MS2噬菌体介导展示猪繁殖与呼吸综合征病毒线性表位的嵌合纳米颗粒制备及免疫原性

MS2 Phage-mediated Preparation and Immunogenicity of Chimeric Nanoparticles with Linear Epitope of Reproductive and Respiratory Syndrome Virus

【目的】以MS2噬菌体外壳蛋白为载体,构建展示猪繁殖与呼吸综合征病毒GP5蛋白上线性表位的嵌合纳米颗粒,并研究其免疫原性,为猪繁殖与呼吸综合征病毒或其他病毒表位展示提供新的方法和思路。【方法】利用重叠延伸PCR将GP5上优势线性表位基因序列插入到MS2噬菌体外壳蛋白基因上,构建重组载体,通过原核表达系统表达嵌合蛋白,目的蛋白经过硫酸铵沉淀和凝胶过滤层析纯化,用动态光散射和电镜对嵌合蛋白进行物理表征,通过蛋白印迹和动物免疫试验研究表位嵌合颗粒的免疫原性。【结果】成功将线性表位基因插入MS2噬菌体外壳蛋白基因,嵌合蛋白在原核表达系统中以水溶性表达,目的蛋白经过纯化,纯度达85%以上。嵌合蛋白在体外自组装形成了均一的、直径为25~31 nm的嵌合表位纳米颗粒,该嵌合颗粒免疫动物后,产生可以和灭活病毒反应的高水平抗体,具有良好的免疫原性。【结论】MS2噬菌体外壳蛋白可以耐受9个外源多肽(猪繁殖与呼吸综合征病毒GP5上线性表位)的插入,在体外自组装形成嵌合病毒样颗粒。该颗粒将外源多肽高密度展示在表面,免疫动物可产生针对该表位的抗体。该技术可为猪繁殖与呼吸综合征病毒其他表位或更长串联表位的展示奠定基础。

【Objective】Using the protein on coat of MS2 bacteriophage as a carrier,chimeric nanoparticles with a linear epitope of porcine reproductive and respiratory syndrome virus,GP5,were constructed,and their immunogenicity determined.【Method】The dominant neutralizing epitope gene sequence on GP5 was inserted into the MS2 protein by overlapping extension PCR.The recombinant vector was to express the chimeric protein through a prokaryotic expression system.The target protein was purified by ammonium sulfate precipitation and gel filtration chromatography followed by characterization with dynamic light scattering and electron microscopy.The immunogenicity of the constructed chimeric nanoparticles was determined by Western blot and animal immunoassay.【Result】The linear epitope was successfully inserted into the target sequence,and the recombinant vector reached a purity greater than 85%.The purified target protein selfassembled in vitro to form uniform chimeric nanoparticles with a diameter of 25-31 nm.After immunizing animals,the chimeric particles produced a high-level of antibodies that reacted with inactivated viruses showing significant immunogenicity.【Conclusion】The MS2 phage coat protein allowed the insertion of 9 exogenous polypeptides(the linear epitope on GP5)and self-assembled in vitro to form the chimeric virus-like particles.Each particle carried the exogenous polypeptides on the surface generating high immunogenicity.The current technology provided a new venue for building other epitopes of porcine reproductive and respiratory syndrome virus or epitopes with longer tandem.