Coxsackie virus a10 strain and its application

A coxsackie virus, A10 technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., to achieve the effects of genetic stability, strong virulence, and good crossover

Active Publication Date: 2022-01-07
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, under the situation that there is no relevant detection standard strain of Coxsackievirus A10 type at home and abroad and the challenge strain used for vaccine protective evaluation, it is more necessary to provide a new Coxsackievirus type A10 strain. To meet the needs of existing technology development

Method used

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  • Coxsackie virus a10 strain and its application
  • Coxsackie virus a10 strain and its application
  • Coxsackie virus a10 strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Preliminary Screening of Coxsackievirus A10 Type Strain

[0087] Processing of clinical samples:

[0088] In a biological safety cabinet, take 0.25 ml of each sample (pharyngeal and anal test sub-sample that has been tested positive for Coxsackievirus A10 by fluorescent quantitative PCR) and add it to a centrifuge tube. 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.

[0089] Virus isolation and culture:

[0090]Take the prepared healthy and non-contaminated RD cells grown 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 of virus culture solution at the same time, place at 35°C 5% CO 2 Adsorption in the incubator for 1h. After that, add 3.5ml of virus culture solution to each well, and place at 35°C 5% CO...

Embodiment 2

[0159] Example 2 Plaque purification

[0160] Inoculate the virus dilutions of the primary screened strains for the first plaque purification.

[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, add cell culture medium (RD cells) to 4 ml, place in 5% CO 2 Incubate at 37±1°C for 48 hours in an incubator until a dense monolayer grows. The original culture medium was discarded, and the cell surface was washed with serum-free maintenance medium (MEM medium) to remove residual bovine serum and dead cells.

[0162] (2) Virus preparation: Dilute the virus solution by 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, and 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 ...

Embodiment 3

[0168] Example 3 Determination of Detecting Candidate Virus Strains

[0169] The virus strain purified by three plaques was amplified to the 5th generation to establish the original seed, and the relevant identification research and passage stability research were carried out on it. Strains with a high degree of infection were used as candidate strains for detection. Unless otherwise specified, the detection method is the same as in Example 1.

[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 process.

[0172] The results of the strain test show that the strain numbe...

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PUM

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Abstract

The invention relates to the field of biotechnology, and specifically discloses a Coxsackie virus A10 strain and an application thereof. The amino acid sequence of the P1 structural protein of the Coxsackievirus A10 strain of the present invention is shown in SEQ ID NO.1. The strain has good cross-neutralization ability, stable genetics, and strong virulence. It provides a challenge strain for the establishment of a stable animal model of infection, and can be used as a detection virus for the determination of the neutralizing antibody titer of Coxsackie virus A10 immune serum. strain, and provided technical support for the development of mono / multivalent vaccines related to Coxsackievirus A10.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to Coxsackie virus A10 strain and application thereof. Background technique [0002] Hand, foot, and mouth disease is an infectious disease caused by enteroviruses. There are more than 20 types of enteroviruses that cause hand, foot, and mouth disease. They mostly occur in children under 5 years old, and manifest as mouth pain, anorexia, low fever, hand, foot, mouth Small herpes or small ulcers appear in other parts of the body, most of the children will recover by themselves in about a week, and a few children may cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. Individual critically ill children develop rapidly and lead to death. There is currently a lack of effective treatment drugs for symptomatic treatment. [0003] In recent years, with the popularization of EV-A71 vaccine, the incidence of CV-A6 and CV-A10 has been increasing year by ye...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K14/085C07K16/10A61K39/12A61P31/14
CPCC12N7/00C07K14/005C07K16/1009A61K39/12A61P31/14C12N2770/32021C12N2770/32022C12N2770/32023C12N2770/32034
Inventor 张改梅李国顺朱凤才樊欢嵇红赵丽丽谢学超马廷涛陈磊肖海峰顾美荣刘建凯
Owner BEIJING MINHAI BIOTECH
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