牛IgG2 Fc受体的真核表达、纯化与结晶
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摘要
【目的】制备牛IgG2 Fc受体(boFcγ2R)蛋白并分析其结构与生物学功能,为进一步研究其免疫学功能奠定基础。【方法】PCR扩增boFcγ2R的胞外区基因片段,将其克隆至真核表达载体pMT/BiP/V5-His A构建重组pMT/BiP-boFcγ2R-His质粒,利用果蝇胚胎Drosophila Schneider 2(S2)细胞表达boFcγ2R胞外区重组蛋白,通过镍柱亲和层析、凝胶过滤层析对目的蛋白进行纯化,利用Dot-blot验证其生物学功能;采用蒸汽扩散坐滴法对目的蛋白进行结晶,对晶体进行X-ray衍射分析;利用去糖基化酶Endo Hf和PNGase F对目的蛋白进行处理,以验证其糖基化修饰与晶体无衍射之间是否存在因果关系。【结果】PCR扩增获得了663 bp的boFcγ2R基因胞外区片段。成功构建了pMT/BiP-boFcγ2R-His载体,将其转染S2细胞后诱导表达出26 ku的boFcγ2R胞外区重组蛋白。SDS-PAGE结果显示,通过镍柱亲和层析、凝胶过滤层析的纯化方法得到了高纯度的boFcγ2R胞外区重组蛋白;Dot-blot结果显示,该蛋白具有较好的生物学功能。蛋白结晶的2个条件是:体积分数15%Tacsimate~(TM)(pH 7.0)、0.1 mol/L HEPES (pH 7.0)、20 g/L PEG 3350和0.1 mol/L HEPES (pH 7.5)、250 g/L PEG 3350。蛋白初筛晶体无衍射,去糖基化试验验证了boFcγ2R胞外区蛋白初筛晶体无衍射可能与其糖基化有关的推测。【结论】得到了高纯度的boFcγ2R胞外区重组蛋白,获得其初筛晶体。
【Objective】 This study prepared cattle IgG2 Fc receptor proteins and analyzed its structure and immunological function to provide basis for further research on its immunological function.【Method】 The synthetic cDNA encoding the ectodomain of bovine IgG2 Fc receptor(boFcγ2R) was cloned into pMT/BiP/V5-HisA to construct the eukaryotic expression plasmid.The recombinant boFcγ2R ectodomain was expressed in Drosophila Schneider 2(S2) cells,purified through Ni-chelating affinity and gel filtration chromatography,assayed with Dot-blot,and crystalized by the sitting-drop vapor diffusion method.The crystals were detected by X-ray diffraction.The deglycosylation enzymes Endo Hf and PNGase F was used to verify the relationship between glycosylation modification and crystal non-diffraction.【Result】 The 663 bp boFcγ2R ectodomain fragment was obtained by PCR amplification.The pMT/BiP-boFcγ2R-His vector was successfully constructed and transfected into S2 cells to induce the expression of the 26 ku recombinant boFcγ2R ectodomain.The SDS-PAGE results showed that the purity of the recombinant boFcγ2R ectodomain was up to 99% through Ni-chelating affinity and gel filtration chromatography.Dot-blot showed the boFcγ2R ectodomain had great biological function.The two conditions for protein crystallization were volume fraction of 15% TacsimateTM(pH 7.0),0.1 mol/L HEPES(pH 7.0),and 20 g/L PEG 3 350 and 0.1 mol/L HEPES(pH 7.5),and 250 g/L PEG 3350 conditions by sitting-drop vapor diffusion method.The primary screening crystals were not diffracted,and the deglycosylation test verified that the non-diffraction of the primary screening crystals of the boFcγ2R ectodomain may be related to its glycosylation.【Conclusion】 High-purity recombinant protein of the boFcγ2R ectodomain and its primary screening crystals were obtained.
