2019新型冠状病毒核衣壳蛋白的表达及在诊断方面的应用  

Expression and purification of 2019-nCoV nucleocapsid protein and application in the diagnosis

作　　者：张黎[1] 郑滨洋 缪连军 余秋凡 高行素 金路 李森 雍金贵 潘红星[1] Zhang Li;Zheng Binyang;Miao Lianjun;Yu Qiufan;Gao Xingsu;Jin Lu;Li Sen;Yong Jingui;Pan Hongxing(Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;General Biosystems(Anhui)Corporation Limited,Chuzhou 239000,China;BioMedomics Corporation Limited,Nanjing 210000,China)

机构地区：[1]江苏省疾病预防控制中心疫苗临床评价所,南京210009 [2]通用生物系统(安徽)有限公司,滁州239000 [3]江苏美克医学技术有限公司,南京210000

出　　处：《中华实验和临床病毒学杂志》2020年第4期374-377,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：江苏省疾病预防控制中心科教强业项目(JKRC2016020)。

摘　　要：目的对2019新型冠状病毒(2019-novel Coronavirus,2019-nCoV)核衣壳蛋白(nucleocapsid protein,NP)进行原核表达、纯化与鉴定,并应用于2019-nCoV血清学诊断。方法合成2019-nCoV NP基因,克隆至pET28a载体中,构建表达质粒,并进行诱导纯化。纯化蛋白经SDS-PAGE、间接ELISA、Western blot(WB)、免疫层析法进行鉴定。优化间接ELISA反应条件,进行血清抗体检测。结果重组NP经镍柱纯化后SDS-PAGE电泳显示相对分子质量约50×10^3,与预期一致;间接ELISA、WB表明其能与2019-nCoV感染患者血清特异性结合;用免疫层析法发现对NP检测限为0.2 ng/ml;间接ELISA检测32份2019-nCoV感染者血清及对照血清,发现二者结果可见明显分群。结论原核表达的NP具有良好的免疫原性,可用于制备血清学诊断试剂。Objective To realize prokaryotic expression,purification and identification of 2019-novel Coronavirus(2019-nCoV)nucleocapsid protein(NP),and apply it to the serological diagnosis.Methods The synthetic 2019-nCoV NP gene was cloned into the prokaryotic expression vector pET28a to construct expression plasmid,and then purified by Ni-chelating affinity.SDS-polyacrylamide gel electrophoresis(SDS-PAGE),indirect enzyme-linked immunosorbent assay(ELISA),Western blot(WB),and immunochromatography were used to test the purified protein.Indirect ELISA reaction conditions were optimized for serum antibody detection.Results The relative molecular mass of recombinant NP was about 50×10^3 after SDS-PAGE electrophoresis,which was consistent with the expectation.Indirect ELISA and WB results showed that it could specifically bind to the serum of patients infected with 2019-nCoV.The detection limit of NP was 0.2 ng/ml by immunochromatography.The sera from 32 patients infected with 2019-nCoV and the control sera were detected by indirect ELISA,and the results showed that they were clearly clustered.Conclusions Prokaryotic expression of 2019-nCoV NP has good immunogenicity and can be used for the development of serological diagnostic reagents.

关 键 词：2019新型冠状病毒 核衣壳蛋白 原核表达 诊断试剂 

分 类 号：R373[医药卫生—病原生物学]