【作者】 肖伟；

【导师】 吴雄文；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 硕士

【摘要】 MHCⅠ类分子由两条多肽链组成,一条为轻链,即β2微球蛋白;另一条为重链,其胞外段由α1、α2及α3区组成,其中α1、α2区共同构成抗原肽结合槽,用于结合特定的抗原肽,形成pMHC,供T细胞表面的TCR识别。H-2复合体为小鼠的MHC,其中H-2K基因座与H-2D、H-2L基因座同属小鼠MHCⅠ类分子复合体。MHCⅠ类分子在抗原提呈和T细胞活化中发挥了重要的作用。可溶性MHC-Ig二聚体技术是近几年发展起来的一种免疫学新技术,主要用于检测抗原特异性T细胞。这种二聚体蛋白具有两个供TCR识别的MHC分子,它们是通过Ig铰链区二硫键的作用而形成。相对于单体分子而言,可溶性MHC-Ig二聚体分子在与TCR的结合中具有更强的稳定性。该二聚体分子MHC段的抗原结合槽可加载MHC限制性的抗原肽,形成抗原肽-MHC(pMHC)二聚体复合物,它可以用来检测抗原特异性T细胞。此外,有文献报道, pMHC二聚体复合物可以结合在微球等非细胞载体上用于活化抗原特异性T细胞。在本课题中,我们成功的构建和表达了可溶性H-2K~d/IgG2aFc二聚体融合蛋白,该二聚体由小鼠H-2K~d的胞外段和IgG2a的Fc段组成。体外实验证实,与可溶性H-2K~d/IgG2aFc二聚体共同孵育后的C57BL/6小鼠(H-2Kb)腹腔巨噬细胞能够被抗H-2K~d的特异性抗体标记。实验结果提示我们,可以利用该二聚体Fc段与Fc受体的作用将H-2K~d分子结合到小鼠的巨噬细胞表面,这一研究成果为进一步探讨T细胞识别巨噬细胞表面抗原肽的机制提供了新的方法,该方法可应用于活化抗原特异性的CTL。与以前的方法相比,该方法与体内CTL天然的活化过程更为相似,具有更大的应用前景。一.H-2K~d/IgG2aFc真核表达载体的构建方法:利用本室前期构建完成的pBS-T-H-2K~d(extra)和pGEM-T-IgG2aFc为模板,通过重组PCR技术,经两轮反应得到H-2K~d/IgG2aFc融合基因,PCR产物经凝胶回收后,通过限制性内切酶HindⅢ和xbalⅠ双酶切与经过相同的酶切后的载体pcDNA3.1(+)连接,形成H-2K~d/IgG2aFc真核表达载体。结果:通过限制性酶切分析、PCR鉴定及核苷酸序列测定证实了H-2K~d/IgG2aFc融合基因与预期序列一致,并成功的连接到载体中形成pcDNA3.1(+)-H-2K~d/IgG2aFc真核表达载体。二.可溶性H-2K~d/IgG2a Fc二聚体融合蛋白的表达及功能研究方法:通过电穿孔法将pcDNA3.1(+)-H-2K~d/IgG2aFc真核表达载体转染到J558L细胞中,通过G418筛选,得到稳定表达的克隆株,通过RT-PCR鉴定H-2K~d/IgG2aFc融合基因在阳性克隆株中的表达。大量提取J558L克隆株的上清,通过葡萄球菌蛋白A(SPA)纯化得到可溶性H-2K~d/IgG2aFc二聚体融合蛋白。通过流式细胞术检测可溶性H-2K~d/IgG2aFc二聚体融合蛋白与巨噬细胞表面Fc受体的结合能力。结果:通过细胞RT-PCR,我们得到一段长度为1602bp的序列,说明H-2K~d/IgG2aFc融合基因能够在转染阳性的J558L克隆株中表达。Western-blot和ELISA结果证实了纯化得到的融合蛋白由H-2K~d的胞外段与IgG2a的Fc段两个部分组成,与我们的预期构想一致。流式细胞术结果证实,可溶性H-2K~d/IgG2aFc二聚体融合蛋白能够与小鼠巨噬细胞表面的Fc受体结合。综上所述:本课题将可溶性H-2K~d/IgG2aFc二聚体通过Fc与Fc受体的作用结合到鼠的巨噬细胞上,这提示我们,结合了二聚体的APC细胞加载抗原肽后可用于活化抗原特异性的T细胞,应用于肿瘤和病毒感染性疾病的过继免疫治疗中。更多还原

