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Coxsackie virus a16 strain and its application

A Coxsackie virus, A16 technology, applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve the problem that there is no standard strain of Coxsackie virus detection, and achieve the effect of good immunogenicity and high titer

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

[0004] However, there are no standard strains for detection of Coxsackievirus A16 and challenge strains for vaccine protection evaluation at home and abroad.

Method used

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  • Coxsackie virus a16 strain and its application
  • Coxsackie virus a16 strain and its application
  • Coxsackie virus a16 strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Preliminary Screening of Coxsackievirus A16 Type Strain

[0084] Processing of clinical samples:

[0085] In a biological safety cabinet, 0.25 ml of each sample (pharyngeal and anal test sub-sample that has been tested positive for Coxsackievirus A16 by fluorescent quantitative PCR) was added 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.

[0086] Virus isolation and culture:

[0087] 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 2 c...

Embodiment 2

[0157] Example 2 Plaque purification

[0158] The virus dilutions of the primary screened strains were inoculated in 6-well cell culture plates for purification.

[0159] (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, supplemented with RD cell culture medium, placed 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.

[0160] (2) Virus preparation: Dilute the virus solution by an appropriate multiple.

[0161] (3) Virus adsorption: inoculate the diluted virus solution, 0.4ml / well, set the 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 15 to 20 minutes to make it touch the entire c...

Embodiment 3

[0166] Example 3 Determination of Detecting Candidate Virus Strains

[0167] 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, see Example 1 for the specific research method operation steps.

[0168] The identification research of original seed strains mainly includes immunogenicity, virus titration, genome sequencing analysis, and cross-neutralization ability research.

[0169] 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.

[0170] The results of the strain test show that th...

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Abstract

The invention relates to the field of biotechnology, and specifically discloses the A16 strain of Coxsackie virus and its application. The amino acid sequence of the P1 structural protein of the Coxsackievirus A16 strain of the present invention is shown in SEQ ID NO.1. The strain has good intra-type and inter-type crossover and stable genetics. It can be used as a strain for testing the titer of serum neutralizing antibodies against Coxsackie virus A16 and provides support for the development of mono- / multivalent vaccines related to Coxsackie virus A16. . The strain has good immunogenicity, high titer, and strong virulence. It provides a challenge strain for the establishment of a stable infection animal model, which is very important for the construction of the CV‑A16 mouse model.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to Coxsackie virus A16 strain and application thereof. Background technique [0002] Hand, foot and mouth disease (HFMD) is an infectious disease caused by enterovirus, which includes more than 20 types, including Coxsackievirus type A16 (Coxsackievirus type A16, CA16) and enterovirus Type 71 (enterovirus71, EV71) is two common and important pathogens that cause hand, foot and mouth disease all over the world. The two are circulating alternately or in one area at the same time. The currently marketed EV-A71 vaccine has no cross-protective effect on CV-A16. Neutralizing antibody detection is one of the key indicators for CV-A16 epidemiological investigation and vaccine immunogenicity evaluation. The accuracy of neutralizing antibody titer detection is closely related to the detection strain used. It is of great significance to establish genotype standard detection strains in line with...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K14/085C07K16/10A61K39/125A61P31/14
CPCC12N7/00C07K14/005C07K16/1009A61K39/12A61P31/14C12N2770/32021C12N2770/32022C12N2770/32023C12N2770/32034
Inventor 张改梅刘建凯梁祁潘红星张黎赵丽丽谢学超陈磊马廷涛顾美荣
Owner BEIJING MINHAI BIOTECH
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