人冠状病毒NL63核壳蛋白和棘突蛋白的表达及其在血清学检测中的初步应用  

Expression and Preliminary Application in the Serological Detection of Nucleocapsid Protein and Spike Protein of Human Coronavirus NL63

作　　者：周为民[1] 张陵林[1] 陆柔剑[1] 王慧娟[1] 谭文杰[1] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《生物技术通讯》2010年第2期154-157,共4页Letters in Biotechnology

基　　金：国家"十一五"重大专项(2008ZX10004-014);国家高技术研究发展计划(2007AA02Z463;2007AA02Z464)

摘　　要：目的:重组表达人冠状病毒NL63(HCoV-NL63)的核壳蛋白(N蛋白)及棘突蛋白(S蛋白),用于检测血清中的相应抗体。方法:用原核表达系统表达HCoV-NL63的N蛋白,建立检测N抗体的Werstern印迹法;用真核表达系统表达HCoV-NL63的S蛋白,建立检测S抗体的间接免疫荧光(IFA)法。结果:经Werstern印迹检测,重组S蛋白和N蛋白表达正确;初步建立了N蛋白纯化方法。利用建立的检测方法,检测了100份正常成人血清,总阳性率为81%。其中S抗体阳性率为66%,N抗体阳性率为38%,S抗体和N抗体均为阳性的占总数的22%,双抗体均为阴性的占总数的19%;S抗体的检出率明显高于N抗体。结论:重组HCoV-NL63N蛋白及S蛋白表达成功;S抗体和N抗体共同检测可获得较好的检测结果,减少漏检。Objective：To express the human coronavirus NL63 （HCoV-NL63） nucleocapsid protein （N protein） and the spike protein （S protein） for the detection of serum antibodies.Methods：The recombinant N protein and S protein of HCoV-NL63 were expressed in the prokaryotic and eukaryotic expression system respectively.The N antibody and S antibody in serum were detected by Werstern blot method and indirect immunofluorescence assay （IFA） based on recombinant N protein and S protein respectively.Results：The expression of recombinant N protein and S protein were confirmed by Werstern blot,and the purified N protein was obtained.The N antibody and S antibody in sera from 100 normal adults were detected by Werstern blot and IFA,and the total positive rate was 81%.Where S antibody positive rate was 66%,N antibody positive rate was 38%,both S antibody and N antibody were positive in 22% of the total,both were negative in 19% of the total.S antibody detection rate was significantly higher than N antibody.Conclusion：The recombinant N protein and S protein of HCoV-NL63 were obtained.The detection sensitivity is higer when both of antibody assays are applied,reducing false negatives.

关 键 词：人冠状病毒NL63 核壳蛋白 棘突蛋白 表达 血清学检测 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]