【作者】 庄羽美；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2005， 硕士

【摘要】 CD40是分子量48～50KD的Ⅰ型跨膜糖蛋白,属肿瘤坏死因子受体(TNFR)超家族成员。CD40广泛表达于许多类型细胞表面,包括不同发育和分化阶段的B淋巴细胞、单核细胞、树突状细胞、造血前体细胞和内皮细胞以及一系列肿瘤细胞等。CD40是免疫应答中关键性的共信号分子,其与配体(CD40L)的相互作用提供T、B细胞活化所必需的第二信号。CD40分子可介导B细胞的增殖、分化、抗体的分泌和类型转换;树突状细胞的激发、分化和成熟;T淋巴细胞的活化及细胞免疫功能的调节;内皮细胞粘附分子的改变,炎症因子的释放,白细胞在炎症部位的聚集等。 近年研究表明,多种疾病的发生发展均与CD40分子相关,如自身免疫性疾病、传染性疾病、移植排斥、肿瘤及动脉粥样硬化等。运用阻断型CD40L抗体在动物模型上可以显著延长移植器官的生存,但仍然存在副作用和安全性的问题,而直接阻断CD40分子的治疗策略有望能改善其缺点。最近发现CD40分子除了膜型的形式还存在着可溶性的形式—sCD40,膜型的CD40分子经金属蛋白酶(MP)裂解,释放至液相成为sCD40,可阻断膜型CD40和CD40L的相互作用,负反馈调节免疫应答。这种可溶性形式的CD40分子在肾脏和肝脏疾病患者的血清中的浓度远高于正常人,和这些疾病的发生、发展有密切的相关性。目前关于CD40阻断型抗体和sCD40方面的报道甚少,基于我室已经成功研制了激发型CD40单克隆抗体,研制识别位点不同、功能不同的阻断型CD40单抗以及sCD40 ELISA试剂盒将在肿瘤、移植排斥反应和自身免疫性疾病中具有重要的理论研究和临床应用价值。 本研究以高表达CD40分子的XG2细胞株为抗原,应用SP2/0作为亲本骨髓瘤细胞,采用细胞融合、单克隆抗体筛选、荧光标记、免疫印迹和竞争抑制等方法获得并建立了一株持续分泌特异性鼠抗人CD40单克隆抗体(mAb)的杂交瘤细胞株,命名为3G3。经体外长期传代培养、液氮冻存半年以上复苏良好,分泌抗体的性能稳定。 杂交瘤细胞经体外扩大培养,诱导同种小鼠产生的腹水型单抗,经Protein G纯更多还原

【Abstract】 CD40, a 48KD cell membrane molecule, is a type I glycoprotein, and belongs to the tumor necrosis factor receptor (TNFR) superfamily. CD40 is expressed in many cell types including B cells, monocytes, dendritic cells, endothelial cells, epithelial ceils and also many carcinoma cells. CD40 is a pivotal costimulatory molecule in immune response, and with its ligand (CD40L) transduces the second signal the activation of T and B cell needs. To date, an increasing number of studies indicate that signals mediated by CD40 will lead to B cell proliferation, Ig secretion and its class switch; dendritic cell activation and maturation; adhesion molecules expression on endothelial cells, inflammatory factors secretion, and the accumulation of white cells to the inflammatory sites.Recent reports show the association of CD40 signaling with many human diseases, including autoimmune diseases, infectious diseases, allograft rejection, cancers and atherosclerosis. The blockade of CD40/CD40L interaction by CD40L antibodies, has been investigated to prolong the survival of experimentally transplanted organs. However, this agent was recently halted because of unexpected complication. Thus blocking CD40 mAb may provide an alternative way to inhibit the CD40 costimulatory signal. The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD40L interaction. sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. The levels of sCD40 increasing in some liver diseases have been used as a serum index of apoptosis in liver. Contin and colleagues by analysis sCD40 in uraemic patients suggested that the sCD40 compromised the humoral immune response in vivo. Although the crucial mechanism triggering the sCD40 production and its role in theprocesses of immune response still needs further investigation, the detection of sCD40 levels has been shown to be a very important tool in the diagnosis of many diseases.In this study we prepared another anti-human CD40 mAb 3G3 by using CD40 highly expressing MM cell line XG2 as immunogen. 3G3 was shown to bind a variety of CD40 positive cells but not CD40 negative cells. Competition experiments showed that 3G3 recognized a different epitope of CD40 molecule from that of 5C11, 3B2 or mAb89. Functional studies showed that 3G3 interfered with the homotypic aggregation induced by the agonistic CD40 mAb 5C11 in lymphoma Daudi cells line. What might be more important, 3G3 inhibited the proliferation of lymphocytes in mixed lymphocyte reaction assay. Thus 3G3 may be of great potential in the design of more effective and safer therapy through manipulation of CD40/CD40L interaction.In our current study, we used 5C11 mAb as capture antibody and 3G3 mAb as detection and set up an ELISA kit. What we found was that significantly higher levels of sCD40 were detected in sera of patients with hyperthyroidism, chronic nephritis, rheumatoid arthritis and lung cancer as compared to that of healthy individuals..In conclusion, we generated a novel blocking monoclonal antibody recognizing both membrane and soluble CD40 molecule. This anti-CD40 mAb has immunosuppressive characteristics via blocking CD40/CD40L interaction, which might be valuable in the design of alternative agent to prevent organ or tissue transplant rejection through the blockage of CD40/CD40L costimulatory signals; the sCD40 kit, composed of two different epitope-recognizing antibodies, provides a sensitive tool for the detection of soluble CD40 in patient serum, which may serve as an index in the clinical diagnosis for a variety of autoimmune diseases and cancers.更多还原