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首页> 外文期刊>Bioengineered >Generation of a MDCK cell line with constitutive expression of the Enteropathogenic E. coli effector protein Map as an in vitro model of pathogenesis
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Generation of a MDCK cell line with constitutive expression of the Enteropathogenic E. coli effector protein Map as an in vitro model of pathogenesis

机译：具有肠致病性大肠杆菌效应蛋白图的组成型表达的MDCK细胞系的生成，作为发病机理的体外模型

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Enteropathogenic E. coli (EPEC) cause diarrhea and are the major cause of mortality in developing countries. EPEC use a type III secretion system to deliver effector proteins into the host epithelial cells. To understand the functions of these effectors, majority of studies on EPEC pathogenesis have relied on infections of animals or cell lines with wild type strains of EPEC or mutant strains deficient in one or more effectors. While these studies have provided valuable data, it can be difficult to assess functions of an individual effector in the presence of other EPEC effectors. Recent studies have reported the use of transient transfections with plasmids encoding various EPEC effectors into different cell lines. However, variable transfection efficiencies and expression levels of the effector proteins coupled with their expression for relatively short periods of time pose a problem if the long term effects of these effectors need to be examined. We have generated a MDCK cell line with constitutive expression of the EPEC effector Map (Mitochondrial associated protein) for efficient stable expression of EGFP-tagged Map. We observed that the constitutive expression of Map increased the permeability of charged and non-charged molecules. We also generated polyclonal antibodies against Map and checked for their specificity in MDCK-Map expressing cells. Map has been reported to contribute to the onset of diarrhea but the underlying mechanism is yet to be identified. The MDCK-Map cell line and the anti-Map antibodies generated by us can be used for in vitro studies to examine the role of Map in EPEC pathogenesis.
机译：肠致病性大肠杆菌。大肠杆菌（EPEC）引起腹泻，是发展中国家的主要死因。 EPEC使用III型分泌系统将效应蛋白输送到宿主上皮细胞中。为了了解这些效应子的功能，有关EPEC发病机理的大多数研究都依赖于野生型EPEC株或缺乏一种或多种效应子的突变株对动物或细胞系的感染。尽管这些研究提供了有价值的数据，但在其他EPEC效应子存在的情况下，可能难以评估单个效应子的功能。最近的研究报道了使用编码各种EPEC效应子的质粒瞬时转染到不同细胞系中的用途。然而，如果需要检查这些效应子的长期效应，那么效应蛋白的可变转染效率和表达水平及其在较短时间内的表达就会带来问题。我们已经生成了具有EPEC效应子图（线粒体相关蛋白）的组成型表达的MDCK细胞系，以有效稳定地表达EGFP标签的图谱。我们观察到，Map的本构表达增加了带电和不带电分子的渗透性。我们还产生了针对Map的多克隆抗体，并检查了它们在MDCK-Map表达细胞中的特异性。据报道，Map有助于腹泻的发作，但其潜在机制尚待确定。我们产生的MDCK-Map细胞系和抗Map抗体可用于体外研究，以检查Map在EPEC发病机理中的作用。

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