肠道病毒EV71新型VLPs疫苗的制备及鉴定

Preparation and identification of novel VLPs vaccine against Enterovirus 71

目的制备肠道病毒71型(EV71)的新型病毒样颗粒(VLPs)疫苗,为手足口病防治奠定基础。方法应用分子克隆技术构建外壳蛋白VP1融合基因cVP1,免疫细胞化学及Western Blot测定该融合基因在HeLa细胞表达情况;将cVP1克隆于杆状病毒表达载体pFastbac1,应用昆虫细胞TN5制备重组杆状病毒cVP1 rBV,再将此重组病毒与本室保存的gag rBV以不同MOI比例共感染昆虫细胞TN5,得到含有VP1的重组嵌合病毒样颗粒cVP1 VLPs,最后以Westem Blot及电镜进行鉴定。结果免疫细胞化学及Western Blot结果显示构建的融合基因cVP1可在真核细胞内表达,并成功制备成cVP1 rBV,该重组杆状病毒和gag rBV共感染昆虫细胞制备的VLPs结构完整。结论成功制备了EV71的新型VLPs疫苗,为后续研究打下了基础。

Objective To prepare a novel VLPs vaccine against Enterovirus 71 （ EV71 ）, so as to lay foundation for treatment of hand, foot and mouth disease （HFMD）. Methods Molecular cloning methods was used to construct cVP1 gene, and the fusion gene expression in HeLa cells was tested using immunocytochemistry and Western Blot. Then cVP1 was cloned into pFastbacl vector, and cVP1 recombinant baculovirus （ cVP1 rBV） was obtained using insect cells TN5. After that cVP1 rBV and gag rBV were co - infected into insect cell TN5 in varied MOI ratios to gain cVP1 virus like particles （VLPs） with VP1 protein. These novel VLPs were identified by Western Blot and electron microscope. Results Immunocytochemistry and Western Blot results showed that cVP1 gene could be expressed in eukaryotic cells, and cVP1 VLPs were obtained by insect cells co-infected with cVP1 rBV and gag rBV. These novel VLPs had complete structure. Conclusion Novel VLPs vaccine based on EV71 are constructed successfully, which lays foundation for the following study.