人BAFF基因转染细胞及其单克隆抗体的生物学特性

发布时间：2018-08-13 18:54

【摘要】：BAFF(B cell-activating factor belong to the TNF family)是1999年发现的新分子,主要表达于单核细胞、巨噬细胞、树突状细胞、活化的T淋巴细胞和中性粒细胞等。其受体包括特异性结合受体BAFF-R、TACI和BCMA,三个受体主要表达于T和B淋巴细胞。BAFF与其受体共为TNF/TNFR超家族中的重要成员。业已发现,BAFF信号对B淋巴细胞的分化、发育及抗体的类型转换,及生发中心的维持具有重要作用;对T淋巴细胞具有共刺激作用;并与多种自身免疫病如SLE、RA和SS等、肿瘤如慢性B淋巴细胞性白血病、B细胞性非何杰金氏病及多发性骨髓瘤等,以及与心脏等器官移植的同种排斥反应相关。但对BAFF的基础研究尚需深入。为研究BAFF信号,本研究首先克隆了人全长BAFF分子,建立了BAFF基因转染细胞,并以此为免疫原,成功研制了一株抗人BAFF单克隆抗体,并深入探讨了其生物学特性。分析了健康人外周血来源和胸水来源T淋巴细胞表面BAFF的表达情况和意义。与此同时,本研究克隆了受体全长人TACI基因,建立了可溶性表达TACI-Fc融合蛋白的基因转染细胞,并探讨了所获得TACI-Fc融合蛋白的生物学效应。一、人BAFF基因转染细胞的构建和BAFF分子对B细胞功能的影响 1.人BAFF基因的克隆和逆转录病毒表达载体的构建根据BAFF基因(OeneBank:NM 006573)序列,利用RT-PCR方法从健康人外周血单个核细胞克隆出全长BAFF基因,装入pMD18-T载体,转化感受态大肠杆菌Top10。重组质粒T-BAFF经测序正确。利用PCR方法,从重组质粒T-BAFF扩增BAFF全长基因并引入酶切位点EcoR I和BamH I,装入逆转录病毒表达载体pSIV1,转化感受态大肠杆菌Top10。重组表达载体pSIV1-BAFF经测序正确。 2.脂质体转染法构建BAFF基因转染细胞 pSIV1-BAFF、辅助病毒载体pHIT456和pHIT60混合后,脂质体转染法转染包装细胞293T细胞,获得具有感染力的重组逆转录病毒。将含有完整BAFF重组逆转
[Abstract]:BAFF (B cell-activating factor belong to the TNF family) is a new molecule found in 1999, mainly expressed in monocytes, macrophages, dendritic cells, activated T lymphocytes and neutrophils. Its receptors include the specific binding receptors BAFF-RnTACI and BCMA.The three receptors are mainly expressed in T and B lymphocytes. BAFF and its receptors are important members of the TNF/TNFR superfamily. It has been found that BAFF signal plays an important role in the differentiation, development and antibody type conversion of B lymphocytes, as well as the maintenance of germinal center, and has co-stimulatory effect on T lymphocytes, and is associated with many autoimmune diseases such as SLERA and SS. Tumors such as chronic B lymphocytic leukemia B cell non Hodgkin's disease and multiple myeloma are associated with allograft rejection of heart and other organs. However, the basic research of BAFF needs to be further studied. In order to study the BAFF signal, we first cloned the human full-length BAFF molecule, established the BAFF gene transfection cells, and used it as the immunogen, successfully developed a monoclonal antibody against human BAFF, and studied its biological characteristics. The expression and significance of BAFF on T lymphocytes from peripheral blood and pleural effusion were analyzed. At the same time, the full-length human TACI gene of the receptor was cloned, and the gene transfected cells expressing TACI-Fc fusion protein were established, and the biological effects of the TACI-Fc fusion protein were discussed. 1. Construction of human BAFF gene transfected cells and effect of BAFF molecule on B cell function. 1. Cloning of Human BAFF Gene and Construction of Retrovirus expression Vector according to the sequence of BAFF Gene (OeneBank:NM 006573), The full-length BAFF gene was cloned from the peripheral blood mononuclear cells of healthy people by RT-PCR method. The gene was inserted into the pMD18-T vector and transformed into the competent E. coli Top10. The recombinant plasmid T-BAFF was sequenced correctly. Using PCR method, the full-length BAFF gene was amplified from the recombinant plasmid T-BAFF, and the restriction sites EcoR I and BamH I were introduced into the retroviral expression vector pSIV1. The recombinant expression vector pSIV1-BAFF was sequenced correctly. 2. BAFF gene transfected pSIV1-BAFF cells were constructed by liposome transfection method. After pHIT456 and pHIT60 were mixed, the packaging cells 293T cells were transfected with liposome transfection method. An infectious recombinant retrovirus was obtained. Will contain full BAFF recombination reversal
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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