GPI锚定蛋白介导嵌合新城疫病毒样颗粒的组装
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摘要
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 heterologous gene which is of multiple disadvantages including uncontrollable gene expression and the inability to quantify the heterologous protein loading.Glycosylphosphatidylinositol(GPI)is a protein anchor which functions through linking associated protein to plasma membrane,and GPI-anchored protein insertion can be quantified.In this study,GPI-anchored soluble protein,type Ⅰ transmembrane protein and type Ⅱ transmembrane protein were generated and linked to VLPs,respectively.The GPI-anchored proteins were with confirmed affinity to eukaryotic cells in a time dependent 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.
Current chimeric VLP constructing strategies mainly depend on the integration of heterologous gene which is of multiple disadvantages including uncontrollable gene expression and the inability to quantify the heterologous protein loading.Glycosylphosphatidylinositol(GPI)is a protein anchor which functions through linking associated protein to plasma membrane,and GPI-anchored protein insertion can be quantified.In this study,GPI-anchored soluble protein,type Ⅰ transmembrane protein and type Ⅱ transmembrane protein were generated and linked to VLPs,respectively.The GPI-anchored proteins were with confirmed affinity to eukaryotic cells in a time dependent 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.
引文
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[2]JENNINGS G T,BACHMANN M F.The coming of age of virus-like particle vaccines[J].Biological Chemistry,2008,389(5):521.
[3]MANOLOVA V,FLACE A,BAUER M,et al.Nanoparticles target distinct dendritic cell populations according to their size[J].Eur J Immunol,2008,38(5):1404-1413.
[4]PANTUA H D,MCGINNES L W,PEEPLES M E,et al.Requirements for the assembly and release of Newcastle disease virus-like particles[J].J Virol,2006,80(22):11062-11073.
[5]钱晶,丛彦龙,袁乾亮,等.含有新城疫病毒M、F、HN基因病毒样颗粒的构建及表达[C]//中国畜牧兽医学会动物传染病学分会第十六次学术研讨会论文集.2015.
[6]袁乾亮,钱晶,薛聪,等.基因Ⅶ型新城疫病毒样颗粒的制备与鉴定[J].中国兽医学报,2016,36(1):18-23.
[7]MCGINNES L W,GRAVEL K A,FINBERG R W,et al.Assembly and immunological properties of Newcastle disease virus-like particles containing the respiratory syncytial virus F and G proteins[J].J Virol,2011,85(1):366-377.
[8]MCGINNES L W,PANTUA H,LALIBERTE J P,et al.Assembly and biological and immunological properties of Newcastle disease virus-like particles[J].J Virol,2010,84(9):4513-4123.
[9]SCHUBERT J.Glycosylphosphatidylinositol(GPI)[J].Clin Exp Immunol,1995,102(1):199-203.
[10]MEDOF M E,NAGARAJAN S,TYKOCINSKI M L.Cell-surface engineering with GPI-anchored proteins[J].FASEB J,1996,10(5):574-586.
[11]PATEL J M,KIM M C,VARTABEDIAN V F,et al.Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses[J].Nanomed Nanotechnol Biol Med,2015,11(5):1097-1107.
[12]MORRISON T G.Newcastle disease virus-like particles as a platform for the development of vaccines for human and agricultural pathogens[J].Future Virol,2011,5(5):545-554.
[13]SCHMIDT M R,MCGINNES L W,KENWARD S A,et al.Long-term and memory immune responses in mice against Newcastle disease virus-like particles containing respiratory syncytial virus glycoprotein ectodomains[J].J Virol,2012,86(21):11654.
[14]熊茂林.GPI锚的结构、功能及GPI锚定B7分子在肿瘤免疫治疗中的应用[J].国际免疫学杂志,2003,26(5):230-233.
[15]张云华,黄瑾.GPI锚定蛋白的合成及功能[J].农垦医学,2012(1):59-62.
[16]高健,郭忠武.糖基磷脂酰肌醇及其锚定蛋白的合成研究进展[J].中国科学(化学),2013(8):964-983.
[17]杨文斌,吕茂民.尼帕病毒基因结构及其产物[J].国际病毒学杂志,2003,10(1):31-32.
[18]CLAUSSE M,D AZ A G,GHERSI G,et al.The vaccine candidate BLSOmp31protects mice against Brucella canis infection[J].Vaccine,2013,31(51):6129-6135.