抗鸡白细胞介素4单克隆抗体的制备及鉴定
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摘要
为了制备鸡白细胞介素4(chIL-4)单克隆抗体,将成熟的chIL-4基因亚克隆至原核表达载体pET-28a和pGEX-6P-1上,然后在大肠杆菌中分别诱导重组蛋白His-chIL-4和GST-chIL-4的表达,并纯化。将纯化后的His-chIL-4作为免疫原免疫BALB/c小鼠,经4次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。将纯化后的GST-chIL-4作为筛选抗原,利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后,获得3株稳定分泌抗chIL-4蛋白的杂交瘤细胞株,分别命名为1G11-3B、2E5-3D和1G11-5H。经ELISA检测,3株单克隆抗体的亚型均为IgG1,亲和力解离常数(Kd)分别为1.79×10~(–9)、1.61×10~(–9)和2.36×10~(–9)。经Western blotting及间接免疫荧光试验鉴定,3株单克隆抗体均能特异性识别原核和真核表达的chIL-4蛋白。Western blotting试验证明1G11-3B、2E5-3D和1G11-5H识别的抗原表位区域分别为chIL-4蛋白N端的第1–40、80–112和40–80位氨基酸。该单克隆抗体的制备为chIL-4的检测和生物学功能研究奠定了基础。
To develop monoclonal antibodies(McAbs) against chicken interleukin 4(chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28 a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas th at stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant(Kd) of these McAbs were 1.79×10~(–9), 1.61×10~(–9), and 2.36×10~(–9), respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1–40, 80–112, and 40–80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.
To develop monoclonal antibodies(McAbs) against chicken interleukin 4(chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28 a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas th at stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant(Kd) of these McAbs were 1.79×10~(–9), 1.61×10~(–9), and 2.36×10~(–9), respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1–40, 80–112, and 40–80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.
引文
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[3]Kubota T,Yokayama M,Kishima M,et al.Gene expression of chicken interleukine-4 by baculovirus.Vet Immunol Immunopathol,2009,128(1/3):309.
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[8]Sun BB.Identification and crystal structure of MDV CTL epitope presenting by chicken B15 and study of its immune protection[D].Beijing:China Agricultural University,2013(in Chinese).孙贝贝.鸡B15 MDV CTL表位的鉴定与晶体结构解析及其免疫保护效果研究[D].北京:中国农业大学,2013.
[9]Luo Z,Liu RC,Zheng SJ.Prokaryotic expression of Listeria monocytogene(LM)hly and development of monoclonal antibodies against listeriolysin O(LLO).Chin J Biotech,2009,25(11):1652-1657(in Chinese).罗正,刘若尘,郑世军.单核增生性李氏杆菌溶血素的原核表达及其单克隆抗体的制备.生物工程学报,2009,25(11):1652-1657.
[10]Yokoyama WM.Current Protocols in Immunology.New York:John Wiley&Sonc,Inc,1995.
[11]Li SJ.Development and application of monoclonal antibodies against the IBDV VP2 and VP5 proteins[D].Beijing:China Agricultural University,2013(in Chinese).李善吉.传染性法氏囊病毒VP2和VP5单克隆抗体的制备鉴定及应用[D].北京:中国农业大学,2013.
[12]Liddell JE,Cryer A.A Practical Guide to Monoclonal Antibodies.New York:John Wiley&Sonc,Inc,1991:110-115.
[13]Seder RA,Paul WE,Davis MM,et al.The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+Tcells from T cell receptor transgenic mice.J Exp Med,1992,176(4):1091-1098.
[14]Yokota T,Arai N,de Vries J,et al.Molecular biology of interleukin-4 and interleukin-5 genes and biology of their products that stimulate B cells,T cells and hemopoietic cells.Immunol Rev,1988,102:137-187.
[15]Estes DM,Hirano A,Heussler VT,et al.Expression and biological activities of bovine interleukin 4:effects of recombinant bovine interleukin-4 on T cell proliferation and B cell differentiation and proliferation in vitro.Cell Immunol,1995,163(2):268-279.
[16]Noelle R,Krammer PH,Ohara J,et al.Increased expression of Ia antigens on resting B cells:an additional role for B-cell growth factor.Proc Natl Acad Sci USA,1984,81(19):6149-6153.
[17]Defrance T,Aubry JP,Rousset F,et al.Human recombinant interleukin-4 induces Fc epsilon receptors(CD23)on normal human B lymphocytes.J Exp Med,1987,165(6):1459-1467.
[18]Ohara J,Paul WE.Up-regulation of interleukin4/B-cell stimulatory factor 1 receptor expression.Proc Natl Acad Sci U S A,1988,85(21):8221-8225.
