抗丙型肝炎病毒非结构蛋白NS5A单链抗体的制备及免疫组织化学研究  

Study of Human Single Chain Fv Antibody Against Hepatitis C Virus NS5A Antigen and Its Application in Immunohistochemistry

作　　者：钟彦伟[1] 成军[1] 焦成松[2] 张忠东[1] 李强[1] 李莉[1] 陈菊梅[1] 

机构地区：[1]中国人民解放军302医院传染病研究所基因治疗研究中心,北京100039 [2]广州军区武汉总医院感染科

出　　处：《中西医结合肝病杂志》2003年第2期89-91,共3页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：国家自然科学基金(C39900130)

摘　　要：目的:研制抗丙型肝炎病毒(HCV)非结构蛋白NS5A的人源噬菌体单链可变区抗体,并探讨其在临床病理学诊断中的应用价值。方法:以大肠杆菌表达的重组HCV非结构蛋白NS5A为固相抗原,利用抗原-抗体亲和性结合的原理,从半合成的人源可变区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)和DNA序列分析,获得HCV NS5A的人源单链抗体;用该抗体对7721转染全长HCV cDNA后筛选的阳性克隆细胞中的HCV NS5A抗原进行免疫组化鉴定。结果:ELISA结果表明,制备的HCV NS5A人源单链抗体能与HCV NS5A抗原特异性结合;免疫组化结果表明,该抗体能够特异性识别HCV NS5A抗原,与正常肝细胞无交叉反应。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,为HCV NS5A病原的检测提供了新的有效的检测手段。Objective: To detect hepatitis C virus NS5A antigen in 7721 cell transfected with HCV cDNA by immunohis-tochemistry method with human single chain Fv antibody. Methods: The recombinant phages were panned by NS5A antigen which was coated in a microtiter plate, after five rouds of biopanning, 35 clones were identified specific to NS5A antigen. The affinity and specificity of scFv was evaluated by ELISA and immunohistochemistry. Results: scFv-NS5A DNA digestion and sequence data showed that the scFv gene is composed of 786 bp. ELISA and irnmunohistochemistry demonstrated that the human single chain Fv antibody against HCV NS5A antigen has a specific combination capacity with hepatitis C virus NS5A antigen. Conclusion: The application of HCV NS5A specific ScFv in irnmunohistochemistry was successfully achieved.

关 键 词：抗丙型肝炎病毒 非结构蛋白 NS5A 单链抗体 制备 免疫组织化学 医学细胞生物学 人体免疫学 

分 类 号：R392[医药卫生—免疫学]