Extraction and Purification of Capsular Polysaccharide from Streptococcus Pneumoniae and Escherichia Coli for Conjucate Vaccines Preperation

Abstract

Glycoconjugate vaccines, in which a cell surface carbohydrate from a micro-organism is covalently attached to an appropriate carrier protein, are proving to be the most effective means to generate protective immune responses to prevent a wide range of diseases (1). In this work we extracted the capsular polysaccharides from S. pneumoniae and E. coli by culturing in soybean casein digest medium and terrific broth respectively. The polysaccharides extracted were purified using tris-magnesium sulphate, DNAse and RNAse for removal of nucleic acid. The removal was confirmed spectrophotometrically by measuring A260/280 ratio. The protein contamination was removed by precipitation of proteins with phenol acetal solution. The residual amount of protein was detected using Bradford assay, the residual amount of protein left in the extracted polysaccharide was found to be 0.5% and 0.45% in case of S. pneumoniae and E. coli respectively. The polysaccharides extracted and purified in this work was finally quantified using sialic acid assay and glucuronic acid assay and was confirmed using FTIR analysis. These polysaccharides extracted can be further used for conjugate vaccine preparation by conjugating along with carrier protein.