Mac-1-FP融合蛋白在CHO細胞中的可調(diào)控性表達、鑒定和FRET技術(shù)對其相互作用的檢測
發(fā)布時間：2018-11-13 07:26
【摘要】：白細胞粘附分子Mac-1(macrophage differentiation antigen associated with complement three receptor function)為位于白細胞表面的整合素家族β2亞族的成員之一,它是白細胞表面參與機體防御功能及免疫反應(yīng)的極為重要的粘附分子。它由α(CD11b)和β(CD18)兩個亞基以非共價鍵的方式形成異二聚體,編碼CD11b和CD18的基因分別位于人的第16號和第21號染色體。Mac-1具有兩種主要功能:一是與活化的內(nèi)皮細胞表面的ICAM-1高親和力的粘附,進而介導(dǎo)白細胞的游走與滲出;二是介導(dǎo)白細胞對iC_3b調(diào)理的微生物或免疫復(fù)合物的細胞毒功能。 迄今為止,Mac-1粘附功能的研究已經(jīng)相當(dāng)深入,但由于方法學(xué)的限制,對于Mac-1的分布、貯存、轉(zhuǎn)位和變構(gòu)的過程,尚未見有活細胞內(nèi)對其全過程動態(tài)實時的定位定量的研究。以往文獻上研究Mac-1的分布、貯存、轉(zhuǎn)位的方法,主要采用的是抗體標(biāo)記Mac-1的流式細胞術(shù)測定或免疫組化技術(shù)顯微鏡下直接觀察Mac-1的細胞內(nèi)分布,其缺點是不可能在單細胞和/或活細胞連續(xù)地觀察,如果要研究在細胞內(nèi)的走向則還需要增大細胞表面通透性,這就難免干擾了細胞的生理狀態(tài)。并且集團平均(ensemble averaging)研究所探測的是細胞內(nèi)大量分子的綜合平均效應(yīng),得到的是由大量對象組成的一個整體所表現(xiàn)出的平均響應(yīng)和平均值,因此從這些實驗所得出的結(jié)果只代表這一測量時間內(nèi)細胞內(nèi)大量分子的平均行為,這一平均效應(yīng)掩蓋了許多特殊的信息。GFP(綠色熒光蛋白)及其突變體BFP(藍色熒光蛋白)、CFP(青色熒光蛋白)、YFP(黃色熒光蛋白)具有發(fā)光不需要底物、穩(wěn)定、無毒性、分子量小、形成融合蛋白后不致影響自身和目的基因產(chǎn)物的空間構(gòu)象和功能等優(yōu)點,已成為研究蛋白質(zhì)分子在活細胞內(nèi)變化的主要標(biāo)記物。近年來,結(jié)合熒光蛋白技術(shù)的熒光共振能量轉(zhuǎn)移(fluorescence resonance energy transfer,FRET)技術(shù)已逐漸運用于活細胞內(nèi)蛋白質(zhì)之間相互作用的研究。FRET的發(fā)生取決于供體熒光和受體熒光的性
[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 偽 (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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q51
本文編號：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 偽 (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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q51
【共引文獻】
相關(guān)碩士學(xué)位論文 前3條
1 顧紅祥;受控凋亡素基因逆轉(zhuǎn)錄病毒載體誘導(dǎo)人肝癌細胞凋亡研究[D];暨南大學(xué);2003年
2 楊俊文;凋亡素基因原核及真核表達載體構(gòu)建研究[D];暨南大學(xué);2004年
3 楊小柯;用于CHO大規(guī)模表達的質(zhì)粒載體的構(gòu)建鑒定及相關(guān)工作建立[D];暨南大學(xué);2005年
，本文編號：2328414
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