多表位丙型肝炎病毒抗原的表达和分析  

Preparation of a Multi-epitope Fusion Antigen for Hepatitis C Virus Antibodies Immunoassay

作　　者：苏秋东[1] 郭敏卓 伊瑶[1] 毕胜利[1] 贾志远[1] 邱丰[1] Su Qiudong;Guo Minzhuo;Yi Yao(Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]北京海关技术中心,100026

出　　处：《医学研究杂志》2020年第6期47-50,55,共5页Journal of Medical Research

基　　金：“十三五”国家科技重大专项基金资助项目(2018ZX10734404)。

摘　　要：目的目前常用的丙型肝炎病毒(hepatitis C virus,HCV)血清学检测试剂都基于3~6个重组蛋白或合成多肽,抗原制备繁琐且诊断效果参差不齐。制备一种诊断效能优良的HCV抗原势在必行。方法利用原核表达系统和层析纯化技术制备涵盖我国HCV主要流行株(1b和2a)的6个免疫显性区域的多表位HCV抗原(命名为H65),并以此建立HCV-IgG的血清学检测方法,通过对部分HCV阴阳性血清的检测,初步评价了H65抗原的免疫活性。结果融合Trx标签的H65抗原为可溶性形式表达,纯化后蛋白浓度可达2.991mg/ml,纯度为94.53%;Western blot法检测实验初步证实了蛋白H65的抗原性;应用某商品化试剂盒与H65抗原血清检测方法,对50份HCV阴阳性血清进行检测,McNemer检验显示,新制备诊断抗原与“金标准”比较,P>0.05,Kappa>0.75,且与某商品化试剂盒比较,P>0.05,Kappa>0.75,提示一致性优异。结论H65具有良好的免疫活性,为进一步应用多表位HCV抗原为基础的HCV-IgG体外诊断试剂提供了依据。Objective Current Hepatitis C virus(HCV)immunoassays used three to six separate recombinant proteins or synthetic peptides with cumbersome preparation of diagnostic antigens and uneven diagnostic efficiency.It is imperative to prepare a HCV antigen with excellent diagnostic efficiency.Methods Prokaryotic expression and chromatographic purification were applied to prepare multi-epitope HCV diagnostic antigens(named H65)covering six immunodominant regions of HCV major epidemic strains(1b and 2a)in China.The immunological activity of H65 antigen was preliminarily evaluated by detection of some hepatitis C virus negative and positive sera.Results With Trx as a tag,the soluble H65 was prepared with the concentration 2.991mg/ml and purity 94.53%.Western blot analysis demonstrated the antigenicity of H65.Based on the data from 50 HCV positive and negative sera,McNemer test suggested that there was no statistical difference between the‘Gold standard’and H65-based immunoassay with a Kappa>0.75,while there was also no statistical difference between a commercial kit and H65-based immunoassay with a Kappa>0.75,indicating excellent consistency.Conclusion H65 had good immunological activity,which provided a basis for further application of multi-epitope HCV antigen-based HCV-IgG immunoassay.

关 键 词：丙型肝炎病毒 多表位诊断抗原 原核表达与层析纯化 免疫检测 

分 类 号：R512.6[医药卫生—内科学]