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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。
Objective To construct the eukaryotic expression plasmid of hemagglutinin (HA) gene of double subtype (H3 / H1) influenza virus and express it in HeLa cells. Methods The gene fragments of HA1 and H3 subtypes of influenza virus H1 subtypes were respectively modified by PCR, and the (G4S) 3 flexible linker and the FMDV 2A protein linker were introduced into the fusion fragments. Using DNAstar combined with bioinformatics software Insight Ⅱ, the spatial conformation was simulated. The H1HA1-H3HA fusion gene fragment was cloned downstream of the eukaryotic expression vector pVAX1 CMV promoter. The HeLa cells were transfected by lipofectamine. The mRNA transcription of target gene was detected by RT-PCR and the expression of target protein in transfected cells was detected by indirect immunofluorescence. Results The recombinant eukaryotic expression plasmid pVAX1-H3HA-H1HA1 expressing the influenza A virus of the double subtype (H3 / H1) influenza virus was confirmed by restriction enzyme digestion and sequencing. HeLa cells transfected with the recombinant plasmids could detect the transcription of the target gene mRNA and the expression of the target protein. Conclusion The eukaryotic expression plasmid pVAX1-H3HA-H1HA1 has been successfully constructed and can be correctly transcribed and expressed in HeLa cells, which lays the foundation for the study of bivalent nucleic acid vaccine of H3 and H1 subtype influenza viruses.