Prokaryotic Expression,Purification and Application of N Protein C-terminal Recombinant Protein in Novel Coronavirus（SARS-CoV-2）

doi:10.13560/j.cnki.biotech.bull.1985.2020-0902

Abstract

Abstract:

The expression and purification of the C-terminal recombinant protein of SARS-CoV-2 Nucleocapsid（N）protein were investigated through the prokaryotic expression system,aiming to provide the effective recombinant protein for the early and rapid diagnosis of COVID-19. The C-terminal gene sequence of the N protein was synthesized,the recombinant vector was constructed,and the recombinant target protein was expressed by the prokaryotic system. The expressed products were purified by Ni²⁺ affinity chromatography and gel filtration chromatography. The purity of recombinant proteins was determined by SDS-PAGE electrophoresis and high performance liquid chromatography. The biological activity of the target protein was verified by Western blot. The purified recombinant protein was used as the envelope antigen. The full-length nucleocapsid protein was labeled with fluorescent microspheres and assembled into fluorescent immunoassay strips to detect the positive and negative serum samples confirmed by PCR. The recombinant vector pET21b-NC was successfully constructed,and 80% of the target protein was expressed in soluble form under 37℃ induction. The purity of the target protein was >90% through Ni²⁺ affinity chromatography and gel filtration chromatography,and the fluorescence immunoassay strip was assembled with the recombinant protein,which distinguished the negative and positive serum. The C-terminal of SARS-CoV-2 N protein can be expressed in Escherichia coli in a soluble form,and the high-purity target protein shows fine reactivity and specificity in fluorescent immunoassay test paper,which can be preliminarily used for early screening of COVID-19.

Key words: SARS-CoV-2, nucleocapsid protein, prokaryotic expression, purification

ZHANG Xi-xi, ZHANG Yi-qing, LI Yu-lin, HAN Xiao, WANG Guo-qiang, WANG Xiao-jun, WANG Xu-dong, WANG Yun-long. Prokaryotic Expression,Purification and Application of N Protein C-terminal Recombinant Protein in Novel Coronavirus（SARS-CoV-2）[J]. Biotechnology Bulletin, 2021, 37(5): 92-97.

Figures/Tables 8

Table 1 Primer sequence

引物名称Primer name	引物序列Primer sequence（5'-3'）
NC-F	GGAATTCCATATGCATCATCACCATCA
NC-R	CCGCTCGAGTTACGGAAAGGTTTTAT

Fig.1 Comparison of N protein C-terminal amino acid sequences of SARS-CoV-2, SARS-CoV and MERS-CoV

Fig.2 N protein C-terminal sequence gene expression vector PCR and double enzyme digestion A: M1: DNA molecular weight standard, 1: PCR amplification product. B: M2: DNA molecular weight standard, 2: Double digestion product

Fig.3 Soluble expression of recombinant protein M: The molecular weight standard of protein. 1：Pre-induced sample. 2: Supernatant induced at 37℃. 3: Precipitation induced at 37℃. 4: Supernatant induced at 25℃. 5: Precipitation induced at 25℃. 6: Supernatant induced at 16℃. 7: Precipitation induced at 16℃

Fig.4 SDS-PAGE analysis of prokaryotic expression and purification of the C-terminal recombinant protein of SARS-CoV-2 N protein M: The molecular weight standard of protein. 1：Pre-induced sample.2: Supernatant induced at 37℃. 3：Ni2+ affinity chromatography purification.4: Ni2+ affinity chromatography purification and gel chromatography purification

Fig.5 High performance liquid phase analysis of SARS-CoV-2 N protein C-terminal recombinant protein gel filter chromatography

Fig.6 Western blotting analysis of SARS-CoV-2 N protein C-terminal recombinant protein M: The molecular weight standard of protein. 1: The purified recombinant protein.2: Pre-induced sample

Fig.7 Serum detection of fluorescent immunomy A: Serum detection for PCR-negative patients. B: Serum detection for PCR-positive patients

