牛Ⅰ型干扰素受体α链与其配体结合活性的鉴定

doi:10.16431/j.cnki.1671-7236.2018.09.034

摘要/Abstract

摘要：

为研究牛Ⅰ型干扰素受体α链（IFNAR1）与其配体结合的生物学性质，本研究采用RT-PCR方法从感染水疱性口炎病毒（vesicular stomatitis virus，VSV）的犊牛原代肾细胞中扩增得到IFNAR1基因，然后构建了表达BoIFNAR1胞外区多肽（BoIFNAR1-EC）的原核表达载体pET30a-BoIFNAR1-EC，在Rosetta^TM（DE3）pLysS宿主菌中表达出重组蛋白rBoIFNAR1-EC，并以纯化后的rBoIFNAR1-EC作为免疫原制备兔抗牛IFNAR1的多克隆抗体，随后鉴定BoIFNAR1与其配体的结合活性。结果表明，试验成功克隆得到1 685 bp的牛IFNAR1基因，编码560个氨基酸，由24个氨基酸组成的信号肽、414个氨基酸组成的胞外区、23个氨基酸组成的跨膜区及99个氨基酸组成的胞内区组成，与其他物种IFNAR1氨基酸序列同源性为63%~91%，在进化上具有高度保守性，其与羊的亲缘关系最近。随后构建了含牛IFNAR1胞外区基因的重组表达载体，并诱导表达了重组牛IFNAR1胞外区蛋白rBoIFNAR1-EC，其在大肠杆菌中几乎全部为可溶性表达，经纯化透析后作为免疫原制备了兔抗牛IFNAR1的特异性抗体，抗体效价高达1：204 800。配体结合试验表明，牛IFNAR1重组蛋白可与牛IFN-αA和IFN-ε结合，其多克隆抗体可阻断牛IFN-αA和IFN-ε与细胞表面的IFNAR1特异性结合，进而阻断牛IFN-αA和IFN-ε的抗病毒信号传导。

关键词: Ⅰ型干扰素受体α 链(IFNAR1); 重组蛋白; 多克隆抗体; 抗病毒活性

Abstract:

In order to study the biological properties between the bovine type Ⅰ interferon receptor alpha (IFNAR1) chain and its binding ligand, the bovine IFNAR1 (BoIFNAR1) was amplified by RT-PCR from primary kidney cells infected with vesicular stomatitis virus (VSV),and the recombinant plasmid pET30a-BoIFNAR1-EC was constructed to express the recombinant protein of BoIFNAR1 extracellular region (rIFNAR1-EC) in Rosetta^TM (DE3) pLysS.Besides,the polyclonal antibody (PAb) against BoIFNAR1 was prepared in rabbits with purified rBoIFNAR1-EC.Then the binding ability of bovine IFNAR1 to its ligands was identified.The results showed that the cloned CDS length of BoIFNAR1 gene was 1 685 bp.Moreover,the open reading frame of BoIFNAR1 encoded 560 amino acids,which consisted of one singal peptide encoded by 24 amino acids,one extracelluar region encoded by 414 amino acids,one transmembrane region encoded by 23 amino acids and one intracelluar region encoded by 99 amino acids.What's more,the homology of amino acid among BoIFNAR1 and other species ranges from 63% to 91%,indicating it was highly conserved in evolution,and especially close to sheep.And then a recombinant expression vector containing the extracellular region of BoIFNAR1 was constructed and the recombinant protein rBoIFNAR1-EC was induced.The results showed that rBoIFNAR1-EC was expressed in E.coli as a soluble form,which could be used as the immnuogen in preparing specific antibody against BoIFNAR1 after purification and dialysis.The titer of PAb obtained here was 1:204 800.Ligand binding experiment showed that rBoIFNAR1-EC could bind with bovine IFN-αA and IFN-ε.The PAb against BoIFNAR1 could block the specific binding area of bovine IFN-αA and IFN-ε to the cell surface IFNAR1, resulted in blocking the antiviral signal transduction of bovine IFN-αA and IFN-ε.

Key words: type Ⅰ interferon receptor alpha chain(IFNAR1); recombinant protein; polyclonal antibody; antiviral activity

中图分类号:

S852.4

王玉姣, 郭永丽, 罗修鑫, 高明春, 王君伟. 牛Ⅰ型干扰素受体α链与其配体结合活性的鉴定[J]. 《中国畜牧兽医》, 2018, 45(9): 2610-2619.

WANG Yujiao, GUO Yongli, LUO Xiuxin, GAO Mingchun, WANG Junwei. Identification of Ligand Binding Activity Between Bovine Type Ⅰ Interferon Receptor α Chain and Its Ligand[J]. , 2018, 45(9): 2610-2619.

