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Method for preparing vaccine for anti-HPV 18 infection by pichia yeast expression system

A technology of human papillomavirus and Pichia pastoris, applied in antiviral agents, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of undisclosed nucleotide sequence recombinant L1 protein activity, etc.

Active Publication Date: 2009-07-22
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] Patent CN1869215A discloses a method for preparing human papillomavirus virus-like particles using the Pichia pastoris expression system. The gene encoding the late protein of human papillomavirus obtained by PCR amplification of cervical cancer pathological tissue specimens is cloned into the Pichia pastoris In the yeast secretion expression vector, the recombinant Pichia cells were constructed; the recombinant Pichia cells were used for fermentation culture and methanol-induced secretion of L1 protein. After the culture supernatant was purified, the obtained L1 protein could self-assemble into virus-like particles, but the The literature does not disclose the nucleotide sequence of the HPV L1 gene synthesized according to the codon preference of Pichia pastoris and the activity of the recombinant L1 protein

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  • Method for preparing vaccine for anti-HPV 18 infection by pichia yeast expression system
  • Method for preparing vaccine for anti-HPV 18 infection by pichia yeast expression system
  • Method for preparing vaccine for anti-HPV 18 infection by pichia yeast expression system

Examples

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Embodiment 1

[0040] Example 1 Synthesis of full-length HPV 18 L1 gene

[0041] The nucleotide sequence of the full-length HPV 18 L1 gene was designed according to the codon bias used by Pichia pastoris. See SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in the sequence listing.

[0042] The above-mentioned full-length genes were commercially synthesized by Jierui Company, and the HPV 18 L1 gene was sequenced using a DNA sequencer #3730.

Embodiment 2

[0043] Example 2 Construction of full-length or truncated HPV 18 L1 Pichia pastoris expression vector

[0044] The two ends of the full-length HPV 18 L1 gene with the sequence obtained in Example 1 as SEQ ID NO.1 were loaded into the pUC18 plasmid (Jie Rui Company) with EcoRI and KpnI restriction sites, named pUC-18 L1, and sent to To Shanghai Sangon Bioengineering Co., Ltd., DNA sequencer 3730 was used for DNA sequencing.

[0045] Entrusted Shanghai Sangon Bioengineering Co., Ltd. to synthesize three primers required to amplify full-length or truncated HPV 18 L1. The two forward primers include a BstBI restriction endonuclease site and have the following nucleotide sequences:

[0046]P1, 5'-TCCCAATCTTCGAAACGATGTGTTTGTACACTAGAGTTT-3' (full length, as shown in SEQ ID NO.7);

[0047] P2, 5'-TCCCAATCTTCGAAACGATGGCTTTGTGGA-3' (truncated, as shown in SEQ ID NO.8).

[0048] The reverse primer includes KpnI restriction endonuclease sites flanking the stop codon and has the nucleot...

Embodiment 3

[0051] Example 3 Construction and Screening of Full-length or Truncated HPV 18 L1 Pichia Expression Strains

[0052] In order to improve the integration efficiency of the recombinant plasmid on the yeast chromosome, single-digest pPICZA-18 L1 plasmid and pPICZA-18 L1' plasmid with SacI restriction endonuclease to linearize it, and perform the same digestion on the empty plasmid pPICZA as a negative control. Add absolute ethanol to the enzyme digestion reaction solution to precipitate DNA, dissolve the linearized DNA fragment with a small amount of double distilled water, and transform Pichia pastoris X-33 (purchased from Invitrogen ). The electroporation conditions were: 5 micrograms of DNA fragments, a voltage of 1500 volts, a resistance of 25 ohms, and an electric shock time of 5 milliseconds. Electroporation products were plated on YPDS agar containing 200 μg / ml zeocycin, isolated single colonies of transformed cells, and re-inoculated on 1000 μg / ml and 1500 μg / ml zeocyci...

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Abstract

The invention discloses a method for preparing a recombinant human papillomavirus 18-typed L1 protein with a pichia pastoris expression system and comprises the steps of loading an HPV 18 L1 gene in accordance with optimal design into an expression carrier, transforming pichia pastoris and cultivating a transformant, thus obtaining the recombinant human papillomavirus 18-typed L1 protein, which is self-assembled into virus-like particles inside a pichia pastoris body; wherein, nucleotide sequences of the HPV 18 L1 gene in accordance with optimal design are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6. The HPV 18 L1protein that is prepared by the method of the invention has high expressed quantity. The invention further discloses a method for preparing a papillomavirus 18-typed infection vaccine with better activity by utilizing the virus-like particles of the recombinant HPV 18 L1 protein.

Description

technical field [0001] The invention relates to a method for preparing recombinant human papillomavirus type 18 L1 protein using a Pichia pastoris expression system and using the L1 protein to prepare a vaccine against human papillomavirus type 18 infection. Background technique [0002] Human papillomavirus (humanpapillomavirus, HPV) is a non-enveloped small double-stranded circular DNA virus belonging to the subfamily Polyomavirinae of Papovaviridae. HPV can be transmitted through close contact between humans, causing common warts and anogenital genital warts and other lesions on the skin of the infected person, and is listed as a sexually transmitted disease. In 1995, the International Cancer Research Center announced the results of a study confirming that HPV has a close causal relationship with cervical cancer. It can be seen that HPV infection has become a pathogen that seriously endangers human health. Therefore, the development of efficient and cheap HPV vaccine is...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/81C07K14/025A61K39/12A61P35/00A61P31/20
Inventor 张高峡雷建强袁靖宇张梦华熊莹华沈琼吴克
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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