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目的构建乙型肝炎病毒e抗原结合蛋白2(HBEBP2)的真核表达载体并筛选人肝细胞中与HBEBP2相互作用的基因。方法通过PCR扩增获得HBEBP2基因,构建真核表达载体pGBKT7-HBEBP2,转化酵母菌AH109并在其中进行表达。随后与预转化了人肝细胞文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基(SD/-Trp/-Leu/-His/-Ade)和铺有X-α-gal的营养缺陷型培养基(SD/-Trp/-Leu/-His/-Ade)上进行双重筛选获得阳性克隆。提取文库质粒pACT2-DNA并与pGBKT7-HBEBP2共同转化AH109酵母菌株,于铺有X-α-gal的营养缺陷型培养基(SD/-Trp/-Leu/-His/-Ade)上进行筛选以排除假阳性克隆。挑取真阳性克隆送测序,并进行生物信息学分析。结果筛选出6种与HBEBP2相互作用的蛋白,其中包括人类线粒体蛋白、人类α-2-糖蛋白1、人类磷酸甘露糖-p-长醇利用缺陷1和人类丝氨酸蛋白酶抑制剂等4个已知功能蛋白及2个未知功能序列。结论筛出人肝细胞中一组与HBEBP2相互作用的蛋白,为进一步探讨HBEBP2在HBV致病过程中的作用奠定了基础。
Objective To construct eukaryotic expression vector of hepatitis B virus e antigen - binding protein 2 (HBEBP2) and to screen for genes that interact with HBEBP2 in human hepatocytes. Methods The HBEBP2 gene was amplified by PCR. The eukaryotic expression vector pGBKT7-HBEBP2 was constructed and transformed into yeast AH109. Subsequently, the yeast cells Y187, which pre-transformed human hepatocyte library plasmid pACT2, were co-cultured in the presence of auxotrophic medium (SD / -Trp / -Leu / -His / -Ade) The positive clones were obtained by double screen on SD / -Trp / -Leu / -His / -Ade. Library plasmid pACT2-DNA was extracted and transformed with pGBKT7-HBEBP2 into AH109 yeast strain and screened on auxotrophic medium (SD / -Trp / -Leu / -His / -Ade) plated with X-α-gal Exclude false-positive clones. Pick the true positive clones for sequencing, and perform bioinformatics analysis. Results Six proteins were identified that interacted with HBEBP2, including four human mitochondrial proteins, human α-2-glycoprotein 1, human mannose-phosphate dehydrogenase 1, and human serine protease inhibitors Functional protein and two unknown functional sequences. Conclusion A group of proteins that interact with HBEBP2 in human hepatocytes are screened out, which lays the foundation for further exploring the role of HBEBP2 in the pathogenesis of HBV.