Gu Jianyou, Ruan Xizhen, Huang Zhenyu, Chen Jigang, Zhou Jiyong
Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China.
Vet Immunol Immunopathol. 2010 Apr 15;134(3-4):230-8. doi: 10.1016/j.vetimm.2009.10.021. Epub 2009 Oct 24.
Interleukin 2 (IL-2) is an essential cytokine that plays a pivotal role in the replication, maturation and differentiation of lymphocytes. In this study, the functional domains of chicken IL-2 (chIL-2) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. Nine neutralizing mAbs to chIL-2 were produced using the recombinant chIL-2 monomer expressed in prokaryotic cells as an immunogen and used to finely map the functional domains of the chIL-2 protein. The mimotopes of nine anti-chIL-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 chIL-2 molecule, that is, N(26)K(27)I(28)H(29)L(30)E(31)L(32)P(35)Q(43)Q(44)T(45)L(46)Q(47)C(48)Y(49)L(50) (domain I), E(68)E(69)F(70)K(79)K(82)S(83)L(84)T(85)G(86)L(87) (domain II) and N(88)H(89)G(91)K(104)F(105)P(106)D(107)E(111)L(112)Y(118)L(119) (domain III). The neutralizing mAbs to chIL-2 inhibited the in vitro lymphocyte proliferation stimulated by three peptide domains of chIL-2. The predicted tertiary structure of chIL-2 reveals that domain I was positioned in the long A-B loop and the N terminal of Helix B, domain II was mostly situated in Helix C, and domain III was distributed in the C-D loop and Helix D. These data demonstrate the functional domains of chIL-2 and provide a clue for elucidating the interaction between chIL-2 and its receptor.
白细胞介素2（IL-2）是一种重要的细胞因子，在淋巴细胞的复制、成熟和分化过程中发挥着关键作用。在本研究中，利用单克隆抗体（mAb）、合成肽和噬菌体展示肽库对鸡白细胞介素2（chIL-2）的功能结构域进行了定位。以原核细胞中表达的重组chIL-2单体作为免疫原，制备了9种针对chIL-2的中和单克隆抗体，并用于精细定位chIL-2蛋白的功能结构域。通过噬菌体展示和肽竞争ELISA分别鉴定了9种抗chIL-2单克隆抗体的模拟表位，包括KIELPSL、EHLDXNDSLYL、NHLXGXY、WHLPPSL、EFKASXL、TENPFPE、SGLYL、AHGYWEL和HHGYWEL。这些模拟表位在chIL-2分子中构成了三个构象功能结构域，即N(26)K(27)I(28)H(29)L(30)E(31)L(32)P(35)Q(43)Q(44)T(45)L(46)Q(47)C(48)Y(49)L(50)（结构域I）、E(68)E(69)F(70)K(79)K(82)S(83)L(84)T(85)G(86)L(87)（结构域II）和N(88)H(89)G(91)K(104)F(105)P(106)D(107)E(111)L(112)Y(118)L(119)（结构域III）。针对chIL-2的中和单克隆抗体抑制了chIL-2三个肽结构域刺激的体外淋巴细胞增殖。chIL-2的预测三级结构表明，结构域I位于长A-B环和螺旋B的N端，结构域II主要位于螺旋C中，结构域III分布在C-D环和螺旋D中。这些数据证明了chIL-2的功能结构域，并为阐明chIL-2与其受体之间的相互作用提供了线索。
