【作者】 李大为；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2003， 硕士

【摘要】 B7-CD28超家族是最具特征的共刺激分子家族，不仅提供重要的正性共刺激信号(B7-1／B7-2-CD28,ICOSL-ICOS)起始和维持T细胞针对抗原的免疫应答，而且也提供负性共刺激信号(B7-1／B7-2-CTLA-4，PD-L1／PD-L2-PD-1)下调和终止T细胞介导的免疫应答。PD-1分子属于CD28家族，是55KDa的Ⅰ型跨膜糖蛋白，胞外区包含一个IgV样结构域。此结构域及胞外段的糖基化被认为在与配体的结合中发挥重要的作用。一系列的研究表明，PD-1通过与其两个配体PD-L1和PD-L2的作用而抑制T、B细胞的活化及细胞因子的产生，在维持机体的外周耐受发挥至关重要的作用。PD-1也参与了一系列疾病的发病。对小鼠的肿瘤和移植排斥模型的体外和体内的研究表明，PD-1可作为肿瘤、自身免疫性疾病和移植排斥中有效的治疗靶点。但是迄今为止，尚无商品化高纯度的PD-1重组蛋白，限制了它的实验研究和临床实验。本实验克隆了PD-1的全长和胞外段基因，并且首次在大肠杆菌中大规模的表达，继而进一步纯化了GST-PD-1融合蛋白，并建立了一个鉴定其生物学活性的方法，为以后的PD-1及相关分子实验研究奠定了基础。一、人PD-1基因全长及胞外段的克隆和载体的构建根据文献报道的人PD-1的基因序列设计合成了胞外段特异性引物。采用RT-PCR方法从人外周血PHA刺激活化的T细胞中扩增了PD-1的胞外段基因，装入T克隆载体，测序正确后，在TOP10宿主菌中增殖PGEX-5X-3-PD-1质粒，经酶切和PCR鉴定后转化BL21(DE3)表达菌株。设计合成PD-1全长基因的特异性引物，经RT-PCR克隆全长片段(含5UTR)，经过对多个克隆的测序和重组PCR重组人GsT-PD一l融合蛋白在大肠杆菌中的高效表达及其生物学特性的研究中文摘要克隆含有酶切位点的全长基因，装入·PcDNA3.1载体，全长基因测序正确。二、人GST一PD一1融合蛋白在大肠杆菌中的表达和鉴定 在Lura细菌培养基中增殖BL21表达菌，采取一系列实验条件，包括降低温度，缩短诱导时间等，均证实不能诱导可溶性GST一PD一1融合蛋白的表达，而表达的蛋白主要集中在包涵体内。表达动力学分析表明，应用Ilnlnol/L IPTG诱导5h可获得最有效的表达。实验室规模增殖表达菌，经超声裂解和离心，沉淀变性及复性，透析后经GST亲和层析柱一步纯化融合蛋白。实验结果表明，纯化后的即一1一GST融合蛋白纯度可达90%以上，定量为Zmg/耐。应用Western一b1Otting对融合蛋白进行生物学鉴定，结果为约46KD的单一条带。三、人GST一PD一1重组蛋白的生物学功能的初步研究 应用本科室建立的PD一L1基因转染的L929成纤维细胞株与人外周血T细胞在不同浓度的PHA存在条件下共育48小时，采用空载体转染的L929细胞株和GST作为对照，检测培养上清中的IL一2和IFN一Y的浓度。结果表明PD一Ll一L929细胞与对照相比可以显著抑制活化T细胞IL一2和IFN一Y的产生。GST一PD一1融合蛋白在较高的浓度下可部分逆转PD一Ll一L929细胞对这两种细胞因子产生的抑制。 以上结果表明，GST一PD一1融合蛋白可在BLZI(D E3)细胞中高效表达，经过变性、复性和纯化可获得高浓度、高纯度的活性蛋白。但其中只有部分具有生物学活性，推断由于复性的条件仍然并非最佳。在以后的实验中要进一步优化复性条件，以期提高其活性成分所占的比例。更多还原

【Abstract】 The B7-CD28 superfamily, one of the best-characterized costimulatory family, not only provides critical positive second signals to initiate and sustain T cell response, but also contributes key negative second signals to downregulate and terminate T cell response. The PD-1 receptor, which belongs to CD28 family, is a 55KDa type I transmembrane protein of Ig superfamily with an extracellular IgV-like domain. The domain plays an important role in binding to ligands. Ligation PD-1 with its two ligands, PD-L1 and PD-L2, triggers inhibitory signals to inhibit T and B cell activation and production of cytokines. PD-L and PD-1 interaction involves in maintenance of peripheral tolerance. In addition, the PD-1 expression and biological effects are closely relatesd to a series of diseases including autoimmune diseases, tumor and trasplantation rejection. Studies on mouse models showed that PD-1 receptor was a promising target in the intervention of human autoimmune disease, transplantation rejection and malignant cancers. Otherwise, human PD-1 recombinant protein is not available so far, which restricts its studies. In our studies, the cDNA fragments edcoding full-length and extra-cellular region of PD-1 were obtained and extra-cellular fragment was cloned into GST fusion protein expression vector. The target protein was expressed in E coli and then purified with higher purity. Furthermore, a method for characterizing its biological activity was set up.1. Cloning the cDNA ecoding PD-1 full-length and extra-cellular fragmentsThe published cDNA sequence for PD-1 was used for specific pimer design. Extra-cellular fragment of PD-1 gene was amplified through RT-PCR from PHA-activated human peripheral blood T cells and was cloned into T-vector plasmid. The derived plasmid was verified by DNA sequence analysis, and then cloned into GST fusion protein expression vector (pGEX-5X-3). Expression vector named pGEX-5X-3-PD-l was transformed into BL21 (DE3) strain of Escherichia coli for further expression of fusion protein. Full-length fragment of PD-1 gene including 5’UTR was amplified at the same time. Recombinant PCR was carried out, and then aquired fragment was cloned into pcDNA3.1 vector. The derived recombinant plasmid was certified by DNA sequence analysis.2. Expression, purification and characterization of GST-PD-1 fusion proteinBL21 expression strain was propagated in lura broth. A series of methods (low temperature induction, shorten of induction time, etc) was tried to induce soluble GST-PD-1 fusion protein but without sucesses. Fusion protein was mainly expressed as inclusion body. Dynamics study indicated that the most high-level protein expression was after 5 hour IPTG induction at Immol/L. The cell slurry was disrupted by sonication, and then fusion protein was purified through GST affinity chromatography column after denaturation, refolding and dialysis. Purity of protein is above 90% with 2mg/ml concentration. Western blotting showed a single band of molecular mass 46KDa.3. Setting up a way to characterize biological activity of GST-PD-1 fusion proteinHuman peripheral blood T cells were cocultured with PD-L1 transfected L929 fibroblast cell strain at different PHA concentration. IL-2 and IFN- Y concentration of supernatant were determined by ELISA after 48 hours. Compared with mock tansfected L929 cells, PD-L1 transfected L929 cells inhibited significantly production of these cytokines of activated T cells. GST-PD-1 fusion protein reversed the inhibitory effects at a higher concentration.These results suggest GST-PD-1 fusion protein can be expressed in BL21 Escherichia coli strain at a high yield. GST-PD-1 fusion protein with higher purity has inhibitory biological effects. Further studies should be concentrated to optimize the process of refolding in order to obtain a nature equal activity of PD-1 molecule.更多还原