Identified Of Enterococcus Faecalis Isolated From Septicemia Geese And Prokaryotic Expression Of Cytolysin Activator

The four strains of pathogenic bacterias were Enterococcus faecalis,that isolated from septicemia geese.The methods included test of characteristic identification, such as observing morphology,test of tolerance, test of virulence factor, biochemical test and so on,and test of molecular biology,such as 16SrDNA detection and phylogenetic analysis.It is positive to make L-pyrrolidonyl-β-naphthylamide,bile esculin,dynamic test,arginine dihydrolase test,;tellurite, pyruvate utilize test; mannitol, sorbitol, sorbose,lactose, salicin fermentation tests.It is negativ to make catalyst, ferment glucose aerogenesis test; sucrose, arabinose, raffinose, xylose fermentation test.They could grow in the condition of pH9.6,10℃,45℃,60℃30min,6.5%NaCl The 16SrDNA analysis indicated that these isolates possess high homology to E.faecalis ATCC29212(99.0%),and the nucleotide homology to E.faecalis GU385352 and others stranins that are published in Genbank are all above 99.6% respectively.Through analysis of vireulence phenotype and determination of LD50,we discovered that the four isolates were middling pathogenicity,and we named it TLME3.To explore the variability of Enterococcus faecalis TLME3 Cytolysin Activator gene. Designed CylA primers according to the senqence of Enterococcus faecalis pathogenicity island in NCBI database Genbank, by the methods of PCR amplification,cloned into pMD19-T vector and detected positive clones by PCR and restriction enzyme digestion.The results was that the homology rate of gene sequences between TLME3 CylA and L37110, AF329367, AF454824, AY032999 was all above 99.0%. The rate of HE1, HE9, HE12, HE30 isolated from swine in China was more than 98.1%,the highest homology rate was 99.8%.The results indicated that the cloned fragment is the seqence of hemolysin CylA gene of Enterococcus faecalis,the gene is highly conserved.In order to study the function of Cytolysin Activator of Enterococcus faecalis which isolated form goose named TLME3. Take CylA cloned into the prokaryotic expression vector pET-28a (+) to construct the recombinant plasmid pET-CylA. Identified by EcoRI, XhoI, and transformed into E. coli BL21 (DE3), induced expression by IPTG. Using the High-Affinity GST Resin purified fusion protein, analysis by SDS-PAGE and Western Blot. The senqence obtained is 1239bp, and the homology rate between Genbank L37110 and other strains were all above 99.0%. The product of SDS-PAGE, Western blot is the CylA, the molecular weight is 65.9kD. The results showed that in BL21 (DE3) strain in CylA TLME3 highly expressed genes.