重组EGFP-aquaporin-4融合蛋白真核表达载体的构建及其在FRT细胞中的表达和定位

发布时间：2018-03-06 11:37

  本文选题：aquaporin-　切入点：转染　出处：《吉林大学学报(医学版)》2013年02期 　论文类型：期刊论文

【摘要】：目的:构建以增强型绿色荧光蛋白(EGFP)为报告基因的重组真核表达载体pEGFP-aquaporin-4,以EGFP示踪aquaporin-4在Fisher大鼠甲状腺滤泡上皮细胞(FRT细胞)中的表达和定位,为进一步研究aquaporin-4提供实验依据。方法:应用RT-PCR方法获得aquaporin-4编码区基因,克隆入真核表达载体pEGFP-N1。经酶切和测序鉴定证实为aquaporin-4后,Lipofectamine 2000脂质体转染重组EGFP-aquaporin-4融合蛋白真核表达载体至FRT细胞中,倒置荧光显微镜下观察aquaporin-4在FRT细胞中的表达和分布,并应用Western blotting法检测aquaporin-4蛋白的表达。同时检测转入FRT细胞内钙黄绿素的相对荧光强度以判断FRT细胞水通透性的状况。结果:EcoRⅠ和KpnⅠ双酶切和测序重组载体结果证实目的基因aquaporin-4成功克隆到真核表达载体pEGFP-N1中。荧光显微镜下观察融合EGFP的aquaporin-4主要在FRT细胞膜上表达;Western blotting结果证实脂质体转染重组载体的FRT细胞表达aquaporin-4。转染重组EGFP-aquaporin-4融合蛋白真核表达载体的FRT细胞水通透性显著高于未转染的FRT细胞,其水通透性是未转染FRT细胞的1.65倍。结论:成功构建aquaporin-4真核表达载体,证实其可在FRT细胞中表达,并具有明显的膜蛋白特性和良好水通透性。
[Abstract]:Aim: to construct a recombinant eukaryotic expression vector pEGFP-aquaporin-4 using enhanced green fluorescent protein (EGFP) as a reporter gene and to trace the expression and localization of aquaporin-4 in Fisher rat thyroid follicular epithelial cells (Fisher). Methods: aquaporin-4 coding region gene was obtained by RT-PCR method. PEGFP-N1 was cloned into the eukaryotic expression vector pEGFP-N1.After aquaporin-4 was confirmed by restriction endonuclease digestion and sequencing, the recombinant EGFP-aquaporin-4 fusion protein was transfected into FRT cells by lipofectamine 2000 liposome. The expression and distribution of aquaporin-4 in FRT cells were observed by inverted fluorescence microscope. The expression of aquaporin-4 protein was detected by Western blotting, and the relative fluorescence intensity of calcium xanthocyanin was detected to determine the water permeability of FRT cells. Results the recombinant vectors of FRT 鈪，

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