抗PEA单抗和单链抗体制备及在绿脓感染中的应用
发布时间：2018-09-01 12:30
【摘要】： 绿脓杆菌(Pseudomonas aeruginodsa PA)是导致外科手术或烧伤细菌感染死亡的最主要的病原细菌之一。绿脓杆菌外毒素A(PEA)是该菌的主要致病因子之一。PEA分为三个(I~III)功能区,I区与易感细胞识别有关;II区与PEA的“跨膜移位”有关;III区具有细胞毒性作用。PEA的毒性是其I区与细胞表面受体结合后,由受体介导的一系列反应引发的。所以抑制I区与受体的结合就可以阻断毒素的全部毒性作用。 为获得绿脓杆菌外毒素A受体结合区蛋白,本研究将PEA受体结合区亚单位基因克隆到表达载体pET28(c)中,构建了原核表达质粒pET-28(c)+B10,表达了重组的绿脓杆菌外毒素A受体结合区蛋白,进行了免疫学实验。进而利用该蛋白免疫Balb/c小鼠制备了抗PEA单克隆抗体,抗PEA(3C6)单克隆抗体经过正辛酸和硫酸铵纯化后,对严重绿脓杆菌感染小鼠进行了疗效实验,效果明显。 在此基础上,采用RT-PCR重叠延伸剪接法方法构建了单链抗体基因,并将基因克隆入pET28a中,筛选得到重组表达质粒pET28a-(PEA-1)ScFv。通过表达、变性、复性、纯化之后,进行了PEA的细胞毒性抑制实验。单链抗体在高浓度下能够起到一定的抑制作用。 本研究旨在寻求治疗烧伤后绿脓杆菌感染的新方法新途径。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginodsa PA) is one of the leading pathogenic bacteria that cause surgical or burn bacterial infection. Pseudomonas aeruginosa exotoxin A (PEA) is one of the main pathogenic factors of P. aeruginosa. PEA is divided into three (I~III) functional regions, region I and susceptible cell recognition related to the "transmembrane translocation" of PEA. After binding to the cell surface receptor, Triggered by a series of receptor-mediated reactions. Therefore, inhibition of the binding of region I to the receptor can block all the toxic effects of the toxin. In this study, the PEA receptor binding subunit gene was cloned into the expression vector pET28 (c), and the prokaryotic expression plasmid pET-28 (c) B10 was constructed to express the recombinant protein of Pseudomonas aeruginosa exotoxin A receptor binding region. Immunological experiments were carried out. Then Balb/c mice were immunized with this protein to prepare monoclonal antibody against PEA. After purification of monoclonal antibody against PEA (3C6) by n-octanoic acid and ammonium sulfate, the therapeutic effect of the monoclonal antibody against severe Pseudomonas aeruginosa infection in mice was studied. On this basis, the single chain antibody gene was constructed by RT-PCR overlapping extension splicing method, and cloned into pET28a. The recombinant expression plasmid pET28a- (PEA-1) ScFv. was screened. After expression, denaturation, renaturation and purification, cytotoxicity inhibition of PEA was studied. Single chain antibody (scFv) can play a certain inhibitory effect at high concentration. The aim of this study was to seek a new method for the treatment of Pseudomonas aeruginosa infection after burn.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
本文编号：2217206
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginodsa PA) is one of the leading pathogenic bacteria that cause surgical or burn bacterial infection. Pseudomonas aeruginosa exotoxin A (PEA) is one of the main pathogenic factors of P. aeruginosa. PEA is divided into three (I~III) functional regions, region I and susceptible cell recognition related to the "transmembrane translocation" of PEA. After binding to the cell surface receptor, Triggered by a series of receptor-mediated reactions. Therefore, inhibition of the binding of region I to the receptor can block all the toxic effects of the toxin. In this study, the PEA receptor binding subunit gene was cloned into the expression vector pET28 (c), and the prokaryotic expression plasmid pET-28 (c) B10 was constructed to express the recombinant protein of Pseudomonas aeruginosa exotoxin A receptor binding region. Immunological experiments were carried out. Then Balb/c mice were immunized with this protein to prepare monoclonal antibody against PEA. After purification of monoclonal antibody against PEA (3C6) by n-octanoic acid and ammonium sulfate, the therapeutic effect of the monoclonal antibody against severe Pseudomonas aeruginosa infection in mice was studied. On this basis, the single chain antibody gene was constructed by RT-PCR overlapping extension splicing method, and cloned into pET28a. The recombinant expression plasmid pET28a- (PEA-1) ScFv. was screened. After expression, denaturation, renaturation and purification, cytotoxicity inhibition of PEA was studied. Single chain antibody (scFv) can play a certain inhibitory effect at high concentration. The aim of this study was to seek a new method for the treatment of Pseudomonas aeruginosa infection after burn.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 才琳;抗黄曲霉毒素B1单克隆抗体和单链抗体的制备及活性研究[D];黑龙江八一农垦大学;2010年
，本文编号：2217206
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