真核表达纯化的SARS-CoV-2 N蛋白在不同人群血清检测中的应用性能分析  

Application and Performance of SARS-CoV-2 N Protein Expressed and Purified by Eukaryotic Expression System in Serum Detection Among Different Populations

作　　者：杨燕 王慧娟[2] 黄梦婧 黄保英[2] 赵莉[2] 邓瑶[2] 沈晓玲 谭文杰[1,2] YANG Yan;WANG Huijuan;HUANG Mengjing;HUANG Baoying;ZHAO Li;DENG Yao;SHEN Xiaoling;TAN Wenjie(Zhejiang Provincial Key Laboratory of Medical Genetics,School of Laboratory Medicine and Life Sciences,Wenzhou Medical University Wenzhou 325035,China;Key Laboratory of Biosafety,National Health Commission of the People's Republic of China,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Department of Microbiology,Basic Medical College,Inner Mongolia Medical University,Hohhot O10010,China)

机构地区：[1]温州医科大学检验医学院生命科学学院,325035 [2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京102206 [3]内蒙古医科大学基础医学院微生物教研室,呼和浩特010010

出　　处：《病毒学报》2023年第2期313-321,共9页Chinese Journal of Virology

基　　金：北京市科学技术委员会(项目号:Z211100002521017),题目:变异毒株生产用疫苗株筛选及有效成分检测方法研究。

摘　　要：通过真核系统表达纯化新型冠状病毒(SARS-CoV-2)的全长核衣壳蛋白(Nucleocapsid protein,N蛋白),分析其抗原性与应用性能。将SARS-CoV-2 N蛋白表达质粒转染HEK-293T细胞后,通过Western Blot和免疫荧光验证蛋白表达以及表达定位。对N蛋白进行纯化后,通过Western Blot和ELISA并采用多种血清样本(WHO参比品血清、正常人血清、新冠灭活疫苗接种后血清、新冠感染者恢复期血清)对该N蛋白在血清抗体检测中的应用性能进行分析。比较真核系统与原核系统表达的N蛋白抗原性上的差异,并分析基于真核系统表达N蛋白构建的IgG抗体ELISA检测体系与获批的商品化新冠RBD (Receptor binding domain)IgG抗体ELISA检测试剂盒及用于检测中和抗体的活病毒微量中和试验结果间的相关性。结果显示,真核系统表达的全长N蛋白主要定位于胞质,作为抗原检测新冠IgG抗体,具有比原核系统表达的N蛋白更高的检测灵敏度。针对新冠感染者恢复期血清,基于哺乳动物细胞表达的N蛋白构建的ELISA体系检测的IgG抗体滴度与试剂盒检测的RBD-IgG抗体滴度以及中和抗体滴度之间具有良好的相关性。本研究表明真核系统表达纯化的SARS-CoV-2 N蛋白具有良好的抗原性与检测性能,为SARS-CoV-2血清学检测方法的建立与优化以及进一步明确N蛋白诱发的免疫反应特点提供了参考。The full length nucleocapsid protein(N protein) of SARS-CoV-2 was expressed and purified in eukaryotic system, and its antigenicity and application properties were analyzed. The expression and localization of SARS-CoV-2 N protein in HEK-293T cells were verified by Western Blot and immunofluorescence. After purification of N protein, application of N protein in serum antibody detection was analyzed by Western Blot、ELISA using various serum samples(WHO reference serum, normal human serum, serum after vaccination with inactivated vaccine, serum of convalescent patients with SARS-CoV-2 infection).The antigenicity of N protein expressed in eukaryotic system was compared with that expressed in prokaryotic system, the correlation between the IgG antibody ELISA detection system constructed with N protein expressed in eukaryotic system and the results of the commercial ELISA kit for detecting IgG antibodies against RBD(Receptor binding domain) and the live virus micro-neutralization test for detecting neutralizing antibodies was analyzed. Results showed that the full-length N protein expressed in eukaryotic system was mainly localized in the cytoplasm, and was more sensitive than the N protein expressed in prokaryotic system for detection of IgG antibody to SARSCoV-2. There was a good correlation between the IgG antibody titers detected by this method and those detected by SARS-CoV-2 RBD-IgG commercial kit and neutralization antibody titers. This study indicates that the purified SARS-CoV-2 N protein expressed in eukaryotic system has good antigenicity and detection performance. This study provides a reference for the establishment and optimization of serological detection methods for SARS-CoV-2 and further understanding of the characteristics of N protein induced immune response.

关 键 词：新型冠状病毒 核衣壳蛋白 真核表达 抗原性 ELISA 

分 类 号：R373.1[医药卫生—病原生物学]