Construction of a Diverse Fab Expression Library from Autoimmunized Mice Based on an Improved Preparation of Cloning Arms from Bacteriophage Vectors: A New Library with Potential for Screening of Biocatalysts

Abstract

This paper describes an improved procedure for the preparation of cloning arms from vectors Lc1 and Hc2 for the construction of cDNA libraries in phage δ for expression in E. coli of the combinatorial Fab fragments of the immunoglobulin repertoire from autoimmunized mice. It was found that annealing, dialysis and dephorphorylation after the restriction enzyme digestion of vectors were critical for the high efficient preparation of cloning arms. The construction of Fab cDNA libraries involves the use of primer-directed PCR amplification products of total RNA from autoimmunized mice. Fab production was confirmed by the immunoassay and sequence analysis. The partial sequenced genes for Fab fragments were different from each other in the FR4, CDR3 and FR3 regions, indicating a diverse nature of the library. This library may be used to screen for catalytic Fab's.

Key Words:

• Catalytic antibodies
• Diverse Fab library
• autoimmunized mice
• cloning arms preparation
• screening