[摘 要] 目的:构建含有甲型流感病毒M2基因的真核表达载体。体外转染真核细胞株HEK293细胞并检测目的蛋白的表达。方法:从接种人流感病毒株A/PR/8/34(H1N1)的鸡胚尿囊液中提取病毒RNA，用特异引物进行RT-PCR，扩增M2基因。通过分子克隆技术将所扩增片段插入真核表达质粒载体pcDNA3.1(+)。经酶切及PCR鉴定后用PolyFect脂质体将其转染到HEK293细胞中，通过免疫荧光技术鉴定其表达。结果:经双酶切、PCR及测序鉴定证实M2基因的真核表达载体构建成功。免疫荧光技术证实流感病毒M2基因的表达。结论:甲型流感病毒M2蛋白是一种具有高保守性，可形成离子通道并影响流感病毒表面蛋白血凝素(HA)天然构象的形成，被认为是具有交叉免疫保护性的结构蛋白。甲型流感病毒M2基因真核表达质粒的构建及成功表达将为甲型流感病毒基因工程疫苗，通用疫苗和核酸疫苗的研究打下基础。

    [关键词] 甲型流感病毒；M2基因；真核表达载体； 免疫荧光技术

    Construction and expression of eukaryotic expressing vector containing M2 gene from influenza A virus

    YANG Jing,ZHANG Wei-dong, CAO Kang,et al.

    (Department of Microbiology,West China school of Preclinical and Forensic Medicine,Sichuan University, Chengdu 610041, China; Department of Microbiology, Yunyang Medical College, Shiyan，Hubei 442000, China; Deparment of Pathobiology , the Basic medicine school of Hebei Medical University, Shijiazhuang 050017, China; Deparment of Pathobiology , Chengdu Medical College, Chengdu 610041,China)

    Abstract:Objective To construct eukaryotic expression plasmid containing M2 gene from influenza A virus , transfer it into HEK293 eukaryotic cell line and detect the expression of target protein.Methods RNA was extracted from allantoic fluid of chicken embryo inoculated with influenza A virus A/PR/8/34. M2 gene was amplified by RT-PCR with specific primer and inserted into the eukaryotic expression vector pcDNA3.1(+) for constructing the recombinant plasmid pcDNA3.1(+)-M2. Then the recombinant plasmid was transfected into HEK293 cells with PolyFect Transfection Reagent after identification by restriction enzyme analysis, PCR and sequencing analysis.Results Restriction enzyme identification, PCR and sequencing analysis demonstrated that the M2 gene was successfully cloned and inserted into pcDNA3.1(+). Immunofluorescence assay confirmed that the M2 gene could be expressed in HEK293 cells.Conclusion M2 protein of influenza A virus is a kind of highly conserved protein in amino acid sequence and its ion channel activity can affect the structural stabilization of haemegglutinin (HA) of influenza virus. So the study provides a basis for exploring genetic engineered vaccine ......