【Abstract】 The MHC classⅠmolecule contains a light chain (β2 microgloblin,β2m) and a heavy chain ,which includedα1、α2 andα3 regions, the antigen-binding groove composes ofα1 andα2 regions with antigenic peptide fragment resident in it. The mouse MHC is H-2, the class I of it comprises H-2K, H-2D and H-2L loci. The MHC classⅠmolecule plays the central role in the antigen presentation and T cell activation.Recently, the soluble MHC-Ig dimer is a new technology developed to detect antigen-specific T cells. This kind of divalent protein composes 2 ligands for specific T cell receptors linked via covalent disulfide of Ig hinge region. Compared to monomeric molecules, the dimeric protein has higher stability to enhance the interaction between the ligand and TCR. The antigen-binding groove of the soluble MHC-Ig dimer can be loaded with a MHC restricted peptide, forming a pMHC dimer, which can be applied to identify antigen-specific T cell. In additon, it has been reported that the activation of antigen-specific T cells is achieved by pMHC dimer bound to non-cell carrier.In this study, we have successfully constructed and expressed the divalent H-2K~d/IgG2aFc fusion protein, which consists of the extracellular domains of H-2K~d and the Fc region of IgG2a. Furthermore, peritoneal Mφof C57BL/6 (H-2Kb) can be stained with H-2K~d specific monoclonal antibody (mAb) after incubated with the H-2K~d/IgG2aFc fusion protein in vitro. This result demonstrates the fusion protein can be used to attach the H-2K~d molecule via Fc-FcR interaction to the surface of murine Mφ, and provides a novel means to manipulate the T-cell recognized peptide on the surface of murine Mφ, which can be applied to activate antigen-specific cytotoxic T lymphocyte (CTL). Compared to the previous means, this method is much more similar to the na?ve activation of T cell, which is made to be more valuable.1. Construction of H-2K~d /IgG2aFc hybrid geneMethods The H-2K~d/IgG2aFc hybrid gene was constructed by recombinant PCR and the recombinant plasmids pBS-T-H-2K~d(extra) and pGEM-T-IgG2aFc were used as stencil-plates. The PCR products were purified and digested by restriction endonucleases HindⅢand Xbal, and ligated into eukaryotic expression vector pcDNA 3.1(+) which was digested by the same restriction endonucleases. Result DNA sequencing indicated that fragment was according with the sequence of GeneBank. The recombinant plasmid pcDNA 3.1(+)-H-2K~d/IgG2aFc was constructed successfully by double digestion of restriction endonucleases analysis.2. Expression and functional test of the divalent H-2K~d/IgG2aFc fusion proteinMethods To generate stable transfectants, the gene of H-2K~d /IgG2aFc was transfected into J558L cell line by electroporation. High expressing clones were selected under G418 and adapted to multiply in serum-free media for collection. The cultural supernatant was passed over a Staphylococcal Protein A (SPA) column to purify the dimeric H-2K~d/IgG2aFc fusion protein. The ability of the fusion protein to bind with Fc receptors on murine macrophages was confirmed by flow cytometry (FCM).Result A 1602bp fragment was obtained from cell RT-PCR and it confirmed that the H-2K~d/IgG2aFc hybrid gene expressed in J558L cells. The fusion protein showed a 58.4kD band as revealed by SDS-PAGE and Western Blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K~d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K~d indicated the fusion protein consists of both Fc portion and H-2K~d. The binding test by FCM showed that peritoneal Mφof C57BL/6 (H-2Kb) can be stained with H-2K~d specific monoclonal antibody (mAb) after incubated with the H-2K~d/IgG2aFc fusion protein. In this study, the divalent H-2K~d/IgG2aFc fusion protein binds to murine macrophage via Fc-FcR interaction to form a single-epitope presemting cell which can be used to activate and expand antigen-specific T cells for adoptive immunotherapy, such as treatment for cancer and virally infected diseases.更多还原