Construction and Expression of Bicistronic Eukaryotic Plasmid Containing Mycobacterium Tuberculosis Ag85B Gene and IL-2 Gene

Objective: To construct the bicistronic eukaryotic plasmid containing Mycobacterium tuberculosis Ag85B gene and IL-2 gene.Methods: The DNA fragments of Ag85B gene were amplified from the genome DNA of Mycobacterium tuberculosis by polymerase chain reaction(PCR) and the cDNA encoding murine IL-2 was obtained by subcloning.The two gene fragments were inserted into eukaryotic coexpression plasmid vector pIRES to construct recombinant plasmid.Then A_(549)cells were transfected with this recombinant plasmid using lipofectamine reagent and the expression of Ag85B and IL-2 were detected by RTPCR.Results: The bicistronic eukaryotic plasmid pIRES-85B-IL-2 was proved to be correct by restriction enezyme digestion and nucleotide sequencing and it could express Ag85B and IL-2 mRNA in vitro.Conclusion: The bicistronic eukaryotic plasmid containing Mycobacterium tuberculosis Ag85B gene and IL-2 gene was constructed successfully,which lays a foundation for the study in animals.