【摘要】 目的构建双亚型(H3/H1)流感病毒血凝素(HA)基因真核表达质粒,并在HeLa细胞中进行表达。方法通过PCR分别改造流感病毒H1亚型HA1和H3亚型HA基因片段,在融合片段间引入(G4S)3柔性linker和口蹄疫病毒2A蛋白linker。采用DNAstar结合生物信息学软件InsightⅡ分析后,对其空间构象进行模拟。将H1HA1-H3HA融合基因片段克隆至真核表达载体pVAX1 CMV启动子下游。通过脂质体法转染HeLa细胞,RT-PCR法检测转染细胞中目的基因mRNA的转录,间接免疫荧光检测转染细胞中目的蛋白的表达。结果表达双亚型(H3/H1)流感病毒血凝素基因的重组真核表达质粒pVAX1-H3HA-H1HA1经酶切和测序证明构建正确。转染重组质粒的HeLa细胞可检测到目的基因mRNA的转录和目的蛋白的表达。结论已成功构建了真核表达质粒pVAX1-H3HA-H1HA1,并可在HeLa细胞中正确转录与表达,为H3、H1亚型流感病毒双价核酸疫苗的研究奠定了基础。更多还原

【Abstract】 Objective To construct a eukaryotic expression vector for dual-subtype(H3 / H1)hemagglutinin of influenza virus and express in HeLa cells.Methods The HA1 gene fragment of influenza H1 virus and HA gene fragment of influenza H3 virus were modified by PCR using the designed primers,between which flexible linker(G4S)3 and foot-and-mouth virus 2A protein linker were introduced.The conformation of expressed protein was stimulated and evaluated by bioinformatics software.The H1HA1H3HA fusion gene was cloned downstream to the CMV promoter of eukaryotic expression vector pVAX1,and the constructed recombinant plasmid pVAX1-H3HA-H1HA1 was transfected to HeLa cells in mediation of liposome.The transcription of mRNA of target gene was determined by RT-PCR,and the expression of target protein by IFA.Results Restriction analysis and sequencing proved that recombinant plasmid pVAX1-H3HA-H1HA1 was constructed correctly.Both the transcription of mRNA of target gene and the expression of target protein were proved in transfected HeLa cells.Conclusion The eukaryotic expression vector pVAX1-H3HAH1HA1 was successfully constructed and transcribed and expressed in HeLa cells,which laid a foundation of development of bivalent influenza virus DNA vaccine.更多还原