Construction and characterization of an attenuate strain apxIC-/p36+ of Actinobacillus pleuropneumoniae serovar 10

Abstract:

Abstract: [Objective] To construct an attenuate Actinobacillus pleuropneumoniae serovar 10 strain apxIC-/p36+ for new vaccine development. [Methods] The mutant was constructed by transconjugation and counter-selection and then verified by PCR, western blot and sequence analysis. A transconjugation plasmid pEICALDH was constructed and transformed into donor strain Escherichia coli X7213. After mixing the donor cells with A. pleuropneumoniae acceptor cells, we cultivated the mixture for 6 hours and plated on solid medium containing chloromycetin. Then the CmR positive clones were picked and inoculated into liquid medium without any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies(SucBR) were selected and considered as mutant. [Results] Compared with parental strain, the mutant have the same growth rate in vitro and reduced virulence in mice; additionally, the animal experiment indicated that the mutant strain can successfully induce as good immune response as the parental strain, despite of deletion of apxIC gene. [Conclusion] In conclusion, we successfully constructed the attenuate strain apxIC-/p36+ of Actinobacillus pleuropneumoniae serovar 10, and this mutation system will facilitate development of live attenuated vaccines.