利用同源重组构建人脑的酵母双杂交cDNA文库

Construction of Yeast Two Hybrid cDNA Library of Human Brain through Homologous Recombination

目的 利用SMART（switching mechanism at 5′end of RNA transcript）技术，通过同源重组的方法，在酵母菌株Y187中构建人脑全长cDNA文库。方法 利用SMART技术，以人脑总RNA为模板，反转录合成cDNA第一链。再在DNA聚合酶下，通过长距离PCR，扩增双链cDNA。同时构建pGADT7-Rec质粒载体。纯化的ds cDNA与线性化pGADT7-Rec质粒载体共同转化酵母菌株感受态Y187，经胞内同源重组形成环状文库质粒。应用营养缺陷的方法在SD／-Leu平板上筛选并收集所有克隆从而获得酵母双杂交的人脑cDNA文库。结果 所获得的文库容量为1．2×10^6，重组子不同插入片断大小在0．3～2kb之间。结论 在酵母菌株Y187成功构建了用于酵母双杂交的人脑cDNA文库。

Objective To construct yeast two hybrid cDNA library of human brain in yeast cell Y187. Methods The first chain of cDNA was synthesized by reverse transcript from human brain total RNA as template by SMART methods. The ds cDNA was amplified by long distance-PCR in existence of DNA polymerase and pGADT7-Rec vector was constructed as well. The purified ds cDNA and the linearized plasmid pGADTT-Rec were co-transformed into the competent yeast Y187. Recombinants plasmids formed through homologous recombination in vivo. All the recombinants were harvested from the SD/- Leu plates and then constituted the human brain cDNA library. Results 1.2×10^6 recombinants were obtained from the human brain cDNA library and the insert fragments of reeombinants were demonstrated to distribute among 0.3-2 kb in size. Conclusion The yeast two hybrid cDNA library of human brain in yeast cell Y187 was successfullv constructed.