鼠抗人4IgB7-H3单克隆抗体的研制及其初步应用
发布时间：2018-09-04 07:16
【摘要】： 协同刺激分子(co-stimulatory molecules)在免疫调节中起着重要的作用,B7-H3是新近发现的协同刺激分子家族成员。人B7-H3有两种不同形式的剪切体:B7-H3a和B7-H3b。B7-H3a胞外段由IgV-IgC两个免疫球蛋白结构域组成,故又称为2IgB7-H3,而B7-H3b胞外段由IgV1-IgC1-IgV2-IgC2四个免疫球蛋白结构域组成,故又称为4IgB7-H3。B7-H3mRNA表达水平极其广泛,在几乎所有淋巴组织及非淋巴组织均能检测到该分子mRNA的转录,其中4IgB7-H3mRNA表达丰度远高于2IgB7-H3mRNA,因此,既往的研究均认为4IgB7-H3是B7-H3的主要表达形式。但由于没有特异性识别人4IgB7-H3的单克隆抗体,所以无法明确4IgB7-H3在组织中的蛋白表达谱。本研究制备了一株可以特异性识别人4IgB7-H3的鼠源性抗人B7-H3单克隆抗体,继而研究和分析了该分子的表达谱。 目的:研制特异性识别鼠抗人4IgB7-H3单克隆抗体并对其进行了初步应用。 方法:以高表达人4IgB7-H3的293T细胞为免疫原,常规免疫小鼠、细胞融合和筛选。L929/4IgB7-H3基因转染细胞为抗体筛选阳性细胞,L929/2IgB7-H3基因转染细胞为抗体筛选阴性细胞,经间接免疫荧光标记和流式细胞术对杂交瘤细胞进行反复筛选和多次亚克隆化,流式细胞术分析抗体对B7家族不同成员基因转染细胞的识别。经位点竞争实验对该抗体抗原表位的识别特异性进行了鉴定。利用两株抗体采用流式细胞术检测4IgB7-H3在肿瘤细胞株和免疫细胞上的表达特性以及免疫组织化学方法检测此两株抗体在肿瘤细胞株和肿瘤组织上的识别情况。 结果:成功地制备了两株持续稳定分泌鼠抗人B7-H3单克隆抗体的杂交瘤细胞株4C3和9C3,其中单抗4C3既能识别2IgB7-H3又能识别4IgB7-H3分子,而单抗9C3只能识别4IgB7-H3。蛋白水平上4IgB7-H3相当局限地表达在一些肿瘤细胞株、人外周血单核细胞以及成熟的树突状细胞;而B7-H3(2IgB7-H3和/或4IgB7-H3)分子广泛地表达在多种肿瘤细胞株、单核细胞以及树突状细胞。此外,免疫组织化学分析表明4IgB7-H3可在脑胶质瘤组织中特异性表达,并随着胶质瘤病理分级的升高而递增。 结论:获得了两株稳定分泌鼠抗人B7-H3单克隆抗体的杂交瘤细胞株4C3和9C3。4IgB7-H3单克隆抗体9C3的成功获得为进一步分析人B7-H3两种剪切体的表达特性以及生物学功能提供了物质基础。
[Abstract]:Costimulator (co-stimulatory molecules) plays an important role in immunomodulation. B7-H3 is a newly discovered member of costimulator family. Human B7-H3 has two different forms of shearing bodies: B7-H3a and B7-H3b.B7-H3a, which are composed of two immunoglobulin domains (IgV-IgC), so they are also called 2IgB7-H3. The extracellular segments of B7-H3b are composed of four immunoglobulin domains (IgV1-IgC1-IgV2-IgC2), so they are also called 4IgB7-H3.B7-H3mRNA. The transcription of mRNA was detected in almost all lymphoid tissues and non-lymphoid tissues, in which the abundance of 4IgB7-H3mRNA expression was much higher than that of 2IgB7-H3mRNAs. Therefore, previous studies have suggested that 4IgB7-H3 is the main expression form of B7-H3. However, there is no specific monoclonal antibody to recognize human 4IgB7-H3, so it is impossible to identify the protein expression profile of 4IgB7-H3 in tissues. In this study, a murine monoclonal antibody against human 4IgB7-H3 was prepared and its expression profile was analyzed. Objective: to develop a specific mouse anti-human 4IgB7-H3 monoclonal antibody and its preliminary application. Methods: 293T cells with high expression of human 4IgB7-H3 were used as immunogen, and mice were immunized by routine immunization. The cells transfected with. L929 / 4IgB7-H3 gene were used as antibody screening positive cells. L929 / 2 IgB7-H3 gene transfected cells were used as antibody screening negative cells. The hybridoma cells were screened repeatedly by indirect immunofluorescence and flow cytometry. The recognition of different members of B7 gene transfection cells was analyzed by flow cytometry. The recognition specificity of the antigen epitope was identified by site competition test. The expression of 4IgB7-H3 in tumor cell lines and immune cells was detected by flow cytometry and the recognition of these two antibodies in tumor cell lines and tumor tissues was detected by immunohistochemical method. Results: two hybridoma cell lines, 4C3 and 9C3, which secreted murine monoclonal antibody against human B7-H3 successfully, were successfully prepared. McAb 4C3 could recognize both 2IgB7-H3 and 4IgB7-H3 molecules, while McAb 9C3 could recognize only 4IgB7-H3. At the protein level, 4IgB7-H3 is expressed in some tumor cell lines, human peripheral blood monocytes and mature dendritic cells, while B7-H3 (2IgB7-H3 and / or 4IgB7-H3) molecules are widely expressed in many tumor cell lines, monocytes and dendritic cells. In addition, immunohistochemical analysis showed that 4IgB7-H3 could be expressed specifically in gliomas and increased with the increase of glioma pathological grade. Conclusion: two hybridoma cell lines, 4C3 and 9C3.4IgB7-H3 monoclonal antibody 9C3, which secrete murine anti-human B7-H3 monoclonal antibody stably, have been successfully obtained, which provide a material basis for further analysis of the expression characteristics and biological functions of human B7-H3.