【摘要】 目的 重组表达丙型肝炎病毒(HCV)非结构蛋白2(NS2),建立并评价基于化学发光法的血清抗NS2抗体的检测方法。方法 以HCV 1b基因型的NS2核苷酸序列质粒(PUC-NS2)为模板，构建包含NS2全序列的重组质粒(pGEX-KG-NS2);诱导NS2蛋白原核表达，将纯化后的NS2融合蛋白包被于微孔板，基于化学发光法制备抗NS2抗体检测试剂盒，并对其方法学指标进行评价。结果 成功诱导获得相对分子质量(M_r)约51 000的NS2融合蛋白；并制备血清抗NS2抗体检测试剂盒，试剂盒方法学评价：精密度试验(批内变异系数4.60%～9.17%,批间变异系数6.62%～10.09%)结果良好，空白限和检出限的相对发光强度比值(RLIR)分别为1.57、4.80(r=0.9870),分析测量范围(AMR)为1.63～44.50 RLIR;准确度试验：平均回收率为99.4%,类风湿因子等阳性血清标本对本试验无交叉反应，试剂盒15个月内稳定，45例HCV感染者血清抗NS2抗体阳性率为20%(9/45)。结论 获得了原核表达的HCV NS2并制备了检测血清抗NS2抗体的试剂盒。更多还原

【Abstract】 Objective To express the recombinant HCV NS2, establish and evaluate the detection method of serum anti-ns2 antibody based on chemiluminescence. Methods Using the NS2 sequence plasmid of HCV 1b genotype(PUC-NS2) as the template, a recombinant plasmid containing the whole NS2 sequence(pGEX-KG-NS2) was constructed. Prokaryotic expression of NS2 protein was induced. The purified NS2 fusion protein was coated on the microplate, and the anti-NS2 antibody detection kit was prepared based on chemiluminescence, and the methodological index was evaluated. ResultsNS2 fusion protein with relative molecular weight(M_r) of about 51 000 was successfully induced and expressed, and a serum anti-NS2 antibody detection kit was synthesized. Methodological evaluation of kit: Precision test showed favorable results(intra batch coefficient of variation [CV] was 4.60%～9.17%, inter batch CV was 6.62%～10.09%). The relative luminous intensity ratio(RLIR) of the blank limit and the detection limit were 1.57 and 4.80(r=0.9870), respectively, and the analytical measurement range(AMR) was 1.63～44.50 RLIR. Accuracy experiments: The average recovery was 99.4%. The positive serum samples such as rheumatoid factor had no cross reaction to this test, and the kit was stable within 15 months. The positive rate of anti-NS2 antibody in serum of 45 HCV infected patients was 20%(9/45). Conclusion The prokaryotic expression of HCV NS2 fusion protein is successfully obtained, and the anti-NS2 antibody detection kit in serum is developed.更多还原