吉林大学学报(医学版)

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anoctamin 1在中国仓鼠卵巢细胞中的表达及其离子通道特性

郝 峰1,王 皓1,许会静1,方 青2,张 丽2,朱杭飞1,张雲乔1,鞠晓红3   

  1. 1. 吉林医药学院生物化学检验教研室,吉林 吉林 132013;2. 吉林医药学院实验中心,吉林 吉林 132013;3. 吉林医药学院病原教研室,吉林 吉林 132013
  • 收稿日期:2014-07-09 出版日期:2014-11-28 发布日期:2015-01-18
  • 通讯作者: 鞠晓红 (Tel:0432-64560542, E-mail:haof863@163.com) E-mail:haof863@163.com
  • 作者简介:郝 峰(1973-),男,吉林省辽源市人,副教授,细胞生物学博士,主要从 事氯离子通道调节剂筛选及其机制研究。
  • 基金资助:

    吉林省教育厅科研基金资助课题(2013-351);吉林省大学生创新创业训练计划资助课题(2013,2014);吉林医药学院大学生科研基金资助课题

Expression of anoctamin 1 in Chinese hamster ovary cells  and properties of its ion channel

HAO Feng1,WANG Hao1,XU Hui-jing1,FANG Qing2,ZHANG Li2,ZHU Hang-fei1,ZHANG Yun-qiao1,JU Xiao-hong3   

  1. 1.Department of Biochemistry Laboratory,Jilin Medical College,Jilin 132013,China;2.Department of Experimental Center,Jilin Medical College,Jilin 132013,China;2.Department of Etiology,Jilin Medical College,Jilin 132013,China)
  • Received:2014-07-09 Online:2014-11-28 Published:2015-01-18

摘要:

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。

关键词: anoctamin 1;离子通道;卵巢细胞, 中国仓鼠

Abstract:

Abstract:Objective To investigate the expression   of anoctamin 1 in Chinesehamster 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 ofanoctamin 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 wasused 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 functionalproperties 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.

Key words: anoctamin 1, ion channel, ovary cells, hamsters

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