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首页> 外文期刊>World Journal of Gastroenterology >Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system.
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Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system.

机译：猪水通道蛋白1水通道的克隆和特性在胃肠道系统中广泛表达。

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AIM: To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system. METHODS: A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells. RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot. RESULTS: An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut. Northern blot showed a single 3.0 kb transcript in selected digestive organs. pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Immunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins. CONCLUSION: pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.
机译：目的：克隆并鉴定胃肠道系统中的猪水通道蛋白（AQPs）。方法：采用基于PCR的克隆策略和RACE技术，从逆转录猪肝cDNA中克隆全长AQP编码序列。停止流光散射和基于YFP的荧光方法用于测量红细胞和稳定转染的CHO细胞的渗透水渗透率。 RT-PCR，Northern印迹和免疫组织化学被用于确定胃肠道表达和克隆的AQPs的本地化。通过Western印迹分析转染的细胞和红细胞中的蛋白质表达。结果：从肝脏mRNA克隆了一个编码271个氨基酸的猪水通道蛋白的813 bp cDNA（命名为pAQP1）（pAQP1与人AQP1的同源性为93％，包含两个在AQP家族中保守的NPA基序，一个用于N联糖基化的共有序列） ，在半胱氨酸191处有一个汞敏感位点）。 RT-PCR分析显示pAQP1 mRNA在猪消化腺和肠道中大量表达。 Northern印迹显示在选定的消化器官中有一个3.0kb的转录本。 pAQP1蛋白通过免疫过氧化物酶定位在小肠中央乳腺，唾液腺微血管以及肝内胆管上皮。在稳定转染了pAQP1 cDNA的猪红细胞和CHO细胞中检测到HgCl2抑制的高渗透水渗透性。猪红细胞和pAQP转染的CHO细胞的免疫印迹分析显示未糖基化的28 ku条带和较大的糖基化蛋白。结论：pAQP1是迄今为止可以分子鉴定的第一种猪水通道蛋白。 pAQP1在猪消化器官的上皮和内皮中的广泛分布可能表明通道介导的水运输在液体分泌/吸收以及消化系统的消化功能和病理生理中起着重要作用。

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