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目的本实验拟通过ＲＴ－ＰＣＲ技术，克隆及表达大鼠心肌线粒体ｓＭｉＭｉ－ＣＫ基因。方法从大鼠心肌线粒体总ＲＮＡ中扩增出ｓＭｉＭｉ－ＣＫＤＮＡ片段（１３３ｋｂ），将该片段克隆到载体ｐＵＣ１９上，对其进行酶切和测序，然后将其克隆到ＰＢＶ２２０上，经温度诱导，使其在大肠杆菌中得以表达，并用ＮＡＤＨ法测其活性。结果ｓＭｉＭｉ－ＣＫ基因序列与国外报导的序列完全一致，表达的ｓＭｉＭｉ－ＣＫ分子量为４５８ＫＤ，占菌体总蛋白的１０７％，其活性为２７４×１０５ＩＵ／Ｌ菌液。结论ｓＭｉＭｉ－ＣＫ基因的克隆及表达，为今后从基因水平上研究心肌缺血性损伤的机制及治疗提供了依据。
Objective To clone and express mitochondrial sMiMi-CK gene in rat myocardium by RT-PCR. Methods The sMiMi-CK DNA fragment (1.33kb) was amplified from the total mitochondrial RNA of rat myocardium. The fragment was cloned into the vector pUC19, digested and sequenced. The fragment was cloned into PBV220 and induced by temperature , So that it can be expressed in E. coli, and its activity was measured by NADH method. Results The sequence of sMiMi-CK gene was exactly the same as that reported in foreign countries. The molecular weight of sMiMi-CK was 458KD, accounting for 107% of the total bacterial proteins. The activity of sMiMi-CK was 2.74 × 105IU / L. Conclusion The cloning and expression of sMiMi-CK gene provide the basis for studying the mechanism and treatment of myocardial ischemic injury at the gene level in the future.