Cloning, Sequencing and Expression of Immunoglobulin Variable Regions of Murine Monoclonal Antibodies Specific to Cucumber mosaic virus coat protein

A mouse monoclonal antibody (mAb) prepared by hybridoma technology could recognize the Cucumber mosaic virus (CMV) coat protein. The Fab fragments (antigen-binding site, one complete light chain, and part of one heavy chain) genes encoding the light chain and the Fd (a monovalent antigen-binding fragment consisting of a complete light chain paired with the variable region and the first constant domain of the heavy chain) region of the heavy chain of the mAb were cloned and expressed in Escherichia coli. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the pepo-4 cell line to reverse transcription-polymerase chain reaction, then subcloning the products in the pGEM-T easy vector. Sequence analyses of the Fab fragment revealed that the light and heavy chains belong to the V 2 and VH1/VHJ558 germline gene family with GenBank accession numbers EF672211 and EF672197, respectively. The pARA7 expression vector was designed for the expression of Fab in the periplasmic space. Recombinant Fab fragments were purified and analyzed by indirect ELISA. These results suggest that the recombinant Fab-4 antibody produced by E. coli acts as a source for the generation of Fab with very stable and specific expression. _____________________________________________________________________________________________________________