V-2A-GFP/V-2A-coexpressing Mycobacterium tuberculosis antigens and GFP

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V-2A-GFP/V-2A-coexpressing Mycobacterium tuberculosis antigens and GFP V-2A-GFP/V-2A-coexpressing Mycobacterium tuberculosis antigens and GFP. (A) Map and principle of V-2A-GFP/V-2A. V-2A-GFP/V-2A-coexpressing Mycobacterium tuberculosis antigens and GFP. (A) Map and principle of V-2A-GFP/V-2A. After the removal of the stop codons of Rv3407, Ag85A, and HspX, these genes were linked to GFP with the FMDV 2A sequence. Expression is under the control of the CMV promoter. Transcription leads to the formation of one long mRNA transcript. Posttranslational cleavage of the 2A sequence causes segmentation of the long translated polypeptide into individual proteins. (B) Protein coexpression by V-2A. HEK293 cells were transiently transfected with V-2A and a control vector. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; transferred to a polyvinylidene difluoride membrane; and probed with biotinylated polyclonal antibodies to Rv3407 (Biogenes, Berlin, Germany), Ag85A, (MPIIB, Berlin, Germany), and HspX (Affinity Bioreagents), followed by addition of streptavidin-conjugated horseradish peroxidase (MPIIB), and then developed with an ECL Western blotting analysis system (Amersham Biosciences). Bands of 11 kDa, 30 kDa, and 14 kDa corresponding to Rv3407, Ag85A, and HspX, respectively, were detected in the lysates of cells transfected with V-2A but not in the lysates of cells transfected with the control vectors. Fayaz-Ahmad Mir et al. Clin. Vaccine Immunol. 2009; doi:10.1128/CVI.00237-09