23F型肺炎球菌多糖与CRM197蛋白结合物制备及特性研究

Preparation and Characterization of PNPS23F-CRM197 Conjugates

目的采用改良胺化还原法,白喉无毒突变体CRM197蛋白作为载体,制备23F型肺炎球菌多糖蛋白结合物。方法在还原胺法的基础上,以1,6-己二酰肼(ADH)为分子臂,在碳二亚胺(EDAC)作用下衍化CRM197蛋白,衍化后蛋白再与多糖上的醛基反应,形成共轭结合;采用高效液相色谱技术(HPLC)、免疫扩散等方法分析结合物,并将获得的结合物免疫NIH小鼠,用ELISA方法检测多糖IgG水平。结果 HPLC图谱和免疫扩散检测结果证明了结合物的获得;动物免疫产生了较多糖高的抗23F肺炎多糖抗体,并有免疫记忆发生。结论采用改良还原氨化法可以制备出动物免疫原性良好的23F型多糖蛋白结合物。

Objective To employ the improved reductive animation （RA） method to prepare Pneumococcal polysaccharide （PN23F polysaccharide） conjugate vaccine, using mutant form of diphtheria toxin CRM197 protein as carrier protein. Methods The oxidized PN23F PS was chemically bound to CRM197 via a linker, adipic acid dihydrazide （ADH）. The conjugates were confirmed by HPLC and immunodiffusion assays. The immunogenicity of the conjugate was evaluated in NIH mice and the sera were analyzed with indirect ELISA. Results The profile of HPLC and immunodiffusion showed the existence of conjugates ; the result of immunogenicity showed higher level of anti - PN23F antibody than native PN23F and memory responses in mice. Conclusion The conjugates developed with improved reductive animation method can produce effective immunogenicity in mice.