Construction Of Phage-displayed Site-directed Random Mutation Combinatorial Library Of Immunoglobulin-binding Molecules

Objective: To construct a phage-displayed site-directed random mutation combinatorial library of immunoglobμLin-binding molecμLes, which can provide reference the study of the in vitro molecμLar evolution of the library with different immunoglobμLins,and the relationship of their structure and function. Methods: The gene fragment encoding the Z domain of Protein A was generated by SOE PCR and cloned into pMD-18T. The random points mutate at the 10th,13th,27th,28th,31st and 32nd amino of Z domain. The KpnⅠrestriction site were introduced into the both ends and a random linking peptide of 3 amino acids into 3′-end. The mutations were digested by KpnⅠand 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 immunoglobμLin-binding molecμLes was established. The library was evaluated by the capacity, titer, and DNA sequence analysis. ResμLts: The site-directed random mutation combinatorial library consisted of about 2.7×107 transformed clones and the phage library titer was 1.4×1012 TU/ml. 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 immunoglobμLin-binding molecμLes has been successfμLly constructed. It has large capacity and ideal diversity and randomicity in primary structure, which can meet the requirements of in vitro molecμLar evolution study.