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

发布时间：2018-05-07 08:23

本文选题：呼吸道合胞病毒 + 融合糖蛋白　；参考：《北京交通大学学报》2016年06期

【摘要】：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.
[Abstract]:To analyze the effect of fusion glycoprotein (DEC205/CD205) of human respiratory syncytial virus (respiratory syncytial virus) on the expression and activity of recombinant F protein. F protein is an important neutralizing antigen of RSV. The encoding gene of extracellular domain of RSV F protein was cloned to the C-terminal of single chain antibody of anti-DEC205 (single chain antibody of anti-DEC205), which could recognize the receptor of mouse dendritic cells. The plasmid pVAX1 / scDECFWestern blot was used to detect the expression of scDECF. The binding activity of scDECF to DEC205 receptor was detected by immunofluorescence and flow cytometry. The expression of scDECF in 293T cells transfected with pVAX1/scDECF was confirmed by Western blot. The obtained supernatants were incubated with CHOmDEC205 cells expressing mouse DEC205 receptor. Immunofluorescence and flow cytometry showed that the scDECF was significantly bound to CHOmDEC205 cells compared with the recombinant F protein scISOF as control. In this study, the recombinant protein scDECF was successfully expressed, and the recombinant protein scDECF had specific binding activity to the cells expressing DEC205 receptor.
【作者单位】：北京交通大学生命科学与生物工程研究院;
【基金】：中央高校基本科研业务费专项资金资助(2009YJS037) 国家重大新药创制专项资金资助(2013ZX09103-003-011)
【分类号】：R373.14

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