辣椒脉斑驳病毒CP基因的原核表达及其抗血清的制备

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-ChiVMV CP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38 kD。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。

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

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.