抗人脂多糖结合蛋白二硫键稳定性Fv抗体的构建表达及生物学活性的初步研究
发布时间：2018-12-11 16:21
【摘要】： Gˉ杆菌感染过程中,其产生的内毒素引起的感染性休克和多器官功能障碍综合症(multiple organs dysfunction syndrome, MODS)被认为是感染及内毒素血症患者死亡的根本原因。细菌内毒素又称为脂多糖(lipopolysaccharide, LPS),是该类病症的主要致病物质。脂多糖结合蛋白(Lipopolysaccharide Binding Protein,LBP)是一种促进LPS与效应细胞受体相结合的关键载体蛋白,在炎症反应过程中具有重要的作用。 研究表明,LBP能显著促进LPS与单核/巨噬细胞膜表面的CD14(membraneCD14 mCD14)结合,形成LPS-LBP-mCD14复合物,经TLR4-MD-MYD88和NF-Kappa B途径,激活单核/巨噬细胞,促进炎症的发生、发展;LBP的N、C端分别为与LPS和mCD14的结合区,因此若能研究获得针对LBP的抗体,特别是针对LBP N端(N-teminatal fragment of human LBP,NH-LBP)的抗体,则可能阻断LPS与LBP的结合,从而降低内毒素介导的炎症反应,降低Gˉ杆菌感染及脓毒性休克的死亡率。 本研究以人LBP及人NH-LBP为抗原,对本课题组构建的人源噬菌体抗体库(Fab)进行筛选,获得针对人源LBP及人源NH-LBP均有高结合力的单克隆抗体菌株;通过mega-PCR引入半胱氨酸(Cys)及酶切位点,并连接表达载体pET-28a(+),使得dsFvV_H链和dsFvV_L链分别在工程菌BL21 star(DE3)中高效表达。抽提其包涵体并纯化,随后使dsFvV_H链和dsFvV_L链蛋白在体外复性,通过半胱氨酸形成二硫键(-S-S-)连接,获得完整的针对LBP特别是NH-LBP的二硫键稳定Fv单克隆抗体(disulfide stabilized Fv fragment antibody dsFv),并初步研究其体内外生物学活性。 本研究的主要结果和结论如下: 1、以人LBP为抗原,以人源噬菌体抗体库(Fab)进行5轮筛选,在筛选过程中使抗体效价由3.8×10~8升高为3.1×10~(13),提高倍数为8.15×10~4倍,证明筛选效果良好。 2、获得的抗体库质粒(pComb3H)切除geneⅢ后,以LBP及NH-LBP为抗原经ELISA法鉴定,获得编码与人LBP及NH-LBP皆有高结合力的单克隆抗体菌株(4号菌),其表达抗体的结合力对于LBP为阴性对照的4.2倍,对于NH-LPB为阴性对照的3.8倍;通过对4号菌的质粒序序列测定及其表达上清的Western-Blotting检测,确定其表达的可溶性抗体Fab与LBP和NH-LBP可良好结合。
[Abstract]:Endotoxin-induced septic shock and multiple organ dysfunction syndrome (multiple organs dysfunction syndrome, MODS) are considered to be the root causes of death in patients with infection and endotoxemia. Bacterial endotoxin, also known as lipopolysaccharide (lipopolysaccharide, LPS), is the main pathogenic substance of this disease. Lipopolysaccharide binding protein (Lipopolysaccharide Binding Protein,LBP) is a key carrier protein that promotes the binding of LPS to effector cell receptors and plays an important role in inflammatory reaction. It has been shown that LBP can significantly promote the binding of LPS to CD14 (membraneCD14 mCD14) on the surface of mononuclear / macrophage cell membrane and form LPS-LBP-mCD14 complex. Through TLR4-MD-MYD88 and NF-Kappa B pathway, mononuclear / macrophage can be activated and inflammation can be promoted. Development; The C terminal of LBP is the binding region of LPS and mCD14, so if we can obtain the antibody against LBP, especially against LBP N terminal (N-teminatal fragment of human LBP,NH-LBP), we may block the binding of LPS to LBP. Thus, the inflammatory response mediated by endotoxin was reduced, and the mortality of infection and septic shock was decreased. In this study, human LBP and human NH-LBP were used as antigens to screen the human phage antibody library (Fab) constructed by our team, and the monoclonal antibody strains with high binding ability to both human LBP and human NH-LBP were obtained. Cysteine (Cys) and endonuclease sites were introduced by mega-PCR and ligated with expression vector pET-28a (), to make dsFvV_H and dsFvV_L chains highly expressed in BL21 star (DE3). The inclusion bodies were extracted and purified, then the dsFvV_H and dsFvV_L chain proteins were renatured in vitro, and disulfide bonds (-S-) were formed by cysteine. The complete disulfide bond of LBP, especially NH-LBP, was obtained