| Riemerella anatipestifer is one of acute and septic pathogens,which infects water fowls including duck,widgeon and stating geese.R.anatipestifer has many serotypes,in China at least 21 serotypes have been reported.Among these different serotypes,little cross reaction and obvious variance in pathogenicity was found.Besides,this bacteria is not active in biochemical reaction,so it need relative higher nutrition condition.First of all,this study attempted to isolate and identify R.anatipestifer strains from the brain tissue of 20 cases suspected of R.anatipestifer infection.The clinical isolates appeared smooth and bluish green halo in the culture medium and showed gram negative after staining.The bacteria present short rod form and bipolar stain in Wright's staining and inactive biochemical reactions.According to these results,the clinical isolates were preliminary estimated to be R.anatipestifer.Then,the clinical isolates were further identified by PCR method of 16 sRNA and the results showed that the homology was higher than 99% between the isolated bacteria and R.anatipestifer reported in NCBI.Through the results of morphological observation,biochemical tests and PCR detection,the clinical isolates were identified as R.anatipestifer.The serotypes of five R.anatipestifer isolated in this study were type 1,2 and 10 through the slide agglutination test.The animal regression experiment showed that the 5 R.anatipestifer were pathogenic with typical symptoms of R.anatipestifer infection,and the lethallty rate was 100%.The pathological sections showed pathological changes on heart,liver,spleen,lung and other organs caused by R.anatipestife.Secondly,the gene sequence of ompA in 5 R.anatipestifer were amplified by PCR method and its protein structure was analyzed using biology software.The results showed that the gene sequences of ompA had a homology of 98% between these 5 strains,which suggests a high homology among different serotypes of R.anatipestifer.Then,B cell epitopes was predicted by PCR amplification and the length of this gene was 408 bp.Furthermore,The recombinant plasmid PET-32a-ompA was constructed by double restriction enzyme digestion and ligation with PET-32 a empty vector.The recombinant plasmid was transfected into E.coli Rostta(DE3)to induce the expression of His-Omp fusion protein.SDA-PAGE gel electrophoresis showed that the fusion protein was 31.4k D.The fusion protein was purified by Ni2 affinity chromatography and detected by Western-bolt.The results showed that the fusion protein with prokaryotic expression had good immunogenicity.Then the purified fusion protein was used as immunogen to immunize SPF mice three times.Spleen cell were collected from immunized mice and fused with myeloma cell to produce mouse monoclonal antibody.The monoclonal antibody was further screened by purified PET-32a-omp fusion protein.Three monoclonal anti-R.anatipestifer ompA were obtained and results of ELISA showed the highest antibody titer reached 1:256000.It was confirmed that the purified PET-32a-omp fusion protein was able to obtain high titer immune serum.Conclusion: in this study,we successfully isolated R.anatipestifer,and got the gene sequence of ompA.Through prokaryotic expression and purification,monoclonal anti-R.anatipestifer ompA were obtained.The results indicated that the expressed protein could be efficiently combined with immune serum,which laid the foundation for further establishment of ELISA assay in vitro. |
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