牛源IL-6基因克隆与生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.11.004

摘要/Abstract

摘要： 试验旨在克隆牛白细胞介素-6（IL-6）基因，并对其编码蛋白进行生物信息学分析，对荷斯坦奶牛进行尾根采血提取RNA后逆转录为cDNA，根据NCBI数据库中已知的牛IL-6基因mRNA序列设计特异性引物，应用RT-PCR技术克隆得到CDS区全长序列，利用生物信息学软件分析牛IL-6基因序列特征、同源性及编码产物的理化性质等。结果表明，试验成功克隆了牛IL-6基因CDS区，全长627 bp，分子质量为23.759 ku，共编码208个氨基酸，理论等电点为7.58，脂溶系数为93.37，属于亲水性蛋白；牛IL-6基因与猪、绵羊、大鼠、人、猫、犬、鸡、小鼠、马和兔的同源性分别为84.4%、96.2%、66.7%、77.5%、75.9%、79.3%、48.5%、67.0%、79.5%和67.5%；系统进化树分析发现，牛与绵羊亲缘关系最近，猪次之，鸡最远；在二级结构预测中，该蛋白无规则卷曲与α-螺旋分别为34.6%和60.1%；亚细胞定位于细胞质；该蛋白含有信号肽和跨膜螺旋结构。本研究为进一步分析牛IL-6基因的功能奠定了一定的理论基础。

关键词: 牛; IL-6基因; 克隆; 生物信息学分析

Abstract: The aim of the experiment was to clone the bovine interleukin-6 (IL-6) gene and analyze its coding protein bioinformatically.RNA was extracted from the tail heel blood of Holstein cows and retrieved into the cDNA.Specific primers were designed according to the known bovine IL-6 gene sequence in NCBI database,the full-length sequence of CDS region was cloned by RT-PCR.The biological characteristics of bovine IL-6 gene sequence,homology and physicochemical properties of coding products were analyzed by bioinformatics software.The results showed that the bovine IL-6 gene CDS region was successfully cloned,the total length was 627 bp,and the molecular weight was 23.759 ku,encoding a total of 208 amino acids.The theoretical isoelectric point was 7.58,the fat soluble coefficient was 93.37,which belonged to the hydrophilic protein.The homologies of Bos taurus IL-6 gene with Sus scrofa,Ovis aries,Rattus norvegicus,Homo sapiens,Felis catus,Canis lupus familiaris,Gallus gallus,Mus musculus,Equus caballus and Oryctolagus cuniculus were 84.4%,96.2%,66.7%,77.5%,75.9%,79.3%,48.5%,67.0%,79.5% and 67.5%,respectively.Phylogenetic tree analysis revealed that cattle and sheep had the closest genetic relationship,followed by pigs,and chickens was farthest.In the secondary structure prediction,the random coil and alpha-helix of the protein were 34.6% and 60.1% respectively.Subcellular localization was located in cytoplasm;The protein contained signal peptide and transmembrane helix structure.This study laid a theoretical foundation for further analysis of bovine IL-6 gene function.

Key words: bovine; IL-6 gene; cloning; bioinformatics analysis

中图分类号:

S858.23

吕亚南, 刘静雨, 姚竞杰, 安志兴, 白跃宇, 张晓建. 牛源IL-6基因克隆与生物信息学分析[J]. 中国畜牧兽医, 2019, 46(11): 3163-3171.

LYU Yanan, LIU Jingyu, YAO Jingjie, AN Zhixing, BAI Yueyu, ZHANG Xiaojian. Cloning and Bioinformatics Analysis of IL-6 Gene in Bovine[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3163-3171.

