基孔肯雅病毒样颗粒的制备

Preparation of Chikungunya virus-like particles

为制备高纯度、均一的基孔肯雅病毒的病毒样颗粒,将基孔肯雅病毒的结构蛋白基因序列(C-E3-E2-6K-E1)人工合成后克隆至杆状病毒-昆虫细胞表达载体中;重组质粒转化DH10Bac感受态细胞,经蓝白斑筛选和PCR鉴定,获得重组杆状病毒杆粒;重组杆状病毒杆粒转染ExpiSf9昆虫细胞,包装获得重组杆状病毒,并再次感染ExpiSf9昆虫细胞表达基孔肯雅结构蛋白;通过细胞超薄切片验证杆状病毒的产生,并用流式细胞仪测定病毒滴度;免疫荧光、蛋白免疫印迹分析目的蛋白的表达,透射电镜鉴定病毒样颗粒的形成;最后,通过蔗糖密度梯度离心纯化病毒样颗粒。结果表明:成功构建了含基孔肯雅病毒结构蛋白的重组杆状病毒杆粒,获得了高滴度的重组杆状病毒,重组杆状病毒表达了基孔肯雅病毒的结构蛋白,结构蛋白自组装成60~70 nm的颗粒。这一研究通过昆虫杆状病毒表达系统成功制备了纯度高和均一性好的基孔肯雅病毒样颗粒,为基孔肯雅病毒的诊断试剂和疫苗研发奠定了基础。

To prepare the high purity and uniformity of virus-like particles(VLPs) of Chikungunya virus(CHIKV). The structural protein gene sequence(C-E3-E2-6 K-E1) of CHIKV was synthesized artificially, and cloned into the baculovirus-insect cell expression vector. The recombinant plasmid was transformed into DH10 Bac competent cells, and the recombinant baculovirus bacmid was screened through blue-white spot screening and PCR. The recombinant baculovirus bacmid was chemically transfected into the ExpiSf9 insect cells, packaged to obtain recombinant baculovirus, and then re-infected with ExpiSf9 insect cells to express the structural protein of CHIKV. The production of baculovirus was verified by ultrathin section and titrated via flow cytometer. Then, the expression of CHIKV structural protein was confirmed by immunofluorescence and Western blot(WB). The formation of VLPs was observed via transmission electron microscopy(TEM). Finally, the VLPs were purified through the sucrose density gradient centrifugation. The recombinant baculovirus plasmid that contained the full structural gene of CHIKV was successfully constructed. High titer of the recombinant baculoviruses was produced. Structural protein of CHIKV was self-assembled to 60-70 nm particles under TEM. In this study, high purity and uniformity of CHIKV VLPs was prepared via the insect baculovirus expression system, which lays the foundation for the research and development of diagnosis methods and vaccines for CHIKV.