| Erysipelothrix rhusiopathiae is a kind of pathogenic bacteria harmful to mammals,birds,reptiles,fish and human beings,it can cause high fever,sepsis,skin lesions,arthritis and other problems.Swine erysipelas caused by this bacterium has been reported in various countries,which poses a great threat to all stages of pig population.In recent years,it has brought serious losses to the Chinese pig industry and belongs to the second class animal disease.In order to provide a scientific basis for the prevention,control and treatment of swine erysipelas in Aksu area,Xinjiang,this experiment aseptically collected the disease materials of pigs with suspected swine erysipelas such as arthritis,sepsis,abortion,etc.Including the heart,liver,spleen,kidney,skin rash,etc.of sick pigs.Bacterial culture was carried out on the diseased materials,and the morphology of the colonies that grew out was observed,and the bacteria were observed by Gram-positive microscopic or slender Campylobacter by gram staining and electron microscope observation.It was further isolated and purified,the bacterial genome was extracted,amplified with bacterial 16 S r RNA universal primers,and the amplified target fragment was connected to the p MD19-T vector for cloning,identification,and then the plasmid was extracted and sequenced.Erysipelothrix rhusiopathiae strain.In order to determine the drug susceptibility characteristics of the isolated Erysipelothrix rhusiopathiae,drug sensitivity experiments were carried out on the strain of Erysipelothrix rhusiopathiae,and the results showed that the isolated strain was highly sensitive to ten β-lactam drugs;Sulfonamides,nitrofurans,and two aminoglycosides were highly sensitive.Different degrees of resistance to lincosamides,tetracyclines,quinolones,macrolides,polypeptides,and chloramphenicol were shown.In order to further clarify the drug resistance characteristics and pathogenicity of the strain,the whole genome sequencing of the strain was carried out,and the full-length sequence with a size of 1913700 bp was obtained,which was uploaded to NCBI,and the gene accession number PRJNA816312 was obtained,which enriched pigs.The whole genome database of Erysipelothrix rhusiopathiae was analyzed,and the full-length sequence of the bacteria was analyzed to clarify its drug resistance characteristics,virulence factors and other gene function analysis.The surface protective protein SPA,one of the virulence factors of Erysipelothrix rhusiopathiae,is highly expressed in the highly pathogenic Erysipelothrix rhusiopathiae,but is rarely expressed in non-pathogenic and weakly pathogenic strains.The structure is relatively stable and has good immunogenicity.In order to provide a scientific basis for the rapid diagnosis and detection of erysipelas in this farm,the surface protection antigen SPA of Erysipelothrix rhusiopathiae was selected as the target protein for research in this experiment.First,the Spa gene was cloned and sequenced,and the DNA of Erysipelothrix rhusiopathiae was used as a template to amplify its sequence to construct a recombinant plasmid p MD19-T-Spa.After identification and sequencing,the size of the Spa gene was 1530 bp.Then,the SPA prokaryotic expression vector was constructed and expressed.The p MD19-T-Spa plasmid DNA was double digested with Bam H and Xho I to obtain the Spa gene fragment,and after connecting with the expression vector,the cloned strain DH5α was transformed,and the recombinant plasmid was extracted.After identification and sequencing by enzyme digestion,the recombinant plasmid p ET-28a-Spa was transformed into the expression host strain E.coli BL21(DE3),and after induction for 1,2,3,4,and 5 h,the recombinant expression strain of Spa and the control strain were treated with The resulting product was analyzed by SDS-PAGE.The results showed that: after induction by IPTG,the expression strain DE3 successfully expressed the target protein,and its size was the same as the expected size,which was56.1KDa. |
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