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目的 重组表达人α 干扰素与乙型肝炎病毒前S2融合蛋白 (IFN PreS2 ) ,探索治疗HBV感染的特异性免疫药物。方法 采用聚合酶链反应 (PCR)技术扩增出人IFN α2b和HBVPreS2基因片段 ,分步克隆获取融合基因 ,构建pBV IFN PreS2重组表达载体 ,转化E .coli后诱导表达IFN PreS2融合蛋白。结果 在E coli中表达了相对分子质量为 2 70 0 0的目标蛋白 ,后者以包含体形式存在 ,表达量占菌体总蛋白的 15 %。经过反相高效液相色谱 (RP HPLC)等步骤纯化后 ,目标蛋白纯度达到 95 %。复性的表达产物在体外测定具有相应的生物活性和免疫学特性 ,干扰素生物比活达到 10 8IU/mg蛋白质。融合蛋白能与抗IFN α抗体、抗HBVPreS2抗体及多聚人血清白蛋白 (PH SA)结合。结论 在原核系统中有效表达了具有高生物活性的IFN PreS2融合蛋白。
Objective To recombinantly express human interferon - alpha (IFN - α) and hepatitis B virus preS2 fusion protein (IFN PreS2) and to explore specific immune drugs for the treatment of HBV infection. Methods Human IFNα2b and HBVPreS2 gene fragments were amplified by polymerase chain reaction (PCR). The fusion gene was cloned by stepwise cloning. The recombinant plasmid pBV IFN PreS2 was constructed and transformed into E.coli to express IFN PreS2 fusion protein. As a result, the target protein with a relative molecular mass of 2 70 0 was expressed in E. coli which was present in inclusion bodies and accounted for 15% of the total bacterial proteins. Purified by reverse-phase high-performance liquid chromatography (RP HPLC) and other steps, the target protein purity of 95%. The refolding expression product has the corresponding biological activity and immunological characteristics in vitro, and the specific biological activity of interferon reaches 10 8 IU / mg protein. The fusion protein can bind to anti-IFNα antibody, anti-HBVPreS2 antibody and poly-human serum albumin (PH SA). Conclusion The IFN PreS2 fusion protein with high biological activity was efficiently expressed in prokaryotic system.