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[目的]构建诱饵载体pGBKT7-Lescpth5,并检测其转录自激活活性及对酵母细胞的毒性。[方法]采用PCR技术扩增Lescpth5基因,连接到诱饵载体pGBKT7上,转化重组载体于酵母感受态细胞AH109中,在营养缺陷型培养基上进行自激活检测和毒性检测。[结果]酶切和测序鉴定结果显示诱饵载体pGBKT7-Lescpth5构建成功,读码框正确。自激活检测结果和毒性检测结果显示诱饵载体对酵母菌株AH109没有转录激活活性,对酵母菌也无毒害作用。[结论]成功构建的诱饵载体可以用于酵母双杂交系统中,为下一步的cDNA文库筛选奠定基础。
[Objective] The bait vector pGBKT7-Lescpth5 was constructed and its transcriptional activation activity and toxicity to yeast cells were tested. [Method] The gene of Lescpth5 was amplified by PCR and ligated into the bait vector pGBKT7. The recombinant vector was transformed into yeast competent cells AH109. The self - activation assay and the toxicity assay were carried out on the auxotrophy medium. [Result] The results of enzyme digestion and sequencing showed that bait vector pGBKT7-Lescpth5 was successfully constructed and the reading frame was correct. The results of self-activation test and toxicity test showed that the bait vector had no transcriptional activation activity on the yeast strain AH109 and no toxic effect on the yeast. [Conclusion] The constructed bait vector could be used in yeast two-hybrid system, which laid the foundation for the next screening of cDNA library.