噬菌体展示免疫球蛋白结合分子定点随机突变组合文库的构建

Construction of phage-displayed site-directed random mutation combinatorial library of immunoglobulin-binding molecules

目的构建噬菌体展示免疫球蛋白结合分子定点随机突变组合文库,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及结构和功能的研究打下基础。方法应用overlapping PCR制备Protein A的Z结构域。用含随机核苷酸序列的引物,制备对Z结构域的第10、13、27、28、31和32位氨基酸进行随机突变的定点随机突变体库。并在突变体片段两端引入KpnⅠ位点,3′端引入3个氨基酸大小随机连接肽的序列。经KpnⅠ酶切后随机连接,克隆于噬菌粒展示载体pCANTAB5S。克隆产物转化大肠杆菌TG1,辅助噬菌体M13K07拯救,构建噬菌体展示定点随机突变组合文库。结果该定点随机突变组合文库的转化子数目为6.4×106个,滴度为1.4×1015TU/L;文库中含0.72以上的阳性克隆,其中有2个单结构域的阳性克隆占0.15;15个样品的序列分析显示各突变位点和随机连接肽的氨基酸序列呈随机性分布。结论成功构建了噬菌体展示免疫球蛋白结合分子定点随机突变组合文库,该库库容量在一级结构上具有良好的随机性和多样性,可满足体外分子进化研究的要求。

Objective To construct a phage-displayed site-directed random mutation combinatorial library of immunoglobulin-binding molecules, which can provide reference the study of the in vitro molecular evolution of the library with different immunoglobulins, and the relationship of their structure and function. Methods The gene fragment encoding the Z domain of Protein A was generated and cloned into pMD-18T. The random points mutate at the 10 th,13 th,27 th,28 th,31 st and 32 nd amino of Z domain. The Kpn Ⅰ restriction site was introduced into the both ends and a random linking peptide of 3 amino acids into 3＇-end. The mutations were digested by Kpn Ⅰ and ligated at random, and then recombined into the vector pCANTAB5S. After the recombinants were transformed into E. coli TG1 and rescued by the helper phage M13KO7, a phage-displayed site-directed random mutation combinatorial library of immunoglobulin-binding molecules was established. The library was evaluated by the capacity, titer, and DNA sequence analysis. Results The site-directed random mutation combinatorial library consisted of about 6.4 × 10^6 transformed clones and the phage library titer was 1.4 × 10^5 TU/L. More than 72% clones in the library were positive and about 15% positive clones contained 2 inserted domains. The sequencing analysis of 15 clones indicated the amino acid mutation frequencies of every mutation site and the nucleotide acids encoding random linking peptide were distributed randomly. Conclusion The phage-displayed site-directed random mutation combinatorial library of immunoglobulin-binding molecules has been successfully constructed. It has large capacity and ideal diversity and randomicity in primary structure, which can meet the requirements of in vitro molecular evolution study.