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Bovine coronavirus VPN (virtal private network) gene, encoded recombinant protein and application thereof

A technology of coronavirus and bovine crown, applied in the field of genetic engineering, can solve problems such as error-prone, complicated operation, expensive equipment, etc., and achieve the effect of accurate results, strong specificity and low cost

Inactive Publication Date: 2018-10-30
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the neutralization test is a fast, accurate, high-sensitivity and low-cost method, it is complex in operation and prone to errors, so it is not suitable for rapid clinical diagnosis
The HI test has disadvantages such as strong subjectivity, difficult to standardize the interpretation of the judgment results, and is not suitable for large-scale detection; the indirect immunofluorescence test requires high technical requirements for operators and expensive equipment, which is not conducive to popularization; The virus is an antigen, and there is a great risk of spreading the virus

Method used

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  • Bovine coronavirus VPN (virtal private network) gene, encoded recombinant protein and application thereof
  • Bovine coronavirus VPN (virtal private network) gene, encoded recombinant protein and application thereof
  • Bovine coronavirus VPN (virtal private network) gene, encoded recombinant protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 bovine coronavirus recombinant VPN protein

[0039] 1. Test materials

[0040] 1. Bovine coronavirus (SWUN7901) was provided by the Animal Medicine Laboratory of Southwest University for Nationalities.

[0041] 2. Bacterial strains and plasmids: Escherichia coli Rosetta (DE3) was purchased from Tiangen Company, and the expression vector BCoV VPN was purchased from Invitrogen Company of the United States.

[0042] 3. Tool enzymes and main reagents: Taq DNA polymerase, restriction enzymes BamHI and XhoI, one-step reverse transcription PCR (RT-PCR) kit were purchased from Dalian Bioengineering Company; tetramethylbenzidine was purchased from Thermo Company; Isopropyl-beta-D-thiogalactopyranoside (Isopropyl-beta-D-thiogalactopyranoside, IPTG) was purchased from SIGMA Company, horseradish peroxidase-labeled goat anti-bovine IgG was purchased from KPL Company of the United States; other Reagents were analytically pure products purchased from co...

Embodiment 2

[0054] Embodiment 2 Application of the recombinant VPN protein of the present invention in the establishment of bovine coronavirus indirect ELISA diagnostic method

[0055] 1. Test materials

[0056] 1. Coating antigen: the recombinant VPN protein purified in Example 1.

[0057] 2. Serum for test: bovine coronavirus positive serum is screened from clinical samples by kit (svanova company), and negative serum is fetal bovine serum purchased from BI company.

[0058] 2. Test method

[0059] The test method adopts the indirect ELISA method, detects 20 bovine coronavirus VPN antiserum, and conducts the indirect ELISA test control with BCoV whole virus-coated plates.

[0060] Carry out on the microtiter plate according to the conventional method. The recombinant VPN protein of the present invention was diluted with 50 mM pH 7.6 phosphate buffer solution and coated on a microtiter plate, 100 μL / well, overnight at 4°C. The coating solution was discarded the next day, and washed t...

Embodiment 3

[0076] Embodiment 3 The specificity test of the indirect ELISA diagnostic method of the present invention

[0077] 1. Test materials

[0078] 1. Coating antigen: 1) the recombinant VPN protein purified in Example 1.

[0079] 2. Serum for the test: The bovine coronavirus positive serum was screened from clinical samples by the kit svanova company, and the negative serum was fetal bovine serum purchased from BI company.

[0080] 3. Viruses: bovine viral diarrhea virus (BVDV); bovine rotavirus ((BRV); bovine enterovirus (BEV); bovine chlamydia (CVM); bovine mycobacterium tuberculosis (M.bovis); Q-fever (Q-Fever).

[0081] 2. Cross-test method

[0082] The test method adopts the indirect ELISA method to detect other 6 kinds of cattle diseases, and uses the BCoV commercial kit to coat the plate to carry out the indirect ELISA test control.

[0083] The recombinant VPN protein of the present invention was diluted with 50 mM phosphate buffer solution of pH 7.6 and coated on a mic...

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Abstract

The invention discloses a bovine coronavirus VPN (virtal private network) gene, encoded recombinant protein and an application thereof. A BCoV (bovine coronavirus) indirect ELISA (enzyme linked immunosorbent assay) method is established by using the recombinant protein. The bovine coronavirus VPN has the advantages of having a good antigenic epitope and high expression in prokaryotic cells, constructing a prokaryotic expression vector, successfully establishing the BCoV indirect ELISA diagnostic method by coating the antigen with the expressed recombinant protein, determining the best conditions for the method to provide a quick and easy serological differential diagnosis method for immunological detection of bovine antibodies and epidemiological investigation, and solving current drawbacks of a diagnosis method of yak coronavirus diarrhea, which is time-consuming, laborious and cannot be diagnosed quickly, and expensive expenses of biological reagents and instruments.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bovine coronavirus VPN gene, encoded recombinant protein and application thereof. Background technique [0002] Bovine coronavirus (Bovne Corona virus, BCoV) belongs to the order of Bovneviridae, Coronaviridae, and one of the members of subgroup 2a of the genus Coronaviridae. It is one of the main pathogens that cause diarrhea and respiratory diseases in cattle. Diarrhea in calves, bloody feces, severe watery diarrhea (sometimes with blood and mucus) in adult cattle in winter, and respiratory infections in cattle of all ages are characteristic of Mebsu et al. (Mebus C A, Stair E L, Rhodes M B, et al. Pathology of neonatal calf diarrhea induced by a coronavirus-like agent [J]. Veterinary Pathology, 1973, 10(1): 45-64.) After the first report of BCoV in the United States, it has been reported in Japan, Ireland, Canada, and Argentina in adult cattle and cal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C07K14/165C12N15/70G01N33/569
CPCC07K14/005C12N15/70C12N2770/20022G01N33/56983
Inventor 汤承岳华阿比克哈莫王远微
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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