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Method for preparing grass carp reovirus (GCRV) gene engineering vaccines

A technology of genetic engineering vaccine and reovirus, applied in the field of genetic engineering/molecular virology, can solve the problems of high cost and unstable titer of virus particle subunit vaccine, improve the immune protection rate of fish, good stability Sexual and immunogenic, high expression effect

Inactive Publication Date: 2014-03-12
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In view of the instability of crude tissue plasma, cell inactivated vaccines and antigen subunit vaccines and genetic material such as residual nucleic acids, as well as the high cost and unstable potency of virus particle subunit vaccines obtained by conventional virus purification methods, etc. , it is necessary to improve the previous preparation method

Method used

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Embodiment Construction

[0034] The grass carp reovirus genetically engineered vaccine provided by the invention is a genetically engineered vaccine that expresses GCRVVP5 and VP7 efficiently in insect cells obtained by a genetic engineering method. The specific preparation steps of the GCRV genetic engineering vaccine are as follows: through sucrose-lined centrifugation, the purified VP5 and VP7 protein complexes are obtained, the purified protein is diluted with 0.65% normal saline and then soaked in fry to obtain an immune protection effect of 90%.

[0035] 1. Propagate the recombinant baculoviruses of the recombined GCRV outer capsid proteins VP5 and VP7 using the sf9 insect cell line, collect the sf9 cells infected with the recombinant virus for 72 hours, centrifuge the cells at a low speed, and remove the supernatant. Rinse 3 times with PBS or phosphate buffered saline to remove bovine serum albumin components, add 3 times the volume of lysate to the low-speed centrifuged cell pellet, mix thoroug...

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Abstract

The invention provides a method for preparing grass carp reovirus (GCRV) gene engineering vaccines. The method comprises the following steps of: selecting recombinant baculoviruses (vAcGCRV-VP5 / VP7) of sf9 insect cell proliferated recombinant GCRV outer capsid proteins VP5 and VP7; separating recombinant protein components by freezing, thawing and cracking recombinant virus infected sf9 cells and adopting sucrose lining centrifuging method, and thus obtaining a purified in vitro expression VP5 and VP7 protein complex; and performing fish protection experiments on the purified VP5 and VP7 proteins to prove that the VP5 and VP7 proteins have good immune protection effect. The GCRV gene engineering vaccines prepared by the method not only have good stability and immunogenicity, but also have good immune protection effect on grass carp fries; the immune protection rate of the GCRV gene engineering vaccines reaches 90 percent; and the method is suitable for large-scale production and application of the vaccines.

Description

technical field [0001] The invention relates to the field of genetic engineering / molecular virology, in particular to the preparation of a genetically engineered vaccine for the outer capsid protein of Grass carp reovirus (GCRV, also known as Grass carp hemorrhagic virus, GCHV) method. The virus strain used in the present invention is grass carp reovirus Hunan Shaoyang strain GCRV873, also known as grass carp haemorrhagic disease virus Hunan Shaoyang strain GCHV873, has been preserved by the China Center for Type Culture Collection, the preservation date is July 23, 2004, and the preservation number is CCTCC NO: V200414. Background technique [0002] Grass carp (Ctenopharyngodon idellus) is one of the four major freshwater aquaculture species in my country. It is cheap and good, and is deeply loved by consumers. Grass carp reovirus is the main pathogen that causes the death of grass carp. When the disease is severe, it can cause more than 85% of grass carp fry to die, thus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/15A61K48/00C12N15/866A61P31/14
Inventor 方勤丁清泉邵玲孙小云
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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