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

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

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。

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＇-ethylearbodiimide 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 CAMP 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 11[, 2010 edition）. The conjugate formed obvious precipitation line with diagnostic serum for group A Neisseria meningitides, by which the in- duced GAMP-specific antibody titer was significantly higher than by GAMP and by mixture of GAMP and CRM197. Conclusion GAMP-ADH-CRMI97 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.