抗PEA單抗和單鏈抗體制備及在綠膿感染中的應(yīng)用
發(fā)布時間：2018-09-01 12:30
【摘要】： 綠膿桿菌(Pseudomonas aeruginodsa PA)是導(dǎo)致外科手術(shù)或燒傷細菌感染死亡的最主要的病原細菌之一。綠膿桿菌外毒素A(PEA)是該菌的主要致病因子之一。PEA分為三個(I~III)功能區(qū),I區(qū)與易感細胞識別有關(guān);II區(qū)與PEA的“跨膜移位”有關(guān);III區(qū)具有細胞毒性作用。PEA的毒性是其I區(qū)與細胞表面受體結(jié)合后,由受體介導(dǎo)的一系列反應(yīng)引發(fā)的。所以抑制I區(qū)與受體的結(jié)合就可以阻斷毒素的全部毒性作用。 為獲得綠膿桿菌外毒素A受體結(jié)合區(qū)蛋白,本研究將PEA受體結(jié)合區(qū)亞單位基因克隆到表達載體pET28(c)中,構(gòu)建了原核表達質(zhì)粒pET-28(c)+B10,表達了重組的綠膿桿菌外毒素A受體結(jié)合區(qū)蛋白,進行了免疫學(xué)實驗。進而利用該蛋白免疫Balb/c小鼠制備了抗PEA單克隆抗體,抗PEA(3C6)單克隆抗體經(jīng)過正辛酸和硫酸銨純化后,對嚴重綠膿桿菌感染小鼠進行了療效實驗,效果明顯。 在此基礎(chǔ)上,采用RT-PCR重疊延伸剪接法方法構(gòu)建了單鏈抗體基因,并將基因克隆入pET28a中,篩選得到重組表達質(zhì)粒pET28a-(PEA-1)ScFv。通過表達、變性、復(fù)性、純化之后,進行了PEA的細胞毒性抑制實驗。單鏈抗體在高濃度下能夠起到一定的抑制作用。 本研究旨在尋求治療燒傷后綠膿桿菌感染的新方法新途徑。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
【引證文獻】
相關(guān)碩士學(xué)位論文 前1條
1 才琳;抗黃曲霉毒素B1單克隆抗體和單鏈抗體的制備及活性研究[D];黑龍江八一農(nóng)墾大學(xué);2010年
，本文編號：2217206
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