羊IL-1Ra基因全长cDNA克隆及生物信息学分析
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摘要
试验旨在对羊白介素1受体颉颃因子(interleukin-1receptor antagonist,IL-1Ra)基因进行全长cDNA克隆及生物学分析。根据布鲁氏菌感染羊白细胞层SSH cDNA文库中的IL-1Ra基因序列信息及已知核苷酸序列(GenBank登录号:KC425613.1)设计引物,利用RT-PCR结合RACE方法扩增并克隆IL-1Ra基因,对其进行序列及相关分子特性分析。结果表明,羊IL-1Ra基因全长为1 228bp,可编码174个氨基酸,含有1个完整的IL-1保守结构域和1个IL-1Ra结构域(PHA02651结构域)。IL-1Ra分子质量为19 765.8u,等电点(pI)为5.72,其分子式为C_(88)5H_(1385)N_(235)O_(256)S_(11),且含有信号肽。二级结构分析显示,IL-1Ra分子存在较多的β折叠及受体结合位点,与三级结构预测结果一致,且与人/鼠IL-1Ra分子的空间结构相似度极高,达到90%以上。羊IL-1Ra有IL-1特有的三叶草结构,其酶结合结构域位于TYR~(47)至GLU~(66)之间。将羊IL-1Ra与牛、虎鲸、宽吻海豚、猪、家犬、人、鼠等14个物种进行蛋白质同源性比对,发现在各物种间存在5个高度同源的半胱氨酸位点。系统进化树分析发现,羊IL-1Ra基因全长cDNA所编码的氨基酸序列同山羊、牛单独形成一个分支。本研究结果为今后深入研究IL-1Ra基因的生物学功能奠定了基础。
This study was aimed to clone ovine interleukin-1 receptor antagonist(IL-1 Ra)gene and analyze the gene structure using bioinformatics.The primers was designed according to the obtained SSH cDNA library IL-1 Ra gene sequence information and the known nucleotide sequence(GenBank accession No.:KC425613.1),IL-1 Ragene was amplified and cloned by RT-PCR and RACE methods,IL-1 Ra sequence and its related molecular characteristics were analyzed.The results showed that the length of IL-1 Ragene was 1 228 bp,and encoded 174 amino acids.It was predicted that IL-1 Ra had a complete IL-1 conserved domain and IL-1 Ra domain(PHA02651 domain).The molecular weight of IL-1 Ra was 19 765.8 u,the isoelectric point(pI)was 5.72,itsmolecular was C_(885)H_(1385)N_(235)O_(256) S_(11). IL-1 Ra contained the signal peptide.The secondary structure of IL-1 Ra had moreβ-fold and receptor binding sites,which were consistent with the predicted results of tertiary structure,and had a similarity to the spatial structure of human/mouse IL-1 Ra,up to more than 90%.The IL-1 Ra contained a clover structure which was peculiar to the IL-1,and its enzyme-binding domain was located between TYR~(47) and GLU66.The results showed that there were five highly homologous cysteines between sheep and the other fourteen species.By establishing the phylogenetic tree of IL-1 Ra,it was found that the full-length cDNA of IL-1 Ra in sheep formed a branch with goat and cattle.This study result could provide the basis for the further study of the biological function of IL-1 Ragene.
