烟草曲茎病毒 AC4基因的原核表达与免疫定位

Prokaryotic expression and immuno localization of AC4 gene of Tobacco curly shoot virus

利用 PCR方法从烟草曲茎病毒 ( Tb CSV) Y3 5分离物的病株中获得 AC4基因 ,将其克隆到原核表达载体 p ET-3 0 a上获得重组质粒 p ET-Y3 5 AC4,重组质粒导入大肠杆菌 BL2 1 ( DE3 ) p Lys S中 ,IPTG诱导后 SDS-PAGE发现 ,AC4蛋白已在大肠杆菌中获得了表达。利用 Ni2 +-NTA亲和树脂纯化了 AC4重组蛋白 ,免疫家兔获得 AC4蛋白的多克隆抗体。利用免疫胶体金技术对感病烟草中AC4蛋白进行定位发现,TbCSV AC4蛋白主要在细胞质的叶绿体和线粒体中积累。

The AC4 gene of Tobacco curly shoot virus (TbCSV)Y35 isolate was obtained from the infected tobacco plants by PCR and cloned into expression vector pET 30a to generate the recombinant plasmid pET Y35AC4. The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLysS, and TbCSV AC4 fusion protein was expressed with the induction by IPTG. The AC4 fusion protein was purified with Ni^(2+)NTA resin and its polyclonal antibody was produced in a rabbit. Immuno gold labeling with the antibody against AC4 fusion protein indicated that TbCSV AC4 protein localized in the chloroplast and mitochondria of infected tobacco cells.