埃博拉病毒重组膜蛋白Gp-Fc表达及免疫原性研究

Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc

本研究利用HEK293F细胞表达了埃博拉病毒重组膜蛋白,并通过免疫小鼠初步研究了其免疫原性。按照人密码子使用频度优化编码埃博拉病毒膜蛋白胞外区基因,合成后将其插入到真核表达载体pXG-Fc中,构建埃博拉病毒糖蛋白和人IgG Fc片段融合蛋白表达质粒pXG-modGP-Fc。利用瞬时转染技术,将融合蛋白表达质粒转染到高密度培养的悬浮HEK293F细胞中,实现了分泌表达。通过Protein A亲和层析,得到了纯化的重组蛋白。将纯化的融合蛋白免疫小鼠,并通过间接ELISA对小鼠抗体效价进行评价。蛋白纯化及分析结果表明,本研究构建的真核表达系统能够有效表达埃博拉重组蛋白GP-Fc,细胞培养上清中重组蛋白以二聚体形式存在。通过间接ELISA分析,纯化的重组蛋白免疫实验动物后,可以在血清中检出高滴度的抗原特异性IgG,显示该重组蛋白具有良好的免疫原性。通过该研究,我们得到了具有良好免疫原性的重组蛋白,同时,该工作为研制基于重组蛋白的埃博拉疫苗以及筛选单克隆抗体打下基础。

We used 293 cells to express the recombinant membrane protein of the Ebola virus.Then,the immunogenicity of the recombinant protein was studied by immunized BALB/c mice.According to the codon use frequency of humans,the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized,synthesized,and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc.To achieve expression,the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology.The recombinant protein was purified by protein A affinity chromatography.BALB/c mice were immunized with the purified fusion protein,and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay（ELISA）.Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively,and that the recombinant protein in the supernatant of the cell culture was present as a dimer.After immunization with the purified recombinant protein,a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA,showing that the recombinant protein had good immunogenicity.These data suggest that we obtained a recombinant protein with good immunogenicity.Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.