鲤鱼诱导型一氧化氮合成酶全长cDNA克隆及其在外周血白细胞中的差异表达分析

doi:10.16431/j.cnki.1671-7236.2016.05.002

摘要/Abstract

摘要： 试验旨在获得鲤鱼诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)cDNA全长序列,并以此为基础探讨在丝裂原刺激下外周血白细胞中iNOS的表达变化。以从鲤鱼正常外周血白细胞cDNA文库中获得的iNOS的EST序列为基础,采用基因文库筛选和cDNA5'末端快速扩增技术(5'-RACE)相结合的方法,成功扩增出鲤鱼iNOScDNA全长序列,然后进行鲤鱼外周血白细胞原代培养,分成对照组和试验组,其中试验组分别为脂多糖(LPS,1.0μg/mL)刺激4、12h,刀豆蛋白A(ConA,1.0μg/mL)刺激4、24h,对照组为相同培养时间无丝裂原刺激的外周血白细胞,根据得到的iNOScDNA全长序列和鲤鱼β-actin序列分别设计特异性引物,应用实时荧光定量PCR方法检测各组外周血白细胞中iNOS在mRNA水平上的表达情况并进行分析。序列分析结果显示,最后获得的cDNA片段共3704bp,包含74bp的5'端非编码区,246bp的3'端非编码区,一个3384bp的完整的开放阅读框(ORF),共编码1127个氨基酸。序列同源性分析结果显示,该序列与鲫鱼iNOS基因同源性高达100%;实时荧光定量PCR结果显示,经LPS、ConA刺激的试验组外周血白细胞中iNOS表达量均升高,且短时间(4h)刺激的表达量高于长时间(LPS12h;ConA24h)刺激的表达量。综上所述,在LPS、ConA刺激过程中,iNOS在外周血白细胞中表达量均有上调,结果提示存在炎症反应的动态变化。

关键词: 鲤鱼; iNOS; cDNA克隆; 外周血白细胞; 差异表达分析

Abstract: The study was aimed to obtain carp iNOS cDNA full-length sequence, and investigate the changes in peripheral blood leukocyte expression of iNOS in the stimulation of different mitogens.Based on EST sequences of iNOS that obtained from carp normal peripheral blood leukocytes cDNA library, full-length cDNA sequence of carp iNOS was successfully amplified by using gene library screening and rapid-amplification of cDNA 5'ends (5'-RACE) method.Carp peripheral blood leukocytes were divided into control and experimental groups and cultured.The experimental groups were stimulated by LPS (1.0 μg/mL) and ConA (1.0 μg/mL) for 4 and 12 h, respectively.And control groups were in the same cultured conditon time without mitogen stimulated.Real-time PCR was used to detect the expression of iNOS in peripheral blood leukocytes of each group.The results showed that the cDNA fragment was total 3 704 bp containing 74 bp 5'untranslated region (UTR), 246 bp 3'UTR and a 3 384 bp ORF encoding 1 127 amino acids.Sequence homology analysis showed that the amino acid sequence of carp iNOS shared 100% identity with Carassius auratus.After LPS and ConA stimulation, the iNOS expression of peripheral blood leukocytes in the experimental groups were increased, and the expression in the short time stimulation condition (4 h) was higher than the long time (LPS 12 h; ConA 24 h).In conclusion, iNOS expression of peripheral blood leukocytes was raised after LPS and ConA stimulation, suggesting that there were dynamic changes in the inflammation.

Key words: Cyprinus carpio; iNOS; cDNA clones; peripheral blood leukocytes; differential expression analysis

中图分类号:

Q786

温振才, 曹津津, 卢强. 鲤鱼诱导型一氧化氮合成酶全长cDNA克隆及其在外周血白细胞中的差异表达分析[J]. , 2016, 43(5): 1133-1139.

WEN Zhen-cai, CAO Jin-jin, LU Qiang. Cloning of Carp Inducible Nitric Oxide Synthase Full-length cDNA and its Differential Expression Analysis in Peripheral Blood Leukocytes[J]. , 2016, 43(5): 1133-1139.

