) without antibiotic, and after formulation, solid tank sterilization was performed in a 30L fermenter (Bioengineering Co.). Sterilizing at 121deg.C for 30 min, and cooling to 30deg.C. Inoculating the activated seed liquid into a tank at a ratio of 1:15. The fermentation temperature is 30.0+ -0.5deg.C, initial pI5.00+ -0.05, initial rotation speed of 300rpm, aeration rate of 0.5vvm, DO (dissolved oxygen value) of 100%, and PTM1 (CuSO) is added 4 ·5H 2 O6.0g，NaI0.008g，MnSO 4 3.0g，NaMoO 4 0.2g，HaBO 3 0.02g，ZnSO 4 20.0g，CoCl 2 0.5g，FeSO 4 ·7H 2 O65.0g，biotin0.2g，H 2 SO 4 5.0mL deionized water was added to 1L) trace salts. The dissolved oxygen value is maintained at not lower than 20% for about 24 hours in the initial proliferation stage, and rapidly rises when the carbon source is consumed, so that the wet weight of the thallus reaches about 100g/L. The initial two hours were supplemented with a 50% volume percent glycerol solution (12 mL of PTM1 per liter added) at a rate of 200mL/h per hour. After two hours of feeding, 300mL/h was used. Through regulating stirring speed, air flow and tank pressure <0.8 bar) maintains dissolved oxygen levels above 30%. After the addition of the culture medium for about 4 hours, the addition of the culture medium was stopped and the dissolved oxygen value was increased when the wet weight of the cells was about 200 g/L. Simultaneously, the pH value is controlled to be 6.00+/-0.05, and methanol (12 mL of PTM1 is added per liter) is added for induction. The initial methanol addition was controlled at 30mL/h. The amount of methanol added was slowly increased, and after 4 hours of methanol induction, the feed rate was set at 90mL/h. Maintaining the dissolved oxygen value higher than 20 percent by volume,the temperature was maintained at 30℃and the pH was controlled at 6.00.+ -. 0.05. And discharging fermentation liquor after the fermentation is finished after the induction is carried out for 40 hours. And (3) centrifugally collecting thalli at the temperature of 4 ℃, wherein the wet weight of the thalli reaches 400g/L.
From the working seed pool of HPV16L 1-expressing genetically engineered bacteria obtained in example 3, 1 strain glycerol cryopreservation tube was taken, 100. Mu.L was aspirated after thawing and inoculated into 5mL YPD medium at 280 revolutions per minute (rpm), and incubated at 30℃for 20 hours. The density of the thallus reaches OD 600 About 1-2. And the microscopic examination has no contamination of miscellaneous bacteria. 1mL of the qualified activating solution was inoculated into 500mL of YPD medium, 280rpm, and incubated at 30℃for 20 hours. The density of the thallus reaches OD 600 About 2-6. And the microscopic examination has no contamination of miscellaneous bacteria. Basic salt culture medium BSM1 (K) for fermentation 2 SO 4 273g，MgSO 4 109g，CaSO 4 ·2H 2 O17.6g，H 3 PO 4 400.5mL, KOH62g, 600g of glycerol, PTM160mL, 1mL of enemy, deionized water to 15L) without antibiotic, and after preparation, sterilization in a 30L fermenter (Bioengineering Co.). Sterilizing at 121deg.C for 30 min, and cooling to 30deg.C. Inoculating the activated seed liquid into a tank at a ratio of 1:15. The fermentation temperature is 30.0+ -0.5deg.C, initial pH is 5.00+ -0.05, initial rotation speed is 300rpm, aeration rate is 0.5vvm, DO (dissolved oxygen value) is 100%, and PTM1 (CuSO) is added 4 ·5H 2 O6.0g，Na10.008g，MnSO 4 3.0g，NaMoO 4 0.2g，H 3 BO 3 0.02g，ZnSO 4 20.0g，CoCl 2 0.5g，FeSO 4 ·7H 2 O65.0g，biotin0.2g，H 2 SO 4 5.0mL deionized water was added to 1L) trace salts. The initial proliferation stage is about 24 hours, the dissolved oxygen value is maintained to be not lower than 20%, and when the carbon source is consumed, the dissolved oxygen value is rapidly increased, and the wet weight of the thallus reaches about 100g/L. The initial two hours were supplemented with a volume percent 50% glycerol solution (12 mL of PTMI per liter) at a rate of 200mL/h per hour. After two hours of feeding, 300mL/h was used. Through regulating stirring speed, air flow and tank pressure<0.8 bar) maintains dissolved oxygen levels above 30%. After the addition of the culture medium for about 4 hours, the addition of the culture medium was stopped and the dissolved oxygen value was increased when the wet weight of the cells was about 200 g/L. At the same time, the pII value is controlled to be 6.00+/-0.05, and methanol (12 mL of PTM1 is added per liter) is added for induction. Initial methanol additionThe amount was controlled at 30mL/h. The amount of methanol added was slowly increased, and after 4 hours of methanol induction, the feed rate was set at 90mL/h. The dissolved oxygen value is maintained to be higher than 20 percent by volume, the temperature is maintained to be 30 ℃, and the pH value is controlled to be 6.00+/-0.05. And discharging fermentation liquor after the fermentation is finished after the induction is carried out for 40 hours. And (3) centrifugally collecting thalli at the temperature of 4 ℃, wherein the wet weight of the thalli reaches 400g/L.
From the working seed pool of HPV18L 1-expressing genetically engineered bacteria obtained in example 3, 1 strain glycerol cryopreservation tube was taken, 100. Mu.L was aspirated after thawing and inoculated into 5mL YPD medium, 280 revolutions per minute (rpm), and incubated at 30℃for 20 hours. The cell density reaches an OD600 of about 1-2. And the microscopic examination has no contamination of miscellaneous bacteria. 1mL of the qualified activating solution was inoculated into 500mL of YPD medium, 280rpm, and incubated at 30℃for 20 hours. The cell density reaches an OD600 of about 2-6. And the microscopic examination has no contamination of miscellaneous bacteria. Basic salt culture medium BSM1 (K) for fermentation 2 SO 4 273g，MgsO 4 109g，CaSO 4 ·2H 2 O17.6g，H 3 PO 4 400.5mL, KOH62g, 600g of glycerol, PTM160mL, 1mL of enemy, deionized water to 15L) without antibiotic, and after preparation, sterilization in a 30L fermenter (Bioengineering Co.). Sterilizing at 121deg.C for 30 min, and cooling to 30deg.C. Inoculating the activated seed liquid into a tank at a ratio of 1:15. The fermentation temperature is 30.0+ -0.5deg.C, initial pH is 5.00+ -0.05, initial rotation speed is 300rpm, aeration rate is 0.5vvm, DO (dissolved oxygen value) is 100%, and PTM1 (CuSO) is added 4 ·5II 2 O6.0g，NaIO.008g，MnSO 4 3.0g，NaMoO 4 0.2g，H 3 BO 3 0.02g，ZnSO 4 20.0g，CoCl 2 0.5g，FeSO 4 ·7H 2 065.0g，biotin0.2g，H 2 SO 4 5.0mL deionized water was added to 1L) trace salts. The initial proliferation stage is about 24 hours, the dissolved oxygen value is maintained to be not lower than 20%, and when the carbon source is consumed, the dissolved oxygen value is rapidly increased, and the wet weight of the thallus reaches about 100g/L. The initial two hours were supplemented with a 50% volume percent glycerol solution (12 mL of PTM1 per liter added) at a rate of 200mL/h per hour. After two hours of feeding, 300mL/h was used. Through regulating stirring speed, air flow and tank pressure<0.8 bar) maintains dissolved oxygen levels above 30%. Supplementing for about 4 hours, and stopping supplementing when the wet weight of the thallus is about 200g/LThe dissolved oxygen value of the material is increased. Simultaneously, the pH value is controlled to be 6.00+/-0.05, and methanol (12 mL of PTM1 is added per liter) is added for induction. The initial methanol addition was controlled at 30mL/h. The amount of methanol added was slowly increased, and after 4 hours of methanol induction, the feed rate was set at 90mL/h. The dissolved oxygen value is maintained to be higher than 20 percent by volume, the temperature is maintained to be 30 ℃, and the pH value is controlled to be 6.00+/-0.05. And discharging fermentation liquor after the fermentation is finished after the induction is carried out for 40 hours. And (3) centrifugally collecting thalli at the temperature of 4 ℃, wherein the wet weight of the thalli reaches 400g/L.
