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目的构建艾滋病病毒核心蛋白(gag)与干扰素(IFNα-2b)融合基因表达质粒,观察其与痘苗病毒共表达的结果,研究其意义。方法利用基因重组技术,将IFNα-2b基因片段插入到gag基因的nt 531位点,经脂质体转染与血凝素阴性蚀斑筛选,挑出重组病毒。经免疫荧光、蛋白印迹分析(Western blot)和斑点酶联免疫(Dot ELISA)鉴定表达产物。结果间接免疫荧光实验显示,转染重组质粒的细胞表面有绿色荧光。免疫印迹实验与Dot ELISA结果均显示,转染重组质粒的细胞裂解物中存在表达的gag/IFNα-2b蛋白。结论成功地构建了重组真核细胞表达质粒,表达的蛋白具有良好的免疫原性与免疫反应性。
Objective To construct the expression plasmid of fusion gene of gag and interferon-α (IFNα-2b) and observe the result of its co-expression with vaccinia virus and to study its significance. Methods The IFNα-2b gene fragment was inserted into nt 531 of gag gene by gene recombination technique. The recombinant plasmid was selected by liposome transfection and hemagglutinin negative plaque screening. The expression products were identified by immunofluorescence, Western blot and Dot ELISA. Results Indirect immunofluorescence assay showed that the surface of transfected plasmid had green fluorescence. Immunoblotting and Dot ELISA results showed that the expressed gag / IFNα-2b protein was present in the cell lysates transfected with the recombinant plasmids. Conclusion The eukaryotic expression plasmid has been constructed successfully. The expressed protein has good immunogenicity and immunoreactivity.