Fingerprint Typing Of Avibacterium Paragal-linarum And Construction Of Aroa Mutants

Avibacterium paragallinarum (Apg) is one of short gram-negative bacillus of Pasteurella families, which could cause the Infectious Coryza of chicken. The common typing methods of Apg are based on its hemagglutinin activity. As the genome sequence of this bacterium is unknown, the reports about the virulence factors are really limited, mostly focused on individual genes.The enterobacterial repetitive intergenic consensus(ERICs) and random amplified polymorphic DNAs (RAPDs) were used as the target sequences of PCR to generate a fingerprint of Avibacterium paragallinarum. The genetic distance calculation and cluster analysis of the fingerprints were done by the POPGENE and MEGA4.0softwares.The results showed that both methods can get rich, stable fingerprint, with a polymorphism of85.7%(18/21). The tested bacteria, including10international standard strains, vaccine strain and4domestic isolates were divided into11types and gathered for4classes, which corresponded perfectly with subtype classified with existing serotyping method.The result showed that the finerprint technology in the prensent study can distinguish different Serological subtypes.Taking an international standard strain145(C-3) of Apg as template, In this experiment, we designed degenerate primers according to aroA gene with CODEHOP software. The improved chromosome walking technology was used to amplify the whole gene sequence of aroA. Studies on the nucleic acid sequence and structure of coding protein have been done. Besides, sequence alignment and molecular evolution analysis with other serotypes and related bacteria were also done. The whole aroA gene of Avibacterium paragallinarum was obtained, which is1293bp, encoding430 amino acids with3transmembrane region and5potential epitopes. The homology of amino acid sequences is88.1%～100%between different serotypes of experiment strains, and the nucleic acid homology is over74.2%between bacteria of the same family.Furthermore, the up and down stream of Apg C-serotype aroA gene were obtained by PCR amplification, approximately0.7kb. Meanwhile, the gene segment of Chloramphenicol resistance of0.9kb was inserted into the restriction enzyme cutting site. The transfer vector was constructed successfuly, named pUC18-U-CM-D. We obtained an nameed by. The mutant strain, termed D0912-CMr, was achieved by the introduction of the transfer vector into Apg serovar C for homologous recombination. Identification and analysis of biological characteristics of this mutant strain were well verified subsequently. The result showed that there was no significant difference in morphology between the wild strain and the mutant strain, and the growth of the mutant strain was not effected distinctly according the bacteria-growth curve.