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采用RT-PCR技术扩增出建兰花叶病毒贵州分离物(CyMV-GZ)的依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)基因保守序列。测序结果表明:该RdRp基因序列长735 bp,编码245个氨基酸残基。构建原核表达载体pET32a(+)-RdRp,将重组质粒转化大肠杆菌BL21(DE3),在37℃以0.3 mmol·L~(-1) IPTG诱导表达分子量约49 kDa重组蛋白。以该重组蛋白为抗原免疫小鼠,制备的抗体效价达1∶204 800。间接ELISA检测显示该多抗与CyMV病叶汁发生特异性免疫反应,而与其他6种同属或不同属病毒叶汁无血清学交叉反应。本实验为进一步研究RdRp的功能以及从分子水平上探讨该病毒的致病机制奠定了基础。
The RNA-dependent RNA polymerase (RdRp) gene conserved sequence of CyMV-GZ was amplified by RT-PCR. The sequencing results showed that the RdRp gene was 735 bp in length and encoded 245 amino acid residues. The prokaryotic expression vector pET32a (+) - RdRp was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced with 0.3 mmol·L -1 IPTG at 37 ℃ to induce the recombinant protein with molecular weight of 49 kDa. The recombinant protein was used as an antigen to immunize mice and the titer of antibody was 1:20 800. Indirect ELISA showed that the polyclonal antibody reacted specifically with the leaf juice of CyMV disease and had no serological cross-reactivity with the other six virus strains of the same or different genus. The experiment lays the foundation for further study of the function of RdRp and the molecular mechanism of this virus.