Expression and purification of mouse Ttyh1 fragments as antigens to generate Ttyh1-specific monoclonal antibodies
Expression and purification of mouse Ttyh1 fragments as antigens to generate Ttyh1-specific monoclonal antibodies
复制标题
表达和纯化小鼠 Ttyh1 片段作为抗原，生成 Ttyh1 特异性单克隆抗体
DOI：
10.1016/j.pep.2016.09.013
复制
发表时间：
2017-02-01
影响因子：
1.6
通讯作者：
Han, Hua
中科院分区：
文献类型：
--
作者：
Cao, Xiu-Li;Zhang, Xing;Han, Hua
Ttyh1是果蝇Tweety的小鼠同源物，被预测为五次跨膜蛋白。Ttyh1 mRNA在小鼠脑组织中以限制性模式表达，并在人脑胶质瘤细胞中表达。Ttyh1蛋白可能作为一个大电导氯离子通道，然而，Ttyh1在正常神经发育和肿瘤发生中的作用在很大程度上是未知的，至少部分是由于缺乏有效的抗体。在这里，我们报告的表达E。大肠杆菌和纯化的两个重组Ttyh 1蛋白片段对应于预测的胞外结构域和小鼠Ttyh 1的羧基末端之一。利用这些Ttyh1蛋白产物，通过使用常规杂交瘤技术建立了一组针对小鼠Ttyh1蛋白的单克隆抗体（mAb）。利用胚胎脑组织和培养的小鼠神经干细胞（NSCs）进行Western印迹和免疫荧光分析，基于其活性确定抗Ttyh1 mAb的特异性。我们还表明，小鼠Ttyh1蛋白表达在培养的神经干细胞，最有可能在膜和细胞质。在小鼠胚胎脑中，Ttyh 1蛋白似乎特异性地表达在脑室区的顶端边缘作为点状结构，如通过使用免疫荧光测定的。本研究为进一步探讨Ttyhl在小鼠体内的生物学功能和病理学意义提供了有用的工具。(C)2016 Elsevier Inc. All rights reserved.
Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1. With these Ttyh1 protein products, a set of monoclonal antibodies (mAbs) against the mouse Ttyh1 protein was established by using conventional hybridoma techniques. The specificity of the anti-Ttyh1 mAbs was determined based on their activities in Western blotting and immunofluorescent analysis using embryonic brain tissues and cultured mouse neural stem cells (NSCs). We also show that the mouse Ttyh1 protein was expressed in cultured NSCs, most likely in membrane and cytoplasm. In mouse embryonic brains, it appeared that the Ttyh1 protein was specifically expressed in the apical edge of the ventricular zone as puncta-like structures, as determined by using immunofluorescence. Taken together, our study provided a useful tool for further exploration of the biological functions and pathological significance of Ttyhl in mice. (C) 2016 Elsevier Inc. All rights reserved.