Construction of the eukaryotic recombinant plasmid expressing the CFP10 protein of Mycobacterium tuberculosis and its expression in vitro

To express the CFP10 gene of Mycobacterium tuberculosis,a pair of primers specific to the CFP10 gene was designed and synthesized according to the nucleotide sequences of M.tuberculosis H37Rv strain available in GenBank.CFP10 gene was amplified by PCR from the DNA of M.tuberculosis and cloned into the pGM-T vector and verified by sequencing.The CFP10gene was subcloned into pEGFP-N1 to construct recombinant plasmid of pEGFP-N1-CFP10.Then the 293T and U937 cells were transfected with pEGFP-N1-CFP10.Western blot assay indicated that the EGFP-CFP10 protein was expressed in 293T and U937cells,respectively,and its molecular weight was 37 ku as expected.The location of CFP10-EGFP fusion protein was observed by confocal laser scanning microscope and the CFP10-EGFP fusion protein was mainly distributed in the cytoplasm.