生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (4): 17-.doi: 10. 3969 / j. issn. 2095-1736. 2021. 04. 017

• 研究报告 • 上一篇    下一篇

SARS-CoV-2核衣壳蛋白预测与原核重组表达应用

  

  1. 1.四川省自然资源科学研究院,成都610015; 2.成都中医药大学药学院西南特色中药资源国家重点实验室,成都611137; 3.成都市第三人民医院血液科,成都610031;4.成都大学医学院(护理学院),成都610106)
  • 出版日期:2021-08-18 发布日期:2021-08-18
  • 通讯作者: 秦小波,副研究员,从事生物化学与分子生物学研究,E-mail: qxb_2003@163.com; 高继海,副教授,博士,从事中药与分子代谢研究,E-mail: 271969318@qq.com
  • 作者简介:秦小波,副研究员,从事生物化学与分子生物学研究,E-mail: qxb_2003@163.com; 高　华,主治医师,硕士,从事血液生物化学研究,E-mail: 22394155@qq.com; 张冰冰,硕士研究生,从事生物化学与分子生物学研究,E-mail: 2425580236@ qq.com
  • 基金资助:
    四川省级公益性科研院所基本科研业务费项目;四川省中医药管理局科研项目(NO. 2016ZY008)

Prediction and prokaryoticex pression of SARS-CoV-2 nucleocapsid protein

  1. 1. Sichuan Natural Resource Institute, Chengdu 610015, China; 2. National Key Laboratory of Chinese Medicine Resources with Southwest Characteristics, School of Pharmaceutical, Chengdu University of Traditional Chinese Medicine,Chengdu 611137, China; 3. Department of Hematology, the Third People′s Hospital of Chengdu, Chengdu 610031, China; 4. Medical School (Nursing School), Chengdu University, Chengdu 610106, China
  • Online:2021-08-18 Published:2021-08-18

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摘要： 为了优化构建SARS-CoV-2核衣壳蛋白原核表达载体并获得大肠杆菌表达的重组融合蛋白,研究利用公开的摘基因组数据与在线各蛋白质预测工具分析SARS-CoV-2核衣壳蛋白与其他β-CoV核衣壳蛋白的异同,以及二、三级结构情况;为合成、构建大肠杆菌表达系统密码子优化的SARS-CoV-2核衣壳蛋白原核表达载体提供辅助。基于生物信息分析,设计并构建含His标签的重组原核载体pET-N,转化BL21大肠杆菌感受态细胞,IPTG诱导表达目的蛋白,亲和层析法纯化重组融合蛋白,并经SDS-PAGE与Western Blot进行鉴定。预测分析了SARS-CoV-2核衣壳蛋白的序列特征与结构情况;并成功获得原核表达重组载体pET-N,发现其重组融合蛋白在上清液与包涵体中都存在,通过破碎包涵体,纯化获取目的蛋白,为间接ELISA法检测感染者血清中抗体所需的ELISA包被抗原提供参考。

关键词: 冠状病毒, 核衣壳蛋白, SARS-CoV-2, 原核表达, 间接ELISA

Abstract: In order to optimize the construction of prokaryotic expression vector of SARS-CoV-2 nucleocapsid protein and obtain the re-combinant fusion protein expressed inEscherichiacoli, we first analyzed the sequential similarities between SARS-CoV-2 nucleocapsidprotein and other β-CoV nucleocapsid proteins, as well as the secondary and tertiary structures using public genomic data and proteinprediction tools. Then, a recombinant vector pET-N with His tag was constructed and transformed into BL21 cells. The target protein was next induced and expressed by IPTG. Finally, the recombinant fusion protein was purified by affinity chromatography and identi-fied by both SDS-PAGE and Western Blot. We found that the recombinant fusion protein existed in both the supernatant and the inclu-sion body, as well the target protein was purified by breaking the inclusion body. The recombinant fusion protein provides an alternativeway of detecting the antibody in the serum of infected persons at the full advantages of the bioinformatics methodology.

Key words: coronavirus, nucleocapsid protein, SARS-CoV-2, prokaryotic expression, indirect ELISA

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