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Non-structural protein gene 3ABC of foot-and-mouth disease virus and its preparation and use

A non-structural protein, foot-and-mouth disease virus technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, sugar derivatives, etc., can solve the problems of live virus escape, spread, incomplete virus inactivation, etc., and achieve low production costs , High biosafety effect

Inactive Publication Date: 2005-04-13
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 3ABC of foot-and-mouth disease virus and its preparation and use
  • Non-structural protein gene 3ABC of foot-and-mouth disease virus and its preparation and use
  • Non-structural protein gene 3ABC of foot-and-mouth disease virus and its preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Cloning of cNDA Sequence of Nonstructural Protein Gene 3ABC of Foot-and-Mouth Disease Virus

[0058] The foot-and-mouth disease virus was inoculated on the passaged hamster kidney cells (BHK) covered with a single layer, and the virus was collected when 80% of the cells showed cytopathic changes. Using the extracted FMDV RNA as a template, 3ABC cDNA of FMDV nonstructural protein gene was amplified by RT-PCR. The primers used for RT-PCR are designed according to the genome sequence of FMDV reported by D.K.J Mackay et al. (Vaccine.1998,16(5):446-459), and its sequence is as follows:

[0059]P1: 5'-TGGATCCATGATTTCAATCCCTTCC-3' (upstream primer)

[0060] P2: 5'-GCGAATTCATCACTCGTGGTGTGG-3' (downstream primer)

[0061] The RT-PCR reaction system was: template RNA 8 μL, 2×Reaction buffer 25 μL, P1 and P2 primers 1.0 each (final concentration 10 pmol / L), Taq / mix 1.0 μL, add DEPC-treated water to 50 μL. The RT-PCR reaction conditions were 50□ for 30 minutes, 94□ for 1 minute;...

Embodiment 2

[0063] Construction of Prokaryotic Expression Vector of Foot-and-Mouth Disease Virus Nonstructural Protein 3ABC Gene

[0064] Digest pT-3ABC and vector pGEX-KG with BamHI and Xba I respectively, recover 3ABC gene and vector pGEX-KG; then connect with T4 DNA ligase, 16□ water bath overnight, transform DH5α competent bacteria, culture at 37□, randomly Multiple single colonies were selected, placed in LB liquid medium and cultured at 37°C for 12 hours, and then the plasmid was extracted from it. After enzyme digestion and identification, a positive recombinant plasmid was screened and named pKG-3ABC.

Embodiment 3

[0066] Construction of recombinant Escherichia coli BL21 / pKG3ABC and Escherichia coli BL21 / pKG

[0067] Transform the recombinant expression vector pKG-3ABC into Escherichia coli BL21 competent cells, spread LB / ampicillin (Amp) plates, pick multiple single colonies and put them in LB liquid medium for 12 hours at 37°C, and then use isopropyl Thio-D-D-galactoside (IPTG) induced expression, followed by SDS-PAGE and Western-blot detection, from which the recombinant Escherichiacoli BL21 / pKG3ABC capable of inducing the expression of foot-and-mouth disease virus non-structural protein 3ABC in Escherichia coli BL21 was screened out .

[0068] Simultaneously, the vector pGEX-KG was transformed into Escherichia coli BL21 cells synchronously, induced and expressed in Escherichia coli BL21 according to the above method, and a blank control recombinant Escherichia coli BL21 / pKG without expression of foot-and-mouth disease virus nonstructural protein 3ABC was screened out.

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Abstract

The present invention relates to non-structural protein gene 3ABC of foot-and-mouth disease virus, prokaryotic expression vector constituted with the gene, recombinant expression strain Escherichia coli BL21 / pKG3ABC obtained through transforming the vector to colibacillus BL21 and screening, method of expressing non-structural protein gene 3ABC of foot-and-mouth disease virus with the expression strain and purifying the expression protein, and the ELISA discrimination and diagnosis method established with the expression protein. The recombinant expression strain Escherichia coli BL21 / pKG3ABC is preserved in CCTCC and in the preservation number of CCTCC M203068.

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 / pKG3ABC) containing the non-structural protein gene 3ABC of foot-and-mouth disease virus, which is used to express the non-structural protein 3ABC of foot-and-mouth disease virus, and to establish an enzyme-linked immunosorbent assay (ELISA) for differential diagnosis The method is used to distinguish animals immunized with inactivated foot-and-mouth disease vaccines from animals infected with wild virus. The invention provides a fusion expression plasmid (pKG-3ABC) containing the nonstructural protein 3ABC gene of foot-and-mouth disease virus (pKG-3ABC), a recombinant Escherichia coli strain (Escherichiacoli BL21 / pKG3ABC), a method for expressing and purifying the 3ABC protein, and a distinguishable foot-and-mouth disease inactivation method established by using the protein Differential diagnostic ...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12N1/21C12N15/70C12P19/34G01N33/569G01N33/68
Inventor 陈焕春顾贫曹胜波钱平唐勇金梅林吴斌方六荣刘正飞吕建强
Owner HUAZHONG AGRI UNIV
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