Preparation method for recombinant coxsackie virus A16 like particle and applications thereof

A Coxsackie virus, A16 technology, applied in the field of preparation of recombinant Coxsackie virus A16 virus-like particles, can solve the problem of specific drugs or vaccines on the market, and achieve the effect of increasing production

Inactive Publication Date: 2012-07-04
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is no specific drug or vaccine against CoxA16 infection on the market. Therefore, the development of CoxA16 preventive vaccine is of great significance for controlling the epidemic of hand, foot and mouth disease in infants and young children.

Method used

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  • Preparation method for recombinant coxsackie virus A16 like particle and applications thereof
  • Preparation method for recombinant coxsackie virus A16 like particle and applications thereof
  • Preparation method for recombinant coxsackie virus A16 like particle and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, Codon optimization and total gene synthesis of Cox A16 P1 gene and Cox A16 3CD gene

[0036] The codon optimization and whole gene synthesis process of Cox A16 P1 gene and Cox A16 3CD gene are described in detail below step by step.

[0037] 1. Codon optimization of Cox A16 P1 gene and Cox A16 3CD gene

[0038] According to the wild-type Cox A16 P1 gene sequence (as shown in sequence 3 in the sequence listing) and the wild-type Cox A16 3CD gene sequence (as shown in sequence 4 in the sequence listing), after design and repeated verification, it is suitable for expression in yeast cells The optimized Cox A16 P1 gene sequence and the optimized Cox A16 3CD gene sequence, that is, the P1 gene sequence and the 3CD gene sequence of the Cox A16 virus are transformed into those containing such as Sharp PM etc. (Sharp PM, Cowe E .Synonymous Codon Usage in Saccharomyces cerevisiae.Yeast, 1991, 7 (7): 657-678.) The optimized sequence of the preferred codon of yeast des...

Embodiment 2

[0042] Example 2, Construction of Yeast Recombinant Expression Vector Carrying Cox A16 P1Y and Cox A16 3CD Y

[0043] 1. Preparation of Escherichia coli DH5α competent cells

[0044] Pick a single colony of DH5α (Takara Company, catalog number D9057S) from a fresh plate cultured at 37°C for 16-20 hours, inoculate it in 5 ml of LB medium without antibiotics, and culture it with vigorous shaking at 37°C overnight (12-16 hours). . On the next day, draw 0.5ml from the above culture and transfer it to 50ml LB medium at a volume ratio of 1:100 to continue culturing for about 3 hours until the OD600 value of the bacterial solution is 3, then transfer the bacteria to a sterile medium under aseptic conditions. , Place in a 50ml centrifuge tube pre-cooled with ice, and bathe in ice for 30 minutes. Centrifuge at 4000rpm for 10min at 4°C, discard the supernatant, invert the tube for 1min to drain the residual culture solution, resuspend the bacterial pellet in 10ml of ice-cooled 100mM C...

Embodiment 3

[0056] Example 3, Mass expression and purification of recombinant CoxA16 virus-like particles in yeast cells

[0057] 1. Preparation of Competent Cells of Saccharomyces cerevisiae

[0058] Pick single-clonal colony INVSc 1 (Saccharomyces cerevisiae, catalog number: C81000, purchased from Invitrogen) from the Saccharomyces cerevisiae plate and inoculate it in 10ml of YPD culture medium (purchased from Beijing Xinjingke Company), and culture it with shaking at 30°C for 16h. Take an appropriate volume of culture and add it to 48ml YPD culture medium and mix well to make its OD600 value 0.5. After shaking and culturing at 30°C for 1 hour, the OD600 value is 0.7. After continuing to cultivate for 30 minutes, transfer the culture to a 50ml sterile centrifuge tube centrifuge at 1500rpm at room temperature for 5min, discard the supernatant, resuspend the pellet with 10ml of solution I (included with the kit) in Sc EasyComp transformation kit (purchased from Invitrogen), centrifuge at ...

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Abstract

The invention discloses a preparation method for a recombinant coxsackie virus A16 like particle, which comprises the following steps: (1) cloning a P1 gene and a 3CD gene of a coxsackie virus A16 to a target plasmid to obtain a recombinant expression vector; (2) transforming a target yeast cell by using the recombinant expression vector obtained in the step (1) to obtain a recombinant yeast cellfor expressing the P1 gene and the 3CD gene; and (3) cracking the recombinant yeast cell obtained in the step (2), and separating to obtain the recombinant coxsackie virus A16 like particle. The recombinant coxsackie virus A16 like particle can be prepared in a yeast expression system by the method provided by the invention. Compared with a wild-type P1 gene and a wild-type 3CD gene, the yield ofthe coxsackie virus A16 like particle in the yeast expression system is greatly increased through the optimization of codons of the P1 gene and the 3CD gene, and the recombinant coxsackie virus A16 like particle can be further used for producing candidate preventive vaccines and pharmaceutical compositions for infant hand-foot-and-mouth diseases.

Description

technical field [0001] The present invention relates to a method for preparing recombinant Coxsackievirus A16 virus-like particles, in particular to the preparation of recombinant Coxsackievirus A16 virus through a yeast expression system by using codon-optimized Coxsackievirus A16 P1 gene and 3CD gene particle-like method. Background technique [0002] Hand foot mouth disease (Hand foot mouth disease, HFMD) is a common infectious disease in infants and young children. Groups 2 and 5; Echo virus (ECHO viruses) and enterovirus 71 (EV 71) infection. Among them, EV 71 and Cox A16 are the most common (Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 1974; 129: 304-309.). CoxA16 infection can lead to complications such as myocarditis, cardiomyopathy, and aseptic meningitis. In addition, CoxA16 is also the causative agent of many human diseases, which can cause respiratory tract infectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C12N15/81C12N7/04C12N15/63C12N5/10C12N1/19A61K39/125A61P31/14
Inventor 盛望陈琳王晓雯曾毅李泽琳
Owner BEIJING UNIV OF TECH
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