Screening exonic genes of proteins in hepatocytes interacting with HBEBP2 by yeast two-hybrid technique / 解放军医学杂志

ABSTRACT

Objective To construct the eukaryotic expression vector of HBEBP2 gene,and screen the exonic genes of proteins in hepatocytes interacting with HBEBP2.Methods The DNA fragment of HBEBP2 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 transformed AH109 were then mated with yeast cells Y187 containing hepatic cDNA library plasmid.The diploid yeast cells were plated on synthetic dropout nutrient medium(SD/-Trp/-Leu/-His/-Ade) containing X-?-gal for the first selection.Library plasmids pACT2-DNA were extracted and co-transformed into yeast cell AH109 together with pGBKT7-HBEBP2.Then the yeast cells were plated on synthetic dropout nutrient medium(SD/-Trp/-Leu /-His/-Ade) containing X-?-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.