鲤鱼2种脂蛋白脂肪酶基因的cDNA克隆、表达及其酶活性

Cloning and expression of two genes encoding lipoprotein lipase of Cyprinus carpio

为了对鲤鱼(Cyprinus carpio)2种脂蛋白脂肪酶(LPL1、LPL2)基因进行c DNA克隆以及表达和酶活性分析,测定了鲤鱼2种脂蛋白脂肪酶基因的组织表达特征,对这2种基因进行了原核表达,采用对硝基苯酚法比较了2种LPL的酶活性。克隆结果表明,鲤鱼LPL1和LPL2基因的开放阅读框分别为1 524 bp和1 503 bp,分别编码507个和500个氨基酸,氨基酸相似性为45.51%,LPL2氨基酸功能位点由^(83)N突变至^(83)K。实时荧光定量PCR结果表明:LPL1和LPL2均在肝脏中表达量最高,其次在心脏、脂肪、肌肉、脑、肾中,前肠表达量最低,在所有组织(器官)中LPL1的表达量比LPL2高。构建的原核表达载体LPLs-p EASY(E2),经IPTG诱导,在Transetta(DE3)细胞中表达了分子量分别为5.55×10~4、5.35×10~4的融合蛋白LPL1和LPL2。酶活性测定结果表明:LPL1和LPL2酶活性最适温度均为为35℃,最适p H均为8.0。LPL1酶活性高于LPL2,推测可能与LPL2的^(83)N位点突变所导致的N-糖基化位点缺失有关。〗

Two genes encoding lipoprotein lipase LPL1 and LPL2 in Cyprinus carpio were cloned and the expression patterns were characterized. The open reading frames of LPL1 and LPL2 were 1 524 bp and 1 503 bp in length, encoding 507 and 500 amino acids respectively. The amino acid shared 45？51% similarity. The functional sites of LPL2？encoded ami-no acid mutated from 83 N to 83 K. The real time fluorescence quantitative PCR revealed that the expression levels of LPL1 and LPL2 in the liver were the highest, followed by heart, fat, muscle, brain, kidney, and in the foregut was the lowest. In all tissues and organs the expression of LPL1 was higher than that of LPL2. By constructing the prokaryotic expression vector LPL1？pEASY （E2） and LPL2？pEASY （E2）, the fusion protein LPL1 with molecular mass of 5.55×104 and LPL2 with molecular mass of 5.35× 104 were obtained from the Transetta （ DE3） after induced by 0？5 mmol/L isopropylβ？D-thiogalactoside （IPTG）. The two enzymes presented the highest activities under the conditions of 35℃ and pH 8. The higher activity of LPL1 may be caused by the loss of N-glycosylation site resulting from the mutation of 83 N site in LPL2.