超正电荷绿荧光蛋白+36GFP作为核酸载体的细胞穿透性分析

Cell penetration of supercharged green fluorescent protein +36GFP as DNA carrier

旨在通过原核表达纯化超正电荷绿色荧光蛋白+36GFP,研究其与核酸的结合作用及作为核酸载体的细胞转导功能。将pET+36GFP-HA2质粒转化到大肠杆菌BL21(DE3)菌株中,然后表达纯化+36GFP蛋白。将得到的目的蛋白在特定浓度下分别转导293细胞、HepG2细胞、A549细胞和B16细胞,流式细胞仪检测+36GFP的转导效率;+36GFP蛋白(100 nmol/L)转导A549细胞,激光共聚焦显微镜观察结果;将+36GFP蛋白与质粒DNA按不同比例孵育,凝胶阻滞实验检测+36GFP与DNA的结合能力;激光共聚焦显微镜和流式细胞仪检测+36GFP蛋白携带质粒DNA转导细胞后报告基因的表达。结果显示,+36GFP蛋白具有较高的细胞转导效率,且随浓度升高转导效率增加,呈浓度依赖性。凝胶阻滞实验显示,+36GFP能够与质粒DNA结合,阻滞DNA在凝胶中迁移,且呈现一定的浓度依赖性。+36GFP包裹质粒转导细胞后,可高效携带质粒DNA转导进入细胞,使质粒报告基因得到表达。本研究成功表达纯化了+36GFP蛋白,证实该蛋白具有较高的细胞转导效率,可将外源核酸携带入细胞使外源基因得到表达。

In this study, we expressed and purified supercharged green fluorescent protein （＋36GFP） that we used to study its combination with nucleic acid and its cell transduction efficiency as carrier of DNA. We transformed pET＋36GFP-HA2 plasmid into Escherichia coli BL21 （DE3）, then expressed and purified the target protein. We used the protein to transduce a variety of mammalian cell lines including B16 cells, 293 cells, A549 cells and HepG2 cells at specified protein concentrations. Transduction efficiency of the protein was analyzed by flow cytometry. Under laser scanning confocal microscope, we observed visually transduction efficiency of ＋36GFP protein （100 nmol/L） to A549 ceils. We incubated ＋36GFP with plasmid DNA and analyzed their binding ability with gel mobility shift assay. Then we transduced cells with the mixture of plasmid DNA/＋36GFP protein at various ratio and detected the expression of reporter gene by using laser scanning confocal microscope and flow cytometry. The experimental results demonstrate that ＋36GFP had high transduction efficiency, and as the concentration increased, the efficiency improved in a dose-dependent manner. Gel mobility shift assay indicates that ＋36GFP could bind to plasmid DNA, blocking the migration of DNA in the gel in a concentration-dependent manner. After the plasmid wrapped by ＋36GFP penetrated into cells, the cells could express target protein efficiently, proving that ＋36GFP had the ability to carry nucleic acids into cells. Sucussful expression and purification of ＋36GFP protein confirms its high efficiency of cell transduction and its ability as carrier to deliver exogenous nucleic acids into cells.