基于酵母双杂交的EgSmadE表达载体构建及自激活检测

发布时间：2018-10-22 07:39

【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。
[Abstract]:Objective to construct the yeast two-hybrid eukaryotic expression vector of SmadE (Echinococcos granulosus SmadE) gene of Echinococcus granulosus, and to detect the toxicity and self-activation activity of the recombinant protein. Methods EgSmadE gene fragment was obtained by PCR and cloned into yeast two-hybrid vector pGADT7 and pGBKT7.. The recombinant vector was introduced into Y2HGold yeast receptive cells by PEG/LiAc method, and its toxicity and self-activation activity were detected by phenotypic screening of solid medium. Results the total length of EgSmadE gene was 1129bp. after being ligated with yeast two-hybrid recombinant vector, pGADT7-EgSmadE and pGBKT7-EgSmadE, were digested and sequenced correctly by PCR, restriction endonuclease. The results showed that the recombinant vector expression product was not toxic to yeast, and the white colony growth was found in SD/-Trp and SD/-Leu medium, while SD/-Leu/X/A and SD/-Trp/X/A had no colony growth, indicating that the recombinant vector expression product had no self-activating activity to the downstream reporter gene. Conclusion the successful construction of yeast two-hybrid vector pGADT7-EgSmadE and pGBKT7-EgSmadE, can be used to identify EgSmadE protein and lay a foundation for the study of molecular mechanism of interaction between host and echinococcus.
【作者单位】：新疆医科大学基础医学院生理学教研室;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;
【基金】：新疆维吾尔自治区自然科学基金项目(No.2015211C029)
【分类号】：R532.32

【参考文献】