[19]Hu-Li J,Shevach EM,Mizuguchi J,et al.B cell stimulatory factor 1(interleukin-4)is a potent costimulant for normal resting T lymphocytes.JExp Med,1987,165(1):157-172.
[20]Romani L,Mencacci A,Grohmann U,et al.Neutralizing antibody to interleukin-4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis.J Exp Med,1992,176(1):19-25.
[21]Puccetti P,Mencacci A,Cenci E,et al.Cure of murine candidiasis by recombinant soluble interleukin-4 receptor.J Infect Dis,1994,169(6):1325-1331.
[22]Kopf M,Le Gros G,Bachmann M,et al.Disruption of the murine IL-4 gene blocks Th2 cytokine responses.Nature,1993,362(6417):245-248.
[23]Bogdan C,Nathan C.Modulation of macrophage function by transforming growth factor beta,interleukin-4,and interleukin-10.Ann N Y Acad Sci,1993,685:713-739.
[24]Thornhill MH,Wellicome SM,Mahiouz DL,et al.Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells.The contribution of vascular cell adhesion molecule-1-dependent andindependent binding mechanisms.J Immunol,1991,146(2):592-598.
[25]Bennett BL,Cruz R,Lacson RG,et al.Interleukin-4 suppression of tumor necrosis factorα-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-κB.J Biol Chem,1997,272(15):10212-10219.
[26]Wondimu A,Veit M,Kohn B,et al.Molecular cloning,expression and characterization of the Canis familiaris interleukin-4.Cytokine,2001,16(3):88-92.
[27]Nuntaprasert A,Mori Y,Fujita K,et al.Expression and purification of recombinant swine interleukin-4.Comp Immunol Microbiol Infect Dis,2005,28(1):17-35.
[28]Chen GH,Jing ZZ,Sun SD.Cloning and expression of porcine IL-4 gene.Chin J Prev Veter Med,2005,27(6):469-473(in Chinese).陈国华,景志忠,孙世铎.猪白细胞介素-4基因的克隆与表达.中国预防兽医学报,2005,27(6):469-473.
[29]Cottrez F,Auriault C,Capron A,et al.Murine interleukin-4 production with baculovirus:an easy and rapid method for a small scale production of functional interleukins.Eur Cytokine Netw,1994,5(5):481-487.
[30]Jayaram B,Devos R,Guisez Y,et al.Purification of human interleukin-4 produced in Escherichia coli.Gene,1989,79(2):345-354.
[2]Avery S,Rothwell L,Degen WD,et al.Characterization of the first nonmammalian T2cytokine gene cluster:the cluster contains functional single-copy genes for IL-3,IL-4,IL-13,and GM-CSF,a gene for IL-5 that appears to be a pseudogene,and a gene encoding another cytokine-like transcript,KK34.J Interferon Cytokine Res,2004,24(10):600-610.
[3]Kubota T,Yokayama M,Kishima M,et al.Gene expression of chicken interleukine-4 by baculovirus.Vet Immunol Immunopathol,2009,128(1/3):309.
[4]Dai H,Zheng JY,Chen JH,et al.Cloning and expression of chicken interleukin-4 gene.Chin JPrev Veter Med,2008,30(6):473-477(in Chinese).戴华,郑佳玉,陈俊华,等.鸡白细胞介素4基因的克隆及其原核表达.中国预防兽医学报,2008,30(6):473-477.
[5]Weng L,Zhang LJ,Zhang QB,et al.Development of a sensitive sandwich ELISA to chicken interleukin-2(Ch IL-2).J Xiamen Univ:Nat Sci,2006,45(S1):142-145(in Chinese).翁凌,张凌晶,张其标,等.抗体夹心酶联免疫吸附法测定鸡白细胞介素-2.厦门大学学报:自然科学版,2006,45(S1):142-145.
[6]Wang XY.Development and preliminary application of double sandwich ELISA for detecting chicken interleukin-18[D].Tai’an:Shandong Agricultural University,2013(in Chinese).王肖祎.鸡白细胞介素18双抗体夹心ELISA检测方法的建立及初步应用[D].泰安:山东农业大学,2013.
[7]Zheng YS.Effects of immunosuppressive virus infection on interferon-gamma production in the splenic leukocytes of SPF chickens[D].Tai’an:Shandong Agricultural University,2006(in Chinese).郑玉姝.免疫抑制性病毒感染对SPF鸡脾细胞IFN-γ产生的影响[D].泰安:山东农业大学,2006.
[8]Sun BB.Identification and crystal structure of MDV CTL epitope presenting by chicken B15 and study of its immune protection[D].Beijing:China Agricultural University,2013(in Chinese).孙贝贝.鸡B15 MDV CTL表位的鉴定与晶体结构解析及其免疫保护效果研究[D].北京:中国农业大学,2013.
