DsbC介导HCV优势抗原表位原核可溶性表达与间接ELISA分析  

Soluble expression of HCV dominant antigen epitope in prokaryotic system mediated by DsbC protein and its indirect ELISA analysis

作　　者：肖彭莹[1] 黄国红 王丽君 孙卫国 张灵霞[2] 侯江厚 XIAO Pengying;HUANG Guohong;WANG Lijun;SUN Weiguo;ZHANG Lingxia;HOU Jianghou(Women′s Health Department,Kunming Matermal and Child Health Hospital,Kunming,Yunnan 650013,China;Army Tuberculosis Prevention and Control Key Laboratory,the Eighth Medical Center of General Hospital of PLA,Beijing 100091,China)

机构地区：[1]昆明市妇幼保健院妇女保健部,云南昆明650013 [2]解放军总医院第八医学中心结核病研究所,北京100091

出　　处：《国际检验医学杂志》2023年第1期63-68,共6页International Journal of Laboratory Medicine

基　　金：昆明创新团队项目(2019-1-R-23676);国家科技重大专研项目(2017ZX10201301-007)。

摘　　要：目的DsbC蛋白与丙型肝炎病毒(HCV)优势抗原表位原核融合表达并纯化,获得一种特异优良的HCV血清抗体诊断抗原。方法通过生物信息学软件,筛选我国HCV主要流行株优势抗原片段并进行串联融合形成组合肽命名为P367,将P367与DsbC蛋白进行融合后在原核系统内可溶性表达并纯化获得重组抗原DsbC-P367,建立HCV-IgG血清学检测方法,通过对HCV阴阳性血清的检测,评价DsbC-P367与P367在血清学诊断差异,获得一种新型HCV-IgG血清学诊断抗原制作方法。结果通过信息学软件筛选获得的优势抗原表位来源于不同基因型,全长367个氨基酸。通过原核表达系统发现,DsbC-P367以可溶性表达形式存在,亲和纯化后其相对分子质量为63×10^(3),纯度为92%,Western blot实验初步证实重组DsbC-P367与P367蛋白均具有抗原性;对100份明确HCV阴阳性血清进行检测,DsbC-P367与P367灵敏度分别为96%、90%,特异度均为100%,与“金标准”比较,McNemer检验显示均P=1.00,Kappa分别为0.95、0.87,提示DsbC-P367相对于P367具有更优的灵敏度。结论重组获得的DsbC-P367融合肽在HCV血清学抗体检测中具有良好的灵敏度和特异度,可开发为HCV-IgG体外诊断试剂盒用于HCV感染的实验室诊断。Objective To obtain a specific and excellent hepatitis C virus(HCV)serum antibody diagnostic reagent by expressing and purifying of HCV dominant antigen epitopes fused with DsbC protein.Methods Through bioinformatics software,the dominant antigen fragments of major HCV epidemic strains in China were screened and fused in tandem to form a combined peptide named P367.After the fusion of P367 and DsbC protein,the recombinant antigen DsbC-P367 was obtained by soluble expression and purification in prokaryotic system.HCV IgG serological detection method was established.The difference between DsbC-P367 and P367 in serological diagnosis was evaluated through the detection of HCV positive and negative serum,and a new method for preparing HCV IgG serological diagnostic antigen was obtained.Results The dominant epitopes obtained by informatics software were from different genotypes,with a total length of 367 amino acids.Through prokaryotic expression system,it was found that DsbC-P367 existed in soluble expression form.After affinity purification,its relative molecular weight was 63×10^(3),and its purity was 92%.Western blot test results preliminarily confirmed that both recombinant DsbC-P367 and P367 proteins had antigenicity.The sensitivity of DsbC-P367 and P367 was 96%and 90%respectively,and the specificity was 100%when 100 positive and negative HCV sera were detected.Compared with the"gold standard",McNemer test showed that all P=1.00,Kappa were 0.95 and 0.87 respectively,suggesting that DsbC-P367 had better sensitivity than P367.Conclusion The recombinant DsbC-P367 fusion peptide has good sensitivity and specificity in the detection of HCV serological antibodies,and could be developed as an in vitro diagnostic kit for HCV IgG for laboratory diagnosis of HCV infection.

关 键 词：丙型肝炎病毒 抗原表位筛选 DsbC蛋白 间接酶联免疫吸附试验 

分 类 号：R373.21[医药卫生—病原生物学]