Construction and expression of bicistronic eukaryotic expression plasmid containing Mycobacterium Tuberculosis Ag85B and ESAT-6 genes

Objective To construct the bicistronic eukaryotic expression plasmid containing Mycobacterium tuberculosis Ag85B and ESAT-6 genes. Methods The DNA fragments of Ag85B gene and ESAT-6 gene amplified from the genome of Mycobacterium tuberculosis by polymerase chain reaction (PCR) were cloned into the co-expression vector pIRES. The recombinant plasmid was analyzed by restriction enzyme digestion and the inserted target genes in the pIRES were verified by nucleotide sequencing. Then A549 cells were transfected with this recombinant plasmid using lipofectamine reagent,and the expression of Ag85B and ESAT-6 were detected by RT-PCR. Results The bicistronic recombinant eukaryotic expression plasmid, which could express Ag85B and ESAT-6 mRNA in vitro, was corrected. Conclusion The bicistronic eukaryotic expression plasmid containing Mycobacterium tuberculosis Ag85B and ESAT-6 genes is constructed successfully and can co-express Ag85B and ESAT-6 in vitro, which lays a foundation for further study in animals.