丙型肝炎病毒丝氨酸蛋白酶抗原性分析及特异性抗体制备  

Analysis of antigenicity of hepatitis C virus serine protease domain and preparation of its specific antibodies

作　　者：杜桂鑫[1] 刘树玲[1] 侯利华[1] 陈万荣[1] 王海涛[1] 

机构地区：[1]军事医学科学院微生物流行病研究所应用分子生物学实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2002年第4期393-397,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 396 30 0 2 0 )

摘　　要：目的　分析丙型肝炎病毒 (HCV)丝氨酸蛋白酶的抗原性并制备特异性抗体。方法　构建表达质粒并且在大肠杆菌中可溶性表达单链型丙型肝炎病毒丝氨酸蛋白酶 ;以纯化后的重组蛋白包被酶联板对 86份献血员和 12份健康人血清中的特异性抗体进行检测 ;将纯化蛋白偶联到Sepha rose 4B上制备亲和纯化柱 ,对丙肝病毒感染者血清中的特异性抗体进行亲和纯化 ;以纯化蛋白免疫小鼠 ,制备特异性单克隆抗体 ,对纯化后的人多抗和鼠单抗进行鉴定。结果　以纯化的重组单链丝氨酸蛋白酶为抗原检测 98份血清 ,结果阳性率为 73.9%,商品试剂盒漏检的 1份血清中也检测出特异性抗体。用重组蛋白从 2 0 0mlHCV感染者血清中亲和纯化到 4.5 7mg多克隆抗体 ,效价达 10 7,特异性很好。制备的 4株IgG类鼠单抗 ,无交叉反应 ,能够被人血清抗体竞争性抑制。结论　表达的HCV丝氨酸蛋白酶具有良好的抗原性 ,在HCV感染人群中该区域抗体的阳性率较高 ,制备了特异性很好的多克隆抗体和单克隆抗体。Objective To analyze the antigenicity of the hepatitis C virus protease domain and to prepare specific antibodies. Methods Constructed and expressed a mono-chain protease of hepatitis C virus in soluble form in E.coli, then the purified protease was used as antigen to detect the specific antibodies in sera from 98 blood donors. The protease was coupled to Sepharose 4B to purify specific antibodies from hepatitis C patients′sera. Using the protease as antigen to immunize mice and the specific monoclonal antibodies were produced. Results 73.9% of 98 serum samples had specific antibodies against HCV serine protease and one sample that had been identified as negative by commercial kit was also positive. 4.57mg polyclonal antibodies with high specificity were purified from 200ml sera of hepatitis C patients and the titer was over 10 7. Four strains of hybridomas that secret specific monoclonal antibodies were established and the specificity was identified by indirect ELISA and Competitive ELISA. Conclusion The antigenicity of a mono-chain hepatitis C virus protease was evident and there were high rates of specific antibodies among HCV carriers.

关 键 词：抗体制备 丙型肝炎病毒 抗原性 单克隆抗体 丝氨酸蛋白酶 HCV 丙型肝炎 

分 类 号：R392[医药卫生—免疫学]