【作者】 顾建友；

【导师】 周继勇；

【作者基本信息】 浙江大学 ， 微生物学， 2009， 博士

【摘要】 白细胞介素2(IL-2)是一种重要的免疫调节细胞因子,在淋巴细胞的生长与分化等方面起着重要的作用。本研究中,利用原核表达的重组鸡IL-2(cIL-2)单体蛋白作为抗原制备了一组抗cIL-2的单克隆抗体(mAb),对具有抑制cIL-2依赖的T淋巴母细胞增殖作用的mAbs进行了表位重叠合成肽扫描和噬菌体表面展示技术分析,结果显示：抗cIL-2单抗所识别的表位均为构象表位,它们的模拟表位分别为KIELPSL、EHLDXNDSLYL、NHLXGXY、WHLPPSL、EFKASXL、TENPFPE、SGLYL、AHGYWEL和HHGYWEL。利用Clustal W序列比对和合成肽竞争ELISA鉴定了其相应的天然表位组成,并据此将其天然表位划分为三个功能域：N26K27I28H29L30E31L32P35Q43Q44T45L46Q47C48Y49L50(功能域Ⅰ)、E68E69F70K79K82S83L84T85G86L87(功能域Ⅱ)和N88H89G91K104F105P106D107E111L112Y118L119(功能域Ⅲ)。淋巴细胞增殖试验显示三个功能域的多肽在体外具有促进活化T淋巴母细胞增殖的作用。通过同源建模方法构建了cIL-2蛋白的三维模拟结构,将其功能域Ⅰ定位于A-B环(包括部分的beta折叠A)和螺旋B的N端部分；功能域Ⅱ主要定位于螺旋C；功能域Ⅲ定位于C-D环(包括部分的beta折叠B)和螺旋D中。利用噬菌体展示技术和合成肽竞争ELISA方法,鉴定了4株具有抑制鸭IL-2(dIL-2)依赖的T淋巴母细胞增殖作用的抗dIL-2 mAbs的结合表位。这些mAbs所识别的表位均为构象表位,其模拟表位分别为LVXGSMPS、KPHKHHXHHSHM、HVPNERYPLR和WXXXKAKP。Clustal W序列比对分析获得了其相应的天然表位组成,分别为L13I14K15G21S23M24P34S41T33P34T37K38E39C40S41W42Q43T44Y48L49、Y32T33p34N35E58R74F103P104L114R115和W42Q43D67E68K69V70K82P88。对这些天然表位进行序列分析,可以发现一个共同的关键功能区域：Y32T33P34N35D36T37K38E39C40S41W42Q43T44(功能域Ⅰ)。通过同源建模方法构建了dIL-2蛋白的三维模拟结构,将其功能域Ⅰ定位于A-B环和螺旋B的N端部分。利用噬菌体展示技术和合成肽竞争ELISA方法,鉴定了5株具有抑制鹅IL-2(gIL-2)依赖的T淋巴母细胞增殖作用的抗gIL-2 mAbs的结合表位。这些mAbs所识别的表位均为构象表位,其模拟表位分别为HHDPWDXLP、ESLSRXXMXXLXP、SHHLPTSXL、HPDPWDAPLSS和HEPWQLXL。Clustal W序列比对分析获得了其相应的天然表位组成,分别为I14K15D16P34W42Q43T44L45Q46、D8T9L10T11K19L20S23M24K25E30L31Y32P34、T11E18K19L20G21T22S23M24L29、T33P34E36S41W42D61P88P89L115R116S117和Y32T33P34W42Q43L45Q46L49。对这些天然表位进行序列分析,可以发现一个共同的关键功能区域：T11I14K15D16E18K19L20G21T22S23M24K25L29E30L31Y32T33P34E36S41W42Q43T44L45Q46(功能域Ⅰ)。通过同源建模方法构建了gIL-2蛋白的三维模拟结构,将其功能域Ⅰ定位于螺旋A、A-B环和螺旋B的N端部分。比较鸡、鸭、鹅IL-2的功能域组成及位置,发现在其分子的N端区域均存在一个关键的功能域,而其中的第28位到第46位序列均为最关键的功能位点,表明禽类IL-2分子的功能域分布具有相似性。利用体外抑制cIL-2依赖的T淋巴母细胞增殖活性方法,分析了3株识别真核细胞表达的鸡IL-2Rα链(cCD25)的mAbs的生物学活性,发现其中的2株具有显著的抑制功能。流式细胞计分析表明cCD25表达在脾单个核细胞的表面。利用噬菌体表面展示技术和合成肽竞争ELISA方法,鉴定了2株抗cCD25功能性mAbs的结合表位。该单抗的噬菌体模拟位基序为PVDRPRD和SLGKXTPVDEPXY。利用Clustal W序列比对和合成肽竞争ELISA鉴定了其相应的天然表位组成,并据此将其天然表位划分为一个功能域：K152W153T154P155V156D157R158P159C160T161(功能域Ⅰ)。通过同源建模方法构建了cCD25蛋白胞外域的三维模拟结构,将其功能域Ⅰ定位于D2结构域的近C末端部分。利用体外抑制cIL-2依赖的T淋巴细胞增殖活性方法,分析了5株识别真核细胞表达的鸡IL-2Rγ链(cCD132)的mAbs的生物学活性,发现其中1株mAb具有显著的抑制功能。流式细胞计分析表明cCD132表达在脾单个核细胞的表面。利用噬菌体表面展示技术和合成肽竞争ELISA方法,鉴定了mAb C10的结合表位。该单抗的噬菌体模拟位基序为QEHQNL,其相应的天然表位组成为Q84E94L95Q96N97L98。与人CD132序列的比对分析显示,Q96可能是个关键的功能位点,随后发现定点突变Q96A的表位多肽完全丧失了结合(?)mAb C10的能力,证明了Q96是个关键的功能氨基酸。通过同源建模方法构建了cCD132蛋白胞外域的三维模拟结构,将其功能域定位于N结构域和C结构域间的肘关节样的连接区。利用噬菌体展示技术,分别分析了鸡白介素2受体α、γ的cIL-2结合模拟表位和鸡白介素2的受体α、γ结合模拟表位,并对这些结合表位及前期鉴定的功能性表位进行了功能鉴定和结构定位,提出了cIL-2和其受体α、γ相互作用的基本模型。cIL-2中的K27～C41直接与其受体α、γ发生强相互作用,提示了cIL-2R的α、γ亚基间存在互相接触的界面,完全不同于人IL-2Ra亚基游离于β和γ亚基组成的聚合物而单独在相反的侧面与IL-2发生相互作用。cCD25中的α99-112主要负责与cIL-227-41作用。cCD132中的γ119-137主要负责与cIL-227-41、cIL-279-96作用,而γ82-101则是第二个特异性与cIL-279-96发生相互作用的位点。cIL-2Rα、γ间的相互作用分析表明cIL-2R的两个亚基间存在着互相作用,该相互作用界面主要由cCD2599-112/cCD132119-137构成,该界面同时也是主要参与cIL.227-41相互作用的结构。cIL-227-41/cCD2599-112/cCD132119-137构成了三聚体复合物的相互作用中心。上述研究结果为阐述cIL-2和cIL-2Rα、γ间的基本相互作用提供了实验证据和结构模型。更多还原

