H7N9禽流感病毒重组质粒构建及应用  

Construction and application of H7N9 avian influenza virus gene recombinant plasmid pGEM-HA-NA-M

作　　者：张锦海[1] 陈文琦[2] 胡丹[1] 王平[1] 吕恒[1] 刘玉[1] 陈凤娟[1] 任浩[3] 王长军[1] 

机构地区：[1]南京军区军事医学研究所,江苏南京210002 [2]南京医科大学附属南京医院 [3]第二军医大学热带医学与公共卫生学系

出　　处：《中国公共卫生》2013年第12期1868-1870,共3页Chinese Journal of Public Health

基　　金：国家重大传染病防治专项(2013ZX10004-103;2013ZX10004-203;2013ZX10004-218;2012ZX10004801-004);全军后勤科研"十二五"重大项目(AWS11C001;AWS11C009);南京军区医学科技重点课题(10Z039;11Z040);第二军医大学军事医学专项(2012JS01);江苏省科技支撑计划(社会发展)项目(BE2012609;BE2013603)

摘　　要：目的构建一种含H7N9禽流感病毒全长血凝素/神经氨酸酶/基质蛋白(HA/NA/M)基因的重组质粒,为核酸检测方法提供一种通用的阳性定量标准品。方法设计H7N9禽流感病毒HA、NA及M抗原基因全长开放阅读框的克隆引物,提取H7N9禽流感病毒总RNA后用实时荧光定量PCR获得相应片段,用3次酶切连接方法,依次插入到pGEM-T easy质粒,进行测序确认;线性化后的重组质粒用T7 RNA聚合酶进行体外转录,RNA转录产物纯化后测定浓度,用实时荧光定量PCR构建标准曲线进行验证。结果 HA、NA和M基因扩增片段大小分别约为1.7、1.3、1.1 kb,与预期相符;构建的重组质粒pGEM-HA-NA-M插入片段的测序结果与GenBank公布序列一致;由重组质粒体外转录获得同时含有H7N9禽流感病毒HA、NA、M全长开放阅读框序列的RNA片段质量浓度为399.5 ng/μL,梯度稀释后用3种实时荧光定量PCR方法均获得了良好的标准曲线。结论成功构建重组质粒pGEM-HA-NA-M,由此质粒体外转录获得的RNA片段可作为H7N9禽流感病毒核酸快速检测方法通用的阳性定量标准品。Objective To construct a combinant plasmid of full length HA/NA/M gene of H7N9 avian influenza virus and to provide quantitative reference for pathogen detection.Methods According to specific sequence of HA/NA/M gene of H7N9 avian influenza virus,the primers were designed and synthesized.Total RNA extracted from H7N9 avian influenza virus and the cDNA of of HA/NA/M were cloned by reverse transcription PCR（RT-PCR）and inserted into pGEM-T easy vector after three times of restriction enzyme assay.The linearized plasmids were used to transcript RNA in vitro by T7 RNA polymerases,then the products were purified and diluted to a series of standard concentrations of cRNA which was used as standard quantitative template of real-time fluorescence quantitative RT-PCR method.Results The amplified fragment by RT-PCR was of expected size and its sequence was in concordance with that published on GenBank.The cRNA including full-length HA/NA/M was obtained by in vitro transcription with the recombinant plasmid and the mass concentration was 399.5 ng/μl.The cRNA were diluted to precise quantification copy number,which were proved by real-time RT-PCR amplification.Conclusion The combinant plasmid pGEM-HA-NA-M was constructed successfully and in vitro transcription products of the plasmid can be used as a quantitative reference for the rapid detection of nucleic acid of H7N9 avian influenza virus.

关 键 词：H7N9禽流感病毒 血凝素 神经氨酸酶 基质蛋白 标准品 

分 类 号：R511.7[医药卫生—内科学]