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  

Phage Display of Random Combinatorial Libraries of Ig-binding Mono-domains of Protein A and Protein L and Ig Affinity Screening

作　　者：徐容[1] 沈毅臖 邓松华[1] 蔡春晓[1] 陈秋莉[2] 贾建安[2] 王锦红[2] 潘欣[2] 潘卫[2] 

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032 [2]第二军医大学基础医学部微生物学教研室,上海200433

出　　处：《生物化学与生物物理进展》2005年第6期535-543,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30472050)~~

摘　　要：ProteinA和proteinL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proteinA的A、B、C、D抗体结合结构域和proteinL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proteinL的单结构域和proteinA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 Sac I sequence were synthesized to amplify A,B,C,D domain of protein A and 133 domain of protein L by PCR respectively. After digestion with restriction enzyme Sac I, 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 Sac I site of phagemid pCANTAB5S. The capacity of the phage library were calculated as 3.4x10(7) clones,and the titer was 6.2x10(10) 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), which consists of the repetition of mono-domain of protein A( MPA) 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.

关 键 词：噬菌体展示 分子进化 结构域 组合文库 PROTEIN A PROTEIN L 

分 类 号：Q78[生物学—分子生物学]