贵州黑山羊、黔北麻羊IL-2基因的克隆与生物信息学分析
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摘要
为获得贵州特色品种黑山羊与黔北麻羊IL-2基因,本试验以Con A刺激的贵州黑山羊与黔北麻羊外周血淋巴细胞为材料,采用RT-PCR方法分别扩增贵州黑山羊与黔北麻羊IL-2基因,进行克隆与生物信息学分析。结果显示,(1)贵州黑山羊与黔北麻羊IL-2基因核苷酸与氨基酸一致性为100%,完整编码阅读框大小为468 bp,编码155个氨基酸;(2)黔北麻羊、贵州黑山羊IL-2基因与Gen Bank中羊源IL-2基因核苷酸一致性为95.9%~100%,氨基酸一致性为91.1%~100%;(3)遗传进化树结果表明,贵州黑山羊与黔北麻羊IL-2基因处于同一进化分支,与羊源IL-2基因亲缘关系较近;(4)生物信息学分析结果显示,IL-2蛋白二级结构中以α螺旋与无规则卷曲较多,该蛋白为亲水性蛋白,含信号肽序列,无跨膜结构,无特征功能结构域;(5)不同动物IL-2蛋白的抗原性分析存在差异,贵州黑山羊/黔北麻羊IL-2蛋白与羊源IL-2蛋白的比较差异较小,与人和其它动物的比较差异较大。由此得出结论,获得贵州黑山羊与黔北麻羊IL-2基因完整编码区序列,二者核苷酸与氨基酸序列一致。贵州黑山羊/黔北麻羊IL-2基因与羊源IL-2基因亲缘关系近,预测其编码的蛋白为亲水性的分泌型蛋白,不同动物IL-2基因抗原性存在差异。
To obtain the IL-2 gene of Black goat and Qianbei ma goat which were two native breed in Guizhou province,this research took the peripheral blood mononuclear cells as a material,which was from Black goat and Qianbei ma goat and stimulated by Con A.And we amplified the IL-2 gene respectively by RT-PCR,then cloning and bioinformatics analysis were carried out.The results showed that the IL-2 gene of Guizhou Black goat and Qianbei ma goat were the same at the nucleotide sequence and amino acid sequence,and its ORF was 468 bp,which encoded 155 amino acids.The nucleotide sequences similarity of two IL-2 genes with the IL-2 gene in Gen Bank were 95.9%~100%,and the amino sequences similarity were 91.1% ~100%.The phylogenetic tree showed that the IL-2 genes in Guizhou Black goat and Qianbei ma goat were from the same evolutionary branch,which were close with the IL-2 gene in Gen Bank.The bioinformatics analyses showed that theα-helix and random coil were major in the IL-2 protein secondary structure.And the protein was hydrophilic with signal peptide sequence,but no transmembrane structure and functional structure domain.Comparative analysis of antigenicity showed that there had difference of the IL-2 protein among different species.The IL-2 protein difference between Guizhou Black goat/Qianbei ma goat and other sheep derived were small,but the difference between Guizhou black goat/Qianbei ma goat and people or other animals was great.The IL-2 coding sequence of Guizhou Black goat and Qianbei ma goat were acquired,indicating the nucleotide and amino acid sequences were similar.They might have a close relationship with the IL-2 gene of sheep derived animals.The coding protein of IL-2 might be the secreting type and possess hydrophily,and the antigenicity of different animals existed difference.
