中国生物工程杂志

Objective To express a new tuberculosis DNA vaccine expressing siRNA, the Mycobacterium tuberculosis fusion antigen gene and the hIL-12 in human embryonic kidney 293 cells. Methods On the basis of the new tuberculosis DNA vaccine expressing siRNA targeted Mcl-1L, the Mycobacterium tuberculosis fusional antigen gene Ag85B and ESAT6(PVAE) and the hIL-12, it is fused with enhanced green fluorescent protein(EGFP) gene and get the eukaryotic expression vector pVAX1-siRNA-PVAE-EGFP-hIL12. Use siEGFP targeted EGFP instead of the siRNA and get the expression vector pVAX1-siEGFP-PVAE-EGFP-hIL12. Then the two recombinant plasmids was transfected 293 cells, observe the expression of Ag85B and ESAT6 fusion antigen gene through the expression of the green fluorescent protein and the siRNA. ELISA analyze the expression of hIL-12. Results Coexpression vector of MTB DNA vaccine was changed successfully by the restriction enzyme analysis and sequencing. Green fluorescent can be detected in the transfected cells which confirm the expression of the fusion antigen gene and the siRNA. The mount of hIL-12 excretion was 1571.63pg/ml supernatant 48h after transfection, 2392.25pg/ml supernatant 72h after transfection. Conclusion The DNA vaccine co-expressing the MTB fusion antigen gene and the hIL-12 was constructed and transiently expressed in 293 cells. It laid a foundation of further study on anti-MTB therapeutic effect of the new DNA vaccine.