HeLa细胞cDNA文库及酵母双杂交诱饵载体pGBKT7-CPn0308的构建

Construction of cDNA Library for HeLa Cells and A Bait Vector of pGBKT7-CPn0308 in Yeast Two-hybrid System

旨在利用酵母双杂交系统构建HeLa细胞的cDNA文库,并构建酵母双杂交诱饵载体pGBKT7-CPn0308。从HeLa细胞中提取总RNA,应用SMARTTM技术,构建以pGADT7-Rec为载体的HeLa细胞酵母GAL4 AD融合cDNA文库。应用PCR技术扩增目的片段CPn0308,并成功克隆该基因到诱饵载体pGBKT7中,将重组质粒转化酵母菌株AH109后,检测诱饵载体有无自激活和细胞毒性作用。结果显示,文库展现良好的多态性,重组诱饵载体pGBKT7-CPn0308不具有毒性且未自主激活报告基因,说明该文库和诱饵质粒可用于酵母双杂交系统。

It was to construct a yeast GAL4 fusion cDNA library for HeLa cells by yeast two-hybrid system and construct yeast two-hybrid system bait vector of pGBKT7-CPn0308.A yeast GAL4 fusion cDNA library from HeLa cells was constructed.Total RNA of HeLa cells was purified by using Trizol reagents,and the cDNAs of the mRNAs contained in the purified total RNA sample were synthesized by SMARTTM technology,which were subsequently cloned into the plasmid pGADT7-Rec.CPn0308 gene was amplified by polymerase chain reaction（PCR）,and successfully cloned into yeast two hybrid bait vector pGBKT7.These recombinant plasmids were transformed into yeast cells AH109,and then self-activation and toxic action of the bait vector pGBKT7-CPn0308 were tested.Result showed the library show satisfactory polymorphism.The pGBKT7-CPn0308 bait vector was identified that neither had the ability of self-activation nor yeast cell toxicity.Therefore,the constructed library and pGBKT7-CPn0308 could be used in yeast two-hybrid system.