参考文献

[1] DITTRICH A,HESSENKEMPER W,SCHAPER F.Systems biology of IL-6,IL-12 family cytokines[J].Cytokine & Growth Factor Reviews,2015,26(5):595-602.
[2] UNVER N,MCALLISTER F.IL-6 family cytokines:Key inflammatory mediators as biomarkers and potential therapeutic targets[J].Cytokine & Growth Factor Reviews,2018,41:10-17.
[3] TANAKA T,NARAZAKI M,MASUDA K,et al.Regulation of IL-6 in immunity and diseases[J].Advances in Experimental Medicine and Biology,2016,941:79-88.
[4] HUNTER C A,JONES S A.IL-6 as a keystone cytokine in health and disease[J].Nature Immunology,2015,16(5):448-457.
[5] RIDKER P M.From C-reactive protein to interleukin-6 to interleukin-1:Moving upstream to identify novel targets for atheroprotection[J].Circulation Research,2016,118(1):145-156.
[6] BRAHMAKSHATRIYA V,KUANG Y,DEVARAJAN P,et al.IL-6 production by TLR-activated APC broadly enhances aged cognate CD4 helper and B cell antibody responses in vivo[J].Journal of Immunology,2017,198(7):2819-2833.
[7] KOTAKE S,SATO K,KIM K J,et al.Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from arthritis patients are responsible for osteoclast-like cell formation[J]. Journal of Bone and Mineral Research,2009,11(1):88-95.
[8] NIU H Q,ZHAO W P,ZHAO X C,et al.Combination of 4-hydroperoxy cyclophosphamide and methotrexate inhibits IL-6/sIL-6R-induced RANKL expression in fibroblast-like synoviocytes via suppression of the JAK2/STAT3 and p38MAPK signaling pathway[J].International Immunopharmacology,2018,61:45-53.
[9] NAKAHARA H,SONG J,SUGIMOTO M,et al.Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis[J].Arthritis & Rheumatology,2014,48(6):1521-1529.
[10] 史明艳,高雪,易力,等.过表达IL-6对山羊毛囊干细胞增殖和迁移的影响[J].畜牧兽医学报,2019,50(6):1154-1161. SHI M Y,GAO X,YI L,et al.Effects of overexpression of IL-6 on proliferation and migration of goat hair follicle stem cells in vitro[J].Chinese Journal of Animal and Veterinary Sciences, 2019,50(6):1154-1161.(in Chinese)
[11] KNÜPFER H,PREISS R.Significance of interleukin-6(IL-6) in breast cancer (review)[J]. Breast Cancer Research and Treatment,2007,102(2):129-135.
[12] HEINRICH P C,CASTELL J V,ANDUS T.Interleukin-6 and the acute phase response[J]. Biochemical Journal,1990,265(Suppl 3):621-636.
[13] LI A,YANG Q,VERMET H,et al.Evaluation of human hepatocytes under prolonged culture in a novel medium for the maintenance of hepatic differentiation:Results with the model pro-inflammatory cytokine interleukin 6[J].Drug Metabolism Letters,2014,8(1):12-18.
[14] KIMURA A,KISHIMOTO T.IL-6:Regulator of Treg/Th17 balance[J].European Journal of Immunology,2010,40(7):1830-1835.
[15] CARRIER Y,GAO W,KORN T,et al.Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells[J].Nature (London),2006,441(7090):235-238.
[16] PETERFI L,YUSENKO M V,KOVACS G.IL6 shapes an inflammatory microenvironment and triggers the development of unique types of cancer in end-stage kidney[J].Anticancer Research,2019,39(4):1869-1874.
[17] KUROYANAGI G,ADAPALA N S,YAMAGUCHI R,et al.Interleukin-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis in a murine model[J].Bone,2018,116:221-231.
[18] 查安东,邢桂玲,高晓雯,等.IL6基因内含子3多态性与仔猪腹泻、生长和公猪繁殖性状的关联分析[J].畜牧兽医学报,2017,48(9):1674-1682. ZHA A D,XING G L,GAO X W,et al.Association analysis between IL6 gene intron 3 polymorphism and piglet diarrhea,growth and boar reproduction traits[J].Chinese Journal of Animal and Veterinary Sciences,2017,48(9):1674-1682.(in Chinese)
[19] 刘梅芬,李勤楠,马震原,等.重组鸡白细胞介素-6/2融合蛋白对新城疫(LaSota株)活疫苗免疫增强作用研究[J].中国畜牧兽医,2019,46(2):590-599. LIU M F,LI Q N,MA Z Y,et al.Immunopotentiation effect of recombinant chicken interleukin-6/2 fusion protein on Newcastle disease virus (LaSota) live vaccine[J].China Animal Husbandry & Veterinary Medicine,2019,46(2):590-599.(in Chinese)
[20] 吕阳,曹阳,高一,等.草原红牛ACSL3基因CDS区克隆、生物信息学分析及组织表达研究[J].中国畜牧兽医,2019,46(4):957-966. LYU Y,CAO Y,GAO Y,et al.Cloning,bioinformatics and tissue expression analysis of ACSL3 gene CDS in red steppe cattle[J].China Animal Husbandry & Veterinary Medicine,2019,46(4):957-966.(in Chinese)
[21] 杨亚军,王震,张洁,等.牛妊娠相关糖蛋白BoPAG1的制备及生物信息学分析[J].生物技术,2019,29(1):29-34. YANG Y J,WANG Z,ZHANG J,et al.Preparation and bioinformatics analysis of pregnancy-associated glycoprotein BoPAG1 in dairy cows[J]. Biotechnology,2019,29(1):29-34.(in Chinese)
[22] 杨玉梅,柴志欣,王会,等.类乌齐牦牛SIRT3基因克隆与生物信息学及差异表达分析[J].中国畜牧杂志,2019,55(6):56-62. YANG Y M,CHAI Z X,WANG H,et al.Cloning and bioinformatics and differential expression analysis of SIRT3 gene in Wuqi-like yak[J].Chinese Journal of Animal Science,2019,55(6):56-62.(in Chinese)
[23] 张灿.牛IL-6基因原核表达及其对口蹄疫病亚单位疫苗的佐剂效应研究[J].中国农学通报,2014,30(20):11-16. ZHANG C.Expression of bovine IL-6 and the adjuvant effects on subunit vaccine of foot-and-mouth virus[J].Chinese Agricultural Science Bulletin,2014,30(20):11-16.(in Chinese)
[24] 王刚,潘丽,张永光.牛IL-6基因克隆及其在大鼠体内活性预测[J].生物学杂志,2011,28(3):1-4. WANG G,PAN L,ZHANG Y G.Clone of bovine IL-6 gene and its activity prediction on rat[J].Journal of Biology,2011,28(3):1-4.(in Chinese)