人甲型H1N1流行性感冒病毒全基因组分析与反向遗传载体的克隆  

Analysis of human influenza A virus HIN1 genome and construction of its reverse genetics vectors

作　　者：王革非[1] 张衡[1] 李卫中[1] 曾俊[1] 张丹桂[1] 陈幼莹[1] 陈小璇[1] 李康生[1] 

机构地区：[1]汕头大学医学院微生物学与免疫学教研室汕头大学医学院广东高校免疫病理重点实验室,515041

出　　处：《中华传染病杂志》2009年第10期577-581,共5页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（30771988,30571674）;广东省自然科学基金资助项目（05008347,8151503102000022）;广东省高校优秀青年创新人才培育资助项目（LYM08056）

摘　　要：目的明确一株人甲型H1N1流行性感冒（流感）病毒的遗传背景，建立流感病毒反向遗传的平台。方法对分离自汕头市儿童的一株人甲型H1N1流感病毒进行空斑纯化；利用甲型流感病毒通用引物，对该病毒全基因组的8条片段（PB2、PB1、PA、HA、NP、NA、M和NS）进行RT-PCR全长克隆、DNA测序及初步生物信息学分析；将其8条全长基因组片段分别插入反向遗传通用载体，构建人甲型H1N1流感病毒的反向遗传系统。结果RT-PCR克隆得到该H1N1毒株的8条全长片段，经测序分析确认该毒株的基因序列，该毒株与2006年至2008年分离的人甲型H1N1流感病毒具有很高的同源性，未出现奥司他韦或金刚乙胺抗药性的遗传突变。PCR与测序证实，插入该菌株8个全长基因组片段的载体序列完全正确。结论成功构建了该毒株的感染性克隆。获得了该毒株全基因组序列，为明确病毒遗传信息、提供流行病学监测数据、建立流感病毒反向遗传平台奠定了研究基础。Objective To identify the genetic background of a human H1N1 influenza virus and construct a reverse genetics system of human influenza. Methods The H1N1 human influenza virus isolated in Shantou city was cultured in chicken embryo eggs and purified by plaque test. The eight fragments of viral full genome （PB2, PB1, PA, HA, NP, NA, M, NS） were cloned by reverse transcriptase-polymerase chain reaction （RT-PCR） using H1N1 virus universal primer and sequenced. Then eight fragments of H1N1 viral genome were inserted into the reverse genetic vector to construct the reverse genetics system of H1N1 human influenza virus. Results The eight fragments of the H1N1 strain were cloned by RT-PCR and identified by sequencing and analysis. The viral sequence revealed the HA had high homology with the other strains isolated from 2006- 2008. Neither oseltamivir nor rimantadine resistant mutation in the genome was found. Polymerase chain reaction （PCR） and sequencing results indicated that the infectious clones of this H1N1 strain, containing 8 full-length genome fragments, were constructed. Conclusions The infectious clones of the H1N1 strain are successfully constructed. The whole genome sequence of the strain is obtained, which provides research basis for identifying viral genetic information, understanding epidemiological data and establishing reverse genetics system of human influenza.

关 键 词：流感病毒A型 H1N1亚型 克隆 分子 遗传学技术 基因组 病毒 序列分析 

分 类 号：R686[医药卫生—骨科学]