This study was aimed to clone ovine interleukin-1 receptor antagonist(IL-1 Ra)gene and analyze the gene structure using bioinformatics.The primers was designed according to the obtained SSH cDNA library IL-1 Ra gene sequence information and the known nucleotide sequence(GenBank accession No.:KC425613.1),IL-1 Ragene was amplified and cloned by RT-PCR and RACE methods,IL-1 Ra sequence and its related molecular characteristics were analyzed.The results showed that the length of IL-1 Ragene was 1 228 bp,and encoded 174 amino acids.It was predicted that IL-1 Ra had a complete IL-1 conserved domain and IL-1 Ra domain(PHA02651 domain).The molecular weight of IL-1 Ra was 19 765.8 u,the isoelectric point(pI)was 5.72,itsmolecular was C_(885)H_(1385)N_(235)O_(256) S_(11). IL-1 Ra contained the signal peptide.The secondary structure of IL-1 Ra had moreβ-fold and receptor binding sites,which were consistent with the predicted results of tertiary structure,and had a similarity to the spatial structure of human/mouse IL-1 Ra,up to more than 90%.The IL-1 Ra contained a clover structure which was peculiar to the IL-1,and its enzyme-binding domain was located between TYR~(47) and GLU66.The results showed that there were five highly homologous cysteines between sheep and the other fourteen species.By establishing the phylogenetic tree of IL-1 Ra,it was found that the full-length cDNA of IL-1 Ra in sheep formed a branch with goat and cattle.This study result could provide the basis for the further study of the biological function of IL-1 Ragene.
引文
[1]张文华,江剑平.白介素-1β在炎性痛中的作用及其机制[J].生命科学,2010,22(3):291-295.ZHANG W H,JIANG J P.Roles and mechanisms of interleukin-1βduring inflammatory pain[J].Chinese Bulletin of Life Sciences,2010,22(3):291-295.(in Chinese)
[2]PRIESTLE J P,SCHAR H P,GRUTTER M G.Crystallographic refinement of interleukin 1 beta at2.0 A resolution[J].Proceedings of the National Academy of Sciences of the United States of America,1989,86(24):9667-9671.
[3]GRAVES B J,HATADA M H,HENDRICKSON W A,et al.Structure of interleukin 1alpha at 2.7-A resolution[J].Biochemistry,1990,29(11):2679-2684.
[4]AREND W P,JOSLIN F G,MASSONI R J.Effects of immune complexes on production by human monocytes of interleukin 1or an interleukin 1inhibitor[J].Journal of Immunology,1985,134(6):3868-3875.
[5]SECKINGER P,KAUFMANN M T,DAYER J M.An interleukin 1inhibitor affects both cell-associated interleukin 1-induced T cell proliferation and PGE2/collagenase production by human dermal fibroblasts and synovial cells[J].Immunobiology,1990,180(4-5):316-327.
[6]晏小艳,马燕红.白介素-1与肌腱挛缩发生的研究进展[J].中国康复医学杂志,2011,26(12):1182-1185.YAN X Y,MA Y H.Research progress of interleukin-1and tendon contracture[J].Chinese Journal of Rehabilitation Medicine,2011,26(12):1182-1185.(in Chinese)
[7]JACQUES C,GOSSET M,BERENBAUM F,et al.The role of IL-1and IL-1Ra in joint inflammation and cartilage degradation[J].Vitamins and Hormones,2006,74(1):371-403.
[8]MANSUR I G,SCHIAVON V,GIUSTINIANI J,et al.Engagement of IL-1receptor accessory protein(IL-1RAcP)with the monoclonal antibody AY19provides co-activating signals and prolongs the CD2-induced proliferation of peripheral blood lymphocytes[J].Immunology Letters,2011,139(1-2):52-57.
[9]DONG H K,CHO S,CHUNG Y H,et al.Expression and function of IL-1R complex governing human Th17responses(BA8P.167)[J].The Journal of Immunology,2015,194(1Suppl):116.
[10]AREND W P.Thebalance between IL-1and IL-1Ra in disease[J].Cytokine and Growth Factor Reviews,2002,13(4-5):323.
[11]HURME M,SANTTILA S.IL-1receptor antagonist(IL-1Ra)plasma levels are co-ordinately regulated by both I L-1Ra and IL-1 beta genes[J].European Journal of Immunology,2015,28(8):2598-2602.
[12]FISCHER E,MARANO M A,BARBER A E,et al.Comparison between effects of interleukin-1alpha administration and sublethal endotoxemia in primates[J].American Journal of Physiology,1991,261(2):442-452.
