建兰花叶病毒RdRp基因的原核表达及多克隆抗体制备

Prokaryotic expression of RdRp protein of Cymbidium mosaic virus and preparation of its polyclonal antibody

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 conserved region of RdRp gene of Cymbidium mosaic virus （CyMV） isolate from Guizhou pro-vince was amplified by RT-PCR, followed by cloning into pMD18-T vector. Sequence analysis showed that RdRp gene was 735 nt in length and encoded 245 amino acids. Furthermore, RdRp gene was cloned into prokar-yotic expression vector pET32a（＋） to generate recombinant plasmid pET32a-RdRp, followed by introducing into Escherichia coli strain BL21（DE3）. A fusion protein with a size of about 49 kD was expressed at 37℃ after addition of 0.3 mmol·L^-1 IPTG. Polyclonal antibodies against RdRp protein were obtained by hypodernal injection of mice with the recombinant protein. The titer of the antiserum was 1∶204 800, and the prepared antiserum reacted specifically with CyMV-infected leaf extract, but not with samples infected by other 6 viruses.