to stabilize the Fv monoclonal antibody (disulfide stabilized Fv fragment antibody dsFv), and its biological activity in vitro and in vivo was preliminarily studied. The main results and conclusions of this study are as follows: 1. Using human LBP as antigen and human phage antibody library (Fab) for 5 rounds, the titer of antibody was increased from 3.8 脳 10 ~ (8) to 3.1 脳 10 ~ (13). The improvement was 8.15 脳 10 ~ (-1) times, which proved that the screening effect was good. 2. The obtained antibody library plasmid (pComb3H) was excised from gene 鈪，
本文编号：2372855
[Abstract]:Endotoxin-induced septic shock and multiple organ dysfunction syndrome (multiple organs dysfunction syndrome, MODS) are considered to be the root causes of death in patients with infection and endotoxemia. Bacterial endotoxin, also known as lipopolysaccharide (lipopolysaccharide, LPS), is the main pathogenic substance of this disease. Lipopolysaccharide binding protein (Lipopolysaccharide Binding Protein,LBP) is a key carrier protein that promotes the binding of LPS to effector cell receptors and plays an important role in inflammatory reaction. It has been shown that LBP can significantly promote the binding of LPS to CD14 (membraneCD14 mCD14) on the surface of mononuclear / macrophage cell membrane and form LPS-LBP-mCD14 complex. Through TLR4-MD-MYD88 and NF-Kappa B pathway, mononuclear / macrophage can be activated and inflammation can be promoted. Development; The C terminal of LBP is the binding region of LPS and mCD14, so if we can obtain the antibody against LBP, especially against LBP N terminal (N-teminatal fragment of human LBP,NH-LBP), we may block the binding of LPS to LBP. Thus, the inflammatory response mediated by endotoxin was reduced, and the mortality of infection and septic shock was decreased. In this study, human LBP and human NH-LBP were used as antigens to screen the human phage antibody library (Fab) constructed by our team, and the monoclonal antibody strains with high binding ability to both human LBP and human NH-LBP were obtained. Cysteine (Cys) and endonuclease sites were introduced by mega-PCR and ligated with expression vector pET-28a (), to make dsFvV_H and dsFvV_L chains highly expressed in BL21 star (DE3). The inclusion bodies were extracted and purified, then the dsFvV_H and dsFvV_L chain proteins were renatured in vitro, and disulfide bonds (-S-) were formed by cysteine. The complete disulfide bond of LBP, especially NH-LBP, was obtained to stabilize the Fv monoclonal antibody (disulfide stabilized Fv fragment antibody dsFv), and its biological activity in vitro and in vivo was preliminarily studied. The main results and conclusions of this study are as follows: 1. Using human LBP as antigen and human phage antibody library (Fab) for 5 rounds, the titer of antibody was increased from 3.8 脳 10 ~ (8) to 3.1 脳 10 ~ (13). The improvement was 8.15 脳 10 ~ (-1) times, which proved that the screening effect was good. 2. The obtained antibody library plasmid (pComb3H) was excised from gene 鈪，
本文编号：2372855
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2372855.html
最近更新
教材专著
