【作者】 朱瑾；

【导师】 吴军；

【作者基本信息】 第三军医大学 ， 外科学， 2002， 博士

【摘要】 背景：抗原特异性T细胞的活化在一些免疫相关疾病(如移植排斥反应、变应性疾病)的发生、发展中起着主导性的作用。而T细胞的活化除需要T细胞受体(TCR)与抗原呈递细胞(Antigen presenting cell,APC)表面的抗原肽-MHC复合物结合所形成的第一信号外，还需要T细胞和APC表面的其它膜分子结合所提供的共刺激信号(亦称第二信号或辅助刺激信号，Costimulatory signal)的参与。共刺激信号决定着T细胞与抗原反应的程度和结果。研究表明B7-CD28通路是最主要的刺激T细胞活化的共刺激信号通路，其不仅参与T细胞活化过程，还影响活化后T细胞的分化、细胞因子产生等。缺乏或阻断B7-CD28共刺激信号将导致T细胞活化增殖障碍、T细胞凋亡(Apoptosis)、克隆排除(Clone depletion)或进入一种无反应状态(无能，Anergy)，从而诱导针对该识别过程中特异性抗原的免疫耐受。CTLA4(Cytotoxic T lymphocyte associated antigen 4)作为一种主要的共刺激负调节剂，可竞争性结合B7分子，阻断B7-CD28途径、并通过产生抑制性信号使T细胞活化障碍。重组人CTLA4Ig融合蛋白已被运用于防治移植排斥反应、自身免疫性疾病和变应性疾病等的实验和临床研究，并取得了肯定的效果。CTLA4Ig融合蛋白是通过基因重组技术将编码CTLA4胞外区1-125个氨基酸序列的DNA片段与编码IgG Fc段的DNA片段融合后表达生成，其功能区是CTLA4胞外区。CTLA4胞外区序列决定了其空间结构和配体结合特异性。CTLA4胞外区DNA的表达蛋白由于不含有外源DNA表达产物，理论上与天然CTLA4蛋白的功能更为一致，也减少 了额外DNA表达产物的副作用。同时，由于CTLA4胞外区片段大小仅有 400hp，较CTLA4Ig融合蛋白＊．ZKb）在表达菌中的表达将更为稳定。GSlls 是毕赤酵母uichia pastoris）的一种菌株，己被用于多种外源蛋白的表达。 其可高密度生长，外源蛋白呈分泌性表达，具有外源蛋白产量高、纯化相 对容易的优点。为此，本实验选择 GSlls作为表达菌。 目的：获取具有生物学活性的重组人CTLA4胞外区蛋白（CTLA4e）， 探讨其在变应性疾病和器官移植排斥反应防治中的作用和机理。 方法：1．重组人 CTLA4胞外区蛋白在酵母 GS 115中的表达和纯化： 首先通过基因操作技术，从pCTLA4八g质粒中扩增400hp的CTLA4胞外 区片段；将CTLA4胞外区片段插人pPICg质粒中获取单拷贝酵母表达质 粒pPICg－CTLA4125并进一步构建多拷贝酵母表达质粒pPICgK（。 利用限制性内切酶分析、＊＊A序列测定在＊＊A水平对质粒进行鉴定。将 pPICgK七 质粒电转化GSlls酵母；通过筛选获取稳定表达CTLA4 胞外区蛋白的表达菌。表达菌甲醇诱导发酵；发酵上清液经超滤、硫酸胺 粗分级分离以及阴离于交换层析，纯化出CTLA4 胞外区蛋白。利用 IJot．ELISA、SDA－PAGE、Western．blot等方法在蛋白水平对CTLA4胞外区 蛋白的表达进行了鉴定。 2．重组人 CTLA4胞外区蛋白离体生物学活性鉴定：我们通过h1dR 掺入法观察了CTLA4胞外区蛋白对人双向混合淋巴细胞反应（Mixed lymphocyte reaction，MLR产 T细胞增殖的影响。 3．重组人 CTLA4胞外区蛋白体内生物学活性研究：l）观察 CTLA4 胞外区蛋白在小鼠变应性鼻炎中的作用。通过卵清蛋白（Ovalbumin，OVA） 致敏和激发诱导小鼠变应性鼻炎；60只小鼠随机分为变应性鼻炎组（OVA 组）、正常对照组（ Saline组）和 CTLA4e组（200 u g／只、l／日 X 7次，IP）， 分别比较首次和一月后再次用抗原激发时各组动物鼻部症状、鼻粘膜形态 学改变，以及血清抗原特异性 IgE水平和鼻腔灌洗液（Nasal lavage fluid， NLF）中EOtaxin（嗜酸粒细胞趋化素）水平。2）观察CTLA4e在移植排 斥反应防治中的作用。30只异基因气管移植大鼠随机分成CTLA4e组（500 ·IX·119／只、l／日 X 3次，IP）、Sgline组和环胞素 A对照组（CSA组，SSg／kg、l／日 X 7次，IM），观察大鼠存活时间和术后 7d移植气管形态学改变。24只异基因皮肤移植小鼠，随机分为 CTLAe组（200n g／只、l／日 X3次，IVLSaline组、CsA组（sing／kg、l／日 X 7次，IM）和重组人 CTLA4Ig组（CTLA4Ig组，200 p g／只、1／日X 3次，IV人 分别观察移植皮肤存活时间和术后7d、14d组织形态学改变。 结果：1．成功构建CTLA4 胞外区蛋白的多拷贝酵母表达质粒pPICgK－CTLA4125；获取整合pPICgK－CTLA4125质粒、并能表达CTLA4胞外区蛋白的 GS表达菌；并从表达菌甲醇诱导发酵液中纯化出分子量在28KDa、单体形式的重组人CTLA4胞外区蛋白。 2．重组人CTLA4胞外区蛋白还可显著抑制MLR中T细胞的增殖，抑制率可高达95％。 3．重组人CTLA4胞外区蛋白可明显减轻变应性鼻炎小鼠的鼻部症状、鼻粘膜形更多还原

