[Construction and characteristics of a recombinant strain apx II C- /apx I A+ of Actinobacillus pleuropneumoniae serotype 7].

An Actinobacillus pleuropneumoniae apx II C mutant was constructed by transconjugation and counterselection method. Briefly, a transconjugation plasmid pEHA1 was constructed, and transformed into donor strain Escherichia coli 32155. After mixed the donor cells with A . pleuropneumoniae acceptor cells, the mixture was cultivated for about 5 hours and plated on solid medium containing chloromycetin. Then the Cm(R) positive clones were picked and inoculated into liquid medium in the absence of any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies (Suc(R)) were selected and considered as mutant. The mutant was verified by PCR, heredity stability, exotoxin secretion and sequence analysis, suggested that the construction of the mutant was sucessful. The biological characteristics of this mutant strain was further investigated. Compared with parental strain, the results indicated that the mutant hold the same growth rate in vitro and reduced virulence on mice. Altogether, this mutation system will facilitate development of live attenuated vaccines and research on functions of novel genes of A. pleuropneumoniae.

[Construction and characterization of delta crp delta asd mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain].

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.