脑膜炎奈瑟菌A群荚膜多糖与B群外膜蛋白复合物偶联物的制备和免疫原性

Preparation and the immunogenicity of the conjugate made from group A capsular polysaccharide and group B outer membrane protein complex of Neisseria meningitidis

目的 脑膜炎奈瑟菌 (Nm)A群荚膜多糖与B群菌株 34 0 7或 5 42 85 2外膜蛋白复合物偶联 ,希望获得 1种对A和B群Nm感染皆有预防效果 ,并可提高A群荚膜多糖对婴幼儿免疫力的偶联物。方法 用饱和硫酸铵沉淀细菌培养上清液 ,沉淀物再经SephacrylS 30 0纯化获得外膜蛋白复合物。通过碳化二亚铵 (EDC)介导的缩合反应将B群外膜蛋白复合物与A群荚膜多糖偶联。偶联物、未偶联的荚膜多糖、B群外膜蛋白复合物及A群荚膜多糖和B群外膜蛋白复合物简单混合物按相同程序免疫小鼠获得抗体 ,经ELISA、杀菌力试验和Westernblotting测定了偶联物的免疫原性。结果 偶联物比未偶联的荚膜多糖或者荚膜多糖同B群外膜蛋白复合物简单混合抗原的免疫原性增强 2 1～32 0倍 ;其中以B群 34 0 7菌株的外膜蛋白复合物与A群荚膜多糖偶联的效果更好。偶联物免疫血清不仅对A群代表菌株 (2 90 19)和B群菌株 (34 0 7,5 42 85 2和 2 90 2 1)均有较强的杀菌活性 ,而且还对所试的具有不同菌型特征的其它 8株B群菌株仍有较强的交叉反应性。Westernblotting初步分析发现上述两种偶联物免疫的血清与各自的外膜蛋白复合物在相对分子质量约 42× 10 3 、39× 10 3 和 2 6× 10 3 处有相同的反应带 ,其中 42× 10 3 左右的条带为 1类外膜蛋白。

Objective To prepare the conjugate of ACPS and outer membrane protein complexes (OMPC) of Neisseria meningitidis ( Nm ) and to study its immunogenicity was OMPC purified from the strain 3407 or 542852. Methods OMPC was purified on Sephacryl S 300 HR after the cultural supernatants were precipitated by 70% ammonium sulfate. ACPS was conjugated to OMPC of serogroup B (BOMPC) by carbodiimide mediated condensation. Mice were respectively immunized by the conjugates, unconjugated ACPS, BOMPC and simple mixture of ACPS and BOMPC in the same procedure, then the immunogenicity of the conjugates was determined by ELISA, bactericidal test and Western blotting. Results The immunogenicity of the conjugates was enhanced by 21 to 320 times as large as the unconjugated ACPS or the simple mixture of ACPS and BOMPC. The effect of conjugation of ACPS to the strain 3407 OMPC was better than that to the strain 542852 OMPC. Antisera evoked by BOMPC ACPS conjugates not only possessed a stronger bactericidal activity to the serogroup A strains (29019) and the serogroup B strains (3407, 542852, 29021) but also showed broadly cross reactions to other eight serogroup B strains of different bacterial types. It was primarily found by Western blotting analysis that the sera elicited by the above conjugates obviously reacted with M ?r?42000, 39000 and 26000 proteins in OMPC. Among the reactive bands, the 42kD protein was classⅠOMP. Conclusion The above conjugates not only possessed strong immunogenicity of Nm serogroup A and serogroup B but also enhanced the immunogenicity of ACPS to mice.