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Preparation method of EV71 vaccine and vaccine prepared by preparation method

A technology for EV71 and vaccines, applied in the field of preparation of virus-like particle vaccines, which can solve the problems of long production cycle, high cost and low expression level

Active Publication Date: 2012-07-11
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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Problems solved by technology

However, the main disadvantage of the insect cell expression system is transient expression. Virus infection will eventually lead to the death of host expression cells, so the infection must be restarted, and each round of protein production must reinfect newly cultured cells, which is expensive and has a long production cycle. Moreover, the expression level of EV71 virus-like particles reported in the literature using insect cells is about 10 mg / 10 9 Cells, with low expression levels, have certain problems in industrial production

Method used

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  • Preparation method of EV71 vaccine and vaccine prepared by preparation method
  • Preparation method of EV71 vaccine and vaccine prepared by preparation method
  • Preparation method of EV71 vaccine and vaccine prepared by preparation method

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Embodiment

[0031] 1. Gene selection and codon optimization design

[0032] Referring to the C4 type Chinese strain BJ08-Z020-1 (GenBank: FJ606449.1), DNA sequences encoding EV71P1 and 3CD were synthesized by techniques known in the art. The DNA sequences of BJ08-Z020-1 wild-type P1 and 3CD are SEQ ID NO: 1 and SEQ 1D NO: 2, and the amino acid sequences encoded by them are SEQ ID NO: 3 and SEQ ID NO: 4. The DNA sequences of wild-type P1 and 3CD were modified, and codons with high frequency in Pichia pastoris were used as codons to obtain optimized sequences SEQ ID NO: 5 and SEQ ID NO: 6.

[0033] 2. Construction of P13CD-pPEXZ recombinant expression vector

[0034] Firstly, the P1 and 3CD genes are obtained by means of gene synthesis known in the art according to the above synthesis method, and then the P1-pPICZaB and 3CD-pPICZaB recombinant vectors are constructed. Then, the AOX1 promoter of 3CD-pPICZaB was replaced with the pYPT1 promoter to obtain 3CD-pYPT1. The specific implementat...

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Abstract

The invention discloses a preparation method of an EV71 vaccine and the vaccine prepared by the preparation method, relating to the preparation method of taking a Pichia yeast as an expression system for co-expression of P1 and 3CD proteins of the EV71 after codon optimization by utilizing different promoters, and obtaining the viral like particle vaccine with immunogenicity, and also relating to the vaccine prepared by the preparation method.

Description

technical field [0001] The invention relates to a method for preparing a virus-like particle vaccine with immunogenicity by using Pichia pastoris as an expression system, co-expressing codon-optimized P1 and 3CD proteins of EV71 using different promoters. It also relates to vaccines prepared by this method. Background technique [0002] Enterovirus 71 (EV71) belongs to the genus Enterovirus in the Picornaviridae family, and mainly causes central nervous system diseases such as hand, foot and mouth disease, aseptic meningitis, brainstem encephalitis, and polio-like paralysis, causing disability and death It is a neurotropic enterovirus second only to poliovirus in terms of damage. EV71 takes humans as the only host, and the main route of transmission is fecal-oral transmission. The population is generally susceptible to EV71, but its main targets are infants and young children under 5 years old. Since it was first reported in 1974, EV71 has caused more than a dozen large-s...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/41C12N15/57C12N15/81C12N1/19A61K39/125A61P31/14C12R1/84
Inventor 沈琼傅文彬雷建强张高峡
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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