【作者】 吴海涛；

【导师】 张双全；

【作者基本信息】 南京师范大学 ， 动物学， 2003， 硕士

【摘要】 人B淋巴细胞刺激因子(hBLyS)是1999年发现的肿瘤坏死因子超家族成员。最早发现其可诱导肿瘤细胞凋亡，之后发现它是一个强有力的B细胞共刺激因子，它在体外及转基因动物体内可明显刺激B细胞的生长，免疫球蛋白分泌，并导致动物自身免疫性改变。BLyS的生物功能，作用机制极其复杂，其缺陷或过量表达均能引起机体的免疫失衡，从而诱发多种疾病。BLyS的过度表达是发生自身免疫疾病，特别是系统性红斑狼疮(SLE)的重要因素之一。因此，对BLyS及其受体的研究有助于阐明SLE等自身免疫疾病，BLyS、其可溶性受体和单克隆抗体有望开发为治疗自身免疫综合症的新型药物。 BLyS是高等动物表达的真核蛋白，有N242糖基化位点。故尝试CHO真核分泌表达。本实验通过3’端互补，进行引物延伸合成EPO信号肽序列：信号肽和hsBLyS基因采用重叠延伸拼接法形成融合基因；融合基因分别插入pcDNA3.0、pcDNA3.1、pEFneo真核载体：引物延伸合成信号肽时，利用亮氨酸同义密码，将信号肽基因的倒数第二个密码突变，在重组载体上的信号肽序列之后，形成BlnⅠ酶切位点，使三种载体成为分泌表达载体。 磷酸钙共沉淀将表达质粒和标志质粒pSV-dhfr，共转染入CHO—dhfr细胞；DMEM选择培养液，筛选dhfr阳性细胞克隆；氨甲喋呤浓度梯度增高，加压扩增表达。获稳定表达hsBLyS的细胞株，经MTX加压扩增，表达量由0.1μg/ml上升至2.0μg/ml。 MTT法知hsBLyS表达上清对B淋巴细胞具有共刺激作用；流式细胞仪分析显示只有在共刺激作用下，B细胞才进入S期。 DNA琼脂糖凝胶电泳和流式细胞仪结果显示，hsBLyS表达上清可诱导K562细胞凋亡，且具有剂量依赖效应。更多还原

【Abstract】 The B Lymphocyte Stim lator(BLyS) also known as BAFF,TALL-1,THANK, zTNF4,is the most recent addition to the tumor necrosis factor family(TNF) ligands.BlyS induces both in vivo and in vitro B cell proliferation,differentiation and immunoglob lin secretion.Meanwhile,it also strongly suppresses the growth of tumor cell lines .The over-production of BLyS is associated with the development of certain autoimmune disease,such as systemic lupus erythaematosus(SLE) ,rheumatoid arthritis,myasthenia gravis, Sj greh’s syndrome. Therefore,BlyS,its receptor or related antagonists may find medical utility in the treatment of B cell disorders associated with autoimmunity,neoplasia,or immunodeficiency syndromes.In this study,EPO signal peptide sequence and hsBLyS gene were linked by SOE method.The fusion product was cloned into eukaryotic plasmids.pcDNA3, pcDNA3.1, pEFneo,respectively.Meanwhile, the EPO signal peptide sequence was mutated so as to form a restriction enzyme cut site :Bin I .Thus the recombinant plasmid can be used as secreting plasmid expressing other gene.The recombinant expression plasmid and marker plasmid:pSV-dhfr were co-transfected into CHO-dhfr- cells.After screened and amplified by selection medium and MTX respectively,we constructed a CHO cell line which secreting express hsBLyS stably.The supernatant containing hsBLyS co-stimulated B cell proliferation highly with anti-IgM by MTT analysis.Flow cytometry results showed only in the function of costim lation,can B cell entered the s phase of cell cycle. Apoptosis was identified by DNA electrophoresis and Flow cytometry .The results showed the supernatant containing hsBLyS induced a does and concentration-dependant increase in apoptosis cells.更多还原