生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (6): 23-.doi: 10.3969/j.issn.2095-1736.2020.06.023

• 研究报告 • 上一篇    下一篇

Cpf1核酸酶的原核表达、纯化及多克隆抗体制备与鉴定

  

  1. 滨州学院 生物与环境工程学院 山东省黄河三角洲野生植物资源开发利用工程技术研究中心， 滨州 256603
  • 出版日期:2020-12-18 发布日期:2020-12-21
  • 作者简介:董彬，博士，讲师，主要从事食品生物技术研究，E-mail: dongbin@bzu.edu.cn
  • 基金资助:
    山东省自然科学基金资助项目（ZR2018PC010，ZR2019MH054，ZR2019PH097）;山东省重点研发计划资助项目(No.2019GSF107010)

Prokaryotic expression and purification of Cpf1 in Escherichia coli and preparation of polyclonal antibody#br#

  1. Shandong Provincial Engineering and Technology Research Center for Wild Plant Resources Development and Application of Yellow River Delta, College of Biological and Environmental Engineering,Binzhou University, Binzhou 256603, China
  • Online:2020-12-18 Published:2020-12-21

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摘要： 将Cpf1核酸酶N端166个氨基酸的DNA编码序列克隆至pET-28a(+)质粒中，在大肠杆菌BL21中进行重组表达，对诱导其表达的IPTG浓度和诱导时间开展条件优化。利用6×His纯化标签得到的重组蛋白作为抗原进行动物免疫，间接ELISA方法测定免疫后抗血清效价达到128 000，并经Protein A纯化后得到抗体，Western Blot分析抗体的特异性，结果显示可以特异性识别大肠杆菌中表达的Cpf1(1-166)和Cpf1，为Cpf1在生物体中的检测提供工具，且将为推动CRISPR/Cpf1系统在生物体中的应用奠定良好的实验基础。

关键词: CRISPR-Cpf1, 大肠杆菌, 原核表达, 蛋白纯化, 多克隆抗体

Abstract: In this study, the DNA encoding sequences of Cpf1 nuclease fragment (N-terminal 166 amino acids) were subcloned into pET-28a (+) vector and expressed in E. coli BL21. The IPTG concentration and induction time were optimized, and the purified protein was used to immunize New Zealand white rabbits. The titer of antiserum reached 128 000 by indirect ELISA method. The antibody was purified by Protein A resin and identified by Western Blot. The results showed that Cpf1(1-166) and Cpf1 expressed in E. coli could be specifically identified. This antibody provids a tool for the detection of Cpf1 in organisms and lay a foundation for promoting the application of CRISPR/Cpf1 system.

Key words: CRISPR-Cpf1, Escherichia coli, prokaryotic expression, protein purification, polyclonal antibody

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