双峰驼CYP2J cDNA全长克隆、序列信息及组织表达分析

doi:10.11843/j.issn.0366-6964.2018.10.009

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (10): 2133-2144.doi: 10.11843/j.issn.0366-6964.2018.10.009

双峰驼CYP2J cDNA全长克隆、序列信息及组织表达分析

贾志鹏^1,2, 彭小彬^1,2, 罗查干^1,2, 哈斯苏荣^1,2,3*

1. 内蒙古农业大学兽医学院, 呼和浩特 010018;
2. 农业部动物疾病临床诊疗技术重点实验室, 呼和浩特 010018;
3. 内蒙古骆驼研究院, 阿拉善右旗 737300

收稿日期:2018-01-31 出版日期:2018-10-23 发布日期:2018-10-23
通讯作者: 哈斯苏荣,教授,博士,主要从事兽医药理学、兽医寄生虫学与药物动力学方面的教学与科研工作,E-mail:bohaas@163.com
作者简介:贾志鹏(1990-),男,蒙古族,内蒙古乌兰察布人,硕士生,主要从事兽医药理学与毒理学研究,E-mail:605490844@qq.com
基金资助:
国家自然科学基金（31560710）

The Full-length Cloning, Sequence Information and Tissues Expression Analysis of CYP2J cDNA in Bactrian Camel

JIA Zhi-peng^1,2, PENG Xiao-bin^1,2, LUO Cha-gan^1,2, HASI Su-rong^1,2,3*

1. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease of Ministry of Agriculture, Hohhot 010018, China;
3. Inner Mongolia Institute of Camel Research, Alxa Right Banner 737300, China

Received:2018-01-31 Online:2018-10-23 Published:2018-10-23

摘要/Abstract

摘要：

旨在研究双峰驼CYP2J基因及其编码蛋白的结构和功能。本试验首先通过RACE和RT-PCR技术，扩增得到双峰驼CYP2J基因cDNA全长。然后利用生物信息学方法，对该基因编码的蛋白特征进行分析，并使用MEGA 6软件构建双峰驼CYP2J蛋白与相关物种CYP2J蛋白的进化树。最后利用实时荧光定量PCR测定CYP2J基因在双峰驼各组织的表达量差异。结果表明，双峰驼CYP2J基因cDNA全长为1 770 bp，其中完整开放阅读框（ORF）为1 509 bp，共编码502个氨基酸。生物信息学分析结果表明，其ORF编码的蛋白为一种稳定的亲水性蛋白，且不具有信号肽识别功能，但有一个跨膜区域（12~35位氨基酸残基），主要定位在内质网上。该蛋白一些重要的理化特性如分子式、等电点（pI）、相对分子质量、正电荷的氨基酸残基数（Arg+Lys）和负电荷的氨基酸残基数（Asp+Glu）分别为C₂₆₄₅H₄₀₉₉N₇₁₃O₇₂₆S₁₅、8.45、57 983、59、56。其二级结构主要由46.41%的α-螺旋、13.15%的延伸链和40.44%的无规则卷曲组成。进一步进化树分析表明，双峰驼CYP2J蛋白与羊和牛的亲缘关系最近。实时荧光定量PCR检测表明，CYP2J基因在所检测的双峰驼各组织均有表达，其中在肝和胰腺的表达量高，其次是肾、肺、心、小肠和血管，而在脾和大肠的表达量较低。因此，本研究成功获得了双峰驼CYP2J基因cDNA的全序列及其开放阅读框（ORF）所编码蛋白的生物学信息，为下一步双峰驼CYP2J蛋白和抗体的制备奠定了基础。

Abstract:

