Construction of an ASD⁺ expression-cloning vector: Stable maintenance and high level expression of cloned genes in a salmonella vaccine strain

An ASD⁺ expression-cloning vector was constructed for the purpose of high-level stable expression of foreign antigen genes in a Salmonella typhimurium vaccine strain. It possesses the asd+ gene of Streptococcus mutans as a unique plasmid marker and is stably maintained in Aasd mutants of 5. typhimurium when using media lacking diaminopimelic acid. The recombinant Asd+ plasmid possessing the spa A gene of Streptococcus sobrinus, which is regulated by the trc promoter, produced the fused SpaA protein at high level in the S. typhimurium vaccine strain. The Asd+ vector/ A asd mutant host constructs represent a balanced lethal combination that eliminates the need for vector drug resistance markers, an essential attribute for live vaccines. This strategy also has applications whenever stable high-level expression by genetically modified bacteria, in the absence of external selection pressures, is desirable.