Extraction, Purfication And Identification Of CP5 From Staphylococcus Aureus

Objective:Staphylococcus aureus(S,aureus) is one of the pathogenic bacterium of bovine mastitis.And the capsular polysaccharide(CP) is one of the major virulence factors.Research reveals that both the serotype 5 and 8 were the major serotype of these pathogenic bacterium.So we selected the CP5 S.aureus to extract,purify and identificatify the CP,ready for the biologically and immunological effectiveness research later.Methods:Bacteria were cultivated on 50 Columbia blood agar base plates for 24h, collected with the PBS(pH 7.4).First,the bacteria was autoclaved twice at 121℃for 1h, Cells were pelleted by centrifugation and the supernatants were pooled,and the nucleic acids and proteins were partially removed by fractionation with 30%ethanol(V/V) at 4℃overnight.After centrifugation at 25,000×g for 30 min,the supernatant was precipitated in 80%(V/V) ethanol at 4℃overnight.The white,flocculent precipitate was suspended in PBS(pH 7.4).Then it was digested overnight at 37℃with DNase I and RNase A, followed by overnight digestion at 37℃with Pronase and Proteinase k.After dialysis(molecular cut off range 12～14KDa).it was treated with 0.05mol/L sodium metaperiodate at room temperature(25℃) in the dark room for 45 min to remove teichoic acid.The reaction was stopped by adding 25%(V/V) ethylene glycol.The sample was then dialyzed against 0.05 mol/L sodium metaperiodate with deionized water.The dialysate was applied to 100KDa and 50KDa centrifugal ultrafiltration to remove the unwanted material,then sifted with a DEAE-Sephacel column(2.0 by 16.0cm),fractions that were positive with Ultraviolet Light Spectrophotometer specific(490nm) absorption value were pooled,desalted,and lyophilized.Result:By the C₆H₅OH-H₂SO₄ Method,the concention of the crued CP is 26.15μg/ml, and 29.88%;by the Nucleinic acid and Protein Detecter,the nucleinic acid and the protein total concentration was 0.1113 mg/ml and 0.0103 mg/ml respectively;the monosaccharide composition of the CP5 determined by the HPLC,there be Man,Fuc, Rham,GluA,Glc,Gal,Xyl and Ara.Conclusions:Determined by the HPLC and 1H-NMR,the extracted and purficated material was consistent with the true of CP5 of S.aureus.