Construction and characteristics of a recombinant strain apxⅡC-/apxⅠA+ of Actinobacillus pleuropneumoniae serotype 7

Abstract:

An Actinobacillus pleuropneumoniae apxⅡC mutant was constructed by transconjugation and counterselection method. Briefly，a transconjugation plasmid pEHA1 was constructed，and transformed into donor strain Escherichia coli β2155. 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.