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 Δasd mutants of S. typhimurium when using media lacking diaminopimelic acid. The recombinant Asd+ plasmid possessing the spaA 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/Δasd 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.