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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,检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(EgTβRⅠintracellular domain,EgTβRⅠ-Ⅰ)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法转化入酵母菌株,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pGBKT7-EgTβRⅠ-Ⅰ真核表达载体,经双酶切,分别得到1 662bp和1 314bp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2mm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酵母菌无毒性作用和无自激活活性;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。
Objective To construct full-length and intracellular eukaryotic expression vectors of EgTβRⅠ (EgT) and to detect the cytotoxicity and self-activation activity of recombinant bait plasmids against yeast strains. Methods The total RNA of Echinococcus granulosus was extracted by TRIzol method. The full-length EgTβRⅠ gene and the EgTβRⅠ-Ⅰ intracellular domain of EgTβRⅠ were amplified by RT-PCR. The pGBKT7-EgTβRⅠ and pGBKT7-EgTβRⅠ-Ⅰ The eukaryotic expression vector was verified by PCR, restriction enzyme digestion and sequence analysis. After the PEG / LiAc method was transformed into yeast, the toxic effect of bait protein on yeast and its self-activation activity were tested. Results The eukaryotic expression vectors pGBKT7-EgTβRⅠ and pGBKT7-EgTβRⅠ-Ⅰ were successfully constructed. The double-digested fragments of 1 662bp and 1 314bp were obtained respectively. The size of the target gene was consistent with the predicted value. The colony formed by the two recombinant plasmids transformed Y2HGold yeast was consistent with the size of the transformed yeast pGBKT7 plasmid and had a diameter of 1.5-2.0 mm. The two recombinant plasmid-transformed yeasts did not grow blue colonies on the SD / -Trp / X / A plate, Colonies of 2 mm in diameter were formed on SD / -Trp plates. Conclusion The eukaryotic expression vector pGBKT7-EgTβRⅠ and pGBKT7-EgTβRⅠ-Ⅰ were constructed successfully. The expressed protein was non-toxic and non-self-activating to Y2HGold yeast. The expression vector could be used in yeast two-hybrid system. The interaction between ligands lays the foundation.