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A kind of coxsackie virus b5 type virus-like particle, its preparation method and application

A coxsackie virus, virus-like technology, applied in the field of genetic engineering and biomedicine, can solve the problems of long production cycle, high production conditions, virus leakage and spread, etc., and achieve the goal of improving protection efficiency, strong immunity and long duration Effect

Active Publication Date: 2021-10-22
GUILIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

EV71 monovalent inactivated vaccine cannot produce cross-protection against other enteroviruses
The preparation technology of enterovirus inactivated vaccine has its advantages, but it also has great defects: 1. The production cycle is long: it takes 5-8 months from the acquisition of new subtype virus strains to the production and marketing of vaccines, and it is difficult to contain new vaccines. outbreak of the epidemic
2. High production conditions: In order to prevent man-made pollution and virus leakage and spread, the whole set of production must be carried out under strict control conditions according to regulations, and the virus inactivation must be completely and thoroughly
However, no enterovirus-associated virus-like particle vaccine is currently on the market

Method used

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  • A kind of coxsackie virus b5 type virus-like particle, its preparation method and application
  • A kind of coxsackie virus b5 type virus-like particle, its preparation method and application
  • A kind of coxsackie virus b5 type virus-like particle, its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The preparation method of Coxsackievirus B5 type virus-like particles of the present embodiment comprises the following steps:

[0075] Step 1: Preparation of pOET5-CB5-P1-3CD recombinant plasmid

[0076] The codon-optimized Coxsackievirus B5 capsid protein P1 gene and 3CD protease gene were synthesized, and then respectively connected to the EcoRI / NotI restriction site and the P10 promoter of the insect baculovirus shuttle vector pOET5. The BamHI / Hind III restriction site behind the Ph promoter to obtain the pOET5-CB5-P1-3CD recombinant plasmid, such as figure 1shown. The main elements of the plasmid are as follows: p10 promoter is a P10 promoter, polyhedrin promoter is a Ph promoter, AmpR is an ampicillin resistance gene, and AmpR promoter is an ampicillin resistance gene promoter.

[0077] Wherein, the nucleotide sequence of the codon-optimized capsid protein P1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the codon-optimized 3CD protease gene is shown ...

Embodiment 2

[0094] This embodiment provides a coxsackievirus type B5 virus-like particle vaccine, the coxsackievirus type B5 virus-like particle vaccine is composed of 50 μg / mL coxsackievirus type B5 virus-like particle obtained in Example 1 and Freud Equal volumes of adjuvant are mixed for liquid injection.

experiment example 1

[0095] Experimental Example 1: Animal Immunization and Serum Antibody Determination and Virus Neutralization Reaction

[0096] It was carried out according to the currently used animal immunization experiment method of enterovirus. The experimental animals were 6-week-old BALB / c female mice without specific pathogens (Specific pathogen Free, SPF). The specific operation is as follows: in the 0th week, the 2nd week and the 4th week, each BALB / c mouse was subcutaneously injected with 0.3ml of coxsackievirus B5 virus-like particles (10 μg total) emulsified in Freund's adjuvant. protein), with the same dose of inactivated Coxsackievirus B5 virus as the positive control, and the PBS group as the negative control, the mice were treated at the 0th week, the 2nd week, the 4th week, the 6th week and the 12th week Blood was collected by tail docking, and serum was taken for ELISA experiment and microneutralization experiment.

[0097] The steps of the ELISA experiment are as follows: ...

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Abstract

The invention discloses a Coxsackie virus B5 type virus-like particle, its preparation method and application, and belongs to the technical fields of genetic engineering and biomedicine. The coxsackie virus type B5 virus-like particle is a recombinant baculovirus that expresses the P1 capsid protein gene and the 3CD protease gene of the coxsackie virus type B5 to infect Sf9 insect cells, and cultures to more than 90% of the Sf9 insect cells were obtained after infection with lesion. The invention also discloses the preparation method and application of the Coxsackievirus B5 type virus-like particle. The coxsackievirus type B5 virus-like particle of the present invention has a similar appearance structure and size to wild virus particles, and its immunogenicity reaches or even exceeds the level of wild inactivated virus, and can be used to prepare coxsackievirus type B5 Virus-like particle vaccines.

Description

technical field [0001] The invention relates to a coxsackievirus type B5 virus-like particle, a preparation method and application thereof, and belongs to the technical fields of genetic engineering and biomedicine. Background technique [0002] Coxsackiecirus B5 (CB5 for short) belongs to the Enterovirus genus of the Picornaviridae family and is an important member of the Enterovirus B group. CB5 is an icosahedral spherical particle with a diameter of 24-30nm, composed of an unfolded capsid and a single-stranded ribonucleic acid of about 7400 nucleotides, and contains an open reading frame with 5' on both sides Untranslated region and 3' untranslated region with poly A tail. It encodes a polyprotein of approximately 2100 amino acids, which is further cleaved to form three precursor proteins, called P1, P2 and P3. P1 is cleaved into four structural viral proteins, namely VP1, VP2, VP3 and VP4. The cleavage of P2 and P3 forms 7 non-structural proteins, namely 2A, 2B and 2C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/085C12N15/866C12N5/10A61K39/125A61P31/14
CPCA61K39/12A61K2039/5258A61K2039/54A61P31/14C07K14/005C12N15/86C12N2710/14043C12N2770/32023C12N2770/32034
Inventor 刘启亮刘洪波章宁漆琪
Owner GUILIN MEDICAL UNIVERSITY
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