Selection of single-chain variable fragments specific for ESAT-6 antigen using ribosome display.

OBJECTIVES

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 6-kDa early secretory antigenic target (ESAT-6) antigen of from a mouse scFv library.

MATERIALS AND METHODS

The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed in BL21 (DE3). The purified recombinant ESAT-6 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-6 VH/k chain library was assembled by joining of VH and k into the VH/k chain with a 72-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-6 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-6 protein. The affinity of the candidate scFv was ~ 3.74×10 M.

CONCLUSION

It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB.