[1]周 杰,俞菊华,李红霞,等.鲤鱼2种脂蛋白脂肪酶基因的cDNA克隆、表达及其酶活性[J].江苏农业学报,2016,(02):414-423.[doi:10.3969/j.issn.1000-4440.2016.02.027]
　ZHOU Jie,YU Ju-hua,LI Hong-xia,et al.Cloning and expression of two genes encoding lipoprotein lipase of Cyprinus carpio[J].,2016,(02):414-423.[doi:10.3969/j.issn.1000-4440.2016.02.027]
点击复制

鲤鱼2种脂蛋白脂肪酶基因的cDNA克隆、表达及其酶活性()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年02期
页码:
414-423
栏目:
畜牧兽医·水产养殖
出版日期:
2016-03-20

文章信息/Info

Title:
Cloning and expression of two genes encoding lipoprotein lipase of Cyprinus carpio
作者:
周 杰1;  俞菊华1; 2;  李红霞2;  李建林2;  唐永凯2;  于 凡2
1.南京农业大学无锡渔业学院,江苏 无锡 214081; 2.中国水产科学研究院淡水渔业研究中心农业部淡水鱼类遗传育种和养殖生物学重点开放实验室,江苏 无锡 214081
Author(s):
ZHOU Jie1;  YU Ju-hua1; 2;  LI Hong-xia2;  LI Jian-lin2;  TANG Yong-kai2;  YU Fan2
1. Wuxi Fishery College, Nanjing Agricultural University, Wuxi 214081, China; 2.Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater, Ministry of Agriculture/Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China
关键词:
鲤鱼;  脂蛋白脂肪酶;  基因克隆;  原核表达;  酶活性
Keywords:
Cyprinus carpio;  lipoprotein lipase;  gene cloning;  prokaryotic expression;  enzyme activity
分类号:
S965.116
DOI:
10.3969/j.issn.1000-4440.2016.02.027
文献标志码:
A
摘要:
为了对鲤鱼(Cyprinus carpio)2种脂蛋白脂肪酶(LPL1、LPL2)基因进行cDNA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83N突变至83K。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-pEASY(E2),经IPTG诱导,在Transetta(DE3)细胞中表达了分子量分别为 5.55×104、5.35×104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pH均为8.0。LPL1酶活性高于LPL2,推测可能与LPL2的83N位点突变所导致的N-糖基化位点缺失有关。
Abstract:
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 amino acid mutated from 83N to 83K. 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 isopropyl β-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 83N site in LPL2.

参考文献/References:

