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Vaccine against human papillomavirus (HPV) as well as preparation method and application thereof

A technology of human papilloma and virus, applied in the fields of genetic engineering and vaccinology, can solve problems such as high production cost, excessive glycosylation, and the inability of Saccharomyces cerevisiae to use high-density fermentation

Active Publication Date: 2013-08-21
SHANGHAI WELLVAC BIOTECHNOLOGIES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in Saccharomyces cerevisiae, the exogenous HPVL1 gene exists in the plasmid, not on the chromosome, so it is easy to lose the plasmid during culture or fermentation production
Saccharomyces cerevisiae is prone to hyperglycosylation, which leads to the production of VLPs that produce adverse allergic reactions in some individuals
Saccharomyces cerevisiae cannot use high-density fermentation, and the culture medium and other reasons lead to high fermentation costs, all of which lead to high production costs

Method used

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  • Vaccine against human papillomavirus (HPV) as well as preparation method and application thereof
  • Vaccine against human papillomavirus (HPV) as well as preparation method and application thereof
  • Vaccine against human papillomavirus (HPV) as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0084] Preparation and fermentation production of engineering bacteria

[0085] The inventive point of the present invention mainly lies in the transformed HPV L1 gene. As for the technologies such as inserting the transformed HPV L1 gene into a plasmid, transforming Pichia pastoris, engineering bacterium, fermentation of engineered bacterium, and separation of products, the conventional methods known in the art are basically carried out (for example, people such as Sambrook, Molecular Cloning: Conditions described in a laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989)).

[0086] Usually, specific restriction sites can be introduced at the 5' end and 3' end of the optimized gene, and the optimized gene can be cloned into an expression vector (such as pPIC9, pPIC9K, etc.) by conventional methods of molecular cloning. Then, transform and integrate into the P. pastoris host cell chromosome. High-copy transformants were selected by conventional methods (suc...

Embodiment 1

[0145] Example 1. Construction of HPV52L1 Pichia pastoris expression system

[0146] 1. Obtaining the target gene

[0147] Optimized sequence: According to the amino acid sequence of natural HPV52L1, optimize the design according to the following factors: Pichia pastoris codon preference, A+T composition in the gene, G+C content, mRNA secondary structure and translation initiation codon AUG flanking sequences, etc.

[0148] The nucleotide sequence of an optimized target gene is shown in SEQ ID NO:1. The amino acid sequence of the gene encoding HPV52L1 protein is shown in SEQ ID NO:2. The optimized gene (SEQ ID NO: 1) was artificially synthesized, and NotI and EcoRI restriction sites were introduced into its 5' end and 3' end respectively.

[0149] Control sequence 1: wild-type HPV52L1 gene (SEQ ID NO: 3), encoding the same HPV52L1 protein.

[0150] Control sequences 2 and 3: nucleotide sequences (SEQ ID NO: 4 and 21) generated by optimizing only according to the codon bias...

Embodiment 2

[0164] Example 2. Construction of HPV58L1 Pichia pastoris expression system

[0165] Example 1 was repeated with the difference that the coding sequence of HPV52 L1 was replaced with the following coding sequence of HPV58 L1:

[0166] The nucleotide sequence of the optimized HPV58 L1 target gene is shown in SEQ ID NO:5. The amino acid sequence of the gene encoding HPV58L1 protein is shown in SEQ ID NO:6. The optimized gene (SEQID NO: 5) was artificially synthesized, and NotI and EcoRI restriction sites were introduced at its 5' end and 3' end, respectively.

[0167] Control sequence 4: wild-type HPV58L1 gene (SEQ ID NO: 7), encoding the same HPV58L1 protein.

[0168] Control sequences 5 and 6: nucleotide sequences (SEQ ID NO: 8 and 22) generated by optimizing only according to the codon bias principle of Pichia pastoris, encoding the same HPV58L1 protein. The control sequence is fully synthesized, and enzyme cutting sites are introduced at both ends.

[0169] As a result, ...

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Abstract

The invention relates to a vaccine against human papillomavirus (HPV) as well as a preparation method and an application thereof. Specifically, the invention provides the encoding sequence of an optimized human papillomavirus capsid protein L1 (HPV L1) suitable for expression in Pichia pastoris, and a method for efficiently producing HPV L1. The optimized HPV L1 gene is very suitable for expression in Pichia pastoris, and has the characteristic of high expression level and high stability. According to the invention, HPV L1 proteins and self-assembled virus-like particles can be obtained in an efficient, simple and cheap way. The invention also provides a vaccine composition comprising HPV virus-like particles.

Description

technical field [0001] The invention relates to the fields of genetic engineering and vaccinology. Specifically, the present invention relates to a vaccine against human papillomavirus and its preparation method and use. More specifically, the present invention relates to the optimized coding sequence of the major capsid protein L1 of human papillomavirus suitable for Pichia pastoris expression, and a method for efficiently producing the major capsid protein. The present invention also relates to HPV virus-like particles (HPV VLP) formed by self-assembly of main capsid protein and vaccine composition containing HPV VLP. Background technique [0002] Cervical cancer is the second most common cancer among women, especially in developing countries. Since the discovery of the correlation between HPV virus infection and cervical cancer in 1983-1984, at least 118 human papillomaviruses have been isolated, and it has been confirmed that HPV infection-induced cancers account for t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/37C12N15/63C12N1/19C12N7/04C12P21/02A61K39/12A61P35/00A61P31/20C12R1/84
Inventor 楼觉人姜自军蔡蓓蓓仝光杰徐绎函
Owner SHANGHAI WELLVAC BIOTECHNOLOGIES CO LTD
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