禽流感病毒H5N1全基因组克隆与分析  

Analysis of avian influenza virus H5N1 genome and construction of its infectious clones

作　　者：王革非[1] 李卫中[1] 张衡[1] 曾俊[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,汕头515041

出　　处：《微生物学免疫学进展》2010年第1期7-13,共7页Progress In Microbiology and Immunology

基　　金：国家自然科学基金(30771988;30972766);广东省自然科学基金(8151503102000022;9451503102003499);广东高校优秀青年创新人才培育项目(LYM08056);农业微生物学国家重点实验室开放课题(AML200910);汕头大学医学院科研基金项目资助

摘　　要：为明确广东地区分离的一株禽流感病毒H5N1的遗传背景,建立流感病毒反向遗传的平台。对该株禽流感病毒进行了空斑纯化与组织细胞培养,检测其在MDCK细胞中的增殖特性;利用H5N1病毒通用引物,通过RT-PCR对该病毒全基因组的8条片段进行全长克隆及测序分析;将H5N1的8条全长基因组片段分别插入反向遗传通用载体中,构建禽流感病毒H5N1的感染性克隆。结果表明,该H5N1毒株在MDCK细胞中可不依赖胰酶进行有效增殖与复制,可使MDCK细胞出现典型细胞病变,具有高致病性禽流感病毒的细胞增殖特征。RT-PCR克隆得到该H5N1毒株的PB2、PB1、PA、HA、NP、NA、M和NS八条全长片段,经测序分析确认该毒株的基因序列,其内部编码序列出现多处突变,其中HA连接肽为多个连续碱性氨基酸,表明该毒株可不依赖胰酶进行有效复制,与细胞培养结果一致,未出现抗药性的遗传突变。PCR与测序证明,插入H5N1八个全长基因组片段的载体序列完全正确,表明成功构建了该毒株的感染性克隆。为明确病毒遗传信息,建立流感病毒反向遗传的平台,为进一步研究禽流感病毒相关疫苗提供了研究基础。To identify the genetic background of an avian H5N1 influenza virus and construct a system for reverse genetics of avian influenza,the H5N1 avian influenza virus isolated in Guangdong province,was cultured in chicken embryo eggs and purified by plaque test,and its proliferative characters were detected in MDCK cells.The 8 fragments of viral full genome were cloned by RT-PCR using H5N1 virus universal primer and sequenced.And then 8 fragments of H5N1 viral genome were inserted into the reverse genetic vector to construct the infectious clones of H5N1 avian influenza virus.The results showed that this H5N1 strain could proliferate effectively without trypsin and produce typical CPE in MDCK cells.The 8 fragments（PB2,PB1,PA,HA,NP,NA,M,NS） of this H5N1 strain are cloned by RT-PCR and identified by sequencing and analysis.The viral sequence reveal some novel mutations in the genome.The HA connecting peptides are multiple successive basic amino acid,can effective duplicating without trypsin,consistent with viral proliferative characters result in cell.Neither oseltamivir nor rimantadine resistant mutation in the genome is found.PCR and sequencing results indicated that the infectious clones of this H5N1 strain,which inserting 8 full-length genome fragments,are constructed.These researches identify the viral genetic background,establish reverse genetics system of avian influenza and provid research basis for further vaccines development of avian influenza.

关 键 词：流感病毒 H5N1 克隆 反向遗传 序列分析 

分 类 号：R373.1[医药卫生—病原生物学] Q939.4[医药卫生—基础医学]