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Anti-African swine fever P30 protein single-domain antibody and ELISA kit for detecting African swine fever virus

An African swine fever virus and single-domain antibody technology, which is applied in the field of ELISA kits, can solve the problems of lack of specific antibodies, etc., and achieve accurate detection, good detection repeatability, and high detection sensitivity.

Active Publication Date: 2020-04-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the current ELISA method for detecting African swine fever virus lacks specific and strong antibodies

Method used

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  • Anti-African swine fever P30 protein single-domain antibody and ELISA kit for detecting African swine fever virus
  • Anti-African swine fever P30 protein single-domain antibody and ELISA kit for detecting African swine fever virus
  • Anti-African swine fever P30 protein single-domain antibody and ELISA kit for detecting African swine fever virus

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Experimental program
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preparation example Construction

[0031] The preparation method of the single domain antibody p30-17 of the anti-African swine fever virus P30 protein preferably comprises the following steps:

[0032] 1) Linking the coding sequence of p30-17 to the expression vector pet-30a expression vector (restriction sites are EcoR I and Xho I), and constructing a prokaryotic expression vector;

[0033] 2) The prokaryotic expression vector was identified by positive colonies, and the positive bacterial solution obtained by screening was added to TB medium at a ratio of 1:100 for expansion and cultivation until the OD value was 3.0, and then IPTG with a final concentration of 0.1mM was added to induce expression, 6h Finally, the bacteria were collected, and after ultrasonic disruption, the precipitate was resuspended with lysate to obtain a resuspension;

[0034] 3) Using different concentrations of imidazole to purify the resuspension to obtain the single domain antibody p30-17 against African swine fever virus P30 protei...

Embodiment 1

[0063] A kind of component that detects the ELISA kit of ASFV of pig is as follows:

[0064] A microtiter plate coated with a capture antibody: the capture antibody is a single domain antibody (p30-17) against the ASFV p30 protein;

[0065] Detection antibody: the detection antibody is a horseradish peroxidase-labeled antibody that is a single-domain antibody (p30-30) against ASFVp30 protein, and the dilution is 1:2000 (1 μg / mL);

[0066] 25× concentrated washing solution: phosphate buffer solution with a pH value of 7.4 containing 0.5% Tween, which needs to be diluted 25 times with distilled water during work (purchased from the Diagnostic Center of Lanzhou Veterinary Research Institute);

[0067] Serum diluent: NaCl 8.5g, NaH 2 PO 4 2H 2 O 0.356g, Na 2 HPO 4 12H 2 0 2.772g, dissolved in distilled water and adjusted to 500mL, pH value is 7.2-7.4;

[0068] Chromogenic solution: TMB two-component chromogenic solution:

[0069] Stop solution: 1M H 2 SO 4 , Add 56mL of ...

Embodiment 2

[0088] The using method of the kit prepared in embodiment 1

[0089] 1. Add sample

[0090] Take out the ELISA plate, add two parallel wells for each negative control and positive control, 100 μl / well of the sample to be tested (the sample is directly added to the bottom of the well, care should be taken to avoid sticking to the wall), seal the plate, and incubate at 37°C for 15 minutes.

[0091] 2. Washing

[0092] Aspirate and discard the samples in the wells of the ELISA plate (do not throw them away at will to avoid contamination), wash with 300 μL / well of washing solution for 3 times, and pat dry on absorbent paper after each wash.

[0093] 3. Add enzyme-labeled secondary antibody

[0094] Add enzyme-labeled antibody working solution at 100 μl / well, seal the plate, and incubate at 37°C for 15 minutes.

[0095] 4. Washing

[0096] Pour out the liquid in the well, wash 5 times with 300 μL / well of washing solution, and pat dry on absorbent paper after washing. If washin...

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Abstract

The invention, which belongs to the technical field of virus detection, provides an anti-African swine fever P30 protein single-domain antibody and an ELISA kit for detecting the African swine fever virus. The amino acid sequence of the single-domain antibody p30-17 for resisting the African swine fever virus P30 protein is as shown in SEQ ID No. 1. In addition, the invention also relates to an ELISA kit for detecting African swine fever virus based on a double-antibody sandwich method. The ELISA kit comprises an elisa plate coated with a capture antibody being a single-domain antibody p30-17,an enzyme-labeled detection antibody being a single-domain antibody p30-30 of the anti-African swine fever virus p30 protein with an amino acid sequence shown as SEQ ID No.2, a standard positive reference substance, a serum diluent, a substrate developing solution A, a substrate developing solution B, a stop solution and a concentrated washing solution. The kit can realize rapid and accurate detection and has good detection specificity.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to an anti-African swine fever P30 protein single domain antibody and an ELISA kit for detecting African swine fever virus. Background technique [0002] African swine fever is an acute, highly contagious infectious disease caused by African swine fever virus, and the fatality rate can reach 100%. The World Organization for Animal Health lists it as an animal disease that must be notified, and my country lists it as a first-class animal disease . African swine fever virus belongs to the double-stranded DNA virus order, African swine fever virus family, African swine fever virus genus, and is the only member of the virus family. African swine fever virus p30 protein is a protein produced in the early stage of virus infection. It is a protein of about 30kD encoded by the CP204L gene. Due to its strong immunogenicity and p30 can induce a strong immune response in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/569
CPCC07K16/081C07K2317/22G01N33/56983G01N2333/01
Inventor 刘志杰赵亚茹杨吉飞牛庆丽刘光远罗建勋关贵全殷宏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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