基于酵母双杂交的EgSmadE表达载体构建及自激活检测  

Construction and identification of DNA-AD and DNA-BD vectors for EgSmadE in a yeast two-hybrid system

作　　者：李静 李亮[2] 张传山[2] 叶建蔚[2] 毕晓娟[2] 吕国栋[2] 林仁勇[2] LI Jing LI Liang ZHANG Chuan-shan YE Jian-wei BI Xiao-juan LV Guo-dong LIN Ren- yong(Departmentof Physiology, PreclinicalMedicine College, XinjiangMedical University, Urumqi 830011, China Xinjiang Key Lab of Echinococcosis, Clinical Medical Research Institute, the First Affiliated Hospital of Xinjiang Medical University)

机构地区：[1]新疆医科大学基础医学院生理学教研室,新疆乌鲁木齐830011 [2]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

出　　处：《中国病原生物学杂志》2017年第6期535-539,共5页Journal of Pathogen Biology

基　　金：新疆维吾尔自治区自然科学基金项目(No.2015211C029)

摘　　要：目的构建细粒棘球蚴SmadE(Echinococcos granulosus SmadE)基因的酵母双杂交真核表达载体,并检测其表达重组蛋白的毒性和自激活活性。方法以PCR技术获得EgSmadE基因片段,并克隆至酵母双杂交载体pGADT7及pGBKT7。应用PEG/LiAc法将重组载体导入Y2HGold酵母感受态细胞,利用固体培养基表型筛选对其毒性和自激活活性进行检测。结果扩增EgSmadE基因全长1 129bp,与酵母双杂交重组载体连接后获得pGADT7-EgSmadE和pGBKT7-EgSmadE,经PCR、限制性内切酶双酶切和测序均正确;连接产物转化酵母菌后经培养基表型鉴定与对照组菌落大小一致,表明重组载体表达产物对酵母菌无毒性;SD/-Trp和SD/-Leu培养基均有白色菌落生长,而SD/-Leu/X/A和SD/-Trp/X/A无菌落生长,表明重组载体表达产物对下游报告基因无自激活活性。结论成功构建酵母双杂交载体pGADT7-EgSmadE及pGBKT7-EgSmadE,可用于鉴定EgSmadE蛋白质,为宿主与棘球蚴相互作用的分子机制研究奠定基础。Objectives To construct a yeast two-hybrid system to express Echinococcus granulosus （Eg） SmadE and to study the toxicity and self-activation of the recombinant protein were studied in order to provide a reference for screen- ing EgSmadE-related proteins. Methods The complete gene encoding EgSmadE from E. granulosus was amplified u- sing PCR. The EgSmadE gene and the yeast two-hybrid vectors pGADT7 and pGBKT7 were digested with two enzymes and joined with T4 ligase. After identification using PCR, restriction enzyme digestion, and sequencing, the recombinant vectors were transformed into the yeast strain Y2HGold using the PEG/LiAc method to detect the toxicity and self-activa- tion of proteins in SD/-Trp, SD/-Leu, SD/-Leu/X/A, and SD/-Trp/X/A plates. Results Vectors for a yeast two-hy- brid system were successfully constructed and were found to contain a gene fragment 1,129 bp in length following EcoR I and BamH I double enzyme digestion and sequencing. The vectors were designated pGADTT-EgSmadE and pGBKT7- EgSmadE. Yeast cells containing the recombinant plasmids were similar in size to positive controls, indicating that the two plasmids were not toxic to yeast cells. In a self-activation experiment, yeast containing the recombinant plasmids pro- duced white colonies on SD/-Trp and SD/-Leu plates, and there was no growth of colonies on SD/-Leu/X/A and SD/- Trp/X/A plates. This indicated that the proteins were not self-activated. Conclusion Vectors for a yeast two-hybrid system were successfully constructed and were used to screen EgSmadE-related proteins. This experiment has laid the foundation for the future study of the molecular mechanism for interaction between a host and parasite.

关 键 词：细粒棘球蚴 EgSmadE 酵母双杂交 自激活 毒性检测 

分 类 号：R383.33[医药卫生—医学寄生虫学]