In order to study the structure and function of CYP2J gene and protein of Bactrian camel, the full-length cDNA sequence of CYP2J gene of Bactrian camel was amplified firstly by RACE and RT-PCR techniques. Then the bioinformatics method was used to analyze the characteristics of the protein encoded by CYP2J gene, and the phylogenetic tree of CYP2J protein between Bactrian camel and related species was constructed by using MEGA 6. Finally, real-time quantitative PCR was used to determine the difference of CYP2J gene expression in various tissues of Bactrian camels. The results showed that the full-length CYP2J cDNA sequence of Bactrian camel was 1 770 bp containing a 1 509 bp complete open reading frame (ORF), which encoded a protein with 502 amino acids. Bioinformatics analysis indicated that the protein encoded by the ORF was predicted to be a stable hydrophilic protein without signal peptide recognition function but had a transmembrane segment (12-35 aa), and was anchored to endoplasmic reticulum. And some important physicochemical properties of the protein such as the molecular formula, isoelectric point, molecular weight, number of positively charged residues (Arg+Lys) and negatively charged residues (Asp+Glu) were C₂₆₄₅H₄₀₉₉N₇₁₃O₇₂₆S₁₅, 8.45, 57 983, 59 and 56, respectively. The secondary structure were mainly composed of 46.41% alpha helix, 13.15% extended strand and 40.44% random coil. Further phylogenetic tree analysis indicated that CYP2J protein of Bactrian camel had the closest genetic relationship with CYP2J protein of sheep and cattle. The real-time quantitative PCR result showed that CYP2J gene expressed in every tissues detected of Bactrian camel and the expression level of CYP2J gene was the highest in liver and pancreas, followed by kidney, lung, heart, small intestine and blood vessels, and the lowest in spleen and large intestine. In conclusion, the full-length cDNA sequence of CYP2J of Bactrian camel and the bioinformation of protein encoded by open reading frame(ORF) were successfully obtained in this study, which laid the foundation for preparing the CYP2J protein and antibody of Bactrian camel.

中图分类号:

S824.2

贾志鹏, 彭小彬, 罗查干, 哈斯苏荣. 双峰驼CYP2J cDNA全长克隆、序列信息及组织表达分析[J]. 畜牧兽医学报, 2018, 49(10): 2133-2144.

JIA Zhi-peng, PENG Xiao-bin, LUO Cha-gan, HASI Su-rong. The Full-length Cloning, Sequence Information and Tissues Expression Analysis of CYP2J cDNA in Bactrian Camel[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2133-2144.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2018.10.009

