【作者】 沈双；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2011， 硕士

【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。更多还原

【Abstract】 GL50 is a new member of B7 family. The interaction of GL50 with ICOS, the specific receptor for GL50, is critically involved in the activation, proliferation, differentiation and cytokine production of T cells as well as in the antibody secretion of B cells during the secondary immune responses. It is well-accepted that the ICOS/GL50 signal pathway plays a pivotal role in the regulation of inflammatory responses, in the prevention of transplantation rejection and tumor development, as well as in the modulation of autoimmune diseases and allergic response. As an important regulatory factor in the signaling pathways, mouse anti-human GL50 monoclonal antibodies provide powerful tools for us to further study the co-stimulatory signal pathway and cross-talking in immune response. So we can investigate the essence of immune response. Thus, establishment of human GL50 transfected cell line and monoclonal antibodies against human GL50 may provide a valuable and potential way for the clinical diagnosis, treatment and prevention of diseases.This article consists of two parts:Part I Establishment of cell line transfected with human GL50 gene and preliminary study on its co-stimulatory effect on T cell activation and proliferation in vitroTotal RNA was extracted from B lymphoma cell line Daudi, which was reverse transcripted into cDNA . The human GL50 gene was then amplified by RT-PCR. The target gene was inserted into vector pIRES2-EGFP and confirmed by sequencing. The recombinant pIRES2-EGFP-GL50 vector was transfected into L929 cells with LipofectamineTM 2000, and the cells was further selected with G418 for a long time. GL50 expressed on L929/GL50 cells was analysed by FCM and RT-PCR. Effect of L929/GL50 cells on T cells proliferation and cytokine production in vitro was studied by methods of CCK-8 and ELISA. In vitro, L929/GL50 cells could obviously promote the proliferation of T cells, while up-regulating the production of IL-4、IL-10 and IL-17.Part II Generation and characteration of mouse anti-human GL50 monoclonal antibody with biological functionThe L929/GL50 transgenic cells were used to immunize Balb/C mice. The immunized splenocytes were fused with murine myeloma cells (SP2/0) by the cell fuse technique. By means of HAT selective culture and repeated screening with L929/GL50 as antibody-screening positive cell and L929/mock as negative control, two hybridoma cell lines (2B4 and 4D11) continuously and steadily secreting specific anti-human GL50 were obtained. The two hybridomas grew well after long-term culture in vitro and storage in liquid nitrogen.The monoclonal antibody was produced according to ascites-inducing procedure in mouse abdominal cavity. The average yield of ascites was about 3.5 milliliter (ml) per mouse. After the ascites was purified by protein G affinity chromatography, the concentration of the protein was above 4 milligram (mg)/ml and the ascitic titer was over 1:1000 dilution by immunofluorescence analysis. Every 1×106 cells required 0.2～2 microgram (μg) purified monoclonal antibody in indirect immunofluorescence. Karyotype analysis result showed that the number of the chromosomes of the hybridoma was more than these of mouse somatic chromosomes, which suggested that they are fused cells.Fast-strip method analysis displayed that monoclonal antibodies 2B4 and 4D11 were mouse IgG1 and IgG2a, and the light chain wereκ. Dot-blot and flow cytometry analysis showed that monoclonal antibodies 2B4 and 4D11 exclusively binded GL50 molecules. The mAbs were also identified by ELISA.The competitive inhibition results showed that monoclonal antibodies (2B4、4D11) and MIH12, a commercial anti-GL50 antibody, recognized different epitopes.Flow cytometry analysis showed that GL50 molecule was highly expressed on cell lines such as Daudi, Raji, HO8910, THP-1 and ECV. B lymphocytes, isolated from peripheral blood hardly express GL50 molecules. L929/GL50 cells could obviously promote the proliferation of T cells. Specific mAbs 2B4 and 4D11 could act as blockers in the proliferation of T cells.更多还原