Haemophilus Parasuis Aroa Gene Identification Analysis And Transfer Vector Construction

Haemophilus parasuis,a non-motile,small,pleomorphic rod in the family Pasteurellaceae, is a commensal organism of the upper respiratory tract of conventional pigs.Under some conditions,it can cause severe systemic diseases.It has recently emerged as one of the major caurses of nursery mortality in swine herds in the whole world.Factors involved in systemic invasion by H.parasuis remain largely unknown.The aroA gene,encoding 5'-enolpyruvylshikimate-3-phosphate synthetase which is a key enzyme in the biosynthesis of aromatic amino acids,has been used successfully for taxonomic purposes and inactivated avirulent mutant.In this paper,this is the first report showing an approximately 3.7 kb fragment containing the whole aroA gene was obtained from the genomic DNA of all serovars of Haemophilus parasuis by PCR and bacterial genome walking techniques.BLAST and DNAStar analysis showed that the size of the whole aroA gene was 1314 bp.The nucleotide sequence similarity was above 97.7%in H.parasuis serovars.And the similarity was 70.6%- 78.9%between 15 serovars of H.parasuis and other members of Pasteurellaceae.The results confirmed that the nucleotide sequence of the aroA gene of all serovars of H.parasuis was well-conserved,as well as in Gram-negative organisms.Based on the research of obtaining the aroA gene of H.parasuis, investigation the relation between the aroA gene and H.parasuis characters,such as the diversity of the serovars and the toxicity.Identification and analysis on the aroA gene will be helpful for researching the function of aroA within H.parasuis and further investigating H.parasuis characters.A 3640 bp fragment containing the aroA gene was amplified from Hps serovar 5 genomic DNA by PCR.Based on cloning and sequencing,the Hindâ…¢site of the aroA gene was inserted with a chloramphenicol resistance gene cassette and the transfer plasmid pTA-Chl~r was constructed.