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African swine fever virus (ASFV) antibody detection kit based on chimeric P54 antigen epitope and application

A technology of African swine fever virus and antigenic epitope, which is applied in the direction of virus/bacteriophage, virus, viral peptide, etc., can solve the problems of sensitivity and specificity limitations, and achieve the goal of improving sensitivity and specificity and avoiding virus spread risk effect

Active Publication Date: 2021-06-29
SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the fact that African swine fever virus is a highly pathogenic and severe swine infectious disease virus, my country's statutory first-class infectious disease virus, in accordance with biosafety management requirements, except for laboratories approved by the Ministry of Rural Agriculture, other laboratories are not allowed to operate involving the isolation of African swine fever virus Therefore, commercialized ELISA kits are coated with single or multiple proteins of prokaryotic or eukaryotic ASFV proteins, and their sensitivity and specificity are limited in the detection of African swine fever virus antibody levels. sex

Method used

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  • African swine fever virus (ASFV) antibody detection kit based on chimeric P54 antigen epitope and application
  • African swine fever virus (ASFV) antibody detection kit based on chimeric P54 antigen epitope and application
  • African swine fever virus (ASFV) antibody detection kit based on chimeric P54 antigen epitope and application

Examples

Experimental program
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Effect test

example 1

[0102] Example 1 Structural Analysis of Bluetongue Virus VP7 Protein and Prediction of Flexible Structure Region

[0103] Bluetongue virus core-like particles are assembled from bluetongue virus structural proteins VP3 and VP7, and 60 VP3 dimers (120 VP3 proteins) assemble to form the inner core of core-like particles with a diameter of 55 nm, and 260 VP7 three Aggregates (780 VP7 proteins) are located in the outer layer of core-like particles similar to a brush coat, and the structure is as follows: figure 1 . For this reason, the present invention takes VP7 protein as the transformation target.

[0104] The invention adopts tools such as Garnier-Robson method, Chou-Fasman method and Karplus-Schulz method to analyze the structure of the bluetongue virus VP7 protein, so as to predict the flexible structural region of the VP7 protein. Finally, the present invention screened out 8 possible flexible structural regions, and their positions and amino acid residue sequences are sh...

example 2

[0107] Example 2 The bluetongue virus VP7 amino acid sequence of the chimeric African swine fever virus P54 epitope

[0108] The research team analyzed the secondary structure of the African swine fever virus P54 protein and determined that 66EDIQFINPYQ75 in the amino acid sequence of the P54 protein is a conserved epitope. According to NCBI comparison analysis, almost all African swine fever virus P54 proteins contain this amino acid sequence; And the research team screened and prepared a monoclonal antibody against this epitope (P54-2E4 MAb). For this reason, the present invention replaces the 8 possible flexible structural regions of the bluetongue virus VP7 protein in case 1 with the EDIQFINPYQ amino acid sequence, thereby obtaining the bluetongue virus VP7 amino acids of 8 chimeric African swine fever virus P54 antigenic epitopes Sequence, see Table 2 for specific alternatives

[0109] Table 2 Alternatives

[0110]

example 3

[0111] Example 3 Preparation and identification of chimeric core-like particles

[0112] (1) Construction of recombinant baculovirus

[0113] Insert the bluetongue virus vp3 gene into the vector pFastBac by gene synthesis and subcloning technology TM Dual, construct the recombinant plasmid pFastBac-Dual-vp3; then the VP7 gene (rvp7(47-51), rvp7(78-84), rvp7(113-119), rvp7(136-142), rvp7(155) after mutation -161), rvp7(175-179), rvp7(182-188)) and rvp7(233-239) were respectively inserted into another multiple cloning site of the vector, thereby constructing and obtaining 8 kinds of recombinant plasmids pFastBac-Dual- vp3-rvp7(47-51), pFastBac-Dual-vp3-rvp7(78-84), pFastBac-Dual-vp3-rvp7(113-119), pFastBac-Dual-vp3-rvp7(136-142)pFastBac-Dual - vp3-rvp7(155-161), pFastBac-Dual-vp3-rvp7(175-179), pFastBac-Dual-vp3-rvp7(182-188) and pFastBac-Dual-vp3-rvp7(233-239). The recombinant plasmids with correct sequencing were transformed into DH10Bac, screened by blue-white spot and co...

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Abstract

The invention discloses an African swine fever virus (ASFV) P54 antibody detection method and kit based on bluetongue virus core-like particles with a chimeric ASFV P54 protein antigen epitope. The chimeric core-like particle is used as a coating antigen, the antigen epitope is better presented in a form similar to that of an original virus, and meanwhile, the monoclonal antibody with the specificity of the antigen epitope is used as a blocking enzyme-labeled antibody, so that better specificity and better sensitivity are achieved. The ASFV P54 antibody detection kit is high in safety, good in sensitivity, high in specificity, high in accuracy and convenient to operate, and compared with an imported kit, the detection coincidence rate reaches 100%.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a blocking ELISA kit for detecting African swine fever virus P54 antibody and an application thereof, which is suitable for specific, rapid and accurate detection of African swine fever virus P54 antibody. Background technique [0002] African swine fever (African Swine Fever, ASF) is a severe, highly contagious animal infectious disease caused by African swine fever virus (African Swine FeverVirus, ASFV). Its acute symptoms are characterized by high fever, reticuloendothelial hemorrhage, and high mortality. All breeds and ages of domestic and wild boars are susceptible. Ornithodoros soft ticks, especially O. moubata and O. erraticus, are the storage and transmission vectors of ASFV. Previously, the disease was limited to Africa, until it spread to Europe, South America and the Caribbean in the middle of the last century. It was introduced to Eastern Eu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N33/569G01N33/543
CPCC07K14/005C07K2319/00C12N2710/12022C12N2710/12023C12N2720/12122C12N2720/12123G01N33/543G01N33/56983
Inventor 黄超华花群义曹琛福吴江史卫军林彦星林永涛翁巧玉曾少灵杨俊兴
Owner SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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