Expression of anoctamin 1 in Chinese hamster ovary cells and properties of its ion channel / 吉林大学学报(医学版)

ABSTRACT

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.