缺血再灌可诱导新基因的克隆及其基本特性

目的 :发现并克隆缺血再灌可诱导基因。方法 :应用mRNA差异显示技术 ,在猪的心肌组织 ,分离缺血再灌可诱导基因的cDNA片段 ,随后筛选猪心脏cDNA文库。经克隆、测序、杂交后 ,分析克隆基因的基本特性。结果 :发现和分离到一个缺血再灌上调基因的cDNA片段 ,通过筛选cDNA文库获得一个编码 6 0 8个氨基酸开放式阅读框架的阳性克隆子。序列分析显示该克隆子的DNA和氨基酸序列与已知基因、蛋白质氨基酸均无同源性。Northern杂交分析显示该基因在缺血再灌猪心肌组织的表达明显增高 ,并且在正常猪的心、肝、肺、肾、脾、肠、脑和骨骼肌组织均可检测到其表达。Southern杂交分析显示该基因具有高度的进化保守性。结论 :克隆的基因为缺血再灌可诱导新基因 ,它在缺血再灌的应答中可能起着重要的作用。

Cloning and the basic characteristics of a novel cDNA induced by ischemia and reperfusion

Objective To identify and clone genes induced by ischemia and reperfusion. Methods cDNA fragments responding to ischemia and reperfusion in porcine myocardium were isolated by mRNA differential display and then the cDNA library from porcine heart was screened. After cloning, sequencing, and hybridization, the basic characteristics of the gene were analyzed. Results One cDNA fragment from a novel gene upregulated by ischemia and reperfusion was identified and cloned subsequently; a novel full-length cDNA containing an open reading frame encoding 608 amino acid was obtained by screening the library. Sequencing results revealed that both DNA and amino acid sequences had no substantial homology to previously described DNA or protein sequences. Northern blot analysis confirmed that this gene expression in ischemia and reperfusion myocardium was significantly increased, and the expression was also able to be detected in normal porcine heart, liver, lung, kidney, spleen, intestine, brain, and skeletal muscle. Southern blot analysis clearly showed that this novel gene had high evolutional conservation in human beings, pigs, rabbits, and mice. Conclusion The cloned gene is a novel one induced by ischemia and reperfusion, and it may play a very important role in the response to ischemia and reperfusion.