【Objective】 This study prepared cattle IgG2 Fc receptor proteins and analyzed its structure and immunological function to provide basis for further research on its immunological function.【Method】 The synthetic cDNA encoding the ectodomain of bovine IgG2 Fc receptor(boFcγ2R) was cloned into pMT/BiP/V5-HisA to construct the eukaryotic expression plasmid.The recombinant boFcγ2R ectodomain was expressed in Drosophila Schneider 2(S2) cells,purified through Ni-chelating affinity and gel filtration chromatography,assayed with Dot-blot,and crystalized by the sitting-drop vapor diffusion method.The crystals were detected by X-ray diffraction.The deglycosylation enzymes Endo Hf and PNGase F was used to verify the relationship between glycosylation modification and crystal non-diffraction.【Result】 The 663 bp boFcγ2R ectodomain fragment was obtained by PCR amplification.The pMT/BiP-boFcγ2R-His vector was successfully constructed and transfected into S2 cells to induce the expression of the 26 ku recombinant boFcγ2R ectodomain.The SDS-PAGE results showed that the purity of the recombinant boFcγ2R ectodomain was up to 99% through Ni-chelating affinity and gel filtration chromatography.Dot-blot showed the boFcγ2R ectodomain had great biological function.The two conditions for protein crystallization were volume fraction of 15% TacsimateTM(pH 7.0),0.1 mol/L HEPES(pH 7.0),and 20 g/L PEG 3 350 and 0.1 mol/L HEPES(pH 7.5),and 250 g/L PEG 3350 conditions by sitting-drop vapor diffusion method.The primary screening crystals were not diffracted,and the deglycosylation test verified that the non-diffraction of the primary screening crystals of the boFcγ2R ectodomain may be related to its glycosylation.【Conclusion】 High-purity recombinant protein of the boFcγ2R ectodomain and its primary screening crystals were obtained.
引文
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[4] Ravetch J V,Bolland,S.IgG Fc receptors [J].Annu Rev Immunol,2001,19:275-290.
[5] Nimmerjahn F,Ravetch J V.Fcgamma receptors as regulators of immune responses [J].Nature Reviews Immunology,2008,8(1):34-47.
[6] Daeron M.Fc receptor biology [J].Annu Rev immunol,1997,15(15):203-234.
[7] Deo Y M,Graziano R F,Repp R,et al.Clinical significance of IgG Fc receptors and FcγR-directed immunotherapies [J].Immunol Today,1997,18(3):127-135.
[8] Wang G,Chopra R K,Royal R E,et al.A T cell-independent antitumor response in mice with bone marrow cells retrovirally transduced with an antibody/Fc-γ chain chimeric receptor gene recognizing a human ovarian cancer antigen [J].Nature Medicine,1998,4(2):168.
[9] Jg V D W,Capel P J.Human IgG Fc receptor heterogeneity:molecular aspects and clinical implications [J].Immunology Today,1993,14(5):215-221.
[10] Kcskovics I.Fc receptors in livestock species [J].Veterinary Immunology & Immunopathology,2004,102(4):351-362.
[11] Zhang G,Young J R,Tregaskes C A,et al.Identification of a novel class of mammalian Fcreceptor [J].J Immunol,1995,155:1534-1541.
[12] Duncan A R,Woof J M,Partridge L J,et al.Localization of the binding site for the human high-affinity Fc receptor on IgG [J].Nature,1988,332(6164):563-564.
[13] Qiao S,Yang Y,Liu Y,et al.Characterization and ligand specificity of sheep IgG2 receptor [J].Immunogenetics,2009,61(8):597-601.
[14] Morton H C,Howard C J,Storset A K,et al.Identification of residues within the extracellular domain 1 of bovine Fcc2R essential for binding bovine IgG2 [J].J Biol Chem,2001,276:47794-47800.
[15] Morton H C,van Zandbergen G,van Kooten C,et al.Immuno-globulin-binding sites of human FcaRI (CD89) and bovine Fcc2Rare located in their membrane-distal extracellular domains [J].J Exp Med,1999,189:1715-1722.
[16] Zhang G,Guo J Q,Zhou J Y.Identification of the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcγ2R) using synthetic peptides [J].FEBS Letters,2006,580:1383-1390.
[17] Sondemann P,Huber R,Oosthuizen V,et al.The 3.2-A° crystal structure of the human IgG1 Fc fragment-FccRIII complex [J].Nature,2000,406:267-273.
