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

A bird flu virus and non-structural protein technology, applied in the field of animal virology, can solve the problems of live virus escape, spread, and incomplete virus inactivation, and achieve the effect of low production cost and high biological safety

Inactive Publication Date: 2007-03-07
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

[0054] Cloning of avian influenza virus non-structural protein gene NS1 cDNA sequence

[0055] 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 obtained by the applicant from a chicken farm infected with avian influenza virus in Hubei Province. Identify the serotype of this strain as H 9 N 2 , The strain is conserved in the China Center for Type Culture Collection (CCTCC), preservation date: May 20, 2004, preservation number: CCTCC-C200410. Collect viruses. Using the viral RNA extracted from the avian influenza virus as a template, the non-structural protein gene cDNA of the avian influenza virus was amplified by RT-PCR. The primers used in RT-PCR are designed according to the NS1 genome sequence reported by NCBI, and the sequence is as follows:

[0056] P1: 5’-GCGTCGACATAATGGATTCCAAC-3’ (upstream primer)

[0057] P2: 5’-CCACTCGAGCTATTTTGGAGAGAG-3’ (downstrea...

Embodiment 2

[0060] Construction of Prokaryotic Expression Vector of Non-structural Protein NS1 Gene of Avian Influenza Virus

[0061] Use Sal and Xhol to digest pT-NS1 and vector pET-28a respectively, and recover NS1 gene and vector pET-28a; then ligate with T4 DNAligase, and bathe overnight at 16°C to transform E. coli DH5α competent cells, culture at 37°C, and randomly A number of single colonies were selected and placed in LB liquid medium at 37°C for 12 hours, and plasmids were extracted from them. After identification by restriction enzyme digestion, a positive recombinant plasmid was obtained and named pET-28a-NS1.

Embodiment 3

[0063] Construction of Recombinant Escherichia coli BL21 / pET-28a-NS1 and Escherichia coli BL21 / pET-28a

[0064]Transform the recombinant expression vector pET-28a-NB1 into E. coli BL21 competent cells, spread LB kanamycin (Kana) plates, select multiple single colonies and place them in LB liquid medium at 37°C for 8 hours and then use them separately Isopropylthio-β-D-galactoside (IPTG) was induced to express, and then SDS-PAGE and dot blot analysis were performed to screen out recombinant large intestine capable of inducing expression of avian influenza virus non-structural protein NS1 in E. coli BL21 Escherichia coli BL21 / pET-28a-NS1.

[0065] At the same time, the vector pET-28a was synchronously transformed into E. coli BL21 cells, and the expression was induced in E. coli BL21 according to the above method, and a blank control recombinant Escherichia coli BL21 / pET-28a with no non-structural protein expression of avian influenza virus was selected. .

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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. Specifically, it relates to a new recombinant Escherichia coli strain (Escherichia coli) BL21 / pET-28a-NS1 containing avian influenza virus non-structural protein gene NS1, which is used to express avian influenza virus non-structural protein NS1 and establish an enzyme-linked immunosorbent assay Test (ELISA) differential diagnosis method is used to distinguish chickens immunized with inactivated avian influenza vaccine from chickens infected with wild virus. The present invention provides an expression plasmid (pET-28a-NS1) containing avian influenza virus non-structural protein gene NS1, recombinant E. coli strain Escherichia coli BL21 / pET-28a-NS1, NS1 protein expression and purification methods, and methods for constructing the protein The differential diagnosis method can distinguish between chickens immunized with inactivated avian influenza vaccine and chickens infected with wild virus. B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/44C12N1/21G01N33/569C12Q1/68C07H21/04
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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