靶向融合防龋DNA疫苗编码蛋白靶向于人树突状细胞的体外研究

Fusion Protein Encoded by Targeted Fusion DNA Vaccine pGJA-P/VAX_1 against Caries can be Targeted to Human Dendritic Cells

目的:体外检测靶向融合防龋DNA疫苗pGJA-P/VAX1编码蛋白的生物学活性,研究蛋白是否具有靶向于抗原递呈细胞如树突状细胞的能力。方法:pGJA-P/VAX1和pVAX1分别转染真核细胞COS-7,收集并浓缩细胞培养上清。利用双抗夹心法,以人的IgG为标准品,检测上清及浓缩液中pGJA-P/VAX1编码蛋白的含量。体外培养人外周血来源的树突状细胞,与两种上清浓缩液孵育一段时间后,通过流式细胞术检测树突状细胞是否结合编码蛋白。实验分A、B、C组,A组树突状细胞与VAX1浓缩液作用,B组中树突状细胞与GJA-P/VAX1浓缩液作用,C组中树突状细胞表面B7分子首先被B7-1和B7-2的单克隆抗体封闭,然后与GJA-P/VAX1浓缩液作用。结果:pGJA-P/VAX1转染细胞后,培养上清的浓缩液中蛋白浓度相当于0.22μg/mL人的IgG。而pVAX1转染上清及浓缩液均未有蛋白检出。流式结果显示B组细胞平均荧光强度及细胞表面编码蛋白阳性率明显高于A组和C组。B组相对A组和C组,细胞直方图峰向右偏移。A、C两组细胞平均荧光强度近似,且两组细胞直方图近乎重合。结论:靶向融合防龋DNA疫苗编码的融合蛋白保持了CTLA-4的活性,可通过B7分子靶向结合树突状细胞。

Objectives： To explore the functional property of fusion protein encoded by pGJA- P/VAX,. Methods： COS -7 cells were transfected with pGJA -P/VAX, or pVAX, using sofast^TM transfect reagent. Two kinds of cultured supernatam were collected and concentrated. Fusion protein in the supernatant was assayed by using ELISA. With or without blocking B7 molecules expressed on cell surface, human dendritic cells （DCs） were incubated with concentrated supernatant from cells transfected with pGJA - P/VAX1. Another group of DCs without blocking B7 molecules were incubated with concentrated supernatant from cells transfected with pVAX1. DCs bound with fusion protein were detected by flow cytometry. Results： Significantly higher fluorescence was observed when DCs incubated with supernatant of pGJA -P/VAX1. Comparably lower fluorescence was observed in other two groups. Conclusion： Protein encoded by pGJA - P/VAX1 retains the activity of CTLA -4 and could bind to B7 molecules on DCs with specificity.