大肠杆菌Nissle 1917株鞭毛展示6His标签的构建及相关生物学特性分析  

Flagellin displaying the 6His tag in probiotic Escherichia coli strain Nissle 1917 and its biological characteristic analysis

作　　者：杨颖[1,2] 区炳明 杨溢[1,2] 张倩[1,2] 杨玮枫[1,2] 夏芃芃[1,2] 朱国强[1,2] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国预防兽医学报》2016年第5期371-375,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30571374;30771603;31072136;31270171);江苏高校优势学科建设工程资助项目;科技部转基因生物新品种培育重大专项(2014ZX08006-001B)

摘　　要：为探索大肠杆菌Nissle 1917株(EcN)鞭毛展示的应用,本研究以重组质粒p UC18-fli C为模板,反向PCR产物经DpnⅠ、Exnase^(TM)Ⅱ重组环化,将6His标签基因分别插入无质粒大肠杆菌Nissle 1917(EcNcured of its two cryptic plasmids p MUT1和p MUT2;EcNc)fli C高变区774 bp^775 bp和792 bp^811 bp之间,构建嵌合鞭毛基因片段fliC01和fliC02。分别将其克隆于自杀性载体pRE112中,构建重组质粒pRE112-fliC01和pRE112-fliC02,并分别转化至EcNc中。通过一次重组和二次重组将嵌合鞭毛基因整合于EcNc基因组中,构建无抗性筛选的重组菌株EcNc fliC01和EcNc fliC02。对重组菌株进行生长状况、鞭毛形成以及体外细胞黏附试验和小鼠体内定植试验检测,结果表明EcNc fliC01和EcNc fliC02的生长未受影响,表达的嵌合鞭毛能够被H1多抗或His-Tag单克隆抗体识别并组装形成鞭毛丝;重组菌对IPEC-J2细胞的黏附能力及小鼠肠道定植能力与亲本株EcNc相比无显著性差异。本实验结果为进一步研究EcNc鞭毛展示技术应用于活疫苗研制奠定了基础。In order to explore the application prospect of flagellin display of Escherichia coil Nissle 1917 （EcN） strain, the 6 His tag encoding sequences were respectively inserted into hypervariable domains 774 bp-775 bp and 792 bp-Sl 1 bp of tliC originally from EcNc （EcN cured of its two cryptic plasmids pMUT1 and pMUT2）, with pUC18-t/iC as the template by using inverse PCR, sequentially the products were cyclized by Dpn I and ExnaseTM II, and the chimeric genes t/iC01 and t/iC02 were respectively inserted into suicide vector pRE112, resulting in the recombinant plasmids ofpRE112-fliC01 and pRE112-t/iC02. The recombinant plasmids were transformed into EcNc, respectively. The chimeric genes were respectively integrated into the chromosome of EcNc after the first recombination and the second recombination reaction, and the recombinant bacteria were named EcNe f/iC01 and EcNc fliC02, respectively. Both recombinant bacteria grew normally as EcNc did and were flagellated which chimeric flagellin could by specifically recognized by the rabbit polyclonal serum against E.coli HI antigen and His-tag

关 键 词：大肠杆菌Nissle 1917 自杀性载体 鞭毛蛋白 6His标签 

分 类 号：S852.61[农业科学—基础兽医学]