HCoV-NL63 N蛋白不同片段的表达纯化及血清学检测应用分析  

Expression and Purification of Different Segments from HCoV-NL63 Nucleocapsid Protein and Their Application in Detection of Antibodies

作　　者：赵敏[1,2] 张庭瑛[1,2] 周为民[2] 赵国霞[1,2] 张陵林[2] 高基民[1] 谭文杰[1,2] 

机构地区：[1]温州医学院医学病毒学研究所,温州325000 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京102206

出　　处：《病毒学报》2011年第3期244-249,共6页Chinese Journal of Virology

基　　金：863课题(2007AA02Z464;2007AA02Z463);十一五重大专项(2008ZX10004-014)

摘　　要：将HCoV-NL63核衣壳蛋白N端（Np1~154aa）、C端（Cp141~306aa）基因片段克隆到原核表达载体pET30a（＋）上进行原核表达,制备相应的纯化蛋白Np、Cp蛋白,利用Np、Cp蛋白建立基于Western-Blot条带印迹的HCoV-NL63抗体检测法,并与基于全长N蛋白（Nf）的HCoV-NL63抗体检测法相平行筛查了50份成年体检血清。结果显示：50份成年体检血清中,采用Nf、Np、Cp分别检出25、27、36份HCoV-NL63抗体阳性血清,检出率分别为50%、54%、72%。不同N蛋白与血清反应抗体阳性谱存在差异,其中Np与Nf检出一致率为64%,Cp与Nf检出一致率为54%,而Np与Cp检出一致率为54%。本研究表明人冠状病毒NL63在我国人群中感染常见,N蛋白C端（Cp）检出率比全长N（Nf）及N端（Np）要高,Nf、Np、Cp在抗体检测上存在不一致性。这为HCoV-NL63血清学试剂研发及免疫学研究提供了依据与实验基础。Prokaryotic expression plasmids carrying N-terminal（1-163aa） and C-terminal（141-306aa） gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a（＋） vector.Consequently,we prepared two purified proteins,Np and Cp,respectively,and established a Western blotting-based line assay（WBLA） for detection of antibodies against HCoV-NL63 using three purified proteins： Np,Cp and Nf,a full-length HCoV-NL63 nucleocapsid protein as previously reported.We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA.The results showed that： 25（50%）,27（54%）,36（72%） of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf,Np and Cp,respectively.Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np,54% between Nf and Cp,54% between Np and Cp.Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.

关 键 词：HCOV-NL63 原核表达 核衣壳蛋白 Western-Blot条带印迹 血清学检测 

分 类 号：R446.61[医药卫生—诊断学]