[13]AREND W P,WELGUS H G,THOMPSON R C,et al.Biological properties of recombinant human monocyte-derived interleukin 1receptor antagonist[J].Journal of Clinical Investigation,1990,85(5):1694-1697.
[14]丁先锋.IL-1及IL-1Ra在变应性接触性皮炎及异位性皮炎发生发展过程中的作用[J].世界最新医学信息文摘,2013,13(1):76-78.DING X F.The role of IL-1and IL-1Ra in the development of allergic contact dermatitis and atopic dermatitis[J].World Latest Medicine Information,2013,13(1):76-78.(in Chinese)
[15]HU J,LI H,CHI G,et al.IL-1RA gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules could alleviate rheumatoid arthritis[J].International Journal of Clinical and Experimental Medicine,2015,8(1):706-713.
[16]BRUS H,VAN D L M,TAAL E,et al.Compliance in rheumatoid arthritis and the roleof formal patient education[J].Seminars in Arthritis and Rheumatism,2017,26(4):702.
[17]刘楠楠.羊peroxiredoxin 6全长cDNA克隆、表达及ELISA检测方法的建立[D].长春:吉林大学,2013.LIU N N.Cloning and expression of full length cDNA of peroxiredoxin 6and establishment of ELISA[D].Changchun:Jilin University,2013.(in Chinese)
[18]李闯,徐云明,唐峰,等.小尾寒羊溶菌酶基因的克隆与差异表达分析[J].中国畜牧兽医,2013,40(3):19-22.LI C,XU Y M,TANG F,et al.Cloning and differential expression analysis of lysozyme gene in Small tail Han sheep[J].China Animal Husbandry&Veterinary Medicine,2013,40(3):19-22.(in Chinese)
[19]FERRARA-BOWENS T M,CHANDLER J K,GUIGNET M A,et al.Neuropathological and behavioral sequelae in IL-1R1and IL-1Ra gene knockout mice after soman(GD)exposure[J].Neurotoxicology,2017,63:43-56.
[20]PRINS M,ERIKSSON C,WIERINCKX A,et al.Interleukin-1βand interleukin-1receptor antagonist appear in grey matter additionally to white matter lesions during experimental multiple sclerosis[J].PLoS One,2013,8(12):e83835.
[21]NEDUMPUN T,WONGYANIN P,SIRISEREEWAN C,et al.Interleukin-1receptor antagonist(IL-1Ra):An early immunomodulatory cytokine induced by porcine reproductive and respiratory syndrome virus(PRRSV)[J].Journal of General Virology,2017,98(1):77-88.
[2]PRIESTLE J P,SCHAR H P,GRUTTER M G.Crystallographic refinement of interleukin 1 beta at2.0 A resolution[J].Proceedings of the National Academy of Sciences of the United States of America,1989,86(24):9667-9671.
[3]GRAVES B J,HATADA M H,HENDRICKSON W A,et al.Structure of interleukin 1alpha at 2.7-A resolution[J].Biochemistry,1990,29(11):2679-2684.
[4]AREND W P,JOSLIN F G,MASSONI R J.Effects of immune complexes on production by human monocytes of interleukin 1or an interleukin 1inhibitor[J].Journal of Immunology,1985,134(6):3868-3875.
[5]SECKINGER P,KAUFMANN M T,DAYER J M.An interleukin 1inhibitor affects both cell-associated interleukin 1-induced T cell proliferation and PGE2/collagenase production by human dermal fibroblasts and synovial cells[J].Immunobiology,1990,180(4-5):316-327.
[6]晏小艳,马燕红.白介素-1与肌腱挛缩发生的研究进展[J].中国康复医学杂志,2011,26(12):1182-1185.YAN X Y,MA Y H.Research progress of interleukin-1and tendon contracture[J].Chinese Journal of Rehabilitation Medicine,2011,26(12):1182-1185.(in Chinese)
[7]JACQUES C,GOSSET M,BERENBAUM F,et al.The role of IL-1and IL-1Ra in joint inflammation and cartilage degradation[J].Vitamins and Hormones,2006,74(1):371-403.
