[Construction of phage-displayed library by DNA shuffling and the screening of potent hirudin variants].

Aim: To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method.

Methods: PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin.

Results: After shuffling and selection, a clone (HV2-N47K) with high antithrombotic activity was obtained.

Conclusion: Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.