From the working seed pool of the genetically engineered bacteria expressing HPV58L1 obtained in example 3, 1 strain glycerol cryopreservation tube was taken, 100. Mu.L was aspirated after thawing and inoculated into 5mL YPD medium, 280 revolutions per minute (rpm), and incubated at 30℃for 20 hours. The cell density reaches an OD600 of about 1-2. And the microscopic examination has no contamination of miscellaneous bacteria. 1mL of the qualified activating solution was inoculated into 500mL of YPD medium, 280rpm, and incubated at 30℃for 20 hours. The cell density reaches an OD600 of about 2-6. And the microscopic examination has no contamination of miscellaneous bacteria. Basic salt culture medium BSM1 (K) for fermentation 2 SO 4 273g，MgSO 4 109g，CaSO 4 ·2H 2 O17.6g，H 3 PO 4 400.5mL, KOH62g, 600g of glycerol, PTM160mL, 1mL of enemy, deionized water to 15L) without antibiotic, and after preparation, sterilization in a 30L fermenter (Bioengineering Co.). Sterilizing at 121deg.C for 30 min, and cooling to 30deg.C. Inoculating the activated seed liquid into a tank at a ratio of 1:15. The fermentation temperature is 30.0+ -0.5deg.C, initial pH is 5.00+ -0.05, initial rotation speed is 300rpm, aeration rate is 0.5vvm, DO (dissolved oxygen value) is 100%, and PTM1 (cuSO) is added 4 ·5H 2 O6.0g，NaI0.008g，MnSO 4 3.0g，NaMoO 4 0.2g，H 3 BO 3 0.02g，ZnSO 4 20.0g，CoCl 2 0.5g，FeSO 4 ·7H 2 065.0g，biotin0.2g，H 2 SO 4 5.0mL deionized water was added to 1L) trace salts. The initial proliferation stage is about 24 hours, the dissolved oxygen value is maintained to be not lower than 20%, and when the carbon source is consumed, the dissolved oxygen value is rapidly increased, and the wet weight of the thallus reaches about 100g/L. The initial two hours were supplemented with a 50% volume percent glycerol solution (12 mL of PTM1 per liter added) at a rate of 200mL/h per hour. After two hours of feeding, 300mL/h was used. By adjusting stirring rotation Fast air flow and tank pressure<0.8 bar) maintains dissolved oxygen levels above 30%. After the addition of the culture medium for about 4 hours, the addition of the culture medium was stopped and the dissolved oxygen value was increased when the wet weight of the cells was about 200 g/L. Simultaneously, the pH value is controlled to be 6.00+/-0.05, and methanol (12 mL of PTM1 is added per liter) is added for induction. The initial methanol addition was controlled at 30mL/h. The amount of methanol added was slowly increased, and after 4 hours of methanol induction, the feed rate was set at 90mL/h. The dissolved oxygen value is maintained to be higher than 20 percent by volume, the temperature is maintained to be 30 ℃, and the pH value is controlled to be 6.00+/-0.05. And discharging fermentation liquor after the fermentation is finished after the induction is carried out for 40 hours. And (3) centrifugally collecting thalli at the temperature of 4 ℃, wherein the wet weight of the thalli reaches 400g/L.
Example 5: HPV L1 protein purification
After collected HPV6L1 thalli are subjected to bacteria breaking (bacteria breaking buffer solution: 200mM MOPS,pH7.0,0.7NaCl,0.05%Tween-80), supernatant after bacteria breaking is taken and purified by a chromatography method to obtain L1 protein which is self-assembled into virus-like particles, and the specific steps are as follows: pichia pastoris cells expressing HPV6L1VLP were isolated as per 1:5 adding a bacteria breaking buffer solution, mixing, and then crushing the cell suspension at high pressure and repeating the operation to crush 90% of cells after fully mixing. The high-pressure broken bacterial liquid is centrifugally separated at 9000rpm for 30min at 10 ℃, and the supernatant is collected after centrifugation. The supernatant of the bacteria after centrifugal clarification is subjected to preliminary purification by a POROS50HS (Applied Biosystems company chromatographic column) by linear gradient elution from 100% buffer A (0.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80) to 100% buffer B (1.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80), and eluted components are collected and detected by SDS-PAGE and Western-blot.
After combining the eluted fractions containing HPV6L1 protein, further purification was performed using a CHT (BIO-RAD type ii) column by: linear gradient elution from 100% buffer A (5nM PB,0.6M NaCl,50mM MOPS,pH6.5,0.05%Tween-80) to 100% buffer B (200mM PB,0.6M NaCl,pH6.5,0.05%Tween-80). And collecting elution components, detecting by SDS-PAGE and Western-blot, and combining the components containing HPV6L1 VLPs to obtain the final purified sample. SDS-PAGE electrophoresis detected the purity of the L1 protein, and scanning showed that the purity of the purified virus-like particles was greater than 90% (FIG. 11). The purified sample was observed by electron microscopy (electron microscopy chamber of the chemical system of double denier university) to present virus-like particles (FIG. 16), which showed particle diameters between 50-100 nm.
After collected HPV11L1 thalli are subjected to bacteria breaking (bacteria breaking buffer solution: 200mM MOPS,pH7.0,0.7NaCl,0.05%Tween-80), supernatant after bacteria breaking is taken and purified by a chromatography method to obtain L1 protein which is self-assembled into virus-like particles, and the specific steps are as follows: pichia pastoris cells expressing HPV11L1VLP are added into a bacteria breaking buffer solution according to the ratio of 1:5, and after the mixture is fully mixed, the cell suspension is broken under high pressure, and the operation is repeated, so that 90% of cells are broken. The high-pressure broken bacterial liquid is centrifugally separated at 9000rpm for 30min at 10 ℃, and the supernatant is collected after centrifugation. The supernatant of the bacteria after centrifugal clarification is subjected to preliminary purification by a POROS50IIS (Applied Biosystems company chromatographic column) by linear gradient elution from 100% buffer A (0.5MNaCl,50mM MOPS,pH7.0,0.05%Tween-80) to 100% buffer B (1.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80), and eluted components are collected and detected by SDS-PAGE and Western-blot.
After combining the eluted fractions containing HPV11L1 protein, further purification was performed using a CHT (BIO-PAD type ii) column by: linear gradient elution from 100% buffer A (5mM PB,0.6M NaCl,50mM MOPS,pH6.5,0.05%Tween-80) to 100% buffer B (200mM PB,0.6M NaCl,pH6.5,0.05%Tween-80). And collecting elution components, detecting by SDS-PAGE and Western-blot, and combining the components containing HPV11L1 VLPs to obtain a final purified sample. SDS-PAGE electrophoresis detected the purity of the L1 protein, and scanning showed that the purity of the purified virus-like particles was greater than 90% (FIG. 11). The purified sample was observed by electron microscopy (electro-microscopy room at the university of eastern China) to present virus stem particles (FIG. 17), which showed particle diameters between 50-100 nm.
After collected HPV16L1 thalli are subjected to bacteria breaking (bacteria breaking buffer solution: 200mM MOPS,pH7.0,0.7NaCl,0.05%Tween-80), supernatant after bacteria breaking is taken and purified by a chromatography method to obtain L1 protein which is self-assembled into virus-like particles, and the specific steps are as follows: pichia pastoris cells expressing IIPV16L1VLP are added into a bacteria breaking buffer solution according to the ratio of 1:5, and after the cells are fully mixed, the cell suspension is broken under high pressure, and the operation is repeated, so that 90% of the cells are broken. The high-pressure broken bacterial liquid is centrifugally separated at 9000rpm for 30min at 10 ℃, and the supernatant is collected after centrifugation. The supernatant of the bacteria after centrifugal clarification is subjected to preliminary purification by a POROS50HS (Applied Biosystems company chromatographic column) by linear gradient elution from 100% buffer A (0.5M NaGl,50mM MOPS,pH7.0,0.05%Tween-80) to 100% buffer B (1.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80), and eluted components are collected and detected by SDS-PAGE and Western-blot.
