金堂黑山羊白介素10基因的克隆及表达分析

doi:10.16431/j.cnki.1671-7236.2018.12.008

摘要/Abstract

摘要：

为探究山羊白介素10（interleukin 10，IL-10）基因的序列特征及在组织中的相对表达情况，试验采用PCR法扩增并克隆金堂黑山羊IL-10基因，用DNAMAN、DNAStar、ExPASy等生物信息学软件进行序列分析，运用实时荧光定量PCR法检测IL-10基因在金堂黑山羊不同组织中的表达情况。结果显示，金堂黑山羊IL-10基因序列长为379 bp，开放阅读框为276 bp，编码91个氨基酸残基。IL-10蛋白二级结构中含有α-螺旋、β-转角和无规则卷曲，分别为76.92%、2.20%和20.88%。IL-10蛋白三级结构模型与人IL-10（1ilk.1.A）基因同源性最高（86.81%）。系统进化分析结果发现，金堂黑山羊IL-10基因与挪威山羊IL-10基因亲缘关系最近。实时荧光定量PCR检测发现，在金堂黑山羊肺脏中IL-10基因表达水平显著高于脾脏、心脏、肌肉和肾脏（P<0.05），在肾脏中IL-10基因表达水平显著高于脾脏、心脏和肌肉（P<0.05），在脾脏、心脏和肌肉中IL-10基因表达水平较低，但差异不显著（P>0.05）。本试验结果揭示了IL-10基因具有高度的保守性，可能参与调控山羊的免疫应答反应。

关键词: 金堂黑山羊; IL-10基因; 克隆; 表达分析

Abstract:

This study was aimed to explore the sequence characteristics of interleukin 10 (IL-10) gene and its relative expression of different tissues in goat.IL-10 gene of Jintang Black goat was amplified and cloned by PCR,the gene sequence was analyzed by bioinformatics softwares such as DNAMAN,DNAStar and ExPASy,then the expression of IL-10 gene in different tissues was detected by Real-time quantitative PCR.The results showed that the sequence of IL-10 gene was 379 bp,and the open reading frame was 276 bp,encoding 91 amino acid residues.The secondary structure of IL-10 protein contained alpha helix(76.92%),beta turn(2.20%) and random coil(20.88%).The tertiary structure model of IL-10 protein had the highest homology with human IL-10 (1ilk.1.A) gene (86.81%).In the phylogenetic tree,IL-10 gene in Jintang Black goat was closely related to Norwegian goat.Real-time quantitative PCR result showed that the expression level of IL-10 gene in lung of Jintang Black goat was significantly higher than that in spleen,heart,muscle and kidney (P<0.05),the expression of IL-10 gene in kidney was significantly higher than that in spleen,heart and muscle (P<0.05),the expressions of IL-10 gene in spleen,heart and muscle were lower,but there was no significant difference (P>0.05).It revealed that IL-10 gene was highly conserved,and it might be involved in regulating the immune response of goats.

Key words: Jintang Black goat; IL-10 gene; cloning; expression analysis

中图分类号:

方庆, 王利. 金堂黑山羊白介素10基因的克隆及表达分析[J]. 《中国畜牧兽医》, 2018, 45(12): 3371-3378.

FANG Qing, WANG Li. Cloning and Expression Analysis of Interleukin 10 Gene in Jintang Black Goat[J]. , 2018, 45(12): 3371-3378.

