【摘要】 目的对人心肌肌钙蛋白I(cTnI)基因实行定点突变并进行原核表达,观察此突变对cTnI表达量的影响。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,设计引物将其第2和第4个密码子突变后插入原核表达载体形成重组体,并导入宿主菌BL21(DE3)中,经IPTG诱导表达,Ni-NTA树脂纯化后行Westernblot鉴定,观察突变对cTnI表达的影响。结果突变后的cTnI基因与对照组相比在大肠埃希菌中得到高效表达,经纯化可获得电泳单点纯的cTnI蛋白。结论成功克隆了cTnI基因,所设计的同义突变可促进cTnl在大肠埃希菌中的高效表达。更多还原

【Abstract】 Objective To induce the site-directed mutation of human cardiac troponin I (cTnI) gene, express the mutant in E. coli, and to study the effects of the mutation on the prokaryotic expression of cTnI. Methods The cDNA encoding cTnI was cloned with RT-PCR from the total RNA extracted from human myocardium tissues. A pair of primers was designed and, after the mutations were induced at the second and the fourth codons, inserted into prokaryotic vector pET-28c (+) and transform the recombinant to BL21 (DE3) bacteria. After purified with Ni-NTA resin, the histidine-tagged fusion protein expressed by IPTG-induced was identified by Western blotting and the expression yield of cTnI protein was investigated. Results The expression of the recombinant carrying processed cTnI cDNA was stronger than that in control group. Conclusion cDNA encoding cTnI was successfully cloned. The recombinant with mutations can be more efficient expressed in E. coli. The cTnI protein can be purified to near homogeneity.更多还原