辣椒脉斑驳病毒CP基因的原核表达及其抗血清的制备  

Expression of Chilli veinal mottle virus Coat Protein and Preparation of a Virus-specific Antiserum

作　　者：吴育鹏[1,2] 王健华[1,2] 冯团诚[1] 张雨良[1] 王小明[1,2] 刘志昕[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所,农业部热带作物生物技术重点开放实验室,海口571101 [2]海南大学环境与植物保护学院,海南儋州570228

出　　处：《园艺学报》2010年第10期1598-1604,共7页Acta Horticulturae Sinica

基　　金：国家科技支撑计划项目(2007BAD48B01)

摘　　要：采用RT-PCR方法克隆了辣椒脉斑驳病毒文昌分离物(ChiVMV-WC)的CP基因,并将其连接到原核表达载体pET-30b(+)上,克隆测序以确定其阅读编码框的正确性,然后将获得的重组质粒pET30b-ChiVMVCP转化大肠杆菌Rosetta(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析结果表明,CP基因在大肠杆菌中获得了高效表达,获得的融合蛋白分子量约为38kD。用Ni2+-NTA琼脂糖亲和层析纯化的融合蛋白免疫兔子并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的ChiVMV-WC编码CP蛋白发生特异性反应。间接酶联免疫吸附法(ID-ELISA)检测抗血清效价为1/106。通过对田间20个样品的ID-ELISA检测,证实了所制备的抗血清与ChiVMV病叶具有良好的反应特异性。The coat protein（CP）gene of Chilli veinal mottle virus Wenchang isolate（ChiVMV-WC） was amplified by RT-PCR,and cloned into the expression vectors pET-30b（＋）.After the reading frame is confirmed by sequencing,the recombinant plasmid pET30b-ChiVMV CP was transferred into E.coli Rosetta（DE3）and the expression of the recombinant CP protein was then induced by IPTG.SDS-PAGE analysis showed a high level expression of the recombinant CP of about 38 kD.The fusion protein was then purified with Ni2＋-NTA agarose affinity chromatography,and used to immune rabbits for antiserum preparation.Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of ChiVMV-WC.The optimal titer of the antiserum in indirect enzyme-linked immunosorbent assay （ID-ELISA）was determined to be 1/106.ID-ELISA testing of a number of field samples demonstrated the sensitivity and specificity of the antiserum described in this paper.

关 键 词：辣椒 辣椒脉斑驳病毒 外壳蛋白 原核表达 抗血清 

分 类 号：S641.3[农业科学—蔬菜学]