To obtain the IL-2 gene of Black goat and Qianbei ma goat which were two native breed in Guizhou province,this research took the peripheral blood mononuclear cells as a material,which was from Black goat and Qianbei ma goat and stimulated by Con A.And we amplified the IL-2 gene respectively by RT-PCR,then cloning and bioinformatics analysis were carried out.The results showed that the IL-2 gene of Guizhou Black goat and Qianbei ma goat were the same at the nucleotide sequence and amino acid sequence,and its ORF was 468 bp,which encoded 155 amino acids.The nucleotide sequences similarity of two IL-2 genes with the IL-2 gene in Gen Bank were 95.9%~100%,and the amino sequences similarity were 91.1% ~100%.The phylogenetic tree showed that the IL-2 genes in Guizhou Black goat and Qianbei ma goat were from the same evolutionary branch,which were close with the IL-2 gene in Gen Bank.The bioinformatics analyses showed that theα-helix and random coil were major in the IL-2 protein secondary structure.And the protein was hydrophilic with signal peptide sequence,but no transmembrane structure and functional structure domain.Comparative analysis of antigenicity showed that there had difference of the IL-2 protein among different species.The IL-2 protein difference between Guizhou Black goat/Qianbei ma goat and other sheep derived were small,but the difference between Guizhou black goat/Qianbei ma goat and people or other animals was great.The IL-2 coding sequence of Guizhou Black goat and Qianbei ma goat were acquired,indicating the nucleotide and amino acid sequences were similar.They might have a close relationship with the IL-2 gene of sheep derived animals.The coding protein of IL-2 might be the secreting type and possess hydrophily,and the antigenicity of different animals existed difference.
引文
Antony G.K.,and Dudek A.Z.,2010,Interleukin 2 in cancer therapy,Current Medicinal Chemistry,17(29):3297-3302
Dai Z.J.,Zhou L.,Zeng Z.Y.,Tang D.Y.,Liang H.Y.,Liu Z.,and Wang W.C.,2015,Molecular cloning and bioinformatics analysis of IL-2,IL-4 and IL-6 genes of Guizhou Baixiang Pig,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(5):931-937(代振江,周莉,曾智勇,汤德元,梁海英,刘钊,王伟丞,2015,贵州白香猪IL-2,IL-4和IL-6基因的克隆及生物信息学分析,基因组学与应用生物学,34(5):931-937)
Goodall J.C.,Emery D.C.,Perry A.C.F.,English L.S.,and Hall L.,1990,c DNA cloning of ovine interleukin 2 by PCR,Nucleic Acids Research,18(19):5883
Hai G.,Liu S.W.,Han Z.X.,and Chen H.Y.,2007,Expression of caprine interleukin-2 and preparation of rabbit antisera against caprine interleukin-2,Zhongguo Yufang Shouyi Xuebao(Chinese Journal of Preventive Veterinary Medicine),29(9):685-689(海岗,刘胜旺,韩宗玺,陈洪岩,2007,山羊IL-2的体外表达及其多克隆抗血清的制备,中国预防兽医学报,29(9):685-689)
Jiang T.,Zhou C.C.,and Ren S.X.,2016,Role of IL-2 in cancer immunotherapy,Oncoimmunology,5(6):e1163462
Jin B.,2007,Cloning and prokaryotic expression of caprine interferon,interleukin-2 and preparation of their antiserum,Northeast Agriculture University,Supervisor:Chen H.Y.,pp.18-20(金标,2007,山羊干扰素-酌与白细胞介素-2的克隆,原核表达及其抗血清的制备,硕士学位论文,东北农业大学,导师:陈洪岩,pp.18-20)