【Abstract】 Backgroud: The activation of antigen-specific T cells plays a vital role in the initiation and development of some immune-related diseases, such as transplant rejection and allergic diseases. Effective activation of antigen-specific T cells not only requires the first signal transduction through T-cell receptor(TCR) binding with peptide-MHC complex on the antigen presenting cell (APC), but also needs the second signal, termed costimulation. Costimulation critical to the degree and consequence of T cell activation is provided by interaction between soluble factors or cell-surface molecules on the T cell and on the APC. It has been demonstrated that B7-CD28 pathway is a prominent costimulation pathway which leads full T cell activation, induces differentiation and regulates the production of cytokines. Lacking and blocking of B7-CD28 pathway will result in blockage of the T cell activation and proliferation. T cell will be apopotosis, clone depletion and /or anergy, in turn inducing an antigen-specific tolerance. CTLA4 (Cytotoxic T lymphocyte associated antigen 4), a prominent negative costimulator, can competitively bind with B7, and block B7-CD28 pathway and T cell activation by producing inhibitory singals. Recombinated human CTLA4Ig fusion protein had been used experimentally and clinically in prevention and treatment of transplant rejection, autoimmune diseases and allergic diseases, and positive curiative results were obtained. CTLA4Ig fusion protein was expressed by genetically fusing the DNA fragment encoding 1-125 amino acid residues of CTLA4 extracellular domain with DNA fragment encoding an IgG Fc region. Itsinfunctional region is the extracellular domain of CTLA4. The sequence of CTLA4 extracellular domain determines its space construction and binding specific with its ligand. Without heterologous DNA, the function of protein of CTLA4 extracellular domain will be more identical to that of natural CTLA4 theoretically. Meanwhile, because the fragment of CTLA4 extracellular domain is only 400bp, its expression will be more stable than that of CTLAIg fusion protein( 1.2kb). Yeast GS115, one kind of Pichia pastoris, has been used to express varied heterologous proteins. GS115 can grow in a high density and produce heterologous proteins by secretion, which are in a high-level and easy to be purified. Hence, yeast GS115 was used to express the target protein in this study.Objection: To obtain the biological activated recombianated protein of huaman CTLA4 extracellular domain(CTLA4e), and to explore its effects on allergic diseases and transplant rejection.Methods: 1.Expression and purification of recombinant human CTLA4 extracellular domain: a 400bp DNA fragment of CTLA4 extracellular domain was obtained from the pCTLA4/Ig plasmid with genetical technique. Then, this DNA fragment was inserted into pPIC9 plasmid to construct the single copy plasmid of yeast expression system (pPIC9-CTLA4125). Furthermore, the multi-copy plasmid (pPIC9K-CTLA4125) was constructed. After the plasmid was assayed for DNA sequence, it was transformed into GS115 by electroporation. The stable expression yeast was obtained by screening and was induced to ferment by methanol. After ultrafiltration and rough separation with ammonium sulfate and anion-exchange chromatography, the recombination protein of CTLA4 extracelluar domain was rectified from the ferment supernatants. The expression of target protein was identified with Dot-ELISA, SDS-PAGE and Western blotting.2. Inspection for the biological activities of recombination protein of?CTLA4 extracellular domain in vitro: We investigated the role of CTLA4e in T cell proliferation of MLR with 3H-TdR incorporation.3. Investigation for the biological activities of recombination protein of CTLA4 extracellular domain in vivo: First, we observed the effects of CTLA4e on allergic rhinitis in mice. After sensitized and challenged by ovalbumin(OVA), 60 mice were equally divided into allergic rhinitis group(OVA group), Saline group and CTLAe group(200 U g/mice,更多还原