表达Ⅶ型新城疫病毒F蛋白的延迟裂解型沙门菌DNA疫苗载体的构建  

Construction of regulated delayed lysis Salmonella DNA vaccine expressing Ⅶ Newcastle disease virus F protein

作　　者：姚心茹 刘晶 王成宇 胡译文 赵维静 杨桂连 姜延龙 王春凤 

机构地区：[1]吉林农业大学动物科学技术学院吉林省动物微生态制剂工程研究中心

出　　处：《中国兽医科学》2018年第3期305-310,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31602092,31672528);国家十三五重点研发计划项目(2017YFD0501000);国家“863”计划项目(2013AA102806);吉林省科技发展计划项目(20160519011JH,20170204034NY);吉林省产业创新专项资金项目(2016C063);吉林省教育厅科学技术研究项目(JJKH20170316KJ)

摘　　要：为了构建表达新城疫病毒Ⅶ型F蛋白的延迟裂解型沙门菌载体,本试验首先通过PCR技术将Flag标签携带到pUC-F-opt片段中,构建出pUC-F-opt-Flag片段,再将构建好的片段克隆到沙门菌载体pYA4545中,构建重组质粒pYA4545-pUC-F-opt-Flag,并将重组质粒转染HEK-293 T细胞,收集细胞总蛋白。通过Western-blot检测目的蛋白的表达情况。同时将真核表达质粒转染Caco2细胞,并以间接免疫荧光试验检测蛋白表达。结果显示,成功构建了重组质粒pYA4545-pUC-F-opt-Flag,经Western-blot和间接免疫荧光法检测目的蛋白成功表达,为后期试验奠定了基础。In order to construct a regulated delayed lysis Salmonella vector expressing F protein of Newcastle disease virus genotype Ⅶ,the Flag tag was firstlyfused with pUC-F-opt fragment by PCR,yielding pUC-Fopt-Flag.The comstructed fragment was then subcloned into the Salmonella vector pYA4545,resulting in recombinant plasmid pYA4545-pUC-F-opt-Flag.The plasmid wasthen transfected into HEK-293 T cells and Caco2 cells,respectively.The expression of target protein was detected by Western-blot and indirect immunofluorescence.The results showed that the recombinant plasmid pYA4545-pUC-F-opt-Flag was successfully constructed and expressed by both Western-blot and indirect immunofluorescence assay,which laid the foundation for the later experiments.

关 键 词：新城疫病毒 F蛋白 延迟裂解 沙门菌 真核表达 

分 类 号：S852.659.5[农业科学—基础兽医学]