Grass carp reovirus(GCRV) is the first aquatic virus isolated and characterized in China. More than 60 aquareoviruses have been identified since the first reovirus-like virus was isolated from aquatic tissues by Meyers in 1979 (12, 16). Many of these isolates cause asymptomatic infection or infections with very minor diseases. It has been recognized that GCRV is the most pathogenic among all the isolates of aquareoviruses reported to date (16). Therefore, GCRV provides a good model system to study aquareovirus replication and pathogenesis and such studies also have significance in the fish farming agriculture.
GCRV has been classified as a member of genus Aquareovirus of family Reoviridea (13, 19). Similar to other Reoviruses, the virion of GCRV has a multilayered spherical structure enclosing a genome consisting of 11 segments of dsRNA. Due to the lack of standard serotypes, the classification of aqua-reovirus into the Aquareovirus genus is currently done mainly based on RNA:RNA hybridization. According to Rangel et al., six different genetic groups (Aquareovirus genogroups A-F) have been identified among aquareovirus isolates based on reciprocal RNA blot hybridization and those not belonging to genogroups A-F, such as GCRV, are classified into an additional group G (16). More recently, genomic sequence and phylogenic analyses of GCRV and three other aquareovirus isolates have suggested that GCRV should be classified as a member of genogroup C. To date, among the isolated aquareoviruses, GCRV and striped bass reovirus (SBRV, Aquareovirus A) have been characterized in the greatest detail (1, 5, 7, 14, 18). Sequence and phylogenic analyses indicate a relatively higher level of sequence homology between aquareoviruses and mammalian orthoreoviruses compared with other members of the family (1, 5).
While considerable fundamental and applied research has been carried out on mammalian or human isolates from the genus Orthoreovirus and Rotavirus (2, 3, 8, 10, 17, 20), very little progress has been made with members of the genus Aquareovirus. According to the complete genome sequence of GCRV and the result of genome analysis, VP7 encoded by GCRV s10 is the outer capsid protein of the virus. To understand the interaction of GCRV infection with its host cells, it is necessary to express the outer capsid protein of GCRV in vitro. The high level expression of GCRV VP7 protein in Prokaryotic Cells was reported at the first time in this study.
High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells
- Received Date: 07 November 2007
- Accepted Date: 25 December 2007
Abstract: Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid，which was named as pR/GCRV-VP7，was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover，the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.