一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .

Screening, Expression and characterization of a Novel Protein Binding to Hepatitis B Surface Antigen

In order to study the functional proteins involved in the process of hepatitis B virus invasion to hepatocytes, a cDNA clone coding a protein binding to hepatitis B surface antigen(HBsAg) was screened out from a human liver cDNA expression phage library by immunoscreening. DNA sequencing analysis showed that the cDNA had an open reading frame encoding 344 amino acid residues without transmembrane domain and belonged to immunoglobulin superfamily. This gene was inserted into expression vector pTriplEx and the recombinant fusion protein of 44 kD was purified from E.coli XL1 blue. The specific binding ability of the recombinant protein to HBsAg was verified by Western blotting and ELISA. Flow cytometry (FCM) analysis showed that purified recombinant protein could enhance the affinity of HBsAg to human hepatoma cells HepG2. This finding suggested that hepatitis B surface antigen binding protein(HBsAg BP) might be a soluble co receptor mediating hepatitis B virus invasion to hepatocytes.