[1] GOLDBERG A P, CHAIT A, BRUNZELL J D. Postprandial adipose tissue lipoprotein lipase activity in primary hypertriglyceridemia [J]. Metabolism,1980, 29(3): 223-229.
[2] MASUNO H, SCHULTZ C J, PARK J W, et al. Glycosylation, activity and secretion of lipoprotein lipase in cultured brown adipocytes of newborn mice. Effect of tunicamycin, monensin, 1-deoxymannojirimycin and swainsonine [J]. Biochem, 1991, 277(3): 801-809.
[3] WONG H, SCHOTZ M C. The lipase gene family [J]. Lipid Res, 2002, 43(7): 993-999.
[4] WION K L, KIRCHGESSNER T G, LUSIS A J, et al. Human lipoprotein lipase complementary DNA sequence [J]. Science, 1987, 235(4796): 1638-1641.
[5] EDWARDS W D, DANIELS S E, PAGE R A, et al. Cloning and sequencing of a full length cDNA encoding ovine lipoprotein lipase [J]. Biochim Biophys Acta, 1993, 1172(1-2): 167-170.
[6] OKU H, OGATA H Y, LIANG X F. Organization of the lipoprotein lipase gene of red sea bream Pagrus Major [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2002, 131(4): 775-785.
[7] JOSE-IBANEZ A, PEINADO-ONSURBE J, SANCHEZ E, et al. Lipoprotein lipase(LPL)is highly expressed and active in the ovary of European seabass(Dicentrarchus labrax), during gonadal development [J]. Comparative Biochemistry and Physiology-Part A: Molecular and Integrative Physiology, 2008, 150(3): 347-354.
[8] 朱择敏,马冬梅,白俊杰,等. 大口黑鲈两种脂蛋白脂肪酶基因cDNA的克隆及表达特征分析[J]. 水产学报, 2013, 37(5): 641-650.
[9] LINDBERG A, OLIVECRONA G. Lipoprotein lipase from rainbow trout differs in several respects from the enzyme in mammals [J]. Gemm, 2002, 292(1-2): 213-223.
[10] GILLER T, BUCHWALD P, BLUM-KAELIN D, et al. Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2: differences in colipase dependence and in lipase activity [J]. Biol Chem, 1992, 267(23): 16509-16516.
[11] 濮剑威,孙成飞,蒋霞云,等. 草鱼两个肌肉生长抑制素cDNA克隆、表达及过量表达对胚胎发育的影响[J]. 生物技术通报, 2011, 27(8): 153-166.
[12] OKU H, KOIZUMI N, OKUMURA T, et al. Molecular characterization of lipoprotein lipase, hepatic lipase and pancreatic lipase genes: Effects of fasting and refeeding on their gene expression in red sea bream Pagrus major[J]. Comp Biochem Physiol, 2006, 145(2): 168-178.
[13] AMAYA A, SANCHEZ-GURMAEHES J, GUTIERREZ J, et a1. Regulation of lipoprotein lipase activity in rainbow trout(Oncorhynchus mykiss)tissues[J]. Gen Comp Endocrino, 2006, 146(3): 226-235.
[14] 周 旋,王志勇,姚翠鸾. 大黄鱼部分脂肪代谢相关基因的克隆及其表达分析[D]. 厦门: 集美大学, 2010.
[15] LIANG X, OKU H, OGATA H Y. The Effect of dietary fatty acids on lipoprotein lipase gene expression in the liver and visceral adipose tissue of fed and starved red sea bream Pagrus major[J]. Comp Biochem Physiol A, 2002, 131: 335-342.
[16] OGATA H Y, SHEARER K D. Influence of dietary fat and adiposity on feed intake of juvenile red sea bream Pagrus major[J]. Aquaculture, 2000, 189(3-4): 237-249.
[17] OGATA H Y, OKU H. The effects of dietary retinoic acid on body lipid deposition in juvenile red sea bream(Pagrus major); a preliminary study[J]. Aquaculture, 2001, 193(3-4): 271-279.
