丙型肝炎病毒核心蛋白人源抗独特型单链抗体在大肠杆菌中的表达  

Expression of soluble human anti-idiotypic single chain Fv antibody to hepatitis C core protein in E. coli

作　　者：钟彦伟[1] 成军[1] 张忠东[1] 李强[1] 李莉[1] 陈菊梅[1] 

机构地区：[1]解放军第302医院传染病研究所基因治疗研究中心,北京100039

出　　处：《解放军医学杂志》2004年第1期10-12,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助课题 (编号 3990 0 1 30 )

摘　　要：目的　在大肠杆菌中表达丙型肝炎病毒核心蛋白人源抗独特型单链抗体。方法　采用噬菌体表面展示技术 ,将丙型肝炎病毒 (HCV)核心蛋白单克隆抗体固相包被于Nunc板。从人源噬菌体单链可变区抗体库中经过 3轮“吸附 洗脱 扩增”的筛选 ,获得结合活性较强的人源HCV核心蛋白抗独特型 (抗 Id)scFv阳性克隆。从阳性克隆中提取质粒 ,经SfiⅠ/NotⅠ酶切鉴定后 ,亚克隆到pCANTAB5E载体 ,转化大肠杆菌XL1 Blue ,应用异丙基硫代 β D 半乳糖苷 (IPTG)诱导表达可溶性的HCV核心蛋白抗独特型单链可变区抗体。酶联免疫吸附法 (ELISA)证实表达的HCV核心蛋白抗 IdscFv具有与HCV核心蛋白单克隆抗体反应的特异性。对转化的大肠杆菌XL1 Blue上清中表达的HCV核心蛋白抗 IdscFv进行硫酸铵沉淀 ,并进行SDS PAGE电泳。结果　筛选得到的HCV核心蛋白抗 IdscFv片段基因由 774bp组成。SDS PAGE电泳表明 ,大肠杆菌XL1 Blue中表达的可溶性HCV核心蛋白抗 IdscFv分子量约 2 8kD。结论　HCV核心蛋白抗 IdscFv与HCV核心蛋白抗 IdscFv单克隆抗体具有较强的结合活性和特异性。HCV核心蛋白抗 IdscFv的筛选和表达成功 ,为今后HCV核心蛋白抗 IdscFv的研究和应用奠定了基础。Objective To express soluble human anti-idiotypic single chain Fv to hepatitis C core protein in E.coli. Methods Using phage display technique, the semisynthetic phage library was panned by HCV core monoclonal antibody which was coated in a microtiter plate. After three rounds of biopanning, 53 clones were identified specific to HCV core antibody. The specificity of anti-idiotypic scFv was determined by ELISA. After digested with Sfi/Not, the selected HCV core anti-idiotypic scFv positive clone was subcloned into the vector pCANTAB5E for the expression of E-tagged soluble anti-idiotypic scFv. The E.coli XL1-Blue was transformed and induced by IPTG. The specificity of anti-Id scFv was evaluated with ELISA. Results HCV core anti-Id scFv DNA digestion and sequence data showed that the scFv gene was composed of 774bp. ELISA results demonstrated that the soluble human HCV core anti-idiotypic scFv to HCV core monoclonal antibody had a specific combination character. The molecular weight of expressed HCV core anti-idiotypic scFv was 28kD as shown by SDS-PAGE. Conclusion HCV core anti-Id scFv has been successfully expressed in E.coli.

关 键 词：C型肝炎样病毒属 病毒核心蛋白质类 抗独特型单链可变区抗体 基因表达 

分 类 号：R512.63[医药卫生—内科学]