CytoTrap酵母双杂交筛选与乙型肝炎病毒HBx蛋白相互作用的肝细胞蛋白

Screening of hepatocyte proteins interacting with hepatitis B virus X protein using CytoTrap yeast two-hybrid technique

目的筛选与乙型肝炎病毒HBx蛋白发生相互作用的肝细胞蛋白。方法构建诱饵重组载体pSos-HBx,经测序正确后将其转化酵母菌cdc25Hα中,检测其在酵母中的表达及有无自激活作用,并利用CytoTrap酵母双杂交的方法筛选与诱饵蛋白HBx相互作用的肝细胞蛋白。结果获得重组诱饵质粒pSos-HBx,验证可以在酵母中表达诱饵蛋白Sos-HBx,且诱饵质粒在此系统中没有自激活作用。利用此系统筛选出6个能与HBx相互作用的肝细胞蛋白,分别为纤连蛋白1、翻译调控肿瘤蛋白1、核受体结合蛋白IQ-WD1、软泡抑素、血清类粘蛋白1、二硫化物异构酶家族A3。结论成功克隆出乙型肝炎病毒HBx蛋白的结合蛋白,为进一步研究HBx蛋白在肝炎或肝癌过程中发挥的作用提供了新线索。

Objective To screen the hepatocyte proteins that interact with hepatitis B virus X protein（HBx）.Methods The recombinant plasmid pSos-HBx was constructed by inserting Sos-HBx fragment into the bait vector,and after sequence verification the plasmid was transformed into competent yeast cells.The expression and self-activation of Sos-HBx protein was detected in the yeast cells.The hepatocyte proteins interacting with the bait protein was screened with CytoTrap yeast two-hybrid technique.Results The reconstructed plasmid harboring HBx gene expressed Sos-HBx protein in the yeast cells without self-activation of the protein.CytoTrap yeast two-hybrid system identified 6 hepatocyte proteins that interacted with HBx,including fibronectin 1,translationally controlled tumor protein,IQ motif and WD repeats 1,follistatin,orosomucoid 1, and disulfide isomerase family A member 3.Conclusion Six HBx-binding hepatocyte proteins have been identified using the CytoTrap yeast two-hybrid system,which provides clues for further investigation of the role of HBx protein in hepatitis and liver cancer.