CONSTRUCTION OF A HUMAN IG COMBINATORIAL LIBRARY FROM GENOMIC-V SEGMENTS AND SYNTHETIC CDR3 FRAGMENTS

A naive combinatorial Ig library was constructed from semi-synthetic V genes consisting of human genomic V segments and synthetic CDR3 fragm ents. V(H) and V(kappa) segments were amplified from human genomic DNA by polymerase chain reaction using V subgroup-specific primers. The a mplified V(H) and V(kappa) segments were combined with synthetic oligo nucleotides containing a J region and CDR3 with amino acid sequence va riations, resulting in complete V genes. These V genes were cloned int o a phagemid expression vector in a single-chain form fused to the car boxyl-terminus of the M13 minor coat protein III. Phagemid particles d isplaying the single chain hybrid proteins on their surface were scree ned with Con A as Ag. Several clones showing specific binding to Con A were obtained after four rounds of selection and were further analyze d for their binding properties and DNA sequences. This method provides a novel way to create a naive combinatorial library without using mRN A from B lymphocytes as template. The method should be useful to isola te human antibodies that react with self-Ag.