Purification of Capsular Polysaccharide Produced by Streptococcus pneumoniae ATCC ۴۹۶۱۹

Background and Aim : Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The capsular polysaccharide (CPS) is the main virulence factor and target antigen in current pneumococcal vaccines. Based on the antigen composition of capsular polysaccharides, Streptococcus pneumoniae is classified into more than ۹۸ serotypes. The purpose of this study was extraction and purification of CPS from Streptococcus pneumoniae ATCC۴۹۶۱۹.Methods : After cultivation of Streptococcus pneumoniae ATCC۴۹۶۱۹ on TSB medium at ۳۷ ºC with ۵% CO۲ for ۲۴ hours, a simplified purification procedure for capsular polysaccharide was carried out after cell lysis with SDS and boiling for ۲۰ minutes at ۱۰۰ºC. The supernatant pH was adjusted to ۴.۵ with ۵ M acetic acid, and capsular polysaccharides were collected from dialysis of cell lysates after using different concentrations of ethanol and proteinase K treatment (۲۰mg/ml) for ۳۰ minutes at ۵۶ºC. In order to measure the percentage of purity and determine the concentration of capsular polysaccharide, the standard tests were performed including carbazole assay, phenol sulfuric acid test, SDS page, Bradford, agarose gel electrophoresis, and sample absorbance measurement at ۲۶۰ and ۲۸۰ nm.Results : This purification procedure rendered purified CPS in a yield of ۵mg from ۳litre cultivation using the result of phenol sulfuric acid which showed the presence of pentose and hexose compounds in CPS. In all concentrations of ethanol precipitation, we have observed the presence of CPS. The highest and lowest concentrations of purified polysaccharides were found in ۸۰% and ۴۰% ethanol fractions, respectively. The dark purple color in the carbazole assay demonstrated that hyaluronic acid was also present in the CPS component. Nucleic acid and protein contaminations of CPS were successfully eliminated by using fractionation with ۳۰–۸۰% ethanol and proteinase K treatment. The results of Bradford, SDS page, and A۲۸۰nm proved the removal of protein contamination, and the results of agarose gel electrophoresis and A۲۶۰nm showed the nucleic acid contamination was eliminated.Conclusion : In this study, we have demonstrated a simple and efficient method for purification of the capsular polysaccharide from Streptococcus pneumoniae ATCC۴۹۶۱۹ which can be used for conjugation with proteins as a candidate for polysaccharide conjugate vaccine.

کلیدواژه ها:

Streptococcus pneumoniae ، Capsular Polysaccharide ، Ethanol precipitation ، Polysaccharide Conjugate Vaccine ، Iran.