细胞毒淋巴细胞相关抗原4融合蛋白靶向树突状细胞作用的体外研究

目的：研究细胞毒淋巴细胞相关抗原4（CTLA-4）靶向重组质粒pGJGLU／GFP在COS-7细胞中的表达水平,并研究CTLA-4融合蛋白的生物学活性,检测融合蛋白能否与体外培养的抗原递呈细胞树突状细胞靶向结合。方法：质粒pGJGLU／GFP体外转染COS-7细胞,荧光显微镜下观察蛋白的表达,利用双抗夹心法,以人IgG作为标准品,检测培养上清中蛋白的表达水平。体外培养人外周血来源的树突状细胞,并与浓缩的上清液作用,通过流式细胞术检测结合在树突状细胞表面的蛋白,在封闭（C组）与未封闭（B组）树突状细胞表面CTLA-4配体,即B7分子的条件下,比较树突状细胞的平均荧光强度,实验设置阴性对照（A组）。结果：转染后的COS-7细胞高表达绿色荧光融合蛋白,ELISA方法检测到浓缩后上清中蛋白浓度约相当于0．62mg／L人IgG。流式细胞术检测到B组细胞平均荧光强度明显高于A组和c组,而A组与C组细胞平均荧光强度没有明显差异。结论：靶向重组质粒pGJGLU／GFP在COS-7细胞中高表达CTLA-4融合蛋白,蛋白具有CTLA-4的活性并与体外培养的树突状细胞特异性结合。

Fusion Protein Encoded a Targeted DNA Construct pGJGLU/GFP Binds to Human Dendritic Cells in Vitro

Objectives: To investigate the expression level in COS-7 cells and to explore the functional property of the fusion protein encoded by a targeted DNA construct pGJGLU/GFP.Methods: COS-7 cells were transfected with pGJGLU/GFP or pEGFP-N1 by using sofastTM transfect reagent.Two kinds of cultured supernatant were collected and concentrated.Fusion protein in the supernatant was assayed by using ELISA.With(Group C) or without(Group B) blocking B7 molecules expressed on cell surface,human dendritic cells(DCs) were incubated with concentrated supernatant from cells transfected with pGJGLU/GFP.Another group(Group A) of DCs without blocking B7 molecules was incubated with concentrated supernatant from cells transfected with pEGFP-N1.DCs bound with fusion protein were detected by flow cytometry.Results: Green fluorescence was observed in COS-7 cells with a high percentage.The Group B had significantly higher mean fluorescence intensity than Group A and Group C.There is no significant difference in MFI between Group A and Group C.Conclusion: Protein encoded by pGJGLU/GFP retains the activity of CTLA-4 and could bind to B7 molecules on DCs with specificity.