Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic se-quencing showed that GCRV shared high degree...Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic se-quencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 ? resolution by electron cryomicro-scopy. Each GCRV capsid has a multilayered organization, consisting of an RNA core, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression re-sulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symme-try. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.展开更多
Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inne...Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.展开更多
An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was trans...An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.展开更多
基金The research in Dr.Zhou’s lab is supported by NIH(Grant Nos.AI46420&CA94809)the Welch Foundation(AU-1492)+1 种基金 the American Heart Association(Grant No.0240216N)This work was supported by the National Natural Science Foundation of China(Grant Nos.30170730&30470074).
文摘Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic se-quencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 ? resolution by electron cryomicro-scopy. Each GCRV capsid has a multilayered organization, consisting of an RNA core, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression re-sulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symme-try. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.
基金National Natural Science Foundation of China (Grant Nos 30470074,30671615)Innovation Project of the Chinese Academy of Sciences (KSCX2-YW-N-021).
文摘Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.
文摘An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.
