Construction And Selection Of Phage Displayed Random Recombinant Library Of IgA-Affibodies

IgA is believed to play an important role for human body. Efficient and selective methods for IgA detection and purication are of major importance for a vast number of medical applications areas. In vitro molecular directed evolution is important to research on the relationship between structure and function of biomacromolecule. It can chage and optimize the properties of biomacromolecule under laboratorial conditions. Phage display and DNA shuffling are important and efficient techniques for directed molecular evolution. In this study, we randomly combined two affinity proteins (affibodies) showing selective binding to human IgA to construct a phage-displayed random combinatorial library of Ig A affibodies, using phage display technique combined with in vitro molecular directed evolutional selection to obtain the strongest binding IgA affibody molecules .By compare the binding affinity of IgA affibody multimers and monomers to explore the relationship between structure and function of IgA-binding molecule, and to lay a foundation for directed improvement of IgA-binding molecule as well. So we carried out the following four-part work.1. The gene synthesis of two IgA affibodies.According to the amino acid sequences of the article, we designed several primers for two IgA affibodies. The coding sequences of two IgA affibodies, A1 and A2, were generated by Overlap PCR, and were individually subcloned into the TA-cloning vector pMD-18T. Sequence analysis showed the coding sequences of two IgA affibodies were correct.2. The construction of a phage-displayed random combinatorial library of IgA affibodies.The two IgA affibodies with the Kpn /recognition site in both ends and a random...