【摘要】：脂肪是生物體重要的儲(chǔ)能物質(zhì),其分解產(chǎn)物脂肪酸又是生物體重要的結(jié)構(gòu)物質(zhì)和活性物質(zhì)。脂蛋白脂肪酶能夠分解脂肪,在生物體脂肪代謝及其相關(guān)脂類調(diào)控中發(fā)揮著重要的作用。在魚類中存在著兩種脂蛋白脂肪酶(LPL1、LPL2)。目前對(duì)于鯉魚LPL結(jié)構(gòu)及功能方面的研究還沒有報(bào)道,所以本文選取鯉魚為研究對(duì)象,闡明了鯉魚LPL基因的表達(dá)及其功能情況。利用RT-PCR的方法分離了鯉魚LPL1和LPL2編碼區(qū)cDNA全長(zhǎng)序列,克隆結(jié)果表明,鯉魚LPL1和LPL2開放閱讀框(ORF)分別為1524 bp和1503 bp,分別編碼507個(gè)和500個(gè)氨基酸。同源分析結(jié)果表明,LPL1和LPL2氨基酸序列相似性為45.51%。氨基酸功能位點(diǎn)分析表明,LPL1、LPL2的N-糖基化位點(diǎn)分別為85N、401N和400N,肝素結(jié)合域分別為321R-323N和319R-321N,催化活性位點(diǎn)分別為174S、198D、283H和172S、196D、281H,二聚體形成的保守疏水殘基位點(diǎn)分別為218A、230G、237G和216A、228G、235G。系統(tǒng)進(jìn)化分析表明,鯉魚LPL1、LPL2與不同綱動(dòng)物的LPL1、LPL2之間的遺傳距離與分類地位基本一致,進(jìn)化樹很好的顯示了魚綱不同科、目的系統(tǒng)發(fā)育水平。利用實(shí)時(shí)熒光定量PCR檢測(cè)了 iPL1、LPL2在鯉魚不同組織中的表達(dá)情況,結(jié)果顯示,LPL1和LPL2在不同組織中均有表達(dá),在肝臟中表達(dá)量最高,其次為心臟、脂肪、肌肉、腦、中腎,在前腸中表達(dá)量最低,在所有組織中,LPL1的表達(dá)量始終高于LPL2的表達(dá)量。利用酶聯(lián)免疫法和BCA全蛋白測(cè)定法測(cè)定了鯉魚心臟、脂肪、肌肉、肝臟等組織中的脂蛋白脂肪酶含量,結(jié)果發(fā)現(xiàn),這與LPL在mRNA水平上的表達(dá)量一致。LPL1和LPL2原核表達(dá)蛋白成功地在大腸桿菌中獲得且使用Ni柱純化。使用對(duì)硝基苯酚法(pNP)測(cè)定了 LPL1和LPL2酶活,結(jié)果表明,LPL1和LPL2酶活的最適溫度均為35℃,最適pH均為8.0,在最適溫度和pH條件下,LPL1和LPL2的酶活分別為22.69U/g、17.4U/g。很顯然,兩者序列差異導(dǎo)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，

本文編號(hào)：2458155