细粒棘球蚴TGF-βⅡ型受体不同结构域酵母双杂交载体的构建和自激活鉴定  

Construction and identification of DNA-AD and DNA-BD vectors with different domains of EgTβRⅡ for ayeast two-hybrid system and testing of the plasmid for autoactivation

作　　者：李亮[1,2] 张传山[1] 杨乐[1] 王丽敏[1] 吕国栋[1] 王俊华[1] 林仁勇[1,2] 

机构地区：[1]新疆医科大学第一附属医院临床医学研究院,新疆包虫病基础医学重点实验室,新疆乌鲁木齐830054 [2]新疆医科大学基础医学院生物化学与分子生物学教研室,新疆乌鲁木齐830011

出　　处：《中国病原生物学杂志》2014年第5期434-437,F0003,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81260252);新疆维吾尔自治区包虫病基础医学重点实验室开放课题(No.XJKLE-2012-4)

摘　　要：目的构建细粒棘球蚴转化生长因子pII型受体全长（EgTβRⅡ）、受体结合域（EgTβRⅡ-A）和激酶域（EgTβRⅡ-K）的酵母双杂交真核表达载体，检测重组融合蛋白对酵母Y2HOold菌株的毒性作用和自激活活性。方法采用Trizol法提取细粒棘球绦虫总RNA，反转录合成cDNA；PCR扩增EgTβRⅡ—A、EgTβRⅡ—K和EgTβIRⅡ基因，分别克隆入pGADT7、pGBKT7载体中，经PCR、限制性内切酶鉴定及序列测定正确后，采用PEG／I。iAc法转入酵母菌，检测重组融合蛋白对酵母菌毒性和自激活活性。结果构建EgTβRⅡ-A、EgTβR1／-K和EgTβRU的pGADT7、pGBKT7重组质粒，经双酶切和1％琼脂糖凝胶电泳鉴定，得到长度为406bp、1023bp和1824bp的目的基因片段，与预期结果一致；重组质粒转化酵母菌后形成的菌落与对照质粒pGBKT7转化菌的菌落生长状况一致，直径1．5～2．0mm，而在SD／-Leu／X／AbA、SD／-Trp／X／AbA平板上无菌落生长。结论成功构建EgTβRⅡ-A、EgTCtRⅡ-K和EgTCtRⅡ基因的pGADT7、pGBKT7真核表达载体，其表达蛋白对酵母菌Y2HGold无毒性作用和自激活活性，重组质粒载体可用于酵母双杂交系统，为鉴定EgTL3RII在TGF-β／Smad通路中的生物学功能奠定了理论基础。Objectives To construct a yeast two-hybrid system of the full-length receptor gene （EgTβRⅡ ）, receptor domain gene （EgTβ Ⅱ-A）, and kinase domain gene （EgTβR Ⅱ-K） of transforming growth factor-β type II receptor （EgTβRⅡ） from Echinococcus granulosus and to test the toxicity and autoactivation activity of the recombinant fusion protein to the Y2HGold yeast strain. Methods EgTβR Ⅱ-A, EgTβR Ⅱ-K, and EgTβR U gene fragments from E. granulosus were amplified using RT-PCR and were then cloned into pGADT7 and pGBKT7 vectors. The recombinant plasmids were identified using PCR, restriction analysis, and sequencing and were then transformed into the Y2HGold yeast strain to test their toxicity and autoactivation activity using a PEG/LiAc-based method. Results The yeast two hybrid expression vectors EgTI3R ]I -A, EgTβR Ⅱ -K, and EgTβR Ⅱ were successfully constructed and respectively includ- ed gene fragments of 426 bp, 1 023 bp and 1 824 hp. The Y2HGold yeast strain that was transformed with the recombi- nant plasmids produced colonies 1.5-2.0 mm in size and the yeast grew well on SD/-Trp or SD/-Leu plates. Colonies were the same size as those of the positive control, but no colonies were detected on SD/-Leu/X/AbA, SD/-Trp/X/AbA plates. Conclusion The recombinant plasmids constructed here could be used in a yeast two-hybrid system for further study of the biological role of EgTβR Ⅱ in the TGF- β/Smad signaling pathway.

关 键 词：细粒棘球蚴 转化生长因子βⅡ型受体 酵母双杂交 

分 类 号：R383.33[医药卫生—医学寄生虫学]