新型冠状病毒N蛋白的原核表达与纯化

樊志浩; 李玉林; 张恒; 王旭东; 王云龙

doi:10.12187/2022.04.005

August 2022

Article Contents

FAN Zhihao, LI Yulin, ZHANG Heng, et al. Prokaryotic expression and purification of SARS-CoV-2 N protein[J]. Journal of Light Industry, 2022, 37(4): 34-40. doi: 10.12187/2022.04.005

Citation: FAN Zhihao, LI Yulin, ZHANG Heng, et al. Prokaryotic expression and purification of SARS-CoV-2 N protein[J]. Journal of Light Industry, 2022, 37(4): 34-40. doi: 10.12187/2022.04.005 shu

Prokaryotic expression and purification of SARS-CoV-2 N protein

FAN Zhihao ^1,2 ,
LI Yulin ² ,
ZHANG Heng ³ ,
WANG Xudong ³ ,
WANG Yunlong ^1,2,4

1. School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China;
2. He'nan Bioengineering Technology Research Center, Zhengzhou 450121, China;
3. He'nan General Hospital, Zhengzhou 450002, China;
4. Department of Bioengineering, Zhengzhou Technical College, Zhengzhou 450121, China

Received Date: 2021-07-19
Accepted Date: 2021-11-02

Abstract

Bioinformatics methods were used to predict the hydrophilicity, hydrophobicity, antigen epitopes and analyse multiple sequence alignment of the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The recombinant plasmid pET28a/N was constructed. In the prokaryotic expression system of Escherichia coli, the solubility and expression level of the protein were improved by adjusting the change of induction temperature and time, and the expressed recombinant N protein was purified and identified. The results showed that SARS-CoV-2 N encoded 419 amino acids, with an isoelectric point (PI) of 10.10, no transmembrane region, no signal peptide sequence, and strong local hydrophilicity. The full-length protein had a high antigenic index and was highly conserved, and its homology with SARS-CoV N protein was 90.5%. After fermentation with Escherichia coli prokaryotic expression system, the engineering strain BL21 (DE3)/pET28a/N was induced at 16 ℃ for 20 h with the final IPTG concentration of 0.2 mmol/L, and the protein was soluble and most pressed at this time, accounting for 70% of the total protein expression. The target protein purified by Ni-NTA affinity chromatography and gel filtration chromatography had a purity of 90% and a molecular weight of 55 kDa, which was specific.
- SARS-CoV-2,
- N protein,
- antigen epitope,
- soluble expression
References
1. [1]
  CHEN Y,LIU Q Y,GUO D Y.Emerging coronaviruses:Genome structure,replication,and pathogenesis[J].Journal of Medical Virology,2020,92(4):418-423.
2. [2]
  GAUNT E R,HARDIE A,CLAAS E C J,et al.Epidemiology and clinical presentations of the four human coronaviruses 229E,HKU1,NL63,and OC43 detected over 3 years using a novel multiplex real-time PCR method[J].Journal of Clinical Microbiology,2010,48(8):2940-2947.
3. [3]
  KROKHIN O,LI Y,ANDONOV A,et al.Mass spectrometric characterization of proteins from the SARS virus:A preliminary report[J].Molecular&Cellular Proteomics,2003,2(5):346-356.
4. [4]
  HE R T,DOBIE F,BALLATINE M,et al.Analysis of multimerization of the SARS coronavirus nucleocapsid protein[J].Biochemical and Biophysical Research Communications,2004,316(2):476-483.
5. [5]
  TILOCCA B,SOGGIU A,SANGUINETTI M,et al.Comparative computational analysis of SARS-CoV-2 nucleocapsid protein epitopes in taxonomically related coronaviruses[J].Microbes and Infection,2020,22(4/5):188-194.
6. [6]
  CHEN N S,ZHOU M,DONG X,et al.Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan,China:A descriptive study[J].Lancet,2020,395(10223):507-513.
7. [7]
  REN S Y,WANG W B,GAO R D,et al.Omicron variant (B.1.1.529) of SARS-CoV-2:Mutation,infectivity,transmission,and vaccine resistance[J].World Journal of Clinical Cases,2022,10(1):1-11.
8. [8]
  KANG S S,YANG M,HONG Z S,et al.Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites[J].Acta Pharmaceutica Sinica B,2020,10(7):1228-1238.
9. [9]
  CONG Y Y,ULASLI M,SCHEPERS H,et al.Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle[J].Journal of Virology,2020,94(4):e01925-19.
10. [10]
  CALLIGARI P,BOBONE S,RICCI G,et al.Molecular investigation of SARS-CoV-2 proteins and their interactions with antiviral drugs[J].Viruses,2020,12(4):445.
11. [11]
  AHMED S F,QUADEER A A,MCKAY M R.Preliminary identification of potential vaccine targets for the COVID-19 coronavirus (SARS-CoV-2) based on SARS-CoV immunological studies[J].Viruses,2020,12(3):254.
12. [12]
  GUO L,REN L L,YANG S Y,et al.Profiling early humoral response to diagnose novel coronavirus disease (COVID-19)[J].Clinical Infectious Diseases,2020,71(15):778-785.
13. [13]
  ZHANG T,WU Q F,ZHANG Z F.Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak[J].Current Biology,2020,30(7):1346-1351.
14. [14]
  WU Y X,MA L,CAI S H,et al.RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-κB hyper-activation and inflammation[J].Signal Transduction and Targeted Therapy,2021,6(1):167.
15. [15]
  潘传燕,冯鹏霏,张永德,等.尼罗罗非鱼PPARδ的原核表达及其多抗制备和纯化[J].福建农业学报, 2019,34(9):1053-1058.
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沈阳化工大学材料科学与工程学院沈阳 110142

Prokaryotic expression and purification of SARS-CoV-2 N protein

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通讯作者: 陈斌, bchen63@163.com