黄瓜绿斑驳花叶病毒外壳蛋白基因原核表达及抗血清制备

Prokaryotic Expression and Antiserum Preparation of Cucumber green mottle mosaic virus Coat Protein Gene

将黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的外壳蛋白(coat protein,CP)基因克隆到原核表达载体pEHISTEV上获得pEHISTEV-CGMMV-CP,转化大肠杆菌Rosetta,经IPTG诱导,表达出分子量为19.5 kD的CGMMV CP融合蛋白。切胶回收诱导表达的CGMMV CP免疫家兔,制备抗血清。Western Blotting检测发现,制备的抗血清能与CGMMV CP发生特异性免疫反应,与同属的烟草花叶病毒(Tobacco mosaic virus,TMV)无反应。琼脂双扩散试验测定,制备抗血清的效价为1∶16,ELISA测定效价为1∶1024。该血清特异性强、效价高,可用于CGMMV的检测。

The coat protein （CP） gene of Cucumber green mottle mosaic virus （CGMMV） was cloned in- to the prokaryotic expression vector pEHISTEV to produce pEHISTEV - CGMMV - CP, which was transformed into competent cells of Escherichia coli Rosetta. Upon induction with IPTG, a fusion protein of 19.5 kD was expressed from the vector pEHISTEV - CGMMV - CP. The fusion protein was recovered from the gel and used to immunize rabbit for antiserum preparation. The resultant antiserum showed strong positive reaction with CP of CGMMV, but not with that of Tobacco mosaic virus, in Western Blotting assay. The titter of antiserum was 1：16 in agarose immuno diffusion and 1：1024 in ELISA. The antiserum prepared in this research had high ti- ter and specificity and could be used in the detection of CGMMV.