基孔肯雅病毒囊膜蛋白假病毒模型的构建

摘要

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 replication-defective HIV-backborn pseudovirus using Chikungunya virus envelope protein and observe its infection in mammalian cells. Methods: The sequence of Chikungunya virus envelope protein gene was synthesized using overlapping PCR method and then was cloned into eukaryotic vector. The above plasmid was co-transfected into 293FT cells with HIV-based lentivirus packaging plasmids. Supernatant was collected and filtered with 0.22 μm filter after 48 hours, then was used to infect fresh 293FT cells in the presence of 8 μg/mL Polybrene. Results: The results of sequencing showed that correct sequence of Chikungunya virus envelope protein gene was synthesized and cloned into eukaryotic vector. The expression of Chikungunya virus envelope protein was observed and packaged into pseudovirus which could infect fresh 293FT cells effectively. Conclusion: The synthesized envelope protein gene of Chikungunya virus can be expressed correctly and packaged into pseudovirus. The packaged pseudovirus can infect 293FT cells effectively, which can be used to study Chikungunya virus infection and transmission, as well as to screen anti-Chikungunya virus agents.