酵母双杂交体系筛选乙型肝炎病毒前S1蛋白相关蛋白

目的 利用酵母双杂交体系筛选乙型肝炎病毒前S1(PreS1)蛋白相关蛋白,进一步探讨PreS1在乙型肝炎病毒(HBV)人胞中的作用机制。方法 以HBV DNA阳性血清为模板扩增含EcoRⅠ与PstⅠ酶切位点的PreS1基因序列,pAS2—1载体及PreS1基因PCR产物经双酶切并连接,对饵载体pAS2—1—PreS1进行序列测定;将pAS2—1—PreS1转化入酵母菌AH109,以western blot法证实其在酵母细胞中的表达。将pAS2—1—PreS1及正常成人肝细胞cDNA文库共转化酵母细胞,通过选择性培养、LacZ活性测定筛选阳性菌落,分离实验及交合实验排除假阳性,扩增阳性克隆的目的基因并进行序列测定,结果提交BLAST程序,在GenBank数据库查询其同源序列。结果 饵载体pAS2—1—PreS1经序列测定含有完整的PreS1基因片段,western blot证实转化的酵母细胞能正确表达完整的PreS1—BD融合蛋白。pAS2—1—PreS1与正常成人肝细胞cDNA文库共转化酵母细胞后,选择性培养共长出97个菌落,筛选出1个阳性克隆,其PCR扩增产物经GenBank数据库查询与人类初期多肽相关复合体α亚单位(NACA)高度同源。结论 成功构建了pAS2-1-PreS1饵载体,并通过酵母双杂交体系筛选出肝细胞内与PreS1相互作用的蛋白NACA。

Screening the hepatitis B virus PreS1 associated protein by the yeast two-hybrid system

Objectives To screen the hepatitis B virus PreS1 associated protein from normal human liver cDNA library by the yeast two-hybrid system and explore the role of PreS1 protein in the infection of hepatitis B virus (HBV). Methods PCR was preformed to amplify the PreSl gene containing EcoRI and PstI from HBV positive serum, and the production was inserted into plasmid pAS2-1 after digesting with the former two restricted endonuclease, then the bait vector pAS2-1-PreS1 was verified by auto-sequencing assay. The PreS1-BD fusion protein expressed in the yeast cells was confirmed by western blot, after pAS2-l-PreSl was transfected into the yeast cell AH109. Yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade2 medium, and the second screening was performed with LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive, then the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed Site NCBI to seek homologous sequence. Results Bait vector pAS2-l-PreSl included the anticipated fragment of PreSl gene. Western blot showed that pAS2-l-PreSl could correctly express PreS1-BD fusion protein in the yeast cells. After yeast cells co-transfected with pAS2-l-PreSl and the normal human liver cDNA library, 97 colonies grew in the selective SC/-trp-leu-his-ade2 medium, only one clone was positive and showed high homology with Homo sapiens nascent-polypeptide-associated complex alpha polypeptide. Conclusions Bait vector pAS2-1-PreS1 is successfully constructed, and nascent-polypeptide-associated complex alpha polypeptide protein expressed in hepatocyte can interact with PreS1 by the yeast two-hybrid system.