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Coxsackie virus A6 type strain and immunogenic composition and application thereof

A coxsackie virus, immunogenic technology, applied in antiviral agents, viruses/phages, biochemical equipment and methods, etc., can solve the problems that CV-A6 strains have not been found, CV-A6 strains cannot grow, etc. , to achieve the effect of strong cross neutralization ability, good immunogenicity and good passive immunity

Active Publication Date: 2022-07-29
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Katsumi Mizuta et al. found that the CV-A6 strain could not grow in Vero cells when they infected different cell lines with the CV-A6 strain (Mizuta, Katsumi et al. "Longitudinal epidemiology of viral infectious diseases combining virus isolation, antigenic analysis andphylogenetic analysis as well as seroepidemiology in Yamagata, Japan between1999 and 2018." Japanese journal of infectious diseases (2019): n. pag.) Bian Lianlian et al. also showed that the separation rate of CV-A6 may be less than 1% (Bian Lianlian, Liu Siyuan, Jiang Wei , Mao Qunying, Gao Fan, Yang Xiaoming, Liang Zhengyu. Multivalent Vaccine for Hand, Foot and Mouth Disease: Reality and Dream[J]. Chinese Journal of Biological Products, 2020,33(01):106-112.DOI:10.13200 / j.cnki. cjb. 002971.)
No CV-A6 strain isolated from Vero cell line has been found in the prior art

Method used

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  • Coxsackie virus A6 type strain and immunogenic composition and application thereof
  • Coxsackie virus A6 type strain and immunogenic composition and application thereof
  • Coxsackie virus A6 type strain and immunogenic composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Isolation and culture of CV-A6 virus strain

[0091] 1. Processing of clinical samples

[0092] The clinical samples were processed according to the guidelines for the prevention and control of hand, foot and mouth disease (2009 edition).

[0093] 2. Virus isolation

[0094] The processed samples were inoculated into 80~90% density of healthy non-polluting African green monkey kidney passage cells (Vero) according to a certain proportion, 35 ℃, 5% CO. 2 Adsorbed in the incubator for more than half an hour, replenished to the culture volume, 35°C, 5% CO 2 The cells were cultured in an incubator, and a cell control without sample was set at the same time. Use an inverted microscope to observe cells every day, such as enterovirus cytopathic effect (CPE): cells become rounded, refraction enhanced and detached from the tube wall, etc., record the changes, and continuously observe the changes in the inoculated wells and control wells within 7 days. CPE (1+~4+). ...

Embodiment 2

[0097] Example 2 Identification and detection of virus strains

[0098] Molecular identification, genome sequencing and titer detection were performed on the fourth-generation virus liquid of Coxsackie virus A6 strain V991 harvested in Example 1.

[0099] 1. Molecular identification and genome sequencing

[0100] The virus was identified by RT-PCR, and the general primers and VP1-specific primers used for enterovirus nucleic acid detection are shown in Table 1.

[0101] Table 1 Universal primers and VP1-specific primers for enterovirus nucleic acid detection

[0102]

[0103] (1) Virus nucleic acid extraction

[0104] Take the fourth-generation virus solution, add reagents and virus samples according to the instructions, then place it in a nucleic acid extractor, extract nucleic acids according to the preset procedure, and store the extracted nucleic acids in a -70°C refrigerator.

[0105] (2) PCR detection of HEV-5UTR universal primers

[0106] Nested PCR was performed...

Embodiment 3

[0140] Example 3 Detection of cross-neutralization ability of CV-A6 strains

[0141] The immune serum of Coxsackie virus A6 strain V991 (hereinafter referred to as CV-A6 serum) was tested for cross-neutralization ability by microcytopathic method. The specific method is as follows: after inactivating the serum in a water bath at 56 °C for 30 minutes, dilute it from 1:8, add it to a 96-well plate, 2 wells per sample, 100 μl / well, after 2-fold dilution, add 32~320 CCID 50 / 0.05ml of virus solution at 36±1℃, 5% CO 2 Incubator, neutralize for 1 to 2 hours. Add RD cell suspension (1~2 × 10 5 pcs / ml), 100 μl / well. Put in 5% CO 2 After culturing for 7 days, the results were judged. Neutralization titers < 8 were judged negative, and ≥ 8 were positive.

[0142] The immune sera of Coxsackie virus A6 strain V991 were cross-neutralized with 12 different CV-A6 strains, of which 1 was the prototype strain (type A) and 11 were circulating genotype (D3) (cross-neutralization). And the ...

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Abstract

The invention relates to the technical field of biology, in particular to a coxsackie virus A6 type strain and an immunogenic composition and application thereof. The amino acid sequences of VP4, VP2, VP3 and VP1 proteins of the coxsackie virus A6 type strain are respectively shown as SEQ ID NO.1, 2, 3 and 4, and the amino acid sequences of 2A, 2B, 2C, 3A, 3B, 3C and 3D proteins are respectively shown as SEQ ID NO.5, 6, 7, 8, 9, 10 and 11. The virus strain is obtained by separating Vero cells, is susceptible to the Vero cells, can obtain higher titer, has the advantages of good immunogenicity, strong cross neutralization capability and the like, and can be used for preparing vaccines or medicines for preventing or treating coxsackie virus A6 infection or diseases caused by the coxsackie virus A6 infection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to Coxsackie virus A6 strains and immunogenic compositions and applications thereof. Background technique [0002] Hand-foot-mouth disease (HFMD) is a common infectious disease in infants and young children caused by a variety of enteroviruses. Children under the age of 5 are a high-risk group. Typical manifestations are rashes on the hands, feet, and mouth. , herpes, ulcers, etc., can be complicated by aseptic meningitis, encephalitis, acute flaccid paralysis, respiratory tract infection and myocarditis, etc. Individual severe cases can lead to disability or death. [0003] The main pathogenic serotypes of hand, foot and mouth disease include Coxsackievirus (Coxsackievirus, CV) A group 4-7, 9, 10, 16 and B group 1-3, 5, part of the serotype of Echovirus (Echovirus) Type 71 (Enterovirus A71, EV-A71), etc., there is no cross immunity between enterovirus types. In recent years, Coxsacki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C07K14/085A61K39/125A61K39/39A61P31/14
CPCC12N7/00C07K14/005A61K39/12A61K39/39A61P31/14C12N2770/32021C12N2770/32022C12N2770/32023C12N2770/32034A61K2039/5258A61K2039/521A61K2039/55505
Inventor 肖霞张德宝顾美荣李国顺陈磊郭林简伟肖海峰徐颖之张改梅刘建凯
Owner BEIJING MINHAI BIOTECH
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