Seow H.F.,Rothel J.S.,Radford A.J.,and Wood P.R.,1990,The molecular cloning of ovine interleukin 2 gene by the polymerase chain reaction,Nucleic Acids Research,18(23):7175
Sha W.L.,Yang G.L.,and Wang C.F.,2013,Progress on the application of interleukin-2 in the prevention and control of animal diseases,Zhongguo Shouyi Zazhi(Chinese Journal of Veterinary Drug),47(4):58-61(沙万里,杨桂连,王春凤,2013,白细胞介素-2在动物疾病防治中的应用进展,中国兽医杂志,47(4):58-61)
Tovey M.G.,and Lallemand C.,2010,Adjuvant activity of cytokines,Humana Press,626:287-309
Ying Q.H.,Li X.R.,and Lu C.P.,2002,Cloning,Identification and sequence analysis of the caprine interleukin-2(IL-2)Gene,Nongye Shengwu Jishu Xuebao(Journal of Agriculature Biotechnology),10(3):250-254(应琦华,李祥瑞,陆承平,2002,山羊IL-2基因c DNA的克隆,鉴定及序列分析,农业生物技术学报,10(3):250-254)
Zhao D.L.,and Shan F.P.,2013,The latest research progress pf Interleukin-2,Weishengwuxue Zazhi(Journal of Microbiology),33(4):77-83(赵定亮,单风平,2013,白细胞介素-2最新研究进展,微生物学杂志,33(4):77-83)
Dai Z.J.,Zhou L.,Zeng Z.Y.,Tang D.Y.,Liang H.Y.,Liu Z.,and Wang W.C.,2015,Molecular cloning and bioinformatics analysis of IL-2,IL-4 and IL-6 genes of Guizhou Baixiang Pig,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(5):931-937(代振江,周莉,曾智勇,汤德元,梁海英,刘钊,王伟丞,2015,贵州白香猪IL-2,IL-4和IL-6基因的克隆及生物信息学分析,基因组学与应用生物学,34(5):931-937)
Goodall J.C.,Emery D.C.,Perry A.C.F.,English L.S.,and Hall L.,1990,c DNA cloning of ovine interleukin 2 by PCR,Nucleic Acids Research,18(19):5883
Hai G.,Liu S.W.,Han Z.X.,and Chen H.Y.,2007,Expression of caprine interleukin-2 and preparation of rabbit antisera against caprine interleukin-2,Zhongguo Yufang Shouyi Xuebao(Chinese Journal of Preventive Veterinary Medicine),29(9):685-689(海岗,刘胜旺,韩宗玺,陈洪岩,2007,山羊IL-2的体外表达及其多克隆抗血清的制备,中国预防兽医学报,29(9):685-689)
Jiang T.,Zhou C.C.,and Ren S.X.,2016,Role of IL-2 in cancer immunotherapy,Oncoimmunology,5(6):e1163462
Jin B.,2007,Cloning and prokaryotic expression of caprine interferon,interleukin-2 and preparation of their antiserum,Northeast Agriculture University,Supervisor:Chen H.Y.,pp.18-20(金标,2007,山羊干扰素-酌与白细胞介素-2的克隆,原核表达及其抗血清的制备,硕士学位论文,东北农业大学,导师:陈洪岩,pp.18-20)
Seow H.F.,Rothel J.S.,Radford A.J.,and Wood P.R.,1990,The molecular cloning of ovine interleukin 2 gene by the polymerase chain reaction,Nucleic Acids Research,18(23):7175
Sha W.L.,Yang G.L.,and Wang C.F.,2013,Progress on the application of interleukin-2 in the prevention and control of animal diseases,Zhongguo Shouyi Zazhi(Chinese Journal of Veterinary Drug),47(4):58-61(沙万里,杨桂连,王春凤,2013,白细胞介素-2在动物疾病防治中的应用进展,中国兽医杂志,47(4):58-61)
Tovey M.G.,and Lallemand C.,2010,Adjuvant activity of cytokines,Humana Press,626:287-309
Ying Q.H.,Li X.R.,and Lu C.P.,2002,Cloning,Identification and sequence analysis of the caprine interleukin-2(IL-2)Gene,Nongye Shengwu Jishu Xuebao(Journal of Agriculature Biotechnology),10(3):250-254(应琦华,李祥瑞,陆承平,2002,山羊IL-2基因c DNA的克隆,鉴定及序列分析,农业生物技术学报,10(3):250-254)
Zhao D.L.,and Shan F.P.,2013,The latest research progress pf Interleukin-2,Weishengwuxue Zazhi(Journal of Microbiology),33(4):77-83(赵定亮,单风平,2013,白细胞介素-2最新研究进展,微生物学杂志,33(4):77-83)
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