山羊IL-9基因克隆表达与单克隆抗体的制备
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摘要
为了研究捻转血矛线虫诱导山羊产生Th9型免疫应答的特点,制备了抗山羊IL-9单克隆抗体。采用基因克隆、表达和纯化得到重组蛋白IL-9,并以重组IL-9为免疫原,重组蛋白IL-9和His-tag为筛选抗原,通过杂交瘤细胞技术,筛出1株稳定分泌抗山羊IL-9单克隆抗体细胞株,命名为6A11A8。将该细胞株接种小鼠腹腔,制备腹水,并将纯化后的抗体与生物素耦合,用流式细胞术检测山羊外周血单核细胞(PBMCs)中表达IL-9的细胞亚类。结果显示:融合蛋白大小约为16 ku,主要分布于包涵体中,筛选出的6A11A8细胞株产生的抗体效价高、特异性强,其细胞上清抗体效价可达1∶2430,其抗体亚型为IgG2bκ,层析柱纯化小鼠腹水后其效价可达1∶512 000,该单抗可识别山羊PBMCs中分泌IL-9的亚类细胞。结论:本研究获得了山羊IL-9单克隆抗体,为研究山羊Th9型免疫反应奠定了基础。
In this study, monoclonal antibodies against goat IL-9 were prepared for further investigating Th9 immune response in goats induced by Haemonchus contortus. The recombinant protein pET30a(+)/IL-9 was obtained by gene cloning, expression and purification, and was used as the immunogen. Then, the recombinant protein and His-tag were used as screening antigens, and the cells were selected by hybridoma. A cell strain stably secreting the anti-goat IL-9 monoclonal antibody was designated as 6A11A8 whose cell line was administrated to BALB/c mice intraperitoneally, and purified by column chromatography. The antibody was bound with Biotin and was used in flow cytometry to track the IL-9 expressing cells in PBMCs of goats. The results were as follows: SDS-PAGE showed that the fusion protein was of 16 ku in size, and was induced and mainly distributed in the inclusion body. The cell line 6A11A8 was found with high specificity. The titer of the cell-culture supernatant was 1:2430. Furthermore, the subtype of the monoclonal antibody expressed by 6A11A8 was IgG2bκ. To obtain sufficient antibody, cell line 6A11A8 was administrated to BALB/c mice intraperitoneally, and the antibody titer reached 1:512000 after purification by column chromatography. The IL-9 expressing cells in goat PBMCs identified by the monoclonal antibody. To sum up, in this study, a monoclonal antibody against goat IL-9 was obtained, which laid the foundation for further research on Th9 immune response.
In this study, monoclonal antibodies against goat IL-9 were prepared for further investigating Th9 immune response in goats induced by Haemonchus contortus. The recombinant protein pET30a(+)/IL-9 was obtained by gene cloning, expression and purification, and was used as the immunogen. Then, the recombinant protein and His-tag were used as screening antigens, and the cells were selected by hybridoma. A cell strain stably secreting the anti-goat IL-9 monoclonal antibody was designated as 6A11A8 whose cell line was administrated to BALB/c mice intraperitoneally, and purified by column chromatography. The antibody was bound with Biotin and was used in flow cytometry to track the IL-9 expressing cells in PBMCs of goats. The results were as follows: SDS-PAGE showed that the fusion protein was of 16 ku in size, and was induced and mainly distributed in the inclusion body. The cell line 6A11A8 was found with high specificity. The titer of the cell-culture supernatant was 1:2430. Furthermore, the subtype of the monoclonal antibody expressed by 6A11A8 was IgG2bκ. To obtain sufficient antibody, cell line 6A11A8 was administrated to BALB/c mice intraperitoneally, and the antibody titer reached 1:512000 after purification by column chromatography. The IL-9 expressing cells in goat PBMCs identified by the monoclonal antibody. To sum up, in this study, a monoclonal antibody against goat IL-9 was obtained, which laid the foundation for further research on Th9 immune response.
引文
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[25] Angkasekwinai P,Srimanote P,Wang Y H,et al.Interleukin-25(IL-25) promotes efficient protective immunity against Trichinella spiralis infection by enhancing the antigen-specific IL-9 response[J].Infect Immun,2013,81(10):3731-3741.
