应用RNA聚合酶Ⅰ反向遗传操作技术拯救汉滩病毒84FLi株微基因组  

Rescue of Hantaan virus 84FLi strain minigenomes by RNA polymerase Ⅰ-driven system

作　　者：张野[1] 李新红[1] 姜泓[1] 黄长形[1] 王平忠[1] 孙利[1] 白雪帆[1] 

机构地区：[1]第四军医大学唐都医院全军感染病诊疗中心,西安710038

出　　处：《中华传染病杂志》2009年第1期6-10,共5页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（30200010）

摘　　要：目的建立基于汉滩病毒（HTNV）84FLi株L片段的RNA聚合酶Ⅰ微基因组拯救体系。方法将含有氯霉素乙酰转移酶（CAT）编码区的cDNA插入含有HTNV 84FLi株L片段5′端和3″端非编码区的质粒内的两个非编码区之间，将此cDNA嵌合体（polⅠ表达盒）克隆人人polⅠ启动子和终止子之间，分别获得正义和反义方向的RNA polⅠ转录报告质粒。用报告质粒转染293T细胞或等量293T和HTNV感染的Vero混合培养细胞，在HTNV的L蛋白和核衣壳蛋白表达质粒共转染后48h收获细胞，检测CAT活性。用上清液感染293T细胞，了解CAT活性的传代能力。结果构建了正义和反义方向的HTNV 84FLi株L片段微基因组RNA聚合酶Ⅰ报告质粒pLvRNA-CAT和pLcRNA-CAT，用此质粒转染细胞后能检测到CAT的表达，且CAT活性能在拯救病毒微基因组中传代。结论应用RNA聚合酶Ⅰ反向遗传操作技术成功拯救了HTNV 84FLi株微基因组。Objective To develop a reverse genetics system for Hantaan virus （HTNV） 84FLi strain by using RNA polymerase Ⅰ （pol Ⅰ ）-mediated transcription. Methods Complementary DNA （eDNA） containing the coding sequence for chloramphenicol acetyltransferase （CAT） was inserted into the 5′ and 3″- terminal untranslated regions of HTNV 84FLi L segment. These chimeric cDNAs （pol Ⅰ expression cassette） were cloned into plasmids and between the human pol Ⅰ promoter and terminator to generate sense and anti-sense RNA pol Ⅰ transcription reporter plasmids. The reporter plasmids were transfected into 293T cells or the 1 ： 1 combination of 293T and HTNV infected Vero cells. These cells were cotransfected with expression plasmids encoding L （RNA dependent RNA polymerase） and N （nucleoprotein） viral proteins. Cells were harvested 48 h post transfection and the CAT activity was detected. The 293T cells were infected with the supernatant to explore the passage ability of CAT activity. Results The reporter plasmids pLvRNA-CAT and pLcRNA-CAT were constructed successfully. CAT activity was detected in transfected cells and could also be serially passaged in the rescued virus minigenomes. Conclusion The RNA polymerase Ⅰ-driven reverse genetics system successfully rescues HTNV 84FLi minigenomes.

关 键 词：汉滩病毒 遗传学技术 微基因组 DNA指导的RNA聚合酶类 

分 类 号：R3[医药卫生—基础医学]