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2221377
[Abstract]:Costimulator (co-stimulatory molecules) plays an important role in immunomodulation. B7-H3 is a newly discovered member of costimulator family. Human B7-H3 has two different forms of shearing bodies: B7-H3a and B7-H3b.B7-H3a, which are composed of two immunoglobulin domains (IgV-IgC), so they are also called 2IgB7-H3. The extracellular segments of B7-H3b are composed of four immunoglobulin domains (IgV1-IgC1-IgV2-IgC2), so they are also called 4IgB7-H3.B7-H3mRNA. The transcription of mRNA was detected in almost all lymphoid tissues and non-lymphoid tissues, in which the abundance of 4IgB7-H3mRNA expression was much higher than that of 2IgB7-H3mRNAs. Therefore, previous studies have suggested that 4IgB7-H3 is the main expression form of B7-H3. However, there is no specific monoclonal antibody to recognize human 4IgB7-H3, so it is impossible to identify the protein expression profile of 4IgB7-H3 in tissues. In this study, a murine monoclonal antibody against human 4IgB7-H3 was prepared and its expression profile was analyzed. Objective: to develop a specific mouse anti-human 4IgB7-H3 monoclonal antibody and its preliminary application. Methods: 293T cells with high expression of human 4IgB7-H3 were used as immunogen, and mice were immunized by routine immunization. The cells transfected with. L929 / 4IgB7-H3 gene were used as antibody screening positive cells. L929 / 2 IgB7-H3 gene transfected cells were used as antibody screening negative cells. The hybridoma cells were screened repeatedly by indirect immunofluorescence and flow cytometry. The recognition of different members of B7 gene transfection cells was analyzed by flow cytometry. The recognition specificity of the antigen epitope was identified by site competition test. The expression of 4IgB7-H3 in tumor cell lines and immune cells was detected by flow cytometry and the recognition of these two antibodies in tumor cell lines and tumor tissues was detected by immunohistochemical method. Results: two hybridoma cell lines, 4C3 and 9C3, which secreted murine monoclonal antibody against human B7-H3 successfully, were successfully prepared. McAb 4C3 could recognize both 2IgB7-H3 and 4IgB7-H3 molecules, while McAb 9C3 could recognize only 4IgB7-H3. At the protein level, 4IgB7-H3 is expressed in some tumor cell lines, human peripheral blood monocytes and mature dendritic cells, while B7-H3 (2IgB7-H3 and / or 4IgB7-H3) molecules are widely expressed in many tumor cell lines, monocytes and dendritic cells. In addition, immunohistochemical analysis showed that 4IgB7-H3 could be expressed specifically in gliomas and increased with the increase of glioma pathological grade. Conclusion: two hybridoma cell lines, 4C3 and 9C3.4IgB7-H3 monoclonal antibody 9C3, which secrete murine anti-human B7-H3 monoclonal antibody stably, have been successfully obtained, which provide a material basis for further analysis of the expression characteristics and biological functions of human B7-H3.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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