烟草曲茎病毒复制蛋白基因的原核表达和免疫定位  

Prokaryotic Expression and Immuno-localization of Replication Protein Gene of Tobacco curly shoot virus

作　　者：崔晓峰[1] 李云琴[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,杭州310029

出　　处：《微生物学报》2004年第6期745-748,共4页Acta Microbiologica Sinica

基　　金：国家自然科学基金 ( 3 0 3 70 0 5 8);国家杰出青年基金 ( 3 0 12 5 0 3 2 )~~

摘　　要：利用PCR方法从烟草曲茎病毒 (TbCSV)Y35分离物的病株中获得复制蛋白 (Rep)基因 ,将其克隆到原核表达载体pGEX 4T 1上获得重组质粒pGEX Y35Rep。重组质粒导入大肠杆菌BL2 1(DE3)pLysS中 ,IPTG诱导表达后发现部分Rep融合蛋白以可溶性形式表达。利用GST Sepharose 4B亲和层析柱纯化了Rep的融合蛋白 ,免疫家兔获得Rep蛋白的抗体。对TbCSV侵染烟草中Rep蛋白的亚细胞分布研究发现 ,Rep蛋白主要分布于含有细胞核的组份中。利用免疫胶体金技术对感病烟草中Rep蛋白进行了定位 。The replication protein (Rep) gene of Tobacco curly shoot virus (TbCSV) Y35 isolate was obtained from the infected tobacco plants by PCR and cloned into expression vector pGEX-4T-1 to generate the recombinant plasmid pGEX-Y35Rep. The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLys S, and TbCSV Rep fusion protein was expressed with induction by IPTG and some products were soluble. The Rep fusion protein was purified with GST-Sepharose 4B affinity chromatography and its polyclonal antibody was produced in a rabbit. Studies on subcellular distribution of TbCSV Rep protein in infected tobacco leaves revealed that Rep protein mainly exists in the fractions containing the nucleus. Immuno-gold labeling with the antibody against Rep fusion protein also indicated that Rep protein localized in the nuclei of infected tobacco cells.

关 键 词：烟草曲茎病毒 复制蛋白 表达 定位 

分 类 号：Q75[生物学—分子生物学]