After combining the eluted fractions containing HPV16L1 protein, further purification was performed using CHT (BTO-RAD type ii) column in the following manner: linear gradient elution from 100% buffer A (5mM PB,0.6M NaCl,50mM MOPS,pH6.5,0.05%Tween-80) to 100% buffer B (200mM PB,0.6M NaCl,pH6.5,0.05%Tween-80). And collecting elution components, detecting by SDS-PAGE and Western-blot, and combining the components containing HPV16L1 VLPs to obtain a final purified sample. SDS-PAGE electrophoresis detected the purity of the L1 protein, and scanning showed that the purity of the purified virus-like particles was greater than 90% (FIG. 13). The purified sample was observed by electron microscopy (electro-microscopy room at the university of eastern China) to present virus-like particles (FIG. 18), which showed particle diameters between 50-100 nm.
After collected HPV18L1 thalli are subjected to bacteria breaking (bacteria breaking buffer solution: 200mM MOPS,pH7.0,0.7NaCl,0.05%Tween-80), supernatant after bacteria breaking is taken and purified by a chromatography method to obtain L1 protein which is self-assembled into virus-like particles, and the specific steps are as follows: pichia pastoris cells expressing HPV18L1VLP are added into a bacteria breaking buffer solution according to the ratio of 1:5, and after the mixture is fully mixed, the cell suspension is broken under high pressure, and the operation is repeated, so that 90% of cells are broken. The high-pressure broken bacterial liquid is centrifugally separated at 9000rpm for 30min at 10 ℃, and the supernatant is collected after centrifugation. The supernatant of the bacteria after centrifugal clarification is subjected to preliminary purification by a POROS50HS (Applied Biosystems company chromatographic column) by linear gradient elution from 100% buffer A (0.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80) to 100% buffer B (1.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80), and eluted components are collected and detected by SDS-PAGE and Western-blot.
After combining the eluted fractions containing HPvl8L1 protein, further purification was performed using a CHT (BIO-RAD type II) column by: linear gradient elution from 100% buffer A (5rmM PB,0.6M NaCl,50mM MOPS,pH6.5,0.05%Tween-80) to 100% buffer B (200mM PB,0.6M NaCl,pH6.5,0.05%Tween-80). And collecting elution components, detecting by SDS-PAGE and Western-blot, and combining the components containing the IIPV18L1 VLPs to obtain a final purified sample. SDS-PAGE electrophoresis detected the purity of the L1 protein, and scanning showed that the purity of the purified virus-like particles was greater than 90% (FIG. 14). The purified sample was observed by electron microscopy (electro-microscopy room at the university of eastern China) to present virus-like particles (FIG. 19), which showed particle diameters between 50-100 nm.
After collected HPV58L1 thalli are subjected to bacteria breaking (bacteria breaking buffer solution: 200mM MOPS,pH7.0,0.7NaCl,0.05%Tween-80), supernatant after bacteria breaking is taken and purified by a chromatography method to obtain L1 protein which is self-assembled into virus-like particles, and the specific steps are as follows: pichia pastoris cells expressing HPV58L1VLP are added into a bacteria breaking buffer solution according to the ratio of 1:5, and after the mixture is fully mixed, the cell suspension is broken under high pressure, and the operation is repeated, so that 90% of cells are broken. The high-pressure broken bacterial liquid is centrifugally separated at 9000rpm for 30min at 10 ℃, and the supernatant is collected after centrifugation. The supernatant of the bacteria after centrifugal clarification is subjected to preliminary purification by a POROS50HS (Applied Biosystems company chromatographic column) by linear gradient elution from 100% buffer A (0.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80) to 100% buffer B (1.5M NaCl,50mM MOPS,pH7.0,0.05%Tween-80), and eluted components are collected and detected by SDS-PAGE and Western-blot.
After combining the eluted fractions containing IIPV58L1 protein, further purification was performed using CIIT (B1O-RAD type II) column in the following manner: linear gradient elution from 100% buffer A (5mM PB,0.6M NaCl,50mM MOPS,pH6.5,0.05%Tween-80) to 100% buffer B (200mM PB,0.6M NaCl,pH6.5,0.05%Tween-80). And collecting elution components, detecting by SDS-PAGE and Western-blot, and combining the components containing HPV58L1 VLPs to obtain the final purified sample. SDS-PAGE electrophoresis detected the purity of the L1 protein, and scanning showed that the purity of the purified virus-like particles was greater than 90% (FIG. 15). The purified sample was observed by electron microscopy (FIG. 20), which shows that the particle diameter was between 50 and 100 nm.
Example 6: measurement of expression level of the recombinant HPVL1 protein of the present invention
In this example, the content of the total protein in the post-fermentation bacterial cell-disrupting supernatant measured by the Bradford method and the expression level of the HPVL1VLP measured by the Elisa sandwich method were calculated to obtain the content of the HPVL1VLP in the total protein after disruption. The method comprises the following specific steps:
1. determination of total protein content in the fermentation broths supernatant using Bradford method
The assay was performed using the K4000Bradford protein quantitation reagent kit commercially available from shanghai lottery biotechnology limited.
To a 1.5ml EP tube, 0. Mu.l, 10. Mu.l, 20. Mu.l, 40. Mu.l, 80. Mu.l, 100. Mu.l BSA standard (0.5 mg/ml) and 40. Mu.l of the bacterial supernatant of the fermentation tubes obtained in example 4 (diluted 100-fold) were added at a time, and the mixture was made up to a total volume of 100. Mu.l with water, followed by mixing. 3 replicates were set for each concentration. 900 mu l Bradford solution was added to each tube, mixed immediately, and left at room temperature for 10 minutes, and OD595 light absorption values were measured. And (3) calculating a linear equation according to a standard curve of protein concentration to absorbance value, which is made according to 6 groups of BSA standard products, and calculating the total protein content of the bacterial supernatant of the fermentation thallus according to the light absorbance value obtained by the bacterial supernatant and the linear equation of the standard curve.
2. Determination of HPVL1VLP content in post-fermentation cell-disruption supernatant by Elisa sandwich method
Purified HPVL1 VLPs were used as standard protein concentration curves, and pre-induction thalli served as negative controls.
The rabbit anti-HPVL 1VLP polyclonal antibody was diluted 2000-fold with coating solution (1.6g Na2CO3,2.95g NaHCO3), and then 0.1ml of diluted rabbit polyclonal antibody was added to each well of the ELISA plate overnight at 4 ℃. The coating was removed, the wells were washed with 0.3ml of PBST (PBS, pH7.0,0.05% Tween-20), and incubated with 0.3ml of blocking solution (5% nonfat milk powder+PBST) at 37℃for 2 hours.
The purified HPVL1VLP obtained in example 5 was diluted in a serial double dilution with dilution (PBS, pIII 7.0) from a concentration of 2. Mu.g/ml to 0.0625. Mu.g/ml as a standard sample. Meanwhile, the bacterial supernatant of the fermentation cells obtained in example 4 was diluted 200 times, then 0.1ml of different concentrations of HPVL1VLP solution after gradient dilution or the diluted bacterial supernatant was added to the wells, respectively, and after incubation at 37℃for 1 hour, the antigen solution was removed and the wells were washed with 0.3ml of PBST. MAB885 murine anti-HPV 52L1VLP MAB (available from CHEMICON) was then 1000-fold diluted with antibody dilution buffer (PBS, pH7.0,2% nonfat milk powder) and added to wells, each well was incubated at 37℃for 1 hour. The mab solution was removed and the wells were washed with 0.3ml PBST. To each well was added 5000-fold dilution of HRP-labeled goat anti-mouse IgGO.1ml with antibody dilution buffer, and incubated at 37℃for 0.5 hours. The antibody solution was removed and the wells were washed with 0.3ml of PBST, and 0.1ml of DAB color development solution (available from Amresco Co.) was added to each well, and allowed to act at room temperature for 20 minutes. 0.05m12M H was added to each well 2 SO 4 Stop solution to stop reaction and use enzyme-labeled colorimeter to determine OD 450 Absorbance values.
OD utilizing gradient diluted HPVLIVLP 450 And (3) preparing a standard protein concentration curve, and converting the standard protein concentration curve to obtain the fermentation expression quantity of the HPVL1 protein.
The results of this example are shown in tables 2-6.
As can be seen from Table 2, the expression level of HPV6L1 gene of the present invention was up to 134.1. Mu.g/mg (HPV 6L1VLP in the supernatant of the strain/total protein of the supernatant of the strain).
Table 2: the expression level of HPV6L1 gene of the present invention
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As can be seen from Table 3, the expression level of HPV11L1 gene of the present invention can reach up to 123.8. Mu.g/mg (HPV 11L1VLP in the supernatant of the bacterium/total protein of the supernatant of the bacterium).
