Extraction, Purification And Identification Of Type 5 Capsular Polysaccharides From Staphylococcus Aureus

One third of cow at lactation period happens to mastitis, especially subclinical mastitis 90% of which is caused by Staphylococcus aureus(S.aureus) to damage the cow industry badly. Capsular polysaccharides(CP) have been identified in clinical isolates of S.aureus, and serological distinction of 11 CP types has been proposed. Two CP types, 5 and 8 which is account for 80% of S.aureus is predominant serotypes, virulence factors and protective antigens. The type 5 capsular polysaccharide(CP5) is extracted and purified, so that it makes foundation for preparing the bispecific antibodies(BsAb) of CP5 and L-selectin of bovine polymorphonuclear leucocytes (PMN) to prevent and cure the bovine S.aureus mastitis.Make the difference of culture conditions which affect growth of S.aureus and the yield of CP5 of S.aureus to get the best production. Depending on the nature of medium containing differernt carbon source, the way of culture including to be still and vibration and state of medium, four medium which are used to culture S.aureus, such as brain heart infusion(BHI), modified Staphylococcus medium(mod110), sheep blood esculin medium and Columbia medium, make up many kinds of culture conditions.Capsular polysaccharides exisits on the surface of S.aureus from transmission electron microscope scanogram. As result of certain celluar resolvase releasing, with the bacterium decomposeing CP falls off after the logarithmic growth. Depending on different absorption value of suspension of S.aureus at 560nm wave length, make growth curve of different culture conditions. According to the indirect competition ELISA, make competition curve to get regression equation: y=-0.7331x+92.123 (R=0.9929), and then compute the yields of CP5 under kinds of culture conditions.According to experimental results, it makes clear that the other three solid mediums is better for growth of S.aureus and the yields of CP than liquid medium under any way of cultuer except Columbia solid medium. It is known that shaking culture with lactose as carbon source is more profitter to the growth of S.aureus than stationary culture with the carbon source of glucose. Although mod110 is best medium for the yield of CP5, considering the procedure of preparing mediums,origin of material and process of extraction and purification, Columbia solid medium is selected to culture S.aureus to get CP5.The superf CP5 of S.aureus was educed through twice autoclaving including 121℃by 45min and 121℃by 60min.And the clear supernatant liquid of CP5 was obtained with high speed centrifugation(20000g×30min) in low temperature(4℃). And then the successive steps were ultrafiltration with a 30kDa cutoff hollowfiber cartridge, treatment by sodium metaperiodate (NaIO4), ultrafiltration with a 100kDa cutoff hollowfiber cartridge under the guide of spectrophotometrically at 220nm, 260nm, and 280nm. Finally, 4500mL ultrafiltrate was get between the molecular mass of two ultrafitration membrane and 2.0000g crude CP5 extract harvested through concentration of lyophilization.Because of the maximum absorption at 206nm for polysaccharides, the crude product containing polysaccharides was proved by utraviolet scan spectra from 100nm to 1000nm.Through DEAE-Sephacel ion exchange chromatography with 0.05M sodium acetate buffer eluant containing 0.15M sodium chloride by the flow fate of 3ml to 4ml per minute(6.2ml per tube) and Sepharose CL-6B agarose gel chromatography with 0.2M sodium chloride eluant by 0.2ml per min(16ml per tube), purify the crude CP5 furthermore. Depending on the absorption value with utraviolet spectrophotometer at 206nm and optical value with indirect ELISA, the former eluant was kept exceeding to 0.300 for absorption value and 0.436 for optical value, and the latter was getted exceeding to 0.300 for absorption value and 0.600 for optical value. At last 100μg CP5 was harvested through concentration of lyophilization.To solve the problem of unbinding to ELISA plates for polysaccharides, the polysaccharides which reacted with cyanuric chloride and then were treated with natrium hydroxydatum( NaOH ) are coupled with polylysine(PLL). While the characteristic absorption peak for polysaccharides places at 206nm, the powerful absorption value for peptid tendines of conjugate of PLL with polysaccharides present at 217nm. It suggests that polysaccharides links to carrier protein(PLL) according to the absorption value of the conjugate leaning to 210nm. It also proves that polysaccharides and PLL is coupled successfully by rate of coupling being 1:37.6.Make the standard curve of glucose with the method of phenylic alcohol and get the regression equation: y=0.0067x+0.0011 (R=0.9709), and then compute the concentrate of polysaccharides in purification (70.18%). The content of protein and nucleic acid is respectively 0.125% and 0.140% with equipment detecting protein and nucleic acid.Sandwiched ELISA containing the first antibody which is rabbit serum of anti-type 5 of S.aureus and valence more than 12800 and the second antibody that is monoclonal antibody(McAb) gived by Max J.Paape of anti-type 5 of S.aureus. Three absorption values of purification were 2.563, 2.568 and 2.570, while nagative value is 0.256. On the whole it illustrates that the purification is CP5 of S.aurues. With high performance liquid chromatogram(HPLC) analysising the structure of CP5 of S.aurues, it indicates that CP5 is made up of mannitose, fucose, glucose, galactose and arabinose. The content of simple sugars above respectively is 1.4652, 6.5428, 7.5363, 5.3510, 2.9732. It also proves that the purification is CP5 of S.aurues.