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Chicken infectivity bursa of Fabricius virus VP3 gene, expressed recombinant protein and application

A chicken infectious bursal disease technology, applied in the field of genetics, can solve the problems of difficult diagnosis and prevention of chicken infectious bursal disease, distinction between inimmunity and wild virus infection, low specificity, etc., to achieve fast and effective Differential diagnosis method, beneficial to purification, high sensitivity effect

Inactive Publication Date: 2007-07-04
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the above-mentioned methods, some have the problem of low specificity, some have the defects of cumbersome steps and high cost, and none of the existing diagnostic methods can distinguish VP2 subunit vaccine immunization from wild virus infection. The diagnosis and prevention of chicken infectious bursal disease have caused certain difficulties

Method used

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  • Chicken infectivity bursa of Fabricius virus VP3 gene, expressed recombinant protein and application
  • Chicken infectivity bursa of Fabricius virus VP3 gene, expressed recombinant protein and application
  • Chicken infectivity bursa of Fabricius virus VP3 gene, expressed recombinant protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] [Example 1] Preparation of Chicken Infectious Bursal Virus Recombinant VP3 Protein

[0045] 1. Test material

[0046] 1.1 Virus The vvIBDV-Gx strain was isolated, identified and preserved by the research group of the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute.

[0047] 1.2 Strains and plasmids Escherichia coli E.coli DH5α was preserved by our laboratory, and the expression vector pPROEX-HTa was purchased from Invitrogen Corporation of the United States.

[0048] 1.3 Tool enzymes and main reagents Ex Taq DNA polymerase, restriction enzymes EcoRI and HindIII, one-step reverse transcription PCR (RT-PCR) kit were purchased from Dalian Bao Biological Engineering Company; o-phenylenediamine and gel recovery reagents The box was purchased from Shanghai Huashun Biological Engineering Co., Ltd.; Isopropyl-beta-D-thiogalactopyranoside (IPTG), horseradish peroxidase-labeled rabbit anti-chicken IgG were purchased from Sigma Company of t...

Embodiment 2

[0064] [Example 2] Application of recombinant VP3 protein of the present invention in the establishment of chicken infectious bursal disease indirect ELISA diagnostic method

[0065] 1. Test materials

[0066] 1. Coating antigen: the recombinant VP3 protein purified in Example 1.

[0067] 2. Serum and animals used in the test: chicken infectious bursal disease standard positive serum and chicken infectious bursal disease virus VP2 protein standard positive serum were all prepared by the inventor using SPF chickens according to conventional methods, and standard negative serum was purchased from Harbin SPF Chicken Serum from Laboratory Animal Center of Veterinary Research Institute.

[0068] 2. Test method

[0069] The test method adopts the indirect ELISA method, and detects 12 antisera of chicken infectious bursal disease virus VP2, and uses the whole IBDV virus-coated plate to carry out the indirect ELISA test control.

[0070] Carry out on the microtiter plate according to...

Embodiment 3

[0083] Embodiment 3 Preparation of the indirect ELISA diagnostic kit of the present invention

[0084] 1. Preparation of ELISA plate coated with antigen

[0085] 1.1 Antigen preparation: the recombinant VP3 protein purified in Example 1 was used.

[0086] 1.2 Coating: The final concentration of the antigen is 2 μg / ml, the coating volume per well is 100 μl, cover the coated microwell plate, and incubate overnight in a refrigerator at 4°C;

[0087] 1.3 Washing: After incubation for 2 hours, discard the coating solution in the ELISA plate, and start washing (the composition and preparation of the washing solution is: 0.01M, PBS with pH 7.4 plus 0.5% Tween 20), wash each well 3 times, The time interval is 3 minutes each time. After washing, the coated board is buckled dry on absorbent paper.

[0088] 1.4 Blocking: blocking with a blocking agent (blocking agent is 0.5% horse serum in PBS), after the blocking is completed, cover the coated microwell plate, and place it in a 37° C....

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Abstract

The invention discloses a section cDNA in full length of chicken infectious bursal disease virus VP3 cDNA, method for preparing recombined protein by using its expressed carrier, an IBDV indirect ELISA diagnosis method by using said recombined protein and kit prepared with said recombined protein. The invention screens and analyzes the full sequence of chicken infectious bursal disease virus VP3 cDNA, chooses a section cDNA sequence that possesses richful epitope and can highly expressed in prokaryotic cells, establishes prokaryotic expression vector for prokaryotic expression, establishes IBDV indirect ELISA diagnosis method by using expressed recombined protein as coating antigen, and decides the optimal reaction condition. It provides a fast and simple serological identification and diagnosis method for immunity chicken antibody detection and epidemiological investigation. The invention also provides kits taking said recombined VP3 protein as coating antigen, and it is demonstrated that the kit is characterized by good sensitivity, spciality, stability and repeatability.

Description

technical field [0001] The present invention relates to a gene, in particular to a chicken infectious bursal virus VP3 gene, the construction and transformation of the gene expression vector, the expressed recombinant protein and the detection or diagnosis of infectious bursal virus prepared by the recombinant protein A virus test kit belongs to the field of genetic engineering. Background technique [0002] Chicken infectious bursal disease (IBD) is an acute highly contagious disease of chickens and turkeys caused by infectious bursal virus (IBDV). It mainly injures chicks and young chickens of 3-12 weeks of age, and brings huge losses to the poultry industry of countries all over the world. The target cells of IBDV are immature B lymphocytes or B prelymphocytes, which undergo degeneration and necrosis soon after infection. Since the bursa is an important organ for the maturation of B cells, after chickens are infected with IBDV, a large number of B cells in the lymphoid ...

Claims

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Application Information

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IPC IPC(8): C12N15/33C12N15/63C07K14/005C12P21/02G01N33/535A61K39/12
Inventor 王笑梅高宏雷高玉龙付朝阳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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