Mac-1-FP融合蛋白在CHO细胞中的可调控性表达、鉴定和FRET技术对其相互作用的检测
发布时间：2018-11-13 07:26
【摘要】：白细胞粘附分子Mac-1(macrophage differentiation antigen associated with complement three receptor function)为位于白细胞表面的整合素家族β2亚族的成员之一,它是白细胞表面参与机体防御功能及免疫反应的极为重要的粘附分子。它由α(CD11b)和β(CD18)两个亚基以非共价键的方式形成异二聚体,编码CD11b和CD18的基因分别位于人的第16号和第21号染色体。Mac-1具有两种主要功能:一是与活化的内皮细胞表面的ICAM-1高亲和力的粘附,进而介导白细胞的游走与渗出;二是介导白细胞对iC_3b调理的微生物或免疫复合物的细胞毒功能。 迄今为止,Mac-1粘附功能的研究已经相当深入,但由于方法学的限制,对于Mac-1的分布、贮存、转位和变构的过程,尚未见有活细胞内对其全过程动态实时的定位定量的研究。以往文献上研究Mac-1的分布、贮存、转位的方法,主要采用的是抗体标记Mac-1的流式细胞术测定或免疫组化技术显微镜下直接观察Mac-1的细胞内分布,其缺点是不可能在单细胞和/或活细胞连续地观察,如果要研究在细胞内的走向则还需要增大细胞表面通透性,这就难免干扰了细胞的生理状态。并且集团平均(ensemble averaging)研究所探测的是细胞内大量分子的综合平均效应,得到的是由大量对象组成的一个整体所表现出的平均响应和平均值,因此从这些实验所得出的结果只代表这一测量时间内细胞内大量分子的平均行为,这一平均效应掩盖了许多特殊的信息。GFP(绿色荧光蛋白)及其突变体BFP(蓝色荧光蛋白)、CFP(青色荧光蛋白)、YFP(黄色荧光蛋白)具有发光不需要底物、稳定、无毒性、分子量小、形成融合蛋白后不致影响自身和目的基因产物的空间构象和功能等优点,已成为研究蛋白质分子在活细胞内变化的主要标记物。近年来,结合荧光蛋白技术的荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术已逐渐运用于活细胞内蛋白质之间相互作用的研究。FRET的发生取决于供体荧光和受体荧光的性
[Abstract]:Leukocyte adhesion molecule (Mac-1 (macrophage differentiation antigen associated with complement three receptor function) is a member of the integrin family 尾 2 subfamily located on the surface of leukocytes. It is a very important adhesion molecule on the surface of leukocytes involved in the defense function and immune response of the body. It is composed of 伪 (CD11b) and 尾 (CD18) subunits to form heterodimers in the form of non-covalent bonds. The genes encoding CD11b and CD18 are located on human chromosomes 16 and 21, respectively. Mac-1 has two main functions: one is to adhere to the high affinity of ICAM-1 on the surface of activated endothelial cells, and then to mediate the migration and exudation of leukocytes; The second is to mediate the cytotoxic function of leukocytes to the microorganism or immune complex mediated by iC_3b. Up to now, the research on the adhesion function of Mac-1 has been very thorough, but due to the limitation of methodology, the distribution, storage, transposition and metamorphism of Mac-1 have been studied. No study has been done on the dynamic and real-time localization of the whole process in living cells. In previous literatures, the distribution, storage and translocation of Mac-1 were studied mainly by flow cytometry with antibody labeled Mac-1 or by direct observation of the intracellular distribution of Mac-1 under the microscope of immunohistochemical technique. The disadvantage is that it is impossible to observe continuously in single cell and / or living cell. If we want to study the trend of cell, we need to increase the permeability of cell surface, which inevitably interferes with the physiological state of cell. And the group average (ensemble averaging) Institute detects the combined average effect of a large number of molecules in the cell, resulting in the average response and average value of a whole made up of a large number of objects. Therefore, the results obtained from these experiments only represent the average behavior of a large number of molecules in the cell during this measurement time. This average effect masked many special information. GFP (green fluorescent protein (GFP) and its mutant BFP (blue fluorescent protein). CFP (cyan fluorescent protein), YFP (yellow fluorescent protein) has the advantages of no substrate, stable, non-toxic, low molecular weight, and the formation of fusion protein will not affect the spatial conformation and function of its own and target gene products. It has become the main marker to study the changes of protein molecules in living cells. In recent years, fluorescence resonance energy transfer (fluorescence resonance energy transfer,FRET) combined with fluorescent protein technique has been gradually applied to the study of protein interaction in living cells. The occurrence of FRET depends on the fluorescence of donor and receptor.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q51
本文编号：2328414
[Abstract]:Leukocyte adhesion molecule (Mac-1 (macrophage differentiation antigen associated with complement three receptor function) is a member of the integrin family 尾 2 subfamily located on the surface of leukocytes. It is a very important adhesion molecule on the surface of leukocytes involved in the defense function and immune response of the body. It is composed of 伪 (CD11b) and 尾 (CD18) subunits to form heterodimers in the form of non-covalent bonds. The genes encoding CD11b and CD18 are located on human chromosomes 16 and 21, respectively. Mac-1 has two main functions: one is to adhere to the high affinity of ICAM-1 on the surface of activated endothelial cells, and then to mediate the migration and exudation of leukocytes; The second is to mediate the cytotoxic function of leukocytes to the microorganism or immune complex mediated by iC_3b. Up to now, the research on the adhesion function of Mac-1 has been very thorough, but due to the limitation of methodology, the distribution, storage, transposition and metamorphism of Mac-1 have been studied. No study has been done on the dynamic and real-time localization of the whole process in living cells. In previous literatures, the distribution, storage and translocation of Mac-1 were studied mainly by flow cytometry with antibody labeled Mac-1 or by direct observation of the intracellular distribution of Mac-1 under the microscope of immunohistochemical technique. The disadvantage is that it is impossible to observe continuously in single cell and / or living cell. If we want to study the trend of cell, we need to increase the permeability of cell surface, which inevitably interferes with the physiological state of cell. And the group average (ensemble averaging) Institute detects the combined average effect of a large number of molecules in the cell, resulting in the average response and average value of a whole made up of a large number of objects. Therefore, the results obtained from these experiments only represent the average behavior of a large number of molecules in the cell during this measurement time. This average effect masked many special information. GFP (green fluorescent protein (GFP) and its mutant BFP (blue fluorescent protein). CFP (cyan fluorescent protein), YFP (yellow fluorescent protein) has the advantages of no substrate, stable, non-toxic, low molecular weight, and the formation of fusion protein will not affect the spatial conformation and function of its own and target gene products. It has become the main marker to study the changes of protein molecules in living cells. In recent years, fluorescence resonance energy transfer (fluorescence resonance energy transfer,FRET) combined with fluorescent protein technique has been gradually applied to the study of protein interaction in living cells. The occurrence of FRET depends on the fluorescence of donor and receptor.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q51
【共引文献】
相关硕士学位论文 前3条
1 顾红祥;受控凋亡素基因逆转录病毒载体诱导人肝癌细胞凋亡研究[D];暨南大学;2003年
2 杨俊文;凋亡素基因原核及真核表达载体构建研究[D];暨南大学;2004年
3 杨小柯;用于CHO大规模表达的质粒载体的构建鉴定及相关工作建立[D];暨南大学;2005年
，本文编号：2328414
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