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目的通过杆状病毒表达系统表达制备基孔肯雅病毒(CHIKV)病毒样颗粒并进行初步鉴定。方法首先将基孔肯雅病毒的结构蛋白基因C-E3-E2-6K-E1克隆入p Fast-Bac~(TM) Dual转移载体中并鉴定;然后将构建正确的重组转移载体转化DH10Bac感受态细胞后提取重组杆粒并鉴定;再将重组杆粒转染Sf9昆虫细胞后获得重组杆状病毒,并利用免疫荧光法(IFA)鉴定其感染Sf9细胞后E2蛋白的表达;最后通过透射电镜(TEM)和Western-Blot鉴定CHIKV病毒样颗粒。结果成功获得了含有CHIKV结构蛋白基因的重组杆状病毒,该重组病毒经过IFA及WB验证在昆虫细胞中能够表达CHIKV的E蛋白,并在电镜下观察到了直径约60~80 nm的CHIKV病毒样颗粒。结论通过杆状病毒表达系统成功表达并获得CHIKV的病毒样颗粒,为CHIKV早期快速检测法的建立奠定基础。
Objective To prepare chikungunya virus (CHIKV) virus-like particles by baculovirus expression system and make preliminary identification. Methods The gene of Chikungunya virus C-E3-E2-6K-E1 was cloned into pFast-Bac ~ (TM) Dual transfer vector and identified. Then, the correct recombinant transfer vector was transformed into DH10Bac competent cells The recombinant bacmid was extracted and identified. The recombinant baculovirus was transfected into Sf9 insect cells to obtain recombinant baculovirus. The expression of E2 protein in Sf9 cells was identified by immunofluorescence (IFA). Finally, the expression of E2 protein was detected by transmission electron microscopy TEM) and Western-Blot to identify CHIKV virus-like particles. Results The recombinant baculovirus containing the CHIKV structural protein gene was successfully obtained. The recombinant virus was verified by IFA and WB to express CHIKV E protein in insect cells. The CHIKV virus-like sample with a diameter of about 60-80 nm was observed under electron microscope Granules. Conclusion The virus-like particles of CHIKV were successfully expressed by baculovirus expression system, which laid the foundation for the establishment of CHIKV rapid early detection method.