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A kind of preparation method of grass carp reovirus assembled in vitro

A technology of reovirus and grass carp, applied in the field of virology, can solve problems such as the research on the membrane penetration mechanism of the outer capsid protein, and overcome the limitations, high infectivity, and packaging that are difficult to implement reverse genetics operation technology high efficiency effect

Inactive Publication Date: 2017-10-03
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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Problems solved by technology

[0003] At present, structural biology data have shown that the outer capsid proteins VP5 and VP7 have important functions in the process of GCRV infection. Given that the GCRV genome is composed of 11 segmented double-stranded RNAs, like other double-stranded segmented RNA viruses, currently It is not yet possible to use reverse genetics, that is, to realize the study of protein function through changes at the gene level, which limits the study of the membrane penetration mechanism mediated by the outer capsid protein during GCRV infection. Therefore, it is necessary to provide an effective A method for revealing the mechanism of GCRV infection mediated by outer capsid proteins VP5 and VP7 at the molecular level

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  • A kind of preparation method of grass carp reovirus assembled in vitro

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[0033] The method for preparing the above-mentioned in vitro assembled grass carp reovirus provided by the invention comprises the following steps:

[0034] (1) CIK cells (grass carp kidney cell line) were used for virus propagation, and a large number of virus suspensions of grass carp reovirus Hunan Shaoyang strain GCRV873 infected with CIK cells were obtained;

[0035] (2) Concentrating and purifying the cell virus suspension infected with GCRV873 by centrifugation;

[0036] (3) Treat the purified virus with trypsin to completely degrade the complete viral coat protein VP5 and VP7 to form viral core particles containing nucleic acid, which are purified by 25-55% (w / v) CsCl density gradient centrifugation at 50% A large number of virus core particles were obtained in the CsCl gradient solution;

[0037] (4) Sf9 cells were infected with recombinant baculovirus vAcGCRV-VP5 / VP7, and a large amount of outer capsid proteins VP5 and VP7 were expressed;

[0038] (5) The Sf9 cells...

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Abstract

The present invention is a preparation method for assembling grass carp reovirus in vitro, specifically a method for assembling in vitro of grass carp reovirus GCRV core and baculovirus expression system for expressing outer capsid proteins VP5 and VP7, the method comprising multiplying GCRV873 1. Acquisition and centrifugal purification of viral core containing nucleic acid, expression of outer capsid proteins VP5 and VP7, in vitro assembly and centrifugal purification steps. The method is simple and convenient to operate, and the purified in vitro assembled virion R-GCRV prepared by the method provides an effective tool for studying the mechanism of VP5 and VP7-mediated virus infecting host cells.

Description

technical field [0001] The present invention relates to the field of virology, in particular to a method for in vitro recombination of purified grass carp reovirus (GCRV) core and outer capsid proteins VP5 and VP7 expressed by a baculovirus expression system to form complete virus particles . The virus strain used is grass carp reovirus Hunan Shaoyang strain GCRV873, which 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 important freshwater aquaculture fishes in my country. It is susceptible to grass carp reovirus when it is young. serious economic loss. The research on the mechanism of virus infection is conducive to finding the target of antiviral drugs, and provides an effective way for the development of antiviral drugs and the prevention of viral diseases. GCRV is a non-enveloped...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C12N7/04C12R1/93
Inventor 方勤颜世翠郭洪张杰闫利明陈清秀张付贤
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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