The Research Of Pathogens Antibiotic Resistance And Antigenic Gene Of Streptococcus Of Bovine Mastitis

Mastitis is one of the most important diseases that affect the dairy industry．The pathogens ofthis disease are mainly focus on bacteria although they are in variety and complexity. Through thestudy on the pathology observation, main pathogenic bacteria, drug resistance investigation andstreptococcus antigenic gene analysis of cow mastitis in Gansu region, screening antigen gene withvaccine value, laid the foundation for the control of dairy cow mastitis.The healthy cow breast tissue samples and the risk of bovine mammary tissue samples weretaken from the slaughterhouse, Though HE staining pathological diagnosis suggested differentmastitis pathogens gave riseto distinguished different histopathological.32strains of Streptococcus agalactiae,15strains of Staphylococcus aureus,13strains ofEscherichia coli and4strains of Streptococcus dysgalactiae were isolated and identified from milksamples of bovine with clinical mastitis in different Gansu regions of dairy farm, then Kirby-Bauermethod was used to study their antibiotic resistance. The results showed that common antibioticshave varying degrees of resistance to four kinds of bacteria of dairy cow mastitis Four Strains weresensitive to florfenicol, enrofloxacin, levofloxacin, ciprofloxacin, cefoperazone/shubatan andcephalosporin with sensitivity of60%～100%and resistant topenicillinstreptomycin,sulfamethoxazole compound.THB selective solid medium and Islam medium was used to selectively culture thestreptococcus agalactiaee, and primers of streptococcus agalactiae16s rRNA gene was designedaccording to the reference strain to identify12strains of suspected streptococcus agalactiae. OnlineBLAST showed that16s rRNA possessed high similarities within the12strains (99.0％-100％)and with their counterparts registered in GenBank(>99.0％).The sip gene is agalactiae important adhesion and colonization factor, with a highly conservednucleic acid sequence and strong immunogenicity, it is an important candidate antigen. The Sipgene was amplified by conventional PCR,it contained a1305bp, which encoded434amino acids.The molecular mass of the deduced amino acid sequences was56ku. Blast analysis showed that itshared high identities (91.0%-99%) with Sip sequences of other GBS registered in GenBank. Thededuced amino acid homology was90.2%-98.8%. Sequence and phylogenetic analysis of Sip genesegments revealed that the Streptococcus agalactiae GS was recent Group B streptococcus isolatedin dairy cattle from China, The Sip gene connected with the pET-28a, successfully constructedrecombinant plasmid pET-28a-sip. BL21IPTG (pET-28a-sip) positive recombinant bacteria SDS-PAGE and Wester-blot showed that expression of target protein of molecular weight is56ku.ORF analysis and Primers of B cell epitopes enriched sequences of CAMP was designedaccording to the published CAMP gene sequences in GenBank, followed by adding doubleenzymatic sites, the Kozak sequence and the start cordon. The target sequence was cloned andsequenced to make sure all elements were correct prior to link with thepcDNATM3.1V5-HisA.transformed into DH5α competent cells, Recombinant plasmid was extractedand identified for the target sequence by double enzymatic digestion and sequencing. The resultsshowed the pcDNA3.1-cfb was successfully constructed, thus facilitated further developing ofStreptococcus agalactiaee genetic engineering vaccine.