噬菌体展示免疫球蛋白结合分子单结构域随机组合文库的构建  

Constructing a phage-displayed random combinatorial library of single domains of immunoglobulin-binding molecules

作　　者：杨华[1] 李连青[2] 王蓓霞[3] 徐容[3] 沈毅珺[3] 贾建安[3] 潘欣[3] 陈秋莉[3] 潘卫[3] 

机构地区：[1]山西医科大学基础医学部微生物学与免疫学教研室,太原030001 [2]山西省临床检验中心,太原030012 [3]第二军医大学基础医学部微生物学教研室,上海200433

出　　处：《第二军医大学学报》2005年第4期396-401,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30472050).

摘　　要：目的:构建由免疫球蛋白结合分子单结构域随机组合的噬菌体展示文库,为应用各种类型免疫球蛋白对该文库进行体外分子进化筛选研究打下基础。方法:PCR扩增制备分别编码Protein A 的A、D结构域、Protein G的B2 结构域和Pro tein L的B3结构域的序列片段,片段两端引入XbaⅠ酶切位点,3′端引入分别编码0、1、2、3个氨基酸大小随机连接肽的序列,并克隆于pMD 18T载体构建重组质粒。以上述16种重组质粒为模板,PCR扩增分别制备各种单结构域片段及两端部分T载体序列,XbaⅠ酶切,各种酶切片段混合后连接,并克隆于噬菌体展示载体pCANTAB5S,构建噬菌体展示免疫球蛋白结合分子单结构域随机组合文库,挑取单克隆进行序列测定,评价文库的随机性。结果:噬菌体展示免疫球蛋白结合分子单结构域随机组合文库库容量为2×107,滴度为1.3×1011;文库中含77%以上的阳性克隆,其中由2个以上单结构域组成的阳性克隆占24%;序列分析显示组合文库包含上述4种单结构域序列,连接方式符合随机连接的特点,随机连接肽的编码核酸也呈随机分布。结论:成功构建了免疫球蛋白结合分子单结构域随机组合文库,该文库库容、多样性和随机性完全可满足进行体外分子进化研究的要求。Objective:To construct a phage-displayed random combinatorial library of single domains of immunoglobulin-binding molecules,providing reference for in vitro molecular evolution study of the library with different immunoglobulins. Methods: Gene fragments encoding the A, D domain of Protein A, the B2 domain of Protein G, and the B3 domain of Protein L were generated by PCR amplification using the primers which introduced recognition site for Xba Ⅰin both ends and nucleotide acid sequences in the 3′-end, which encoding random linking peptide containing 0, 1, 2 or 3 amino acids. All fragments were individually subcloned into the TA-cloning vector pMD-18T. With these 16 recombined plasmids as templates, long fragments were amplified by PCR using the universal primers of pMD-18T, then the PCR products were digested with Xba Ⅰand ligated into the Xba Ⅰsite of the phagemid pCANTAB5S to construct a phage-displayed random combinatorial library. The combinatorial library was evaluated by sequence analysis. Results: The combinatorial library consisted of about 2×10 7 clones and the phage library titer was 1.3×10 11. More than 77% clones in the library were positive and about 24% positive clones contained inserted fragments which had more than 2 domains. Sequence analysis showed that inserted fragments of clones in the combinatorial library were all composed of the 4 single domains ligated randomly. The nucleotide acids encoding random linking peptide also distributed randomly. Conclusion: The random combinatorial library of the single domains of Ig-binding molecules has been successfully constructed. The capacity, variety and randomicilty of the library meet the requirements of in vitro molecular evolution study.

关 键 词：免疫球蛋白结合分子 噬菌体展示 组合文库 结构域 随机连接肽 

分 类 号：Q785[生物学—分子生物学]