人和羊IgG对噬菌体展示细菌免疫球蛋白结合分子单结构域随机组合文库的体外进化筛选

发布时间：2018-06-14 09:25

本文选题：细菌噬菌体 + 免疫球蛋白G　；参考：《安徽医科大学学报》2013年08期

【摘要】：目的使用人和羊不同物种IgG来诱导噬菌体展示免疫球蛋白结合分子单结构域随机组合文库进行体外分子进化筛选,判断不同的Fc段构像是否具有特异的结合优势,同时探索其优势结合序列的组合特点。方法通过PCR获得金黄色葡萄球菌蛋白A(SpA)的A、B、D、E和链球菌(C和G群)蛋白G(SpG)的B2、B3各单结构域片段,构建由该6个片段随机组合的噬菌体展示文库。再分别使用人和羊IgG对文库进行体外亲和筛选,以期能够获得具有特异优势结合能力的组合分子,并在Phage ELISA的水平上进一步验证筛选结果。结果成功构建了符合进行体外进化筛选要求的噬菌体展示单结合结构域随机组合文库后,经过6轮人IgG诱导筛选,文库的展示片段大小统一为3个结构域组合分子,E-B-B3;经过6轮羊IgG诱导筛选,文库的展示片段大小也统一为4个结构域组合分子,D-A-B3-B3。Phage ELISA进一步验证最终组合序列的对人和羊IgG的结合能力。结论首次得到两种天然SpA、SpG分子中不存在的,且分别特异地与人和羊IgG具有强结合作用的新型组合分子E-B-B3、D-A-B3-B3,在人和羊IgG的纯化和检测具有很大的应用前景。
[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.
【作者单位】：安徽医科大学病理生理学教研室;第二军医大学微生物学教研室上海市医学生物防护重点实验室;安庆医药高等专科学校;
【基金】：安徽省教育厅自然基金(编号:KJ2010A117) 国家自然科学基金项目(编号:30872405、30872246、30972632、30972799) 上海市基础研究重点项目(编号:08JC1405200)
【分类号】：R392.7

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