摘要
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516bp,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0.4mmol·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。
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.
作者
万晴姣
刘倩
李欲轲
龚记熠
张宇斌
乙引
洪鲲
WAN Qing-jiao;LIU Qian;LI Yu-ke;GONG Ji-yi;ZHANG Yu-bin;YI Yin;HONG Kun(School of Life Sciences,Guizhou Normal University,Guiyang 550001,China;Guizhou Key Laboratory of Plant Physiology and Developmental Regulation,Guiyang 550001,China)
出处
《植物病理学报》
CAS
CSCD
北大核心
2019年第1期20-26,共7页
Acta Phytopathologica Sinica
基金
教育部长江学者和创新团队发展计划资助(PCSIRT1227)
贵州省重点实验室项目[黔科合计Z字(2011)4005号]
贵州省农业攻关项目[黔科合NY字(2014)3036]
关键词
齿兰环斑病毒
RDRP
原核表达
多克隆抗体
Odontoglossum ringspot virus
RdRp
prokaryotic expression
polyclonal antibodies
