人GL50基因转染细胞的构建及功能性鼠抗人GL50单克隆抗体的研制
发布时间:2018-03-29 15:33
本文选题:GL50 切入点:基因转染 出处:《苏州大学》2011年硕士论文
【摘要】:GL50(CD275, inducible costimulator ligand)分子是B7家族的新成员,与其特异性受体ICOS(CD278, inducible costimulator)分子结合所提供的共刺激信号在T细胞的活化、增殖、细胞因子的分泌以及B细胞的增殖、分化、抗体产生等方面发挥着重要的调节作用。GL50信号参与了机体的移植排斥反应、抗肿瘤免疫、抗感染免疫、自身免疫性疾病和过敏反应等众多免疫过程。转染人GL50基因细胞及鼠抗人GL50单克隆抗体(monoclonal antibody, mAb)的研制能够为进一步研究GL50在机体免疫应答中的作用以及与其他共刺激信号通路之间的关系提供有力的工具,从而深入探讨机体免疫应答的本质,同时也为临床相关的疾病诊断、治疗和预防提供了新的手段。 第一部分转染人GL50基因细胞株的建立及其对T细胞体外增殖与活化的促进作用 从人B淋巴瘤细胞株Daudi中抽提总RNA,采用RT-PCR的方法,把总RNA逆转录成cDNA并大量扩增目的基因。将目的基因插入pIRES2-EGFP载体中并测序,通过脂质体转染技术将重组载体pIRES2-EGFP-GL50转染L929细胞。用G418筛选并通过流式细胞术与RT-PCR的方法反复鉴定,最终获得了稳定高表达GL50分子的L929/GL50基因转染细胞。功能分析表明L929/GL50基因转染细胞能够促进T细胞增殖,并可以显著地促进T细胞分泌细胞因子IL-10、IL-4和IL-17,而不影响IL-2的分泌。 第二部分功能性鼠抗人GL50单克隆抗体的研制 以L929/GL50为免疫原,免疫Balb/c小鼠,采用经典杂交瘤技术,将免疫后鼠的脾脏细胞与小鼠骨髓瘤细胞株SP2/0进行融合并经HAT培养基选择培养。以L929/GL50细胞作为阳性筛选细胞株,经流式细胞术分析,对抗体分泌阳性孔内细胞反复筛选并经多次的克隆化培养,最终获得2株稳定、持续分泌鼠抗人GL50单克隆抗体的杂交瘤细胞株,分别命名为2B4、4D11。这两株杂交瘤细胞株经体外连续传代(50代)培养,在液氮冻存半年后复苏,仍然生长状态良好,分泌抗体性能稳定。 采用本科室建立的腹水诱生的方法制备单克隆抗体,腹水的平均产量为3~4ml/只小鼠。经Protein G亲和层析柱分离纯化,两株单克隆抗体纯化后蛋白浓度在3~4mg/ml之间,间接免疫荧光标记法分析表明,其效价在1:5000以上,用于间接免疫荧光分析时抗体蛋白的用量为0.2~2μg/1×106细胞。单克隆抗体核型分析的结果显示,两株杂交瘤2B4与4D11的染色体数目都超过小鼠B细胞与SP2/0的染色体数目,约为100条左右,表明这两株杂交瘤2B4与4D11为融合体。 使用快速定性试纸条鉴定,单克隆抗体2B4与4D11的重链分别为IgG1、IgG2a,两者轻链同为κ链。流式细胞术,Dot-blot与酶联吸附的方法分析表明,单克隆抗体2B4与4D11均能够与人GL50分子特异性结合。抗体的抗原表位竞争抑制实验结果表明,单克隆抗体2B4与4D11识别相同的抗原表位,与商品化抗体MIH12识别的抗原表位不同。 经单抗2B4与4D11鉴定表明,人GL50分子高表达于B淋巴瘤细胞系中的Daudi和Raji,卵巢癌细胞株HO8910,单核细胞来源细胞株THP-1,血管内皮细胞株ECV等。但是,健康人外周血中的B淋巴细胞上基本无GL50分子的表达。功能分析表明,特异性GL50mAb 2B4和4D11均可阻断GL50与ICOS结合从而抑制T细胞在体外的增殖。
[Abstract]:GL50 (CD275 inducible, costimulator ligand) is a new member of the B7 family, and its specific receptor ICOS (CD278 inducible costimulator) combined with molecular costimulatory signal in the activation of T cell proliferation, provided, cytokine secretion and B cell proliferation, differentiation, antibody generation plays an important regulatory role of.GL50 signaling in the body of the transplant rejection, anti tumor immunity, infection, autoimmune diseases and allergic reactions and other immune process. GL50 transfected cells and mouse anti human GL50 monoclonal antibody (monoclonal antibody, mAb) for us to further study on the immune response in GL50 the role of other costimulatory signaling pathway provides a powerful tool, so as to explore the nature of the immune response, but also for the clinical diagnosis of diseases related to the treatment. New means of treatment and prevention are provided.
