title: Selection of single-chain variable fragments specific forMycobacterium tuberculosisESAT-6 antigen using ribosome display.
abstract: OBJECTIVE
  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 ofMycobacterium tuberculosisfrom a mouse scFv library.
  
  
  METHODS
  The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed inEscherichia coliBL21 (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 ~ 3.74×108M-1.
  
  
  CONCLUSIONS
  It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB.
authors.affiliation: Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Shahrzad
authors.initials: S
authors.lastname: Ahangarzadeh
authors.affiliation: Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Bandehpour
authors.affiliation: Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Kazemi
keywords: Antibody; ESAT-6; Mycobacterium tuberculosis; Ribosome display; scFv