细粒棘球蚴TGF-βⅠ型受体全长和胞内域酵母双杂交载体的构建及自激活鉴定

Full-length and intracellular domain constructs of transforming growth factor-βtypeⅠreceptor fromEchinococcus granulosus for use in a yeast two-hybrid system and testing of the bait plasmid for autoactivation

目的构建细粒棘球绦虫（Eg）转化生长因子I型受体（EgTβRI）全长及胞内域酵母双杂交真核表达载体，愉测重组诱饵质粒对酵母菌株的毒性作用及自激活活性。方法采用TRIzol法提取细粒棘球蚴总RNA；采用RT—PCR扩增EgTβRI全长基因及EgTβRI胞内域（EgTβRIintracellulardomain，EgTβRII）基因片段，分别构建pGBKT7-EgTβRI和pGBKT7-EgTβRI—I真核表达载体，经PCR、限制性酶切鉴定及序列测定正确后，PEG／LiAc法转化人酵母菌株，检测诱饵蛋白对酵母菌的毒性作用及其自激活活性。结果成功构建了pGBKT7-EgTβRI及pGBKT7EgTβRI—I真核表达载体，经双酶切，分别得到1662bp和1314bp目的基因片段，大小与预测值相符。两重组质粒转化Y2HGold酵母菌形成的菌落与pGBKT7质粒转化酵母菌菌落大小一致，直径为1．5～2．0mm；两重组质粒转化酵母菌在SD／Trp／X／A平板上无蓝色菌落生长，在SD／-Trp平板上形成直径为2mill的菌落。结论成功构建了pGBKT7-EgTβRI及pGBKT7-EgTβRI—I真核表达载体，其表达蛋白对Y2HGold酵母菌无毒性作刷和无自激活活性；该表达载体可以用于酵母双杂交系统，为进一步研究EgTβRI与其配体之间的交互作用奠定了基础。

Objectives To prepare a full-length construct of transforming growth factor-t？ type I receptor from Echhm coccus granulosus （EgTIβR I ） and an intraeellular domain construct （EgTβR I - I ） for a yeast two-hybrid system and to test the recombinant bait plasmid for toxicity and autoactivation activity. Methods EgTI3R I and EgTI3R ] I gene fragments from Eg RNA were amplified with RT-PCR, and then sequences were cloned into a eukaryotic expression vec tor. After identification by PCR, restriction analysis, and sequencing, the recombinant plasmid was transformed into the yeast strain Y2HGold via PEG/LiAc-mediated transformation, and its toxicity and autoactivation activity were deter mined. Results The yeast two hybrid expression vectors pGBKTT-EgTI3R I and pGBKT7 EgTI3R [ - I were success fully constructed. Two gene fragments with a length of 1,662 bp and 1,314 bp were obtained by restriction enzyme diges tion analysis. The results were consistent with the predicted values. The yeast strain Y2HGoId that was transformed with the recombinant plasmids grew well on SD-Trp plates. Colonies ranged in size from 1.5 -2.0 mm and were the same size as those of the positive control. Colonies nearly 2.0 mm in size were present on SD/-Trp plates, but blue colonies were not present on SD/-Trp/X/A plates. Conclusion The eukaryotic expression vectors constructed in this study can bc used in a yeast two hybrid system. This work has laid the foundation for further study of the interaction between TβR Ⅰ and its ligands.