乙型肝炎病毒X蛋白与去唾液酸糖蛋白受体2突变体相互作用的研究

Cloning of a gene coding for novel mutant of asialoglycoprotein receptor 2 binding to hepatitis B virus X protein in hepatocytes

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。

AIM: The pathogenesis of HBV-induced malignant trans- formation is incompletely understood. The X protein of hepatitis B virus (HBxAg) is a multifunctional protein that can influence a variety of signal transduction pathways within the cell and is essential for establishing natural viral infection, it also has been implicated in the development of liver cancer associated with chronic infection. Further understanding of the interaction between HBxAg and proteins in hepatocytes is of great significance for the prevention of the development of hepatocellular carcinoma (HCC). METHODS: HBxAg bait plasmid was constructed by ligating the HBxAg gene with a yeast expression vector pGBKT7, then transformed into yeast AH109 (a type). The transformed yeast cells were amplified and mated with yeast cells Y187 (α type) containing liver cDNA library plasmid pCAT2 in 2×YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and syn- thetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection twice. Rasmid of ture positive blue colonies was extracted and analysed by DNA sequencing and blast in genbank. After the complete sequence of the novel mutant of asialoglycoprotein receptor 2 (ASGPR2) was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR)and cloned into pGADT7 vector, the recombined plasmid was translated by using reticulocyte lysate and analysed by im- munoprecipitation technique in vitro together with HBxAg. RESULTS: Eighteen genes in forty-one positive colonies were obtained, one of them is a novel mutant of ASGPR2, which is 80 % homologous to natural ASGPR2. The complete sequence of the mutant was amplified from the mRNA of HepG2 cell by RT-PCR successfully. The interaction between HBx and ASGPR2 mutant was further confirmed by immu- noprecipitation technique. CONCLUSION: Interaction between HBx and ASGPR2 mutant can be observed in both yeast cell and in vitro.