人心肌肌钙蛋白I基因原核表达载体的构建与鉴定

目的构建人心肌肌钙蛋白I(cTnI)基因的原核表达重组体并鉴定。方法利用RT-PCR方法从人心肌细胞的总RNA中克隆出编码人cTnⅠ的cDNA片段,设计引物将其第二和第四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,采用双酶切及PCR方法鉴定后测序。结果经PCR扩增成功获得699bp的cTnI基因,重组体酶切分析及PCR显示结果与预想一致,测序正确。结论成功克隆了cTnI基因并构建了原核表达重组体。

Constructing and Identificating Prokaryotic Vector of Human Cardiac Troponin I Gene

Objective To construct and identify prokaryotic expression plasmid of cTnI gene.Methods The cDNA encoding cTn1 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.The recombinant plasmid was indentified by restriction endonuclease,PCR and DNA sequencing.Results Conclusion cloned gene was 699 bp in length.Enzyme dissection analysis,PCR and DNA seguencing showed that the products was the same as the expected gene.Conclusion cDNA encoding cTnI was successfully cloned and prokaryotic expression plasmid of cTnI was suceessfully constructed.