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目的在昆虫细胞中表达中国流行株 B亚型 HIV-1 Gag蛋白,制备重组 Gag蛋白。方法将中国 株 B亚型 HIV-1的 Gag全基因序列,克隆到杆状病毒表达载体 pfastbacI中,构建了重组质粒 pfastGag。经转座及平板 筛选,提取重组穿梭质粒 Bacmid。经转染Sf9昆虫细胞后,获得了重组杆状病毒。用 SDS-PAGE、Western blot和间接免疫荧光实验分析表达的目的蛋白。结果重组病毒在昆虫细胞 Sf9中高效表达了中国株 B亚型 HIV-1 Gag蛋 白,表达的重组蛋白具有良好的抗原活性,可与HIV-1标准阳性血清及p24单克隆抗体发生较强的免疫反应。结论 重组表达产物可用于艾滋病的免疫诊断和疫苗研究。
Objective To express HIV-1 Gag protein of Chinese subtype B strain in insect cells and prepare recombinant Gag protein. Methods The full-length Gag gene of HIV-1 of Chinese strain B was subcloned into the baculovirus expression vector pfastbacI to construct the recombinant plasmid pfastGag. After transposition and screening, the recombinant shuttle plasmid Bacmid was extracted. After transfected into Sf9 insect cells, a recombinant baculovirus was obtained. The expressed proteins were analyzed by SDS-PAGE, Western blot and indirect immunofluorescence assay. Results The recombinant virus highly expressed HIV-1 Gag protein of Chinese strain B in Sf9 insect cells. The expressed recombinant protein had good antigenic activity and could be immunized with HIV-1 positive serum and p24 monoclonal antibody reaction. Conclusion The recombinant expression product can be used in immunodiagnosis and vaccine research of AIDS.