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Recombined aquatic bird flu virus H5 subtype hemagglutinin, its preparation method and uses

An influenza virus and hemagglutinin technology, applied in the biological field, can solve problems such as confusion of influenza antibody detection methods, and achieve the effects of rapid and easy promotion, easy production and high purity

Inactive Publication Date: 2006-06-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the particularity of the species of waterfowl, the detection method of its influenza antibody has always been a problem that perplexes the evaluation of the immune effect of waterfowl vaccines and the diagnosis of waterfowl influenza.

Method used

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  • Recombined aquatic bird flu virus H5 subtype hemagglutinin, its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Expression and preparation method of recombinant waterfowl influenza virus H5 subtype hemagglutinin antigen:

[0029] 1. The cultivation of H5N1 subtype AIV and the extraction of RNA

[0030] Take the H5N1 virus and inoculate it into 9-11-day-old SPF chicken embryos through the allantoic cavity, incubate in a 35°C incubator, collect the allantoic fluid of dead chicken embryos after 24 hours, purify the virus by differential centrifugation, and resuspend the virus in TEN buffer , RNA extraction was carried out according to the instructions of the Trizol kit; the extracted RNA was used for RT-PCR.

[0031] Synthesis of primers for amplifying the AIV hemagglutinin gene

[0032] According to the hemagglutinin gene sequence of AIV / H5 subtype in Genebank, a pair of primers at the 5' end and 3' end of the ORF of the hemagglutinin gene were designed. The 5' end primer and the 3' end primer introduced BamH I and Hind III restriction sites, respectively. Synthesize the followi...

Embodiment 2

[0055] Establishment of indirect ELISA detection technology for H5 subtype waterfowl influenza virus hemagglutinin antibody:

[0056] 1. Preparation of waterfowl AIV / H5 antibody detection plate

[0057] Use 0.05MTris-HCl buffer solution with pH 8.5 as the coating solution, dilute water bird influenza virus H5 / HA protein to 10μg / ml, add 100μl / well to a detachable 96-well microtiter plate, 37°C for 2 hours, and then 4 ℃ coated overnight, blocked with PBS containing 5% skimmed milk at 37 ℃ for 2 hours, fully washed with PBS (pH 5.4) containing 0.05% Tween-20, and spin-dried. Then add 20% sucrose phosphate buffer solution to protect at room temperature for 3 hours, dry in a drying room, and then use for assembly of H5 subtype waterfowl influenza virus hemagglutinin antibody indirect ELISA detection kit.

[0058] 2. Preparation of rabbit anti-waterfowl IgG polyclonal antibody

[0059] The whole blood of waterfowl was collected, the serum was separated, and the IgG of waterfowl wa...

Embodiment 3

[0073] Identification of H5 subtype influenza antibodies using AGIP (AGIP):

[0074] 1. Equipment used in the experiment

[0075] Glass slide (2.6×7.6cm), level, capillary dropper, hole punch (commonly used drawing tip with a diameter of 3mm), wet box, etc.

[0076] 2. Experimental reagents

[0077] pH8.6 0.1M barbiturate buffer: barbital sodium 10.3g, barbiturate 1.84g, thimerosal 100mg, distilled water to 500.0ml. 1-1.2% agar gel: 1.1-1.2g of high-quality agar powder, 50.0ml of distilled water, boil and dissolve in a water bath, add 50.0ml of the above-mentioned warm barbiturate buffer, mix well, and pack (each test tube 3.5-4.0 ml, poured on a glass slide after melting), and stored at 4°C for later use.

[0078] 3. Method

[0079] 1. Plate making: Put the glass slide on the water platform, boil the melted agar in the water bath and pour the plate (one test tube of agar pours one glass slide).

[0080] 2. Punching: After the agar is cooled, use a puncher to punch holes,...

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Abstract

The invention discloses the recombinant water avian influenza virus H5 hypotype hemagglutinin antigen and preparing method and application. The recombinant water avian influenza virus H5 hypotype hemagglutinin antigen adopts the colibacillus pronucleus expression system to express the hemagglutinin gene of water avian influenza virus segregation strain in our country. Utilizing the expression hemagglutinin protein as antigen, H5 hypotype influenza virus and anticoagulant protein antibody are detected. The diagnostic technique on the base of recombinant water avian influenza virus hemagglutinin protein antigen can detect the infection of duck and goose. The recombinant resin protein produced by the invention is not only simple operation, but also high purity and good specificity and sensibility. After the recombinant resin protein is purified, it can be used in pack the polystyrene micro reaction plate to set up ELISA diagnose method whose sensitivity is higher.

Description

technical field [0001] The invention relates to the field of biotechnology, and further relates to a recombinant waterfowl influenza virus H5 subtype hemagglutinin antigen, a preparation method and an application thereof. Background technique [0002] Avian influenza (avian influenza), the full name of avian influenza virus infection, is an infectious disease of poultry caused by type A influenza virus of Orthomyxoviridae and Influenza virus, from respiratory system infection to sepsis, etc., poultry The fatality rate after infection is high. Bird flu was first discovered in Italy by Perroncito in 1878. Its pathogen was discovered for the first time in 1900, and it was only confirmed by serology in 1955 that it belonged to type A (A) influenza virus. Since then, avian influenza viruses have been prevalent in poultry around the world, with varying degrees of impact. In particular, the outbreak of bird flu in Hong Kong in 1997 has already posed a threat to human health. So...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K16/28C12N1/21C12N15/13C12N15/70G01N33/53
Inventor 周继勇吴建祥陈洪勋
Owner ZHEJIANG UNIV
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