禽流感病毒HA-M2融合表位与EGFP蛋白在鸭胚成纤维细胞中的表达

摘要

本研究选择禽流感病毒H9N2株的HA的两个保守T细胞表位基因和M2基因胞外保守序列(M2e),经人工合成并通过PCR扩增获得HA-M2融合表位基因,将其与含有增强型绿色荧光蛋白的载体pEGFP-N1连接,构建了重组真核表达质粒HA-M2-pEGFP-N1.采用脂质体法转染体外培养的鸭胚成纤维细胞(DEF)后,通过RT-PCR、直接荧光观察和间接免疫荧光检测,结果表明,成功构建了重组真核表达质粒HA-M2-pEGFP-N1,且以HA抗原表位和保守的M2e序列为基础构建的重组蛋白能够在鸭胚成纤维细胞中成功表达.本试验为研制新型的禽流感通用疫苗,进一步探索HA基因与靶细胞相互作用及AIV的感染机理等奠定了基础.%In this study, two T cell epitopes genes of hamagglutinin conserved domains of avian influenza virus H9N2 strain and Ml gene of extracellular domain were synthetized and joined as HA-M2 multiple-epitopes fusion gene of avian influenza virus by RT-PCR. The recombinant eukaryotic expression plasmid of HA-M2 fusion gene containing enhanced green fluorescent protein (EGFP) report gene was constructed, and transfected by lipofectin 10 prepared duck embryo fibroblasts (DEF). The fluorescence expression was directly detected with fluorescence microscope, and the expression of HA-M2 was tested by RT-PCR and indirect immunofluorescence assay (IFA) respectively. The results indicated the successful construction of the eukaryotic expression plasmid HA-M2-pEGFP-Nl, and showed that HA-M2 gene was expressed efficiently in transfected DEF. It provided a basis for the study of new universal influenza vaccines and further research on interaction of HA gene and its target cells and as for infection mechanism of AIV.