Hemmati et al. Table 1: Primers and vector used for cloning of esat-6, cfp-10 and esat-6/cfp-10a Antigen Primer sequence( 5' to 3' ) Forward (F) Cloned between or Reverse (R) ESAT-6 TAAGGATCCATGACAGAGCAGGAGTG F BamHI and ECORI GCGAATTCTGCGAACATCCCAGTG R CFP-10 TAAGGATCCATGGCAGAGATGAAGAAC F BamHI and ECORI GCGAATTCGAAGCCCATTTGCGAGGA R TAAGGATCCATGACAGAGCAGGAGTG F BamHI ESAT-6/CFP-10 GGAACCTGGAGATGCGAACATCCCAGTG R TCTCCAGGTTCCATGGCAGAGATGAAGAAC F AAGGAATTCGAAGCC CATTTGCGAGGA R ECORI GGATCC : BamHI site, GAATTC :ECORI site GGAACCTGGAGA: Linker TCTCCAGGTTCC: linker a Restriction sites are underlined. Vector pGEX4T1 pGEX4T1 pGEX4T1 pGEX4T1 (DH5α). Positive clones were evaluated to find the correct insertion. Expression and purification of the recombinant pro- teins were performed by standard protocols.5,7 E. coli BL21 (DE3) transformed by pGEX4T1 (+) was grown in LB medium containing 100 µg/ml ampicillin. Ex- pression was induced at mid-log phase by adding IPTG. Cell pellets were lyzed in lysis buffer comprises of 1% of Triton X-100, 1 mM EDTA (pH=8.0), aprotinin, leupeptin and pepstain A (1 µg/ml of each) in PBS. The cell lysates were sonicated, and the suspension was cen- trifuged.5 The mixture was allowed to bind to glutathi- one agarose beads. The lysate-bead mixture was incu- bated with reduced glutathione reagent to elute GST- tagged proteins. After dialysis, purity of proteins was analyzed by SDS-PAGE and western blotting. For pro- ducing pure recombinant proteins without GST-tag, thrombin cleavage of the proteins was performed. The SDS-PAGE of recombinant protein products was performed in the presence of 0.1% SDS in the gel. Protein samples (with and without GST) were prepared by mixing with reducing sample buffers13 and boiling in a sand bath for 5 minutes. Gel was run at a constant voltage of 200 V. Recombinant proteins transferred to nitrocellulose membranes (Millipore, Bi- omanufacturing and Life Science Research) using mini Trans-blot Cell (Bio-Rad). The blots were incubated with an appropriate dilutions of primary anti-ESAT-6, anti-CFP-10 and anti-GST antibodies and then were incubated with horseradish peroxidase-conjugated sec- ondary antibody solution. Finally blots were processed using Enhanced chemiluminescence (ECL).5 The Far-UV CD spectra were used to determine the secondary structure of the recombinant proteins in an Avir circular dichroism spectropolarimeter model 215. Protein samples were dissolved in buffer 50 mM Tris-HCl pH 8.0, 180 mM NaCl, 1 mM Dithiothreitol (DTT) and 10% v/v glycerol. CD Spectra of the pro- teins were recorded from 190 to 260 nm, each spec- trum representing an average of 3 accumulations. Interferon gamma ELISPOT assay for detection and enumeration of IFN-γ producing T-cells against myco- bacterial antigens was performed by using a commer- cially available anti IFN-γ antibody pair and black spot developing reagents from U-Cytech (U-CyTech, Neth- erland).11,12 Briefly, peripheral blood mononuclear cells (PBMCs) (2×105 cells/well) were added to duplicate wells pre-coated with anti- IFN-γ antibody. Following the overnight stimulation with ESAT-6, CFP-10, ESAT- 6/CFP-10, commercial ESAT-6 and PPD, plain medium (as negative control), and phytohemagglutinin (PHA, as positive control), the wells were washed, and the pres- ence of IFN-γ was detected by biotinylated anti-IFN-γ and φ-labeled goat anti-biotin antibody. Afterward addi- tion of activators I and II precipitated silver on φ and revealed the spots of IFN-γ secreting cells. The spots were enumerated with a stereomicroscope and photo- graphed digitally. The number of spots in the back ground control wells was subtracted from the number in the test wells and a response was considered positive if the number of spots per test well was greater than the cutoff point for each antigen and at least twice the value found in the background control wells. The means of the duplicates were used and all results were expressed as number of IFN-γ spot-forming cells (SFC) per million PBMC for comparison.14,15 The statistical significance of differences between experimental and control groups were determined by Mann-Whitney U test (SPSS software, version 16.0, Chicago, IL, USA). Data were considered statistically significant at p<0.05. For evaluation of antigen re- sponses, cutoff values were calculated for each 558 WWW.ircmj.com Vol 13 August 2011