【作者】 白利雄；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2011， 硕士

【摘要】 协同刺激分子在免疫调节中起着重要的作用,PD-1(CD279)所介导的负性信号能导致T细胞的凋亡和功能衰竭,PD-L2(B7-DC,CD273)作为PD-1的第二配体,与PD-1作用抑制T细胞活化和IFN-γ的分泌。PD-L2为B7超家族成员,在蛋白结构上包含有IgV样区、IgC样区、跨膜区和一个短而保守的胞浆区尾部。最近研究表明,PD-L2与血吸虫病、肺结核和过敏性哮喘的发病密切相关。PD-L2在同种异体反应、自身免疫性疾病中也发挥重要作用。研究表明,树突状细胞表面PD-L2与活化T细胞上PD-1相互作用后可显著抑制效应性T细胞的生物学功能和IL-2等细胞因子的产生,PD-L2分子的缺失导致PD-1/PD-L2抑制途径发生异常,进而引发机体产生自身免疫性疾病。许多协同刺激分子能分别以细胞膜型和可溶性两种形式存在,包括OX40L、CD40L、B7-H3和PD-1等。可溶性分子可以通过蛋白水解酶裂解细胞上的膜型分子而形成,也可以由免疫细胞直接分泌。许多可溶性协同刺激分子具有临床诊断价值,然而,可溶性PD-L2(sPD-L2)分子在机体中扮演何种角色,在疾病的发生发展中发挥何种生物学作用,目前尚缺乏深入研究。本课题研究获得了一株新型鼠抗人PD-L2功能性单克隆抗体,并对其生物学特性进行了初步研究;在此基础上建立了特异性检测人sPD-L2的ELISA方法,并对该ELISA检测体系的稳定性和精确性等进行分析、评价;利用sPD-L2的ELISA方法对健康人和支气管哮喘患者外周血中的sPD-L2表达水平及特性进行分析。一、鼠抗人PD-L2单克隆抗体的制备及生物学特性的研究【目的】研制特异性识别人PD-L2(B7-DC)的鼠单克隆抗体,并对其生物学特性和PD-L2分子的表达特性进行初步分析【方法】以高表达人PD-L2分子的基因转染细胞L929/PD-L2作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L2作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-L2单克隆抗体的杂交瘤细胞株;采用Western blot、Ig亚型快速定性试纸法、间接免疫荧光法和竞争结合抑制实验对单抗进行生物学特性的分析,继而利用该单抗进行免疫荧光标记和流式细胞术检测PD-L2在肿瘤细胞株和免疫细胞上的表达特性。【结果】通过多次融合和反复筛选,成功获得一株特异性鼠抗人PD-L2的杂交瘤,该杂交瘤分泌的单克隆抗体能特异识别人PD-L2分子。继而利用上述研制获得的单克隆抗体8F2进行免疫荧光标记和流式细胞检测发现,PD-L2表达在THP-1、SHI-1、U937等单核来源的肿瘤细胞株,以及上调性表达在成熟的树突状细胞和调节性T细胞上。【结论】成功地获得一株特异性鼠抗人PD-L2单克隆抗体,并对其生物学特性和表达谱进行初步分析,证明其识别抗原表位不同于商品化抗体,是一株新型鼠抗人PD-L2单抗,这为进一步研究PD-1/PD-L2信号通路在免疫应答中的生物学作用提供了有价值的物质基础。二、特异性检测人可溶性PD-L2蛋白ELISA试剂盒的研制及哮喘患者外周血sPD-L2表达水平的检测分析【目的】研制特异性检测人sPD-L2的ELISA试剂盒,为定量分析不同来源的临床标本和研究sPD-L2的功能提供有效的检测手段。利用该ELISA试剂盒检测健康人和哮喘患者外周血sPD-L2蛋白的表达,分析sPD-L2在哮喘发生发展过程中的作用机制。【方法】利用anti-PD-L2 mAb(8F2)作包被抗体在碳酸盐缓冲液中预包被ELISA板,biotin-anti-PD-L2 mAb(10D6)作为检测抗体识别与8F2相结合的抗原,再与Streptavidin-HRP反应,最后用TMB底物进行显色反应,建立双抗体夹心法检测sPD-L2的酶联检测试剂盒,并对该试剂盒的特异性进行分析。利用上述建立的ELISA试剂盒检测了80例健康人和110例哮喘患者外周血中sPD-L2的表达。【结果】成功研制出检测人sPD-L2的ELISA方法,该方法能特异性测定人sPD-L2蛋白,而与其他蛋白无交叉反应。试剂盒检测的标准曲线在抗原浓度为1.56-100ng/ml范围内有良好的线性关系,并具有良好的稳定性、准确性和特异性。利用该sPD-L2 ELISA方法对哮喘患者及健康人的外周血中该因子的表达水平进行定量分析显示,哮喘患者血清中sPD-L2的含量显著升高。【结论】特异性检测人sPD-L2 ELISA试剂盒的建立为定量分析sPD-L2蛋白因子提供了有效的检测手段。与健康对照组相比,哮喘患者外周血sPD-L2的表达水平明显升高。综上所述,本课题成功研制出能稳定分泌特异性鼠抗人PD-L2单克隆抗体,在此基础上研制出检测人sPD-L2的ELISA试剂盒,并且利用该试剂盒检测了哮喘病人外周血sPD-L2的表达。更多还原

【Abstract】 Costimulatory molecules play an important role in immune regulation, PD-1(CD279) mediated negative signals can lead to T cell apoptosis . As a second ligand of PD-1 , PD-L2(CD273) inhibit T cell activation and secretion of IFN-γ. Being one of the B7 superfamily, the structure of PD-L2 contains IgV, IgC-like area, transmembrane domain and a short cytoplasmic tail. Recent studies have shown, PD-L2 is closely related with schistosomiasis, tuberculosis and allergic asthma. PD-L2 also plays an important role in the allogeneic reaction and autoimmune diseases. Studies also show that PD-L2 expressing on the surface of dendritic cells interact with PD-1 on the T cells ,can inhibit the biological function of T cells.The absence of PD-L2 led to abnormal PD-1/PD-L2 pathway, and trigger the body to be suffered autoimmune disease.Mounting data demonstrated that many costimulatory molecules assume two forms of expression. Except membrane-bound forms, soluble forms have been found for several members of B7 family including OX40L,CD40L,B7-H3 and PD-1. The soluble protein is either shed from the membrane or produced by the special mRNA. Many soluble proteins are valuable for clinical diagnosis. However, the functions and biological significance of