Non-structural protein gene NS1 of avian influenza virus and its preparation method and use

A non-structural protein, avian influenza virus technology, applied in the field of animal virology, can solve the problems of proliferation, escape of live virus, incomplete virus inactivation, etc., and achieve the effect of high biological safety and low production cost.

Inactive Publication Date: 2005-12-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has incomplete virus inactivation, resulting in the potential danger of live virus escape and spread.

Method used

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  • Non-structural protein gene NS1 of avian influenza virus and its preparation method and use
  • Non-structural protein gene NS1 of avian influenza virus and its preparation method and use
  • Non-structural protein gene NS1 of avian influenza virus and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of NS1cNDA Sequence of Nonstructural Protein Gene of Avian Influenza Virus

[0054] Avian influenza virus (Avian influenza vir, as the original avian influenza virus strain of the present invention) was inoculated on 9-11 day-old chicken embryos, which was isolated by the applicant from a certain chicken farm infected with avian influenza virus in Hubei Province. The serotype of the strain was identified as H 9 N 2 , the strain was preserved in China Center for Type Culture Collection (CCTCC), date of preservation: May 20, 2004, preservation number: CCTCC-V200410. Us) to collect viruses. The avian influenza virus non-structural protein gene cDNA was amplified by RT-PCR using the viral RNA extracted from the avian influenza virus as a template. The primers used in RT-PCR were designed according to the NS1 genome sequence reported by NCBI, and its sequence is as follows:

[0055] P1: 5'-GCGTCGACATAATGGATTCCAAC-3' (upstream primer)

[0056] P2: 5'-CCACTCGAGCTA...

Embodiment 2

[0059] Construction of Prokaryotic Expression Vector of Nonstructural Protein NS1 Gene of Avian Influenza Virus

[0060] Digest pT-NS1 and vector pET-28a with SalI and XhoI, respectively, recover NS1 gene and vector pET-28a; then connect with T4 DNAligase, 16°C water bath overnight, transform Escherichia coli DH5α competent cells, culture at 37°C, randomly Multiple single colonies were selected and put into LB liquid medium for 12 hours of culture at 37°C, and then the plasmid was extracted from it. After identification by enzyme digestion, a positive recombinant plasmid was screened and named pET-28a-NS1.

Embodiment 3

[0062] Construction of recombinant Escherichia coli BL21 / pET-28a-NS1 and Escherichia coli BL21 / pET-28a

[0063]Transform the recombinant expression vector pET-28a-NS1 into Escherichia coli BL21 competent cells, spread LB kanamycin (Kana) plates, pick multiple single colonies and put them in LB liquid medium for 8 hours at 37°C, then use Isopropylthio-β-D-galactoside (IPTG) induced expression, followed by SDS-PAGE and dot blot analysis, from which a recombinant large intestine capable of inducing the expression of avian influenza virus non-structural protein NS1 in Escherichia coli BL21 was screened Bacillus Escherichia coli BL21 / pET-28a-NS1.

[0064] At the same time, the vector pET-28a was transformed into Escherichia coli BL21 cells synchronously, and the expression was induced in Escherichia coli BL21 according to the above method, and the blank control recombinant Escherichia coli BL21 / pET-28a without the expression of non-structural proteins of avian influenza virus was s...

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Abstract

The invention discloses the cDNA sequences of the non-structural protein gene NS1 of avian influenza virus, its preparation process and use, wherein gene is employed for constructing prokaryotic expression vectors, the expression vectors are transformed into bacillus coli to obtain recombinant strains (Escherichia coli BL21/pET-28a-NS1), the strains are utilized to express the non-structural protein NS1 of the avian influenza virus, the expression proteins are used to establish a differential diagnosis method through enzyme linked immunosorbent assay for differentiate vaccinum avian influenzae inactivatum immune chicken from infected chicken. The invention also relates to a differential diagnosis reagent kit and its application.

Description

technical field [0001] The invention relates to the technical field of animal virology. It specifically relates to a new recombinant Escherichia coli strain (Escherichia coli) BL21 / pET-28a-NS1 containing the non-structural protein gene NS1 of the avian influenza virus, which is used to express the non-structural protein NS1 of the avian influenza virus and to establish an enzyme-linked immunosorbent assay Test (ELISA) differential diagnosis method for distinguishing chickens immunized with avian influenza inactivated vaccine from chickens infected with wild virus. The present invention provides an expression plasmid (pET-28a-NS1) containing the non-structural protein gene NS1 of avian influenza virus, recombinant E. A differential diagnosis method that can distinguish chickens immunized with inactivated avian influenza vaccine from chickens infected with wild virus. Background technique [0002] Avian influenza (Avian influenza virus, AIV), also known as true fowl plague o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11
Inventor 金梅林陈焕春赵思婷王贵华张瑞华唐勇李红超刘正飞钱平郭爱珍方六荣曹胜波吴斌
Owner HUAZHONG AGRI UNIV
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