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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 To clone the cDNA of cardiac troponin I (cTnI) from human cardiac muscle tissue and construct a recombinant plasmid which can be introduced into a specific host bacterium in order to express large quantities of cardiac troponin I in high purity. Myocardial damage and prognosis provide diagnostic reagent material. Methods The cDNA encoding human cardiac troponin I was cloned from total RNA of human cardiomyocytes by RT-PCR and inserted into the prokaryotic expression vector to form a recombinant plasmid. The recombinant plasmid was introduced into host strain BL21 (DE3) Thio-β-D-galactoside (IPTG) induced expression of six histidine tag fusion protein, Ni-NTA resin purified by Western blot identification. Results The cDNA of human cTnI was successfully obtained and was highly expressed in Escherichia coli. The product obtained after Ni-NTA resin purification can react with its specific monoclonal antibody. Conclusion The cloned cTnI gene was successfully cloned, and the constructed recombinant was highly expressed in Escherichia coli. After purified, cTnI protein was obtained.