HBsAg诱饵载体的构建、表达及人胰腺cDNA文库的扩增、纯化和转化

Construction and expression of the bait vector of HBsAg in yeast and amplification,purification and transformation of a human pancreas cDNA library

目的构建HBsAg诱饵真核表达载体,并在AH109酵母菌中进行表达,同时扩增、纯化、鉴定及转化人胰腺cDNA文库。方法通过PCR扩增HBsAg基因,克隆到pGEM-T载体,测序正确后酶切并连接至表达载体pGBKT7中,转化酵母菌AH109,在色氨酸缺陷型培养基(SD/-Trp/Kana)上筛选阳性菌落,提取重组蛋白,经SDS-PAGE电泳后进行Western blot分析。扩增、纯化人胰腺cDNA文库并进行酶切鉴定及生物信息学分析,醋酸锂法将其转入Y187酵母菌。结果成功构建酵母表达载体pGBKT7-HBsAg、SDS-PAGE和Western blot显示重组蛋白在酵母细胞中正确表达;成功构建人胰腺cDNA文库,其容量为4×109 CFU/L,插入片段大小为0.5～2.0kb,长度不均,重组率为100%。结论成功构建HBsAg真核载体并在AH109酵母菌中表达,同时获得高质量的胰腺cDNA文库并转化入Y187酵母菌,为通过酵母双杂交筛选与HBsAg相互作用蛋白基因奠定了基础。

Objective To construct and express the eukaryotic expression vector of HBsAg in yeast strain AH109,and to amplify,purify,evaluate,and transform a pancreas cDNA library.Methods PCR was performed to amplify the HBsAg gene and it was then cloned into a pGEM-T vector.After sequencing,the correct DNA fragment was cut and inserted into the yeast expression vector pGBKT7.It was then transformed into yeast strain AH109 and screened on SD/-Trp/Kana.Finally,it was detected by SDS-PAGE and Western blotting.A human pancreas cDNA library was subsequently amplified,purified,evaluated,and transformed into yeast strain Y187 by LiAc.Results pGBKT7-HBsAg was successfully constructed and expressed according to SDS-PAGE and Western blotting.The titer of the cDNA library was 4×109 CFU/L with inserted fragments ranging from 0.5-2.0 kb,and the recombinant rate was 100%.Conclusion The successful expression of HBsAg protein in AH109 yeast cells and the provision of a quality pancreas cDNA library have laid the foundation for screening proteins with a yeast two-hybrid system in a pancreas cDNA library interacting with HBsAg.