ObjectiveTo construct a phage-displayed random combinatorial library of IgA affibodies and to analyze its directed in vitro evolution,so as to study the relationship of IgA affibody structure with its function.MethodsThe coding sequences of two affibodies,ZA1 and ZA2,were generated by overlapping PCR.The affibodies with a random linking peptide coding sequence in the 3′ terminal were randomly ligated and cloned into the KpnⅠsite of the phagemid pCANTAB5S to construct a combinatorial phage library.Totally four rounds of in vitro human IgA directed evolution were conducted,and selected phage clones were prepared individually to test the IgA binding activity by ELISA technology.ResultsThe combinatorial phage library was successfully constructed;it contained about 3.4×107 clones with a titer of 1.6×1012 TU/L,and the positive clones accounted for more than 79%.Sequence analysis showed that the two single affibodies were randomly linked by random linking peptides.The composition of the phage clones displaying three or four affibodies in tandem increased remarkably along with the rounds of selection,which indicated the successful IgA directed evolution.Three new arrangements of two,three or four affibodies in tandem were obtained.ELISA results demonstrated a significantly enhanced IgA binding activity of three or four affibodies in tandem.ConclusionA series of new IgA binding recombinants have been obtained by directed in vitro evolution of combinatorial phage library displaying randomly linked IgA affibodies.The three or four affibodies in tandem have much higher IgA binding activity than others,indicating that the in vitro molecular evolution is an effective way to produce IgA binding proteins with high activity.