A phagemid system enabling easy estimation of the combinatorial antibody library size.

Abstract

We developed a new phagemid system for the generation of combinatorial antibody libraries. This system allows the recombination efficiency to be estimated easily, which aids the accurate measurement of the antibody library size. Two phagemids were constructed. pRTCB bears the structural tetracycline-resistant gene (tetR) and pRTCA bears its promoter region (Ptet). pRTCA and pRTCB were constructed so that recombinase-mediated chain exchange (RMCE) in Cre-expressing bacteria results in the simultaneous acquisition of Tet resistance and newly recombined single chain variable fragment (scFv) genes. PCR and restriction enzyme analysis of randomly selected tetR phagemids showed that all Tet-resistant phagemids have an active tetR gene and a recombined scFv gene. RMCE efficiency was measured by the ratio of the tetR colonies to the total colonies. The scFv proteins expressed from the recombined phagemid vectors were functional. Thus, our phagemid vector system may be useful for making combinatorial antibody libraries.