重组表达丙型肝炎病毒非结构蛋白2(NS2)及其血清抗NS2抗体检测方法的建立与评价

Detection of serum anti-NS2 antibody and recombinant hepatitis C virus nonstructural protein 2(NS2):its development and evaluation

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51000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。

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 51000 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.