Table 3: the expression level of HPV11L1 gene of the present invention
As can be seen from Table 4, the expression level of the HPVI6L1 gene of the present invention can reach 135.8. Mu.g/mg (HPV 16L1VLP in the supernatant of the strain/total protein of the supernatant of the strain) at the highest.
Table 4: the expression level of IIPV16L1 gene of the present invention
As can be seen from Table 5, the expression level of HPV18L1 gene of the present invention was up to 125.4. Mu.g/mg (HPV 18L1VLP in the supernatant of the strain/total protein of the supernatant of the strain).
Table 5: the expression level of HPV18L1 gene of the present invention
As can be seen from Table 6, the expression level of HPV58L1 gene of the present invention was up to 132.1. Mu.g/mg (HPV 58L1VLP in the supernatant of the strain/total protein of the supernatant of the strain).
Table 6: the expression level of HPV58L1 gene of the present invention
Example 7: HPV L1 vaccine preparation
The purified HPVL1 protein obtained in example 5 was adsorbed to an aluminum phosphate adjuvant to prepare an HPV vaccine having immunogenicity, referring to the methods in the pharmacopoeia of the people's republic of China (2005 edition).
Example 8: determination of immunogenicity of HPV L1 Gene expression products
Determination of immunogenicity of HPV6L1 Gene expression products
SPF-grade BALB/c mice (Shanghai Sipulbika laboratory animals Co., ltd.) of 6-8 weeks of age were selected and divided into 4 groups of 8 mice each. Groups 1 to 3 were each injected with 0.5mL of VLPs (as detection groups) adsorbed with aluminum adjuvant at a concentration of 2. Mu.g/mL, 0.2. Mu.g/mL, 0.02. Mu.g/mL, and group 4 mice were immunized (as negative control group) with 0.1mL of a buffer (0.32M sodium chloride, 0.01% Tween-80,0.01M histidine, pH 6.5) containing aluminum adjuvant at five subcutaneous injections at the abdomen for 0 day and blood was collected 28 days after immunization. Placing the collected blood at 37 ℃ for 2 hours, centrifuging at 8000rpm for 5 minutes, absorbing the supernatant to obtain the mouse immune serum, storing at-20 ℃, and detecting the positive transfer rate of the mouse serum, wherein the specific method comprises the following steps: purified Pichia pastoris-expressed HPV6L1 to 1. Mu.g/mL was diluted with coating solution, 96-well ELISA plates were coated, 0.1mL per well was added overnight at 4 ℃. The coating was removed and washed 3 times with 0.3mL of BST, followed by 3 washes with 0.3mL of blocking solution (5% nonfat milk powder+PBST) incubated at 37℃for 2 hours. The serum to be tested was diluted 1:1000 with dilution buffer (2% nonfat milk powder+PBST) per well, 100. Mu.l/well, and the ELISA plate was added in duplicate wells and incubated at 37℃for 1 hour. Washing 6 times, diluting HRP-labeled goat anti-mouse IgG with diluent 1:5000, adding ELISA plate 100 μl/well, incubating at 37deg.C for 0.5 hr, washing 6 times, adding 100 μl/well TMB, developing at 37deg.C for 10 min, adding 2M H 2 SO 4 50 μl of the reaction was stopped. Determination of OD with an enzyme-labeled colorimeter 450 Reading, OD 450 The values are shown in Table 7. The results of the positive transfer rates of the three test groups are shown in Table 8.
TABLE 7 detection of serum positive transfer rate (OD) from HPV6L1 immunized mice 450 Reading out
| Grouping of different doses | Group 1 μg | Group 0.1 μg | Group 0.01 μg |
| Rate of positive rotation | 100％ | 100％ | 12.5％ |
TABLE 8HPV6L1 positive conversion results
Negative average: 0.007; cutoff value: 0.014
Note that: cutoff value is OD of serum antibody to be tested in adjuvant group 450 The average of the values multiplied by 2.1, OD 450 Mouse serum with a value greater than the Cutoff value was judged positive, OD 450 Mouse sera with values less than the Cutoff value were judged negative.
Determination of immunogenicity of HPV11L1 Gene expression products
SPF-grade BALB/c mice (Shanghai Sipulbika laboratory animals Co., ltd.) of 6-8 weeks of age were selected and divided into 4 groups of 8 mice each. Groups 1 to 3 were each injected with 0.5mL of VLPs (as detection groups) adsorbed with aluminum adjuvant at a concentration of 2. Mu.g/mL, 0.2. Mu.g/mL, 0.02. Mu.g/mL, and group 4 mice were immunized (as negative control group) with 0.1mL of a buffer (0.32M sodium chloride, 0.01% Tween-80,0.01M histidine, pH 6.5) containing aluminum adjuvant at five subcutaneous injections at the abdomen for 0 day and blood was collected 28 days after immunization. Placing the collected blood at 37 ℃ for 2 hours, centrifuging at 8000rpm for 5 minutes, absorbing the supernatant to obtain the mouse immune serum, storing at-20 ℃, and detecting the positive transfer rate of the mouse serum, wherein the specific method comprises the following steps: purified Pichia pastoris-expressed HPV11L1 to 1. Mu.g/mL was diluted with coating solution, 96-well ELISA plates were coated, 0.1mL per well was added overnight at 4 ℃. The coating was removed, washed 3 times with 0.3mL of LPBST, and then with 0.3mL of blocking solution (5% degreasing) Milk powder + PBST) was incubated at 37 ℃ for 2 hours and washed 3 times. The serum to be tested was diluted 1:1000 with dilution buffer (2% nonfat milk powder+PBST) per well, 100. Mu.l/well, and the ELISA plate was added in duplicate wells and incubated at 37℃for 1 hour. Washing 6 times, diluting HRP-labeled goat anti-mouse IgG with diluent 1:5000, adding ELISA plate 100 μl/well, incubating at 37deg.C for 0.5 hr, washing 6 times, adding 100 μl/well TMB, developing at 37deg.C for 10 min, adding 2M H 2 SO 4 50 μl of the reaction was stopped. Determination of OD with an enzyme-labeled colorimeter 450 Reading, OD 450 The values are shown in Table 9. The results of the positive conversion rates of the three test groups are shown in Table 10.
TABLE 9 detection of serum positive transfer rate (OD) from HPV11L1 immunized mice 450 Reading out
| Grouping of different doses | Group 1 μg | Group 0.1 μg | Group 0.01 μg |
| Rate of positive rotation | 100％ | 100％ | 25.0％ |
TABLE 10HPV11L1 positive conversion results
Negative average: 0.007; cutoff value: 0.014
Note that:cutoff value is OD of serum antibody to be tested in adjuvant group 450 The average of the values multiplied by 2.1, OD 450 Mouse serum with a value greater than the Cutoff value was judged positive, OD 450 Mouse sera with values less than the Cutoff value were judged negative.
Determination of immunogenicity of HPV16L1 Gene expression products
SPF-grade BALB/c mice (Shanghai Sipulbika laboratory animals Co., ltd.) of 6-8 weeks of age were selected and divided into 4 groups of 8 mice each. Groups 1 to 3 were each injected with 0.5mL of VLPs (as detection groups) adsorbed with aluminum adjuvant at a concentration of 2. Mu.g/mL, 0.2. Mu.g/mL, 0.02. Mu.g/mL, and group 4 mice were immunized (as negative control group) with 0.1mL of a buffer (0.32M sodium chloride, 0.01% Tween-80,0.01M histidine, pH 6.5) containing aluminum adjuvant at five subcutaneous injections at the abdomen for 0 day and blood was collected 28 days after immunization. Placing the collected blood at 37 ℃ for 2 hours, centrifuging at 8000rpm for 5 minutes, absorbing the supernatant to obtain the mouse immune serum, storing at-20 ℃, and detecting the positive transfer rate of the mouse serum, wherein the specific method comprises the following steps: purified Pichia pastoris-expressed HPV16L1 to 1. Mu.g/mL was diluted with coating solution, 96-well ELISA plates were coated, 0.1mL per well was added overnight at 4 ℃. The coating was removed and washed 3 times with 0.3mL of BST, followed by 3 washes with 0.3mL of blocking solution (5% nonfat milk powder+PBST) incubated at 37℃for 2 hours. The serum to be tested was diluted 1:1000 with dilution buffer (2% nonfat milk powder+PBST) per well, 100. Mu.l/well, and the ELISA plate was added in duplicate wells and incubated at 37℃for 1 hour. Washing 6 times, diluting HRP-labeled goat anti-mouse IgG with diluent 1:5000, adding ELISA plate 100 μl/well, incubating at 37deg.C for 0.5 hr, washing 6 times, adding 100 μl/well TMB, developing at 37deg.C for 10 min, adding 2M H 2 SO 4 50 μl of the reaction was stopped. Determination of OD with an enzyme-labeled colorimeter 450 Reading, OD 450 The values are shown in Table 11. The results of the positive conversion rates of the three test groups are shown in Table 12.
