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  

In vitro evolutional selection of a combinatorial phage library displaying randomly-rearranged various binding domains of SpA and SpG with human and goat IgG

作　　者：吴莉莉[1] 祁培培[2] 章萍萍[1] 王锦红[2] 张婧[1] 何婷[2] 潘卫[2] 邓松华[1,3] 

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032 [2]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,上海200433 [3]安庆医药高等专科学校,安庆246052

出　　处：《安徽医科大学学报》2013年第8期867-872,共6页Acta Universitatis Medicinalis Anhui

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)

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。Objective Using human IgG and goat IgG direct evolutional selection of a combinatorial phage library displaying randomly-rearranged various binding domains of protein A and protein G, and showing whether combina- tions of Ig-binding domains of various IBPs could produce useful novel binding properties. Methods Six DNA fragments of A, B, D and E domains of Staphylococcal protein A, B2 and B3 domains of Streptococcal protein G were obtained by PCR. Constructed a combinatorial phage library displaying these fragments and directed molecular evolution in vitro of this library by human IgG and goat IgG respectively to generate novel combination moleculars with special binding advantagese. Results We constructed the combinatorial phage library. Six rounds molecular e- volution of this library directed by human IgG and goat IgG respective generated E-B-B3 and D-A-B3-B3 respec- tively. The phage binding assays confirmed that the two molecules possessed binding advantagese with human IgG and goat IgG. Conclution In this work, two novel combination molecules of Ig-binding domains, E-B-B3 and D- A-B3-B3, are obtained from molecular evolution in vitro of a combinatorial phage library displaying six DNA frag- ments of protein A and protein G and presenting special binding abilities with human IgG and goat IgG which pro- vide a novel candidate molecule for the purification, production and detection of human IgG and goat IgG antibodies.

关 键 词：细菌噬菌体 免疫球蛋白G 定向分子进化 基因文库 

分 类 号：R392.7[医药卫生—免疫学]