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目的:以HIV为骨架构建单次复制性的含基孔肯雅病毒囊膜蛋白的假病毒模型,观察其对哺乳动物细胞的侵染性。方法:PCR合成基孔肯雅病毒囊膜蛋白基因,克隆到真核表达载体上,与HIV慢病毒包装系统质粒共转染293FT细胞,48 h后收培养上清,在8μg/mL Polybrene存在下感染293FT细胞,感染48 h后在荧光显微镜下观察结果。结果:PCR合成了基孔肯雅病毒囊膜蛋白基因并克隆到真核表达载体上,测序结果正确;共转染293FT细胞后,检测到基孔肯雅病毒囊膜蛋白的表达并包装成假病毒,感染新鲜293FT细胞后能够检测到绿色荧光蛋白。结论:合成的基孔肯雅病毒囊膜蛋白基因能正确表达并包装成假病毒,含基孔肯雅病毒囊膜蛋白的假病毒能感染293FT细胞并表达绿色荧光蛋白,可用该假病毒模型进一步研究基孔肯雅病毒的感染性,筛选评价抗基孔肯雅病毒药物。
OBJECTIVE: To construct a single replicative pseudovirion model of chikungunya virus envelope protein with HIV as its framework and observe its infectivity on mammalian cells. Methods: The gene of Chikungunya virus envelope protein was synthesized by PCR and cloned into the eukaryotic expression vector. The 293FT cells were co-transfected with the HIV lentiviral packaging plasmid. After 48 h, the supernatant was harvested and cultured in the presence of 8 μg / mL Polybrene 293FT cells were infected and observed under a fluorescence microscope 48 hours after infection. Results: The Chikungunya virus envelope protein was synthesized by PCR and cloned into the eukaryotic expression vector. The sequencing result was correct. After transfected 293FT cells, the expression of chikungunya virus envelope protein was detected and packaged into false Virus, green fluorescent protein can be detected after infection with fresh 293FT cells. CONCLUSION: The synthetic chikungunya virus envelope protein gene can be correctly expressed and packaged into pseudoviruses. The pseudovirion containing the chikungunya virus envelope protein can infect 293FT cells and express green fluorescent protein. The pseudovirion model can be further used To study the infectivity of Chikungunya virus, screening anti-Chikungunya virus drugs.