【Abstract】 Interleukin 2 (IL-2) is an essential cytokine that plays a pivotal role in the growth and differentiation of lymphocytes. In this study, the functional domains of chicken IL-2 (cIL-2) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. Nine neutralizing mAbs to cIL-2 were produced using the recombinant cIL-2 monomer expressed in prokaryotic cells as an immunogen and used to finely map the functional domains of the cIL-2 protein. The mimotopes of nine anti-cIL-2 mAbs, including KIELPSL, EHLDXNDSLYL, NHLXGXY, WHLPPSL, EFKASXL, TENPFPE, SGLYL, AHGYWEL and HHGYWEL, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute three conformational functional domains in the cIL-2 molecule, that is, N26K27I28H29L30E31L32P35Q43Q44T45L46Q47C48Y49L50 (domainⅠ), E68E69F70K79K82S83L84T85G86L87 (domainⅡ) and N88H89G91K104F105p106D107E111 L112Y118L119 (domainⅢ). The neutralizing mAbs to cIL-2 inhibited the in vitro lymphocyte proliferation stimulated by three peptide domains of cIL-2. The predicted tertiary structure of cIL-2 reveals that domainⅠwas positioned in the long A-B loop and the N terminal of Helix B, domainⅢwas mostly situated in Helix C, and domainⅢwas distributed in the C-D loop and Helix D.The functional domains of duck IL-2 (dIL-2) were finely mapped with four neutralizing mAbs to dIL-2. The mimotopes of four anti-dIL-2 mAbs, including LVXGSMPS, KPHKHHXHHSHM, HVPNERYPLR and WXXXKAKP, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute one functional domain in the dIL-2 molecule, that is, Y32T33P34N35D36T37K38E39C40S41W42Q43T44 (domainⅠ).The predicted tertiary structure of dIL-2 reveals four-helix bundle structure without two beta sheets and domain I was positioned in the long A-B loop and the N terminal of Helix B.The functional domains of goose IL-2 (gIL-2) were finely mapped with five neutralizing mAbs to gIL-2. The mimotopes of five anti-gIL-2 mAbs, including HHDPWDXLP, ESLSRXXMXXLXP, SHHLPTSXL, HPDPWDAPLSS and HEPWQLXL, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute one functional domain in the gIL-2 molecule, that is,T11I14K15D16E18K19L20G21T22S23M24K25L29E30L31Y32T33P34E36S41W42Q43T44L45 Q46 (domain I). The predicted tertiary