發(fā)布時間：2018-02-09 20:49

  本文關(guān)鍵詞： 載體構(gòu)建 scFv-Fc 抗體表達文庫 篩選 大規(guī)模發(fā)酵 全分子抗體 畢赤酵母　出處：《吉林大學》2007年博士論文　論文類型：學位論文

【摘要】： 本研究利用基因重組技術(shù)構(gòu)建了可在真核表達系統(tǒng)畢赤酵母中穩(wěn)定有效表達人小分子抗體scFv-Fc的表達載體pPICZα/scFv-Fc,建立了一套可表達篩選人小分子抗體scFv-Fc的真核表達體系。應用該表達體系構(gòu)建人抗狂犬病毒小分子抗體scFv-Fc的表達文庫,利用抗體的抗原特異結(jié)合活性從中篩選人抗狂犬病毒小分子抗體scFv-Fc,結(jié)果獲得了新的具有狂犬病毒抗原結(jié)合活性的抗體可變區(qū)序列,其中克隆RS3(輕鏈可變區(qū)為κ鏈)和RS9(輕鏈可變區(qū)為λ鏈)具有較好的scFv-Fc小分子抗體表達及狂犬病毒抗原結(jié)合活性。進而在80L發(fā)酵條件下,對畢赤酵母工程菌RS3表達條件進行了優(yōu)化,并建立一種適合于大規(guī)模純化人抗狂犬病毒小分子抗體scFv-Fc的方法。 另外,我們應用基因重組技術(shù),將篩選獲得的抗狂犬病毒小分子抗體RS3的重鏈和輕鏈可變區(qū)基因重組到本實驗室已構(gòu)建的完整人重鏈及輕鏈表達載體pPICZαCH及pPICZαCκ,利用分步整合法轉(zhuǎn)化X33酵母菌對抗狂犬病毒全分子抗體進行分泌表達,制備具狂犬病毒抗原結(jié)合活性的完整人抗體,鑒定重組抗體的生物學活性。結(jié)果表明,在畢赤酵母菌中可經(jīng)甲醇誘導產(chǎn)生具抗原結(jié)合活性的完整分泌型抗體。本實驗的創(chuàng)新之處在于:1)利用畢赤酵母構(gòu)建了人小分子抗體scFv-Fc的表達篩選體系并獲得了新的具有狂犬病毒抗原結(jié)合活性的抗體可變區(qū)序列,國內(nèi)外尚未見報道;2)建立了大規(guī)模發(fā)酵和純化人小分子抗體scFv-Fc的方法,國內(nèi)尚未見報道;3)利用分步整合法電轉(zhuǎn)化X33酵母菌,制備得到具狂犬病毒抗原結(jié)合活性的完整人抗體。
[Abstract]:In this study, the expression vector pPICZ 偽 -scFv-Fc, which can express human small molecule antibody scFv-Fc stably and effectively in Pichia pastoris, was constructed by gene recombination technique, and a set of eukaryotic expression system was established to express and screen human small molecule antibody scFv-Fc. The expression library of human anti-rabies virus small molecule antibody scFv-Fc was constructed by using this expression system. Using the antigen-specific binding activity of antibodies to human rabies virus small molecule antibody scFv-Fc, we obtained a new variable region of antibody with rabies virus antigen-binding activity. Among them, RS3 (light chain variable region is 魏 chain) and RS9 (light chain variable region is 位 chain) have good expression of scFv-Fc small molecule antibody and rabies virus antigen binding activity. The RS3 expression conditions of Pichia pastoris were optimized and a method was established for the large-scale purification of human anti-rabies virus small molecule antibody (scFv-Fc). In addition, we use gene recombination technology, The genes of heavy chain and light chain variable region of anti-rabies virus small molecule antibody RS3 were recombined into the complete human heavy chain and light chain expression vectors pPICZ 偽 Ch and pPICZ 偽 C 魏. The X33 yeast was transformed into X33 yeast by stepwise integration. The whole molecular antibody of canine virus is secreted and expressed. A complete human antibody with rabies virus antigen-binding activity was prepared and the biological activity of the recombinant antibody was identified. In Pichia pastoris, complete secretory antibodies with antigen-binding activity can be induced by methanol. The innovation of this experiment is that we constructed a screening system for the expression of human small molecule antibody scFv-Fc using Pichia pastoris and obtained a new one. A variable region of antibodies with rabies virus antigen-binding activity, The method of large-scale fermentation and purification of human small molecule antibody (scFv-Fc) has not been reported at home and abroad, and no report has been reported in China (3) the whole human antibody with rabies virus antigen-binding activity has been prepared by using stepwise integration method to electrotransform X33 yeast.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

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本文編號：1498829