霍乱O139血清群多糖结合疫苗的生物合成研究  

Study of Vibrio cholerae O139 Serotype Polysaccharides Bio-Conjugated Vaccine

作　　者：黄竞[1] 董岩[2] 潘超[1] 胡显文[1] 曾明[2] 吴军[1] 朱力[1] 王恒樑[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050

出　　处：《生物技术通讯》2017年第3期281-285,346,共6页Letters in Biotechnology

基　　金：国家自然科学基金(81373316);国家重点基础研究发展计划(2013CB910804)

摘　　要：目的:霍乱是由霍乱弧菌引起的一种烈性传染病,其防治已成为一个全球性的公共卫生问题。其中1992年出现的O139血清群是除O1外另一种病原体,发病数日益增多。因此,有必要寻找一种安全有效适用于各种人群的疫苗。方法:敲除霍乱弧菌O139血清群93-3株脂多糖合成途径中O抗原连接酶基因waa L,在周间质产生游离的多糖,之后转入包含来自脑膜炎奈瑟球菌的糖基转移酶和霍乱毒素B亚单位(CTB)编码序列的共表达载体,经IPTG诱导后制备全菌蛋白样品,利用抗His抗体检测糖蛋白的表达,利用Ni柱和离子交换柱对糖蛋白进行纯化,并对其进行糖定量和蛋白定量。结果:以未糖基化、相对分子质量约为14×103的底物蛋白CTB为对照,当共表达CTB和糖基转移酶Pgl L时,通过Western印迹可检测到相对分子质量约为20×103的糖基化蛋白,经Ni柱及阳离子交换柱纯化,得到纯度较高的O139群霍乱O抗原多糖结合蛋白,其纯度约为84.2%,并计算得其糖-蛋白比为0.103∶1。结论:通过生物法合成了一种霍乱O139血清群的多糖结合疫苗,为后续进行动物评价打下了基础。Objective： Cholera is still a major global health problem, Vibrio cholerae O139 serotype emerged as a second aetiologic agent of cholera in 1992. It is necessary to find a safe and effective vaccine to control it.Methods： We constructed V.cholerae O139 group 93-3 strain containing a delection in O-antigen ligase waaL, and free polysaccharides were produced in the periplasm. Then introduced the co-expression plasmid of Neisseria meningitidis O-oligosaccharyltransferase PglL and cholerae toxin B subunit（CTB） to the genetically modified strain.After IPTG induction, the samples were prepared and the expression of glycoprotein was detected by anti His antibody. The target protein was further purified by Ni affinity column and cationic exchange column and quantify polysaccharide and protein content.Results： The substrate protein CTB, which was non glycosylation and relative molecular weight of about 14 kD, was used as control. When the co-expression of CTB and glycosyltransferase PglL, through the Western blot can detect the relative molecular weight of glycosylated protein is about 20 kD. Purified by Ni affinity column and cationic exchange column, we obtained O139 group of cholera O antigen polysaccharide binding protein with purity of 84.2%, and calculate the sugar protein ratio was 0.103∶1.Conclusion： We synthesized a polysaccharide conjugate vaccine by biological method, which laid the foundation for the subsequent animal evaluation.

关 键 词：霍乱弧菌 多糖结合疫苗 O-糖基化 糖蛋白 

分 类 号：Q78[生物学—分子生物学]