乙型肝炎病毒e抗原结合蛋白1“饵”载体的构建及在酵母中的表达

Construction and expression of a novel HBeAg binding protein 1 of hepatitis B virus in yeast.

目的 构建乙型肝炎病毒e抗原结合蛋白1（ HBEBP1）真核表达载体,并在酵母细胞中进行表达.方法 以HepG2细胞来源的eDNA为模板,PCR扩增获得HBEBP1基因,克隆至pGEM-T载体.测序正确后双酶切pGEM-T-HBEBP1,回收目的片段并连接至酵母表达载体pGBKT7中,转化酵母菌AH109,在色氨酸缺陷型培养基（SD/-Trp/卡那霉素）上筛选阳性菌落后大量表达并提取重组蛋白,再进行SDS-PAGE和Western Blot分析.结果 成功构建酵母表达载体pGBKT7-HBEBP1,Western Blot结果显示重组蛋白表达产物存在于胞内,条带清晰、大小正确,相对分子质量约33 × 103.结论 利用真核表达载体在酵母细胞中成功表达HBEBP1蛋白,为研究HBEBP1蛋白的生物学功能奠定了基础.

Objective To construct the eukaryotic expression vector of HBEBP1 gene and express HBEBP1 recombinant protein in yeast.Methods PCR was performed to amplify the gene of HBEBP1 from the cDNA template origining from HepG2,and the gene was cloned into pGEM-T vector.After sequencing,the correct DNA fragment was cut from pGEM-T-HBEBP1 and inserted into yeast expression plasmid pGBKT7.The reconstructed plasmid pGBKT7-HBEBP1 was transformed into yeast cell AH109 and screened on the synthetic dropout nutrient medium （SD/-Trp/Kana）.The yeast protein was isolated and analyzed with SDS-PAGE and Western Blot.Results The eukaryotic expressive vector was constructed successfully.The results of Western Blot showed HBEBPi protein was existed within yeast cells and the molecular weight of it was about 33 × 103.Conclusions The successful expression of HBEBP1 protein in yeast cells lay the foundation for studying biological function of HBEBP1.