参考文献

[1] IVASHKIV L B,DONLIN L T.Regulation of type Ⅰ interferon responses[J].Nature Reviews Immunology,2014,14(1):36.
[2] 马飞.干扰素的研究进展[J].中国畜牧兽医,2005,32(12):36-37.MA F.Research progress of interferon[J] China Animal Husbandry & Veterinary Medicine,2005,32(12):36-37.(in Chinese)
[3] UZÉ G,SCHREIBER G,PIEHLER J,et al.The receptor of the type Ⅰ interferon family[J]. Current Top Microbiology Immunology,2007,316:71-95.
[4] HO A S,LIU Y,KHAN T A,et al.A receptor for interleukin 10 is related to interferon receptors[J].Proceedings of the National Academy of Sciences,1993,90(23):11267-11271.
[5] BAZAN J F.Haemopoietic receptors and helical cytokines[J].Immunology Today,1990,11(10):350-354.
[6] MOGENSEN K E,LEWERENZ M,REBOUL J,et al.The typeⅠ interferon receptor:Structure,function, and evolution of a family business[J].Journal of Interferon and Cytokine Research,1999,19(10):1069-1098.
[7] KOTENKO S V,LANGER J A.Full house:12 receptors for 27 cytokines[J].International Immunopharmacology,2004,4(5):593-608.
[8] CRPZE E,RUSSEL-HARDE D,WAGNER T C,et al.The human type Ⅰ interferon receptor.Identification of the interferon beta-specific receptor-associated phosphoprotein[J].The Journal of Biological Chemistry,1996,271(52):33165-33168.
[9] NOVICK D,COHEN B,RUBINSTEIN M.The human interferon alpha/beta receptor:Characterization and molecular cloning[J].Cell,1994,77(3):391-400.
[10] HAN C S,MATHIALAGANA N,KLEMANN S W, et al.Molecular cloning of ovine and bovine typeⅠ interferon receptor subunits from uteri, and endometrial expression of messenger ribonucleic acid for ovine receptors during the estrous cycle and pregnancy[J]. Endocrinology,1997,138(11):4757-4767.
[11] CUTRONE E C,LANGER J A.Contributions of cloned typeⅠ interferon receptor subunits to differential ligand binding[J]. FEBS Letters,1997,404(2-3):197-202.
[12] LANGER J A,YANG J,CARMILLO P,et al.Bovine type Ⅰ interferon receptor protein BoIFNAR-1 has high-affinity and broad specificity for human type Ⅰ interferons[J]. FEBS Letters,1998,421(2):131-135.
[13] 崔子寅.具抗病毒活性无冗余氨基酸牛α干扰素(A亚型)表达策略研究[D].哈尔滨:东北农业大学,2012.CUI Z Y.Expression strategies research of antiviral activities and amino acid with no redundancy bovine interferon alpha A[D].Harbin:Northeast Agricultural University,2012.(in Chinese)
[14] GUO Y,GAO M,BAO J,et al.Molecular cloning and characterization of a novel bovine IFN-epsilon[J].Gene,2015,558(1):25-30.
[15] 王庭柱,郭永丽,高明春,等.牛IFN-ε基因克隆、表达与抗病毒活性分析[J].东北农业大学学报,2015, 46(12):39-44.WANG T Z,GUO Y L,GAO M C,et al.Cloning,expression and antiviral activity analysis of bovine IFN-ε[J].Journal of Northeast Agricultural University,2015,46(12):39-44.(in Chinese)
[16] PLATANIAS L C.Mechanisms of type-Ⅰ-and type-Ⅱ-interferon-mediated signalling[J]. Nature Reviews Immunolog,2005,5(5):375-386.
[17] 王建举,夏平安,尹彦涛,等.鸡干扰素的研究进展[J].中国畜牧兽医,2008,35(5):152-154.WANG J J,XIA P A,YIN Y T,et al.Research progress of chicken interferon[J].China Animal Husbandry & Veterinary Medicine,2008,35(5):152-154.(in Chinese)
[18] 段花玲,韩方正,程笑,等.Ⅰ型干扰素受体在慢性乙型肝炎中的研究进展[J].世界最新医学信息文摘,2016,18:47-48.DUAN H L,HAN F Z,CHENG X,et al.Research progress of type Ⅰ interferon receptor in chronic hepatitis B[J].World Latest Medicine Information,2016,18:47-48.(in Chinese)
[19] 宋舟,张立艳,董海兵,等.JAK-STAT信号通路研究进展[J].中国畜牧兽医,2012,39(6):128-132.SONG Z,ZHANG L Y,DONG H B,et al.Research progress of JAK-STAT signaling pathway[J].China Animal Husbandry & Veterinary Medicine,2012,39(6):128-132.(in Chinese)
[20] LIM J K,LANGER J A.Cloning and characterization of a bovine alpha interferon receptor[J].Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression,1993,1173(3):314-319.
[21] 杨青原,张军杰,秦运杰,等.猪Ⅰ型干扰素受体Ⅱ胞外域基因的克隆表达及其多克隆抗体制备[J].河南农业大学学报,2013,47(4):420-424.YANG Q Y,ZHANG J J,QIN Y J, et al.Cloning and expression of the porcine type Ⅰ interferon receptor Ⅱ extracellular domain gene preparation of polyclonal antibody[J].Journal of Henan Agricultural University,2013,47(4):420-424.(in Chinese)
[22] 胡向阳,杜远立,陈涛.核因子-κB受体及其配体信号通路在肿瘤中的表达及其研究进展[J].癌变·畸变·突变,2017,29(1):78-81.HU X Y,DU Y L,CHEN T.Nuclear factor-κB receptor and its ligand signaling pathways in tumors and their research progress[J].Carcinogenesis,Teratogenesis and Mutagenesis,2017,29(1):78-81.(in Chinese)
[23] 冯春花,岳锋.猪瘟病毒受体与配体研究进展[J].中国兽医杂志,2015,51(9):75-78.FENG C H,YUE F.Research progress on classical swine fever virus receptors and its ligands[J].Chinese Journal of Veterinary Medicine,2015,51(9):75-78.(in Chinese)
[24] DE WEERD N A,MATTHEWS A Y,PATTIE P R,et al.A hot spot on interferon alpha/beta receptor subunit 1(IFNAR1) underpins its interaction with interferon-beta and dictates signaling[J].The Journal of Biological Chemistry,2017,292(18):7554-7565.