HIVⅠ型外膜蛋白/干扰素A-2b的构建与表达
Construction and expression of recombinant plasmid and expression of fusion of HIV-1 env/IFNα-2b
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摘要:目的 构建艾滋病病毒外膜蛋白(env)与干扰素(IFNα-2b)融合基因表达质粒, 观察其在痘苗病毒中共表达结果。方法 利用基因重组技术, 将IFNα-2b基因片段插入到env基因的nt8383位点, 经脂质体转染与血凝素阴性蚀斑筛选, 挑出重组痘苗病毒。经免疫荧光、免疫印迹(Western blot)和斑点酶联免疫吸附试验(Dot-ELISA)鉴定表达产物。结果 间接免疫荧光实验结果显示, 转染重组质粒的细胞表面有绿色荧光。Western-blot与Dot-ELISA结果均显示, 重组质粒转染细胞的裂解物中存在表达的env/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。Abstract:Objective To construct an eukaryotic expression plasmid and express fusion of HIV-1 env/IFNα-2b in BHK 21 cell.Methods An eukaryotic expression plasmid pSFJ16env/IFNα-2b gene into vaccine vector plasmid pSFJ16 and transfected to BHK 21 cell in vitro by liposome-mediated method.The expressed product was detected by indirect immunfluorescence assay(IFA), western blot and Dot-ELISA.Results Indirect IFA showed green fluorescence on the surface of transfected cell.Both western blot and Dot-ELISA proved that the lysate of transfected cells contained env/IFNα-2b protein.Conclusion An eukaryotic expression plasmid for HIV-1 env/IFNα-2b was successfully constr uced, and the expressed pro duct showed good reactinogenicity and immunogenicity.
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