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Monoclonal antibody of bluetongue virus (BTV) and preparation method and application thereof

A monoclonal antibody, bluetongue virus technology, applied in the biological field, can solve the problem of poor specificity of AGID, and achieve the effect of high affinity, strong specificity and high titer of ascites

Inactive Publication Date: 2009-12-09
花群义
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  • Claims
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AI Technical Summary

Problems solved by technology

Among BLU detection methods, Agar Gel Immuno Diffusion (AGID) has been widely used in the world because of its simple operation and low cost. It is also one of the methods recommended by OIE so far. However, the antigen used in this method There is a certain cross-reactivity with related viruses such as Epizootic hemorrhagic disease virus of deer (EHDV), so the specificity of AGID detection is poor

Method used

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  • Monoclonal antibody of bluetongue virus (BTV) and preparation method and application thereof
  • Monoclonal antibody of bluetongue virus (BTV) and preparation method and application thereof
  • Monoclonal antibody of bluetongue virus (BTV) and preparation method and application thereof

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Embodiment

[0025] 1. Purification of virus and preparation of immunogen

[0026] After the BHK-21 cells grow into a single layer, they are inoculated with BTV and reacted at 37°C for 1 hour. Serum-free basal medium was added to continue culturing, and the virus was harvested when the cytopathic pathology (CPE) reached more than 75% after 2-3 days. The harvested virus liquid was repeatedly frozen and thawed 3 times, centrifuged at 8000r / min for 30min, and the supernatant was taken. Ultracentrifuge at 28000r / min at 4°C for 3h. The precipitate was dissolved with 1 / 100 of the original volume of PBS, and then 20%, 60% (w / v) discontinuous sucrose density gradient 20,000r / min was used for ultracentrifugation at 4°C for 3h, and the purified virus band was harvested as an immune antigen, and DU800 nucleic acid was used to The protein content of bluetongue virus purified by differential centrifugation and sucrose density gradient centrifugation was determined to be 3.268g / L by a protein analyzer...

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Abstract

The invention relates to the biotechnology field. A monoclonal antibody against a BTV VP7 protein of a bluetongue virus (BTV) of the invention is prepared by the following steps: adopting the hybridoma cell technology, taking splenocytes of BALB / c mice immunized with purified BTV for fusion with a mouse myeloma cell (SP2 / 0), after culturing the cells with an HAT selective medium, carrying out screening with indirect ELISA coated by a purified BTV antigen, a BTV VP7 protein antigen expressed by a gene engineering and a control antigen of a normal hamster kidney continuous cell (BHK21), carrying out screening and cloning by limiting dilution to obtain a hybridoma cell line which has the capacities of stable continuous culture and secretion of the monoclonal antibody (McAb) against the specific BTV VP7 protein and preparing McAb mouse ascites of the BTV VP7 protein. The monoclonal antibody against the BTV VP7 protein features strong specificity, high ascites titer, high affinity and simple preparation method, can be used in the detection method of the BTV antibody and antigen and provides an important technical means for prevention and control of bluetongue in China.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the bluetongue virus VP7 protein monoclonal antibody, its preparation method and application. Background technique [0002] Bluetongue disease (BLU) is a severe animal infectious disease caused by bluetongue virus (BTV) of the reoviridae family (reoviridae) orbivirus genus (Obivirusgenus), and is transmitted by Culicoides , mainly harm sheep, goats, cattle and other livestock, and is widely prevalent in the world. The World Organization for Animal Health (OIE) lists BLU as an important animal infectious disease. In many national animal trade agreements, BLU is one of the infectious diseases that must be quarantined. In recent years, with climate warming, the incidence of BLU has increased in many European countries, seriously affecting international animal trade. [0003] There are 24 serotypes of BTV, and the BTV genome contains 10 double-stranded RNA fragments (dsRNA), consisting...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/06C12P21/08G01N33/577
Inventor 花群义杨俊兴詹爱军阮周曦曾少灵曹琛福吕建强陈兵秦智锋陶虹周晓黎艾军
Owner 花群义
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