TABLE 11 detection of serum positive transfer rate (OD) from HPV16L1 immunized mice 450 Reading out
| Grouping of different doses | Group 1 μg | Group 0.1 μg | Group 0.01 μg |
| Rate of positive rotation | 100％ | 100％ | 25.0％ |
TABLE 12HPV16L1 positive conversion results
Negative average: 0.007; cutoff value: 0.014
Note that: cutoff value is OD of serum antibody to be tested in adjuvant group 450 The average of the values multiplied by 2.1, OD 450 Mouse serum with a value greater than the Cutoff value was judged positive, OD 450 Mouse sera with values less than the Cutoff value were judged negative.
Determination of immunogenicity of HPV18L1 Gene expression products
SPF-grade BALB/c mice (Shanghai Sipulbika laboratory animals Co., ltd.) of 6-8 weeks of age were selected and divided into 4 groups of 8 mice each. Groups 1 to 3 were each injected with 0.5mL of VLPs (as detection groups) adsorbed with aluminum adjuvant at a concentration of 2. Mu.g/mL, 0.2. Mu.g/mL, 0.02. Mu.g/mL, and group 4 mice were immunized (as negative control group) with 0.1mL of a buffer (0.32M sodium chloride, 0.01% Tween-80,0.01M histidine, pH 6.5) containing aluminum adjuvant at five subcutaneous injections at the abdomen for 0 day and blood was collected 28 days after immunization. Standing the collected blood at 37deg.C for 2 hr, centrifuging at 8000rpm for 5min, and collecting supernatant to obtain mouse immune serum The cells were stored at-20deg.C and tested for positive turnover rate in murine serum as follows: purified Pichia pastoris-expressed HPV18LI was diluted to 1. Mu.g/mL with coating solution, 96-well ELISA plates were coated, 0.1mL per well was added, and 4℃overnight. The coating was removed and washed 3 times with 0.3mL of BST, followed by 3 washes with 0.3mL of blocking solution (5% nonfat milk powder+PBST) incubated at 37℃for 2 hours. The serum to be tested was diluted 1:1000 with dilution buffer (2% nonfat milk powder+PBST) per well, 100. Mu.l/well, and the ELISA plate was added in duplicate wells and incubated at 37℃for 1 hour. Washing for 6 times, adding HRP-labeled goat anti-mouse IgG with cotton release solution 1:5000, adding ELISA plate at 100 μl/well, incubating at 37deg.C for 0.5 hr, washing for 6 times, adding 100 μl/well TMB, developing at 37deg.C for 10 min, adding 2M H 2 SO 4 50 μl of the reaction was stopped. Determination of OD with an enzyme-labeled colorimeter 450 Reading, OD 450 The values are shown in Table 13. The results of the positive conversion rates of the three test groups are shown in Table 14.
TABLE 13 detection of serum positive transfer rate (OD) from HPV18L1 immunized mice 450 Reading out
| Grouping of different doses | Group 1 μg | 0.i μg group | Group 0.01 μg |
| Rate of positive rotation | 100％ | 100％ | 12.5％ |
TABLE 14HPV18L1 positive conversion results
Negative average: 0.007; cutoff value: 0.014
Note that: cutoff value is OD of serum antibody to be tested in adjuvant group 450 The average of the values multiplied by 2.1, OD 450 Mouse serum with a value greater than the Cutoff value was judged positive, OD 450 Mouse sera with values less than the Cutoff value were judged negative.
Determination of immunogenicity of HPV58L1 Gene expression products
SPF-grade BALB/c mice (Shanghai Sipulbika laboratory animals Co., ltd.) of 6-8 weeks of age were selected and divided into 4 groups of 8 mice each. Groups 1 to 3 were each injected with 0.5mL of VLPs (as detection groups) adsorbed with aluminum adjuvant at a concentration of 2. Mu.g/mL, 0.2. Mu.g/mL, 0.02. Mu.g/mL, and group 4 mice were immunized (as negative control group) with 0.1mL of a buffer (0.32M sodium chloride, 0.01% Tween-80,0.01M histidine, pH 6.5) containing aluminum adjuvant at five subcutaneous injections at the abdomen for 0 day and blood was collected 28 days after immunization. Placing the collected blood at 37 ℃ for 2 hours, centrifuging at 8000rpm for 5 minutes, absorbing the supernatant to obtain the mouse immune serum, storing at-20 ℃, and detecting the positive transfer rate of the mouse serum, wherein the specific method comprises the following steps: purified Pichia pastoris-expressed HPV58L1 to 1. Mu.g/mL was diluted with coating solution, 96-well ELISA plates were coated, 0.1mL per well was added overnight at 4 ℃. The coating was removed and washed 3 times with 0.3mL of BST, followed by 3 washes with 0.3mL of blocking solution (5% nonfat milk powder+PBST) incubated at 37℃for 2 hours. The serum to be tested was diluted 1:1000 with dilution buffer (2% nonfat milk powder+PBST) per well, 100. Mu.l/well, and the ELISA plate was added in duplicate wells and incubated at 37℃for 1 hour. Washing 6 times, diluting HRP-labeled goat anti-mouse IgG with diluent 1:5000, adding ELISA plate 100 μl/well, incubating at 37deg.C for 0.5 hr, washing 6 times, adding 100 μl/well TMB, developing at 37deg.C for 10 min, adding 2M H 2 SO 4 50 μl of the reaction was stopped. Determination of OD with an enzyme-labeled colorimeter 450 Reading, OD 450 The values are shown in Table 15. The results of the positive conversion rates of the three test groups are shown in Table 16.
TABLE 15 detection of serum positive transfer rate (OD) from HPV58L1 immunized mice 450 Reading out
| Grouping of different doses | Group 1 μg | Group 0.1 μg | Group 0.01 μg |
| Rate of positive rotation | 100％ | 100％ | 12.5％ |
TABLE 16HPV58L1 positive conversion results
Negative average: 0.006; cutoff value: 0.012
Note that: cutoff value is OD of serum antibody to be tested in adjuvant group 450 The average of the values multiplied by 2.1, OD 450 Mouse serum with a value greater than the Cutoff value was judged positive, OD 450 Mouse sera with values less than the Cutoff value were judged negative.
In summary, the main capsid protein L1 gene of the human papillomavirus provided by the invention is an optimized L1 gene, and has the following advantages: the optimized gene is suitable for expressing target protein in yeast host with high efficiency, and can meet the requirement of industrial production; meanwhile, the human papillomavirus vaccine provided by the invention can self-assemble to form VLPs structure, after purified VLPs adsorb adjuvant, the vaccine can generate stronger immunogenicity in mice through the measurement of serum positive transfer rate, and the method has the following advantages due to the adoption of a Pichia pastoris expression system: low cost, high yield and more uniform and stable product property.
