Phage Display Immunoglobulin Combined With A Series Of Studies Of The Molecular Structure And Function

Objective: To construct a phage-displayed site-directed mutation combinatorial library of Staphylococcal Protein A(SPA) and screen it in vitro with different immunoglobulins, for obtaining new Ig-binding molecules with new Ig-binding characteristics, and also laying a foundation for studying interaction mechanism between Ig and Ig-binding molecules.Methods: The gene fragment encoding the Z domain of Protein A(Protein A-Z) was generated by Overlap PCR amplification, and was cloned into TA-cloning vector pMD-18T. The random point mutations at the site of 10,13,27,28,31and 32 of Protein A-Z were generated by Overlap PCR amplification with the random nucleotide primers(NNS). By using designed primers, two recognition sites for KpnⅠwere introduced into the mutations′both ends and a random linking peptide of 3 amino acids(NNS) into 3′-end. The mutations were digested with KpnⅠa nd ligated at random, and then recombined into the phagemid 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 Protein A-Z was established. The library was evaluated by the capacity, titer and DNA sequence analysis. Then the molecular directed evolutional affinity selections in vitro of the library were processed about 4 times using individually human IgG and IgA antibody molecules as panning targets. The affinity state during screening was monitored by binding-experiment and the dynamic distribution of displayed fragments in the library was monitored by PCR amplification.Results: The site-directed random mutation combinatorial library consisted of about 2.4×107 transformed clones and the phage library titer was 1.4×1012 TU/ml. The sequencing results of random monoclones indicated the amino acid mutation frequencies of every mutation site and the nucleotide acids encoding random linking peptide distributing randomly. But after IgG and IgA affinity screening, we did not obtain positive clones.Conclusion: With 6 amino acids sites of Protein A-Z being mutated and a random linking peptide of 3 amino acids introduced, there are over 1010 clones to be contained in the library theoretically. So, in order to obtain different kinds of Ig-binding molecules, we need a phage-displayed library of larger capacity. However, the design of experiment is a scientific exploration to obtain various of new Ig-binding molecules with high specificity and activity. The study provides a new path in application of phage-display technology realizing molecular evolution of proteins.