利用武汉野田村病毒衣壳蛋白展示登革病毒保护性抗原的研究

Due to the unique capsid structure of nodaviruses, the nodaviral capsid protein is long believed to be an ideal foreign protein/peptide presentation system. Wuhan nodavirus (WhNV) is the first identified nodavirus in China by our group, and this virus was characterized and studied in considerable details by us. In this project, we aim to establish a novel WhNV-based surface presentation system, and further display the Dengue virus (DENV) surface protective antigen Protein E Domain III (EDIII). For this purpose, the potential insertion sites within WhNV capsid protein will be identified through bioinformatically analyzing the three-dimensional structure of WhNV capsid. After that, the enhanced green fluorescent protein (EGFP) will be inserted into these potential sites in order to screen the sites that can both maintain the native conformation of the inserted foreign proteins and the capability of WhNV capsid to assemble virus-like particles (VLPs). Using the information obtained from the EGFP screening, the WhNV recombinant VLP library expressing four serotypes of DENV EDIII will be constructed, following by the screening with the correct spatial conformation and an effective immune activity of these antigens. The successful accomplishment of this project will lay the foundation for developing a novel effective DENV vaccine, while a stable and efficient WhNV foreign protein/peptide presentation system will greatly promote the development of a number of novel vaccines, and promote the public health and biomedical research in our country. In the aspect of basic research, this project will also generate lots of useful information for our future studies on the structure and function of WhNV capsid, which should deepen our understanding of the mechanism by which WhNV and other non-enveloped positive-strand RNA viruses invade host cells.

本申请项目希望建立一种以武汉野田村病毒（WhNV）衣壳蛋白为依托的新型异源多肽与蛋白展示系统，并以此展示登革病毒（DENV）表面保护性抗原EDIII。我们将通过生物信息学手段，分析模拟WhNV衣壳蛋白三维结构，对其潜在可插入位点进行初步分析，并在此基础上利用插入增强型绿色荧光蛋白EGFP初步筛选出数个可使插入蛋白展示其自然构象的位点。以此筛选为基础，构建融合四种血清型DENV EDIII的WhNV重组VLP库，筛选出具有正确空间构象并具有高免疫活性的VLP。本项目的成功进行将为新型高效登革疫苗的研发打下基础，同时建立稳定高效的WhNV异源多肽与蛋白展示系统将极大推动对其他新型疫苗及解毒剂的研发，具有相当的应用前景，极大推动生物医学的发展。在基础研究方面，本项目也将对研究WhNV衣壳蛋白的结构及功能提供大量信息，为我们深入研究WhNV及其它正义RNA无囊膜病毒入侵宿主细胞的机制打下基础。

近年来，野田村病毒作为一种新型高效的表面展示系统已经获得了国际上的广泛认可。对野田村病毒科中的Flock House virus（FHV）衣壳蛋白结构的研究显示该蛋白表面有6个茎环，在这些茎环中插入异源多肽时不会引起较大的结构变化。插有异源多肽的衣壳蛋白能够组装成重组的VLP，如果在适合的位点插入异源多肽或蛋白，它们就能够在VLP表面呈现出其天然的构象。我们已经运用生物信息学手段，分析模拟出WhNV衣壳蛋白的三维结构，确定了其茎环位置并对其潜在的异源蛋白可插入位点进行了初步分析，并在此基础上利用插入增强型绿色荧光蛋白(EGFP)的方法初步筛选出数个可以使插入的蛋白展示其自然构象的位点。同时，我们使用真核表达系统获得了WhNV野生型的VLP，优化了其提纯条件。进一步构建、筛选及鉴定插入了EGFP及四种血清型DENV EDIII的WhNV VLP，制备展示高拷贝多效价EDIII的新型VLP候选疫苗，以及免疫原性检验实验打下了良好的工作基础。本项目的成功为新型高效登革病毒疫苗的研发打下基础，对我国的公共卫生事业产生巨大的社会效益。同时建立稳定高效的武汉野田村病毒异源多肽和蛋白展示系统将极大推动对多种疫苗及解毒剂的研发，具有相当的应用前景，极大推动生物医学的发展。在基础研究方面，本项目对武汉野田村病毒衣壳蛋白的结构及功能的基础研究提供大量有益的信息，为我们深入研究WhNV及其它正义RNA无囊膜病毒入侵宿主细胞的机制打下基础。