Isolation And Identification Of Canine Bordetella Bronchiseptica And Cloning, Expression And Immunogenicity Analysis Of FimN Gene

Canine Infectious Tracheobronchitis is caused by Bordetella bronchiseptica(Bb) canine adenovirus(CAV),canine distemper virus(CDV) or/and canine parainfluenza virus (CPiV).One hundred and sixty-four nasal swab dogs suffering from cough from November 2006 to January 2008 have been collected from Nanjing and Beijing area.Sixteen Bb isolates were identified by morphology,physiology,biochemistry and serological tests and PCR.The results of Drug sensitivity test showed that the biological characteristics and antibiotics sensitivity of 16 Bb isolates were almost identical.CAV,CDV,CpiV mixed infection were detected in the 16 Bb positive nasal swab samples,and accounted for 81%.The fimN gene sequence of sixteen canine Bordetella bronchiseptica(CBb) isolates in Nanjing and Beijing area and one rabbit Bordetella bronchiseptica(RBb) reference strain were cloned and sequenced.By the MegAlign(DNAStar) software analysis,the 99% nucleotide homology of sixteen Bb isolates is compared with the fimN gene sequence of CBb reference strain in GenBank and one RBb reference strain.The results suggest that the diversity of fimN gene in Bordetella bronchiseptica had not found.The 630bp fragment of fimN gene of Bb was digested from the plasmid pMD 18T-fimN with EcoRⅠ+HindⅢand cloned into the expressing plasmid vector pET-32a(+).The recombinant was transformed into the host strain BL21 and induced to express by 1.0mM IPTG at 37℃.The expression product of a 39.4kDa was identified by SDS-PAGE and Western blot with Bb antiserum.The rabbit antiserum was prepaired by the Purified expressed and its precipitation valence was at 1:40.The results revealed that in vitro expression of the fragment of fimN gene had the critical antigenitie epitopes of Bb,it provided a candidate material for the development of this subunit vaccine.