靶向树突状细胞的呼吸道合胞病毒F重组蛋白的构建与体外活性分析

分析以人呼吸道合胞病毒(Human respiratory syncytial virus,RSV)融合糖蛋白(fusion glycoprotein,F)为抗原靶向DEC205/CD205受体策略对重组F蛋白表达及活性的影响.F蛋白是RSV的重要中和抗原,克隆RSV F蛋白胞外区的编码基因至可识别鼠树突状细胞(Dendritic cells,DCs)DEC205受体的单链抗体(single chain antibody of anti-DEC205,scDEC)的C端,构建可表达重组F蛋白(scDECF)的质粒pVAX1/scDECF,Western blot方法检测scDECF的表达,免疫荧光法和流式细胞术检测表达的scDECF与DEC205受体的结合活性.经Western blot证实在pVAX1/scDECF转染的293T细胞成功表达了scDECF.将所获得的细胞培养上清与表达鼠DEC205受体的细胞CHOmDEC205孵育,用免疫荧光和流式细胞术证实:与作为对照的重组F蛋白scISOF相比,所获得的scDECF与CHOmDEC205细胞呈现明显的结合.本研究成功表达了重组蛋白scDECF,且所获得的重组蛋白scDECF具有与表达DEC205受体的细胞特异性结合的活性.

Construction of respiratory syncytial virus fusion glycoprotein targeting dendritic cells and its in vitro activity analysis

This investigation was implemented by fusing the human respiratory syncytial virus (RSV)fusion glycoprotein (F)extracellular domain to DEC205 single chain antibody and detec-ting the activity of the recombinant protein in vitro.F protein is one important antigen of RSV and targeting F protein to dendritic cells (DCs)may help to enhance the immune efficacy of vac-cine.After cloning the gene encoding the extracellular domain of the F protein to 3' end of the single chain antibody specific for mouse DEC205 receptor,distributed on the surface of mouse dendritic cells,the expression of the recombinant protein scDECF was confirmed by Western blot assay and the DEC205 binding activity was analyzed by immunofluorescence and flow cytometry. Data showed that the recombinant protein scDECF was successfully expressed in eukaryotic cells, and exhibited the specific binding activity to DEC205 receptor on CHOmDEC205 cells.