GPI锚定蛋白介导嵌合新城疫病毒样颗粒的组装

Assembly of GPI-anchored protein mediated chimeric Newcastle disease virus-like particles

当前嵌合病毒样颗粒(chimeric VLPs,cVLPs)策略主要基于融合序列的基因转染水平,但该方法存在多基因表达操控困难、锚定蛋白无法定量等问题。糖基磷脂酰肌醇(glycosylphosphatidylinositol,GPI)是一种蛋白与细胞膜结合的方式,改造的GPI锚定蛋白可以被定量嵌合至真核细胞膜表面。因此,本研究以可溶性蛋白、Ⅰ型跨膜蛋白、Ⅱ型跨膜蛋白为嵌合对象,以GPI锚定方式将外源蛋白嵌合至新城疫病毒样颗粒(NDV VLPs)表面。流式细胞术检测可见GPI锚定蛋白可锚定至各真核细胞表面,并且与孵育时间呈正相关性;各组装GPI-cVLPs在电镜观察下具有完整的病毒粒子结构,与野生型NDV相似;经Western blot检测分析各GPI-cVLPs与NDV阳性血清和His单抗发生反应。综上,该GPI锚定策略能有效将外源蛋白嵌合至NDV VLPs,可为NDV VLP载体平台的应用奠定基础。

Current chimeric VLP constructing strategies mainly depend on the integration of heter- ologous gene which is of multiple disadvantages including uncontrollable gene expression and the inability to quantify the heterologous protein loading. Glycosylphosphatidylinositol（GPI） is a pro- tein anchor which functions through linking associated protein to plasma membrane,and GPI-an- chored protein insertion can be quantified. In this study, GPI-anchored soluble protein, type I trans- membrane protein and type II transmembrane protein were generated and linked to VLPs,respec- tively. The GPI-anchored proteins were with confirmed affinity to eukaryotic cells in a time de- pendent manner. All the GPI-cVLPs possessed an intact virus-like structure similar to wild type Newcastle disease virus（NDV）. The bioactivity of heterologous protein on GPI-cVLPs was verified through Western blot coated with NDV positive serum or anti-his tag monoclonal antibodies. In general,the GPI-anchored heterologous proteins can be efficiently anchored to NDV VLPs, which is a promising platform carrying various antigens.