烟草曲茎病毒复制相关蛋白基因原核表达条件优化  

Optimization of prokaryotic expression of Tobacco curly shoot virus replication-associated protein gene

作　　者：潘滨[1] 吴建祥[1] 李桂新[1] 周雪平[1] 

机构地区：[1]浙江大学生物技术研究所,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2007年第1期24-28,共5页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(30370058).

摘　　要：为了提高烟草曲茎病毒(Tobacco curly shoot virus,TbCSV)复制相关蛋白(replication-associated protein,Rep)基因在大肠杆菌中表达量,研究了大肠杆菌菌株、温度、诱导时间以及诱导剂IPTG浓度等不同条件对GST-Rep融合蛋白表达量的影响.结果表明,大肠杆菌菌株Rosetta在37℃培养3h后,使用终浓度为0.5mmol·L-1的IPTG在28℃诱导培养4h时,GST-Rep融合蛋白表达量最高,表达的GST-Rep融合蛋白可占细菌总蛋白的42%以上.用GST-Sepharose 4B亲和层析柱对GST-Rep融合蛋白进行纯化,SDS-PAGE表明GST-Rep融合蛋白大小约为66kD,与预计的分子量大小一致,Western blot分析表明其能与GST多克隆抗体起特异性反应.Rep基因的优化表达为研究该基因的作用机理打下了基础.In order to improve the expression level of Tobacco curly shoot virus（TbCSV） replicationassociated protein gene＇（Rep） in Escherichia coli, the effects of various factors on the protein expression were studied, which included bacterial strain, culture temperature, inducing time, and the final concentration of inductor IPTG etc. The results indicated that the expression level of GST-Rep fusion protein reached highest using Rosetta as bacterial strain and induced with 0.5 mmol· L^-1 IPTG as final concentration for 4 h at 28℃ after cultured for 3 h at 37℃, and the expression GST-Rep fusion protein accounted for 42 % of the total cell lysates with the above culture conditions. The Rep fusion protein was purified with GST-Sepharose 4B affinity chromatography, SDS-PAGE analysis showed that the molecular mass of GST-Rep fusion protein was about 66 kD, and Western blot analysis indicated the GST polyclonal antibody could specifically bound to purified GST-Rep fusion protein. The optimized expression conditions of Rep will be useful for future research on function of Rep.

关 键 词：Q786烟草曲茎病毒 复制相关蛋白 原核表达 

分 类 号：S432.4[农业科学—植物病理学]