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African swine fever virus antibody detection kit

A technology for detecting African swine fever virus and antibodies, which is applied in the field of animal infectious disease detection, and achieves the effect of low manufacturing cost and fast detection speed

Active Publication Date: 2020-03-10
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no kit that can efficiently detect antibodies against African swine fever

Method used

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  • African swine fever virus antibody detection kit
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  • African swine fever virus antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 coating antigenic protein

[0031] 1. Construction of P54E Gene Recombination Vector

[0032] Referring to the full gene sequence of the first African swine fever P54 in my country (MH766894, E183L, 162222-162776) published by the NCBI database, the gene of the recombinant polypeptide tandem protein R10 (SEQ ID NO: 1) was designed, and the amino acid sequence of the recombinant polypeptide tandem protein R10 was designed As shown in SEQ ID NO:2. The gene of the recombinant polypeptide tandem protein R10 conforms to the tropism of Escherichia coli. The gene of the recombinant polypeptide tandem protein R10 was inserted between the polyclonal restriction sites EcoRI and HindIII of the vector pET-28a(+) by gene synthesis (provided by Nanjing GenScript Biotechnology Co., Ltd.), and the recombinant plasmid pET- 28a-R10.

[0033] By "heat shock" method, the recombinant plasmid pET-28a-R10 was transformed into E.coli BL21(DE3) competent cells ...

Embodiment 2

[0041] Example 2 Kit composition optimization and optimization of the method of use

[0042] 1. Double antigen coated plate

[0043] Determination of the optimal coating concentration of a single antigen: the recombinant polypeptide tandem protein R10 and recombinant protein PS273R were used to coat the microplate respectively, and the negative and positive reference sera of African swine fever antibody diluted 1:100 were detected. The specific method is as follows: the gradiently diluted recombinant polypeptide tandem protein R10 and recombinant protein PS273R were respectively diluted with a coating solution (0.05 mol / L carbonate buffer solution, pH value = 9.6), and then coated with a 96-well microtiter plate, 100 μL per well, incubate at 37°C for 1 hour, then act at 2-8°C for 14-18 hours. Discard the coating solution in the well, add 250 μl of PBST (PBS solution containing 0.05% Tween-20, the concentration of the PBS buffer solution is 0.01mol / L, pH value=7.2~7.4) to each...

Embodiment 3

[0069] The preparation of embodiment 3 African swine fever virus antibody detection kit

[0070] 1. Composition of the African swine fever virus antibody detection kit

[0071] According to the ELISA detection method determined in Example 2 Title 8, the composition of the African swine fever virus antibody detection kit (abbreviated as the kit of the present invention) is determined, specifically comprising: African swine fever antibody positive control serum, African swine fever antibody Negative control serum, double antigen coated plate, concentrated washing solution (10×), sample diluent, HRP-goat anti-pig IgG enzyme-labeled antibody (30×), TMB substrate color development solution and stop solution. details as follows:

[0072] (1) Double antigen coated plate

[0073] Prepare a solution containing 1.25 μg / ml recombinant polypeptide tandem protein R10 and 2.5 μg / ml recombinant protein PS273R with 0.05 mol / L carbonate buffer solution with pH value = 9.6, and coat a 96-well...

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Abstract

The invention provides an African swine fever virus antibody detection kit, relates to the field of animal infectious disease detection, and specifically relates to an African swine fever virus antibody detection kit. The African swine fever virus antibody detection kit comprises an elisa plate coated with a recombinant polypeptide tandem protein R10 and a recombinant protein PS273R, wherein the amino acid sequence of the recombinant polypeptide tandem protein R10 is shown as SEQ ID NO: 2, and the amino acid sequence of the recombinant protein PS273R is shown as SEQ ID NO: 4. The African swinefever virus antibody detection kit provided by the invention can be used for rapidly and accurately detecting the African swine fever virus antibody with high specificity.

Description

technical field [0001] The invention relates to the field of animal infectious disease detection, in particular to an African swine fever virus antibody detection kit. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious disease caused by African swine fever virus (ASFV). Suids and ticks are susceptible animals to ASF, among which domestic pigs are highly susceptible, with a mortality rate as high as 100%, and there is no obvious difference in breed, age and sex. The ASFV genome includes 160-175 open reading frames, encoding 150-200 proteins. [0003] Currently, there is no effective vaccine against ASF, and a positive antibody test indicates that the animal is or has been infected with the virus. With the large-scale spread of ASFV after invading our country, ASF is likely to become another common major swine disease in our country, so antibody detection is of great significance in the control and diagnosis of the disease. In th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68
CPCG01N33/56983G01N33/6854G01N2333/01
Inventor 冯志新白昀邵国青熊祺琰郝飞华利忠陈蓉谢青云谢星甘源杨浩
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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