百日咳鲍特菌脂寡糖的制备及寡糖结合物在小鼠体内免疫原性研究  

Preparation of lipo-oligosaccharides from Bordetella pertussis and investigation of related conjugate immunogenicity in mice

作　　者：李鑫 高晶晶 罗树权 李燕婷 乔瑞洁 刘方蕾 谢贵林 LI Xin;GAO Jing-jing;LUO Shu-quan;LI Yan-ting;QIAO Rui-jie;LIU Fang-lei;XIE Gui-lin(The First Research Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第一研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2018年第5期21-27,共7页Progress In Microbiology and Immunology

摘　　要：目的提取纯化百日咳鲍特菌(简称百日咳杆菌)脂寡糖(lipo-oligosaccharides,LOS),制备百日咳杆菌多糖蛋白结合物,并分析其理化性质及免疫原性。方法通过改良热酚水法提取百日咳杆菌的LOS,采用酶解法纯化LOS,通过1%冰醋酸水解获得寡糖(oligosaccharides,OS),产物经Tricine-SDS-PAGE及核磁共振氢谱(~1H NMR)鉴定。OS的羟基经1-氰基-4-二甲氨基吡啶四氟硼酸酯(1-cyano-4-dimethylamino pyridinium tetrafluoroborate,CDAP)活化后,与破伤风类毒素己二酸酰肼衍生物(TT_(AH))共价结合,制备多糖蛋白结合物,经Sephacryl S-300层析柱纯化后收集结合物原液,并对结合物的理化性质、抗原性进行分析,采用两种剂量(2.5μg/剂和5.0μg/剂)免疫BALB/c小鼠,分析其免疫原性。结果纯化后LOS纯度>90%,核磁共振结果表明OS结构正确,功能基团得到保留。制备的结合物抗原性未发生改变,经3针免疫后,两种剂量下结合物均有剂次加强效应(P<0.05),且两者差异无统计学意义(P>0.05)。结论将百日咳杆菌OS与TT_(AH)结合是制备百日咳杆菌多糖蛋白结合疫苗的可行途径,并且结合物在小鼠体内具有良好的免疫应答。Objective To purify lipo-oligosaccharides（ LOS） from Bordetella pertussis and preparation of the related conjugates,and to investigate the physical and chemical properties and immunogenicity of the conjugates in mice. Methods The lipo-oligosaccharides were extracted from Bordetella pertussis with modified hot phenol water method,and purified by enzymatic hydrolysis,oligosaccharide（ O-SP） was obtained by 1% glacial acetic acid hydrolysis,and the product was identified by Tricine-SDS-PAGE and NMR（^1H NMR）. The hydroxyl of hydrolysate was activated by 1-Cyano-4-dimethylamino pyridinium tetrafluoroborate（ CDAP） and then combined with TTAHin preparation of the conjugate. The conjugates were purified by Sephacryl S-300 chromatography. The physical and chemical characteristics and antigenicity of the conjugate were analyzed. Balb/C mice were immunized with two doses（ 2.5 μg/doge and 5 μg/doge） of the conjugates and the immunogenicity was analyzed. Results The purity of oligosaccharides was more than 90%. The NMR results showed that the structure of oligosaccharides was correct and functional groups were retained. The antigenicity of the prepared conjugates did not change. There were boost responses by the three conjugate immunizations with two doses（ P 〈0.05）,and there was no a statistical difference between the two doses. Conclusion It was a feasible way in preparation of conjugate vaccine against Bordetella pertussis infection by covalent linkage between LOS and carrier proteins of TTAH,and the prepared conjugates could generate highly immune responses in mice.

关 键 词：百日咳鲍特菌 脂寡糖 结合物 免疫原性 

分 类 号：R378.42[医药卫生—病原生物学]