志贺氏I型菌O-特异多糖与蛋白结合物的制备及其抗原性

PREPARATION AND IMMUNOGENICITY OF CONJUGATES COMPOSED OF THE O-SPECIFIC POLYSACCHARIDE OF SHIGELLA DYSENTERIAE TYPE 1 BOUND TO TETANUS TOXOID

目的：制备志贺氏Ｉ型菌的Ｏ－ 特异多糖（Ｏ－ ＳＰ） 与破伤风类毒素（ＴＴ） 的结合物Ｏ－ ＳＰ－ ＴＴ，并通过动物实验检测其抗原性和血清中抗Ｏ－ ＳＰＩｇＧ、ＩｇＭ 抗体水平及抗体杀菌活性。方法：提取纯化志贺氏Ｉ型菌的Ｏ－ ＳＰ，以己二酸二肼（ＡＤＨ） 为连接剂，以破伤风类毒素为载体，制备Ｏ－ ＳＰ－ ＴＴ结合物。对提取物、中间产物、结合物的特性进行检测。结果：Ｏ－ ＳＰ、Ｏ－ ＳＰ－ ＡＨ、Ｏ－ ＳＰ－ ＴＴ均可与兔超免疫血清形成清晰的沉淀线。单独使用Ｏ－ ＳＰ免疫小鼠后抗体无显著性升高。Ｏ－ ＳＰ－ ＴＴ免疫注射后第１ 、２ 次间，第２、３ 次间ＩｇＧ抗体及抗ＴＴ抗体水平均有显著性升高；Ｏ－ ＳＰ－ ＴＴ能刺激高水平抗Ｏ－ ＳＰＩｇＧ、ＩｇＭ 抗体，而死菌对ＩｇＧ的作用较差。Ｏ－ ＳＰ－ ＴＴ免疫后血清中抗体可激活补体产生杀菌作用，杀菌效价为１∶１６０ 。结论：本实验采用的化学方法均未破坏抗原的特异性；Ｏ－ ＳＰ单独使用无免疫原性；Ｏ－ ＳＰ－ ＴＴ注射后针对Ｏ－ ＳＰ、ＴＴ 均加强了免疫应答，并较死菌对ＩｇＧ的作用强，其刺激产生的抗体具有杀菌活性；本实验结果为Ｏ－ ＳＰ－ ＴＴ结合物的进一步应用研究提供了一定的基础资料。

Objective:In this experiment, O-Specific Polysaccharide was extracted from Shigella dysenteriae type 1 and purified, then bound to tetanus toxoid(TT)composed conjugates (O-SP-TT).Immunogenicity of conjugates were measured by animal experiment.Methods:The theoretic basis for vaccine development:protective immunity to enteric disease caused by Shigella may be conferred by serum IgG to antibodies to O-SP of its LPS. Use cyanogen bromide (CNBr) to activate the hydroxyls of polysaccharides (ADH) as spacer, 1-ethy1-3- (3-dimethylamine-propyl) carbodimide (EDAC) to activate carboxyl of protein. Therefore, O-SP and TT are bounded to be conjugate (O-SP-TT). In order to evaluate immunogenicity of O-SP-TT, NIH female mice were injected with 2.5μg O-SP alone or as conjugate O-SP-TT in a saline three times 2 weeks apart.Serum O-SP and TT antibodies were measured by enzyme linked immunosorbent assay.Resuls:The results showed O-SP of Shigella dysenteriae of type 1 alone did not elicit detectable serum antibodies, while O-SP-TT injected as a saline solution elicited a statistically significant rise in both IgG and IgM antibodies after each injection. The resultant O-SP-TT conjugate could elicit high levels of IgG O-SP antibodies and booster responses after the second injection. TT-Abs were elicited by conjugate after the second and third injections. The bactericidal activity of serun O-SP antibody was measured by complement dependent bactericidal experiments. The results confirmed that serum O-SP antibodies elicited by conjugates had stronger bactericidal activity.Conclusion:These data provide the basis for evaluating the effect of this conjugate to prevent Shigellosis.