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Method for detecting neutralizing antibody in coxsackievirus A6 (CV-A6), and recombinant virus applied in method

A technology of Coxsackie virus, group A, applied in the direction of virus/phage, biochemical equipment and method, virus, etc., can solve the problems of long time, large impact, cumbersome operation, etc., and achieve simple cost and easy operation. Effect

Active Publication Date: 2018-05-22
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The result of this method is reliable, but the disadvantage is that the live virus is prone to antigenic drift during the passaging process, and more importantly, it is dangerous during the test operation, so the operation and safety equipment of technicians are required to be high.
Moreover, the operation is cumbersome, and it takes a long time to observe cell lesions. The interpretation of the results is greatly affected by subjective factors, and only semi-quantitative test results can be obtained.

Method used

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  • Method for detecting neutralizing antibody in coxsackievirus A6 (CV-A6), and recombinant virus applied in method
  • Method for detecting neutralizing antibody in coxsackievirus A6 (CV-A6), and recombinant virus applied in method
  • Method for detecting neutralizing antibody in coxsackievirus A6 (CV-A6), and recombinant virus applied in method

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Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1, detection of enterovirus CV-A6 neutralizing antibody detection

[0053] 1. Construction of CV-A6 nucleocapsid protein expression plasmid and CV-A16 replicon plasmid

[0054] (1) Construct CV-A6 nucleocapsid protein expression plasmid

[0055] Using the full-length infectious cloning plasmid of CV-A6 as a template, the structural protein coding segment of CV-A6 (including VP4, VP2, VP3, VP1) was obtained by PCR amplification, and green fluorescent protein (GFP) was inserted into the N-terminal of the viral genome structural protein ( EGFP) gene. The gene sequence of EGFP was spliced ​​with the gene sequence of all structural proteins of CV-A6, and the restriction site of 2A protease was inserted into the C-terminus of the EGFP gene, and spliced ​​to obtain the fusion gene ( figure 1 B), its nucleotide sequence is shown in SEQ ID No.2, wherein the 1st to 717th is the coding gene of green fluorescent protein (the amino acid sequence of green fluorescent pro...

Embodiment 2

[0081] Embodiment two, check the consistency of the method of the present invention and classical live virus neutralization test

[0082] In parallel, the neutralizing antibody titer of a group of human plasma samples was tested with the wild-type CV-A6 live virus and the CV-A6 pseudovirus of the present invention. The wild-type CV-A6 virus strain is CV-A6-2014-XM, and the test sample comes from the phase III clinical serum sample of EV-A71 vaccine (from the China Institute for Food and Drug Control). Correlation analysis was performed on the two groups of test results, such as figure 2 As shown, the results show that the pseudovirus system of the present invention has good consistency with the classic live virus neutralization test when detecting serum neutralizing antibodies.

Embodiment 3

[0083] Embodiment three, test the specificity of CV-A6 pseudovirus system

[0084] In order to test the specificity of the CV-A6 pseudovirus system, a mouse-derived specific antiserum against CV-A6 (serum from SPF grade BALB / c mice immunized with CV-A6(XM) inactivated virus ), mouse-derived specific antiserum against EV-A71 (FY) (from the serum of SPF grade BALB / c mice immunized with EV-A71 (FY) inactivated virus), mouse-derived specific antiserum against CV -B3(112)-specific antiserum (serum from SPF-grade BALB / c mice immunized with CV-B3(112) inactivated virus), mouse-derived specific for CV-A16(G10) Antiserum (from SPF grade BALB / c mice immunized with CV-A16(G10) inactivated virus), mouse-derived specific antiserum against CV-B5(417) (from CV-B5 (417) The serum of SPF level BALB / c mice immunized with inactivated virus) and mouse-derived specific antiserum against hepatitis E HEV (from the hepatitis E vaccine immunized with the Hepatitis E vaccine produced by Wantai Canghai...

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Abstract

The invention discloses a method for detecting a neutralizing antibody in coxsackievirus A6 (CV-A6), and a recombinant virus applied in the method. According to the method, the neutralizing antibody is detected by using a pseudovirus system packaged by two types of recombinant plasmids; by adopting a single-cycle infected pseudovirus, the safety problem caused by use of live viruses is avoided. The results of a plurality of tests prove that the pseudovirus detection system is a CV-A6 neutralizing antibody detection method which is safe, sensitive, rapid, specific, simple and convenient, and low in cost. Based on the characteristics, the pseudovirus detection system is very suitable for the tests for rapid and large-scale detection of the neutralizing antibody, and has an import applicationvalue for development of a CV-A6 viral vaccine and detection of CV-A6 specific neutralizing antibody level of patients and populations.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a detection method for detecting coxsackievirus group A type 6 neutralizing antibodies and a special pseudovirus thereof, in particular to two recombinant plasmids for packaging special pseudoviruses, a method for preparing the plasmid, Special pseudovirus and rapid detection kit. Background technique: [0002] Coxsackievirus group A type 6 (Coxsackievirus A6, CV-A6) belongs to the genus Enterovirus of the family Picornaviridae. CV-A6 is a single-stranded positive-strand RNA virus. The virus particle is a spherical structure with icosahedral stereosymmetry, about 30 mm in diameter, without envelope and protrusions. The total length of the CV-A6 genome is about 7400bp, and the genome consists of an open reading frame and two non-coding regions (5'-UTR and 3'-UTR). The open reading frame is a polyprotein consisting of 2202 amino acids, which is further hydrolyzed into 3 precursor pro...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12N7/01G01N33/569
CPCC12N7/00C12N15/63C12N15/65C12N2770/32321G01N33/56983G01N2469/20
Inventor 吴星苏瑶梁争论陈盼董方玉孙世洋毛群颖高帆卞莲莲姜崴胡亚林
Owner NAT INST FOR FOOD & DRUG CONTROL
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