鲤鱼2种脂酰辅酶A脱氢酶基因cDNA克

发布时间：2018-07-21 20:24

【摘要】：为探究脂酰辅酶A脱氢酶对脂肪酸β氧化的调控机制,使用RT-PCR在鲤鱼肝脏克隆了脂酰辅酶A脱氢酶家族中的MCAD和LCAD全长c DNA序列,阅读框分别为1 275,1 326 bp,编码424,441个氨基酸,分析表明他们均具备ACADs家族的特征序列:FAD结合位点、底物结合位点、催化位点等,与斑马鱼MCAD和LCAD的一致性分别为93.16%和94.56%。实时定量RT-PCR结果表明,MCAD和LCAD主要在肝脏和心脏表达;不同脂肪源对MCAD的表达没有明显影响,但鱼油对LCAD的表达有明显的刺激作用。此外,构建了原核表达载体MCADs-p EASY(E2)和LCADs-pEASY(E2),经IPTG诱导获得了原核表达蛋白,分子量分别为43.0,43.6 kDa。经检测这2个蛋白在370,450 nm处有明显的吸收峰,表明他们能自然结合FAD辅基。吩嗪硫酸甲酯反应法结果表明:MCAD和LCAD酶活性最适温度均为28℃,酶活分别为9.12,10.29 U/g。结果表明,与哺乳动物类似,鲤鱼MCAD和LCAD在肝脏和心脏表达量高;高不饱和脂肪酸对LCAD的表达有促进作用。
[Abstract]:In order to investigate the regulation mechanism of lipoacylcoenzyme A dehydrogenase on fatty acid 尾 oxidation, the full-length cDNA sequences of MCAD and LCAD in lipopolysaccharide A dehydrogenase family were cloned in carp liver by RT-PCR. The reading frame was 1 275 1 326 BP, encoding 424 441 amino acids, respectively. The results showed that they all had the characteristic sequence of ACADs family, such as: FAD binding site, substrate binding site, catalytic site, etc. The consistency with MCAD and LCAD of zebrafish was 93.16% and 94.56%, respectively. The results of real-time quantitative RT-PCR showed that MCAD and LCAD were mainly expressed in liver and heart, but different fat sources had no significant effect on the expression of MCAD, but fish oil could stimulate the expression of LCAD. In addition, prokaryotic expression vectors MCADs-p EASY (E2) and LCADs-pEASY (E2) were constructed. The prokaryotic expression proteins were induced by IPTG and their molecular weights were 43.0 ~ 43.6 kDa. The apparent absorption peaks of the two proteins at 370450 nm showed that they could bind naturally to the fad cogroups. The results of phenazine methyl sulfate reaction showed that the optimum temperature of enzyme activity of MCAD and LCAD was both 28 鈩，

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