参考文献

[1] 卢强,丰培金,李瑞莲,等.有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库的构建[J].中国兽医学报,2004,24(6):568-570.
[2] Pautz A,Art J,Hahn S,et al.Regulation of the expression of inducible nitric oxide synthase[J].Nitric Oxide,2010,23(2):75-93.
[3] Suschek C V,Schnorr O,Kolb-Bachofen V.The role of iNOS in chronic inflammatory processes in vivo:Is it damage-promoting,protective,or active at all[J].Current Molecular Medicine,2004,13(4):763-775.
[4] Gambaryan S,Tsikas D.A review and discussion of platelet nitric oxide and nitric oxide synthase:Do blood platelets produce nitric oxide from l-arginine or nitrite[J].Amino Acids,2015,47(7):1779-1793.
[5] Saeij J P,Stet R J,Groeneveld A,et al.Molecular and functionalch aracterization of a fish inducible-type nitric oxide synthase[J].Immunogen etics,2000,51(4):339-346.
[6] Wang T,Ward M,Grabowski P,et al.Molecular cloning,gene organ ization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene[J].Biochem J,2001,358(3):747-755.
[7] Gerondakis S,Grumont R J,Banerjee A.Regulating B-cell activation and survival in response to TLR signals[J].Immunology and Cell Biology,2007,85(6):471-475.
[8] Gibson F C,Ukai T,Genco C A.Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis[J].Frontiers in Bioscience,2008,13(9):2041-2059.
[9] 卢强,丰培金,李莲瑞,等.正常鲤外周血白细胞cDNA文库的构建[J].中国水产学报,2004,28(5):585-588.
[10] Kleinert H,Pautz A,Linker K,et al.Regu lation of the expression of inducible nitric oxide synthase[J].Eur J Pharm Aco,2004,24(3):255-266.
[11] 杨绍松,陶凯,逯芳芳,等.iNOS在LPS诱导活化小胶质细胞中的表达变化[J].神经解剖学杂志,2014,36(6):666-670.
[12] Chiaki I,Toshinori N.Inducible nitric oxide synthase in the respiratory system:Their roles in inflammation[J].Current Allergy and Asthma Reports,2008,8(1):7-13.
[13] 贾爱荣,王传娟,张晓华.大菱鲜组成型热体克蛋白70的全长cDNA克隆及表达分析[J].水生生物学报,2010,34(3):547-552.
[14] 付保忠,卢强,谭叶平,等.鲤外周血白细胞蛋白酶体激活因子PA28I全长cDNA的克隆、鉴定及其差异表达分析[J].水产学报,2008,32(6):831-836.
[15] 孙真,贾生美,冯祥汝,等.鲤鱼外周血白细胞TLR5M全长cDNA克隆及序列分析[J].中国畜牧兽医,2013,40(9):41-45.
[16] Alvarez-Pellitero P.Fish immunity and parasite infections:From innate immunity to immunoprophylactic prospects[J].Veterinary Immunology and Immunopathology,2008,126:171-198.
[17] 林海珊,欧敏.诱导型一氧化氮合成酶及其相关分子的研究进展[J].现代医学,2015,43(5):644-647.
[18] Tan W,Xue-bin C,Tian Z,et al.Effects of simvastatin on the expression of inducible nitric oxide synthase and brain-derived neurotrophic factor in a lipopolysaccharide-induced rat model of Parkinson disease[J].International Journal of Neuroscience,2016,126(3):561-582.
[19] 张浩,咎金行,刘培勋.诱导型一氧化氮合酶抑制剂的研究进展[J].中国新药杂志,2013,22(6):665-669.
[20] 施开创,陈宏备,胡杰,等.小鼠IL-6、iNOS TaqMan荧光定量PCR检测方法的建立及应用[J].中国畜牧兽医,2011,38(9):44-51.