人MTERF3蛋白的原核表达与多克隆抗体制备

Prokaryotic expression of human mitochondrial transcription termination factor 3 and preparation of its polyclonal antibody

目的人线粒体转录终止因子3(mitochondrial transcription termination factor 3,MTERF3)是线粒体基因表达及能量代谢的负调控因子。文中旨在研究大肠埃希菌中原核表达MTERF3蛋白及制备鼠抗人MTERF3多克隆抗体。方法RT-PCR扩增人MTERF3基因c DNA的完整开放阅读框,克隆至原核表达载体p ET28b,转化大肠埃希菌感受态细胞BL21(DE3),IPTG诱导6×his融合蛋白质表达,Ni2+-NTA琼脂糖凝胶柱亲和层析纯化重组人MTERF3蛋白,以纯化的重组蛋白为免疫原免疫BALB/c小鼠制备其特异性多克隆抗体,用ELISA、Western blot和细胞免疫荧光检测抗体的效价和特异性。结果成功在大肠埃希菌细胞中原核表达重组人MTERF3蛋白,并获得高质量的鼠抗人MTERF3多克隆抗体。ELISA结果显示抗体的效价为1∶105,Western blot和免疫荧光结果显示鼠抗人MTERF3能特异性识别天然的MTERF3抗原。结论原核表达的重组人MTERF3蛋白具有良好的免疫原性,其免疫小鼠后获得的多克隆抗体具有高效价和高度的特异性。人MTERF3蛋白的原核表达及多克隆抗体制备为进一步研究人MTERF3的功能奠定了实验基础。

Objective Human mitochondrial transcription termination factor 3 （ MTERF3 ） is a negative regulator of mitochondrial gene expression and energy metabolism. This study was to construct a prokaryotic expression system for MTERF3 in Escherichia coli （ E. coli ） and prepare its mouseantihuman polyclonal antibody. Methods The complete open reading frame （ ORF） of human MTERF3 cDNA was amplified by RTPCR and subcloned into prokaryotic expression vector pET28b. Then the recombinant plasmid pET28bMTERF3 was transformed into competent E.coli BL21（DE3） and IPTG induced the expression of 6×his fusion protein. The recombinant human MTERF3 protein was purified through Ni^2＋NTA agarose gel column affinity chromatography and the purified recombinant protein was used as immunogen to immunize the BALB/c mice to prepare its specific polyclonal antibody. The titer and specificity of the antibody were analyzed by ELISA, Western blot and cellular immunofluorescence, respectively. Results The recombinant human＆amp;nbsp;MTERF3 protein was successfully expressed in E. coil and the mouseantihuman MTERF3 polyclonal antibody with high quality was successfully prepared. ELISA showed that the titer of the antibody was 1：10^5 . Western blot and immunofluorescence detection revealed that the mouseantihuman MTERF3 antibody could recognize the native MTERF3 antigen specifically. Conclusion Human MTERF3 expressed in the prokaryotic system has strong immunogenicity and the polyclonal antibody obtained from immunizing mice has high titer and specificity. The prokaryotic expression of human MTERF3 and the preparation of its antibody lay the foundation for further function research of human MTERF3.