东方粘虫中肠V-ATP酶a亚基的克隆及原核表达  

Gene Cloning and Prokaryotic Expression of V-ATPase Subunit a in the Midgut of Mythimna separata(Walker)

作　　者：金朵[1,2] 王高振 吴元元 吴文君[1,2] 祁志军[1,2] JIN Duo;WANG Gaozhen;WU Yuanyuan;WU Wenjun;QI Zhijun(Institute of Pesticide Science,College of Plant Protection,Northwest A~F University,Yangling Shaanxi 712100,China 2.Key Laboratory of Botanical Pesticide R＆D of Shaanxi Province,Yangling Shaanxi 712100,China)

机构地区：[1]西北农林科技大学植物保护学院农药研究所,陕西杨凌712100 [2]陕西省植物源农药研究与开发重点实验室,陕西杨凌712100

出　　处：《西北农业学报》2018年第6期915-922,共8页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31371958);杨凌示范区科技计划(2017NY-09)~~

摘　　要：对东方粘虫[Mythimna separata(Walker)]中肠V-ATPase a亚基(MS-VATPa)cDNA全长序列进行克隆、原核表达及纯化研究。采用RT-PCR和RACE技术克隆MS-VATPa基因,再亚克隆于原核表达载体pET-22b(+)以构建重组载体pET22b-MS-VATPa,将鉴定正确的重组质粒转入E.coli BL21(DE3)进行IPTG诱导表达,采用Ni-NTA柱亲和层析法纯化目标蛋白,并进行SDS-PAGE和Western blot验证。结果表明:成功克隆东方粘虫中肠MS-VATPa基因的cDNA全长序列,并实现MS-VATPa基因在大肠杆菌原核表达系统中的可溶性表达。The full-length cDNA of V-ATPase subunit a（MS-VATPa）in the midgut of Mythimna separata had been cloned,expressed and purified in this study.The MS-VATPa gene was cloned by using RT-PCR and RACE techniques and subcloned into prokaryotic expression vector pET-22 b（＋）to construct recombinant vector pET22 b-MS-VATPa,the correct recombinant plasmid was transformed into E.coli BL21（DE3）and induced with IPTG.The target protein was purified by Ni-NTA affinity chromatography and the purified protein was verified by SDS-PAGE and Western blot assay.The results showed that the full-length cDNA of MS-VATPa in the midgut of Mythimna separata was successfully cloned,and expressed in the E.coli expression system.

关 键 词：东方粘虫 昆虫V-ATP酶 基因克隆 原核表达 蛋白纯化 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]