CLONING, EXPRESSION AND T CELL EPITOPE PREDICTION OF FBPA AND FBPB GENES OF MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES

Abstract

The effective treatment and accurate diagnosis of tuberculosis (TB) are not established yet. The Bacillus Chalmette-Guerin vaccine did not provide significant results in the prevention of TB and had only 0-80% efficacy. The fbpA and fbpB genes of M. tuberculosis are antigenic proteins and considered to be virulence factors. They are capable of stimulating immune responses in TB patients. In this study, we observed cloning, expression and T-cell epitope prediction of fbpA and fbpB genes from clinical isolates. The isolates of MultiDrug-Resistant (MDR-TB) were cultured and extracted. The fresh Polymerase Chain Reaction (PCR) products of the fbpA and fbpB genes were inserted into pET SUMO plasmids and transformed into Escherichia coli BL21 (DE3), then expressed in LB medium induced by 1.0 μM of IPTG. Sample sequences were analyzed by ClustalW and NCBI BLAST programs. The T-cell epitope prediction was analyzed by GENETYX vers 8.0. The PCR results were 1071 bp (fbpA gene) and 978 bp (fbpB gene). The SDS-PAGE and Western blotting weighed 48-kDa (fbpA gene) and 46-kDa (fbpB gene). We obtained seven specific T-cell epitopes based on IAd Pattern Position on both genes. Based on Rothbard/Taylor Pattern Position, we discovered twenty-three and sixteen specific T-cell epitopes for fbpA and fbpB genes, respectively. The fbpA and fbpB genes that encode Ag85A and Ag85B proteins have epitopes that are recognized by lymphocyte T-cells and are potentially subunit TB vaccine candidates in the future.