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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 a eukaryotic expression vector for HBsAg decoy and express it in AH109 yeast, and amplify, purify, identify and transform human pancreatic cDNA library. Methods The HBsAg gene was amplified by PCR, cloned into pGEM-T vector, sequenced correctly, ligated into expression vector pGBKT7, and transformed into yeast AH109. The expression of HBsAg was detected in tryptophan-deficient medium (SD / -Trp / Kana) Positive colonies were screened and the recombinant proteins were extracted and analyzed by SDS-PAGE after Western blotting. The cDNA library of human pancreas was amplified, purified and identified by restriction enzyme digestion and bioinformatics analysis. The recombinant plasmid was transformed into Y187 yeast by lithium acetate method. Results The yeast expression vector pGBKT7-HBsAg was successfully constructed. The recombinant protein was correctly expressed in yeast cells by SDS-PAGE and Western blot. The human pancreas cDNA library was successfully constructed with a capacity of 4 × 109 CFU / L and an insert size of 0.5-2.0 kb, uneven length, recombination rate of 100%. Conclusion The eukaryotic vector of HBsAg was successfully constructed and expressed in AH109 yeast. At the same time, a high quality pancreas cDNA library was obtained and transformed into Y187 yeast for the purpose of screening the protein genes interacting with HBsAg through yeast two-hybrid system.