Purification Of Pneumococcal Capsular Polysaccharides Type 14

Streptococcus pneumoniae is Gram-positive bacteria with virulence capsular.It is a major cause of morbidity and mortality worldwide leading community acquired pneumonia, acute otitis media, meningitis and sepsis. Nowadays, 23-Valent pneumococcal polysaccharide vaccine and 7-valent, 13-valent pneumococcal conjugated vaccine which utilize the capsular polysaccharide to provide protection had been introduced in many countries.The first, we utilized the conventional bacteriological test to screen out Pn14 strain with the munber of 31601-5 as working seed which has typical biological characteristics, complete capsular apperence and strong agglutination. Using the bacterial fermentation cylinder, fermentation process was analysed and its properties of each ingredient was studied according to bacteria density and CPS yield. Pn 14 grew better in one of the batches that bacteria density OD600 was up to 13.068 and CPS yield reached to 0.4 g/L, which Pn14 in medium contained the aspartic acid and glutamine was cultured with 37 ℃, pH7.2, 10 % inoculation concentration, CO2 2 L/min and compressed air 3 L/min.The second, we observed the effect of the concentration of lysis agent, the time of lysis and the temperature of lysis on the degree of lysis. If the optical density of fermentation was less then 12, we added 0.12% sodium deoxycholate, then standed 12 to 18 hours at 18 to 22 degree centigrade.The lysate was purified by ethanol or CTAB, respectively. The sample was analyzed including the content of hexosamine, the content of protein, the content of nucleic acid and antigenicity. The experimental result showed that ethanol precipitation was suitable for purification of Streptococcus pneumoniae capsular polysaccharide type 14.Crude polysaccharide was treated by phenol extraction or sodium deoxycholate precipitation, respectively. After fully analysed the obtained polysaccharide, sodium deoxycholate precipitation was superior to phenol extraction, as the deproteinization method in purification process of Streptococcus pneumoniae capsular polysaccharide type 14. The technological parameters were as follows: the final concentration of sodium deoxycholate was 0.5%, the pH was 5.0 or less, the centrifugal supernatant was ultrafiltrated by 100 Kd membrane.The last, we Optimized the ultrafiltration process by response surface methodology. The performance of membrane was investigated with respect to three factors including transmembrane pressure, cross-flow rate and polysaccharide concentration. All factors and their interactive effects on the performance of membrane were also investigated with Box-Behnken Design. The predictive model of regression equation was analyzed by Design Expert software. The optimum ultrafiltration conditions were TMP=12.8Psi, Q=66L/h, CPs=0.86g/L and diavolumes=5.