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Expression of recombinant human papilloma virus subtype 6 and 11 protein in pichia pastoris

A human papilloma and expression vector technology, applied in antiviral agents, viral antigen components, recombinant DNA technology, etc., can solve the problems of low HPV protein expression efficiency, unsatisfactory particle immune effect, low protein activity, etc. The effect of large-scale industrial production, low cost and high expression

Active Publication Date: 2020-01-17
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although some vaccines against HPV have been developed in the prior art, there are generally problems such as low expression efficiency of HPV protein, low activity of the expressed protein, inability to assemble into virus-like particles or unsatisfactory immune effect of the assembled particles.

Method used

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  • Expression of recombinant human papilloma virus subtype 6 and 11 protein in pichia pastoris
  • Expression of recombinant human papilloma virus subtype 6 and 11 protein in pichia pastoris
  • Expression of recombinant human papilloma virus subtype 6 and 11 protein in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 HPV6 and 11 L1 codon optimization design

[0055] There are 64 genetic codes, but most organisms tend to use some of these codons. Pichia pastoris and human genes have their own preferences for codon usage. Since the genetic code is degenerate, each amino acid is encoded by more than one codon, and the codons of the same amino acid are used differently in wild-type genes. The codon preference of Pichia pastoris may lead to low translation efficiency and expression level of recombinant protein. The present inventors are concerned about the wild-type HPV 6 L1 gene (Genebank FR751337.1) and the wild-type HPV 11 L1 gene (Genebank HE611271.1). Carry out transformation: All its amino acids adopt the most frequently used codons. The frequency of Pichia yeast codon usage is shown in Table 1 (see http: / / www.kazusa.or.jp / codon / ). Then, on this basis, and in order to avoid the excessively high GC ratio of the translated mRNA, the secondary structure of the mRNA affects t...

Embodiment 2

[0062] Example 2 Construction of HPV6 and 11 L1 recombinant expression vectors

[0063] The synthesized HPV6 L1 sequence of SEQ ID NO: 2 was cloned into the pPICZalphaB vector (Invitrogen) by the following method.

[0064] 6 L1 DNA fragments with BstBI and KpnI on both ends were amplified by PCR. PCR program: 94°C for 5 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and 50 seconds, cycle 30 times, 72°C for 10 minutes, 10°C for 10 minutes, and the operation ends. The PCR products were identified by agarose gel electrophoresis and a 1500bp band was recovered (Qiagen gel extraction kit). The recovered fragments and pPICZalphaB were digested with BstBI and KpnI (New England Biolab), identified by agarose gel electrophoresis, and 1500bp and 3600bp fragments were recovered respectively. After recovery, 6L1 and pPICZalphaB were ligated with T4 ligase (Takara) overnight at 16°C, and the ligation product was transformed into E.coli DH5α the next day, spread on an LB ...

Embodiment 3

[0067] Example 3 Construction and expression of HPV6 and 11 L1 recombinant expression strains

[0068] Linearize pPICZ6L1 with SacI. After the enzyme digestion reaction, remove the protein from phenol: chloroform, then add 2.5 times volume of absolute ethanol, 1 / 10 volume of 3M NaAc (pH 5.2) to precipitate DNA, and the resulting precipitate is washed with 75% ethanol and dried After a small amount of sterile ddH 2 O dissolves the precipitate, electrotransforms the Pichia X-33 strain (Invitrogen), spreads it on a YPDS plate (containing 200μg / ml Zeocin), cultivates at 30°C for 3 days to obtain hundreds of clones. Pick dozens of clones and inoculate them on YPD plates (containing 1500μg / ml Zeocin), select high-copy plasmid strains, and culture them at 30°C for 2 days. Some clones grow faster. Pick the best clones and inoculate them in 4ml YPD liquid medium. Replace the BMMY medium 24 hours later, and collect the bacteria after 48 hours of induction with 0.5% methanol. After the bac...

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Abstract

The invention discloses a codon-optimized human papilloma virus main capsid protein L1 coding gene, which can efficiently express human papilloma virus main capsid protein L1 after being transferred into yeast cells. The invention further discloses a macromolecule with immunogenicity, which is mainly generated by the expression of the codon-optimized human papilloma virus main capsid protein L1 coding gene in yeast cells. The invention further discloses an application and a composition of the macromolecule with immunogenicity.

Description

[0001] This application is a divisional application for an invention patent whose application date is December 26, 2013, the application number is 201310731077.1, and the invention title is "Expression of recombinant human papillomavirus 6 and 11 subtype proteins in Pichia pastoris". Technical field [0002] The present invention belongs to the field of bioengineering. Specifically, the present invention relates to the main capsid protein of human papilloma virus, its coding gene, and its preparation method and application. Background technique [0003] Human papilloma virus (HPV) is a small, non-enveloped double-stranded circular DNA virus in the subfamily Polyomavirus of the family Papillomavirus. HPV can be spread through close contact between humans, causing common warts and anogenital condyloma acuminata on the skin of infected persons, which is classified as a sexually transmitted disease. In 1995, the research results published by the International Cancer Research Center co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/37C07K14/025C12N15/81C12N1/19A61K39/12A61P31/20A61P35/00C12R1/84
Inventor 丛薇张梦华魏健田平生
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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