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CN 41-1437/TS  ISSN 2096-1553

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

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樊志浩, 李玉林, 张恒, 等. 新型冠状病毒N蛋白的原核表达与纯化[J]. 轻工学报, 2022, 37(4): 34-40. doi: 10.12187/2022.04.005
引用本文:樊志浩, 李玉林, 张恒, 等. 新型冠状病毒N蛋白的原核表达与纯化[J]. 轻工学报, 2022, 37(4): 34-40.doi:10.12187/2022.04.005
shu
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

新型冠状病毒N蛋白的原核表达与纯化

  • 1. 郑州大学 生命科学学院, 河南 郑州 450001;

  • 2. 河南省生物工程技术研究中心, 河南 郑州 450121;

  • 3. 河南省职工医院, 河南 郑州 450002;

  • 4. 郑州职业技术学院 生物工程系, 河南 郑州 450121

    • 作者简介:樊志浩(1997—),男,河南省郑州市人,郑州大学硕士研究生,主要研究方向为生物医药。E-mail:biofzh@126.com;

  • 基金项目:河南省新型冠状病毒防控应急科研攻关项目(201100310300)

  • 中图分类号:Q78

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

  • 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

    CLC number:Q78

  • 摘要:采用生物信息学方法对新型冠状病毒(SARS-CoV-2)核衣壳蛋白(N蛋白)进行亲疏水性、抗原表位预测及多序列对比分析,构建重组质粒pET28a/N,在大肠杆菌原核表达体系下通过调整诱导温度和时间,提升蛋白溶解度和表达量,并对表达出的重组N蛋白进行纯化和鉴定。结果表明:SARS-CoV-2 N蛋白编码419个氨基酸,等电点( PI)为10.10,无跨膜区,无信号肽序列,局部亲水性较强,全长蛋白抗原指数较高,高度保守,与SARS冠状病毒(SARS-CoV) N蛋白同源性为90.5%;采用大肠杆菌原核表达体系发酵,工程菌BL21(DE3)/pET28a/N在IPTG终浓度为0.2 mmol/L、16℃低温条件下诱导20 h,蛋白呈可溶性表达,且此时蛋白的表达量最高,占总蛋白表达量的70%;通过Ni-NTA亲和层析、凝胶过滤层析纯化后的目的蛋白纯度达90%,分子质量为55 kDa,具有特异性。
    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 coliprokaryotic 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.
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    樊志浩, 李玉林, 张恒, 等. 新型冠状病毒N蛋白的原核表达与纯化[J]. 轻工学报, 2022, 37(4): 34-40. doi: 10.12187/2022.04.005
    引用本文:樊志浩, 李玉林, 张恒, 等. 新型冠状病毒N蛋白的原核表达与纯化[J]. 轻工学报, 2022, 37(4): 34-40.doi:10.12187/2022.04.005
    shu
    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

    新型冠状病毒N蛋白的原核表达与纯化

      作者简介:樊志浩(1997—),男,河南省郑州市人,郑州大学硕士研究生,主要研究方向为生物医药。E-mail:biofzh@126.com
    • 1. 郑州大学 生命科学学院, 河南 郑州 450001;
    • 2. 河南省生物工程技术研究中心, 河南 郑州 450121;
    • 3. 河南省职工医院, 河南 郑州 450002;
    • 4. 郑州职业技术学院 生物工程系, 河南 郑州 450121
    • 收稿日期: 2021-07-19
    • 录用日期: 2021-11-02
    基金项目:河南省新型冠状病毒防控应急科研攻关项目(201100310300)

    摘要:采用生物信息学方法对新型冠状病毒(SARS-CoV-2)核衣壳蛋白(N蛋白)进行亲疏水性、抗原表位预测及多序列对比分析,构建重组质粒pET28a/N,在大肠杆菌原核表达体系下通过调整诱导温度和时间,提升蛋白溶解度和表达量,并对表达出的重组N蛋白进行纯化和鉴定。结果表明:SARS-CoV-2 N蛋白编码419个氨基酸,等电点(PI)为10.10,无跨膜区,无信号肽序列,局部亲水性较强,全长蛋白抗原指数较高,高度保守,与SARS冠状病毒(SARS-CoV) N蛋白同源性为90.5%;采用大肠杆菌原核表达体系发酵,工程菌BL21(DE3)/pET28a/N在IPTG终浓度为0.2 mmol/L、16℃低温条件下诱导20 h,蛋白呈可溶性表达,且此时蛋白的表达量最高,占总蛋白表达量的70%;通过Ni-NTA亲和层析、凝胶过滤层析纯化后的目的蛋白纯度达90%,分子质量为55 kDa,具有特异性。

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