摘要
采用RT-PCR技术从感染齿兰环斑病毒贵州分离物(ORSV-GZ)的苋色藜叶片中扩增出该病毒的运动蛋白(MP)基因。测序结果表明:该MP基因全长912 bp,编码303个氨基酸残基。构建原核表达载体pET32a(+)-MP,将重组质粒转化大肠杆菌BL21(DE3),在25℃以1.0 mmol/L IPTG诱导表达重组蛋白基因。SDS-PAGE分析表明,重组蛋白分子量约为54 kDa,与预测相符合。以该重组蛋白为抗原免疫新西兰大白兔,制备的抗体效价达1:25600。该多抗与ORSV病叶汁发生特异性免疫反应,而与其他5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该蛋白的结构和功能奠定基础。
The movement protein(MP) gene of Odontoglossum ringspot virus(ORSV) was obtained by RT-PCR from the Chenopodium amaranticolar leaves infected with ORSV Guizhou(China) isolate. DNA sequencing showed that the MP gene length was 912 bp and encoded 303 amino acid residues. The MP gene was subcloned into a prokaryotic expression vector pET32a(+). By inducing with 1.0 mmol/L IPTG at 25℃ the recombinant MP gene was expressed in E. coli BL21(DE3) and its product identified as a specific band of 54 kDa by SDS-PAGE. Multiclonal antibodies against recombinant MP was obtained by hypodermal injection of rabbits. The titre of antiserum was 1:25 600, and the antiserum reacted specifically with ORSV infected leaf extract, but not with other 5 virus-infected samples.
出处
《植物检疫》
北大核心
2013年第6期60-63,共4页
Plant Quarantine
基金
长江学者和创新团队发展计划资助(PCSIRT)
贵阳市星火计划项目[(2010)筑科农合同字第1-农-23号]
贵州省社发攻关项目[黔科合SZ字(2011)3100号]
贵州省农业攻关项目[黔科合NY字(2010)3031]
贵州省科技联合基金[黔科合J字LKS(2010)17]
关键词
齿兰环斑病毒
运动蛋白
原核表达
多克隆抗体
Odontoglossum Ringspot Virus
movement protein
prokaryotic expression
multiclonal antibodies
