Study On The Molecular Mechanism Of Pathogenesis For Brucella Ovis

Object: Brucellosis is a zoontic disease caused by members of the genus Brucella. Infected pateits demons- trate melta fever and livestock is susceptiable to abortion after infection. Outer membrane proteins (OMPs) and typeⅣsecretion system (TFSS) proteins are putative virulent factors. OMPs and TFSS play an imp- ortant role in intracellular survival and multiplication. It is necessary to explore the mechanism of brucella parasitism and interaction between brucella virulent factors and macrophage target proteins. In this study, It is useful to describe protein-protein net between surface proteins of brucella and prey proteins of sheep macrophages, to screen specific candidate drugs to brucellosis, to give a upstream work for breedings of anti-brucellosis ovis aries.Methods: (1) Brucella ovis 019 strain was identified by conventional bacterial examination and HOOF- Print technique. Software DNAMAN was used to analyze phylogenic tree for B. ovis 019 and reference strains to determine its location in classification of Brucella. (2) The whole ORF length objective genes including omp31, virB2, virB3, virB5 and virB8 were cloned, sequenced and inserted into pGBKT7 vector containing GAL4 DNA-binding domain to become bait plasmids. The bait plasmids were transformed into S. cerevisiae strain Y187 and further examined wether all bait plasmids can activate transcription of down- stream report gene in singles. (3) Sheep alveolar macrophages were isolated and cultured. The model of macropgages infected with Brucella ovis 019 strain was built. cDNA liberary of sheep macrophages was built. (4) S. cerevisiae Y187 strain containing bait plasmids mated S. cerevisiae AH109 strain containing cDNA plasmids. The positive colonies were screened out by yeast two-hybrid system. The partial prey protein were proofed by coimmunoprecipitation.Results: (1) Compared with 8 sequences of HOOF-Prints, only locus 1, locus 6 and locus 8 of B. ovis 019 strain were common to the that of B. ovis 63/290 strain. Phylogenetic tree showed that the similarity between B. ovis 019 and B. ovis 63/290 was higher than that between B. ovis 019 and the other reference strains, which was in accordance with the results by conventional bacteriological methods. (2) The sequenced objective genes including omp31, virB2, virB3, virB5, virB8 conveied to us was significant difference between B. ovis 019 and the reference strains. (3) The cDNA library contained 1.364×105 colonies with 95.65% combinant rate. (4) Yeast two-hybrid system showed that pGBKT7-omp31 bait plasmid obtained 2 positive pGADT7-cDNA, which were more similar to Bos taurus galactose specific lectin and chemokine ligand 16. pGBKT7-virB2 bait plasmid obtained 3 positive pGADT7-cDNA, which were more similar to Bos taurus CD48 molecule, chemokine ligand 2 and lysosomal accessory protein 2. pGBKT7-virB3 bait plasmid obtained 2 positive pGADT7-cDNA, which were more similar to Bos taurus lysosomal accessory protein 2 and hypothetical protein. pGBKT7-virB5 bait plasmid obtained 3 positive pGADT7-cDNA, which were more similar to Bos taurus Purine nucleoside phosphorylase, ferritin heavy polypeptide 1 and 26S proteasome non-ATPase regulatory subunit 8. pGBKT7-virB8 bait plasmid obtained 9 positive pGADT7-cDNA, which were more similar to Bos taurus ISG15 ubiquitin-like modifier, tumor necrosis factor-inducible protein, fatty acid binding protein 4, C19 orf10-like protein, chemokine ligand 16, SGT1, steroidogenic acute regulatory protein, N-myc interactor and 2 hypothetical protein. Co-IP showed that OMP31 interacted ovis aries chemokine ligand 16 and galactose-specific lectin, that VirB5 interacted ferritin heavy polypeptide 1 (FTH1).Conclusions: (1) B. ovis 019 strain is an atypical Brucella spp. (2) The objective genes was significant diff- errence from that of the reference strains The built bait plasmids couldn't activate transcription of downstr- eam report gene in singles. (3) The cDNA library was beyond the request of parameter: 1.0×105 colonies with 95% combinant rate, which meets the wants of built library. (4) 19 prey proteins of macrophages were screened out by years two-hybrid system and 3 target proteins were proved to be interacted with 2 Brucella surface proteins by Co-IP.