structure of gIL-2 reveals four-helix bundle structure with two beta sheets and domain I was positioned in the Helix A, A-B loop and the N terminal of Helix B.Compare the functional domains of poultry IL-2 (cIL-2, dIL-2 and gIL-2), one common key domain was situated in the long A-B loop. The N terminal of poultry IL-2s were important for their bioactivities.The functional domains of chicken CD25 (cCD25) were mapped with two neutralizing mAbs to cCD25, which were identified by inhibition of IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that cCD25 molecules are expressed on the surface of splenic mononuclear cells. The mimotopes of two anti-cCD25 mAbs, including PVDRPRD and SLGKXTPVDEPXY, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute one functional domain in the cCD25 molecule, that is, S119V120G121K152W153T154P155V156D157R158P159C160T161 (domainⅠ) The predicted tertiary structure of cCD25 shows that functional domain I is positioned at the interdomain, paritial Sheet I and the subsequent connected-peptide with membrane.The functional domains of chicken CD132 (cCD132) were mapped with one neutralizing mAb to cCD132, which were identified by inhibition of IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that cCD132 molecules are expressed on the surface of splenic mononuclear cells. The functional domain of cCD132 that binds to chicken interleukin 2, Q84E94L95Q96N97L98, was found through phage display and peptide-competitive ELISA, and its critical residue Q96 was further identified. A tertiary structure model shows that the functional domain is positioned at the elbow-like junction of domain 1 and domain 2.Using phage display and competitive ELISA, a key binding epitope of cIL-2 to cIL-2R was identified and situated in the cIL-227-41 positioned in the long A-B loop. This fact implies that the interaction between cIL-2Ra and y subunits could be present. The binding epitopes of cIL-2Ra and y to cIL-2 were also identified by phage display and competitive ELISA.α99-112 of cCD25 was primary to bind cIL-227-41.γ119-137 of cCD132 was primary to bind cIL-227-41 and cIL-279-96.γ82-101 was the second site binding to cIL-279-96. The key binding epitopes of cIL-2R to cIL-2, namelyα99-112 andγ119-137, formed the common interaction interface to bind the cIL-227-41. The interaction between cIL-2 and cIL-2R (a and y) was further confirmed by the inhibition of lymphocyte proliferation in vitro. These results provide experimental evidences and structure models for elucidating the interaction between cIL-2 and cIL-2R.更多还原