antigen as the means of spot forming cells + two standard deviation (SD) obtained with the PBMCs from 30 healthy BCG-vaccinated donors. Results In this study, we amplified esat-6, cfp-10 and esat- 6/cfp-10 fusion genes from native M. tuberculosis C genomic DNA by PCR. All PCR products, 288 bp for esat-6, 303 bp for cfp-10 and 591 bp for esat6/cfp10 were cloned into pGEX4T1 expression vector. DNA sequencing of the genes showed that all cloned genes were identical to genes reported on gene bank (esat-6, Accession Nos.RV3875; cfp-10, Accession Nos. Rv3874). Esat-6 and cfp-10 genes were completely inserted in the fusion gene. Recombinant antigens in diagnosis of TB The recombinant proteins were all purified from cell-free supernatants by affinity chromatography on GST-Agarose. The expressed products were subject- ed to SDS-PAGE using mini format vertical electro- phoresis (BIO-RAD, USA). Gels were run in tricine- tris running buffer. Expression and purification re- sults for ESAT-6, CFP-10 and ESAT6/CFP10 pro- teins were shown in Figure 1. The theoretical molecu- lar weight of pure ESAT-6, CFP-10, and ESAT- 6/CFP-10 is about 10, 13 and 25 KDa, respectively in E. coli expression system. The resolved proteins were transferred to nitrocellulose membrane to identify expression, by western immunoblotting using mini trans–blot cell (Bio-Rad, USA). ESAT-6/CFP-10 fu- sion protein could be recognized by mouse anti GST, anti ESAT-6 and anti-CFP-10 monoclonal antibodies. Western blot results were shown in Figure 2. Fig. 1: Expression and purification of ESAT-6, CFP-10 and ESAT-6/CFP-10 proteins. Samples (Lane 1-6) were resolved in a 16% (w/v) polyacrylamide gel, under reducing conditions. 1) Un-induced cell; 2) induced cell; 3) glu- tathione-agarose flow through (unbound); 4) washed glutathione agarose showing bound protein; 5) thrombin cleaved supernatant; 6) washed glutathione agarose after thrombin cleavage. Molecular weight standards are shown to the left of lane 1. Fig. 2: Western blots of ESAT-6/CFP-10 fusion protein probed with mouse anti-GST (A), mouse anti-ESAT-6 (B), and mouse anti-CFP-10 (C) monoclonal antibodies. Lanes1and 2 represent ESAT-6/CFP-10 protein with and without thrombin cleavage. WWW.ircmj.com Vol 13 August 2011 559