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  本文選題：細菌噬菌體 + 免疫球蛋白G��； 參考：《安徽醫(yī)科大學學報》2013年08期

【摘要】：目的使用人和羊不同物種IgG來誘導噬菌體展示免疫球蛋白結(jié)合分子單結(jié)構(gòu)域隨機組合文庫進行體外分子進化篩選,判斷不同的Fc段構(gòu)像是否具有特異的結(jié)合優(yōu)勢,同時探索其優(yōu)勢結(jié)合序列的組合特點。方法通過PCR獲得金黃色葡萄球菌蛋白A(SpA)的A、B、D、E和鏈球菌(C和G群)蛋白G(SpG)的B2、B3各單結(jié)構(gòu)域片段,構(gòu)建由該6個片段隨機組合的噬菌體展示文庫。再分別使用人和羊IgG對文庫進行體外親和篩選,以期能夠獲得具有特異優(yōu)勢結(jié)合能力的組合分子,并在Phage ELISA的水平上進一步驗證篩選結(jié)果。結(jié)果成功構(gòu)建了符合進行體外進化篩選要求的噬菌體展示單結(jié)合結(jié)構(gòu)域隨機組合文庫后,經(jīng)過6輪人IgG誘導篩選,文庫的展示片段大小統(tǒng)一為3個結(jié)構(gòu)域組合分子,E-B-B3;經(jīng)過6輪羊IgG誘導篩選,文庫的展示片段大小也統(tǒng)一為4個結(jié)構(gòu)域組合分子,D-A-B3-B3。Phage ELISA進一步驗證最終組合序列的對人和羊IgG的結(jié)合能力。結(jié)論首次得到兩種天然SpA、SpG分子中不存在的,且分別特異地與人和羊IgG具有強結(jié)合作用的新型組合分子E-B-B3、D-A-B3-B3,在人和羊IgG的純化和檢測具有很大的應用前景。
[Abstract]:Objective to use IgG from different species of human and sheep to induce phage display immunoglobulin binding molecular single domain random combination library for in vitro molecular evolution screening to determine whether different FC conformation has specific binding advantage. At the same time, it explores the combination characteristics of its advantage combination sequence. Methods the single domain fragments of staphylococcus aureus protein Agna spa E and streptococcal G group C and G group were obtained by PCR, and the phage display library was constructed by random combination of the six fragments. Then human and sheep IgG were used to screen the library in vitro in order to obtain the combination molecules with specific dominant binding ability and to further verify the screening results at the level of Phage Elisa. Results the phage display mono-binding domain random combination library was successfully constructed after six rounds of human IgG induction screening. The displayed fragment size of the library was unified into three domain assemblage molecules, E-B-B3, which were screened by six rounds of sheep IgG induction. The displayed fragment size of the library was also unified into four domain combinators, D-A-B3-B3.Phage Elisa, to further verify the binding ability of the final combination sequence to human and sheep IgG. Conclusion for the first time, two kinds of natural SpAG-free molecules, E-B-B3D-A-B3-B3, which are non-existent and strongly bound to human and sheep IgG, are obtained for the first time, which have great application prospect in the purification and detection of human and sheep IgG.
【作者單位】： 安徽醫(yī)科大學病理生理學教研室;第二軍醫(yī)大學微生物學教研室 上海市醫(yī)學生物防護重點實驗室;安慶醫(yī)藥高等�？茖W校;
【基金】：安徽省教育廳自然基金(編號:KJ2010A117) 國家自然科學基金項目(編號:30872405、30872246、30972632、30972799) 上海市基礎研究重點項目(編號:08JC1405200)
【分類號】：R392.7

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