Construction And Application Of Crpˉ, Asdˉ Deleted Mutants Host-Vector Balanced Lethal System Of Salmonella Choleraesuis C500 Strain

S. Choleraesuis is the main pathogen of Piglet's Paratyphoid for 2-4 -month-old piglets and has an important status in swine industry. In the meanwhile, it holds significance in public health because it causes human food poisoning through the infected animals or the contaminated products. Vaccination is the most efficient method to control paratyphoid fever. S. choleraesuis C500 strain was an effective attenuated paratyphoid fever vaccine strain, attenuated by chemical method. Although the vaccine has a good immunogenicity, it remains some residual virulence, unkown of background and risk of back mutation.Mucosal immunization and multivalent vaccination are the main trend to prevent infectious diseases in the future. Salmonella as the vector of vaccine has received wide attention in medical and veterinary medicine. The major advantage of Salmonella is that it can be administered by mucosal route (oral or intranasal), easily genetically manipulated and cause little stimuli to animals. In addition, as an intracellular invasive bacterium, Salmonella can effectively present antigen, stimulate specific humoral and cellular immune response against both Salmonella and heterologous antigen, stimulate mucosal and systemic immune at the same time. The mostly studied serotype as a live vector is S. typhimuriurn. S. choleraesuis was seldomly reported as a live vector. Edema disease of swine, ED happened majorly in piglets after weaning 1-2 weeks, caused by certain serotype E. coli and is one of dieases which harm weaning piglets fairly severely. Shiga-like toxin type II variant (SLT— Ⅱv) is the major causative agent of ED, composed with one A subunit and five B subunits. Subunit A is the virulent part of the toxin. Subunit B determines the specificity of the toxin is the immunogenic part of the toxin.Based on above considerations, this study was aimed to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization. C500 strain was used as the parent strain. The crp (cAMP receptor protein) gene was deleted in the C500 genome to decrease virulence and remove the risk of virulence return. Then the asd (aspartate beta-semialdehyde) gene was deleted to construct asd host-vector balanced lethal system for stably expressing heterologous genes. This system was sucessfully used to express gfp (Green Fluorescence Protein) gene and Shiga-like toxin type II variant B (SLT- II vB) gene of 0139 E. coli ED3 strain, and eatablished groundwork for developing novel Piglet's Paratyphoid-ED bivalent vaccine. The main research was described as follows: 1. Cloning of crp and asd gene and construction of recombination suicide plasmidsThe crp and asd gene were amplified and cloned from C500 genome according to the...