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Expression and application of bovine viral diarrhea virus (BVDV) type I E2 protein

A bovine viral diarrhea and virus technology, applied in the direction of viruses, viral peptides, viruses/bacteriophages, etc., can solve the problems of no correct structure and poor activity

Inactive Publication Date: 2016-09-21
CHINA INST OF VETERINARY DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic has adopted multiple ways to carry out prokaryotic expression to it (as: Xu Xingran etc., the cloning of bovine viral diarrhea virus Changchun184 strain E2 gene and the highly efficient expression in large intestine bar, Chinese Journal of Preventive Veterinary Medicine, 2005, 27 (2) : 98-101; Yang Youwu et al., Expression of Bovine Viral Diarrhea Virus E2 Protein and Preparation of Polyclonal Antibody, Advances in Animal Medicine, 2013, 34(9): 128-132), but the prokaryotic expression product was inclusion body without correct structure , poor activity

Method used

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  • Expression and application of bovine viral diarrhea virus (BVDV) type I E2 protein
  • Expression and application of bovine viral diarrhea virus (BVDV) type I E2 protein
  • Expression and application of bovine viral diarrhea virus (BVDV) type I E2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] ──Construction of recombinant baculovirus

[0101] 1. Transposition to obtain recombinant bacmid DNA

[0102] The bacmid DNA constructed by transforming pFB-ME2-His into DH10Bac was named: bME2-His.

[0103] Reagent preparation:

[0104] SOC medium: 2% peptone, 0.5% yeast extract, 10.0mM Nacl, 2.5mM KCl, after autoclaving the above components, add the filtered Mg 2+ solution and glucose solution, adjusted to a concentration of: 20mM MgSO 4 , 10mM MgCl 2 , 20 mM glucose.

[0105] LA-Bac resistant agar plate: tryptone 10g, yeast extract 5g, Nacl 10g, agar 12g, dissolved in 1000ml deionized water, autoclaved, after cooling, add kanamycin to 50μg / ml, gentamicin to 7 μg / ml, tetracycline to 10 μg / ml, IPTG to 40 μg / ml, Bluo gal to 100 μg / ml.

[0106] Resistant LB medium: tryptone 10g, yeast extract 5g, Nacl 10g, dissolved in 1000ml deionized water, autoclaved, cooled to about 55°C, added kanamycin to 50μg / ml, gentamicin to 7μg / ml, tetracycline poison to 10μg / ml.

[01...

Embodiment 2

[0126] ──BVDV indirect immunofluorescence detection kit detection

[0127] Collect 180 samples of bovine serum in the field, subculture the MDBK cell line in the logarithmic growth phase, and add it to a 24-well plate. When the cells grow to 60% to 80% full, blot the medium and inoculate the diluted bovine serum sample , each sample was inoculated into 4 wells, 0.5 mL per well, 37 °C 5% CO 2 After 1.5 hours of adsorption in the incubator, replace with 2% FBS MEM maintenance solution per well 0.5mL, 37°C 5% CO 2 After culturing in the incubator for 48 hours, blot the culture medium dry, add 0.5 mL of pre-cooled acetone:methanol (1:1), fix for 30 min, blot dry, add 0.5 mL PBS to each well for rinsing once after air drying. Using the BVDV indirect immunofluorescence detection kit established in this study and the AHVLAMcAb staining solution, 180 field bovine serum samples were detected simultaneously, with 2 wells for each detection, and the detection rate of BVDV was counted. ...

Embodiment 3

[0129] ——Comparison of BVDV antibody indirect ELISA kit and IDEXX kit

[0130] 749 bovine serum samples were detected with the BVDV antibody indirect ELISA kit developed in this study, and the coincidence rate of the two was counted. Results The positive coincidence rate between the self-made BVDV antibody indirect ELISA kit and the IDEXX ELISA kit was 92.9%, the negative coincidence rate was 97.7%, and the total coincidence rate was 95.7% (Table 2).

[0131] Table 2 Concordance rate between self-made BVDV antibody indirect ELISA kit and IDEXX ELISA kit

[0132]

[0133]

[0134]

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Abstract

The invention relates to expression and application of bovine viral diarrhea virus (BVDV) type I E2 protein. The expression and the application have the advantages that (1) an expression and purification method for efficiently obtaining BVDV E2 protein is established and can be used for preparing the pure BVDV E2 protein; (2) a rabbit is immunized by the BVDV E2 protein to prepare an antibody; compared with an antibody prepared by whole-virus immune cattle, the antibody has good specificity and shallow background and is easy to determine; a secondary antibody is marked by FITC (Fluorescein Isothiocyanate) and an indirect fluorescence method is established, so that the sensitivity is better; the operation is simple: a pre-mixed solution is adopted so that operation time is shortened; (3) the pure BVDV E2 protein is used for covering, sealing conditions, blood serum and antibody preserving fluid are optimized, and a BVDV antibody indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method is established; a BVDV antibody indirect ELISA kit, which is sensitive and low in price, is assembled and a situation of dependence on imported BVDV antibody ELISA kits for a long period can be changed.

Description

technical field [0001] The invention relates to the expression of bovine viral diarrhea virus type I E2 protein and the assembly and application of the bovine viral diarrhea virus antibody indirect ELISA kit and the bovine viral diarrhea virus indirect immunofluorescence detection kit, belonging to the field of veterinary biological products. technical background [0002] Bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) are members of the genus Pestivirus in the family Flaviviridae. BVDV has two biotypes, type I and type II, and type I is the main type in my country. In recent years, it is very common for cattle to be infected with BVDV and continue to carry the virus. Live swine fever vaccines use materials such as bovine serum, trypsin, and bovine testes in the production of live vaccines. If these materials contain low-titer BVDV, they will cause contamination of swine fever cell vaccines. In addition, the use of bovine serum containing BVDV ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18G01N33/569
CPCC07K14/005C07K2319/02C07K2319/21C12N2770/24322G01N33/56983G01N2469/10G01N2469/20
Inventor 范学政徐璐赵启祖宁宜宝丁家波姚文生王芳王琴邹兴启朱元源朱良全蒋卉
Owner CHINA INST OF VETERINARY DRUG CONTROL
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