【作者】 孙京臣；

【导师】 张景强；

【作者基本信息】 中山大学 ， 生物物理学， 2004， 博士

【摘要】 RNA 依赖的RNA 聚合酶(RNA-dependent RNA polymerase,RDRP)是RNA病毒增殖复制的重要酶类,具有转录酶和聚合酶的双重活性,即一方面参与mRNA 的转录,指导合成病毒增殖复制所需要的蛋白质和酶类;另一方面参与以病毒RNA 为模版的子代病毒基因的复制。家蚕质多角体病毒(Bombyx mori Cytoplasmic Polyhedrosis Virus,BmCPV)是呼肠孤病毒科,质多角体病毒属的代表种,为双链RNA 病毒。已有的BmCPV 冷冻电镜三维重构的研究结果表明,在病毒衣壳五重轴的内表面各有一花形结构,位于B-突起(B-spike)的近内侧,推测为转录酶复合物(TEC),可能是BmCPV 结构蛋白VP2 的对应成分。应用RT-PCR和分子克隆等技术成功地从BmCPV 中国株的dsRNA中分三段克隆了RDRP 基因,大小分别为1442bp,827bp,1675bp,进行基因全序列的拼接后,获得了全长完整的BmCPV(C) RDRP 基因(GenBank 序列号为:AY496445),该基因全长3691bp,GC 含量为41.99%,读码框为1-3678bp,共编码1225 个氨基酸(GenBank 序列号为:AAR88092)。应用Vector NT 和DNAtools 等软件对其氨基酸序列一级结构进行预测和分析,其等电点为7.67,属弱碱性蛋白,推导分子量为138648.25Da,含有6 个ASN 糖基化位点,6 个CAMP 依赖磷酸化位点,7 个TYR 蛋白激酶磷酸化位点,4 个Tyrosine sulfation site。二级结构分析含有43.59%的α螺旋,13.55%的β折叠,其余为42.86%的无规则卷曲。在线Blast 分析表明,与BmCPV-1,DpCPV-1,LdCPV-1,LdCPV-14,TnCPV-15 核苷酸同源性分别为89%,81%,81%,54.1%和50.9%,氨基酸同更多还原

【Abstract】 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.更多还原