柔嫩艾美耳球虫(Eimeria tenella)属于顶复器门原虫,严重危害养鸡业。顶膜抗原1(Apical membrane antigen-1,AMA1)是顶复器门原虫入侵宿主细胞前形成运动连接环的重要组成部分,其包含了DⅠ、DⅡ、DⅢ及信号肽四个结构域。为了探究AMA1不同结构域对球虫子孢子入侵的影响,本实验体外表达AMA1 DⅠ结构域,人工合成DⅢ和信号肽结构域,并制备相应抗体,对不同结构域进行抗体入侵抑制实验。结果显示,虫体入侵率都有明显降低,表明Et AMA1中的DⅠ、DⅢ、信号肽在虫体入侵过程中均发挥了一定作用。通过构建重组真核表达质粒p Bi FC-VN155-Et AMA1-DⅠ、p Bi FC-VC155-Et RON2,转染BHK细胞,利用间接免疫荧光(IFA)对其表达情况进行鉴定,结果检测到特异性的绿色荧光,表明构建的重组质粒p Bi FC-VN155-Et AMA1-DⅠ、p Bi FCVC155-Et RON2在真核细胞BHK中表达。通过Bi FC技术对Et RON2与Et AMA1-DⅠ相互作用关系进行验证,结果显示共转染的p Bi FC-VN155-Et AMA1-DⅠ和p Bi FC-VC155-Et RON2组、p Bi FC-b Jun VN173和p Bi FCb Fos VC155阳性对照组均出现特异性绿色荧光,而阴性对照组未激发出绿色荧光,说明Et AMA1-DⅠ和Et RON2之间存在互作关系。因此,推测Et AMA1-DⅠ与Et RON2发生互作形成复合体,在球虫入侵宿主细胞中发挥重要功能。已有研究表明,AMA1能与多个虫体蛋白形成复合物,是虫体成功入侵细胞的关键。但至今尚无AMA1与宿主蛋白互作蛋白的报道。通过构建的原核表达质粒GST-Et AMA1进行重组蛋白的表达与纯化,将GST-Et AMA1蛋白、GST蛋白和PBS分别与谷胱甘肽琼脂糖共孵育后,分别与DF-1细胞蛋白共孵育,洗脱液经SDS-PAGE分析筛选出差异蛋白条带并质谱分析,结果显示三者之间的条带明显不同,质谱分析蛋白质肽段与Uniprot中鸡蛋白质数据库进行Blast比对,初步获得14个Et AMA1胞外域与DF-1细胞之间存在相互作用的潜在蛋白,包括肌动蛋白、微管蛋白、鸟嘌呤核苷酸结合蛋白、核糖体蛋白以及一些保守蛋白等。以上研究结果为深入研究球虫子孢子入侵宿主细胞的分子机制提供理论基础。
Compared to other second messengers, calcium diffuses throughout the cytoplasm quite slowly, a property that favors its ability to act in a highly localized fashion. In contrast, the c AMP molecule is highly diffusibl...
Compared to other second messengers, calcium diffuses throughout the cytoplasm quite slowly, a property that favors its ability to act in a highly localized fashion. In contrast, the c AMP molecule is highly diffusible, but nevertheless can also be generated and detected in subcellular compartments to permit localized signaling events. This is thought to improve the specificity and energetic efficiency of the signal transduction process. This lecture will focus on the properties of localized c AMP signals in and around organelles such the primary cilium, and how these signals are modified by calcium. The pitfalls and challenges of monitoring signaling events in these tiny comparments using optical approaches will also be addressed.
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