基于果蝇S2系统的流感病毒样颗粒的制备及鉴定

The formation and activation of influenza virus-like particles with Drosophila S2 expression system

目的应用果蝇s2表达系统制备流感病毒样颗粒（Virus—likeparticles，VLPs）并鉴定其活性。方法将前期构建的果蝇持续性表达载体pAc—V5-HA、NA、MI与pCoblast共转染至S2细胞，应用杀稻瘟菌素（Blasticindin）结合限制性稀释法筛选共整合流感HA、NA、M1三种基因的单克隆s2细胞株。利用PCR技术鉴定细胞内三种基因整合，Western—Blot技术鉴定细胞上清中VLPs，通过血凝试验评估HA蛋白的凝集效价，通过水解唾液酸的能力评价NA活性。结果应用PCR技术从单克隆s2细胞株基因组中成功扩增流感HA、NA和M1基因；Western—Blot证实细胞上清含有整合HA、NA和M1蛋白的流感VLPs；血凝试验证实流感VLPs能有效凝集鸡红细胞（效价1：160）；NA活性测定发现，流感VLPs能有效水解唾液酸，且呈剂量依赖性，在高剂量（250ng、500ns）条件下VLPs和野生型病毒的NA活性相近。结论成功建立整合流感HA、NA、M1基因的单克隆s2细胞株，该细胞可以持续产生流感VLPs，VLPs具有良好的HA和NA活性，可以作为有效的新型流感疫苗。

Objective To produce influenza virus-like particles（VLPs） with drosophila S2 cell line and identify its bioaetivity. Methods The persistent expression vector sof pAc-V5-HA,NA and M1 suitable for drosophila S2 cell line were constructed at the earlier stage, which were co-transfected into S2 cell with pCoblast vector. Then we screened monoclonal S2 cell lines which integrated HA, NA and M1 genes through blasticindin combined with limited dilution methods. We used PCR assay to identify whether HA, NA and M1 genes were successfully integrated into genome, and used Western-Blot assay to identify whether there was VLPs in the supernatant. The HA activity was evaluated with hemagglutination test, and NA activity was reflected with the ability to hydrolyze sialic acid. Results The HA, NA and M1 genes were successfully amplified from the monoclonalS2 cell using PCR assay. The VLPs integrated HA,NA and M1 protein was identified with Western-Blot assay. The hemagglutination test confirmed influenza VLPs could agglutinate chicken red blood cells with titer of 1： 160. The NA could dose-dependent hydrolyze sialic acid, and no significance was observed at the high dose（250 ng,500 ng）betweeu wild type virus and VLPs. Conclusion We successfully established the monoclonal S2 cell line which integrated influenza HA,NA and M1 genes, and it could persistently produce influenza VLPs with perfect HA and NA activity, VLPs could be considered as effective influenza vaccine candidate in the future.