【摘要】：為探究脂酰輔酶A脫氫酶對脂肪酸β氧化的調(diào)控機制,使用RT-PCR在鯉魚肝臟克隆了脂酰輔酶A脫氫酶家族中的MCAD和LCAD全長c DNA序列,閱讀框分別為1 275,1 326 bp,編碼424,441個氨基酸,分析表明他們均具備ACADs家族的特征序列:FAD結(jié)合位點、底物結(jié)合位點、催化位點等,與斑馬魚MCAD和LCAD的一致性分別為93.16%和94.56%。實時定量RT-PCR結(jié)果表明,MCAD和LCAD主要在肝臟和心臟表達;不同脂肪源對MCAD的表達沒有明顯影響,但魚油對LCAD的表達有明顯的刺激作用。此外,構(gòu)建了原核表達載體MCADs-p EASY(E2)和LCADs-pEASY(E2),經(jīng)IPTG誘導(dǎo)獲得了原核表達蛋白,分子量分別為43.0,43.6 kDa。經(jīng)檢測這2個蛋白在370,450 nm處有明顯的吸收峰,表明他們能自然結(jié)合FAD輔基。吩嗪硫酸甲酯反應(yīng)法結(jié)果表明:MCAD和LCAD酶活性最適溫度均為28℃,酶活分別為9.12,10.29 U/g。結(jié)果表明,與哺乳動物類似,鯉魚MCAD和LCAD在肝臟和心臟表達量高;高不飽和脂肪酸對LCAD的表達有促進作用。
[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，

本文編號：2136752