A novel B cell epitope in cold-shock DEAD-box protein A from Mycobacterium tuberculosis

Abstract

In this study, a hybridoma-based technique and phage display technology were used to obtain mouse monoclonal antibodies (mAb) against cold-shock DEAD-box protein A (CsdA) from Mycobacterium tuberculosis and to determine the location of the relevant epitope. One highly specific mAb, named A3G5, was developed against the recombinant CsdA protein (rCsdA) and could detect rCsdA protein in enzyme-linked immunosorbent assays (ELISA) and Western blot assays. By screening a phage displayed library of random 12-mers (Ph.D.-12), 10 positive phage clones were randomly selected after three rounds of bio-panning and identified by ELISA. Eight of these clones were sequenced, and their amino acid sequences were deduced. One B-cell epitope (-APDPPLSRR-) in the rCsdA protein was identified with mAb A3G5. A synthetic peptide (-MAPDPPLSRR-) (Cpep) matched well with the CsdA sequence at 443-451 aa and was confirmed by affinity ELISA, competitive inhibition assays and the development of an immune response in mice. These results may be of great potential value in the further analysis of the function and structure of the CsdA protein from M. tuberculosis.