烟草脉带花叶病毒cp基因的原核表达及抗血清制备

 利用马铃薯Y病毒属病毒的简并引物, 通过RT-PCR技术获得了云南烟草病毒分离物YND的3'端约1.7 kb片段, 序列分析证明该分离物为烟草脉带花叶病毒(Tobacco vein banding mosaic virus, TVBMV)。将TVBMVcp基因克隆到表达载体pET-22b (+)上, 在大肠杆菌BL21(DE3)中诱导表达出分子量为35.0 kD的融合蛋白。利用该融合蛋白制备了TVBMV的多克隆抗体, ELISA测定抗血清效价为1/4 096。Western blotting和DIBA分析结果表明, 获得的抗血清和原核表达的病毒蛋白及植物病汁液中的病毒均有特异性反应, 可用于TVBMV的快速检测。

Expression of Tobacco vein banding mosaic virus coat protein in E. coli and preparation of antiserum

The 3'-terminal 1.7 kb fragment of YND, a virus isolate collected from Yunnan tobacco was amplified by RT-PCR with degenerate primers. The sequence analysis indicated that YND was an isolate of Tobacco vein banding mosaic virus (TVBMV). The coat protein (CP) gene of TVBMV YND was cloned into expression vector pET22b (+) and transferred into E.coli BL21(DE3). SDS-PAGE showed TVBMV cp gene was expressed as a 35.0 kD fusion protein to high level when induced with IPTG. Antiserum against TVBMV CP was obtained with the fusion protein, and resulting titer was 1:4 096 by ELISA. Western blotting and dot-immunobinding assay (DIBA) results showed that the acquired antiserum could specifically react with both TVBMV CP expressed in E.coli and extracts from diseased plants.