Cloning, Ex Pression And Location Study Of The RNA-dependent RNA Polymerase Gene From Bombyx Mori Cytoplasmic Polyhedrosis Virus

RNA-dependent RNA polymerase(RDRP) is the important enzyme involved in the proliferation of the RNA virus for the activity of both transcriptase and polymerase.It can transcribe the mRNA to synthesize proteins and enzymes needed in virus proliferation and participates in genome replication using the virus RNA as templates. Cytoplasmic Polyhedrosis Virus Bombyx mori I(BmCPV-I) is the typical genus in cypovirus, reoviridae with double strands RNA. CryoEM results of BmCPVs revealed that flower shape structures exist in the inner surface of all the five-fold axis, close to the B-spike inner face, which were deduced to be the TEC(Transcription Enzyme Complex) and probably the component corresponding to structural protein VP2 of BmCPV. Step-by-step RT-PCR and molecular cloning technique were applied to successfully clone the three segments (1442bp, 827bp, 1675bp) of the RDRP gene of the BmCPV (China strain). The whole RDRP gene of 3.7 kb was cloned and was sequenced.(GenBank Accession Number:AY496445).Amino sequence was analyzed by Vector NT and DNAtools. Its isoelectric point is 7.67, and it is a weak basic protein. Its molecular weight is predicted to be 1386483.25Da. There are six ASN glycosylation sites, six CAMP-dependent kinase sites, seven tyrosine kinase phosphorylation sites and four tyrosine sulfation sites. Secondary structure analysis showed that it has 43.59%αhelix,13.55%βsheets and 42.86% random coils. Identities of the nucleotide sequences between BmCPV-C and BmCPV-1,DpCPV-1,LdCPV-1,LdCPV-14,TnCPV-15 were 89%,81%,81%,54.1% and 50.9% respectively and those of the amino acid sequences were 96.5%,93.1%,92.9%, 41.1% and 33.5% respectively. Phylogenetic trees created from the alignment of the nucleotide sequences and the alignment of amino acid sequences show a good agreement .Three conserved regions in RDRPs(acid region,binding site of the nucleotides and catalytic core region) were located using software Clustalx. Additional conserved regions were found in N-terminal of the amino acid sequence which were believed to be the important parts of the RDRP in its replication functions. Three pairs of primers were designed according to the analysis of the restriction enzyme sites on the RDRP gene and pET-28b vector. The whole RDRP gene of 3.7 kb was cloned by RT-PCR and other methods and was sequenced. pET-28b(+) vector was for the first time applied to construct the expression vector pET28b-RDRP,which was then transformed into the E.coli BL21(DE3). The BL21(DE3) strain, containing pET28b-RDRP recombinant plasmid, expressed about 138kDa fusion proteins after the induction with IPTG(1 mmol/L). Western blot analysis indicated that the fusion protein was with the 6×His tags. And the fusion proteins were purified and used to raise antiserum. Western blot analysis indicated that the antiserum could specifically react with the induced protein as well as the BmCPV total proteins at 138kDa which indicated the successful construction of the pET28b-RDRP and its expression in prokaryotic cell. The first antibody was rabbit antibody prepared using recombinant protein and the second antibody was 15nm colloidal gold labeled goat anti rabbit IgG. The third instar silkworms were infected by BmCPV in the midgut reer part and thenimmobilized by 3% polyformaldehyde -0.1% glutaraldehyde mixture, low-temperature embedded by K4M before it was ultraviolet polymerized and prepared as antigen plate using ultrathin sectioning and immuno marked by 1:200 first antibody and 1:40 second antibody .Colloidal gold bound mostly to the virion that dispersedly located in virus generation matrix in the pillar cell of the midgut of silkworms and that located in polyhedron with a higher marking ratio while the average marking ration is about 15%. The results demonstrated that TEC complex does locate at the capsid of BmCPV with relative few copies that coincides with the deduction from the cryoEM results of BmCPV(Zhang et al,1999) Through molecular biology technique and immuno-electron microscopy, cloning ,expression and location study of the RDRP gene of BmCPV was successfully carried out. The results provided the theoretical basis of the combination of the cryoEM results of the BmCPV with those from molecular biology techniques to find out the activity center of RDRP and the replication mechanism of the virus in vivo as well as the activity of the enzymes. Short polypeptides CADD(Computer-aided Drug Design) can also be carried out according to the information of the activity center of RDRP.