酵母双杂交系统筛选HBV PreS1相互作用蛋白

目的利用Sos招募系统(SRS),构建含HBV PreS1基因的酵母双杂交诱饵载体,筛选人肝细胞与HBV PreS1蛋白相互作用的蛋白,进一步探讨HBV侵入肝细胞的机制。方法以HBV ayw亚型全长质粒PCP10为模板,PCR扩增HBV PreS1基因,克隆到酵母表达载体pSos中,构建诱饵质粒pSos-PreS1,将其转化cdc25酵母感受态细胞,提取酵母蛋白质进行Western blot分析,证实其在酵母细胞中的表达;将pSos-PreS1分别与pMyr Lamin C、pMyr SB共转化酵母,证实诱饵蛋白无自激活作用。将pSos-PreS1与人肝cDNA文库共转化cdc25酵母感受态细胞,通过营养及温度选择性培养筛选阳性菌落,扩增阳性菌落目的基因并测序。结果成功构建了HBV PreS1基因酵母双杂交诱饵载体pSos-PreS1,筛选得到5个准阳性克隆,序列分析表明其分别与人类钾通道调制因子1(KCMF1)、细胞色素C、VitD结合蛋白、人源去唾液酸糖蛋白受体(ASGPR)和人白蛋白具有高度同源性。结论利用SRS筛选出5个可能与HBV PreS1蛋白相互作用的蛋白,为进一步了解HBV侵入肝细胞的机制奠定了基础。

Screening the hepatitis B virus PreS1 associated proteins in yeast two-hybrid system

Objective To screen the interacted protein hepatitis B virus(HBV) PreS1 with human hepatocytes from normal human liver cDNA library by sos-recruitment system(SRS) and explore the mechanism of HBV endocytosis.Methods PCR was performed to amplify the gene of HBV PreS1 from the plasmid PCP10/ HBV ayw subtype containing the whole fragment of HBV;the PCR product was cloned into yeast expression plasmid pSos,and reconstituted plasmid was tested by auto-sequencing assay and named pSos-PreS1.The pSos-PreS1 fusion protein expressed in the yeast cells was confirmed by Western blot after pSos-PreS1 was transfected into the yeast cell cdc25.Self-activation of the bait protein was determined by cotransformation of pSos-PreS1 and pMyr-Lamin C,and the cytoplasmic localization of the bait protein was verified by cotransformation of pSos-PreS1 and pMyr SB.Yeast cells co-transfected with pSos-PreS1 and the normal human liver cDNA library grew in selective nutrition and temperature.The true positive clones were submitted for sequencing.The results were submitted to the BLAST notebook of NCBI to seek homologous sequence.Results The yeast expression vector of HBV PreS1 gene was constructed successfully and its expression in yeast was verified.The recombinant bait plasmid did not have self-activation and toxicity to yeast cdc25H cells.Furthermore,the cytoplasmic localization of the bait protein was verified correctly.After yeast cells were co-transfected with pSos-PreS1 and the normal human liver cDNA library,5 clones were positive and showed high homology with KCMF1,cyt C,vitamin D binding protein,Homo sapiens albumin(ALB) and Homo sapiens asialoglycoprotein receptor(ASGPR).Conclusion We obtained 5 proteins which may interact with HBV PreS1 protein by SRS.This is helpful for exploring the mechanism of HBV endocytosis.