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

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

Example Embodiment

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

[0039] 1. Construction of recombinant bacteria for the preparation of 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, and the amino acid sequence of the recombinant protein CD2v is shown in shown in SEQ ID NO:2. The gene of the recombinant protein CD2v was obtained by gene synthesis (by Nanjing GenScript Biotechnology Co., Ltd.), and the gene of the recombinant protein CD2v was inserted between the polyclonal restriction sites EcoRI and HindIII of the vector pET-32a(+). , the recombinant plasmid pET-32a-CD2v was obtained.

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

Example Embodiment

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

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

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

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

[0053] The quantum dot microsphere suspension with a concentration of 1.2 μg / μL (purchased from Beijing Nano Crystal Biotechnology Co., Ltd., product number is FM610C, hydrated particle size is 120 nm) and a concentration of 0.02 M, pH 7.0 MES buffer 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), incubat...

Example Embodiment

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

[0061] like Figure 3-5 , the test strips for detecting African swine fever virus CD2v and MGF360 mucosal antibodies include a bottom plate 2, and a sample pad 3, a nitrocellulose membrane 4 and a water absorption pad 7 are arranged on the bottom plate in sequence. The nitrocellulose membrane 4 is provided with a detection line 5 and a quality control line 6. The detection line 5 is set near the side of the sample pad, and the detection line is coated with mouse anti-pig SC protein monoclonal antibody; the quality control line 6 is close to the absorbent pad 7 Set on one side, the quality control line is coated with purified protein from African swine fever positive serum.

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

[0063] (1) Sample preparation

[0064]Using recombinant protein CD2v and recombinant protein MGF360 as immunogens, adult New Zealand white rab...

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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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