新型冠状病毒（SARS-CoV-2）N蛋白C端重组蛋白的原核表达、纯化及应用

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

摘要/Abstract

摘要：

通过原核表达系统探究SARS-CoV-2 核衣壳（Nucleocapsid,N）蛋白C端重组蛋白表达、纯化的制备方式以及提供有效的重组蛋白用于COVID-19的早期快速诊断。合成N蛋白C端基因序列,构建重组载体,利用原核系统表达重组目的蛋白。利用Ni²⁺亲和层析和凝胶过滤层析的方法对表达产物进行纯化,SDS-PAGE电泳和高效液相色谱鉴定重组蛋白的纯度。通过Western blot验证目的蛋白生物活性,用纯化的重组蛋白作为包被抗原,核衣壳全长蛋白标记荧光微球,组装成荧光免疫层析试纸条检测PCR确证的阳性和阴性血清样本。成功构建重组载体pET21b-NC,工程菌株在37℃诱导条件下,80%目的蛋白以可溶形式表达。通过Ni²⁺亲和层析和凝胶过滤层析,目的蛋白纯度高达90%以上,利用重组蛋白组装荧光免疫层析试纸,该试纸条可区分阴阳血清。SARS-CoV-2 N蛋白C端可在大肠杆菌中以可溶性形式表达,高纯度的目的蛋白在荧光免疫层析检测试纸中表现出较好的反应原性和特异性,可初步用于COVID-19的早期初筛。

关键词: SARS-CoV-2, 核衣壳蛋白, 原核表达, 纯化

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

张西西, 张怡青, 李玉林, 韩笑, 王国强, 王晓军, 王旭东, 王云龙. 新型冠状病毒（SARS-CoV-2）N蛋白C端重组蛋白的原核表达、纯化及应用[J]. 生物技术通报, 2021, 37(5): 92-97.

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.

图/表 8

表1 引物序列

Table 1 Primer sequence

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

图1 SARS-CoV-2、SARS-CoV、MERS-CoV 的 N 蛋白 C 末端氨基酸序列对比

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

图2 N 蛋白 C 端序列基因表达载体的 PCR 及双酶切鉴定 A：M1 ：DNA 分子标准质量,1：PCR 产物 ;B：M2 ：DNA 分子标准质量,2：双酶切产物

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

图3 重组蛋白可溶性表达 M：蛋白分子标准质量;1：诱导前样品;2：37℃诱导后上清;3 ：37℃诱导后沉淀;4：25℃诱导后上清;5：25℃诱导后沉淀;6：16℃诱导后上清; 7：16℃诱导后沉淀

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℃

图4 SARS-CoV-2 N 蛋白 C 端重组蛋白原核表达和纯化的SDS-PAGE 分析 M：蛋白分子标准质量;1：诱导前样品;2：37℃诱导后上清;3：Ni2+亲和层析纯化;4：Ni2+亲和层析纯化和凝胶层析纯化

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

图5 SARS-CoV-2 N 蛋白 C 端重组蛋白凝胶过滤层析纯化的高效液相分析

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

图 6 SARS-CoV-2 N 蛋白 C 端重组蛋白免疫印迹分析 M：蛋白分子标准质量;1：纯化后的重组蛋白;2：诱导前样品

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

图7 荧光免疫层析试剂血清检测 A：PCR 阴性病人血清检测;B：PCR 阳性病人血清检测

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

参考文献 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