His标记的小鼠线粒体转录因子A的克隆和表达纯化及其多克隆抗体的制备  

Cloning,expression and purification of His-tagged mouse mitochondrial transcription factor A and preparation of its polyclonal antibody

作　　者：刘志锋[1] 邵紫韫[1] 徐佳[1] 刘靖华[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学广东省功能蛋白质组学重点实验室,广东广州510515

出　　处：《第一军医大学学报》2005年第7期774-777,共4页Journal of First Military Medical University

基　　金：863课题(2001AA234061);973计划项目(2002CB513005);海外青年学者合作研究基金(30128009);广东省科技计划项目(A1090202)~~

摘　　要：目的表达和纯化带His标记的小鼠线粒体转录因子A(mitochondrialtranscriptionfactorA,mtTFA)融合蛋白,并制备mtTFA的特异性多克隆抗体。方法提取正常BALB/c小鼠肝脏组织总RNA,通过RT-PCR方法扩增出无信号肽的mtTFA编码序列,克隆入经KpnⅠ和BamHⅠ酶切后的pET14b。酶切及测序鉴定正确后转化大肠杆菌BL21(DE3),经IPTG诱导表达后用镍离子亲和树脂(Ni2+-NTAHis·BindResin)纯化融合蛋白。用纯化的蛋白免疫新西兰大白兔,制备多克隆抗体。结果成功克隆出了mtTFA的编码序列,并构建了带His标记的小鼠mtTFA融合蛋白的重组表达质粒pET14b/His-mtTFA。表达的融合蛋白经亲和树脂纯化后得到大量的高纯度目的蛋白,获得了高特异性兔抗血清。结论获得了His标记的小鼠mtTFA原核表达载体,纯化得到高纯度的目标蛋白,并制备出高特异性兔抗血清,对进一步研究其生物学功能及调节细胞核同线粒体之间功能协同性的信号通路有重要意义。Objective To express and purify the fusion protein of His-tagged mouse mitochondrial transcription factor A (mtTFA) in prokaryocytes and prepare rabbit anti-mtTFA polyclonal antibody.Methods The total RNA was extracted from mouse liver cells,and the coding sequence of mtTFA without signal peptides was amplified with reverse transcriptional (RT)-PCR.The PCR product was then cloned into the prokaryotic expression vector pET14b.After enzyme digestion and DNA sequencing,the plasmid was transformed into BL21(DE3) competent cells,and IPTG was used to induce the expression of His-tagged mtTFA.The expressed fusion protein was purified with Ni2+-NTA His-bind resin and quantified with Bradford method.Results The amplified product was confirmed as the coding sequence of mtTFA without signal peptides by DNA sequencing,and the recombinant expression vector could express His-tagged mtTFA in E.coil following the induction by IPTG.The fusion protein of high purity and rabbit anti-mtTFA serum with high specificity were obtained.Conclusions The prokaryotic expression vector for mtTFA is successfully constructed and His-tagged mtTFA protein and specific polyclonal antibody are obtained,which can be helpful for further investigation of the biological function and signaling pathways involved in the regulation of functional cooperation between the nucleus and mitochondria.

关 键 词：线粒体转录因子A 融合蛋白 基因克隆 蛋白表达 

分 类 号：R346[医药卫生—基础医学]