[18] Burmeister W P,Huber A H,Bjorkman P J.Crystal structure of the complex of rat neonatal Fc receptor with Fc [J].Nature,1994,372(6504):379-383.
[19] 郭军庆.牛IgG Fc受体线性配体结合表位鉴定 [D].杭州:浙江大学,2005.Guo J Q.Identification of linear ligand binding epitopes of bovine IgG Fc receptor [D].Hangzhou:Zhejiang University,2005.
[20] 侯婕,李睿,马红芳,等.猪繁殖与呼吸综合征病毒入侵受体唾液酸黏附素的表达与纯化 [J].河南农业科学,2016,45(3):130-134.Hou J,Li R,Ma H F,et al.Expression and purification of Sialoadhesin,a putative receptor of Procine Reproduction and Respiratory Syndrom Virus [J].Henan Academy of Agricultural Sciences,2016,45(3):130-134.
[21] 史平玲,王爱萍,乔松林,等.家畜IgG Fc受体研究进展 [J].河南农业科学,2010(1):124-128.Shi P L,Wang A P,Qiao S L,et al.Research progress of IgG Fc receptor in domestic animals [J].Henan Academy of Agricultural Sciences,2010(1):124-128.
[22] 席俊,张改平,何礼洋,等.牛IgG2Fc受体(boFcγ2R)胞外区基因原核表达载体的构建及其表达 [J].细胞与分子免疫学杂志,2008(2):178-180.Xi J,Zhang G P,He L Y,et al.The construction and expression of ectodomain of bovine IgG2 Fc receptor [J].Chinese Journal of Cellular and Molecular Immunology,2008(2):178-180.
[23] 李青梅,郭军庆,张改平,等.牛IgG2 Fc受体在COS-7细胞表面的稳定表达 [J].中国兽医科学,2007,37(2):121-124.Li Q M,Guo J Q,Zhang G P,et al.Stable expression of bovine IgG2 Fc receptor cDNA on the surface of COS-7 cells [J].Chinese Veterinary Science,2007,37(2):121-124.
[24] Ma H,Jiang L,Qiao S,et al.The crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine CD163 reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection [J].J Virol,2017,91(3):JVI.01897-16.
[25] 马红芳,乔松林,黄明东,et al.Crystal structure of a CD163 scavenger receptor cysteine-rich domain produced in pichia pastoris [J].结构化学,2017,36(9):1409-1417.Ma H F,Qiao S L,Huang M D,et al.Crystal structure of a CD163 scavenger receptor cysteine-rich domain produced in pichia pastoris [J].Chinese Journal of Structure and Chemistry,2017,36(9):1409-1417.
[2] Gessner J E,Schmidt R E.Fcγ receptors [J].Zeitschrift Für Rheumatologie,2013,72(1):68-70.
[3] Takai T.Fc receptors and their role in immune regulation and autoimmunity [J].J Clin Immunol,2005,25(1):1-18.
[4] Ravetch J V,Bolland,S.IgG Fc receptors [J].Annu Rev Immunol,2001,19:275-290.
[5] Nimmerjahn F,Ravetch J V.Fcgamma receptors as regulators of immune responses [J].Nature Reviews Immunology,2008,8(1):34-47.
[6] Daeron M.Fc receptor biology [J].Annu Rev immunol,1997,15(15):203-234.
[7] Deo Y M,Graziano R F,Repp R,et al.Clinical significance of IgG Fc receptors and FcγR-directed immunotherapies [J].Immunol Today,1997,18(3):127-135.
[8] Wang G,Chopra R K,Royal R E,et al.A T cell-independent antitumor response in mice with bone marrow cells retrovirally transduced with an antibody/Fc-γ chain chimeric receptor gene recognizing a human ovarian cancer antigen [J].Nature Medicine,1998,4(2):168.
[9] Jg V D W,Capel P J.Human IgG Fc receptor heterogeneity:molecular aspects and clinical implications [J].Immunology Today,1993,14(5):215-221.
