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Test strip for detecting CD2v and MGF360 mucous membrane antibodies of African swine fever viruses and application of test strip

A technology of African swine fever virus and African swine fever, which is applied to test strips for detecting African swine fever virus CD2v and MGF360 mucosal antibodies and its application field, which can solve the problem of difficult real-time detection, low sensitivity and high cost in grassroots farms and other problems, to achieve the effect of high uniformity, high sensitivity and good stability

Pending Publication Date: 2020-11-10
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sensitivity of IFA is low, requiring fluorescent operators and fluorescent microscopes, and the high cost limits the application in rapid on-site detection.
ELISA uses the principle of antigen-antibody specific reaction to detect whether animals have African swine fever virus antibodies. It has the advantages of high sensitivity and strong specificity, but the operation is cumbersome and time-consuming. In addition, it requires equipment such as microplate readers and incubators. It is difficult to carry out real-time detection in grass-roots farms

Method used

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  • Test strip for detecting CD2v and MGF360 mucous membrane antibodies of African swine fever viruses and application of test strip
  • Test strip for detecting CD2v and MGF360 mucous membrane antibodies of African swine fever viruses and application of test strip
  • Test strip for detecting CD2v and MGF360 mucous membrane antibodies of African swine fever viruses and application of test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Preparation of recombinant protein CD2v and recombinant protein MGF360

[0039] 1. Construction of recombinant bacteria for preparing recombinant protein CD2v

[0040] Referring to the complete gene sequence of the first African swine fever SY18 in my country (MH766894, EP402R, 73369-74451) published by the NCBI database, the gene of the recombinant protein CD2v was designed. The sequence is shown in SEQ ID NO: 1. The amino acid sequence of the recombinant protein CD2v is shown in Shown in SEQ ID NO:2. The gene of recombinant protein CD2v was obtained by gene synthesis (provided by Nanjing KingScript Biotechnology Co., Ltd.), and the gene of recombinant protein CD2v was inserted between EcoRI and HindIII of the polyclonal restriction site of vector pET-32a(+) , to obtain the recombinant plasmid pET-32a-CD2v.

[0041] By "heat shock" method, the recombinant plasmid pET-32a-CD2v was transformed into E.coli BL21(DE3) competent cells to obtain the recombinant ...

Embodiment 2

[0049] Embodiment 2 Preparation of sample diluent of the present invention

[0050] The sample diluent of the present invention: contains the African swine fever virus double protein-marker. The African swine fever virus dual protein-marker is a mixture of recombinant protein CD2v labeled with quantum dot microspheres and recombinant protein MGF360 labeled with quantum dot microspheres.

[0051] The preparation method of sample diluent of the present invention comprises the steps:

[0052] (1) Preparation of recombinant protein CD2v labeled with quantum dot microspheres

[0053] Quantum dot microsphere suspension with a concentration of 1.2 μg / μL (purchased from Beijing Najing Biotechnology Co., Ltd., product number FM610C, hydrated particle size is 120nm) and MES buffer with a concentration of 0.02M and pH 7.0 were mixed by volume. Mix at a ratio of 1:1, then add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 10 mg / mL NHS (N -Hydroxysuccinimide) and ...

Embodiment 3

[0060] The preparation of embodiment 3 test strips of the present invention

[0061] Such as Figure 3-5 , the test strip for detecting African swine fever virus CD2v and MGF360 mucosal antibodies includes a bottom plate 2, on which a sample pad 3, a nitrocellulose membrane 4 and an absorbent pad 7 are sequentially arranged. A test line 5 and a quality control line 6 are arranged on the nitrocellulose membrane 4, and the test line 5 is set near the side of the sample pad, and the test line is coated with a mouse anti-pig SC protein monoclonal antibody; the quality control line 6 is close to the water-absorbing pad 7 One side is set, and the quality control line is coated with purified protein of African swine fever positive serum.

[0062] 1. Preparation of purified protein from African swine fever positive serum

[0063] (1) Sample preparation

[0064]Recombinant protein CD2v and recombinant protein MGF360 were used as immunogens, and adult New Zealand white rabbits (1-2Kg...

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Abstract

The invention provides a test strip for detecting African swine fever virus CD2v and MGF360 mucous membrane antibodies and application of the test strip, and belongs to the field of production and technology of veterinary biological diagnosis products. The test strip comprises a nitrocellulose membrane provided with a detection line and a quality control line, wherein the detection line is coatedwith a mouse anti-swine SC protein monoclonal antibody; the quality control line is coated with purified protein of positive serum of African swine fever. the sample diluent contains quantum dot microsphere labeled recombinant protein MGF360 and recombinant protein CD2v; the sequence of the recombinant protein MGF360 is as shown in SEQ ID NO:4; the sequence of the recombinant protein CD2v is shownas SEQ ID NO:2. The test strip and the sample diluent are used for detecting the African swine fever virus CD2v and MGF360 mucous membrane antibodies, the test strip can be used for early detection of African swine fever infection, and have the advantages of strong specificity, high sensitivity and good stability.

Description

technical field [0001] The invention belongs to the production and technical field of veterinary biodiagnostic products, and in particular relates to a test strip for detecting African swine fever virus CD2v and MGF360 mucosal antibodies and an application thereof. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV) infection. , and is also a legally notifiable animal disease of the World Organization for Animal Health (OIE). [0003] ASFV is a complex, enveloped, double-stranded DNA virus with a diameter of 170-190kb, with terminal cross-linking and inverted repeat regions, which encode 151-167 proteins, and mature virions contain about 50 kinds of proteins structural protein. [0004] ASFV invades pigs mainly through the respiratory and digestive tracts, and the pigs respond to the infection. In addition to produci...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558G01N33/58G01N33/543G01N33/52
CPCG01N33/577G01N33/56983G01N33/558G01N33/54313G01N33/588G01N33/52G01N2333/01G01N2469/20
Inventor 冯志新刘斐白昀李佳豪陈蓉张磊韦艳娜张越李悦刘蓓蓓华利忠谢青云熊祺琰邵国青单衍可陆雨楠王丽
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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