Team:Nanjing China Bio/Safety2

To confirm the distribution of VNP20009 in mouse tissues and to verify the tumor-specific gene induction, we detected GFP expression in tumor, liver, and spleen by fluorescence microscopy. We found the VNPpLacZ-GFP (with constitutive promoter) could induce expression not only in necrotic tumor but also in spleen and liver tissue. However, VNPpNirBeGFP (with tumor specific promoter) could only be induced in hypoxic and necrotic tumor tissue.


Fluorescence microscopy shows the preferential accumulation of VNP20009 pNirBeGFP in necrotic and hypoxic areas inB16F10 tumors. The nirB promoter could be induced specifically in this area but not in liver or spleen. (N, necrotic tumor area; V, vital tumorcells) This experiment was carried out in triplicate.
We designed plasmid constructs expressinggenes of interest for Salmonella- mediated tumor-targeted therapy using the nirB promoter. Our fluorescence microscopy revealed that the nirB promoter could effectively drive theexpression of GFP in S. typhimuriumstrain VNP20009 carryingthe plasmid construct pNirBeGFP under hypoxia,suggesting that the nirB promoter was a potent promoter.


S. typhimurium strain VNP20009 carryingpNirBeGFP or peGFP was grown for 24 h under anaerobic or aerobic conditions. Expression of GFP was examined by fluorescence microscope



Immunoblotting studies further showed that TRAIL was effectivelyexpressed in VNP20009 carrying the plasmid constructpTRAILgrowing under hypoxia.



Bacteriallysates were prepared from VNP20009 carrying construct pTRAIL grown in anaerobic or aerobic jars and were subjected to immunoblottingassays using anti-TRAIL antibodies.
These findings indicated thatgenes of interest for Salmonella- mediated tumor-targeted therapy could be effectively expressed in S. typhimurium strainVNP20009 carrying appropriate expression vectors driven bythe nirB promoter.


Fluorescence intensity was determined. Bar represents the mean ± SD of five independent experiments (n =5;*P<0.001).
Researches revealed that some auxotrophic strains can better colonize in tumors, indicating that special nutrient-rich environment can compensate for the nutrition the strains lack for. Therefore, we knocked out one gene in amino acid metabolism pathways of VNP.


We found CFU (Colony Forming Units) of auxotrophic strains 5 and 6 could reach 109, while that of VNP can only reach 105 at most in normal tissues. The ratio of number of bacteria in tumor tissues to that in liver on the 4th day shows that after knocking out related genes to the metabolism to synthesize certain amino acid, the ability of tumor targeting was highly enhanced, nearly 10 times compared to that of VNP.