A stable and antibiotic-free plasmid expression system for Salmonella typhimurium VNP20009 in tumor therapy

<i>Salmonella typhimurium</i> VNP20009 is a promising tumor-targeted vector for gene therapy, but conventional plasmid systems rely on antibiotic resistance markers and are prone to plasmid loss. To address this, we engineered and compared standardized plasmid expression vectors with different replicons, transcriptional controls, and stabilization modules in VNP20009. Among four replicons tested, pMB1 provided the best balance of stable expression and minimal effects on bacterial physiology. The incorporation of tunable promoter-RBS combinations enabled over 100-fold dynamic control of gene expression. Plasmid stabilization with the axe/txe toxin-antitoxin system maintained plasmid retention and protein expression without impairing growth. In murine tumor models (LLC and B16F10), the pMB1-AT system sustained therapeutic gene expression and enhanced tumor suppression when delivering Endostatin. This work establishes an antibiotic-free and durable plasmid platform for <i>Salmonella</i>-based therapies, offering a framework for reliable gene delivery <i>in vivo</i> and expanding opportunities for synthetic biology and cancer treatment.