Selection of single-chain variable fragments specific for Mycobacterium tuberculosis ESAT-۶ antigen using ribosome display

Objective(s): Tuberculosis (TB) is still one of the problematic infectious diseases in developing countries, especially in Iran. In the present study, we applied ribosome display technique to select single chain variable fragments (scFvs) specific for the ۶-kDa early secretory antigenic target (ESAT-۶) antigen of Mycobacterium tuberculosis from a mouse scFv library. Materials and Methods: The gene encoding ESAT-۶ was cloned into pET۲۲b(+) plasmid and expressed in Escherichia coli BL۲۱ (DE۳). The purified recombinant ESAT-۶ protein was injected into female BALB/c mice for immunization, and then m-RNA was extracted from the spleen of immunized mice. The anti-ESAT-۶ VH/k chain library was assembled by joining of VH and k into the VH/k chain with a ۷۲-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-۶ using a single round of ribosome display via a rabbit reticulocyte lysate system. Results: ELISA assay showed that one of the selected scFvs had higher affinity against the recombinant ESAT-۶ protein. The affinity of the candidate scFv was ̴ ۳.۷۴×۱۰۸ M-۱. Conclusion: It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB.

Keywords:

antibody , ESAT-۶ , Mycobacterium tuberculosis , Ribosome display , scFv