福氏2a志贺菌O-特异性多糖蛋白结合物的制备及其免疫原性

Preparation and immunogenicity of O-specific polysaccharideprotein conjugate of Shigella flexneri 2a

目的制备福氏2a志贺菌O-特异性多糖蛋白结合物,并检测其免疫原性。方法水解的福氏2a志贺菌O-特异性多糖在碳二亚胺[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDAC]的介导下,与己二酰二肼(adipic acid dihydrazide,ADH)缩合,制备衍生物,再与破伤风类毒素(tetanus toxoid,TT)结合制备福氏2a志贺菌O-特异性多糖蛋白结合物(以下简称结合物),并检测各项生化指标。分别用多糖、本实验制备结合物、氰基活化法和氨还原法制备的结合物免疫小鼠,0.2 ml/只(含2μg多糖),共免疫3次,每次免疫2周后检测小鼠血清抗体滴度,并计算抗体几何平均滴度(GMT)。结果结合物游离多糖和游离蛋白含量均较低,内毒素含量合格,KD值由约0.85(多糖)降至0.02(结合物)。各结合物均可诱导小鼠产生特异性抗体,且各组间GMT差异无统计学意义(P>0.05),各针次间GMT均有显著提高(P<0.05)。结论本实验制备的福氏2a志贺菌O-特异性多糖蛋白结合物具有良好的免疫原性,且制备方法步骤简单,反应条件温和,适合用于规模化生产。

Objective To prepare the O-specific polysaccharide-protein conjugate of Shigella flexneri 2a and determine its immunogenicity. Methods O-specific polysaccharide was hydrolyzed and derivatived with adipic acid dihydrazide（ADH） in mediation of 1-（3-dimethylaminopropyl）-3-ethylcarbodiimide hydrochloride（EDAC）, and the prepared derivative was conjugated to tetanus toxoid（TT）. The obtained conjugate was tested for biochemical indexes. Mice were immunized with polysaccharide and the conjugates prepared by the method in this study as well as those by cyan activation method and by ammonia reduction method for 3 times, 0. 2 ml（containing 2 μg polysaccharide） for each,and determined for serum antibody titer 2 weeks after each immunization, based on which the genomic mean titer（GMT） was calculated. Results Both the free polysaccharide and protein content in conjugates were low, while the endotoxin content met the relevant requirements. The KDvalue of polysaccharide was 0. 85, while that of conjugate increased to 0. 02. All the conjugates induced specific antibody in mice. The GMTs of antibody induced by various conjugates showed no significant difference（each P〉0. 05）, which increased significantly with increasing doses（each P〈0. 05）. Conclusion The prepared O-specific polysaccharide-protein conjugate of Shigella flexneri 2a showed good immunogenicity. The method for preparation was simple, and the reaction condition was moderate, which were suitable for large-scale production.