<110> Shanghai ze Biotech Co., ltd
<120> recombinant human papillomavirus protein expression
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<212> PRT
<213> Human papillomavirus type 6
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Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro
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Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Thr Arg Thr Asn Ile
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Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr
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Phe Ser Ile Lys Arg Ala Asn Lys Thr Val Val Pro Lys Val Ser Gly
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Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe
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Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu Val
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Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val
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Gly Val Ser Gly His Pro Phe Leu Asn Lys Tyr Asp Asp Val Glu Asn
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Ser Gly Ser Gly Gly Asn Pro Gly Gln Asp Asn Arg Val Asn Val Gly
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Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro Pro
145 150 155 160
Leu Gly Glu His Trp Gly Lys Gly Lys Gln Cys Thr Asn Thr Pro Val
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Gln Ala Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile Gln
180 185 190
Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala Asp
195 200 205
Leu Gln Thr Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Gly Thr Thr
210 215 220
Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro Tyr Gly Asp
225 230 235 240
Arg Leu Phe Phe Phe Leu Arg Lys Glu Gln Met Phe Ala Arg His Phe
245 250 255
Phe Asn Arg Ala Gly Glu Val Gly Glu Pro Val Pro Asp Thr Leu Ile
260 265 270
Ile Lys Gly Ser Gly Asn Arg Thr Ser Val Gly Ser Ser Ile Tyr Val
275 280 285
Asn Thr Pro Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe Asn
290 295 300
Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile Cys
305 310 315 320
Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr
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Asn Met Thr Leu Cys Ala Ser Val Thr Thr Ser Ser Thr Tyr Thr Asn
340 345 350
Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu Glu Tyr Asp Leu Gln
355 360 365
Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser Ala Glu Val Met Ala
370 375 380
Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe Gly
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Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr Val
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Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu Lys
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Pro Asp Pro Tyr Lys Asn Leu Ser Phe Trp Glu Val Asn Leu Lys Glu
435 440 445
Lys Phe Ser Ser Glu Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu
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Leu Gln Ser Gly Tyr Arg Gly Arg Ser Ser Ile Arg Thr Gly Val Lys
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Arg Pro Ala Val Ser Lys Ala Ser Ala Ala Pro Lys Arg Lys Arg Ala
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Lys Thr Lys Arg
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<210> 2
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<212> PRT
<213> Human papillomavirus type 11
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<309> 2013-06-18
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Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro
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Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Lys Arg Thr Asn Ile
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Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr
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Tyr Ser Ile Lys Lys Val Asn Lys Thr Val Val Pro Lys Val Ser Gly
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Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe
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Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu Val
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Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu Gly Val
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Gly Val Ser Gly His Pro Leu Leu Asn Lys Tyr Asp Asp Val Glu Asn
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Ser Gly Gly Tyr Gly Gly Asn Pro Gly Gln Asp Asn Arg Val Asn Val
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Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro
145 150 155 160
Pro Leu Gly Glu His Trp Gly Lys Gly Thr Gln Cys Ser Asn Thr Ser
165 170 175
Val Gln Asn Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile
180 185 190
Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala
195 200 205
Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Leu Asp Ile Cys Gly Thr
210 215 220
Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro Tyr Gly
225 230 235 240
Asp Arg Leu Phe Phe Tyr Leu Arg Lys Glu Gln Met Phe Ala Arg His
245 250 255
Phe Phe Asn Arg Ala Gly Thr Val Gly Glu Pro Val Pro Asp Asp Leu
260 265 270
Leu Val Lys Gly Gly Asn Asn Arg Ser Ser Val Ala Ser Ser Ile Tyr
275 280 285
Val His Thr Pro Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe
290 295 300
Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile
305 310 315 320
Cys Trp Gly Asn His Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
325 330 335
Thr Asn Met Thr Leu Cys Ala Ser Val Ser Lys Ser Ala Thr Tyr Thr
340 345 350
Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu Glu Phe Asp Leu
355 360 365
Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser Ala Glu Val Met
370 375 380
Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe
385 390 395 400
Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr
405 410 415
Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu
420 425 430
Lys Gln Asp Pro Tyr Lys Asp Met Ser Phe Trp Glu Val Asn Leu Lys
435 440 445
Glu Lys Phe Ser Ser Glu Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe
450 455 460
Leu Leu Gln Ser Gly Tyr Arg Gly Arg Thr Ser Ala Arg Thr Gly Ile
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Lys Arg Pro Ala Val Ser Lys Pro Ser Thr Ala Pro Lys Arg Lys Arg
485 490 495
Thr Lys Thr Lys Lys
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<210> 3
<211> 505
<212> PRT
<213> Human papillomavirus type 16
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<313> (1)..(505)
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Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
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Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn
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Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro
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Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys
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Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro
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Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln
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Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro
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Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp
115 120 125
Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg
130 135 140
Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly
145 150 155 160
Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr
165 170 175
Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn
180 185 190
Thr Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met
195 200 205
Asp Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile
210 215 220
Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu
225 230 235 240
Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe
245 250 255
Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro
260 265 270
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser
275 280 285
Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala
290 295 300
Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu
340 345 350
Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu
355 360 365
Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr
370 375 380
Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu
385 390 395 400
Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp
405 410 415
Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr
420 425 430
Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu
435 440 445
Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu
450 455 460
Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe
465 470 475 480
Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr
485 490 495
Thr Ala Lys Arg Lys Lys Arg Lys Leu
500 505
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<212> PRT
<213> Human papillomavirus type 18
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<313> (1)..(568)
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Met Cys Leu Tyr Thr Arg Val Leu Ile Leu His Tyr His Leu Leu Pro