[8]MANSUR I G,SCHIAVON V,GIUSTINIANI J,et al.Engagement of IL-1receptor accessory protein(IL-1RAcP)with the monoclonal antibody AY19provides co-activating signals and prolongs the CD2-induced proliferation of peripheral blood lymphocytes[J].Immunology Letters,2011,139(1-2):52-57.
[9]DONG H K,CHO S,CHUNG Y H,et al.Expression and function of IL-1R complex governing human Th17responses(BA8P.167)[J].The Journal of Immunology,2015,194(1Suppl):116.
[10]AREND W P.Thebalance between IL-1and IL-1Ra in disease[J].Cytokine and Growth Factor Reviews,2002,13(4-5):323.
[11]HURME M,SANTTILA S.IL-1receptor antagonist(IL-1Ra)plasma levels are co-ordinately regulated by both I L-1Ra and IL-1 beta genes[J].European Journal of Immunology,2015,28(8):2598-2602.
[12]FISCHER E,MARANO M A,BARBER A E,et al.Comparison between effects of interleukin-1alpha administration and sublethal endotoxemia in primates[J].American Journal of Physiology,1991,261(2):442-452.
[13]AREND W P,WELGUS H G,THOMPSON R C,et al.Biological properties of recombinant human monocyte-derived interleukin 1receptor antagonist[J].Journal of Clinical Investigation,1990,85(5):1694-1697.
[14]丁先锋.IL-1及IL-1Ra在变应性接触性皮炎及异位性皮炎发生发展过程中的作用[J].世界最新医学信息文摘,2013,13(1):76-78.DING X F.The role of IL-1and IL-1Ra in the development of allergic contact dermatitis and atopic dermatitis[J].World Latest Medicine Information,2013,13(1):76-78.(in Chinese)
[15]HU J,LI H,CHI G,et al.IL-1RA gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules could alleviate rheumatoid arthritis[J].International Journal of Clinical and Experimental Medicine,2015,8(1):706-713.
[16]BRUS H,VAN D L M,TAAL E,et al.Compliance in rheumatoid arthritis and the roleof formal patient education[J].Seminars in Arthritis and Rheumatism,2017,26(4):702.
[17]刘楠楠.羊peroxiredoxin 6全长cDNA克隆、表达及ELISA检测方法的建立[D].长春:吉林大学,2013.LIU N N.Cloning and expression of full length cDNA of peroxiredoxin 6and establishment of ELISA[D].Changchun:Jilin University,2013.(in Chinese)
[18]李闯,徐云明,唐峰,等.小尾寒羊溶菌酶基因的克隆与差异表达分析[J].中国畜牧兽医,2013,40(3):19-22.LI C,XU Y M,TANG F,et al.Cloning and differential expression analysis of lysozyme gene in Small tail Han sheep[J].China Animal Husbandry&Veterinary Medicine,2013,40(3):19-22.(in Chinese)
[19]FERRARA-BOWENS T M,CHANDLER J K,GUIGNET M A,et al.Neuropathological and behavioral sequelae in IL-1R1and IL-1Ra gene knockout mice after soman(GD)exposure[J].Neurotoxicology,2017,63:43-56.
[20]PRINS M,ERIKSSON C,WIERINCKX A,et al.Interleukin-1βand interleukin-1receptor antagonist appear in grey matter additionally to white matter lesions during experimental multiple sclerosis[J].PLoS One,2013,8(12):e83835.
[21]NEDUMPUN T,WONGYANIN P,SIRISEREEWAN C,et al.Interleukin-1receptor antagonist(IL-1Ra):An early immunomodulatory cytokine induced by porcine reproductive and respiratory syndrome virus(PRRSV)[J].Journal of General Virology,2017,98(1):77-88.