Expression and purification of 2019-nCoV nucleocapsid protein and application in the diagnosis
10.3760/cma.j.cn112866-20200324-00087
- VernacularTitle:2019新型冠状病毒核衣壳蛋白的表达及在诊断方面的应用
- Author:
Li ZHANG
1
;
Binyang ZHENG
;
Lianjun MIAO
;
Qiufan YU
;
Xingsu GAO
;
Lu JIN
;
Sen LI
;
Jingui YONG
;
Hongxing PAN
Author Information
1. 江苏省疾病预防控制中心疫苗临床评价所，南京 210009
- Keywords:
2019-nCoV;
Nucleocapsid protein;
Prokaryotic expression;
Diagnostic reagent
- From:
Chinese Journal of Experimental and Clinical Virology
2020;34(4):374-377
- CountryChina
- Language:Chinese
-
Abstract:
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.
