人心肌肌钙蛋白I基因的克隆及其在大肠埃希菌中的表达

Cloning and Expression of Human Cardiac Troponin I Gene in E.coli

目的从人心肌组织中克隆出心肌肌钙蛋白I(cardiac troponin I,cTnI)的cDNA,构建成表达重组体导入特定宿主菌,以期大量表达并获得高纯度的心肌肌钙蛋白I,为临床检测心肌损伤及预后提供诊断试剂材料。方法利用RT-PCR方法从人心肌细胞的总RNA中克隆出编码人心肌肌钙蛋白I的cDNA片段,将其插入原核表达载体形成重组体并导入宿主菌BL21(DE3)中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,表达出带6个组氨酸标签的融合蛋白,Ni-NTA树脂纯化后行Western blot进行鉴定。结果成功获取了人cTnI的cDNA,并在大肠埃希菌中高效表达。经Ni-NTA树脂纯化后获得的产物可与其特异性单克隆抗体反应。结论成功克隆了cTnI基因,构建的重组体能够在大肠埃希菌中高效表达,经纯化可获得电泳单点纯的cTnI蛋白。

Objective The cloned cDNA of human cardiac troponin Ⅰ （cTnI） from human heart tissueswas inserted into pET-28c （＋） to construct an prokaryotic expression vector, This recombinant was then transformed into E. coli in order to obtain cTnI protein as materials used in clinical diagnosis and prognosis of myocardial injury. Methods cDNA fragment was amplified by RT-PCR from human cardiac total RNA and inserted into pET-28c （＋） to construct a prokaryotie expressing vector, The recombinant was transformed into BL21 （DE3） bacteria and the expressed histidine-tagged fusion protein induced by IPTG was identified by Western blot after being purified by Ni NTA resin. Results The constructor carrying cTnI cDNA could be expressed in E. coli and the protein after purified by Ni-NTA resin could react with specific monoclonal antibody. Conclusion cDNA encoding cTnI was successfully cloned and the recombinant could be expressed in E. coli. The protein could be purified to near homogeneity.