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Recombinant water fowl flu rius H7 subtype hemagglutinin antigen and its prepn process and use

A technology of influenza virus and hemagglutinin, applied in the direction of viral peptides, recombinant DNA technology, pharmaceutical formulations, etc., can solve problems such as the confusion of influenza antibody detection methods, and achieve the effects of rapid and easy promotion, easy purification, and simple operation

Inactive Publication Date: 2006-05-17
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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  • Recombinant water fowl flu rius H7 subtype hemagglutinin antigen and its prepn process and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Expression and preparation method of recombinant waterfowl influenza virus H7 subtype hemagglutinin antigen:

[0028] 1. The cultivation of H7N1 subtype AIV and the extraction of RNA

[0029] Take the H7N1 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.

[0030] Synthesis of primers for amplifying the AIV hemagglutinin gene

[0031] According to the hemagglutinin gene sequence of AIV / H7 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 BglII and HindIII restriction sites respectively. Synthesize the following ...

Embodiment 2

[0054] Establishment of indirect ELISA detection technology for H7 subtype waterfowl influenza virus hemagglutinin antibody:

[0055] 1. Preparation of waterfowl AIV / H7 antibody detection plate

[0056] Use 0.05M Tris-HCl buffer solution with pH 8.5 as the coating solution, dilute water bird influenza virus H7 / HA protein to 10 μg / ml, add 100 μl / well to a detachable 96-well microtiter plate, 37 ° C for 2 hours, and then Coat overnight at 4°C, block with PBS containing 5% skimmed milk at 37°C for 2 hours, wash thoroughly with PBS (pH7.4) containing 0.05% Tween-20, and spin dry. Then add 20% sucrose phosphate buffer solution to protect at room temperature for 3 hours, put it in a drying room to dry, and then use it for the assembly of the H7 subtype waterfowl influenza virus hemagglutinin antibody indirect ELISA detection kit.

[0057] 2. Preparation of rabbit anti-waterfowl IgG polyclonal antibody

[0058] The whole blood of waterfowl was collected, the serum was separated, an...

Embodiment 3

[0072] Identification of H7 subtype influenza antibodies by AGIP:

[0073] 1. Equipment used in the experiment

[0074] Glass slide (2.6×7.6cm), level, capillary dropper, hole punch (commonly used drawing tip with a diameter of 3mm), wet box, etc.

[0075] 2. Experimental reagents

[0076]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.

[0077] 3. Method

[0078] 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).

[0079] 2. Punching: After the agar is cooled, use a puncher to punch holes, usually in...

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Abstract

The present invention discloses one kind of recombinant waterfowl flu virus H7 subtype hemagglutinin antigen and its preparation process and use. The recombinant waterfowl flu virus H7 subtype hemagglutinin antigen adopts colibacillus prokaryotic expression system to express the hemagglutinin gene of separated Chinese waterfowl flu virus strain, and the expressed hemagglutinin protein may be used as the antigen for detecting H7 subtype flu virus and infection generated hemagglutinin resisting protein antigen. Hemagglutinin antigen is the basis for classifying and diagnosing flu virus, so that the diagnosis technology established based on recombinant waterfowl flu virus hemagglutinin protein antigen may be used in detecting the waterfowl flu virus infection of duck, goose, etc. The recombinant membrane protein of the present invention has simple production, simple operation, high purity, excellent specificity and sensitivity, and may be purified for establishing even sensitive ELISA diagnosis method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant waterfowl influenza virus H7 subtype hemagglutinin antigen, a preparation method and use 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 far, the bird fl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11A61K39/145C12N15/09
Inventor 周继勇吴建祥陈洪勋
Owner ZHEJIANG UNIV
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