Isolation And Identification Of H.Parasuis And Bioinformaticsa Nalysis Of Its Outer Membrane Protein P2Gene

The trial isolated three suspected Hameophilus parasuis from lung, pericardial fluid, synovial fluid and other diseased tissues of pigs infected Glaser’s disease,and make susceptibility test to them,got the high sensitivity drugs to the isolates.Amplified outer membrane protein P2gene of Haemophilus parasuis isolates by PCR,The results showed that This test isolates HS I and HS II of serotype4, respectively, are at nucleotide homology of97.3%,97%with the type reference strains SW124of serotype4,and the difference is2.8%,3.1%.The5serotype isolate HW I and type reference strains Nagasaki is in the nucleotide homology of97.1%and a difference of2.9%.A11of these visible Haemophilus parasuis outer membrane protein P2gene is highly conserved, and little effects the serotype.Ⅰ. Isolation and identification of Hameophilus parasuisHandle the diseased tissues of lungs, pericardial fluid, synovial fluid of the suspected infection leather Glaser’s disease swine aseptically collected from Sichuan Nanchong, Suining.Vaccinate the homogenate into liquid medium and culture overnight,then coat the production onto solid medium.Pick the signal colony similars to H.parasuis in biological characteristic onto solid medium, and isolated three strains suspected H.parasuis.After several times of passage purification,then make a series microbial characterization of straining,biochemical tests and drug susceptibility tests,at the last determine them as H.parasuis using16SrDNA PCR.Serotype the three isolates as4,4and5through Antigen-antibody agglutination test.The results of animal experiments show that the three isolates are virulent.The isolates are sensitive to some drugs such as aminoglycosideantibiotic gentamicin, amikacin and neomycin, cephalosporin,claforan of Ceftizoxime,gatifloxacin antibacterials of ofloxacin, levofloxacin,C norfloxacin,macrocyclic erythromycin and azithromycin, florfenicol, cotrimoxazole.Ⅱ. Cloning and sequence analysis of ompP2geneAmplified outer membrane protein P2gene of Haemophilus parasuis isolates by PCR,connect PCR product with cloning vector pMD19-T Simple Vector and import to competent cells DH5a,sequencing and analysis of the positive colonies after PCR and restriction enzyme digestion and the recombinant plasmid cloning referred to as pMD-ompP2.Sequence analysis showed that the cloned genes lengthed1086bpbp、1086bp、1092bp and encoding360,361,363amino acids.The nucleotide sequence and amino acid sequence homology between HS Ⅰ ompP2,HS Ⅱ ompP2,and HW IompP2are at the high level of more than98.1%.Compared with different sources of HPS sequences from the United States, Germany, Japan and China and different serotypes HPS sequence in1,2,4,5,7included in the GenBank,their nucleotide sequence homologyare all at the high level of more than95.0%,but the difference is respectively4.7%and4.8%;the deduced amino acid sequences homology is with a minimum of90.8%and89.7%, the maximum difference is respective at10.1%and10.5%.This test isolates HS I and HS II of serotype4, respectively, are at nucleotide homology of97.3%,97%with the type reference strains SW124of serotype4,and the difference is2.8%,3.1%.The5serotype isolate HW I and type reference strains Nagasaki is in the nucleotide homology of97.1%and a difference of2.9%.All of these visible Haemophilus parasuis outer membrane protein P2gene is highly conserved, and little effects the serotype.III. Bioinformatics analysis of Haemophilus parasuis’s ompP2geneSignalP4.1Server software analysis showed that three of Haemophilus parasuis isolates the first20amino acids of the protein ompP2is the signal peptide, nucleotide excision should be located at25bp.OmpP2nucleotide sequence encoding the mature protein obtained according to the cloning, sequencing, using the DNAStar software to analyze the nucleotide content and speculated that the respective amino acid sequence, Protean prediction analysis showed that the surface,the pro-aqueous and antigen of the outer membrane protein P2encoded by the isolates bacteria are highly conserved:The entire amino acid sequence to remove36,70,120to130,141,180,209to216,222to228of about65amino acid residues, almost all higher hydrophilic region;The surface projections indicate42to64,83to94,101to105,148to157,188to198,231to246,299to313,323to326,388to349area located more likely on the surface of the protein;Epitope prediction results show that the entire amino acid sequence of the antigen that uniformly distributed is in the higher index.Gamier-Robson and Chou-Fasman secondary structure prediction shows that the type4HS I and HS II ompP2is very similar in the mature protein,and have nuances to the type HW I strains:HS I and HS II respectively has12and9alpha-helical regions,24and15beta-folding areas,18and28beta-corner region;a total of28alpha-helical region of the60to71,73to93,127to147,150to165with15wide sectors in them, total beta-fold region of27to106,142to152,208to218,273to296,319to327,353to363;HS I has22flexible regions, the HS II have23and each section is wide.Serotype5HW I to ompP2I mature protein respectively has11and8alpha-helical regions,23and28beta-folding area,18and28beta-corner regions.Be different from type4in alpha-helical,β-folding and beta-corner area,HW I has31total alpha-helical regions of50to95,130to180,245to292,325to365,23total beta-fold regions of25to90,105to120,145to152,180to190,275to300,and23large section of the flexible regions.