Preparation method and application of recombination coxsackie virus B3-type virus-like particles

A technology of coxsackie virus and type 3 virus, which is applied in the field of preparation of recombinant coxsackie virus type B3 virus-like particles, which can solve the problems of incomplete inactivation, illness and danger of vaccinators

Inactive Publication Date: 2012-12-19
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are dangerous and may not even stimulate the body to produce the desired immune response
For example, attenuated or inactivated virus vaccines risk reversion to a virulent form or are not fully inactivated, resulting in disease in recipients
At present, there is no specific drug or vaccine to prevent CVB3 infection on the market. Therefore, the development of a CVB3 preventive vaccine is of great significance for controlling the prevalence of viral myocarditis

Method used

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  • Preparation method and application of recombination coxsackie virus B3-type virus-like particles
  • Preparation method and application of recombination coxsackie virus B3-type virus-like particles
  • Preparation method and application of recombination coxsackie virus B3-type virus-like particles

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Embodiment 1

[0036] Codon optimization and total gene synthesis of embodiment 1, CVB3P1 gene and CVB33CD gene (company synthesis)

[0037] The procedure of codon optimization and whole gene synthesis of CVB3P1 gene and CVB33CD gene is described in detail below step by step.

[0038] 1. Codon optimization of CVB3P1 gene and CVB33CD gene

[0039] According to the wild-type CVB3P1 gene sequence (as shown in sequence 3 in the sequence listing) and the wild-type CVB33CD gene sequence (as shown in sequence 4 in the sequence listing), the optimized CVB3P1 suitable for expression in yeast cells was obtained after design and repeated verification Gene sequence and optimized CVB33CD gene sequence, that is, the P1 gene sequence and 3CD gene sequence of the CVB3 virus will be transformed into one containing such as Sharp PM, Cowe E.Synonymous Codon Usage in Saccharomyces cerevisiae.Yeast without changing the amino acid sequence. , 1991,7(7):657-678.) The optimal sequence of yeast preferred codons des...

Embodiment 2

[0044] Embodiment 2, the construction of the yeast recombinant expression vector carrying CVB3P1Y and CVB33CD Y

[0045] 1. Preparation of Escherichia coli DH5α competent cells

[0046] Pick a single DH5α colony (Takara company, catalog number D9057S) from a fresh plate cultured at 37°C for 16-20h, inoculate it in 5ml of LB medium without antibiotics, and culture it at 37°C with vigorous shaking overnight (12-16h). . The next day, draw 0.5ml from the above-mentioned 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, and 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, and use 10ml of ice-cooled 100mM CaCl 2 The bacterial pellet was...

Embodiment 3

[0065] Example 3, Mass expression and purification of recombinant CVB3 virus-like particles in yeast cells

[0066] 1. Preparation of Competent Cells of Saccharomyces cerevisiae

[0067] Pick a single clone colony INVSc 1 (Saccharomyces cerevisiae, catalog number: C81000, purchased from Invitrogen) from the Saccharomyces cerevisiae plate and inoculate it in 10ml of YPD medium (purchased from Beijing Xinjingke Co., Ltd.), shake and culture 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 room temperature at 1500rpm 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 room temperature ...

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Abstract

The invention discloses a preparation method of recombination coxsackie virus B3-type virus-like particles. The method disclosed by the invention comprises the following steps that 1, the P1 gene and 3CD gene of the coxsackie virus B3 type are cloned to a target plasmid to obtain a recombination expression vector; 2, the target yeast cell is transformed by the recombination expression vector which is obtained in the step 1 to obtain a recombination yeast cell which expresses the P1 gene and the 3CD gene; and 3, the recombination yeast cell which is obtained in the step 2 is cracked, and the recombination coxsackie virus B3-type virus-like particles are obtained after the separation. Experiments prove that after the method disclosed by the invention is utilized, the recombination coxsackie virus B3-type virus-like particles are successfully produced in the yeast expression system, and can be further used for candidate prophylactic vaccine and medicine combination for vital myocarditis and diabetes mellitus type I.

Description

technical field [0001] The invention relates to a method for preparing recombinant Coxsackievirus B3 virus-like particles, in particular to the preparation of recombinant Coxsackievirus B3 virus through a yeast expression system by utilizing codon-optimized Coxsackievirus B3 P1 gene and 3CD gene particle-like method. Background technique [0002] Viral myocarditis (Vital myocarditis, VMC) is a non-specific inflammatory lesion of the myocardium caused by a variety of viruses. Among the viruses that can cause viral myocarditis, the most common are enteroviruses and adenoviruses (Yajima T, Knowlton KU. Viral myocarditis: from the perspective of the virus. Circulation. 2009, 119(19): 2615-2624.), Coxsackievirus B3 (CVB3) among enteroviruses is the main pathogen (Yuan J, Yu M, Lin QW et al. Th17cells contribute to viral replication in coxsackievirus B3-induced acute viral myocarditis. J Immunol. 2010,185(7):4004-4010.). In myocarditis and its late stage, dilated cardiomyopathy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C12N15/81C12N7/04C12N5/10C12N1/15C12N1/19C12N1/21A61K39/125A61P31/14
Inventor 盛望张潇王晓雯赵淼曾毅李泽琳刘伟
Owner BEIJING UNIV OF TECH
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