[2] Neurath M F,Kaplan M H.Th9 cells in immunity and immunopathological diseases[J].Semin Immunopathol,2017,39(1):1-4.
[3] Kaplan M H.Th9 cells:differentiation and disease[J].Immunol Rev,2013,252(1):104-115.
[4] 路敏敏,金世禄.Th9/IL-9与炎性疾病研究进展[J].中华临床医师杂志(电子版),2015(2):300-303.
[5] Licona-Limon P,Arias-Rojas A,Olguin-Martinez E.IL-9 and Th9 in parasite immunity[J].Semin Immunopathol,2017,39(1):29-38.
[6] Sun B,Zhang Y.Overview of orchestration of CD4+ T cell subsets in immune responses[J].Adv Exp Med Biol,2014,841:1-13.
[7] 田洁,王胜军,许化溪.Th9细胞:一种新型效应性CD4+ T细胞亚群[J].细胞与分子免疫学杂志,2009(9):853-855.
[8] Jia L,Wu C.Differentiation,regulation and function of Th9 cells[J].Adv Exp Med Biol,2014,841:181-207.
[9] Kaplan M H,Hufford M M,Olson M R.The development and in vivo function of T helper 9 cells[J].Nat Rev Immunol,2015,15(5):295-307.
[10] Eller K,Wolf D,Huber J M,et al.IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression[J].J Immunol,2011,186(1):83-91.
[11] Stephens G L,Swerdlow B,Benjamin E,et al.IL-9 is a Th17-derived cytokine that limits pathogenic activity in organ-specific autoimmune disease[J].Eur J Immunol,2011,41(4):952-962.
[12] Wilhelm C,Hirota K,Stieglitz B,et al.An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation[J].Nat Immunol,2011,12(11):1071-1077.
[13] Jones T G,Hallgren J,Humbles A,et al.Antigen-induced increases in pulmonary mast cell progenitor numbers depend on IL-9 and CD1d-restricted NKT cells[J].J Immunol,2009,183(8):5251-5260.
[14] Russell S M,Johnston J A,Noguchi M,et al.Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3:implications for XSCID and XCID[J].Science,1994,266(5187):1042-1045.
[15] 程航,余为一.鸡CD4基因片段原核表达及其单抗制备[J].中国家禽,2011(8):22-24.
[16] 豆思远,豆玲,周峰,等.重组牛IFN-γ蛋白单克隆抗体的制备[J].畜牧兽医杂志,2017(6):9-11.
[17] 冯春燕,宋晓晖,仇松寅,等.非洲猪瘟病毒p54蛋白的表达及单抗制备[J].中国动物检疫,2016(5):80-84.
[18] 杨维维,许媛媛,荣超,等.山羊血管紧张素转换酶2基因的克隆表达与生物信息学分析[J].南京农业大学学报,2015(1):120-126.
[19] 郭玲玲,张晓磊,张进顺,等.刚地弓形虫ROP18基因的克隆及生物信息学分析[J].中国病原生物学杂志,2015(5):414-419.
[20] Licona-Limon P,Henao-Mejia J,Temann A U,et al.Th9 cells drive host immunity against gastrointestinal worm infection[J].Immunity,2013,39(4):744-757.
[21] Anuradha R,George P J,Hanna L E,et al.IL-4,TGF-beta,and IL-1-dependent expansion of parasite antigen-specific Th9 cells is associated with clinical pathology in human lymphatic filariasis[J].J Immunol,2013,191(5):2466-2473.
[22] Anuradha R,Munisankar S,Bhootra Y,et al.IL-10- and TGF-beta-mediated Th9 responses in a human helminth infection[J].PLoS Negl Trop Dis,2016,10(1):e4317.
[23] Anuradha R,Munisankar S,Bhootra Y,et al.Systemic cytokine profiles in Strongyloides stercoralis infection and alterations following treatment[J].Infect Immune,2016,84(2):425-431.
[24] Grencis R K,Hultner L,Else K J.Host protective immunity to Trichinella spiralis in mice:activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes[J].Immunology,1991,74(2):329-332.
[25] Angkasekwinai P,Srimanote P,Wang Y H,et al.Interleukin-25(IL-25) promotes efficient protective immunity against Trichinella spiralis infection by enhancing the antigen-specific IL-9 response[J].Infect Immun,2013,81(10):3731-3741.
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