H7-HCP-Tir-Intimin高效降低小鼠粪便中大肠杆菌O157:H7排菌  

Effectively decreased fecal shedding of Escherichia coli O157:H7 following subcutaneous vaccination with H7-HCP-Tir-Intimin

作　　者：张雪寒[1] 何孔旺[1] 叶青[1] 俞正玉[1] 倪艳秀[1] 温立斌[1] 王小敏[1] 李彬[1] 周俊明[1] 汪伟[1] 

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《中国兽医学报》2014年第3期465-470,共6页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划资助项目(2012BAK08B07);国家自然科学基金资助项目(31201919)

摘　　要：构建BL21(PcoldⅠ-fliC-hcpA-tir-eae),并评价对小鼠的免疫原性,以期研制更加有效的候选免疫原,最大限度降低E.coli O157:H7对食物、环境和人类的危害。将946、423、309、811bp的fliC,hcpA,tir和eae基因被依次克隆,中间添加8个氨基酸的间隔序列,构建重组菌BL21(PcoldⅠ-fliC-hcpA-tir-eae)。进口白油和ISA50V作为佐剂与重组蛋白乳化,制备2种油乳剂疫苗,分别接种4周龄BALB/c雄鼠,二免21d,口服攻击E.coli O157:H7109 CFU/只,每天采集粪便,检测排菌。结果显示,2种重组疫苗均能够诱导高滴度血清IgG。ISA50V佐剂组抗体产生早于进口白油佐剂组,但进口白油佐剂组抗体滴度一致性更好。所有免疫组小鼠排菌极度降低,第1天排菌低于104 CFU/g,第5天检测不到排菌,整个检测期无反弹,而对照组在整个检测期排菌持续很高(>104 CFU/g)。结果表明,四价重组蛋白H7-HCP-Tir-Intimin能够高效降低口服大肠杆菌O157:H7后的排菌,极显著降低细菌的定植,很大程度减少病原传播,对环境和人类的威胁。This study aimed to construct BL21（Pcold I -fliC-hcpA-tir-eae） , examine its immuno- genicity in mice,and develop a better candidate immunogen,reducing harms of IT.. coli O157：H7 on food,environment and human. The fliC,hcpA,tir and eae of 946 bp,423 bp,309 bp and 811 bp were cloned one by one to build recombinant BL21（Peold I -fliC-）. The spacer of eight amino acids was inserted into hcpA, tit and eae genes to guarantee their correct expression. Imported mineral oil and ISA50V as adjuvants were emulsified with the recombinant protein to prepare wa- ter-in-oil vaccine,respectively. Vaccines was inoculated to 4-week-old male BALB/c mice with 3 week interval. After boosted, all mice were challenged with E. coli O157：H7 of 109 CFU/mouse and detected the fecal shedding every 2 days. Both vaccine elicited high-titer IgG in sera without sIgA in feces. The vaccine with ISA50V adjuvant induced antibodies earlier than vaccine with mineral oil,in contrast,the vaccine with mineral oil stimulated better uniformity antibody titers. All immunized mice dramatically decreased fecal shedding less than 104 CFU/g on the first sample day and no detectable microorganism on day 5. Compared to the immunized, mice incontrol group maintained a high level （〉104 CFU/g） shedding in entire sample duration. In a nutshell, systemic vaccination with H7-HCP-Tirqntimin was demonstrated to be an effective means to reduce overallshedding levels of EHEC O157：H7 following oral challenge,which will ultimately reduce the bac- terial transmission to humans or environmental contamination.

关 键 词：E coli 0157 H7 鞭毛 Ⅳ菌毛 紧密素转位受体蛋白 紧密素 疫苗 排菌 小鼠 

分 类 号：S852.61[农业科学—基础兽医学]