抗HBsAg单链抗体靶向干扰素的构建及原核表达  

Construction and expression of humanized anti-HBsAg scFv targeting interferon-α in escherichia coli

作　　者：夏小兵[1] 成军 杨继珍[2] 钟彦伟[2] 王刚[2] 方宏清[3] 刘妍[2] 李克[2] 董菁[2] 

机构地区：[1]中国人民解放军第三O二医院传染病 研究所基因治疗研究中心,北京100039 [2]中国人民解放军第三O二医院传染病研究所基因治疗研究中心,北京100039 [3]军事医学科学院生物工程研究所

出　　处：《中华肝脏病杂志》2002年第1期28-30,共3页Chinese Journal of Hepatology

摘　　要：目的 构建抗ＨＢｓＡｇ人源单链抗体与人干扰素α的融合分子－抗ＨＢｓＡｇ单链抗体靶向干扰素，并在大肠杆菌中表达出活性融合蛋白。方法 利用限制性内切酶分别从含有抗ＨＢｓＡｇ人源单链抗体与人干扰素α的质粒中切出目的基因，连接到ｐＥＴ２２ｂ质粒中，构建成单链抗体靶向干扰素表达载体。利用ＳＤＳ—ＰＡＧＥ电泳、竞争性抑制实验与抗病毒实验对表达产物进行分析鉴定。结果 构建出含有抗体靶向干扰素融合基因的表达质粒，诱导１２ｈ后，在ＳＤＳ—ＰＡＧＥ上可以观察到相对分子质量约４５×１０４的目的蛋白，竞争性抑制实验与抗病毒实验表明表达的融合蛋白保留了单链抗体的ＨＢｓＡｇ特异结合活性与干扰素的抗病毒活性。结论 抗ＨＢｓＡｇ单链靶抗体靶向干扰素融合分子的构建及在大肠杆菌中的表达是成功的。Objective To develop a bacteria expression system to produce the fusion protein of humanized antiHBsAg scFV and interferon- a . Methods The expression vector was constructed after cleaving the plasmids harboring the humanized anti-HBsAg scFv and interferon a respectively and ligating to linearized pET22b subsequence. The expression of fusion protein in E.coli was analyzed by SDS-PAGE. The binding activity and antiviral activity of the fusion protein was characterized by competing inhibition test and cytopathic effect reduction. Results The plasmid harboring the in frame arranged fusion gene was constructed and identified. After induction for 12 h, a new band close to 4.5×10~4 was observed using SDS-PAGE. Results of competing ELISA and cytopathic effect reduction showed the fusion protein retained its specific binding activity and antiviral activities. Conclusions The construction and expression of the fusion gene of humanized anti-HBsAg scFv and interferon in E.coli are successful.

关 键 词：融合蛋白 靶向干扰素α 单链抗体 构建 抗HBsAg单链抗体 原核 肿瘤 基因治疗 

分 类 号：R730.5[医药卫生—肿瘤]