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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 Shigella flexneri 2a O-specific polysaccharide conjugate and test its immunogenicity. Methods Hydrolyzed Escherichia coli 2a Shigella O-specific polysaccharides were mixed with adipic acid dihydrazide (EDAC) mediated by EDAC, , ADH) to prepare derivatives, and then combined with tetanus toxoid (TT) to prepare Shigella flexneri 2a O-specific polysaccharide protein conjugate (hereinafter referred to as conjugate), and to detect various biochemical indexes . The mice were immunized with polysaccharides, conjugates prepared in this experiment, and the conjugates prepared by the cyano-activated and ammonia reduction methods. The mice were immunized with 0.2 ml / mouse (containing 2 μg of polysaccharides) for 3 times. After 2 weeks of immunization, the mice Serum antibody titers were calculated and the geometric mean antibody titer (GMT) was calculated. As a result, the content of free polysaccharide and free protein in the conjugate was low, the endotoxin content was acceptable, and the KD value was reduced from about 0.85 (polysaccharide) to 0.02 (conjugate). Each combination could induce specific antibodies in mice, and there was no significant difference in the GMT between the groups (P> 0.05). The GMT of each needle was significantly increased (P <0.05). Conclusions The conjugate of O-specific polysaccharide conjugate of 2a Shigella flexneri prepared in this experiment has good immunogenicity, and the preparation method has the advantages of simple steps and mild reaction conditions, and is suitable for large-scale production.