HCV跨膜蛋白(P7)重组表达及化学发光法检测HCV感染者血清中抗P7抗体的建立与评价  

Establishment and evaluation of recombinant expression of HCV transmembrane protein(P7)and detection of anti-P7 antibody in serum of HCV infected patients by chemiluminescence

作　　者：吴婕 戴玉柱 张英杰(指导) 成军(指导) WU Jie;DAI Yuzhu;ZHANG Yingjie;CHENG Jun(School of Laboratory Medicine,Bengbu Medical College,Bengbu 233000,China)

机构地区：[1]蚌埠医学院检验医学院,蚌埠233000 [2]联勤保障部队第903医院临床检验中心,杭州310013

出　　处：《中国免疫学杂志》2022年第13期1614-1620,共7页Chinese Journal of Immunology

基　　金：南京军区医学科技创新重大专项基金项目(15DX025);浙江省杭州市科技局社会发展科研项目(20160533B75);浙江省医药卫生科研项目(2016KYB250,2021433791);浙江省自然科学基金公益技术研究计划项目(LGF21H200003)资助。

摘　　要：目的:建立化学发光法检测丙型肝炎病毒(HCV)感染者血清抗跨膜蛋白P7(anti-P7)抗体的方法,并进行应用评价。方法:以含有HCV 1b基因型P7核酸序列(Accession No:AJ238800)的质粒PUC-P7为模板,PCR扩增P7基因,构建重组质粒pGEX-KG-P7,P7融合蛋白经原核诱导表达、提取纯化后,间接法包被微孔板、制备化学发光法检测抗P7抗体试剂盒,并对其方法学指标进行评价。结果:质粒pGEX-KG-P7构建正确,经原核诱导表达获得相对分子质量约34 kD的P7融合蛋白,制备的化学发光法检测抗P7抗体试剂盒方法学指标为:精密度试验(批内变异系数2.97%~4.09%,批间变异系数3.75%~5.52%),空白限和检出限分别为1.56、5.99 RLIR,相关系数r=0.9989,分析测量范围(AMR)为1.252~18.774 RLIR,平均回收率为97.8%,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P7抗体阳性率为20%(9/45)。结论:化学发光法检测HCV感染者血清中抗P7抗体试剂盒符合方法学要求,可用于HCV感染者筛查诊断、病情监测、预后评估、疾病机制和流行病学研究,P7蛋白在HCV感染者体内存在免疫应答现象。Objective:To establish a chemiluminescence method for detecting anti-P7 antibody in serum of patients with hepatitis C virus(HCV)infection and to evaluate its application.Methods:P7 gene was amplified by PCR by plasmid PUC-P7 containing P7 nucleic acid sequence of HCV 1b genotype(Accession No:AJ238800)as template.Recombinant plasmid pGEX-KG-P7 was constructed.P7 fusion protein was expressed in E.coli,extracted and purified.Microplate was coated by indirect method and kit for detecting anti-P7 antibody was prepared,and methodological indexes were evaluated.Results:Construction of plasmid pGEX-KG-P7 was confirmed,P7 fusion protein with relative molecular weight of 34 kD was obtained by prokaryotic expression.Methodological indexes of kit were as follows:precision test(coefficient of variation 2.97%~4.09%for within run,coefficient of variation 3.75%~5.52%for between run),blank limit and detection limit were 1.56 RLIR and 5.99 RLIR,correlation coefficient r=0.9989,analysis and measurement range(AMR)was 1.252~18.774 RLIR,average recovery was 97.8%,rheumatoid factor and other positive serum samples had no cross reaction to the test,the kit was stable within 15 months.Positive rate of anti-P7 antibody in 45 patients with HCV infection was 20%(9/45).Conclusion:The kit meets requirements of methodology,and can be used in screening diagnosis,disease monitoring,prognosis evaluation,disease mechanism and epidemiological study of HCV infection.P7 protein has immune response in HCV infected patients.

关 键 词：丙型肝炎病毒 跨膜蛋白P7 抗体 检测 评价 

分 类 号：R392-33[医药卫生—免疫学]