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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 hepatitis C virus (HCV) serine protease and to prepare specific antibodies. Methods The expression plasmid was constructed and the single chain hepatitis C virus serine protease was expressed in Escherichia coli. The purified recombinant protein coated ELISA plate was used to detect the specific antibodies in serum of 86 donors and 12 healthy volunteers ; Purified protein was coupled to Sepharose 4B to prepare affinity purification column for affinity purification of specific antibody in serum of hepatitis C virus infected patients; Specific monoclonal antibody was prepared by immunizing mice with purified protein, Human polyclonal antibody and mouse monoclonal antibody were identified. Results The purified recombinant single-chain serine protease was used as the antigen to detect 98 sera. The positive rate was 73.9%. One of the serums missed by the commercial kit also detected specific antibodies. The recombinant protein was affinity purified from serum of 200 ml HCV infected sera to 4.5 7 mg polyclonal antibody with a titer of 10 7 and good specificity. The prepared 4 monoclonal IgG monoclonal antibodies did not cross-react and could be competitively inhibited by human serum antibodies. Conclusion The expressed HCV serine protease has good antigenicity. The positive rate of antibody in this area is high in HCV infected population, and the polyclonal antibodies and monoclonal antibodies with good specificity were prepared.