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目的:乙型肝炎病毒e抗原(HBeAg)对HBV的感染和复制都不是必须的,普遍认为其与HBV引起免疫耐受、免疫系统功能障碍有关。筛选并克隆人肝细胞cDNA文库中与HBeAg相互作用蛋白的基因,明确其具体作用机制。方法:应用酵母双杂交系统3,将多聚酶链反应(PCR)法扩增的HBeAg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pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和X-α-半乳糖(X-α-gal)上进行双重筛选阳性菌落,提取阳性酵母菌落的质粒转化大肠杆菌氨苄青霉素-LB平板上选择并测序,结果在GenBank中进行生物信息学分析,并根据GenBank中的序列信息设计引物从并克隆到另一酵母表达载体pGADT7中,体外免疫共沉淀方法再次验证二者之间的结合作用。结果:成功克隆出HBeAg基因并在酵母细胞中表达,与肝文库配合后选出既能在四缺(SD/-Trp-Leu-His-Ade)培养基又能使X-α-gal变成蓝色的真阳性菌落39个,其中有5个未知基因,在genbank中未找到同源序列,在HepG2细胞的mRNA中成功扩增出该基因的全序, 体外免疫共沉淀方法证明HBeAg与新基因E-19表达的蛋白质在体外也有结合作用。结论:成功克隆出HBeAg的肝细胞结合蛋白,发现与HBeAg有相互作用的未知蛋白新基因,为HBeAg的功能研究提出新线索。
OBJECTIVE: Hepatitis B virus e antigen (HBeAg) is not necessary for HBV infection and replication. It is generally accepted that HBV e antigen is associated with immune tolerance and immune system dysfunction. Screening and cloning of human hepatocyte cDNA library with HBeAg-interacting protein gene, clear its specific mechanism of action. Methods: The HBeAg gene amplified by polymerase chain reaction (PCR) was ligated into the yeast expression vector pGBKT7 to construct the bait plasmid and transformed into yeast AH109 by yeast two-hybrid system.3. Library plasmid pACT2 Yeast cells Y187 were combined to double screen positive colonies on auxotrophic medium and X-α-galactose (X-α-gal), and plasmids from positive yeast colonies were transformed into E. coli ampicillin-LB The results of the bioinformatics analysis were performed in GenBank and the primers were designed according to the sequence information in GenBank and cloned into another yeast expression vector pGADT7. The co-immunoprecipitation method was used to verify the binding between the two effect. Results: The HBeAg gene was successfully cloned and expressed in yeast cells. After being combined with the liver library, the recombinant plasmid was selected to be able to transform both X-α-gal and SD--Trp-Leu-His- There were 39 true-positive blue colonies, of which 5 were unknown genes. No homologous sequences were found in genbank. The full-length sequence of this gene was successfully amplified in HepG2 cells. Co-immunoprecipitation in vitro demonstrated that HBeAg and new Proteins expressed by the gene E-19 also have a binding function in vitro. Conclusion: The successful cloning of hepatocyte-binding protein of HBeAg and the discovery of a novel gene with unknown interaction with HBeAg provide new clues for the functional study of HBeAg.