Preparation method and application of Coxsackie virus B3 virus-like particles

A technology of coxsackie virus and type 3 virus, applied in the direction of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as danger, incomplete inactivation, and disease of the inoculated

Inactive Publication Date: 2013-01-30
BEIJING UNIV OF TECH
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  • 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 Coxsackie virus B3 virus-like particles
  • Preparation method and application of Coxsackie virus B3 virus-like particles
  • Preparation method and application of Coxsackie virus B3 virus-like particles

Examples

Experimental program
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Embodiment 1

[0043] Example 1. Codon optimization and full gene synthesis of CVB3 P1 gene

[0044] The following is a detailed description of the codon optimization of CVB3 P1 gene and the process of full gene synthesis in steps.

[0045] 1. Codon optimization of CVB3 P1 gene

[0046] According to the wild-type CVB3 P1 gene sequence (named CVB3P1W gene, as shown in sequence 6 in the sequence table), after design and repeated verification, an optimized CVB3 P1 gene sequence suitable for expression in yeast cells (named CVB3P1Y gene, such as Sequence 1 is shown in the sequence list), that is, the P1 gene sequence of the CVB3 virus is transformed into a containing such as Sharp PM (Sharp PM, Cowe E. Synonymous Codon Usage in Saccharomyces cerevisiae. Yeast, 1991, 7 ( 7): 657-678.) The optimized sequence of yeast preferred codons described, thereby increasing the expression level of CVB3 VP1, VP2, VP3 and VP4 in the yeast cell culture environment.

[0047] According to the above method, the optimized...

Embodiment 2

[0051] Example 2. Construction of yeast recombinant expression vector carrying CVB3 VP1, VP2, VP3 and VP4 genes

[0052] 1. Preparation of E. coli DH5α competent cells

[0053] 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 5ml of LB medium without antibiotics, and cultivate overnight at 37°C with vigorous shaking (12-16h) . The next day, transfer 0.5ml from the above culture and transfer it to 50ml LB medium at a volume ratio of 1:100 for about 3 hours. When the OD600 value of the bacterial solution is 3, transfer the bacteria to a sterile medium under aseptic conditions. , Use ice pre-cooled 50ml centrifuge tube, ice bath for 30 minutes. Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, invert the tube for 1 min to drain the remaining culture solution, and use 10 ml of 100 mM CaCl pre-cooled with ice 2 Resuspend the bacterial pellet in the solution in an ice bath for 30 m...

Embodiment 3

[0076] Example 3. Mass expression and purification of recombinant CVB3 virus-like particles in yeast cells

[0077] 1. Preparation of Saccharomyces cerevisiae competent cells

[0078] Pick the monoclonal colony INVSc1 (Saccharomyces cerevisiae, product catalog number: C81000, purchased from Invitrogen) from the Saccharomyces cerevisiae plate, inoculate it in 10ml YPD culture medium (purchased from Beijing Xinjingke Company), shake culture at 30℃ for 16h, take Add a suitable volume of culture to 48ml YPD culture medium and mix to make the OD600 value 0.5. After shaking at 30℃ for 1 hour, the OD600 value is 0.7. After culturing for 30 minutes, transfer the culture to a 50ml sterile centrifuge tube Centrifuge at 1500 rpm for 5 min at room temperature, discard the supernatant, resuspend the pellet with 10ml Sc EasyComp transformation kit (purchased from Invitrogen) in solution I (included in the kit), centrifuge at 1500 rpm at room temperature for 5 minutes, carefully discard the super...

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Abstract

The invention discloses a preparation method of Coxsackie virus B3 virus-like particles. The method provided by the invention comprises the following steps: 1) cloning a VP1 gene and a VP4 gene of the Coxsackie virus B3 to a target plasmid 1 to obtain a recombinant expression vector 1; and cloning a VP2 gene and a VP3 gene to a target plasmid 2 to obtain a recombinant expression vector 2; 2) cotransforming the recombinant expression vector 1 and the recombinant expression vector 2 obtained in the step 1 ) to a yeast cell to obtain a recombinant yeast cell expressing the VP1 gene, the VP2 gene, the VP3 gene and the VP4 gene; and 3) cracking the recombinant yeast cell obtained in the step 2 ), and separating to obtain the Coxsackie virus B3 virus-like particles. Experiments show that Coxsackie virus B3 virus-like particles are successfully prepared by the method provided by the invention in the yeast expression system, and can be further used in candidate preventive vaccines and pharmaceutical compositions for virus myocarditis and diabetes type I.

Description

Technical field [0001] The present invention relates to a preparation method of Coxsackie virus B3 type virus-like particles, in particular to the preparation of Coxsackie virus B3 type P1 (VP1, VP2, VP3 and VP4) genes through a yeast expression system that has been codon optimized Methods and applications of virus B3 virus-like particles. Background technique [0002] Viral myocarditis (VMC) is a non-specific inflammatory lesion of the myocardium caused by a variety of viruses. The course of the disease is long and can be recovered within a few months, and a small number of patients will become chronic. Among the viruses that can cause viral myocarditis, enteroviruses and adenoviruses are the most common (Yajima T, Knowlton KU. Viral myocarditis: from the perspective of the virus. Circulation. 2009,119(19):2615-2624.) , And Coxsackievirus B3 (CVB3) in enterovirus is its main pathogen (Yuan J, Yu M, Lin QW et al. Th17 cells contribute to viral replication in coxsackievirus B3-in...

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 盛望张潇赵淼王晓雯曾毅李泽琳刘伟
Owner BEIJING UNIV OF TECH
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