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Competitive ELISA method based on foot-and-mouth disease type A vp1 protein and its monoclonal antibody

A technology of FMDV-A-VP1 and foot-and-mouth disease, which is applied in the field of animal immunology detection, can solve the problems of troublesome detection operation, high detection cost, large error of results, etc., and achieve the effect of good stability, strong specificity and high efficiency

Inactive Publication Date: 2016-01-27
广西壮族自治区动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a competitive ELISA method based on the foot-and-mouth disease type A VP1 protein and its monoclonal antibody in view of the shortcomings of the existing detection technology for foot-and-mouth disease type A serum, such as high detection cost, troublesome detection operation, and large error in results. To the preparation of monoclonal antibody to type A foot-and-mouth disease protein VP1, and its application in the detection of pathogens and antibodies and the development of vaccines, etc.; established a monoclonal antibody competition ELISA method to detect type A antibody, and formed a type A foot-and-mouth disease immune monitoring technology platform

Method used

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  • Competitive ELISA method based on foot-and-mouth disease type A vp1 protein and its monoclonal antibody
  • Competitive ELISA method based on foot-and-mouth disease type A vp1 protein and its monoclonal antibody
  • Competitive ELISA method based on foot-and-mouth disease type A vp1 protein and its monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Obtaining of embodiment 1 foot-and-mouth disease type A VP1 protein

[0043] (1) Primer design

[0044] According to the gene sequence of foot-and-mouth disease virus type A published in GenBank, design a pair of cloning primers (C1, C2) to amplify the sequence containing FMDV-A-VP1 gene, the size is 886bp; design a pair of primers for prokaryotic expression vector and target gene sequence The complete VP1 gene (639bp) is specifically expressed with primers (E1, E2) and inserted into EcoRI and XholⅠ restriction sites. The primer sequences were sent to Dalian Bao Biological Engineering Co., Ltd. for synthesis.

[0045] Table 1 amplifies the primer nucleotide sequence of FMDV-A-VP1 gene

[0046]

[0047] (2) Gene cloning

[0048] Using the gene DNA of foot-and-mouth disease type A virus as a template, use the first pair of cloning primers C1 and C2 to amplify the sequence (886bp) containing the FMDV-A-VP1 gene, and then use the second pair of expression primers E1...

Embodiment 2

[0053] The acquisition of embodiment 2 foot-and-mouth disease type A VP1 protein monoclonal antibody

[0054] Step a) Animal immunization: immunize 7-week-old BALB / c female mice with the foot-and-mouth disease A-type VP1 protein 1 induced by the expression plasmid PET-32a-VP1. 1:12800 in mice.

[0055] Step b) cell fusion: get the splenocytes of the immunized mice obtained in step a), carry out cell fusion with myeloma cell SP2 / 0, and use the foot-and-mouth disease A-type VP1 protein 2 induced by the expression plasmid pGEX-6p-1-VP1 as The antigen was coated, and the fused cells were screened by indirect ELISA to obtain hybridoma cells.

[0056] Step c) A large number of cloning of monoclonal antibodies: inject the hybridoma cells obtained in step b) into the peritoneal cavity of mice, feed the injected mice for 1 week, collect ascites from mice with enlarged abdomen, and purify the ascites to obtain foot-and-mouth disease A Type VP1 protein monoclonal antibody.

[0057] ...

Embodiment 3

[0058] The competitive ELISA detection method of embodiment 3 porcine foot-and-mouth disease type A serum

[0059] Take the sera of 6 pigs suspected of being infected with FMD type A, and determine whether the sera are negative or positive. Value, data processing, in the competition ELISA detection method:

[0060] Step 1) Sample dilution: the serum sample was diluted to 4 times the original volume to obtain the serum sample to be tested.

[0061] Step 2) Antigen coating: the coating antigen used was FMD A-type VP1 protein 2 induced by the expression plasmid pGEX-6p-1-VP1, and the coating concentration of the coating antigen was 0.625ug / mL.

[0062] Step 3) Blocking: Block the antigen coated in step 2) with 1% BSA solution blocking solution for 40 minutes to obtain a blocked ELISA plate.

[0063] Step 4) Adding sample and adding monoclonal antibody: add the serum sample to be tested obtained in step 1) to the blocked microtiter plate obtained in step 3) to bind the serum a...

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Abstract

The invention relates to a competitive ELISA method based on the foot-and-mouth disease type A VP1 protein and its monoclonal antibody, and also relates to the preparation of the foot-and-mouth disease type A VP1 protein and its monoclonal antibody, belonging to the technical field of animal immunology detection. The present invention utilizes primer pair C1, C2 and primer pair E1, E2 to amplify the gene sequence of the foot-and-mouth disease type A VP1 protein, constructs the expression plasmid, introduces the expression plasmid into a prokaryotic expression host, induces and purifies the foot-and-mouth disease type A VP1 protein, and obtains the foot-and-mouth disease type A VP1 protein. FMD type A VP1 protein was used as an antigen, and the FMD type A VP1 protein monoclonal antibody was obtained by hybridoma technology, and a competitive ELISA method for detecting FMD type A antibody was established based on the FMD type A VP1 protein and its monoclonal antibody. The detection method of the invention has strong specificity and good stability, and can be used for the detection of serum antibody of foot-and-mouth disease type A, and the result is compared with the liquid-phase blocking ELISA detection kit, and the coincidence rate is 95.8%.

Description

technical field [0001] The invention belongs to the technical field of animal immunology detection, and in particular relates to a foot-and-mouth disease type A VP1 protein and a preparation method thereof, and a competition ELISA method based on the prepared foot-and-mouth disease type A VP1 protein and a monoclonal antibody thereof. Background technique [0002] Foot-and-mouth disease is a highly contagious and economically important animal disease caused by foot-and-mouth disease virus (FMDV), infecting cattle, pigs, sheep, goats and other artiodactyls. After animals are infected with foot-and-mouth disease, blisters will appear in the mouth, hooves, nipples and other parts to form erosions, and the diseased animals are generally benign. Although the death rate of foot-and-mouth disease is low, due to its high incidence rate of almost 100% and its rapid spread, it restricts the development of animal husbandry and the participation of countries in international trade of an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/09C07K16/10C12N15/42C12N15/70G01N33/577
CPCC07K14/005C07K16/1009C07K2319/00C12N15/62C12N15/70C12N2770/32122G01N33/56983G01N33/577G01N2333/09
Inventor 熊毅蒋家霞冯淑萍颜健华
Owner 广西壮族自治区动物疫病预防控制中心
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