[18] 温茹淑,郑清梅,刘兴隆,等. 复合微生物制剂对草鱼生长与消化酶活性的影响[J]. 安徽农业科学, 2007(13): 3880-3881.
[19] JOSÉ I A, PEINADO-ONSURBE J, SÁNCHEZ E, et al. Lipoprotein lipase(LPL)is highly expressed and active in the ovary of European sea bass(Dicentrarchus labrax L.), during gonadal development[J]. Comp Biochem Physiol A, 2008, 150(3): 347-354.
[20] SEMENKOVICH C F, LUO C C, NAKANISHI M K, et al. In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion[J]. J Biol Chem, 1990, 265(10): 5429-5433.
[21] 韩春艳,郑清梅,冯丽娜. 饲料中不同的脂肪来源对奥尼罗非鱼体脂沉积、MDH和LPL活性及mRNA表达的影响[C]// 中国水产学会.2011年中国水产学会学术年会论文摘要集, 北京: 海洋出版社, 2011.
[22] 韩 庆,刘良国,张建平,等. 温度和pH对洞庭鲇鱼消化酶活性的影响[J]. 水生生物学报, 2011, 35(1): 22-29.
[23] 周 莉,汪 洋,桂建芳. 鱼类特有的基因组复制[J]. 动物学研究, 2006, 27(5): 525-532.
[24] AUWERX J, LEROY P, SCHOONJANS K. Lipoprotein lipase: Recent contributions from molecular biology[J]. Critical Reviews in Clinical Laboratory Sciences, 1992, 29(3-4): 243-268.
[25] 周 冰,曾武威,孙国涛,等. 树鼩脂蛋白脂肪酶的cDNA克隆组织分布及活性测定[J]. 中国动脉硬化杂志, 2007, 15(11): 801-804.
[26] 梁旭方,白俊杰,劳海华,等. 真鲷脂蛋白脂肪酶基因表达与内脏脂肪蓄积营养调控定量研究[J]. 海洋与湖沼, 2003, 34(6): 625-631.
[27] CHENG H L, MENG X P, SUN S P, et al. Cloning and expression analysis of a cDNA encoding lipoprotein lipase from the liver of adult grass carp(Ctenopharyngodon idella)[J]. Aquaculture Research, 2009, 40(16): 1838-1848.
[28] 高 涵,薛利军,刘小北,等. 幽门螺杆菌BabA蛋白N段的基因克隆、表达纯化及体外粘附活性评价[J]. 中国生物工程杂志, 2013, 33(3): 34-40.
[29] GASPARIAN M E, OSTAPCHENKO V G, YAGOLOVICH A V. Overexpression and refolding of thioredoxin TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase[J]. Biotechnology Letters, 2007, 29(10): 1567-1573.
[30] 曹燕娟,胡东生,刘志刚,等. 牛奶主要过敏αs1酪蛋白全长与片段区基因的克隆表达、纯化及免疫原性鉴定[J]. 中国免疫学杂志, 2010, 26(7): 601-605.
[31] 江慧芳,王雅琴,刘春国. 三种脂肪酶活力测定方法的比较及改进[J]. 化学与生物工程, 2007(8): 72-75.
[32] JOHN E H. Fish nutrition [M]. California: Academic Press Inc, 1987:332-423.
[33] 李加儿,张建强,许晓娟,等. 温度和pH值对斜带髭鲷蛋白酶、淀粉酶活性的影响[J]. 渔业科学进展, 2009, 30(3): 7-12.
[34] 桂远明,刘 垠,刘焕亮,等. 温度对草鱼、鲤、鲢、鳙主要消化酶活性的影响[J]. 大连水产学院学报, 1993, 7(4): 2-8.
[35] 沈文英,胡洪国,潘雅娟. 温度和pH值对南美白对虾消化酶活性的影响[J]. 海洋与湖沼, 2004, 35(6): 543-548.
[36] MOYANO F J, SARASQUETE M C. A screening on some digestive enzyme activities of gilt head seabream(Sparus aurata)larvae [J].Oostende Belgium European Aquaculture Sac, 1993, 4: 4163.
[37] KOLODZEISKAYA M V, VEREBKA S V. Chromatography of trypsin and chymotrypsin-like salmon proteases on sawdust [J]. Appl Biochem Microbiol, 1990, 25(5): 528-531.
[38] HIDALGO M C, UREA E, SANZ A. Comparative study of digestive enzymes in fish with different nutritional habits. Photo lytic and amylase activities [J]. Aquaculture, 1999, 170: 267-283.
[39] ZHANG L Y, LOOKENE A, WU G S, et al. Calcium triggers folding of lipoprotein lipase into active dimers [J]. Journal of Biological Chemistry, 2005, 280(52): 42580-42591.

备注/Memo

备注/Memo:
收稿日期:2015-11-19 基金项目:江苏省水产三新工程项目(Y2015-4); 江苏省自然科学基金项目(BK20141096); 中央级基本科研业务费项目(2015JBFM10) 作者简介:周 杰(1989-),男,江苏东台人,硕士研究生,主要从事遗传育种与分子生物学研究。(Tel)18806172172,(E-mail)714203849@qq.com 通讯作者:俞菊华,(Tel)0510-85554198;(E-mail)dnase@163.com
更新日期/Last Update: 2016-03-20