人源化氨基端脂多糖结合蛋白（NH－LBP）基因工程抗体的构建及其生物学活性研究
发布时间：2018-11-08 14:39
【摘要】：背景: 内毒素血症(endotoxemia)是临床创(烧)伤病人最常见的并发症之一。LPS是G~-细菌细胞壁外膜的主要成分,当细菌死亡时LPS释放入血,由肝细胞产生的LBP将其传递给靶细胞膜上的CD14(mCD14)或血液循环中的可溶性CD14(sCD14),三者形成LPS/LBP/CD14三联复合体,再经过TLR4-MD2-MYD88跨膜受体识别复合物向靶细胞内传递信号,促使胞浆内的NF-κB易位至胞核,导致一系列转录因子的活化,进而释放大量炎性介质和细胞因子,诱发全身炎症反应综合征(SystematicInflammatory Response Syndrome,SIRS)、多器官功能不全综合征(Multiple OrganDysfunction Syndrome,MODS)。在生理状态下循环血液中只有5—10μg/ml的LBP,在内毒素血症时LBP的浓度可以增高10倍左右,低浓度的LBP可将LPS与CD14的结合放大3-4个数量级。经定点突变以及LBP衍生肽的竞争性抑制研究,提示LBP结合LPS的位点定位于LBP氨基末端部分的第91-100位氨基酸残基。因此推测阻断LBP与LPS的结合有可能中断或减弱LPS活化靶细胞的效应,能够阻断这种保留与LPS结合序列的截断型人氨基末端LBP(NH-Lipopolysaccharide bmdmg protein,NH-LBP)的抗体对全长LBP可能具有同样的阻断效应。 目前针对拮抗LBP的研究如动物源性的单克隆抗体,小分子多肽等还处于实验室阶段,研究结果显示动物源性的抗体存在以下局限性:制备工艺复杂,难以大量生产;杂交瘤细胞的稳定性较差;容易产生人抗鼠抗体(HAMA);难以制备融合蛋白等。全长抗体由于分子量较大,在组织内的渗透性较差,从血液循环中清除速度慢。近年来随着分子生物学和分子免疫学的迅速发展出现了基因工程抗体,使人们得以对动物源性的单克隆抗体进行改造,构建人鼠嵌合抗体或全人源化抗体,而且抗体的形式也多样化如scFv,dsFv,双功能抗体,Fab',Fab'_2和miniantibody等。基因工程抗体既可从杂交瘤细胞中制备,也可从未经免疫的脾淋巴细胞或人外周血单核细胞中产生:可
[Abstract]:Background: endotoxemia (endotoxemia) is one of the most common complications in patients with clinical trauma (burn). LPS is the main component of cell wall adventitia, and LPS is released into blood when bacteria die. LBP produced by hepatocytes was transferred to CD14 (mCD14) on target cell membrane or soluble CD14 (sCD14) in blood circulation, which formed LPS/LBP/CD14 triplex complex. The transmembrane receptor recognition complex of TLR4-MD2-MYD88 transduces signals into the target cells, which promotes the translocation of NF- 魏 B in the cytoplasm to the nucleus, resulting in the activation of a series of transcription factors and the release of a large number of inflammatory mediators and cytokines. Induced systemic inflammatory response syndrome (SystematicInflammatory Response Syndrome,SIRS) and multiple organ dysfunction syndrome (Multiple OrganDysfunction Syndrome,MODS). Under physiological conditions, only 5-10 渭 g/ml of LBP, in circulating blood could increase the concentration of LBP by about 10 times during endotoxemia, and low concentration of LBP could amplify the binding of LPS and CD14 by 3-4 orders of magnitude. The results of site-directed mutagenesis and competitive inhibition of LBP derived peptides suggest that the site of LBP binding to LPS is located at the 91-100 amino acid residues in the amino terminal part of LBP. Therefore, it is speculated that blocking the binding of LBP to LPS may interrupt or attenuate the effect of LPS activation on target cells, and block the truncated human amino-terminal LBP (NH-Lipopolysaccharide bmdmg protein,) that retains the LPS binding sequence. NH-LBP) may have the same blocking effect on full-length LBP. At present, the studies of antagonistic LBP such as animal-derived monoclonal antibodies, small molecular peptides and so on are still in the laboratory stage. The results show that the animal-derived antibodies have the following limitations: the preparation process is complex, it is difficult to produce in large quantities; The stability of hybridoma cells is poor, and it is difficult to prepare fusion protein by producing human anti-mouse antibody (HAMA);. Due to its high molecular weight, full-length antibodies have poor permeability in tissues and slow clearance from blood circulation. In recent years, with the rapid development of molecular biology and molecular immunology, genetic engineering antibodies have emerged, which make it possible to modify the animal-derived monoclonal antibodies and construct human-mouse chimeric or full-humanized antibodies. And the forms of antibodies are diverse, such as scFv,dsFv, bifunctional antibodies, Fab',Fab'_2 and miniantibody. Genetically engineered antibodies can be produced either from hybridoma cells, from unimmunized splenic lymphocytes or from human peripheral blood mononuclear cells
