本文選題：aquaporin-　切入點(diǎn)：轉(zhuǎn)染　出處：《吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2013年02期 　論文類型：期刊論文

【摘要】：目的:構(gòu)建以增強(qiáng)型綠色熒光蛋白(EGFP)為報(bào)告基因的重組真核表達(dá)載體pEGFP-aquaporin-4,以EGFP示蹤aquaporin-4在Fisher大鼠甲狀腺濾泡上皮細(xì)胞(FRT細(xì)胞)中的表達(dá)和定位,為進(jìn)一步研究aquaporin-4提供實(shí)驗(yàn)依據(jù)。方法:應(yīng)用RT-PCR方法獲得aquaporin-4編碼區(qū)基因,克隆入真核表達(dá)載體pEGFP-N1。經(jīng)酶切和測(cè)序鑒定證實(shí)為aquaporin-4后,Lipofectamine 2000脂質(zhì)體轉(zhuǎn)染重組EGFP-aquaporin-4融合蛋白真核表達(dá)載體至FRT細(xì)胞中,倒置熒光顯微鏡下觀察aquaporin-4在FRT細(xì)胞中的表達(dá)和分布,并應(yīng)用Western blotting法檢測(cè)aquaporin-4蛋白的表達(dá)。同時(shí)檢測(cè)轉(zhuǎn)入FRT細(xì)胞內(nèi)鈣黃綠素的相對(duì)熒光強(qiáng)度以判斷FRT細(xì)胞水通透性的狀況。結(jié)果:EcoRⅠ和KpnⅠ雙酶切和測(cè)序重組載體結(jié)果證實(shí)目的基因aquaporin-4成功克隆到真核表達(dá)載體pEGFP-N1中。熒光顯微鏡下觀察融合EGFP的aquaporin-4主要在FRT細(xì)胞膜上表達(dá);Western blotting結(jié)果證實(shí)脂質(zhì)體轉(zhuǎn)染重組載體的FRT細(xì)胞表達(dá)aquaporin-4。轉(zhuǎn)染重組EGFP-aquaporin-4融合蛋白真核表達(dá)載體的FRT細(xì)胞水通透性顯著高于未轉(zhuǎn)染的FRT細(xì)胞,其水通透性是未轉(zhuǎn)染FRT細(xì)胞的1.65倍。結(jié)論:成功構(gòu)建aquaporin-4真核表達(dá)載體,證實(shí)其可在FRT細(xì)胞中表達(dá),并具有明顯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，

本文編號(hào)：1574677