anoctamin 1在中国仓鼠卵巢细胞中的表达及其离子通道特性

摘要

Objective To investigate the expression of anoctamin 1 in Chinese hamster ovary cells (CHO)and to analyze the functional properties of its ion channel,and to provide experimental basis for study on the physiological function of calcium-activated chloride channel.Methods The whole sequence of anoctamin 1 was obtained by PCR technique and was subcloned into pcDNA3.1 to construct the expression vector pcDNA3.1-anoctamin 1 was transfected into CHO by liposome-mediated transfection and the CHO stably expressing anoctamin 1 were aquired by selection with zeocin. The expression and distribution of anoctamin 1 in CHO were measured by RT-PCR technique and inverted fluorescence microscope.The functional properties of anoctamin 1 were measured with halide-sensitive fluorescence proteins YFP-H148Q/I152L.The PBS buffer solution with calcimycin and high concentration of iodine ion was used as experimental group,andthe PBS buffer solution without calcimycin and high concentration of iodine ion was used as control group.Results The results of digestion and sequencing confirmed that anoctamin 1 was cloned into pcDNA3.1 by electrophoresis and blast. The results of RT-PCR and inverted fluorescence microscope indicated that anoctamin 1 was expressed in CHO. The results of I- influx as measured by halide-sensitive fluorescence proteins YFP-H148Q/I152L showed that anoctamin 1 had the more functional properties of trans-epithelial transporting I-,and the transporting speed in experimental group was higher than that in control group (P<0.05).Conclusion Anoctamin 1 can be expressed highly in the CHO;Anoctamin 1 expressed in CHO has the characteristics of calcium-activated chloride channel.%目的：探讨 anoctamin 1在中国仓鼠卵巢细胞（CHO）中的表达情况，分析其离子通道特性，为研究钙激活氯离子通道的生理功能提供实验依据。方法：PCR 方法获得 anoctamin 1编码区基因，构建 pcDNA3.1-anoctamin 1真核表达载体。脂质体转染 anoctamin 1至 CHO中，抗生素筛选获取稳定表达 anoctamin 1的 CHO株。应用 RT-PCR技术和倒置荧光显微镜检测 anoctamin 1于 CHO中的表达和分布情况。应用卤族元素敏感的黄色荧光蛋白双突变体 YFP-H148Q/I152L检测 anoctamin 1离子通道的功能。以加入 calcimycin和高浓度碘离子的PBS缓冲液的为实验组，未加入 calcimycin和高浓度碘离子的 PBS缓冲液的为对照组。结果：限制性内切酶酶切后的琼脂糖凝胶电泳和测序，目的基因 anoctamin 1克隆至真核表达载体 pcDNA3.1；RT-PCR和倒置荧光显微镜观察，CHO在mRNA和蛋白水平表达 anoctamin 1，且 anoctamin 1蛋白主要表达于 CHO膜上；荧光淬灭动力学实验，CHO表达的 anoctamin 1具有转运碘离子的作用，且实验组碘离子转运速度显著高于对照组（P＜0.05）。结论：anoctamin 1可高效表达于CHO膜；CHO表达的 anoctamin 1具有经典钙激活氯离子通道特性。