甲型流感病毒血凝素基因HA和HA_1真核载体的构建及其表达研究

Construction of Eukaryotic Expressing Plasmids for HA and HA_1 of Influenza A Virus and Their Transient Expression in HEK293 Cells

目的构建甲型流感病毒A/PR/8/34(H1N1)血凝素基因HA和HA1真核表达载体,并检测其在HEK293细胞中的表达。方法利用RT-PCR技术克隆流感病毒株血凝素HA和HA1基因片段并插入真核表达载体pcDNA3.1(+)。经酶切、PCR和测序鉴定后用PolyFect脂质体转入HEK293细胞,通过免疫荧光技术检测其瞬时表达。结果血凝素基因HA和HA1的真核表达载体构建成功。转染后免疫荧光技术检测显示两者皆可在HEK293细胞中正确表达。结论成功构建的流感病毒HA和HA1真核表达载体为深入研究病毒的作用机制及核酸疫苗的研究奠定了基础。

Objective To construct influenza A virus （A/PR/8/34） HA and HA1 eukaryotic expressing plasmids and study their expression in HEK293 cells. Methods HA and HA1 genes were cloned by RT-PCR and then inserted into pcDNA3. 1 （＋）. After identification of restriction enzyme digestion, PCR and sequencing analysis, HA and HA1 eukaryotic expressing plasmids were transfected into HEK293 cells with PolyFect Transfection Reagent. Immunofluorescence assay was used to observe the transient expressing result. Results It was confirmed that the construction of HA and HA1 eukaryotic expressing plasmids was made successfully. The stronger fluorescence signals were detected in transfected HEK293 cells with these two kinds of plasmids by immunofluorescence assay. Conclusion The experiment is a success in the construction of eukaryotic expressing plasmoids for HA and HA1, thus providing a basis for further probing into the mechanism of virus infection and exploring DNA vaccine.