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Colloidal gold immune chromatography fast differential diagnosis kit of chicken avian influenza vaccine immunity and virus strain infection and application

An avian influenza virus and influenza vaccine technology, which is applied in the fields of immunology and animal molecular biology, can solve the problems of false positives, increase the difficulty of avian influenza monitoring, and does not involve avian influenza vaccine immunization and differential diagnosis technology of wild virus infection. The effect of low production cost and high biosafety

Inactive Publication Date: 2007-08-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since 2004, H5N1 subtype highly pathogenic avian influenza has occurred successively in some Asian countries and regions, and my country has not been spared, which has caused huge economic losses to my country's poultry industry. Therefore, the Chinese government has a high degree of control over avian influenza. Attention has been paid to it, and a series of prevention and control measures have been put forward successively, requiring poultry to be immunized with avian influenza vaccine. However, with the widespread use of inactivated avian influenza vaccines, it has also increased the difficulty of monitoring avian influenza and is prone to false positives. Therefore, It is an urgent problem to establish a differential diagnosis method that can distinguish chicken avian influenza virus wild virus infection from vaccine immunity
[0005] Retrieval of existing literature For avian influenza virus antigen, antibody detection and colloidal gold immunochromatography technology, several invention patents and applications have been published, such as the patent number: ZL200410088716.8 "A method for detecting H5N1 subtype avian influenza virus The method and its special kit, the patent application number is 200510042631.0 "Avian influenza virus antigen detection method and its gold label rapid diagnostic kit and preparation method, the patent application number is 200510057023.7 "Avian influenza immune colloidal gold diagnostic test strip and "Test card" discloses several preparation methods for detecting avian influenza virus antigens, but does not involve the detection of avian influenza antibodies; The document ZL200510011521.8 "A Monoclonal Antibody, a Detection Kit Containing the Monoclonal Antibody and Its Application" discloses the detection method of avian influenza virus serum antibody, but does not involve the differential diagnosis technology of avian influenza vaccine immunization and wild virus infection ;Patent application "Method for Differential Diagnosis of Avian Influenza Naturally Infected Chickens by ELISA (Application No. 200510027316.0)" and "A Non-Structural Protein Gene NS1 of Avian Influenza Virus and Its Preparation and Application (Patent No. ZL200410009157. 7)" and "a kit for detecting natural infection of avian influenza virus and its preparation method (application number is 200310117066.0)" disclosed the ELISA method for the differential diagnosis of avian influenza vaccine immunization and wild virus infection, but did not involve Colloidal gold immunochromatography

Method used

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  • Colloidal gold immune chromatography fast differential diagnosis kit of chicken avian influenza vaccine immunity and virus strain infection and application
  • Colloidal gold immune chromatography fast differential diagnosis kit of chicken avian influenza vaccine immunity and virus strain infection and application
  • Colloidal gold immune chromatography fast differential diagnosis kit of chicken avian influenza vaccine immunity and virus strain infection and application

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Experimental program
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Effect test

Embodiment 1

[0034] 1. Preparation and purification of monoclonal antibodies

[0035] A monoclonal hybridoma cell line secreting anti-chicken IgG Fc fragment (preservation number CCTCC-C200501) prepared by the applicant was injected into the peritoneal cavity of Balb / C mice to induce ascites and produce monoclonal antibodies. Purify the monoclonal antibody with reference to the method in Zhu Liping's "Common Experimental Methods in Immunology" People's Military Medical Publishing House 2000 Edition: Take 5ml of the obtained mouse ascites and mix it with an appropriate amount of silicon dioxide, add an equal volume of barbiturate buffer, After shaking at room temperature for 1 hour, let stand at room temperature for 30 minutes, take the supernatant in a clean centrifuge tube, and centrifuge at 3000 r / m for 10 minutes at 4°C; take 8ml of the supernatant, add 16ml of 0.06mol sodium acetate buffer, and adjust the pH value to 4.5 with HCL After fully stirring, slowly add 132 μl of octanoic acid...

Embodiment 2

[0037] Preparation of recombinant chicken avian influenza virus NS1 protein:

[0038] For the preparation method, please refer to the literature: Sambrook J, Fritsch EF, Manny Attis T, editors (translated by Jin Dongyan, Li Mengfeng, etc.). Molecular Cloning Experiment Guide. Second Edition, Beijing Science Press, 1992 The method provided in .

[0039] 1. Cloning and sequencing of NS1 gene

[0040] The NS1 gene involved in the present invention is cloned by the applicant himself, and the specific method of cloning and sequencing of the NS1 gene is: use an avian influenza strain A / chicken / China / HSS2004 (H9N2) [GeneBank accession number is AY788915, virus isolation and identification see literature: Li Zili et al. Diagnosis of avian influenza in Hubei Province and isolation and preliminary identification of virus strains, Chinese Journal of Preventive Veterinary Medicine, 2001, 23(3): 146 -148] Inoculate AIV into the allantoic cavity of 9-11-day-old healthy chicken embryos, d...

Embodiment 3

[0046] Preparation of rabbit anti-mouse IgG antibody:

[0047] Refer to the method taught by He Zhaoyang et al., "Experimental Technology of Animal Immunology" Changchun, Jilin Science and Technology Press (2002 edition), extract Balb / C mouse IgG immune healthy rabbits, and prepare high-specificity, high-potency rabbit anti-mouse For IgG hyperimmune serum, saturated ammonium sulfate precipitation method is used for crude extraction of hyperimmune serum, and high-purity rabbit anti-mouse IgG antibody is obtained after passing through G-200 column. Use this antibody as a quality control line.

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Abstract

The invention discloses a rapid identifying diagnostic agent box and usage of flock influvaxin and virus and making method of BL21 / pKG-NS1 ( CCTCC NO: M207007) to express recombinant NS1 protein, which comprises the following parts: box, enzyme marking board, indicator paper and dilute liquid of sample, wherein the indicator paper is composed of adsorbing pad, cellulose nitrate film with detecting line of non-structural protein NS1 and quality control line of anti-mouse lgG, metal marking pad coated by anti-cock lgG Fc monoclone antibody, sample pad on the non-adsorbing support film sequently.

Description

technical field [0001] The invention belongs to the field of immune application and relates to related fields such as animal molecular biology and immunology. Specifically, it is an immunochromatographic reaction with colloidal gold labeling and color development, which is used for the rapid differential diagnosis of bird flu vaccine immunization in chickens and wild virus infection. Background technique [0002] As we all know, avian influenza, Newcastle disease, bursa, infectious bronchitis, infectious laryngotracheitis, Marek, egg drop syndrome and other diseases are important diseases that endanger the global poultry industry, especially avian influenza. Since 2004, H5N1 subtype highly pathogenic avian influenza has occurred successively in some Asian countries and regions, and my country has not been spared, which has caused huge economic losses to my country's poultry industry. Therefore, the Chinese government has a high degree of control over avian influenza. Attenti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21G01N33/68G01N33/577G01N33/569G01N33/53C07K16/18A61K35/74A61K39/02C12R1/19A61K35/741
Inventor 毕丁仁胡思顺郑兴华王喜亮张万坡肖运才刘梅李自力石德时许青荣金秀娥
Owner HUAZHONG AGRI UNIV
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