[9]Luo Z,Liu RC,Zheng SJ.Prokaryotic expression of Listeria monocytogene(LM)hly and development of monoclonal antibodies against listeriolysin O(LLO).Chin J Biotech,2009,25(11):1652-1657(in Chinese).罗正,刘若尘,郑世军.单核增生性李氏杆菌溶血素的原核表达及其单克隆抗体的制备.生物工程学报,2009,25(11):1652-1657.
[10]Yokoyama WM.Current Protocols in Immunology.New York:John Wiley&Sonc,Inc,1995.
[11]Li SJ.Development and application of monoclonal antibodies against the IBDV VP2 and VP5 proteins[D].Beijing:China Agricultural University,2013(in Chinese).李善吉.传染性法氏囊病毒VP2和VP5单克隆抗体的制备鉴定及应用[D].北京:中国农业大学,2013.
[12]Liddell JE,Cryer A.A Practical Guide to Monoclonal Antibodies.New York:John Wiley&Sonc,Inc,1991:110-115.
[13]Seder RA,Paul WE,Davis MM,et al.The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+Tcells from T cell receptor transgenic mice.J Exp Med,1992,176(4):1091-1098.
[14]Yokota T,Arai N,de Vries J,et al.Molecular biology of interleukin-4 and interleukin-5 genes and biology of their products that stimulate B cells,T cells and hemopoietic cells.Immunol Rev,1988,102:137-187.
[15]Estes DM,Hirano A,Heussler VT,et al.Expression and biological activities of bovine interleukin 4:effects of recombinant bovine interleukin-4 on T cell proliferation and B cell differentiation and proliferation in vitro.Cell Immunol,1995,163(2):268-279.
[16]Noelle R,Krammer PH,Ohara J,et al.Increased expression of Ia antigens on resting B cells:an additional role for B-cell growth factor.Proc Natl Acad Sci USA,1984,81(19):6149-6153.
[17]Defrance T,Aubry JP,Rousset F,et al.Human recombinant interleukin-4 induces Fc epsilon receptors(CD23)on normal human B lymphocytes.J Exp Med,1987,165(6):1459-1467.
[18]Ohara J,Paul WE.Up-regulation of interleukin4/B-cell stimulatory factor 1 receptor expression.Proc Natl Acad Sci U S A,1988,85(21):8221-8225.
[19]Hu-Li J,Shevach EM,Mizuguchi J,et al.B cell stimulatory factor 1(interleukin-4)is a potent costimulant for normal resting T lymphocytes.JExp Med,1987,165(1):157-172.
[20]Romani L,Mencacci A,Grohmann U,et al.Neutralizing antibody to interleukin-4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis.J Exp Med,1992,176(1):19-25.
[21]Puccetti P,Mencacci A,Cenci E,et al.Cure of murine candidiasis by recombinant soluble interleukin-4 receptor.J Infect Dis,1994,169(6):1325-1331.
[22]Kopf M,Le Gros G,Bachmann M,et al.Disruption of the murine IL-4 gene blocks Th2 cytokine responses.Nature,1993,362(6417):245-248.
[23]Bogdan C,Nathan C.Modulation of macrophage function by transforming growth factor beta,interleukin-4,and interleukin-10.Ann N Y Acad Sci,1993,685:713-739.
[24]Thornhill MH,Wellicome SM,Mahiouz DL,et al.Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells.The contribution of vascular cell adhesion molecule-1-dependent andindependent binding mechanisms.J Immunol,1991,146(2):592-598.
[25]Bennett BL,Cruz R,Lacson RG,et al.Interleukin-4 suppression of tumor necrosis factorα-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-κB.J Biol Chem,1997,272(15):10212-10219.
[26]Wondimu A,Veit M,Kohn B,et al.Molecular cloning,expression and characterization of the Canis familiaris interleukin-4.Cytokine,2001,16(3):88-92.
[27]Nuntaprasert A,Mori Y,Fujita K,et al.Expression and purification of recombinant swine interleukin-4.Comp Immunol Microbiol Infect Dis,2005,28(1):17-35.
[28]Chen GH,Jing ZZ,Sun SD.Cloning and expression of porcine IL-4 gene.Chin J Prev Veter Med,2005,27(6):469-473(in Chinese).陈国华,景志忠,孙世铎.猪白细胞介素-4基因的克隆与表达.中国预防兽医学报,2005,27(6):469-473.
[29]Cottrez F,Auriault C,Capron A,et al.Murine interleukin-4 production with baculovirus:an easy and rapid method for a small scale production of functional interleukins.Eur Cytokine Netw,1994,5(5):481-487.
[30]Jayaram B,Devos R,Guisez Y,et al.Purification of human interleukin-4 produced in Escherichia coli.Gene,1989,79(2):345-354.