陕西烟区烟草脉带花叶病毒外壳蛋白基因的克隆和原核表达  

Cloning and prokaryotic expression of the coat protein gene of tobacco vein banding mosaic virus from Shaanxi province

作　　者：刘萍[1] 纪玲玲[1] 郝兴安[1] 吴云锋[1] 成巨龙 

机构地区：[1]西北农林科技大学植保学院与旱区作物逆境生物学国家重点实验室,杨凌712100 [2]陕西省烟草研究所,西安710061

出　　处：《中国烟草学报》2011年第5期87-89,95,共4页Acta Tabacaria Sinica

基　　金：中国烟草总公司科技重点项目(110200902046);陕西省烟草公司科学研究与技术开发项目(KJ-19-2008);高等学校学科创新引智计划(No.B07049)

摘　　要：根据烟草脉带花叶病毒(TVBMV)CP序列合成上下游引物,利用RT-PCR方法获得了(TVBMV)CP基因。序列分析表明,TVBMV CP基因全长813nt,编码271个氨基酸;与GenBank中已报道的9个分离CP基因相比,其核苷酸及氨基酸序列同源性分别为90.28%-98.15%和97.05%-100%。将TVBMV CP基因经BamHI/NotI双酶切定向插入到pET30a载体中,构建了原核表达载体pET30-TVBMV CP,重组质粒经BamHI和NotI双酶切鉴定及基因测序验证正确后,用IPTG进行诱导表达。结果表明:TVBMV CP在大肠杆菌中获得了高效表达,表达产物的分子量为37 kD。用表达产物为抗原免疫家兔制备了特异性抗血清,其效价为1/2048。用Western-blot检测田间样品,其结果表明,制备的抗血清可用于检测田间的发病植株。The coat protein （CP） gene of Tobacco Vein Banding Mosaic Virus （TVBMV） from Shaanxi province was obtained by RT-PCR. Sequence analysis showed that TVBMV CP gene was 813 nt long and encoded a 271 amino acid protein. Homology analyzed between TVBMV CP and other 9 strains in GenBank were 90.28%-98.15% at nucleotide level, and 97.05%- 100% at amino acid level. The CP gene was cloned into pMD18-T Simple Vector and inserted into pET30a which were cleaved with restrict endonuclease BamH I/Not I. The expression vector of pET30-TVBMV CP was constructed and transformed into BL21 host strain of E. coli and induced expression by IPTG. The special antisera were prepared by immunizing rabbit with expressed product as antigen. Results showed that TVBMV CP was highly expressed in BL21 and molecular weight of expression product was about 37 kD. The antisera gave a titer of 1/2048. Western blot revealed that the prepared antisera can be used to detect tobacco infected by TVBMV.

关 键 词：烟草脉带花叶病毒 外壳蛋白 原核表达 抗血清 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]