| The culture medium of Staphylococcus aureus?J581?isolated from mastitis cows was screened and optimized by single factor test and orthogonal experiment.Transmission electron microscopy?TEM?was used to observe the bacterial capsule before and after optimization.The results showed that Columbia liquid culture medium was optimal for the growth of Staphylococcus aureus,and an increase of 20 % of cells was obtained compared with former ferment condition.In addition,thicker capsular layer was also observed by TEM.A method for estimate colony-forming units by absorbance of fermentation broth was studied.Probit analysis in SPSS showed that the half lethal dose(LD50)of Staphylococcus aureus was was 4.83×108CFU to mice.Precipitation of Staphylococcus aureus was autoclaved at 121 ? to extract capsule polysaccharide.Then crude polysaccharide was treated by phenol extraction method to remove proteins in CP.1.534 g with 28.15% polysaccharide was obtained and the content of protein was3.44% after the removal of protein by phenol extraction method.Purified CP was activated by CNBr,and derivatized with adipic dihydrazide?ADH?.The derivation was then conjugated with TT under the action of ethyl-dimethyl-amino-propyl carbodiimide?EDAC?.Multi-wavelength scanning was used to identify the conjugate materials.The results showed that the final yield of CP was 34.79 mg/L,the content of protein was 0.74%,0.92% for nucleic acid and 12.29% of sialic acid.Compared with the maximum absorption peak of polysaccharide,the absorption peak of the conjugate was shifted,which indicated that the coupling of multiple glycoproteins was successful.In conclusion,CB was finally selected as best culture medium for Staphylococcus aureus.The cell yield increased by 20% due to optimization of culture conditions,and CP was thicker than before.The purification process of CP for Staphylococcus aureus was established,and the yield of CP was 50.55mg/L.Besides,our results provide a proof of successful conjugate between CP and TT. |
PDF Full Text Request