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Coxsackievirus A6-type strain and application thereof

A Coxsackie virus and strain technology, applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve the problems of no Coxsackie virus, no vaccine attack strains, and poor susceptibility of the strains, and achieve Strong virulence, improved accuracy and repeatability, and good immunogenicity

Active Publication Date: 2021-10-29
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no standard test strain of Coxsackievirus type A6, nor is there a challenge strain for the evaluation of vaccine protection
[0003] In addition, because mice are generally less susceptible to clinically isolated strains, only a few clinically isolated viruses can infect mice, and most of them are mouse-adapted mutant strains.

Method used

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  • Coxsackievirus A6-type strain and application thereof
  • Coxsackievirus A6-type strain and application thereof
  • Coxsackievirus A6-type strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Preliminary Screening of Coxsackievirus Type A6 Strains

[0088] (1) Processing of clinical samples

[0089] Add 0.25 ml of each sample to a centrifuge tube in a biosafety cabinet. Add 2.5 μl of penicillin and streptomycin solution, mix well, and place overnight at 4°C. Centrifuge at 2000 rpm for 20 min, and store the supernatant at 2-8°C for inoculation.

[0090] (2) Virus isolation and culture

[0091] Take healthy and non-contaminated RD cells that grow to 80-90% density, and discard the cell culture medium. Inoculate 0.2ml / well of the processed sample into a 6-well plate, inoculate 1 well for each sample, and add 0.2ml / well virus culture solution at the same time, place at 35°C, 5% CO 2Adsorption in the incubator for 1h. After that, add 3.5ml of virus culture solution to each well, and place at 35°C, 5% CO 2 cultured in an incubator. Set 2 wells of well-grown cells that were not inoculated with specimens as cell controls.

[0092] (3) Virus harvest...

Embodiment 2

[0159] Example 2 plaque purification

[0160] Inoculate the virus dilutions of the six strains with good immunogenicity in the cross-neutralization ability study in Example 1 into a 6-well cell culture plate for purification. The specific method is as follows:

[0161] (1) Cell preparation: RD cells that had grown into a monolayer were washed and digested and inoculated in a 6-well cell culture plate, 7×10 5 cells / well in 5% CO 2 Incubate at 37±1°C in an incubator until a dense monolayer grows. Discard the original culture medium, wash the cell surface, and wash away the residual bovine serum and dead cells.

[0162] (2) Virus preparation: Dilute the virus solution to an appropriate multiple.

[0163] (3) Virus adsorption: inoculate the diluted virus solution, 0.4ml / well, set virus solution control and cell control at the same time, place in 5% CO 2 Adsorb in an incubator at 35°C for 1 to 2 hours, during which time the cell plate was gently shaken several times every 15 to...

Embodiment 3

[0168] Embodiment 3 detects the determination of candidate virus strains

[0169] Amplify the virus strain purified by three plaques to the 5th generation to establish the original seed, and conduct relevant identification research and passage stability research (the detection method is the same as in Example 1), and select the cross-protection range according to the passage stability research results The strains with broad spectrum, good genetic stability and high titer were used as candidate strains for detection.

[0170] The identification research of original seed strains mainly includes immunogenicity, virus titration, genome sequencing analysis, and cross-neutralization ability research.

[0171] The research on the stability of subculture is mainly to subculture the original seed virus solution on RD cells in a certain proportion until the 15th generation, and carry out virus titration and genome sequence analysis on each generation of strains during the subculture pro...

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Abstract

The invention relates to the technical field of biology, in particular to a coxsackievirus A6-type strain and application thereof. The amino acid sequence of P1 structural protein of the coxsackievirus A6-type strain is shown as SEQ ID NO.1. The strain has high cross-neutralization capacity in genotypes and between genotypes, is high in toxicity, has high pathogenic and lethal capacity on mice, and has good immunogenicity and high titer and stability. The strain can be used for immunogenicity evaluation or protective evaluation of coxsackievirus A6-type vaccines, the accuracy and repeatability of vaccine immunogenicity evaluation are improved, and the strain can also be used for preparing coxsackievirus infection animal models and has good application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to Coxsackie virus A6 strain and application thereof. Background technique [0002] Coxsackievirus A6 (CV-A6) is a human enterovirus that can cause hand, foot and mouth disease. In the early stage, enterovirus 71 (Enterovirus71, EV-A71) and Coxsackievirus A16 (Coxsackievirus A16, CV-A16) dominated the epidemic, and the vast majority of severe cases and deaths were related to EV-A71. However, in recent years, with the promotion of EV-A71 vaccine, the incidence of CV-A6 and CV-A10 has increased year by year. In the epidemiological investigations in recent years, CV-A6 has become one of the main pathogens causing HFMD. Neutralizing antibody detection is one of the key indicators for CV-A6 epidemiological investigation and vaccine immunogenicity evaluation. The accuracy of neutralizing antibody titer detection is closely related to the detection strain used. At present, the CV-A6 epidem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C07K14/085C07K16/10A61K39/125A61P31/14
CPCC12N7/00C07K14/005C07K16/1009A61K39/12A61P31/14C12N2770/32021C12N2770/32022C12N2770/32023C12N2770/32034A61K2039/5252A61K2039/5254A61K2039/53A61K2039/55505A61K2039/5258
Inventor 张改梅顾美荣胡家垒胡月梅赵丽丽谢学超陈磊马廷涛刘建凯
Owner BEIJING MINHAI BIOTECH
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