Construction Of A Mutant Strain Of Enterococcus Faecalis Hemolysin CylA Gene In Bovine Mastitis And Study On Antigenicity Of Hemolysin

This experiment was conducted to construct a mutant strain of Enterococcus faecalis hemolysin CylA gene and to explore the hemolysin antigenicity of bovine mastitis.The sequence of Enterococcus faecalis CylA gene with sequence number AY032999.1 published on Genbank was used as a template to design primers for the upstream and downstream homologous arm sequences respectively.Replacement of the CylA gene intermediate sequence with the chloramphenicol resistance gene Chl in the p ACYC184 plasmid to reduce the sensitivity of the experimental strain to chloramphenicol for use in antibiotic resistance screening.The X,Chl and Y sequences were sequentially ligated and transferred into the temperature-sensitive suicide plasmid p SET4 S to construct the Enterococcus faecalis CylA gene deletion recombinant plasmid p SET4S-X-Chl-Y.The recombinant plasmid was electrotransferred to Enterococcus faecalis hemolysis-positive strain,and the mutant strain of Enterococcus faecalis hemolysin CylA gene was screened by antibiotic resistance after several bacterial passages.The Enterococcus faecalis hemolysin CylA gene was also cloned for prokaryotic expression to explore its immunogenicity.The results of PCR and single and double digestion showed that the CylA gene deletion recombinant plasmid was successfully established.The results of PCR and hemolysis phenotype identification showed that the mutant strain of Enterococcus faecalis hemolysin CylA gene was successfully obtained,which laid the foundation for the study of the effect of Enterococcus faecalis CylA gene on the pathogenesis of hemolysin.CylA gene cloning and sequencing results showed that a target DNA band of 1239 bp was successfully cloned.The constructed p ET-30a-CylA recombinant plasmid was induced to be expressed using IPTG and identified by SDS-PAGE electrophoresis,which showed that the relative molecular mass of the expressed encoded protein was37 k D,and the protein content obtained after purification by a nickel-coupled affinity chromatography column was 1.5 mg/m L.The expressed antigen was emulsified with Freund’s adjuvant and immunized rabbits with ELISA antibody titers,which showed antigenicity at 1:5 000 at 28 d after the first immunization,providing a candidate subunit antigen for further development of a genetically engineered vaccine for dairy mastitis.