人源化氨基端脂多糖結(jié)合蛋白（NH－LBP）基因工程抗體的構(gòu)建及其生物學(xué)活性研究
發(fā)布時(shí)間：2018-11-08 14:39
【摘要】：背景: 內(nèi)毒素血癥(endotoxemia)是臨床創(chuàng)(燒)傷病人最常見(jiàn)的并發(fā)癥之一。LPS是G~-細(xì)菌細(xì)胞壁外膜的主要成分,當(dāng)細(xì)菌死亡時(shí)LPS釋放入血,由肝細(xì)胞產(chǎn)生的LBP將其傳遞給靶細(xì)胞膜上的CD14(mCD14)或血液循環(huán)中的可溶性CD14(sCD14),三者形成LPS/LBP/CD14三聯(lián)復(fù)合體,再經(jīng)過(guò)TLR4-MD2-MYD88跨膜受體識(shí)別復(fù)合物向靶細(xì)胞內(nèi)傳遞信號(hào),促使胞漿內(nèi)的NF-κB易位至胞核,導(dǎo)致一系列轉(zhuǎn)錄因子的活化,進(jìn)而釋放大量炎性介質(zhì)和細(xì)胞因子,誘發(fā)全身炎癥反應(yīng)綜合征(SystematicInflammatory Response Syndrome,SIRS)、多器官功能不全綜合征(Multiple OrganDysfunction Syndrome,MODS)。在生理狀態(tài)下循環(huán)血液中只有5—10μg/ml的LBP,在內(nèi)毒素血癥時(shí)LBP的濃度可以增高10倍左右,低濃度的LBP可將LPS與CD14的結(jié)合放大3-4個(gè)數(shù)量級(jí)。經(jīng)定點(diǎn)突變以及LBP衍生肽的競(jìng)爭(zhēng)性抑制研究,提示LBP結(jié)合LPS的位點(diǎn)定位于LBP氨基末端部分的第91-100位氨基酸殘基。因此推測(cè)阻斷LBP與LPS的結(jié)合有可能中斷或減弱LPS活化靶細(xì)胞的效應(yīng),能夠阻斷這種保留與LPS結(jié)合序列的截?cái)嘈腿税被┒薒BP(NH-Lipopolysaccharide bmdmg protein,NH-LBP)的抗體對(duì)全長(zhǎng)LBP可能具有同樣的阻斷效應(yīng)。 目前針對(duì)拮抗LBP的研究如動(dòng)物源性的單克隆抗體,小分子多肽等還處于實(shí)驗(yàn)室階段,研究結(jié)果顯示動(dòng)物源性的抗體存在以下局限性:制備工藝復(fù)雜,難以大量生產(chǎn);雜交瘤細(xì)胞的穩(wěn)定性較差;容易產(chǎn)生人抗鼠抗體(HAMA);難以制備融合蛋白等。全長(zhǎng)抗體由于分子量較大,在組織內(nèi)的滲透性較差,從血液循環(huán)中清除速度慢。近年來(lái)隨著分子生物學(xué)和分子免疫學(xué)的迅速發(fā)展出現(xiàn)了基因工程抗體,使人們得以對(duì)動(dòng)物源性的單克隆抗體進(jìn)行改造,構(gòu)建人鼠嵌合抗體或全人源化抗體,而且抗體的形式也多樣化如scFv,dsFv,雙功能抗體,Fab',Fab'_2和miniantibody等�；蚬こ炭贵w既可從雜交瘤細(xì)胞中制備,也可從未經(jīng)免疫的脾淋巴細(xì)胞或人外周血單核細(xì)胞中產(chǎn)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
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
本文編號(hào)：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
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 魏麟;豬LBP和BPI基因多態(tài)性及其蛋白質(zhì)N端功能研究[D];湖南農(nóng)業(yè)大學(xué);2011年
，本文編號(hào)：2318771
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