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目的:构建hERG钾离子通道蛋白(human ether-a-go-go-related gene potassium channel)shRNA表达载体质粒,获得稳定转染干扰质粒的人骨肉瘤细胞系MG-63、SOSP-9607。方法:将4对合成的寡核苷酸链退火形成双链,连接入pGPU6/GFP/Neo表达载体,并测序鉴定。使用脂质体法将重组的质粒转染至MG-63、SOSP-9607,通过G418筛选建立稳定转染的两种细胞系,采用免疫印迹(Western blot)技术检测hERG蛋白的表达。结果:测序结果证实shRNA与载体连接正确,免疫印迹实验证实hERG蛋白表达显著降低。结论:成功构建了hERG shRNA真核表达载体,获得了稳定表达hERG shRNA的人骨肉瘤细胞系MG-63和SOSP-9607。
OBJECTIVE: To construct the shRNA plasmid of human ether-a-go-go-related gene potassium channel, and to obtain the human osteosarcoma cell line MG-63, SOSP-9607 stably transfected with the interference plasmids. Methods: Four pairs of synthetic oligonucleotide strands were annealed to form a double strand, ligated into pGPU6 / GFP / Neo expression vector and sequenced. Recombinant plasmids were transfected into MG-63 and SOSP-9607 by lipofectamine. Stable transfected cell lines were established by G418 screening and hERG protein expression was detected by Western blot. Results: The sequencing results confirmed that the shRNA was ligated with the vector correctly, and the expression of hERG protein was significantly reduced by Western blotting. Conclusion: The eukaryotic expression vector of hERG shRNA was successfully constructed and the human osteosarcoma cell lines MG-63 and SOSP-9607 stably expressing hERG shRNA were obtained.