参考文献

[1] MOORE K W,DE WAAL MALEFYT R,COOFFMAN R L,et al.Interleukin-10 and the interleukin-10 receptor[J].Annual Review of Immunology,2001,19(1):683-765.
[2] FIORENTINO D F,BOND M W,MOSMANN T R,et al.Two types of mouse T helper cell.IV.Th2 clones secrete a factor that inhibits cytokine production by Th1 clones[J].The Journal of Experimental Medicine,1989,170(6):2081-2095.
[3] THOMSON A W,LOTZE M T.The Cytokine Handbook,Two-volume Set[M].4th Edition.London:Guif Professional Publishing,2003.
[4] KOTENKO S V.The family of IL-10-related cytokinesand their receptors:Related,but to what extent[J].Cytokine & Growth Factor Reviews,2002,13(3):223-240.
[5] 郑维兵,慕鹏飞,丁连帅,等.大黄鱼(Larimichthys crocea)IL-10基因的克隆与表达分析[J]. 海洋学报,2017,39(4):50-60. ZHENG W B,MU P F,DING L S,et al.Cloning and expression analysis of large Yellow croaker (Larimichthys crocea) IL-10 gene[J].Acta Oceanologica Sinica,2017,39(4):50-60.(in Chinese)
[6] 万小颖,李挺,秦立志,等.白细胞介素-10研究进展[J].畜牧与兽医,2013,45(3):90-95. WANG X Y,LI T,QING L Z,et al.Research progress of interleukin-10[J].Animal Husbandry andVeterinary Medicine,2013,45(3):90-95.(in Chinese)
[7] URBONAS V,EIDUKAITE A,TAMULIENE I,et al.Increased interleukin-10 levels correlate with bacteremia and sepsis in febrile neutropenia pediatric oncology patients[J].Cytokine,2012,57(3):313-315.
[8] SCUMPIA P O,MOLDAWER L L.Biology of interleukin-10and its regulatory roles in sepsis syndromes[J].Critical Care Medicine,2005,33(12):468-471.
[9] 潘志鹏,伦永志.JAK/STAT信号通路在机体免疫及相关疾病中的作用机制[J].中国免疫学杂志,2016,35(9):1395-1399. PAN Z P,LUN Y Z.The mechanism of JAK/STAT signaling pathway in organ immunity and related diseases[J].Chinese Journal of Immunology,2016,35(9):1395-1399.(in Chinese)
[10] VIEIRA P,DE WAAL-MALEFYT R,DANG M N,et al.Isolation and expression of human cytokine synthesis inhibitory factor cDNA clone:Homology to Epstein-Barr virus open reading frame BCRFI[J].Proceedings of the National Academy of Science of the United States of America,1991,8(4):1172-1176.
[11] VILLINGER F,BRAR S S,MAYN A,et al.Comparative sequence analysis of cytokine genes from human and nonhuman primates[J].The Journal of Immunology,1995,155(8):3946-3954.
[12] HASH S M,BROWN W C,RICE-FICHT A C,et al.Characterization of a cDNA encoding bovine interleukin 10:Kinetics of expression in bovine lympho-cytes[J].Gene,1994,139(2):257-261.
[13] DARWICH L,SEGALES J,RESENDES A,et al.Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2(PCV2)[J].Research in Veterinary Science,2008,84:194-198.
[14] 仇华吉,金吉东,兰文升,等.高效表达重组猪IL-10的基因工程菌株的构建[J].畜牧兽医学报,2003,34(4):385-388. QIU H J,JIN J D,LAN W S,et al.High-levelexpression of recombinant porcine interleukin-10 in E.coli[J].Chinese Journal of Animal and Veterinary Sciences,2003,34(4):385-388.(in Chinese)
[15] 阳玉梅,郑小龙,黄维义,等.广西沼泽型水牛白细胞介素-10和干扰素-γ基因的克隆及其序列分析[J].黑龙江畜牧兽医,2007,12:12-16. YANG Y M,ZHENG X L,HUANG W Y,et al.Cloning and sequencing of interleukin-10 and interferon-γ gene of Guangxi swamp type buffalo[J].Heilongjiang Animal Science and Veterinary Medicine,2007,12:12-16.(in Chinese)
[16] 李晓琪,郭万柱,黄亚平,等.荣昌猪白细胞介素-10(IL-10)基因的克隆及原核表达[J].安徽农业科学,2007,3(15):4446-4447. LI X Q,GUO W Z,HUANG Y P,et al.Cloning and prokaryolic expression of rongchang pig porcine IL-10[J].Journal of Anhui Agricultural Sciences,2007,3(15):4446-4447.(in Chinese)
[17] 李倬.猪白介素-10的表达及其主动免疫对PRRSV灭活疫苗初次免疫效果的影响[D].南京:南京农业大学,2010. LI Z.The expression of porcine IL-10 and the immunological enhancement of exogenous porcine IL-10 active immunization on inactivated PRRSV vacine[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)
[18] 孟庆玲,才学鹏,乔军.中国白兔白介素-10基因的克隆、表达及其抗体的制备[J].中国实验动物学报,2007,15(3):203-207. MENG Q L,CAI X P,QIAO J,et al.Expression and cloning of IL-10 gene in Chinese White rabbits andpreparation of antibodies against recombinant IL-10[J].Acta Laboratorium Animalis Scientia Sinica,2007,15(3):203-207.(in Chinese)
[19] 肖周婷,黄郁聪,简纪常,等.斜带石斑鱼IL-10基因的克隆及其原核表达[J]. 生物技术通报,2015,31(9):131-137. XIAO Z T,HUANG Y C,JIAN J C,et al.Cloning and prokaryotic expression of IL-10 gene of Epinephelus coioides[J].Biotechnology Bulletin,2015,31(9):131-137.(in Chinese)
[20] OUYANG W J,RUTZ S,CRELLIN N K,et al.Regulation and functions of the IL-10 family of cytokines in inflammation and disease[J].Annual Review of Immunology,2001,29(1):71-109.
[21] LOUIS H,LE MOINE,PENY M O,et al.Production and role of interleukin-10 in concanavalin A-inducedhepatitis in mice[J].Hepatology,1997,25:1382-1389.
[22] MITSUYAMA K,TOMIYASU N,TAKAKI K,et al.Interleukin-10 in the patho-physiology of inflammatory bowel disease:Increased serum concentrations during the recovery phase[J].Mediators of Inflammation, 2006,2006:1-7.