Monoclonal antibody of epizootic hemorrhagic disease virus of deer (EHDV) and preparation method and application thereof

An epidemic hemorrhagic fever and monoclonal antibody technology, applied in the biological field, can solve the problems of restricted export trade, economic loss of EHDV, etc., and achieve the effects of simple preparation method, high titer of ascites, and high affinity

Inactive Publication Date: 2009-12-09
花群义
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no effective vaccine and treatment for this disease at present. It takes a lot of manpower and material resources to control and eliminate EHD, and the export trade is also limited, which brings great economic losses to the countries where EHDV is endemic.

Method used

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  • Monoclonal antibody of epizootic hemorrhagic disease virus of deer (EHDV) and preparation method and application thereof
  • Monoclonal antibody of epizootic hemorrhagic disease virus of deer (EHDV) and preparation method and application thereof

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Embodiment Construction

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

[0026] After the BHK-21 cells grow into a monolayer, they are inoculated with EHDV 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 analyzer determined that the EHDV protein content purified by differential centrifugation and sucrose density gradient centrifugation was 2g / L, and it was stored at ...

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Abstract

The invention relates to the biotechnology field. A monoclonal antibody of an epizootic hemorrhagic disease virus of a deer (EHDV) of the invention is an immunoglobulin G-IgG and is composed of a heavy chain and a light chain, which is secreted by a hybridoma cell obtained after fusion of a B cell formed by splenic lymphocyte differentiation of BALA / c mice immunized with EHDV and a mouse myeloma cell (SP2 / 0) and only targets at specific antigen determinants of EHDV. The monoclonal antibody provided by the invention features strong specificity, high ascites titer, high affinity and simple preparation method and can be used in the detection method of the EHDV antibody. The monoclonal antibody also has important application value in serodiagnosis of EHDV because of only targeting at a single antigen epitope of EHDV and can provide powerful technology and product supports for EHDV diagnosis, quarantine and detection as well as epidemiological survey.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a deer epidemic hemorrhagic fever virus monoclonal antibody, a preparation method and an application thereof. Background technique [0002] Epizootic haemorrhagic disease of deer (EHD) is a seasonal, non-contact viral infectious disease. Deer epidemic hemorrhagic disease virus (EHDV), a member of the genus Orbivirus in the Reoviridae family, is a double-stranded RNA virus transmitted by arthropods (mainly Culicoides). Bluetongue virus (BTV) is very similar, and serologically, it has obvious crossover with bluetongue disease. Under natural conditions, deer epidemic hemorrhagic disease virus can cause cattle, sheep and other livestock as well as white-tailed deer (Odocoileus virginianus), mink, eland antelope and other domesticated and wild ruminants infection. The virus has been isolated in the United States from white-tailed deer, mule deer, sheep, goats, cattle, bison, and Culicoi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/06C12P21/08G01N33/577
Inventor 花群义杨俊兴林庆燕阮周曦陈兵曾少灵曹琛福吕建强秦智锋卢体康董俊
Owner 花群义
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