PTDeGFP△E250 mFoxp3融合蛋白表达及穿膜能力鉴定.docVIP

PTDeGFP△E250 mFoxp3融合蛋白表达及穿膜能力鉴定 　 作者：蔺昕 宗扬勇 许逊 田雨香 陈勋 舒新军 夏圣 王胜军 许化溪 邵启祥 【摘要】 目的： 构建、表达并纯化含有蛋白转导结构域（protein transduction domain，PTD）、eGFP的小鼠Foxp3突变体（PTDeGFP△E250 mFoxp3）融合蛋白，为研究小鼠Foxp3的功能奠定基础。方法： 利用重叠延伸PCR方法构建pET28aPTDeGFP△E250 mFoxp3原核表达载体，并在大肠埃希菌Rosetta (DE3)中表达此融合蛋白，经Ni2+柱纯化，通过流式细胞仪和激光共聚焦显微镜观察其穿膜效率。结果： 成功构建了pET28aPTDeGFP△E250 mFoxp3原核表达载体，并表达和纯化了PTDeGFP△E250 mFoxp3融合蛋白，流式细胞仪和激光共聚焦显微镜检测结果显示融合蛋白能很好地穿过细胞膜进入细胞内，并定位于细胞质和细胞核。结论： 成功制备了具有穿膜活性的PTDeGFP△E250 mFoxp3融合蛋白，为更好地研究小鼠Foxp3的功能与特性奠定了实验基础。 【关键词】 蛋白质转导结构域； Foxp3； 突变； 穿细胞膜活性 ［Abstract］ Objective: To express and purify a mutant mouse Foxp3 fusion protein containing a protein transduction domain from TAT and an enhanced green fluorescence protein (PTDeGFP△E250 mFoxp3), furthermore to investigate its′s efficiency and ability transmembrane and laid the groundwork for future research about the function of mouse foxp3.Methods： The pET28aPTDeGFP△E250 mFoxp3 vector was constructed by inserting the PCR products of mouse mutant Foxp3 into pET28aPTDeGFP vector. The fusion protein was expressed by E. coli Rosetta (DE3) and purified by BioRad Profinity IMAC Nicharged Resin. The efficiency and ability of transmembrane of PTDeGFP△E250 mFoxp3 were detected by FCM and confirmed by confocal microscopy. Results： The vector of pET28aPTDeGFP△E250 mFoxp3 was constructed correctly and the fusion protein was expressed and purified efficiently. The results of FCM and confocal microscopy investigation indicated that PTDeGFP△E250 mFoxp3 fusion protein transduce into EL4 cell efficiently. Conclusion： The PTDeGFP△E250 mFoxp3 protein can transduce into cells efficiently and may be a useful tool to study the biological functions of Foxp3. ［Key words］ protein transduction domain；Foxp3；mutant；transmembrane ability CD4+CD25hi调节性T细胞(regulatory T cells，Treg) 是一群具有免疫调节功能的成熟T细胞亚群，其通过抑制潜在的自身反应性CD4+CD25-T细胞，而在自身免疫耐受的形成、维持中发挥着重要的作用［1］。Foxp3(forkhead box P3 )属于Fox转录因子家族成员，2001年由Brunkow等首次报道［2］。2003年Rudensky