Expression of GST-transactivator of transcriptionenhanced green fluorescence protein fusion protein and its effect on the blood labyrinth barrier
融合蛋白GST-反式转录激活因子蛋白-增强型绿色荧光蛋白表达及对血迷路屏障作用
作者: DU YanshunLI YingYANG JunZHANG WeiZHANG LuoDepartment of Otolaryngology Head and Neck Surgery, Beijing Tongren Hospital,Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery(Capital Medical University), Ministry of Education,Beijing Institute of Otolaryngology
刊名: Chinese Archives of Otolaryngology-Head and Neck Surgery, 2015(11), pp.578-580
中文刊名: 中国耳鼻咽喉头颈外科, 2015(11), pp.578-580
来源数据库: CJFDTEMP_U
DOI: CNKI:SUN:EBYT.0.2015-11-012
关键词: Recombinant Fusion ProteinsLabyrinthblood labyrinth barriertransactivator of transcriptionenhanced green fluorescence protein
中文关键词: 重组融合蛋白质类迷路血迷路屏障反式转录激活因子蛋白增强型绿色荧光蛋白
英文摘要: OBJECTIVE To explore whether the protein transduction domain of TAT can mediate the TATEGFP fusion protein passing through the blood labyrinth barrier in order to provide the experimental basis for treatment of inner ear diseases.METHODS The GSTTAT EGFP fusion protein was expressed in E.coli and purified with affinity chromatography by GST resin.10 guinea pigs were divided into 2 groups.One group was injected with GST-TAT EGFP,and another group was injected with GST-EGFP.After 2-4 hours injection,the animals were anesthetized and decapitation.The temporal bones were fixed with 2%paraformaldehyde,and decalcified with 10%EDTA(pH7.4) and then embedded in OCT.Then the fusion protein was examined in frozen section of guinea pig cochlea by immunohistochemisty and fluorescence microscope.RESULTS...
中文摘要: 目的表达并纯化融合蛋白GST-反式转录激活因子蛋白-增强型绿色荧光蛋白(GST-transactivator of transcription-enhanced green f luorescence protein,GSTTAT-EGF P),并研究其是否能跨越血迷路屏障(blo o d labyrinth barrir,BLB)进入内耳。方法用基因重组技术将编码TAT蛋白11个氨基酸短肽基因和EGFP基因重组,分别构建p GEX-6p-1-TAT-EGFP和p GEX-6p-1-EGFP表达载体,将载体分别转化大肠杆菌BL21(DE3),经IPTG诱导表达融合蛋白,用GST树脂柱亲和层析纯化融合蛋白。将10只豚鼠随机分为两组,分别肌肉注射GST-EGFP和GST-TATEGFP;2~4 h后豚鼠经麻醉、断头取耳蜗等组织。用2%多聚甲醛固定,耳蜗经10%EDTA(p H7.4)脱钙、OCT包埋,制备颞骨冰冻切片,采用免疫组化技术和荧光显微镜技术观察TAT短肽是否可以携带分子量55 k D的GST-EGFP跨越BLB进入内耳。结果 GST-TAT-EGFP进入内耳后主要分布于血管纹和螺旋器,而对照组EGFP不能进入内耳。结论短肽TAT可转导大分子蛋白通过BLB进入内耳,为外源性蛋白药物治疗内耳疾病提供新手段。
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