3种方法提取的B群脑膜炎奈瑟菌外膜囊泡组分的免疫原性、毒性及杀菌活性

Immunogenicity and Toxicity of Outer Membrane Vesicle of Serogroup B Neisseria meningitides Extracted by Three Methods

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 extract the outer membrane vesicle（OMV） of serogroup B Neisseria meningitides（MenB）,and analyze its immunogenicity,toxicity and bactericidal activity.Methods The OMV of MenB was extracted by detergent ＋ EDTA,EDTA and centrifugation methods,and named as dOMV,nOMV and sOMV,respectively.The OMVs were analyzed for main protein component by SDS-PAGE,for protein concentration by Lowry method,and for endotoxin content by LAL test.Mice were immunized with the OMVs,then determined for IgG titer in sera by ELISA,and for complement dependent bactericidal response by bactericidal test.Results The main protein components of dOMV,nOMV and sOMV were basically identical,while the endotoxin contents were 5.0 × 103,7.5 × 104 and 4.0 × 106 EU / mg,the outputs were 11.640,10.210 and 4.135 μg / 1010 bacteria,respectively.The serum antibody titers of mice induced by dOMV,nOMV and sOMV were 3.98 × 106,2.51 × 107 and 5.62 × 104,which showed significant difference with those in negative control group（P 0.05）,while the bactericidal antibody titers were 501.70,697.11 and 55.39,respectively.Conclusion The OMV of MenB was successfully prepared by three methods and evaluated for immunogenicity,toxicity and bactericidal activity,which provided a theoretical basis for development of OMV vaccine against MenB.