A群脑膜炎球菌多糖-白喉毒素无毒变异体CRM197结合物的制备及其初步评价

目的制备A群膜炎球菌多糖(Group A meningococcal polysaccharide,GAMP)-白喉毒素无毒变异体CRM197(Cross-reactive material 197)结合物,并分析其理化性质及免疫学特性。方法白喉棒状杆菌C7 M1菌株经发酵培养表达CRM197,表达产物经DEAE-4FF层析柱进行纯化,纯化产物经SDS-PAGE及Western blot鉴定。用溴化氰(CNBr)活化GAMP,己二酰肼(ADH)为连接剂,在碳二亚胺(EDAC)催化下将GAMP与CRM197共价结合,制备GAMP-ADH-CRM197结合物,经Sepharose 4FF层析柱纯化后,进行各项生化检测,采用免疫双扩散检测结合物的抗原性,将结合物皮下注射ICR小鼠,间接ELISA法检测小鼠血清中抗GAMP的抗体滴度,分析其免疫原性。结果发酵培养20 h时,菌液上清中的CRM197蛋白表达量最高;CRM197以分泌形式表达,纯化后纯度达95%以上,可与白喉类毒素单抗发生特异性反应;GAMP-ADH-CRM197结合物各项生化指标均符合《中国药典》三部(2010版)要求,与A群流脑诊断血清形成明显的沉淀线,结合物诱生的多糖特异性抗体滴度显著高于多糖组和混合物组。结论成功制备了GAMP-ADH-CRM197结合物,其具有良好的抗原性和免疫原性,为以CRM197为蛋白载体制备脑膜炎球菌结合疫苗奠定了基础。

Preparation and preliminary assessment of group A meningococcal polysaccharide-nontoxic diphtheria toxin mutant CRM197 conjugate

Objective To prepare group A meningococcal polysaccharide(GAMP)-nontoxic diphtheria toxin mutant CRM197 conjugate and analyze its physico-chemical properties and immunological characters.Methods Corynebacterium diphtheriae C7 M1 strain was fermented,and the expressed CRM197 was purified by DEAE-4FF column chromatography and identified by SDS-PAGE and Western blot.GAMP was activated with cyanogen bromide(CNBr) and coupled to CRM197 using 1,6-adipic acid dihydrazide(ADH) as a linkers and N(-3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDAC) as a catalyst.The prepared GAMP-ADH-CRM197 conjugate was purified by Sepharose 4FF chromatography,then subjected to biochemical test,and determined for antigenicity by double immunodiffusion test.ICR mice were injected s.c.with the purified GAMP-ADH-CRM197 conjugate,and determined for serum antibody titer against GAMP by indirect ELISA to analyze the immunogenicity.Results The expression level of CRM197 in supernatant of C7M1 strain reached the maximum 20 h after fermentation.CRM197 was expressed in a secretory form,reached a purity of more than 95% after purification,and showed specific reaction with monoclonal antibody against diphtheria toxoid.All the biochemical indexes of GAMP-ADH-CRM197 conjugate met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition).The conjugate formed obvious precipitation line with diagnostic serum for group A Neisseria meningitides,by which the induced GAMP-specific antibody titer was significantly higher than by GAMP and by mixture of GAMP and CRM197.Conclusion GAMP-ADH-CRM197 conjugate was successfully prepared,which showed high antigenicity and immunogenicity.It laid a foundation of preparation of meningococcal conjugate vaccine using CRM197 as a carrier protein.