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
本文编号：2318771
[Abstract]:Background: endotoxemia (endotoxemia) is one of the most common complications in patients with clinical trauma (burn). LPS is the main component of cell wall adventitia, and LPS is released into blood when bacteria die. LBP produced by hepatocytes was transferred to CD14 (mCD14) on target cell membrane or soluble CD14 (sCD14) in blood circulation, which formed LPS/LBP/CD14 triplex complex. The transmembrane receptor recognition complex of TLR4-MD2-MYD88 transduces signals into the target cells, which promotes the translocation of NF- 魏 B in the cytoplasm to the nucleus, resulting in the activation of a series of transcription factors and the release of a large number of inflammatory mediators and cytokines. Induced systemic inflammatory response syndrome (SystematicInflammatory Response Syndrome,SIRS) and multiple organ dysfunction syndrome (Multiple OrganDysfunction Syndrome,MODS). Under physiological conditions, only 5-10 渭 g/ml of LBP, in circulating blood could increase the concentration of LBP by about 10 times during endotoxemia, and low concentration of LBP could amplify the binding of LPS and CD14 by 3-4 orders of magnitude. The results of site-directed mutagenesis and competitive inhibition of LBP derived peptides suggest that the site of LBP binding to LPS is located at the 91-100 amino acid residues in the amino terminal part of LBP. Therefore, it is speculated that blocking the binding of LBP to LPS may interrupt or attenuate the effect of LPS activation on target cells, and block the truncated human amino-terminal LBP (NH-Lipopolysaccharide bmdmg protein,) that retains the LPS binding sequence. NH-LBP) may have the same blocking effect on full-length LBP. At present, the studies of antagonistic LBP such as animal-derived monoclonal antibodies, small molecular peptides and so on are still in the laboratory stage. The results show that the animal-derived antibodies have the following limitations: the preparation process is complex, it is difficult to produce in large quantities; The stability of hybridoma cells is poor, and it is difficult to prepare fusion protein by producing human anti-mouse antibody (HAMA);. Due to its high molecular weight, full-length antibodies have poor permeability in tissues and slow clearance from blood circulation. In recent years, with the rapid development of molecular biology and molecular immunology, genetic engineering antibodies have emerged, which make it possible to modify the animal-derived monoclonal antibodies and construct human-mouse chimeric or full-humanized antibodies. And the forms of antibodies are diverse, such as scFv,dsFv, bifunctional antibodies, Fab',Fab'_2 and miniantibody. Genetically engineered antibodies can be produced either from hybridoma cells, from unimmunized splenic lymphocytes or from human peripheral blood mononuclear cells
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
【引证文献】
相关博士学位论文 前1条
1 魏麟;猪LBP和BPI基因多态性及其蛋白质N端功能研究[D];湖南农业大学;2011年
，本文编号：2318771
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