齿兰环斑病毒RdRp基因的原核表达及多克隆抗体制备

 采用RT-PCR法从感染齿兰环斑病毒(Odontoglossum ringspot virus, ORSV)贵州分离物的苋色藜叶片中扩增出病毒的依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase, RdRp)基因保守序列。测序结果显示,该保守片段长516 bp,编码171个氨基酸残基。构建了该片段的原核表达载体pET32a-ORSV RdRp并转化BL21(DE3)菌株,在25℃以0.4 mmol·L^-1 IPTG诱导表达重组蛋白。SDS-PAGE分析表明,重组蛋白分子量约为36 kDa,与预测相符合。将该重组蛋白作为抗原免疫BABL/C小鼠,制备的多克隆抗体效价达1∶102 400。间接ELISA结果表明,该多抗具有较强的特异性,能检测到ORSV感染的病叶汁液中的RdRp,而与其它感染4种同属或不同属病毒的病叶汁液不发生血清学交叉反应,本实验为进一步研究RdRp的结构和功能以及从分子水平上探讨该病毒的致病机制奠定基础。

Prokaryotic expression of RdRp gene of Odontoglossum ringspot virus and preparation of its polyclonal antibody

The conserved region of RdRp (RNA-dependent RNA polymerase) gene of Odontoglossum ringspot virus (ORSV) was amplified by RT-PCR from the Chenopodium amarantcolar leaves infected with ORSV Guizhou (China) isolate. DNA sequencing showed that the RdRp gene fragment was 516 bp in length and encoded 171 amino acid residues. The RdRp gene fragment was subcloned into a prokaryotic expression vector pET32a(+). By inducing with 0.4 mmol·L^-1 IPTG at 25℃ the recombinant RdRp gene was expressed in E. coli BL21(DE3) and its product identified as a specific band of 36 kDa by SDS-PAGE. Polyclonal antibodies against the recombinant RdRp were obtained by hypodernal injection of mice. The titer of the prepared polyclonal antibodies was 1∶102 400, and the antibodies reacted specifically with ORSV infected leaf extract, but not with other 4 virus-infected samples.