贵州医科大学学报

2019, v.44;No.226(07) 757-761+766

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Cas9蛋白的克隆表达、分离纯化及多克隆抗体制备
Cloning Expression,Purification and Polyclonal Antibody Preparation of Cas9 Protein

刘芳;卢婷;蔡梦迪;吴芳草;陈相好;王彩霞;崔古贞;陈峥宏;
LIU Fang;LU Ting;CAI Mengdi;WU Fangcao;CHEN Xianghao;WANG Caixia;CUI Guzhen;CHEN Zhenghong;Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University;Key Laboratory of Medical Microbiology and Parasitology of Higher Learning of Guizhou;College of Laboratory Medicine,Guizhou Medical University;

摘要(Abstract):

目的:获得高纯度Cas9蛋白,制备Cas9蛋白特异性抗体。方法:以Cas9基因序列为模板,设计特异性引物,PCR扩增获得Cas9基因全长序列,利用无缝拼接技术构建pET28a-Cas9重组表达载体;转化E.coli BL21(DE3),经IPTG诱导表达Cas9蛋白,并利用His-Tag技术分离纯化Cas9蛋白;将纯化获得的Cas9蛋白免疫新西兰大白兔,制备Cas9蛋白多克隆抗体。结果:pET28a-Cas9重组表达载体转化的E.coli BL21(DE3)可表达Cas9蛋白,纯化的Cas9蛋白免疫家兔得到的多克隆抗体效价>1∶256 K。结论:本研究获得了高质量Cas9蛋白及多克隆抗体,为后续CRISPR-Cas9基因编辑的应用奠定了良好的基础。
Objective:To obtain Cas9 protein with high-purity and prepare Cas9 protein-specific polyclonal antibody.Methods:The Cas9 gene sequences were used as a template to design specific primers,and the full-length sequence were obtained by PCR.The recombinant expression vector pET28 a-Cas9 was constructed by seamless splicing technique,and then transformed into E.coli BL21(DE3).The Cas9 protein was expressed by IPTG induction and purified by His-Tag technology.The purified Cas9 protein was finally used to immunize New Zealand white rabbits and to prepare polyclonal antibody.Results:The Cas9 protein was successfully expressed in E.coli BL21(DE3) and purified by His-Tag column.The polyclonal antibody was also successfully prepared with a titer of more than 1∶256 K.Conclusion:This study obtained Cas9 protein with high quality and polyclonal antibody,which laid a good foundation for the subsequent application of CRISPR-Cas9 gene editing.

关键词(KeyWords): Cas9蛋白;蛋白表达与纯化;多克隆抗体
Cas9 protein;protein expression and purification;polyclonal antibody

Abstract:

Keywords:

基金项目(Foundation): 贵州省研究生科研基金立项项目(11348);; 国家自然科学基金项目(31760318,31500078,31560318,31601012,81460314);; 贵州省科技计划项目[(2018)1132];; 大学生创新训练项目(DC201710660022)

作者(Author): 刘芳;卢婷;蔡梦迪;吴芳草;陈相好;王彩霞;崔古贞;陈峥宏;
LIU Fang;LU Ting;CAI Mengdi;WU Fangcao;CHEN Xianghao;WANG Caixia;CUI Guzhen;CHEN Zhenghong;Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University;Key Laboratory of Medical Microbiology and Parasitology of Higher Learning of Guizhou;College of Laboratory Medicine,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2019.07.003

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