RESUMO
Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid, attenuated by chemical method. Although the vaccine has good immunogenicity, it remains some residual virulence. In order to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization, delta crp delta asd double deletion mutant was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp (cAMP receptor protein) gene and sacB (sucrose-sensitive gene) gene was constructed and conjugated with C500. The unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR. Then the asd (beta-aspartic semialdehyde dehydrogenase) gene was further deleted in the delta crp strain by the same method. Foreign DAP (diaminopimelic acid) must be supplied for delta crp delta asd mutant to grow. The phenotype, growth properties and virulence in mice of delta crp mutant were further characterized. In conclusion, the delta crp delta asd double-deletion mutant was successfully constructed. The delta crp delta asd mutant can be used as a live vector to express foreign genes and to develop potential oral multivalent vaccines.