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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (3): 31-37    DOI: 10.13523/j.cb.1908061
研究报告     
人Nek2蛋白原核表达纯化及其多克隆抗体制备 *
陈秋利1,杨丽超1,李辉1,温莎1,李刚1,何敏1,2,**()
1 广西医科大学公共卫生学院 南宁 530000
2 广西医科大学实验动物中心 南宁 530000
Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein
CHEN Qiu-li1,YANG Li-chao1,LI Hui1,WEN Sha1,LI Gang1,HE Min1,2,**()
1 School of Public Health Guangxi Medical University, Nanning 530000,China
2 Guangxi Medical University Laboratory Animal Center,Nanning 530000,China
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摘要：

目的通过原核表达系统表达人Nek2蛋白,优化表达条件并纯化Nek2蛋白,制备抗Nek2多克隆抗体.方法 将Nek2基因片段构建到原核表达载体pET30a(+)上,转化大肠杆菌BL21 (DE3);加入诱导剂IPTG诱导表达,对诱导温度,诱导剂IPTG终浓度,诱导时间等条件进行优化;利用12% SDS-PAGE后250mmol/L KCl染色切胶纯化蛋白质,将纯化后的Nek2蛋白进行质谱鉴定;纯化Nek2蛋白免疫BALB/c小鼠制备多克隆抗体,运用ELISA,Western blot和免疫荧光实验检测多克隆抗体效价和特异性.结果 构建了pET30a(+)-Nek2重组原核表达质粒,诱导的重组人Nek2蛋白主要以包涵体的形式存在;蛋白质的最适诱导表达条件为28℃,180r/min条件下加入终浓度为0.2mmol/L IPTG诱导32h;质谱分析纯化后的蛋白质为Nek2蛋白,最终获得浓度为1.35mg/ml纯化后的Nek2蛋白;纯化蛋白免疫小鼠,多克隆抗体效价大于1∶243 000,且具有良好的抗原特异性;免疫荧光实验显示Nek2主要定位于细胞质和细胞核.结论: 利用重组人Nek2蛋白获得具有良好抗原特异性的抗Nek2多克隆抗体.
关键词： Nek2;  原核表达;  多克隆抗体;  KCl染色    
Abstract:

Objectives: To express human Nek2 protein by prokaryotic expression system, optimize expression conditions and purify Nek2 protein and prepare anti-Nek2 polyclonal antibody.Methods: The fragment of Nek2 gene was constructed on the prokaryotic expression vector pET30a(+) and transformed into E. coli BL21 (DE3).Expression of the recombinant protein was induced with IPTG and the conditions such as induction temperature, IPTG inducer concentration and induction time were optimized.The protein was purified by staining with 250mmol/L KCl after 12% SDS-PAGE, and the purified Nek2 protein was identified by mass spectrometry.Polyclonal antibody was prepared by immunizing BALB/c mice with purified Nek2 protein, and the titer and specificity of polyclonal antibody was detected by ELISA, Western blot and immunofluorescence assays.Results: The recombinant prokaryotic expression plasmid pET30a(+)-Nek2 was constructed and the recombinant human Nek2 protein was mainly expressed in the form of inclusion bodies.Optimal induction conditions were 28℃,180r/min,IPTG concentration 0.2mmol/L and 32h induction for expression of recombinant protein.The purified protein by mass spectrometry was Nek2 protein, and the concentration of the purified Nek2 protein was 1.35mg/ml.The purified Nek2 protein was used to immunize mice. The titer of polyclonal antibody was greater than 1:243 000 and the polyclonal antibody exhibited a perfect antigenic specificity.Immunofluorescence assay showed that Nek2 was mainly localized in the cytoplasm and nucleus.Conclusion: The recombinant Nek2 protein was used to obtain a anti-Nek2 polyclonal antibody with perfect antigenic specificity.
Key words: Nek2    Prokaryotic expression    Polyclonal antibody    KCl stain
收稿日期: 2019-08-30 出版日期: 2020-04-18
ZTFLH:  Q816  
基金资助: * 国家自然科学基金(81760612);广西重点研发项目(桂科AB16380351)
通讯作者: 何敏     E-mail: hemin@gxmu.edu.cn
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引用本文:

陈秋利,杨丽超,李辉,温莎,李刚,何敏. 人Nek2蛋白原核表达纯化及其多克隆抗体制备 *[J]. 中国生物工程杂志, 2020, 40(3): 31-37.

CHEN Qiu-li,YANG Li-chao,LI Hui,WEN Sha,LI Gang,HE Min. Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein. China Biotechnology, 2020, 40(3): 31-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1908061    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/31

图1  pET30a(+)-Nek2载体构建
图2  重组人Nek2蛋白表达及条件优化
图3  不同条件下电泳条带的灰度值
图4  重组人Nek2蛋白纯化
图5  Nek2质谱鉴定Mascot比对结果
图6  Nek2多克隆抗体效价检测结果
图7  Nek2多克隆抗体特异性检测结果
图8  Nek2亚细胞定位
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