https://www.xmsyxb.com/CN/Y2018/V49/I10/2133

参考文献

[1] DAIKH B E,LASKER J M,RAUCY J L,et al.Regio-and stereoselective epoxidation of arachidonic acid by human cytochromes P4502C8 and 2C9[J].J Pharmacol Exp Ther,1994,271(3):1427-1433.
[2] WU S,MOOMAW C R,TOMER K B,et al.Molecular cloning and expression of CYP2J2,a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart[J].J Biol Chem,1996,271(7):3460-3468.
[3] SCARBOROUGH P E,MA J X,QU W,et al.P450 subfamily CYP2J and their role in the bioactivation of arachidonic acid in extrahepatic tissues[J].Drug Metab Rev,1999,31(1):205-234.
[4] CAMPBELL W B,HARDER D R.Endothelium-derived hyperpolarizing factors and vascular cytochrome P450 metabolites of arachidonic acid in the regulation of tone[J].Circ Res,1999,84(4):484-488.
[5] WU S,CHEN W N,MURPHY E,et al.Molecular cloning,expression,and functional significance of a cytochrome P450 highly expressed in rat heart myocytes[J].J Biol Chem,1997,272(19):12551-12559.
[6] YAO M,LV D,HUO J,et al.Cerebral small vessel disease is associated with genetic variations in CYP2J[J].Curr Neurovasc Res,2017,14(4):378-384.
[7] LI Q,ZHAO J H,MA P J,et al.Association of CYP2J2 gene polymorphisms with ischemic stroke[J].Int J Clin Exp Med,2015,8(5):8163-8167.
[8] WANG S Y,XING P F,ZHANG C Y,et al.Association of CYP2J2 gene polymorphisms with ischemic stroke and stroke subtypes in Chinese population[J].Medicine,2017,96(10):e6266.
[9] BELOIARTSEV A,DA GLÍRIA RODRIGUES-MACHADO M,ZHOU G L,et al.Pulmonary hypertension after prolonged hypoxic exposure in mice with a congenital deficiency of Cyp2j[J].Am J Respir Cell Mol Biol,2015,52(5):563-570.
[10] KARKHANIS A,HONG Y J,CHAN E C Y.Inhibition and inactivation of human CYP2J2:Implications in cardiac pathophysiology and opportunities in cancer therapy[J].Biochem Pharmacol,2017,135:12-21.
[11] JIANG J G,CHEN C L,CARD J W,et al.Cytochrome P4502J2 promotes the neoplastic phenotype of carcinoma cells and is up-regulated in human tumors[J].Cancer Res,2005,65(11):4707-4715.
[12] SPIECKER M,LIAO J K.Vascular protective effects of cytochrome P450 epoxygenase-derived eicosanoids[J].Arch Biochem Biophys,2005,433(2):413-420.
[13] LEE C A,JONES J P,KATAYAMA J,et al.Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity[J].Drug Metab Dispos,2012,40(5):943-951.
[14] HASHIZUME T,IMAOKA S,MISE M,et al.Involvement of CYP2J2 and CYP4F12 in the metabolism of ebastine in human intestinal microsomes[J].J Pharmacol Exp Ther,2002,300(1):298-304.
[15] XIE F,DING X X,ZHANG Q Y.An update on the role of intestinal cytochrome P450 enzymes in drug disposition[J].Acta Pharm Sin B,2016,6(5):374-383.
[16] UEHARA S,UNO Y,INOUE T,et al.Marmoset cytochrome P4502J2 mainly expressed in small intestines and livers effectively metabolizes human P4502J2 probe substrates,astemizole and terfenadine[J].Xenobiotica,2016,46(11):977-985.
[17] KASPERA R,KIRBY B J,SAHELE T,et al.Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo[J].Biochem Pharmacol,2014,91(1):109-118.
[18] LEE E,WU Z,SHON J C,et al.Danazol inhibits cytochrome P4502J2 Activity in a substrate-independent manner[J].Drug Metab Dispos,2015,43(8):1250-1253.
[19] PHUC N M,WU Z X,LEE E Y,et al.Potential inhibitory effect of 4-hydroxytoremifene on CYP2J2 activities in human liver microsomes[J].Adv Environ Biol,2015,9(19):77-82.
[20] XU M,JU W,HAO H,et al.Cytochrome P4502J2:distribution,function,regulation,genetic polymorphisms and clinical significance[J].Drug Metab Rev,2013,45(3):311-352.
