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首页> 外文期刊>Chinese journal of digestive diseases >Construction of a recombinant adenovirus carrying endothelial nitric oxide synthase gene and its expression and control in esophageal smooth muscle cells
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Construction of a recombinant adenovirus carrying endothelial nitric oxide synthase gene and its expression and control in esophageal smooth muscle cells

机译:携带内皮一氧化氮合酶基因的重组腺病毒的构建及其在食管平滑肌细胞中的表达与调控

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OBJECTIVE: Nitric oxide (NO) is a major inhibitory neurotransmitter, and its deficiency plays an important role in the pathogenesis of motility disorders of the gastrointestinal tract, The present study was designed to generate a recombinant adenovirus containing the tetracycline (Tet)-regulated endothelial nitric oxide synthase (eNOS) gene and to detect the controllable expression of the gene in esophageal smooth muscle cells (ESMC).METHODS: The construction of the recombinant adenovirus was completed in three steps: (1) a Tet-responsible expression cassette was made by cloning the full-length cDNA encoding eNOS into a pTRE-Shuttle Vector, which can be regulated by tetracycline or its analogs, such as doxycycline (Dox); (2) the expression cassette was transferred to Adeno-X viral DNA to form a recombinant adenoviral plasmid (pAd-eNOS) by means of an in vitro ligation reaction; and (3) the Ad-eNOS was packaged into infectious adenoviral particles (Adeno-X-TRE-eNOS) by transfecting human embryonic kidney (HEK) 293 cells. Cultured ESMC were coinfected by Adeno-X-TRE-eNOS and regulation virus (Adeno-X Tet-off virus), and the Dox-regulated eNOS expression was detected by RT-PCR and western blot.
机译:目的:一氧化氮(NO)是一种主要的抑制性神经递质,其缺乏在胃肠道运动性疾病的发病机理中起着重要作用。本研究旨在产生一种含有四环素(Tet)调节的内皮细胞的重组腺病毒。一氧化氮合酶(eNOS)基因并检测该基因在食管平滑肌细胞(ESMC)中的可控表达。方法:重组腺病毒的构建分三个步骤完成:(1)制备了一个Tet负责表达的表达盒通过将编码eNOS的全长cDNA克隆到pTRE-Shuttle载体中,该载体可以由四环素或其类似物(例如强力霉素(Dox))调节; (2)通过体外连接反应将表达盒转移至腺病毒X病毒DNA上,形成重组腺病毒质粒pAd-eNOS。 (3)通过转染人胚肾(HEK)293细胞将Ad-eNOS包装入感染性腺病毒颗粒(Adeno-X-TRE-eNOS)中。培养的ESMC被Adeno-X-TRE-eNOS和调节病毒(Adeno-X Tet-off病毒)共同感染,并通过RT-PCR和western blot检测Dox调节的eNOS表达。

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