诺如病毒病毒样颗粒的表达及其与HBGAs受体的结合研究

Expression of GⅡ-4 strain NoV virus-like particles and analysis of its binding activity with HBGAs receptor

目的:以杆状病毒为载体,在昆虫细胞sf9中表达GⅡ-4型野生株诺如病毒(Noroviruses,NoV)GZ121的衣壳蛋白ORF2,并验证其与组织血型抗原(Histoblood group antigen,HBGAs)受体的结合活性。方法:双酶切质粒PMD18-T-GZ121 ORF2酶切,连接入线性化载体PFastBacTM1,获得重组转座载体pFastBac-GZ121 ORF2,利用Bac-to-Bac杆状病毒表达系统获得重组杆状病毒,将其转染昆虫sf9细胞进行蛋白表达,用蔗糖超离方法纯化以及免疫印迹、透射电镜等方法鉴定表达产物,用EIA法检测NoV-VLP与HBGAs的结合特性。结果:成功表达了分子量约为58 kD的NoV衣壳蛋白,大小约30 nm与预期相符,NoV-VLP与A、B、O型均能结合,与非分泌型唾液不结合,结合方式与rVA387相一致。结论:我国优势流行株GⅡ4型NoV病毒样颗粒(Virus-like particles,VLP)的表达成功及NoV-VLP和HBGAs受体结合模型的建立,为我国NoV疫苗的开发和防治药物的研究奠定了基础。

Objective： To express the capsids of the predominant strain NoV（GⅡ-4） of our country and explore its binding activity and pattern model with HBGAs receptor.Methods： GZ121 NoV ORF2 gene was constructed into baculovirus expression system.After the transfection to sf9 cells,the recombinant proteins were purified by sucrose ultra centrifugation,and then were identified by SDS PAGE,Western blot and TEM.And the binding patterns of NoV to HBGAs antigens were identified by EIA.Results： VLPs expression was confirmed by using SDS PAGE,Western blot and could bind to all secretors（OD2.5）,but not to all nonsecretors（OD≈0.3）.Conclusion： The GⅡ-4 NoV VLP was successfully expressed and HBGAs receptor binding assays were established.These data will help us to explore the host adaptation character of NoV and apply the assays to screen drugs for antivirals against Noroviruses.The results obtained in the present work will also lay a foundation for the development of the immunological diagnostic reagents as well as vaccines for Norovirus.