鲤鱼两种脂蛋白脂肪酶基因的cDNA克

发布时间：2019-04-15 12:27

【摘要】：脂肪是生物体重要的储能物质,其分解产物脂肪酸又是生物体重要的结构物质和活性物质。脂蛋白脂肪酶能够分解脂肪,在生物体脂肪代谢及其相关脂类调控中发挥着重要的作用。在鱼类中存在着两种脂蛋白脂肪酶(LPL1、LPL2)。目前对于鲤鱼LPL结构及功能方面的研究还没有报道,所以本文选取鲤鱼为研究对象,阐明了鲤鱼LPL基因的表达及其功能情况。利用RT-PCR的方法分离了鲤鱼LPL1和LPL2编码区cDNA全长序列,克隆结果表明,鲤鱼LPL1和LPL2开放阅读框(ORF)分别为1524 bp和1503 bp,分别编码507个和500个氨基酸。同源分析结果表明,LPL1和LPL2氨基酸序列相似性为45.51%。氨基酸功能位点分析表明,LPL1、LPL2的N-糖基化位点分别为85N、401N和400N,肝素结合域分别为321R-323N和319R-321N,催化活性位点分别为174S、198D、283H和172S、196D、281H,二聚体形成的保守疏水残基位点分别为218A、230G、237G和216A、228G、235G。系统进化分析表明,鲤鱼LPL1、LPL2与不同纲动物的LPL1、LPL2之间的遗传距离与分类地位基本一致,进化树很好的显示了鱼纲不同科、目的系统发育水平。利用实时荧光定量PCR检测了 iPL1、LPL2在鲤鱼不同组织中的表达情况,结果显示,LPL1和LPL2在不同组织中均有表达,在肝脏中表达量最高,其次为心脏、脂肪、肌肉、脑、中肾,在前肠中表达量最低,在所有组织中,LPL1的表达量始终高于LPL2的表达量。利用酶联免疫法和BCA全蛋白测定法测定了鲤鱼心脏、脂肪、肌肉、肝脏等组织中的脂蛋白脂肪酶含量,结果发现,这与LPL在mRNA水平上的表达量一致。LPL1和LPL2原核表达蛋白成功地在大肠杆菌中获得且使用Ni柱纯化。使用对硝基苯酚法(pNP)测定了 LPL1和LPL2酶活,结果表明,LPL1和LPL2酶活的最适温度均为35℃,最适pH均为8.0,在最适温度和pH条件下,LPL1和LPL2的酶活分别为22.69U/g、17.4U/g。很显然,两者序列差异导致了蛋白酶活不同。
[Abstract]:Fat is an important energy storage substance in organism, and fatty acid is also an important structure and active substance in organism. Lipoprotein lipase can decompose fat and play an important role in lipid metabolism and related lipid regulation. There are two kinds of lipoprotein lipase (LPL1,LPL2) in fish. At present, there is no report on the structure and function of carp LPL. Therefore, the expression and function of LPL gene in common carp have been clarified by choosing carp as the object of study in this paper. The full-length cDNA sequence of common carp LPL1 and LPL2 coding region was isolated by RT-PCR. The cloning results showed that the open reading frame (ORF) of carp LPL1 and LPL2 were 1524 bp and 1503 bp, respectively, encoding 507 amino acids and 500 amino acids. Homology analysis showed that the similarity of amino acid sequence between LPL1 and LPL2 was 45.51%. The amino acid functional site analysis showed that the N-glycosylation sites of LPL1,LPL2 were 85N, 401N and 400N, the heparin binding domains were 321R-323N and 319Rx321N, and the catalytic activity sites were 174S, 198D, 283H and 172s, 196D, 281H, respectively. The conserved hydrophobic residue sites formed by the dimer are 218A, 230G, 237G and 216A, 228G, 235G, respectively. Phylogenetic analysis showed that the genetic distance and taxonomic status between the LPL1,LPL2 of carp and the LPL1,LPL2 of different class animals were basically the same. The phylogenetic tree showed well the level of phylogenetic development of different families of fishes. The expression of iPL1,LPL2 in different tissues of Cyprinus Carpio was detected by real-time fluorescence quantitative PCR. The results showed that LPL1 and LPL2 were expressed in different tissues, the highest expression was in liver, followed by heart, fat, muscle, brain and mesonephros. The expression level of LPL1 was the lowest in foregut and was higher than that of LPL2 in all tissues. The contents of lipoprotein lipase in carp heart, fat, muscle and liver were determined by enzyme linked immunosorbent assay (Elisa) and BCA whole protein assay. This was consistent with the expression of LPL at mRNA level. The prokaryotic expression protein LPL1 and LPL2 were successfully obtained in E. coli and purified by Ni column. The enzyme activities of LPL1 and LPL2 were determined by (pNP) with p-nitrophenol method. The results showed that the optimum temperature and pH of LPL1 and LPL2 were 35 鈩，

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