References 15

[1]	Liu WB, Liu L, Kou GM, et al. Evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against SARS-CoV-2[J]. J Clin Microbiol, 2020,58(6):e00461-20.
[2]	Sarma P, Shekhar N, Prajapat M, et al. In-silico homology assisted identification of inhibitor of RNA binding against 2019-nCoV N-protein(N terminal domain)[J]. J Biomol Struct Dyn, 2021,39(8):2724-2732. doi: 10.1080/07391102.2020.1753580 URL
[3]	Vasan S, Cui H, Gao Z, et al. Structural genomics of SARS-CoV-2 indicates evolutionary conserved functional regions of viral proteins[J]. Viruses, 2020,12(4):360. doi: 10.3390/v12040360 URL
[4]	Ahmed SF, Quadeer AA, Mckay MR. 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. doi: 10.3390/v12030254 URL
[5]	Kandeel M, et al. From SARS and MERS CoVs to SARS-CoV-2:moving toward more biased codon usage in viral structural and nonstructural genes[J]. J Med Virol, 2020,92(6):660-666. doi: 10.1002/jmv.v92.6 URL
[6]	Yamaoka Y, Jeremiah SS, Miyakawa K, et al. Whole nucleocapsid protein of SARS-CoV-2 may cause false positive results in serological assays[J]. Clin Infect Dis, 2020, 23:ciaa637.
[7]	Mu J, Xu J, Zhang L, et al. SARS-CoV-2-encoded nucleocapsid protein acts as a viral suppressor of RNA interference in cells[J]. Sci China Life Sci, 2020,63(9):1413-1416. doi: 10.1007/s11427-020-1692-1 URL
[8]	Wen F, Yu H, Guo J, et al. Identification of the hyper-variable genomic hotspot for the novel coronavirus SARS-CoV-2[J]. J Infect, 2020,80(6):671-693.
[9]	Gopal GJ, et al. Strategies for the production of recombinant protein in Escherichia coli[J]. Protein J, 2013,32(6):419-425. doi: 10.1007/s10930-013-9502-5 URL
[10]	Yin X, Wang W, Tian R, et al. Prokaryotic expression, purification and antigeNi²+city identification of recombinant human survivin protein[J]. Chinese Journal of Cellular & Molecular Immunolog, 2013,29(8):877-881.
[11]	王云龙, 米亚双, 等. CA19-9时间分辨荧光免疫层析检测方法的建立[J]. 生物工程学报, 2018,34(6):1012-1018.
	Wang YL, Mi YS. et al. Establishment of a time-resolved fluorescence immunochromatography assay for CA19-9[J] Chinese Journal of Biotechnology. 2018,34(6):1012-1018.
[12]	石英杰, 等. HPV16/18 E6蛋白荧光免疫层析联合检测方法的建立[J]. 免疫学杂志, 2018,34(6):534-538, 546.
	Shi YJ, Wang YL, Ming L, et al. Establishment of a combined method for detection of HPV16/18 E6 protein by fluorescence immunochromatography[J]. Immunological Journal, 2018,34(6):534-538, 546.
[13]	Burbelo PD, Riedo FX, et al. Detection of nucleo capsid antibody to SARS-CoV-2 is more sensitive than antibody to spike protein in COVID-19 patients[J]. J Infect Dis, 2020 : 20071423.
[14]	Zeng W, Liu G, Ma H, et al. Biochemical characterization of SARS-CoV-2 nucleocapsid protein[J]. Biochem Biophys Res Commun, 2020,527(3):618-623. doi: 10.1016/j.bbrc.2020.04.136 URL
[15]	Tilocca B, Soggiu A, Sanguinetti M, et al. Comparative computational analysis of SARS-CoV-2 nucleocapsid protein epitopes in taxonomically related coronaviruses[J]. Microbes Infect, 2020,22(4-5):188-194. doi: 10.1016/j.micinf.2020.04.002 URL