抗丙型肝炎病毒非结构蛋白NS3单链抗体的制备及免疫组织化学研究

The preparation of human single chain Fv antibody specifically against hepatitis C virus NS3 antigen and its application in immunohistochemistry

目的 研制抗丙型肝炎病毒 (HCV)非结构蛋白NS3的人源噬菌体单链抗体 ,并探讨其在临床诊断中的应用价值。方法 以重组的丙型肝炎病毒非结构蛋白NS3为固相抗原 ,利用亲和筛选的原理 ,从噬菌体抗体库中经过 5轮“吸附 洗脱 扩增”的筛选过程及酶联免疫吸附试验 (ELISA)、斑点免疫杂交试验和DNA序列分析 ,获得HCVNS3的人源单链抗体 ;用该抗体与不同来源的HCVNS3抗原进行反应 ;对 10例石蜡包埋的丙型肝炎患者肝组织进行免疫组化鉴定。结果 ELISA结果表明 ,所制备的HCVNS3人源单链抗体能与不同来源的HCVNS3抗原特异性结合 (吸光度A值为1 38) ;免疫组化结果表明 ,该抗体能够特异性识别丙型肝炎患者肝组织HCVNS3抗原 ,与正常肝组织及乙型肝炎病毒 (HBV)的表面抗原均无交叉反应。结论 此法制备的单链抗体亲和性好 ,特异性强 ,且制备方法简便 ,周期短 。

Objective Immunohistochemistry identification of human single chain Fv antibody (ScFv) against hepatitis C viral NS3 antigen.Methods The recombinant phages were panned by NS3 antigen which was coated on a microtiter plate,after five rounds of biopanning,66 phage clones were identified specific to NS3 antigen.The affinity and specificity of ScFv were evaluated by ELISA,dot blot hybridization and immunohistochemistry.Results The data of ScFv\|NS3 DNA digestion and DNA sequencing showed that the ScFv gene is composed of 747 bp.ELISA,dot blot hybridization and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C NS3 antigen has a specific combination character with hepatitis C virus NS3 antigen of different sources and paraffin\|embedded patients tissues,it didn't react with liver tissue and HBsAg.Conclusion\ The application of HCV NS3 specific ScFv in immunohistochemistry was successfully achieved.