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目的采用3种方法提取B群脑膜炎奈瑟菌(Serogroup B Neisseria meningitidis,MenB)的外膜囊泡(Outer mem-brane vesicle,OMV),并分析其免疫原性、毒性和杀菌活性。方法分别采用去污剂+EDTA、EDTA、从培养上清液中离心提取3种方法获得MenB的OMV,即dOMV、nOMV和sOMV,SDS-PAGE分析OMV主要蛋白组分,Lowry法测定蛋白浓度,LAL试验测定内毒素含量。将3种OMV免疫小鼠,采用ELISA法测定小鼠血清中IgG抗体滴度,杀菌力试验测定3种OMV诱导针对MenB补体依赖的杀菌反应。结果 dOMV、nOMV和sOMV的主要蛋白成分基本一致,内毒素含量分别为5.0×103、7.5×104和4.0×106 EU/mg,产率分别为11.640、10.210和4.135μg/1010个细菌;dOMV、nOMV和sOMV组小鼠血清抗体滴度分为3.98×106、2.51×107和5.62×104,与阴性对照组相比,差异均有统计学意义(P<0.05),杀菌抗体滴度分别为501.70、697.11和55.39。结论用3种不同的方法成功制备了3种MenB的OMV,并对其免疫原性、毒性和杀菌活性进行了比较,为研发B群脑膜炎OMV疫苗提供了理论依据。
Objective To investigate the immunogenicity, toxicity and bactericidal activity of Outer mem-brane vesicle (OMV) from serogroup B Neisseria meningitidis (MenB) by three methods. Methods The OMV of MenB was obtained by centrifugation with EDTA + EDTA and EDTA. The OMV, nOMV and sOMV of MenB were obtained by centrifugation. The main protein components of OMV were analyzed by SDS-PAGE, the protein concentration of OMV was determined by Lowry method, LAL test to determine endotoxin content. Three kinds of OMV were immunized with mice, and the IgG antibody titers in the serum of the mice were determined by ELISA. The bactericidal test of the three kinds of OMV induced the complement-dependent bactericidal response to MenB. Results The main protein components of dOMV, nOMV and sOMV were basically the same. The endotoxin contents were 5.0 × 103, 7.5 × 104 and 4.0 × 106 EU / mg, respectively, and the yields were 11.640, 10.210 and 4.135 μg / The serum antibody titers of mice in nOMV and sOMV groups were 3.98 × 106, 2.51 × 107 and 5.62 × 104, respectively, which were significantly different from those in the negative control group (P <0.05). The titer of bactericidal antibody was 501.70 , 697.11 and 55.39. Conclusion Three kinds of MenB OMV were successfully prepared by three different methods. The immunogenicity, toxicity and bactericidal activity of OMV were compared. The results provide a theoretical basis for the development of meningitis B meningitis OMV vaccine.