【摘要】 为制备携肠球菌胶原蛋白黏附素Ace抗原表位的铁蛋白纳米颗粒,并评价其免疫效果,利用生物信息学软件预测Ace抗原表位,合成相应的基因片段,分别构建含有相应抗原表位基因与铁蛋白H亚基基因的融合表达载体,同时构建融合表达Ace蛋白和H亚基的质粒pET-32a-ace-H,分别转入大肠杆菌中进行融合表达。利用Western blot鉴定各重组融合蛋白的反应原性,并利用透射电镜观察目的蛋白表征。用重组铁蛋白纳米颗粒分别制备免疫原进行家兔接种试验,并定期检测家兔血清特异性抗体水平。结果显示,正确构建了6个Ace蛋白抗原表位基因与H亚基基因的融合表达载体pET-32a-ace1-H～pET-32a-ace6-H,其中pET-32a-ace1-H、pET-32a-ace3-H、pET-32a-ace5-H和pET-32a-ace6-H为可溶性表达载体。Western blot检测显示可溶性表达的4种融合蛋白均具有较好的反应原性。透射电镜图像显示,4种融合蛋白均形成了具有中空结构的铁蛋白纳米笼,其直径与天然铁蛋白类似;而融合蛋白Ace-H纳米笼没有中空结构,且直径比天然铁蛋白大。家兔免疫试验结果显示,6种融合蛋白均在家兔体内诱导产生了特异性抗体,而且,携带单个Ace表位的铁蛋白纳米颗粒Ace1-H、Ace3-H、 Ace5-H与携带多表位的Ace重组蛋白以及Ace-H纳米颗粒相比,其诱导的抗体效价和消长规律均一致,其中以铁蛋白纳米粒Ace5-H的免疫效果为最佳。本研究结果表明铁蛋白纳米颗粒能有效增强蛋白质抗原的免疫原性,有望作为候选表位疫苗佐剂得到应用。更多还原

【Abstract】 To prepare ferritin nanoparticles carrying antigen epitopes of collagen-associated adhesin(Ace) of Enterobacter faecalis and evaluate their immunological effects, bioinformatic softwares were used to predict Ace epitopes, and corresponding gene fragments were synthesized and then fusion expression vectors containing corresponding epitopes and ferritin H subunit genes were constructed respectively; Meanwhile plasmid pET-32 a-ace-H, which fused to express Ace protein and H subunit, was constructed and transferred into Escherichia coli respectively for fusion expression. Western blot was used to identify the reactivity of the fusion proteins, and transmission electron microscopy was used to observe the characterization of the target proteins. Western blot was used to verify the immunogenicity of the recombinant fusion proteins, and transmission electron microscopy was used to observe the morphology of the target proteins. The rabbit was inoculated with immunogen prepared by recombinant ferritin nanoparticles and the serum specific antibody level was measured regularly. The results showed that six epitope-carrying fusion vectors were obtained, designated as pET-32 a-ace1-H to pET-32 a-ace6-H, among which pET-32 a-ace1-H, pET-32 a-ace3-H, pET-32 a-ace5-H and pET-32 a-ace6-H turned out to be fusion expression vectors. Western blot indicated that these four fusion proteins showed strong immunogenicity. TEM images indicated that five H-fusion proteins were characterized by ferritin nano-cages, four of which were hollow, with similar diameter sizes to those of natural ferritin; pET-32 a-ace-H, whereas, did not form a hollow center, whose diameter was larger. The ELISA results indicated that all of the six fusion proteins successfully induced specific antibody in rabbit body, and compared with Ace recombinant proteins and Ace-H nanoparticles carrying multiple epitopes, the antibody valence and dynamics of the single-epitope-carrying Ace1-H, Ace3-H, Ace5-H and Ace3-H showed good consistency, among which the immunological effect of ace5-H, a ferritin nanoparticle, was the best. The research results shows that ferritin nanoparticles can effectively enhance the immunogenicity of the protein antigens, and might become a promising immune adjuvant for epitope vaccines.更多还原