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草鱼呼肠孤病毒的复制特性及其RNA聚合酶的表达研究
Alternative TitleReplication Characterization of Grass Carp Reovirus(GCRV) and Expression of its RNA Polymerase
方勤
Subtype博士
Thesis Advisor朱作言
2002
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Keyword草鱼呼肠孤病毒 结构蛋白 Cdna文库构建 Rna聚合酶
Abstract该研究首先对1999年从湖南长沙新分离到的一株致病性强的草鱼呼肠孤病毒991毒株(命名为GCRV<,991>)进行了体外复制与生物学特性研究.在对GCRV生物学特性认识的基础上,该研究进行了该病毒复制相关基因第1、2、3基因片段的cDNA文库构建与序列分析.
Other AbstractGrass Carp Hemorrhage Virus(GCHV) was first isolated fish virus in Mainland China. The agent provoked severe infectious hemorrhage disease in about 85% of fingerling and yearling populations of grass carp and caused a heavy economic loss in freshwater culture. It was assigned to newly established Aquareovirus genus in family Reoviridea based mainly on its natural host, its non-enveloped double capsid layer morphological structure and 11 dsRNA segments genome properties in the 5th report of the international committee on taxonomy of viruses, and formally named as Grass Carp Reovirus(GCRV) at the same time. GCRV is a highly pathogenic aquareovirus, this hints that latent disaster of hemorrhage disease is still remained, so effective prevention and antivirus research have to be improved. In this study, we mainly focused on its replication characterization and expression on its RNA polymerase in E.coli for the further functional analysis to reveal the interaction between the virus structure and proliferation in host cells. To understand viral replication cycle, it is necessary to reveal its biological properties and genome structure, which is related to coding functional protein involved in virus replication. The biological properties of a new powerful pathogenic grass carp reovirus GCRV991 strain, which was isolated from Shaoyang, Hunan province in 1999, was first investigated in this study. It is showed that ClK(Ctenopharyngodon idellus kidney ) and FHM (Fathead minnow) cell lines are sensitive to GCRV991 strain. In addition, the virus could be neutrialized against original GCRV isolate (GCRVg73 ) antibody, and also the immunocompound was detected by electron microscope. Polyacrylamide gel electrophoresis revealed that GCRV991 genome was composed of 11 segments of dsRNA, and its structural protein was consisted of 5 major plus 2 minor structural polypeptides, which both of the properties were also shared by GCRVg73.The molecular weight of 5 major and 2 minor structural protein were 136kDa,132 kDa,65 kDa,43 kDa,34kDa, and 138kDa, 82kDa,respectively. It is suggested the new GCRV991 isolate may show same antigen properties with GCRVg73. Further more, a Singapore isolate Threadfin reovirus(TFV) was also compared with GCRV991 in its RNA genome and structural protein properties as well as cell infection sensitivities. It is interesting to point out based on serological related Western blot analysis that antiserum against GCRV873 could cross detecting some of the proteins found in TFV, but not all of the proteins. This suggests that GCRV and TFV may share some similar epitopes. GCRV genome is composed of 11 dsRNA with no poly(A) tailing structure in its segment 3'terminal , so the genetic study of viruses is hampered by the technical difficulty of dsRNA complete sequence determination. A strategy of 3 step GCRV cloning was improved in this study, which was taking random hexamers induced TR-PCR at first ,and then get 5'( 2 step )and 3'(3 step) terminal sequence next. As such full-length genome sequences of GCRV segments 1, 2 and 3 , classified as a tentative species of genus Aquareovirus, family Reoviridae, was realized. Segments 1, 2 and 3 were found 3949, 3877 and 3702 nucleotides long, respectively. Conserved motifs were identified at the 5' (GUUAUUU) and (UUCAUQ3' ends of each segment. Each segment contains a single ORF and analysis of the negative strand do not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VP1 might represent the viral guanylyl/methyl transferase ( involved in the capping process of RNA transcripts ) and VP3 "the NTPase/helicase (involved in the transcription and capping of viral RNAs); The highest amino acid identities (26%-41%) were found with Orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses and orthoreoviruses. It should also permit to precise the taxonomic status of these different viruses and address the replication mechanism of the virus. RNA-dependent RNA polymerase (RdRp) is a key replicase in RNA virus replication. To get expressed RNA polymerase, there are 3 different fragments of GCRV-RdRp Gene(GCRV Segment 2 ) was gained by using RT-PCR amplification. The amplified fragments were cloned into T7 promoted prokaryotic expression system pRSET vector and successfully constructed 3 different recombinants ,which were pR/RRp, pR/RRpN and pR/RRpC . The 3 expressed recombinants were demonstrated in frame with the N-terminal tag and in the proper orientation expression by SDS-PAGE. The highly expressed fusion protein was produced by inducing with lnmol /L IPTG , and their molecular weight are about 55kDa, 98kDa, 103kDa, which was in the right size corresponding to predicted values .It indicated the fused proteins were produced in the form of inclusion body with their yields remained steady more than 60% of total bacterial protein. 6xHis-tagged GCRV RNA polymerase was easily purified by affinity chromatography and it also showed all the expressed proteins were able to bind immunologically to rabbit anti GCRV-VP2 serum. Our data provide evidence to further investigate GCRV RdRp activities, and reveal the structure and function relationship in the virus replication.
Pages136
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12668
Collection学位论文
Recommended Citation
GB/T 7714
方勤. 草鱼呼肠孤病毒的复制特性及其RNA聚合酶的表达研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2002.
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