应用酵母双杂交技术筛选与乙型肝炎病毒e抗原结合蛋白2结合的肝细胞蛋白编码基因

Screening exonic genes of proteins in hepatocytes interacting with HBEBP2 by yeast two-hybrid technique

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 the eukaryotic expression vector of HBEBP2 gene, and screen the exonic genes of proteins in hepatocytes interacting with HBEBP＇2. Methods The DNA fragment of HBEBP＇2 was amplified by PCR. The eukaryotic expression vector pGBKT7 HBEBP2 was constructed successfully by yeast-two hybrid system 3 and then transformed into yeast cells AH109. The trans- formed AH109 were then mated with yeast cells Y187 containing hepatic cDNA library plasrnid. The diploid yeast cells were plated on syn thetic dropout nutrient medium （SD/-Trp/-I2eu/ His/ Ade） containing X-α-gal for the first selection. Library plasmids pACT2 DNA were extracted and co transformed into yeast cell AH109 together with pGBKT7 HBEBP＇2. Then the yeast cells were plated on synthetic dropout nutrient medium （SD/ Trp/ Leu / His/ Ade） containing X-α-gal for the second screening to eliminate false positive clones. The real positive clones were sequenced and analyzed by bioinformatics. Results Six proteins binding to HBEBP2 were screened, including human sapiens lactate dehydrogenase D, human sapiens mitochondrion, human sapiens mannose, human sapiens aldehyde oxidase,human sapiens serpin peptidase inhibitor and other two unknown proteins. Conclusions A novel class of proteins in hepatocytes interacting with HBEBP2 has been obtained. It is presumed that HBEBP2 protein is correlated with glycosylation, lipid metabolism and cell proliferation, etc.