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cDNA Cloning, Expression of vp5 and vp7 Genes and Subcecullar Localization of VP5 and VP7 Proteins in Grass Carp Reovirus

Author: WangYa
Tutor: WuZaoHe;JianJiChang
School: Guangdong Ocean University
Course: Marine biology
Keywords: Grass carp reovirus cloning prokaryotic expression VP5 VP7 subcellular localization
CLC: S941.41
Type: Master's thesis
Year: 2011
Downloads: 23
Quote: 0
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Abstract


Grass carp reovirus (GCRV) a double-stranded RNA (dsRNA) virus, is a member of the aquareovirus genus in Reoviridae family. GCRV is an important pathogen of grass carp (ctenopharyngodon idella) involved in hemorrhagic disease, mainly affecting fingerling and yearling grass carp and causing hemorrhagic disease, so it is a threat to the aquaculture industry. In this study, vp5 and vp7 cDNAs were cloned usiong homology cloning technique from GCRV096 isolated from diseased grass carp. Then vp5 and vp7 genes were expressed and subcellullar localization of VP5 and VP7 proteins were studied.Results showed that vp5 cDNA was 1981 bp in length. The open reading frame (ORF) sequence had 1947 nucleotides, encoding 648 amino acids with a calculated molecular mass of 68.65 kDa and an estimated isoelectric point of 6.13. Multiple alignment analysis showed that the deduced amino acid sequence of VP5 shared an overall identity more than 90% with Golden shiner reovirus (GSRV) VP4 amino acid sequence, Grass carp hemorrhagic virus (GCHV) and Grass carp reovirus 873 strain (GCRV873) VP5 amino acid sequences, respectively. Prokaryotic expression vector pET-VP5 was constructed and expressed VP5 fusion protein in E.coli successfully. The VP5 fusion protein expressed in the form of inclusion body and used His Trap? HP affinity colum for purification. Western-blot analysis revealed that the expressed protein was our target protein. The recombined plasmid of pEGFP-VP5 was successfully constructed and transfected into CIK cells by liposome. The cell transfection results demonstrated that VP5 fusion protein was located in the nuclear compartment.The length of vp7 cDNA was 852 bp long with an 831 bp ORF, comprising 276 amino acids with a calculated molecular mass of 29.85 kDa and an estimated isoelectric point of 5.56. The deduced amino acid sequence of VP7 shared an overall identity of 100% with Channel catfish reovirus (CCRV) VP7 amino acid sequence. Prokaryotic expression vector pET-VP7 was constructed and expressed VP7 fusion protein in E.coli successfully. The VP7 fusion protein was expressed as inclusion body and purification by HisTrap? HP affinity colum. Western-blot analysis indicated that the expressed protein was the product of vp7 gene. The result of subcellcular location showed that VP7 fusion protein was located in all cell.This study enriched the research field of GCRV, providing valuable information not only on further investigating of biological character of VP5 and VP7 protein but also the mechanism of the virus penetration into host cells. Furthermore, the study can offer scientific basis for further study of pathogenesis of GCRV on molecular level as well as the prevention and treatment of grass carp hemorrhagic disease.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Fish Diseases > Microbial fish diseases > Viral fish diseases
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