[21] PARK H G,LIM Y R,HAN S,et al.Expression and characterization of truncated recombinant human cytochrome P4502J2[J].Toxicol Res,2014,30(1):33-38.
[22] GRAVES J P,GRUZDEV A,BRADBURY J A,et al.Quantitative polymerase chain reaction analysis of the mouse Cyp2j subfamily:Tissue distribution and regulation[J].Drug Metab Dispos,2015,43(8):1169-1180.
[23] KACZENSKY,ADIYA A,VON WEHRDEN H,et al.Space and habitat use by wild Bactrian camels in the Transaltai Gobi of southern Mongolia[J].Biol Conserv,2014,169(100):311-318.
[24] 萨仁图娅,斯仁达来,付龙霞,等.中国双峰驼种质资源保护调查研究[J].中国草食动物科学,2015,35(6):62-65,78.
SAREN T Y,SIREN D L,FU L X,et al.Investigation on the protection of germplasm resources of Chinese Bactrian Camel[J].China Herbivore Science,2015,35(6):62-65,78.(in Chinese)
[25] 薛亚东,吴三雄,孙志成,等.野骆驼的研究和保护:现状与展望[J].四川动物,2014,33(3):476-480.
XUE Y D,WU S X,SUN Z C,et al.Research and conservation of wild camel:Present status and future prospects[J].Sichuan Journal of Zoology,2014,33(3):476-480.(in Chinese)
[26] ZHAO M L,WILLMS W D,HAN G D,et al.Bactrian camel foraging behaviour in a Haloxylon ammo-dendron (C A Mey) desert of Inner Mongolia[J].Appl Anim Behav Sci,2006,99(3-4):330-343.
[27] 贺丽虹,赵淑娟,胡之璧.植物细胞色素P450基因与功能研究进展[J].药物生物技术,2008,15(2):142-147.
HE L H,ZHAO S J,HU Z B.Gene and function research progress of plant cytochrome P450s[J].Pharmaceutical Biotechnology,2008,15(2):142-147.(in Chinese)
[28] 哈斯苏荣,李擎,玉斯日古楞,等.双峰驼CYP基因家族的比较研究[J].畜牧兽医学报,2015,46(7):1150-1156.
HASI S R,LI Q,YU S R G L,et al.Comparative study of CYP gene family in Bactrian camel (Camelus bactrianus)[J].Acta Veterinaria et Zootechnica Sinica,2015,46(7):1150-1156.(in Chinese)
[29] DE MELIS L E,WHITEMAN P H,STEVENSON T W.Isolation and characterisation of a cDNA clone encoding cinnamyl alcohol dehydrogenase in Eucalyptus globulus Labill[J].Plant Sci,1999,143(2):173-182.
[30] 徐建华,朱家勇.生物信息学在蛋白质结构与功能预测中的应用[J].医学分子生物学杂志,2005,2(3):227-232.
XU J H,ZHU J Y.Bioinformatics and its application on protein structure and function prediction[J].Journal of Medical Molecular Biology,2005,2(3):227-232.(in Chinese)
[31] MESSINA A,SINISCALCO A,PUCCINELLI E,et al.Cloning and tissues expression of the pig CYP1B1 and CYP2J34[J].Res Vet Sci,2012,92(3):438-443.
[32] WANG L,YAO J H,CHEN L,et al.Expression and possible functional roles of cytochromes P4502J1(zfCyp 2J1) in zebrafish[J].Biochem Biophys Res Commun,2006,352(4):850-855.
[33] YU A M,HAINING R L.Expression,purification,and characterization of mouse CYP2d22[J].Drug Metab Dispos,2006,34(7):1167-1174.
[34] GOTOH O.Substrate recognition sites in cytochrome P450 family 2(CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences[J].J Biol Chem,1992,267(1):83-90.
[35] YUDOWSKI G A,EFENDIEV R,PEDEMONTE C H,et al.Phosphoinositide-3 kinase binds to a proline-rich motif in the Na⁺,K⁺-ATPase α subunit and regulates its trafficking[J].Proc Natl Acad Sci U S A,2000,97(12):6556-6561.
[36] SAKAIRI Y,JACOBSON H R,NOLAND T D,et al.5,6-EET inhibits ion transport in collecting duct by stimulating endogenous prostaglandin synthesis[J].Am J Physiol,1995,268(5 Pt 2):931-939.

双峰驼CYP2J cDNA全长克隆、序列信息及组织表达分析

The Full-length Cloning, Sequence Information and Tissues Expression Analysis of CYP2J cDNA in Bactrian Camel

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

本文评价

[1]	方艳, 周俊文, 关伟军, 蒋琳, 浦亚斌, 赵倩君, 何晓红, 马月辉. 基于瘤胃转录组探究双峰驼沙漠适应性分子机制[J]. 畜牧兽医学报, 2021, 52(1): 77-87.
[2]	凌宇, 齐昱, 曹俊伟, 王申元, 周欢敏, 张焱如. 转录组揭示禁水环境下双峰驼结肠组织适应机制[J]. 畜牧兽医学报, 2018, 49(11): 2359-2370.