一种高效快速肺炎球菌多糖蛋白结合疫苗纯化方法的建立

An effective and rapid purification method for pneumococcal capsular polysaccharide-protein conjugate vaccine

目的建立一种高效快速肺炎球菌多糖蛋白结合疫苗CPS9V-CRM_(197)的纯化方法。方法将9V型肺炎球菌多糖(CPS9V)与载体蛋白(CRM197)通过胺还原法进行偶连结合,制备成多糖蛋白结合物CPS9V-CRM_(197)。通过超滤及阴离子层析两步法纯化后,经皮下免疫BALB/c小鼠,5μg/只,首次免疫后14d进行第2次免疫。于末次免疫后0、7、14、21d经颈动脉采血,分离血清,ELISA法检测血清中的IgG及IgM抗体滴度。结果纯化的CPS9V-CRM_(197)结合物纯度达100%,多糖的总回收率为95%,分子半径为8.4nm,CL-4B色谱洗脱体积为50.32ml,酶解敏感性高于载体蛋白CRM197。末次免疫后7、14、21d时,CPS9V-CRM_(197)免疫小鼠血清中IgG及IgM的滴度均明显高于CPS9V组(P<0.05)。结论本实验建立的肺炎多糖结合物两步纯化法具有高效、快速等优点,可用于进一步提高实际工业化生产中的生产效率。

Objective To develop a highly effective and rapid purification method for pneumococcal capsular poly-saccharide-protein conjugate CPS9V-CRM（197）. Methods The capsular polysaccharide of Steptococcus pneumonia type 9V（CPS9V） was conjugated to carrier protein CRM197 by amine reduction. The prepared conjugate CPS9V-CRM（197） was purified by ultrafiltration and anion-exchange chromatography, with which BALB/c mice were immunized s. c. at a dosage of 5 μg for 2 times at an interval of 14 d. The serum samples of mice were collected 0, 7, 14 and 21 d after the second immunization and determined for Ig G and Ig M titers by ELISA. Results The purified CPS9V-CRM（197） conjugate reached a purity of 100%. The total recovery rate of polysaccharide was 95%, while the molecular radius was 8. 4 nm, and the elution volume of CL-4B chromatography was 50. 32 ml. The sensitivity of the conjugate to enzyme digestion was higher than that of CRM197. Both the Ig G and Ig M titers in sera of mice 7, 14 and 21 d after the second immunization with CPS9V-CRM（197） conjugate were significantly higher than those with CPS9V（P 〈 0. 05）. Conclusion The developed twostep purification method for pneumococcal capsular polysaccharide-protein conjugate was effective and rapid, which might be used for further improvement of efficacy of industrial production.