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Cloning, Expression of vp6 and ns38 Genes and Immunogenicity of VP6 and NS38 in Grass Carp Reovirus

Author: XiongLingFang
Tutor: WuZaoHe;JianJiChang
School: Guangdong Ocean University
Course: Aquaculture
Keywords: GCRV096 vp6 ns38 Cloning Prokaryotic expression immunogenicity
CLC: S941.41
Type: Master's thesis
Year: 2011
Downloads: 22
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Abstract


Grass carp reovirus (GCRV) is a member of the Aquareovirus genus in the family Reovirida. The C-terminal of Grass carp reovirus genome did not contain poly(A) sequence and a specific repeated conserved sequence is in C-terminal and N-terminal. GCRV genome contained 11 double stranded RNA (dsRNA) encoded 12 proteins(seven structure proteins and five nonstructural proteins). VP6, the structural protein of GCRV, was encoded by S8 and VP6 played an important role in transcription and replication of GCRV. NS38, the non-structural protion in GCRV, was encoded by S9 and NS38 could play an important role in the early stages of morphogenesis in GCRV. It was shown that VP6 could induce the highest neutralizing antibodies titers, followed by NS38. In the study, vp6 and ns38 genes were cloned by homology cloning and RT-PCR from grass carp reovirus strian 096 which was isolated from diseased grass carp. Then vp6 and ns38 genes were expressed in E.coli and the immunogenicity of VP6 and NS38 proteins were studied. The vp6 gene contains a 1236 bp ORF encoding VP6 protein with 412 amino acids.The calculated molecular mass of VP6 protein was 44 kDa with an estimated isoelectric point of 8.5. There was the high similarity between vp6 gene sequence of GCRV096 and the congeneric DNA sequences of the other Grass Carp Reovirus. The vp6 gene was subcloned into pET-28a(+) to construct recombinant plasmids pET-vp6. SDS-PAGE and Western blot analysis indicated that the recombinant plasmids were successfully expressed in E.coli BL21 (DE3). The recombinant proteins were purified by affinity chromatography on Ni2+-IDA resin and the anti-VP6 serum was prepared by injecting purified recombinant VP6 into rabbits. The titer of anti-VP6 serum is 1:32000 tested by ELISA. After anti-VP6 serum with CIK cells were incubated for 48 h, CIK cells were infected by GCRV096. The Expression of GCRV096 vp6 gene in the CIK cell was quantified by quantitative PCR, in order to analyze the immunogenicity of VP6. The results showed that the amount of GCRV096 in the experimental groups were significantly less than the control group. So, it was shown that VP6 protein had the strong immunogenicity.The ns38 gene contains a 1059 bp ORF encoding NS38 protein with 352 amino acids. The calculated molecular mass of NS38 protein was 37.7 kDa with an estimated isoelectric point of 7.5. There was high similarity between ns38 gene sequence of GCRV096 and the congeneric DNA sequences of the other Grass Carp Reovirus. The ns38 gene was subcloned into pET-28a(+) to construct recombinant plasmids pET-ns38. SDS-PAGE and Western blot analysis indicated that the recombinant plasmids were successfully expressed in E.coli BL21 (DE3). The recombinant proteins were purified by affinity chromatography on Ni2+-IDA resin and the anti-NS38 serum was prepared by injecting purified recombinant NS38 into rabbits.The titer of anti-NS38 serum is 1:38400 tested by ELISA.After anti-NS38 serum and CIK cells were incubated for 48 h, CIK cells were infected by GCRV096. Expression of GCRV096 ns38 gene in the CIK cell was quantified by quantitative PCR, in order to analyze the immunogenicity of NS38. The results showed that the amount of GCRV096 in the experimental groups were significantly less than control group. So it was shown that NS38 protein had the strong immunogenicity.When CIK cells were infected by GCRV096 for 4 h, the ratio between experimental group and control group of expression of GCRV096 ns38 gene was the minimum. NS38 protein played a role in stronger immunogen at 4 h ,for which CIK cells had been infected by GCRV096.In this study, the characters of vp6, ns38 genes and VP6, NS38 proteins were analyzed. And the immunogenicity of VP6 and NS38 proteins were explored. This study provided the theoretical basis and establish the foundation for the prevention and treatment of grass carp hemorrhage.

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