人α-干扰素与乙型肝炎病毒前S2嵌合蛋白的表达纯化和鉴定  

Expression, purification and identification of human IFN-α 2 b/HBV Pre S2 fusion protein

作　　者：徐东平[1] 韩凤连[1] 施红[1] 王福生[1] 程云[1] 金磊[1] 雷周云[1] 刘明旭[1] 

机构地区：[1]解放军302医院生物工程研究室,北京100039

出　　处：《中华实验和临床病毒学杂志》2000年第1期69-72,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：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 express a fusion protein of human interferon α 2b and HBV Pre S2 in E.coli for the purpose of investigating anti HBV immunomodulatory protein.Methods Human interferon α2b and HBV Pre S2 encoding genes were amplified from plasmid templates through PCR, then fused and cloned into plasmid pBV220 through engineering technique to generate expression plasmid pBV IFN Pre S2. The plasmid was transfected into E.coli to produce fusion protein. RP HPLC and ion exchange chromatography were employed to purify fusion protein.Results 27 kDa fusion protein was expressed up to 15% of total bacterial protein in E.coli. After purification, the purity of fusion protein reached 95% of total protein. Anti viral assay showed that IFN Pre S2 protein induced a VSV resistant activity of 1 25×10 8 IU/mg protein in Wish cell line, similar to the bioactivity of original recombinant human IFN α 2 b. ELISA data showed that IFN Pre S2 protein had antigenicities of both human IFN α 2b and HBV Pre S2. In addition, fusion protein showed the feature of binding to polymeric human serum albumin (PHSA).Conclusion The bifunctional fusion protein was efficiently expressed in E.coli. The work provided initial evidence for studying PHSA receptor targeting IFN α 2b,which might have potential application for the treatment of HBV infection.

关 键 词：乙型肝炎病毒 干扰素Α 病毒融合蛋白质类 

分 类 号：R373[医药卫生—病原生物学]