Cloning And Prokaryotic Expression Of The Gene Cyla Of Enterococcus Faecalis Hemolysin In Milk

To clone the Cyla gene of Enterococcus faecalis,construct the Recombinant Prokaryotic expression vector of the Cyla gene antigen predominant sequence,and express it into the BL21 of Escherichia coli,and identify the immune reaction activity of the protein expressed,which is good for establishing the epidemic detection of the cow fecal enterococci.Experimental basis.The milk samples of the mastitis,isolate the strains with hemolytic phenotype,and extract the genomic DNA and identify the Enterococcus faecalis through 16 S rDNA sequence molecules.According to the Cyla gene sequence designed by GenBank,1 pairs of cloned primers are designed,PCR amplification of the dominant sequence of the Cyla gene antigen,and the construction of the recombinant expression plasmid pET-30a(+)-Cyla,transformed into Escherichia coli BL21 cells,was induced,expressed and optimized by IPTG,and was identified by SDS-PAGE electrophoresis,Ni2+ affinity chromatography,and Western-blot to identify the immunoreactivity of the expressed protein.Sequencing results showed that the hemolytic Cyla gene of Enterococcus faecalis was successfully cloned and the sequence length was 1239 BP,encoding a total of 413 amino acid residues.Sequencing and enzyme digestion analysis showed that the recombinant plasmid pET-30a(+)-Cyla was successfully constructed and expressed efficiently in the BL21 cells of Escherichia coli,the relative molecular mass of the induced product was 44 Ku,the purified protein content was 2.44 mg/ mL,and the recombinant protein had a good immune response by the Western-blot result.Original nature.The protein encoded by the epitope sequence of the Cyla gene of the mastitis has good immune response activity and can be used as the active antigen for the epidemic detection of the dairy cow enterococci mastitis.