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Leu Tyr Gly Pro Leu Tyr His Pro Arg Pro Leu Pro Leu His Ser Ile
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Leu Val Tyr Met Val His Ile Ile Ile Cys Gly His Tyr Ile Ile Leu
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Phe Leu Arg Asn Val Asn Val Phe Pro Ile Phe Leu Gln Met Ala Leu
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Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro Ser Val Ala
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Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser Ile Phe Tyr
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His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg
100 105 110
Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys Val Ser Ala
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Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe
130 135 140
Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val
145 150 155 160
Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val
165 170 175
Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser
180 185 190
Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val
195 200 205
Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro
210 215 220
Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro
225 230 235 240
Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu
245 250 255
Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser
260 265 270
Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser
275 280 285
Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly
290 295 300
Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His
305 310 315 320
Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu
325 330 335
Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr
340 345 350
Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe
355 360 365
Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val
370 375 380
Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
385 390 395 400
Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln
405 410 415
Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr
420 425 430
Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp
435 440 445
Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp
450 455 460
Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr
465 470 475 480
Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro
485 490 495
Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp
500 505 510
Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg
515 520 525
Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro
530 535 540
Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala Lys
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Arg Val Arg Val Arg Ala Arg Lys
565
<210> 5
<211> 524
<212> PRT
<213> Human papillomavirus type 58
<300>
<308> Genebank/BAA31851.1
<309> 2007-12-07
<313> (1)..(524)
<400> 5
Met Val Leu Ile Leu Cys Cys Thr Leu Ala Ile Leu Phe Cys Val Ala
1 5 10 15
Asp Val Asn Val Phe His Ile Phe Leu Gln Met Ser Val Trp Arg Pro
20 25 30
Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val
35 40 45
Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala Gly
50 55 60
Ser Ser Arg Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys Ser
65 70 75 80
Pro Asn Asn Asn Lys Lys Val Leu Val Pro Lys Val Ser Gly Leu Gln
85 90 95
Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Phe
100 105 110
Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala
115 120 125
Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Val
130 135 140
Ser Gly His Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu Thr Ser Asn
145 150 155 160
Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys Leu Ser Met
165 170 175
Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Thr
180 185 190
Gly Glu His Trp Gly Lys Gly Val Ala Cys Asn Asn Asn Ala Ala Ala
195 200 205
Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile Glu Asp Gly
210 215 220
Asp Met Val Asp Thr Gly Phe Gly Cys Met Asp Phe Gly Thr Leu Gln
225 230 235 240
Ala Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys Lys
245 250 255
Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly Asp Ser Leu
260 265 270
Phe Phe Phe Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe Asn
275 280 285
Arg Ala Gly Lys Leu Gly Glu Ala Val Pro Asp Asp Leu Tyr Ile Lys
290 295 300
Gly Ser Gly Asn Thr Ala Val Ile Gln Ser Ser Ala Phe Phe Pro Thr
305 310 315 320
Pro Ser Gly Ser Ile Val Thr Ser Glu Ser Gln Leu Phe Asn Lys Pro
325 330 335
Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly
340 345 350
Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met
355 360 365
Thr Leu Cys Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys Asn Asp Asn
370 375 380
Phe Lys Glu Tyr Val Arg His Val Glu Glu Tyr Asp Leu Gln Phe Val
385 390 395 400
Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu Ile Met Thr Tyr Ile
405 410 415
His Thr Met Asp Ser Asn Ile Leu Glu Asp Trp Gln Phe Gly Leu Thr
420 425 430
Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe Val Thr Ser
435 440 445
Gln Ala Ile Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu Lys Glu Asp
450 455 460
Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe
465 470 475 480
Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln
485 490 495
Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr Thr
500 505 510
Arg Ala Pro Ser Thr Lys Arg Lys Lys Val Lys Lys
515 520
<210> 6
<211> 1500
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of full-length HPV 6 L1 protein Gene Using Pichia pastoris preference codon
<400> 6
atgtggagac catccgactc cactgtctac gttccaccac caaacccagt ctctaaggtt 60
gtcgccactg acgcctacgt cacccgtacc aacatctttt accacgcttc ttcctctcgt 120
ttgcttgccg tcggacaccc ttacttctcc atcaaaagag ccaacaagac cgtcgtccca 180
aaggtctccg gttatcagta tagagttttt aaagtcgtcc ttccagaccc taacaagttc 240
gctttgccag actcctcttt gttcgatcca accacccagc gtttggtctg ggcctgtact 300
ggtttggagg ttggtagagg tcagcctctt ggtgttggag tctccggaca ccctttcctt 360
aacaagtacg acgacgtcga gaactccggt tccggtggta acccaggtca ggataaccgt 420
gtcaacgttg gtatggacta caagcagacc cagctttgta tggtcggatg tgctccacca 480
cttggtgagc actggggaaa gggtaaacaa tgcaccaaca ccccagtcca ggctggtgac 540
tgtccacctt tggagcttat cacctccgtt atccaggacg gtgacatggt cgataccggt 600
ttcggagcca tgaattttgc cgaccttcag actaacaagt ccgatgtccc tatcgacatc 660
tgcggtacta cctgcaagta cccagactac cttcagatgg ccgccgaccc atacggagat 720
cgtttgtttt tctttttgcg taaggagcag atgttcgccc gtcacttctt taaccgtgct 780
ggagaggtcg gtgagcctgt cccagacacc cttatcatca aaggttccgg taacagaacc 840
tccgtcggtt cctccatcta cgttaacact ccatctggtt cccttgtttc ctccgaggcc 900
cagttgttta acaagcctta ctggttgcag aaggcccagg gtcacaataa cggtatttgc 960
tggggaaatc agttgtttgt caccgtcgtt gacaccaccc gttctaccaa catgaccctt 1020
tgtgcctccg tcaccacttc ctccacctat actaactccg actacaagga gtatatgcgt 1080
cacgtcgagg aatacgactt gcagtttatt tttcagttgt gttccatcac cttgtccgcc 1140
gaggtcatgg cctacattca taccatgaac ccatccgtct tggaggactg gaacttcgga 1200
ttgtctcctc cacctaatgg aaccttggag gatacctacc gttacgttca gtcccaagcc 1260
attacttgcc aaaagcctac ccctgagaag gaaaaacctg acccatacaa aaatctttct 1320
ttttgggagg ttaatcttaa agaaaaattc tcctccgaat tggaccagta cccattggga 1380
cgtaagtttt tgttgcaatc cggttatcgt ggacgttcct ctatccgtac cggagttaag 1440
cgtcctgccg tttctaaggc ctccgccgcc cctaagagaa aacgtgctaa aaccaagaga 1500
<210> 7
<211> 1515
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of full-length HPV 16 L1 protein Gene Using Pichia pastoris preference codon
<400> 7
atgtccttgt ggttgccatc cgaagccact gtttacttgc ctcctgtccc agtctccaag 60
gtcgtttcta ccgacgagta cgtcgcccgt accaatatct actaccatgc cggaacctcc 120
cgtttgcttg ccgtcggtca tccatacttc cctattaaaa agccaaataa taacaagatc 180
cttgtcccaa aggtctccgg acttcagtac cgtgtcttcc gtattcatct tccagaccct 240
aacaaatttg gtttcccaga cacctccttc tacaaccctg atacccagag acttgtctgg 300
gcctgcgtcg gagttgaggt cggtagaggt caacctttgg gagtcggtat ctccggtcac 360
ccacttttga acaagttgga cgacaccgaa aacgcctctg cttacgccgc taacgctggt 420
gttgacaacc gtgagtgcat ttccatggac tacaaacaga cccagttgtg ccttatcgga 480
tgcaaaccac caatcggaga gcactggggt aagggttccc cttgcaccaa cgttgccgtc 540
aacccaggag actgcccacc tttggagctt atcaacaccg ttatccagga cggtgatatg 600
gtcgatactg gattcggagc catggacttt accacccttc aggccaataa gtccgaagtc 660
ccacttgaca tttgtacctc tatttgtaaa tacccagact acatcaaaat ggtctccgag 720
ccttacggtg actccttgtt tttttatctt cgtagagagc agatgtttgt ccgtcacctt 780
ttcaaccgtg ctggtgctgt cggtgagaac gtcccagacg acctttacat caagggttcc 840
ggttccaccg ccaatttggc ctcctccaac tatttcccaa ctccatccgg ttccatggtc 900
acctccgacg cccaaatctt taataaacca tactggcttc agagagccca gggacataac 960
aacggtatct gctggggtaa ccagttgttt gtcaccgtcg ttgacaccac tcgttccacc 1020
aacatgtcct tgtgcgccgc catctctacc tctgaaacca cctataagaa tactaatttc 1080
aaagagtatt tgagacacgg tgaggagtac gatttgcagt ttatctttca actttgcaag 1140
atcaccttga ccgccgacgt catgacctac atccattcta tgaactccac cattttggaa 1200
gactggaact tcggacttca acctccacct ggtggaactt tggaggacac ctaccgtttc 1260
gtcacttctc aggccattgc ctgtcagaag catacccctc ctgccccaaa agaagatcca 1320
ttgaagaaat atactttttg ggaggttaat cttaaggaga agttctccgc cgacttggat 1380
caattccctc ttggacgtaa gtttttgctt caggccggac ttaaggccaa gcctaagttt 1440
accttgggaa agcgtaaggc cacccctact acctcttcca cctccaccac tgctaagcgt 1500
