Random recombination of antibody single chain Fv sequences after fragmentation with DNasel in the presence of Mn~(2+)

摘要

Material for shuffling was generated by PCR of two scFv DN A sequences; one designated B3M that consists of Mab B3 VH and Mab B5 VL sequences (9.10), and one designated Bl that consists of Mab B1 VH and VL sequences (10). Each was in the VH-linker-VLorientation, with an identical linker sequence. The two sequences have 88% DNA sequence identity, coding for scFvs that differ by 49 amino acids. PCR was done with the primer pair B35' and B5VLFX (for B3M) or the primer pair B15' and B1VL-FX (for Bl). The primers contain Sfil and Notl restriction sites for subcloning into phage display vectors, and a sequence coding for a factor X site to permit proteolytic cleavage of the Fv from its viral protein fusion partner (11). All PCR reactions were done on a Biometro Trio-thermoblock thermocycler (Biometro Inc., Tampa. FL). PCR products were purified with Wizard PCR Preps DNA Purification System (Promega) before DNasel digestion.