人线粒体转录终止因子2在大肠杆菌表达及多克隆抗体的制备与鉴定

Expression of human MTERF2 in E. coli and preparation and characterization of mouse-anti-human MTERF2 antibody

[目的]构建人线粒体转录终止因子2(mitochondrial transcription termination factor 2,MTERF2)基因原核表达载体并在大肠杆菌(Escherichia coli,E.coli)Rosetta细胞中表达目的蛋白质,制备及鉴定鼠抗人MTERF2多克隆抗体。[方法]利用RT-PCR技术从HeLa细胞中扩增出人MTERF2基因cDNA开放阅读框(open reading frame,ORF)序列,将目的基因插入pET-28b原核表达载体中,转化大肠杆菌Rosseta细胞,IPTG诱导人MTERF2融合蛋白质的表达,经包涵体的洗涤、亲和层析法纯化、质谱鉴定后,用纯化出的包涵体蛋白免疫BALB/c小鼠制备鼠抗人MTERF2多克隆抗体。用酶联免疫吸附法检测抗体的效价,Western Blot检测抗体的特异性。[结果]构建了含有人MTERF2基因cDNA ORF序列的原核表达载体pET-28b-h MTERF2,并成功制备高质量的鼠抗人MTERF2多克隆抗体。酶联免疫吸附法分析结果显示抗体的效价约为1∶10 240,Western Blot检测结果显示鼠抗人MTERF2多克隆抗体能识别原核表达的人MTERF2蛋白以及HeLa、U251细胞内源的人MTERF2蛋白。[结论]在大肠杆菌细胞成功地表达人MTERF2蛋白,制备的鼠抗人MTERF2多克隆抗体具有高效价和高特异性,为进一步研究人MTERF2蛋白的功能提供了基础。

[ Objective] To construct the prokaryotic expression vector of human mitochondrial transcription termination factor 2 （ MTERF2）, express it in E. coli optimally, and prapare its mouse - anti - human polyclonal antibody. [ Methods ] The open reading frame （ORF） of human MTERF2 cDNA was amplified by RT- PCR and subcloned into prokaryotic expression vetor pET - 28b. Then the recombinant vector pET - 28b - MTERF2 was transformed into compentent E. coli BL21 （ DE3 ） and IPTG induced the expression of human MTERF2 fusion protein. The recombinant human MTERF2 protein was purified through agar- ose gel column affinity chromatography and indentified by mass spectrometry. The purified recombinant protein was used as im- munogen to immunize the BALB/c mice to prepare its specific polyclonal antibody. The titer of the antibody were analyzed by ELISA and the specificity of the antibody were analyzed by Western Blot. [ Results] The recombinant human MTERF2 protein was successfully expressed in E. coli and the mouse - anti - human MTERF2 polyclonal antibody with high quality was success- fully prepared. ELISA showed that the titer of the antibody was 1 ： 10 240. Western Blot detection revealed that the mouse - anti -human MTERF2 antibody could recognize the native MTERF3 antigen specifucally. [ Conclusion ] Human MTERF2 ex- pressed in the prokaryotic system has strong inmmunogenicity and the polyclonal antibody obtained from iMmunizing mice has high titer and specificity. The prokaryotic expression of human MTERF2 and the preparation of its polyclonal antibody may shed light on the further function research of human MTERF2 protein.