棉铃虫核型多角体病毒orf27基因的表达和细胞内定位  

Expression of the Open Reading Frame 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedro virus in Escherichia coli and Its Cell Localization

作　　者：刘艳荷[1] 陈艳霞[1] 章亦卿[1] 徐海君[1] 张传溪[1] 

机构地区：[1]浙江大学昆虫科学研究所

出　　处：《农业生物技术学报》2007年第3期503-507,共5页Journal of Agricultural Biotechnology

基　　金：高等学校全国优秀博士学位论文作者专项资金(No.20055);国家自然科学基金(No.30070032)资助

摘　　要：棉铃虫核型多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedro virus,HaSNPV)orf27编码区全长768bp,编码255个氨基酸残基组成的多肽,预计分子量29.5kD。PCR扩增获得该基因,克隆到融合表达载体pGEX-4t-2中,表达GST-DRF27融合蛋白分子量55.5kD,表达量约占细菌总蛋白的9.2%。用纯化融合蛋白免疫家兔,制备多克隆抗体。将该基因重组到杆状病毒表达载体Bacmid,并在粉蚊夜蛾(Trichopolusia ni)细胞Tn-5B1-4表达。表达蛋白的分子量为32kD,表达量约占细胞总蛋白的2.9%。将HaSNPVorf27在昆虫细胞中表达产物固定,免疫电镜法确定表达蛋白主要存在于细胞的细胞质中。The open reading frame （orf） 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedro virus is 768 bp in length and potential to encode a polypeptide of 255 amino acids, with a molecular weight of about 29.5 kD. The coding region of orf27 was amplified from HaSNPV genome DNA by PCR, and cloned into the expression vector pGEX-4t-2 for bacterial expression. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. The molecular weight of expressed GST-ORF27 fusion protein was 55.5 kD. Western blotting analysis showed that the recombinant protein could react with GST antibody. The fusion protein was retrieved from the SDS-PAGE gel and used to immunize the rabbits to raise the antibody against HaSNPV ORF27. orf27 gene was also recombined into the baculovirus vector Bacmid form the recombinant Bacmid-orf27 and then expressed in Tn-5B1-4 cells of cabbage looper （Trichopolusia ni）. SDS-PAGE analysis showed the expressed His-ORF27 was 32 kD and identical to the predicted molecular weight. Western blotting analysis with anti-ORF27 further confirmed the correct expression. Immunoelectron microscopy demonstrated His-ORF27 fusion protein was mainly localized in the cytoplasm.

关 键 词：棉铃虫核型多角体病毒 orf27基因 表达 定位 

分 类 号：S186[农业科学—农业基础科学] S188