国家自然科学基金资助项目(30472050).
This work was supported by a grant from The National Natural Sciences Foundation of China (30472050).
Protein A 和 protein L 是细菌产生的两种结构和功能均不同的免疫球蛋白 (immunoglobulin , Ig) 结合分子,在细菌的致病中起重要作用 . 用含 SacⅠ位点的特定引物 PCR 分别扩增制备 protein A 的 A 、 B 、 C 、 D 抗体结合结构域和 protein L 的 B3 抗体结合结构域,各结构域 DNA 片段经 SacⅠ酶切后,再随机连接形成各种不同长度的分子组合文库,将该文库呈现在噬菌体表面构建了噬菌体展示 Ig 结合分子单结构域随机组合文库,所建组合文库容量为 2.3×106个菌落形成单位,滴度为 4.1×1011TU/ml , 包含各种单结构域片段,并以随机方式连接 . 用人 Ig 对该文库进行 4 轮亲和筛选,随机挑选 36 个代表性的阳性克隆进行序列测定分析表明,亲和筛选获得了多种非天然形式存在的新的 Ig 结合分子结构,其中 32 个克隆具有由 protein L 的单结构域和 protein A 的单结构域间隔重复排列而成的特征性 (MDPL-MDPA)n 结构 . 对噬菌体展示 Ig 结合分子单结构域随机组合文库的体外分子进化研究的尝试,为 Ig 结合分子的结构和功能研究提供了一新的途径,也为 Ig 结合分子的定向改造打下基础 .
Protein A and protein L are bacterial cell wall proteins of importance in pathogenesis which have different crystal structures and bind different sites of host immunoglobulin (Ig). A pair of primers containing SacⅠ sequence were synthesized to amplify A,B,C,D domain of protein A and B3 domain of protein L by PCR respectively. After digestion with restriction enzyme SacⅠ , these PCR prepared Ig-binding domains were ligated randomly with each other to come into being a combinatorial molecular library. The library was displayed on phage surface by cloning into SacⅠ site of phagemid pCANTAB5S. The capacity of the phage library were calculated as 3.4×107 clones,and the titer was 6.2×1010 TU/ml. The sequence analysis showed that the displayed DNA fragments in the library comprise of various Ig-binding domains ligated in random. After three or four rounds affinity selection with human Ig, 36 positive clones were sequenced at random to analyze structure of the recombinant molecules. The sequence analysis showed 3 kinds of new molecular structures existed in the selected molecules which were totally different from its natural molecules. The characteristic structure of (MDPL-MDPA)n which consists of the repetition of mono-domain of protein A(MDPA) and mono-domain of protein L (MDPL) existed predominantly in 32 of 36 positive clones. The effort to proceed molecular evolution study of Ig-binding domains combinatorial molecular library by phage display not only provides potent approach for research involved in the relationship between structure and function of Ig-binding molecules, but also a basis for Ig-binding molecules rebuilding.
徐 容,沈毅珺,邓松华,蔡春晓,陈秋莉,贾建安,王锦红,潘 欣,潘 卫.噬菌体展示 protein A 及 protein L Ig 结合单结构域随机组合文库及 Ig 亲和筛选[J].生物化学与生物物理进展,2005,32(6):535-543 XU Rong, SHEN Yi-Jun, DENG Song-Hua, CAI Chun-Xiao, CHEN Qiu-Li, JIA Jian-An, JIA Jian-An, PAN Xin, PAN Wei. Phage Display of Random Combinatorial Libraries of Ig-binding Mono-domains of Protein A and Protein L and Ig Affinity creening[J]. Progress in Biochemistry and Biophysics,2005,32(6):535-543
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