大豆花叶病毒东北3号株系NIb蛋白表达、纯化及单克隆抗体制备  

Expression and Purification of a NIb Protein of the SMV Strain 3 from the Northeastern Regions of China and Preparation of Its Monoclonal Antibody

作　　者：张淋淋[1,2] 李小宇[2] 张春雨[2] 尤晴 张正坤[2] 李启云[2] 张俊华[1] 王永志[2] 

机构地区：[1]东北农业大学,哈尔滨150030 [2]吉林省农业科学院植物保护研究所,东北作物有害生物综合治理重点实验室,吉林省农业微生物重点实验室,吉林公主岭136100 [3]吉林农业大学,长春130118

出　　处：《东北农业科学》2016年第3期62-66,共5页Journal of Northeast Agricultural Sciences

基　　金：吉林省自然科学基金项目(20150414044GH);农业部转基因重大专项(2011ZX08004-004)

摘　　要：本研究利用PCR方法从大豆花叶病毒阳性叶片中成功克隆出大豆花叶病毒东北3号株系nib基因。序列分析表明,大豆花叶病毒东北3号株系与Ar33株系亲缘关系最近,序列同源性高达99.48%,其次为WS160、G4、G2、L株系,同源性分别为98.97%、94.88%、90.95%、90.35%,与6202-2、6067-1株系亲缘关系最远,分别为83.82%、84%;利用大肠杆菌中表达NIb蛋白,并通过亲和层析获得纯化的NIb蛋白,将纯化的NIb蛋白免疫小鼠,制备了2G3、1D6、6F9和6B2共4株NIb蛋白单克隆抗体,为大豆花叶病毒基因nib的功能研究及抗病育种提供材料。A nib gene of Soybean mosaic virus （SMV） strain 3 in northeastern regions of China was cloned from the SMV-infected soybean leaves. Sequence analysis of the nib gene exhibited the highest of 99.48% similarity with that from Ar33, followed by WS160, G4, G2, and L with 98.97%, 94.88%, 90.95% and 90.35%, respectively. The gene sequence also showed a distant phylogenetic relationship with that from 6202-2 and 6067-1 with 83.82% and 84% of similarity respectively. The recombinant Nlb protein was expressed in E.coliand purified using affinity chromatograph. Four monoclonal antibodies against NIb protein, 2G3, 1D6, 6F9 and 6B2 were prepared, by immunizing mice with the purified recombinant NIb protein. The results might lay the foundation for the function investigation of the SMV nib gene and soybean SMV resistance breeding.

关 键 词：大豆花叶病毒 复制酶蛋白 蛋白表达 单克隆抗体 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]