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: 2015-03-11
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 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
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, Codon optimization and total gene synthesis of CVB3 P1 gene

[0044] The codon optimization of CVB3 P1 gene and the process of whole gene synthesis are described in detail below step by step.

[0045] 1. Codon optimization of CVB3 P1 gene

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

Embodiment 2

[0051] Embodiment 2, the construction of the yeast recombinant expression vector carrying CVB3 VP1, VP2, VP3 and VP4 genes

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

[0053] 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...

Embodiment 3

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

[0077] 1. Preparation of Competent Cells of Saccharomyces cerevisiae

[0078] Pick a monoclonal colony INVSc1 (Saccharomyces cerevisiae, catalog number: C81000, purchased from Invitrogen) from a S. Add a suitable volume of culture into 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 room temperature at 1500rpm for 5min, carefully discard the supernatant, and use 1ml of solution II ( The kit comes with) to resuspend the pellet, put 50 μl / tube into a sterilized 1...

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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 method for preparing coxsackie virus type B3 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 by using codon-optimized coxsackie virus type B3 P1 genes Methods and applications of virus-type B3 virus-like particles. 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.) , and Coxsackievirus B3 (CVB3) in enteroviruses is its main pathogen (Yuan J, Yu M, Lin QW et al.Th17 cells contribute to viral replication in coxsackievirus B3-induced acute viral myocarditis.J Immunol. 2010, 185(7): 4004-4010.). ...

Claims

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Application Information

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Patent Type & Authority Patents(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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