The first part of the transfected human GL50 gene cell line and its promoting effect on the proliferation and activation of T cells in vitro
From human B lymphoma cell line Daudi total RNA was extracted by RT-PCR method, the total RNA reverse transcription into cDNA and amplification of the target gene. The target gene was inserted into pIRES2-EGFP vector and sequenced, the recombinant vector pIRES2-EGFP-GL50 was transfected into L929 cells by liposome transfection technique. Screened by G418 and by FCM and RT-PCR. The repeated identification, finally obtained stable high expression of GL50 L929/GL50 gene transfected cells. Functional analysis showed that L929/GL50 gene transfection can promote the proliferation of T cells and T cells can significantly promote the secretion of cytokines IL-10, IL-4 and IL-17, and does not affect the secretion of IL-2.
Development of the second part of functional mouse anti human GL50 monoclonal antibody
Using L929/GL50 as immunogen to immunize Balb/c mice, using classical hybridoma technique, the immune rats spleen cells and mouse myeloma cell line SP2/0 were fused and the HAT selective culture medium. L929/GL50 cells were used as positive screening cell lines by flow cytometry analysis of antibody secreting positive cell repeatedly screening and cloning by repeated training, finally obtained 2 strains of hybridoma cell lines stably, continuous secretion of mouse anti human GL50 monoclonal antibody, named 2B4,4D11. two hybridoma cell lines were passaged (50 generation) cultured, cryopreserved in liquid nitrogen after half a year, still good growth state, secretion antibody stability.
Methods by using an established ascites induced by monoclonal antibody, the average yield of ascites were 3~4ml/ mice. By Protein G affinity chromatography column separation and purification, two strains of monoclonal antibody purified protein concentration between 3~4mg/ml, indirect immunofluorescence analysis showed that, the titer was more than 1:5000, used for indirect immunofluorescence analysis when the antibody concentration is 0.2 ~ 2 g/1 * 106 cells. The results showed that the karyotype analysis of monoclonal antibodies, chromosome number and chromosome number of two hybridoma cell lines 2B4 and 4D11 are more than B cells in mice with SP2/0, about 100 or so, that these two strains of hybridoma 2B4 and 4D11 fusion.
The use of rapid qualitative test strip identification, heavy chain of monoclonal antibody 2B4 and 4D11 were IgG1 and IgG2a, both with light chain kappa chain. Flow cytometry, Dot-blot method and enzyme-linked immunosorbent analysis showed that monoclonal antibodies 2B4 and 4D11 were capable of binding with specific GL50 molecular epitopes. Competitive inhibition experiment showed that 2B4 and 4D11 identify the same monoclonal antibody epitopes, and commercialization of MIH12 antibodies recognize different epitopes.
The monoclonal antibody 2B4 and 4D11 identification showed that GL50 molecule is highly expressed in B lymphoma cell lines Daudi and Raji ovarian cancer cell lines HO8910, monocyte derived cell lines THP-1 and ECV in vascular endothelial cells. However, healthy human peripheral blood B lymphocytes in the fundamental expression of GL50 molecules function analysis showed that the specific GL50mAb 2B4 and 4D11 inhibited GL50 binding with ICOS to inhibit the proliferation of T cells in vitro.
【学位授予单位】:苏州大学
【学位级别】:硕士
【学位授予年份】:2011
【分类号】:R392
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