[10] Kcskovics I.Fc receptors in livestock species [J].Veterinary Immunology & Immunopathology,2004,102(4):351-362.
[11] Zhang G,Young J R,Tregaskes C A,et al.Identification of a novel class of mammalian Fcreceptor [J].J Immunol,1995,155:1534-1541.
[12] Duncan A R,Woof J M,Partridge L J,et al.Localization of the binding site for the human high-affinity Fc receptor on IgG [J].Nature,1988,332(6164):563-564.
[13] Qiao S,Yang Y,Liu Y,et al.Characterization and ligand specificity of sheep IgG2 receptor [J].Immunogenetics,2009,61(8):597-601.
[14] Morton H C,Howard C J,Storset A K,et al.Identification of residues within the extracellular domain 1 of bovine Fcc2R essential for binding bovine IgG2 [J].J Biol Chem,2001,276:47794-47800.
[15] Morton H C,van Zandbergen G,van Kooten C,et al.Immuno-globulin-binding sites of human FcaRI (CD89) and bovine Fcc2Rare located in their membrane-distal extracellular domains [J].J Exp Med,1999,189:1715-1722.
[16] Zhang G,Guo J Q,Zhou J Y.Identification of the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcγ2R) using synthetic peptides [J].FEBS Letters,2006,580:1383-1390.
[17] Sondemann P,Huber R,Oosthuizen V,et al.The 3.2-A° crystal structure of the human IgG1 Fc fragment-FccRIII complex [J].Nature,2000,406:267-273.
[18] Burmeister W P,Huber A H,Bjorkman P J.Crystal structure of the complex of rat neonatal Fc receptor with Fc [J].Nature,1994,372(6504):379-383.
[19] 郭军庆.牛IgG Fc受体线性配体结合表位鉴定 [D].杭州:浙江大学,2005.Guo J Q.Identification of linear ligand binding epitopes of bovine IgG Fc receptor [D].Hangzhou:Zhejiang University,2005.
[20] 侯婕,李睿,马红芳,等.猪繁殖与呼吸综合征病毒入侵受体唾液酸黏附素的表达与纯化 [J].河南农业科学,2016,45(3):130-134.Hou J,Li R,Ma H F,et al.Expression and purification of Sialoadhesin,a putative receptor of Procine Reproduction and Respiratory Syndrom Virus [J].Henan Academy of Agricultural Sciences,2016,45(3):130-134.
[21] 史平玲,王爱萍,乔松林,等.家畜IgG Fc受体研究进展 [J].河南农业科学,2010(1):124-128.Shi P L,Wang A P,Qiao S L,et al.Research progress of IgG Fc receptor in domestic animals [J].Henan Academy of Agricultural Sciences,2010(1):124-128.
[22] 席俊,张改平,何礼洋,等.牛IgG2Fc受体(boFcγ2R)胞外区基因原核表达载体的构建及其表达 [J].细胞与分子免疫学杂志,2008(2):178-180.Xi J,Zhang G P,He L Y,et al.The construction and expression of ectodomain of bovine IgG2 Fc receptor [J].Chinese Journal of Cellular and Molecular Immunology,2008(2):178-180.
[23] 李青梅,郭军庆,张改平,等.牛IgG2 Fc受体在COS-7细胞表面的稳定表达 [J].中国兽医科学,2007,37(2):121-124.Li Q M,Guo J Q,Zhang G P,et al.Stable expression of bovine IgG2 Fc receptor cDNA on the surface of COS-7 cells [J].Chinese Veterinary Science,2007,37(2):121-124.
[24] Ma H,Jiang L,Qiao S,et al.The crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine CD163 reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection [J].J Virol,2017,91(3):JVI.01897-16.
[25] 马红芳,乔松林,黄明东,et al.Crystal structure of a CD163 scavenger receptor cysteine-rich domain produced in pichia pastoris [J].结构化学,2017,36(9):1409-1417.Ma H F,Qiao S L,Huang M D,et al.Crystal structure of a CD163 scavenger receptor cysteine-rich domain produced in pichia pastoris [J].Chinese Journal of Structure and Chemistry,2017,36(9):1409-1417.