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目的:筛选并克隆鉴定人肝细胞中与乙型肝炎病毒(HBV)X蛋白(HBxAg)相互作用蛋白的基因,明确HBxAg在HBV感染及致癌过程中的具体作用。方法:用多聚酶链反应(PCR)法扩增HBxAg基因,连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人肝cDNA文库质粒的酵母细胞Y187进行配合,在营养缺陷型培养基上进行双重筛选阳性菌落,PCR从中扩增出阳性目的片段并测序,进行生物信息学分析。根据Genbank中的序列信息设计引物,从HepG2细胞的mRNA中逆转录出去唾液酸蛋白受体2(ASGPR2)突变体的完整序列,克隆到另一酵母表达载体pGADT7中,体外免疫共沉淀再次证明HBxAg与ASGPR2突变体的结合作用。结果:成功克隆出HBxAg基因并在酵母细胞中表达,配合后选出既能在四缺(SD/-Trp-Leu-His-Ade)培养基又能分解X-α-半乳糖(X-α-gal)变成蓝色的真阳性菌落41个,其中有一个是ASGPR2的新突变体。HepG2细胞的mRNA中能逆转录出ASGPR2的全基因序列, 体外免疫共沉淀结果证实该突变体与HBxAg在体外也有结合作用。结论:成功克隆出HBxAg的肝细胞结合蛋白,发现一新的ASGPR2突变体,并证实HBxAg与ASGPR2突变体在体外及酵母细胞内均有结合作用。
OBJECTIVE: To screen and clone the genes of human hepatocyte protein interacting with hepatitis B virus (HBV) X protein (HBxAg) and clarify the specific role of HBxAg in HBV infection and carcinogenesis. Methods: The HBxAg gene was amplified by polymerase chain reaction (PCR) and ligated into the yeast expression vector pGBKT7 to construct the bait plasmid. The bait plasmid was transformed into AH109 and transformed into yeast cell Y187 transformed with human liver cDNA library plasmid With the double positive screening of auxin-positive colonies on auxotrophic medium, the positive fragments were amplified by PCR and sequenced for bioinformatics analysis. According to the sequence information in Genbank, primers were designed and the complete sequence of ASGPR2 was reverse transcribed from HepG2 cells mRNA and cloned into another yeast expression vector pGADT7. Co-immunoprecipitation demonstrated that HBxAg Binding to ASGPR2 mutants. Results: The HBxAg gene was successfully cloned and expressed in yeast cells. After mating, the HBxAg gene was expressed in both SD--Trp-Leu-His-Ade medium and X-α-galactose -gal) into the blue true-positive colonies 41, of which one is a new mutant ASGPR2. The full-length ASGPR2 gene was reverse transcribed in mRNA of HepG2 cells. Co-immunoprecipitation in vitro showed that this mutant had a binding function with HBxAg in vitro. CONCLUSION: The HBxAg hepatocyte-binding protein was successfully cloned and a new ASGPR2 mutant was found. It was confirmed that both HBxAg and ASGPR2 mutants had a binding activity in vitro and in yeast cells.