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An expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof

A technology for bovine viral diarrhea and a kit, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of time-consuming, labor-intensive, and unsuitable for use in pastoral areas, saving labor and time, avoiding difficulties in renaturation, and having wide application prospects. Effect

Active Publication Date: 2022-07-05
内蒙古元山生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation, serum neutralization experiments, and other methods are time-consuming and laborious. RT-PCR, electron microscope observation, etc. require professionals, and are not suitable for use in pastoral areas

Method used

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  • An expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof
  • An expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof
  • An expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof

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Experimental program
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Effect test

preparation example Construction

[0059] Optionally, the preparation method of the expressed protein at least includes the following steps:

[0060] (1) Query the E2 gene sequence of bovine viral diarrhea virus, optimize the E2 gene sequence according to the preferred codon table of Escherichia coli, add the signal peptide sequence and synthesize it, and obtain the optimized SP-E2 gene sequence, SP-E2 gene sequence The nucleotide sequence is shown in SEQ ID NO: 1;

[0061] (2) Construction of E2 gene expression strain: The optimized nucleotide sequence of SP-E2 gene is connected to the prokaryotic expression vector pET-32a(+) to obtain the recombinant plasmid pET-32a-SP-E2, and the constructed recombinant plasmid pET-32a-SP-E2 was transformed into Escherichia coli BL21 cells, and the recombinant expression strain of SP-E2 gene was constructed;

[0062] (3) Induction, expression and purification of the expressed protein: IPTG was used to induce the expression of the target protein, and the target protein in th...

Embodiment 1

[0083] A kind of ELISA kit for detecting bovine viral diarrhea virus, its composition is as shown in table 1:

[0084] Table 1

[0085] name composition Quantity (marked quantity) Sample Diluent 0.5wt% PEG 6000 1 bottle (250ml) 10× Concentrated Washing Solution PBST with 0.5% (v / v) Tween 20 1 bottle (250ml) enzyme-labeled antibody HRP-labeled rabbit anti-bovine IgG 1 bottle (30ml) Substrate chromogen TMB 1 bottle (30ml) Stop solution 2mol / L dilute sulfuric acid 1 bottle (30ml) 96-well plate coated with BVDV antigen — 5 pieces positive control Standard positive serum 1 bottle (2ml) negative control standard negative serum 1 bottle (2ml) Sealing film — 10 sheets manual — 1 serving

Embodiment 2

[0086] Example 2: Bioinformatics analysis and expression of BVDV-E2 gene

[0087] 1 Materials and methods

[0088] 1.1 Test material

[0089] Escherichia coli BL21 is a product of Quanxingjin Company, DNA restriction enzymes are purchased from NEB Company, DNA gel recovery and purification kit is a product of AXYGENG Company, and a plasmid mini kit is purchased from QIAGEN Company; yeast powder, tryptone, agar The powder was a product of Oxoid Company, the rabbit anti-bovine IgG labeled with horseradish peroxidase was purchased from Sigma Co., Ltd., and the BCA protein quantitative detection kit was purchased from TAKARA Company.

[0090] 1.2 Test method

[0091] 1.2.1 Bioinformatics analysis of E2 gene

[0092] The BVDV(DQ088995.2) E2 gene was analyzed by DNAstar, Swiss and other software and online server, and the size, signal peptide, secondary structure, tertiary structure, antigenicity and hydrophobicity of the protein encoded by the gene were predicted.

[0093] 1.2.2 ...

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Abstract

The invention relates to the field of biological products, in particular, to an expression protein for detecting bovine viral diarrhea virus antibody, an ELISA kit and a preparation method and application thereof. The present invention relates to an expressed protein for detecting bovine viral diarrhea virus antibody. The nucleotide sequence encoding the expressed protein is shown in SEQ ID NO: 1, and the expressed protein is supernatant protein. The present invention also relates to an ELISA kit for detecting bovine viral diarrhea virus antibody, the ELISA kit comprising an ELISA plate coated with the expressed protein. The kit of the invention has the technical advantages of high biological safety, low cost, high coincidence rate, good repeatability, and high specificity and sensitivity.

Description

technical field [0001] The invention relates to the field of biological products, in particular, to an expression protein for detecting bovine viral diarrhea virus antibody, an ELISA kit and a preparation method and application thereof. Background technique [0002] Bovine viral diarrhea virus (BVDV) is the main pathogen causing bovine viral diarrhea disease (BVD). The clinical symptoms are mainly diarrhea, growth and reproduction disorders, immunosuppression, acute or chronic mucosal disease and persistent infection. BVDV belongs to the genus Pestivirus of the family Flaviviridae. It has high homology with the other two swine fever viruses and border disease viruses in the genus Pestivirus, has structural and antigenic correlations, and has serological cross-reaction. According to different classification criteria, BVDV can be divided into one serotype, two biotypes (cytopathic biotype, cp) and non-cytopathic biotype (ncp) and three genotypes (BVDV-1, BVDV-2 and BVDV-3)....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/558
CPCG01N33/56983G01N33/558
Inventor 温永俊月英温永胜杨洋何芳
Owner 内蒙古元山生物科技有限公司
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