摘要
目的:构建ABH1重组蛋白原核表达载体及建立ABH1蛋白纯化技术。方法:应用PCR技术从人的cD-NA文库扩增出ABH1基因片段,并加入限制性内切酶位点和终止子,进而将其插入到原核表达质粒pET-15b中,构建ABH1/15b原核表达克隆,最后用IPTG诱导其在大肠杆菌BL21(DE3)中表达,用镍柱亲和纯化,离子交换层析及凝胶过滤层析三步法纯化ABH1蛋白,并采用电泳迁移率实验(EMSA)检测所纯化蛋白的DNA结合活性。结果:PCR鉴定和诱导表达阳性结果表明ABH1基因被成功构建入载体pET-15b中,测序分析显示,插入片段全长为1189bp,插入位点、终止位点及阅读框都完全正确。经IPTG诱导表达和纯化后,从转入ABH1/15b质粒的BL21菌株中提取并纯化了45000的ABH1融合蛋白,经EMSA检测ABH1是一个核酸结合蛋白。结论:成功构建了重组ABH1/15b的原核表达载体,最终得到了高纯度蛋白,为进一步研究ABH1的功能和活性机制提供了实验材料。
Aim：To construct prokaryotic expression system for expressing ABH1 and establishing purification technology of it.Methods：With human cDNA as template,ABH1 gene fragments were obtained by PCR.The ABH1 cDNA fragment was inserted into the multi-cloning site of pET-15b to construct ABH1/15b.ABH1/15b expression in E.coli BL21（DE3）was induced by IPTG and the fusion protein was purified by Ni-NTA affinity chromatography,iron exchange chromatography and gel filtration chromatography,and DNA binding activity was tested by electrophoretic mobility shift assay（EMSA）.Results：PCR screen and test expression confirmed that the cDNA sequence of ABH1 had been correctly inserted into the multi-cloning site of pET-15b.DNA sequencing showed that in the prokaryotic expression cassette,the inserted sequence,inserted site,stop codon and reading frame of ABH1 were all correct.ABH1（45 000）was successfully extracted and purified from E.coli BL21 after IPTG induction,and the result of EMSA showed that ABH1 was a DNA-binding protein.Conclusion：Prokaryotic expression vector for expression of ABH1 was successfully constructed.The target protein of high purity was obtained.It provided the materials for further investigation of the function and mechanism of ABH1.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2011年第4期553-556,共4页
Journal of Zhengzhou University(Medical Sciences)
关键词
ABH1
表达纯化
核酸结合蛋白
ABH1
expression and purification
DNA-binding protein
