【作者】 陈菁；

【导师】 刘树滔；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 Tat(Trans-activator transcription)蛋白的蛋白转导区域(Protein transduction domain,PTD)——PTD-Tat 能介导其融合的蛋白的转导及细胞内定位。就理论而言,较之 N 端融合,PTD-Tat 的 C 端融合更有利于外源蛋白的表达。因此,对融合在外源蛋白 C 端的跨膜递送作用进行探讨具有研究意义。 本研究用 DNA 重组技术将 PTD-Tat 融合在绿色荧光蛋白(green fluorescence protein ,GFP)的羧基(C)端,构建重组表达质粒 pGEX-GFP-Tat;转化大肠杆菌 BL21(DE3),IPTG 诱导其表达后,采用谷胱甘肽-Sepharose 4B(GS-4B)亲和层析,获得纯化的谷胱甘肽 S-转移酶(GST)融合的重组蛋白GST-GFP-Tat;用于研究融合在外源蛋白 C 端的 PTD-Tat 的跨膜递送作用。 通过融合蛋白 GST-GFP-Tat 与体外培养的 Hela 细胞共培养,荧光显微镜观察和流式细胞仪分析表明:其一,融合蛋白 GST-GFP-Tat 能高效地跨膜进入细胞,且定位于细胞核周围。其二,其跨膜递送与时间、浓度有依赖关系,即 37℃条件下 15min 细胞内可观察到绿色荧光,且荧光强度随着蛋白浓度的增高而增强。其三,温度对跨膜递送作用影响较大,37℃条件下有高跨膜效率的融合蛋白 4 ℃时跨膜递送作用几乎完全被抑制。其四,细胞膜表面的硫酸乙酰肝素(Heparin sulfate ,HS)对 GST-GFP-Tat 的跨膜递送具有重大影响,而代谢抑制剂并不影响其跨膜递送。其五,MTT 实验表明该融合蛋白对细胞无毒性。 此外,本研究通过融合蛋白 GST-GFP-Tat 与 GFP 氨基(N)端含有 PTD-Tat的融合蛋白 GST-Tat-GFP 的阳性对照组的对比发现:流式细胞仪的分析显示 4.0 μmol/L 的蛋白浓度下 GST-GFP-Tat 的转导效率高达 80.0 %,而 GST-Tat-GFP的转导效率只有 32.9 %;荧光显微镜的观察结果与其一致。 最后,动物实验证明该融合蛋白具有跨膜递送的作用:其一,小鼠的皮肤涂抹实验表明 GST-GFP-Tat 能沿着毛囊进到皮肤的真皮层。其二,小鼠的腹腔注射实验则表明 GST-GFP-Tat 能有效递送到小鼠体内各个组织,甚至跨越小鼠的血脑屏障;与阳性对照组的 GST-Tat-GFP 的比对显示其荧光强度较强,尤其两种融合蛋白在跨越血脑屏障方面存在明显差异。其三,融合蛋白喂食线虫发现蛋白进入其整个消化道及其原体腔,且与喂食时间与蛋白浓度呈正相关关系。 通过对融合在 GFP 之 C、N 端的 PTD-Tat 跨膜递送的比较分析,表明 C 端融合之 PTD-Tat 同样具有介导外源蛋白的跨膜递送作用,不仅跨膜递送进入细胞,且实现组织水平上的成功应用;而且 C 端融合之 PTD-Tat 甚至表现出更强的跨膜活性;此外,融合蛋白对细胞未造成毒性。这些结论将为 PTD-Tat 在蛋白药物递送方面的有效应用提供理论指导。更多还原

【Abstract】 The protein transduction domain (PTD) of Tat (Trans-activator transcription) protein is sufficient for intracellular transduction of Tat protein and subcellular location. PTD-Tat fused at the carboxyl-terminus of heterologous protein was theoretically superior than that fused at the amino-terminus of heterologous protein in the expression of the fusion protein. So, it is necessary to study the transmembrane delivery of PTD-Tat fused at the carboxyl-terminus of heterologous protein. The expression plasmid pGEX-GFP-Tat was constructed by genetically fusing PTD-Tat to the carboxyl-terminus of green fluorescence protein (GFP),which was in the right downstream of the coding sequence of glutathione-S-transferase(GST). The fusion protein GST-GFP-Tat was expressed in E.coli BL21(DE3) and purified by Glutathione Sepharose 4B affinity chromatography. The fusion protein will be used for studying the transmembrane delivery of PTD-Tat fused at the carboxyl-terminus of heterologous protein. When HeLa cells in vitro were co-cultured with fusion protein GST-GFP-Tat,the analysis of fluorescence microscope and flow-cytometry showed the results of the experiment as follows.(A)Fusion protein GST-GFP-Tat could internalize into HeLa cells in vitro efficiently and localize to both the cytoplasm and nucleus.(B)The transmembrane of PTD-Tat into cultured HeLa cells increased in a dose/time-dependent manner. The fluorescence intensity could be detected within 15min at 37℃. (C)It seemed that the temperature had a great influence on the uptake of PTD-Tat fusion protein. A rightward shift of the signal was observed signifying a reduction of the uptake of the fusion protein GST-GFP-Tat at 4℃compared to its cellular uptake at 37℃.(D)Heparin sulfate(HS)on the cell membrane affected the internalization of GST-GFP-Tat greatly. However, metabolic inhibitors didn’t induce the internalization of GST-GFP-Tat.(E)MTT colorimetric assay found no cytotoxic effect of GST-GFP-Tat to HeLa cells. Furthermore, flow-cytometry analysis found the transduction efficiency at dose 4.0 μmol/L of GST-GFP-Tat was 80.0 %. In contrast,the transduction efficiency reduced to 32.9 % at dose 4.0 μmol/L of GST-Tat-GFP of positive control, which was GST-Tat-GFP with PTD-Tat fused at the amino-terminus of GFP. The observation of fluorescence microscope was in agreement with results of flow-cytometry analysis. Finally, the animal experiments also showed the transmembrane activity of the fusion protein.(A)GST-GFP-Tat was proved to be delivered into dermis of mice by the result of the skin painting experiment.(B) The fusion protein GST-GFP-Tat could be detected in different tissues, even passed through blood-brain barrier of mice with intraperitoneal injection. And the observation of fluorescence microscope indicated the fluorescence intensity was stronger than that of GST-Tat-GFP, especially in the brain.(C)When the fusion protein was co-culture with nematode,the analysis of fluorescence microscope showed the digestive system and the coelom of nematode could be detected green fluorescence, and the fluorescence intensity was time-and protein concentration-dependent. The research findings indicated that PTD-Tat fused at the carboxyl-terminus of heterologous protein not could internalize into cells in vitro, but the transduction was successfully applied in the tissues of mice by the comparison of PTD-Tat fused at the carboxyl/ amino-terminus of GFP. Furthermore,the transmembrane activity of PTD-Tat fused at the carboxyl-terminus displayed stronger. And the fusion protein showed no cytotoxic effect to the cells. These results will provide theoretical instruction for the efficient application of PTD-Tat in the field of the delivery of protein drugs.更多还原