sPD-L2 remain unknown.In this study, we intend to generate and characterize monoclonal antibodies against human PD-L2. Then the mAbs were used to develope sandwich enzyme-linked immunosorbent assay (ELISA) systems for the detection and quantification of sPD-L2.Part 1 Generation and characterization of monoclonal antibodies against human PD-L2Objective : To prepare functional monoclonal antibodies against human PD-L2 molecule and analysis of their biological characteristics. Method : A stable human PD-L2 transfected cell line L929/PD-L2 was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning and repeated screening with L929/PD-L2 as target cells while L929/mock was used as the negative control, the hybridoma specifically secreting mouse anti-human PD-L2 monoclonal antibody was generated. Then its biological characterization was investigated by Western bloting, rapid murine Ig subclass typing method, indirect immunofluorescene, mutual competitive inhibition test.Results: After multiple fusion technique and repeated screening, One mouse anti-human PD-L2 monoclonal antibody was generated successfully, the monoclonal antibody (named as 8F2) could bind to human PD-L2 specially, and it recognized a new epitope of PD-L2. Furthermore, we use FCM to analyze the expression patterns of PD-L2 on human different cell lines.The datas indicate that PD-L2 express in THP-1, SHI-1, U937 monocytes and other sources of tumor cell lines, as well as increased expression in mature dendritic cells and activated T cells.Conclusion: One mouse anti-human PD-L2 monoclonal antibody was generated successfully, which recognize different epitopes. The PD-L2 monoclonal antibody provide the initial material for further study of the role of human PD-1/PD-L2 signaling pathway in the biological role of immune response.PartПDevelopment of sandwich ELISA systems for evaluating human soluble PD-L2Objective : To establish specific ELISA systems with high sensitivity to detect human soluble PD-L2 proteins respectively. Then detect The level of sPD-L2 of patients with asthma by the sPD-L2 ELISA .Method : Mouse anti-human PD-L2 mAb (8F2), used as coating antibody, was precoated in the ELISA plate with the CBS. Another biotin-anti-PD-L2 mAb (10D6) was used as detecting antibody to recognize the 8F2-bound soluble PD-L2 protein. Then the Streptavidin-HRP was added to the reaction system. Finally, TMB substrate was added for colorable reaction. The absorbance was measured by the microplate reader.Results: The results showed that the ELISA system for detecting human sPD-L2 was established successfully. It could be used to detect human sPD-L2 protein specially without any cross-reaction with other proteins. Standard curve of the system displayed favorable linear correlation when the concentration of sPD-L2 was 1.56-100ng/ml. The ELISA system had favorable stability, precision and specificity. The result showed that levels of sPD-L2 were significantly increased in patients with asthma serum.Conclusion: Two ELISA systems with high specificity and sensitivity provide useful methods to detect human sPD-L2. And levels of sPD-L2 were significantly increased in serum of patients with asthma.In conclusion, one monoclonal antibodies for human PD-L2 have been generated. Base on the mAbs, a ELISA assay for the detection of soluble PD-L2 with high stability, veracity and specificity was developed.更多还原