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  

Construction of EGFP-aquaporin-4 fusion protein and its expression and location in FRT cells

作　　者：李凤娥[1] 郝峰[2] 藏雨轩[2] 马睿泽[3] 孔繁利[4] 刘磊[2] 李艳[2] 

机构地区：[1]北华大学附属医院神经二科,吉林吉林132013 [2]吉林医药学院生物化学检验教研室,吉林吉林132013 [3]吉林医药学院基础护理教研室,吉林吉林132013 [4]北华大学基础医学院病理生理学教研室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2013年第2期273-277,428,共5页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅自然科学基金资助课题(201015241);吉林省教育厅基金资助课题(2011-131;2013-162;2013-351);吉林医药学院大学生科研基金资助课题(吉医学科字[2012]第12号)

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。Objective To construct the eukaryotic expression vector of pEGFP-aquaporin-4 and observe the expreesion and location of aquaporin-4 in FRT cells for its EGFP fusion protein as a reporter by fluorescence microscope and to provide experimental basis for further study on aquaporin-4.Methods The full-length coding sequence of aquaporin-4 was amplified by PCR,and subcloned into pEGFP-N1 for the expression in FRT cells.The FRT cells were transfected with aquaporin-4 using Lipofectamine 2000 Transfection Reagent with different concentrations.The expression of the fusion protein was detected by Western blotting and its location was observed by fluorescence microscope.The osmotic water permeability of the FRT cells was measured using a calcein fluorescence quenching method.Results The results of electrophoresis after restriction enzyme cleavage and sequencing indicated that the EGFP-aquaporin-4 eukaryotic expression vectors were identified.The results of fluorescence microscope and Western blotting confirmed the FRT cells expressed aquaporin-4 after EGFP-aquaporin-4 transfection.The osmotic water permeability of the FRT cells transfeced with recombinant eukaryotic expression vectors of EGFP-aquaporin-4 was 1.65 times higher than the control.Conclusion The recombinant eukaryotic expression vector of EGFP-aquaporin-4 is established successfully and it is proved that aquaporin-4 can express on FRT plasma membranes by Lipofectamine 2000 transfection.The FRT cells transfeced with aquaporin-4 display high osmotic water transport performance across cell membranes.

关 键 词：AQUAPORIN-4 转染 FRT细胞 载体 

分 类 号：R361[医药卫生—病理学]