aagaaaagaa agctt 1515
<210> 8
<211> 1503
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of full-length HPV 11 L1 protein Gene Using Pichia pastoris preference codon
<400> 8
atgtggagac cttctgattc caccgtctac gtcccacctc caaacccagt ctccaaagtc 60
gtcgccactg acgcctacgt taaaagaact aatatcttct accacgcttc ttcctccaga 120
cttttggccg ttggacaccc ttactactcc attaaaaagg ttaacaagac cgtcgtccct 180
aaggtctccg gttatcagta ccgtgtcttc aaagtcgtct tgccagaccc aaacaagttc 240
gccttgcctg attcctccct tttcgaccct accactcaga gattggtctg ggcctgcacc 300
ggacttgaag tcggtagagg tcaaccattg ggtgtcggag tttccggaca cccattgctt 360
aacaaatacg acgacgttga aaactccggt ggatacggag gtaaccctgg tcaggacaac 420
agagtcaacg tcggtatgga ctacaagcag acccaacttt gcatggtcgg ttgcgctcca 480
cctcttggag agcactgggg taagggaact cagtgctcca acacctctgt ccagaacgga 540
gattgcccac cattggagtt gatcacttct gtcatccagg acggtgacat ggtcgatact 600
ggtttcggtg ccatgaactt cgccgacttg cagactaaca agtccgacgt ccctttggac 660
atttgcggaa ccgtctgcaa gtacccagac tatcttcaga tggctgccga tccatgcgga 720
gaccgtttgt tcttttactt gagaaaggaa caaatgttcg ctcgtcactt cttcaatcgt 780
gccggaaccg ttggagaacc agtcccagac gacttgcttg tcaaaggtgg aaacaaccgt 840
tcctccgttg cttcctccat ctatgtccat accccttccg gttctttggt ctcctccgag 900
gcccaacttt tcaacaagcc ttactggctt cagaaggctc agggacacaa caacggaatc 960
tgttggggta accacctttt cgtcaccgtt gttgacacca ccagatccac caacatgacc 1020
ttgtgcgcct ctgtctccaa atctgctacc tacaccaact ccgactataa ggagtacatg 1080
agacacgtcg aagagtttga tttgcagttt atttttcaac tttgctccat caccctttct 1140
gccgaggtca tggcctacat tcacaccatg aacccatccg tcttggagga ctggaacttt 1200
ggtttgtccc caccacctaa cggaaccctt gaggacacct atcgttatgt ccagtcccag 1260
gccatcacct gtcagaagcc aaccccagag aaggagaagc aggaccctta caaagatatg 1320
tctttttggg aggtcaactt gaaagaaaag ttctcttccg agcttgacca attccctctt 1380
ggtagaaagt ttttgttgca atccggttac cgtggacgta cctccgccag aaccggtatc 1440
aaacgtccag ccgtttccaa gccatccact gctcctaagc gtaaacgtac caagaccaag 1500
aaa 1503
<210> 9
<211> 1521
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of full-length HPV 18 L1 protein Gene Using Pichia pastoris preference codon
<400> 9
atggctttgt ggcgtccttc cgacaacact gtctaccttc caccaccttc cgttgctaga 60
gtcgtcaata ccgacgacta cgtcacccgt acctccatct tctatcatgc cggttcctct 120
cgtttgttga ccgtcggtaa cccatacttc cgtgttccag ccggtggtgg taataaacag 180
gacatcccta aggtctccgc ttaccagtat cgtgtcttca gagtccagtt gcctgaccct 240
aacaagttcg gtcttccaga caactccatc tacaaccctg agacccaacg tcttgtttgg 300
gcctgcgccg gagttgagat cggacgtggt caaccacttg gtgtcggtct ttccggacac 360
cctttctaca acaagttgga tgacactgaa tcttcccacg ctgccacttc taacgtctcc 420
gaagacgtcc gtgacaatgt ctccgttgac tacaagcaga cccagttgtg catcttgggt 480
tgtgctcctg ctatcggtga gcactgggct aagggaactg cctgtaagtc ccgtccattg 540
tcccagggag actgcccacc acttgagttg aagaacaccg tcttggagga cggagacatg 600
gtcgataccg gttacggagc catggacttt tccacccttc aggacactaa gtgcgaggtc 660
cctcttgaca tctgccagtc catttgcaag tacccagatt acttgcagat gtccgccgac 720
ccatacggtg actccatgtt cttttgcttg cgtcgtgagc agttgtttgc ccgtcatttc 780
tggaatcgtg ccggaaccat gggtgacact gtccctcagt ccttgtatat caagggaacc 840
ggtatgcgtg cttccccagg ttcttgcgtc tactctcctt ccccttccgg ttctatcgtt 900
acttccgatt cccaactttt caataagcca tattggttgc acaaggccca aggtcacaac 960
aacggtattt gctggcataa ccagttgttc gtcaccgtcg ttgacactac cagatccacc 1020
aacttgacca tctgtgcctc cactcagtcc cctgtccctg gtcagtacga cgccaccaag 1080
ttcaagcagt actcccgtca cgtcgaggaa tacgatttgc agttcatttt tcaactttgc 1140
accatcacct tgaccgccga cgttatgtct tacatccatt ctatgaattc ctccatcttg 1200
gaggactgga acttcggagt ccctccacca cctaccacct cccttgttga cacctacaga 1260
ttcgtccaat ccgtcgccat cacctgtcaa aaggacgccg cccctgccga aaacaaagat 1320
ccttatgaca agcttaagtt ctggaacgtt gacttgaagg agaagttttc tcttgacttg 1380
gaccaatacc ctcttggtag aaagttcctt gtccaggccg gattgcgtcg taagcctact 1440
atcggtcctc gtaagagatc cgccccttcc gccactacct cttctaagcc tgccaagcgt 1500
gtcagagtta gagccagaaa a 1521
<210> 10
<211> 1494
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of full-length HPV 58 L1 protein Gene Using Pichia pastoris preference codon
<400> 10
atgtccgttt ggagaccttc cgaggccact gtctacttgc caccagttcc agtctccaag 60
gttgtttcca ctgacgagta cgtctcccgt acctccatct actactacgc cggttcttcc 120
agattgttgg ccgtcggtaa cccttacttc tccatcaagt ccccaaacaa caacaagaaa 180
gtccttgttc ctaaagtttc cggtcttcag taccgtgtct tccgtgttcg tttgcctgac 240
ccaaacaagt tcggattccc tgacacttcc ttctacaacc ctgacaccca gagacttgtc 300
tgggcctgtg tcggacttga gattggtcgt ggacaacctc ttggtgttgg agtttccggt 360
cacccttact tcaacaagtt cgacgacacc gagacctcca accgttaccc agcccaacca 420
ggttccgaca accgtgagtg cttgtccatg gactacaaac aaacccaatt gtgcttgatc 480
ggttgtaaac caccaactgg tgagcactgg ggaaagggtg tcgcctgcaa caacaacgct 540
gctgccaccg attgtccacc attggagttg tttaactcta ttatcgagga cggagacatg 600
gttgacaccg gattcggttg catggacttc ggaaccttgc aagccaacaa gtccgatgtc 660
ccaatcgaca tctgcaactc cacctgcaag taccctgact acttgaagat ggcctccgag 720
ccatacggtg actctttgtt cttcttcctt cgtagagagc agatgttcgt ccgtcacttc 780
ttcaaccgtg ctggaaagtt gggtgaggcc gtcccagatg acttgtatat caagggttct 840
ggtaataccg ccgtcatcca gtcctctgcc ttcttcccaa ctccatccgg ttccatggtt 900
acctccgagt cccagctttt caacaagcct tattggttgc agcgtgccca gggtcataac 960
aacggaatct gctggggaaa ccagcttttc gtcaccgtcg ttgacaccac ccgttccact 1020
aatatgacct tgtgcaccga ggtcaccaaa gagggaacct acaaaaacga caacttcaaa 1080
gagtatgtcc gtcacgtcga agagtacgac ttgcagttcg tctttcagtt gtgtaaaatc 1140
accttgaccg ctgaaattat gacctatatt cacactatgg actccaacat tttggaagat 1200
tggcagttcg gacttacccc acctccatct gcttccttgc aggacactta ccgtttcgtc 1260
acctcccagg ccatcacttg tcagaagacc gctccaccaa aggagaaaga agaccctttg 1320
aacaaataca ccttttggga ggttaacttg aaggagaagt tttctgccga cttggaccag 1380
ttcccattgg gtagaaaatt cttgcttcag tctggtttga aggccaagcc tcgtttgaag 1440
agatccgccc caaccactcg tgccccatct actaagcgta agaaggttaa gaaa 1494
<210> 11
<211> 29
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 11
cgatggaact tcgaaacgat gtggagacc 29
<210> 12
<211> 32
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 12
gacctgggta ccctattatc tcttggtttt ag 32
<210> 13
<211> 29
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 13
cgatggaact tcgaaacgat gtggagacc 29
<210> 14
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 14
gacctgggta ccctattatt tcttgg 26
<210> 15
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 15
cgatggaact tcgaaacgat gatgtccttg 30
<210> 16
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 16
gacctgggta ccctattaaa gctttc 26
<210> 17
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 17
cgatggaact tcgaaacgat ggctttgtgg 30
<210> 18
<211> 27
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 18
gacctgggta ccctattatt ttctggc 27
<210> 19
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 19
cgatggaact tcgaaacgat gtccgtttgg 30
<210> 20
<211> 27
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 20
gacctgggta ccctattatt tcttaac 27
Claims (8)
1. An isolated gene encoding human papilloma major capsid protein L1, wherein said gene has a codon preferred by pichia pastoris, and wherein said gene has the nucleotide sequence shown in SEQ ID No. 6.
2. An expression vector comprising the sequence of the gene of claim 1.
3. A genetically engineered host cell comprising the expression vector of claim 2, or having integrated into its genome the gene of claim 1.
4. The host cell of claim 3, wherein the cell is a pichia cell.
5. The host cell of claim 4, wherein the pichia pastoris is selected from pichia pastoris strains X-33, GS115, KM71 and SMD 1168.
6. A method for preparing an immunogenic macromolecule having a diameter of 50-80nm, self-assembled from human papillomavirus major capsid protein L1, said method comprising:
(1) Culturing the host cell of claim 3, thereby expressing the human papillomavirus major capsid protein L1 in the host cell and assembling to form a macromolecule having immunogenicity;
(2) Isolating said immunogenic macromolecule.
7. The method of claim 6, wherein in step (2) comprises:
(a) Disrupting the host cells obtained in step (1) to obtain a supernatant containing the macromolecules having immunogenicity; and
(b) Purifying the supernatant obtained in the step (a) by adopting ion exchange column chromatography and hydroxyapatite column chromatography in sequence, thereby obtaining the macromolecule with immunogenicity.
8. A method for expressing HPV6L1 gene in pichia pastoris, comprising the steps of:
(1) Cloning HPV6L1 gene with optimized codon into expression vector, the sequence of the gene is the nucleotide sequence shown in SEQ ID NO. 6;
(2) Transforming the expression vector obtained in the step (1) into a pichia pastoris strain;
(3) Screening the transformed strains obtained in the step (2) by using antibiotics to obtain one or more strains with the best growth condition;
(4) Further screening the strain obtained in the step (3) by testing the expression level of HPV6L1 genes to obtain one or more strains with the highest expression level;
(5) And (3) performing expression by using the strain obtained in the step (4) to obtain HPV6L1 protein.
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| CN104845985A (en) | 2015-08-19 |
| CN117187262A (en) | 2023-12-08 |
| CN111154777A (en) | 2020-05-15 |
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