摘要
目的建立稳定表达TRPA1的HEK-293T细胞模型,并鉴定模型构建是否成功。方法构建TRPA1真核表达质粒,采用脂质体转染法将其转入HEK-293T细胞中,经G418筛选稳定表达株,采用RT-PCR、免疫组化技术,检测HEK-293T细胞TRPA1基因的转录和蛋白的表达。结果经酶切、测序证明,TRPA1基因的真核重组表达质粒已成功构建;PCR、免疫组化检测结果表明,将此重组质粒转入HEK-293T细胞可稳定表达TRPA1基因。结论成功构建了能稳定表达TRPA1通道的HEK-293T细胞株,为体外研究TRPA1生理病理功能和筛选相关TRPA1通道调节剂奠定了基础。
Aim To establish a HEK-293 T cell line model stably expressing TRPA1 channel,and to verify the successful establishment of the model.Methods The eukaryotic expression plasmid of TRPA1 was constructed and transfected into HEK-293 T cells by liposome transfection method,the stable expression strain was screened by G418,and the transcription and protein expression of TRPA1 gene in HEK-293 T cells were detected by RT-PCR and immunohistochemical techniques. Results After restriction enzyme digestion and sequencing,it proved recombinant cloning plasmid of TRPA1 gene was successfully constructed; the results of PCR and immunohistochemistry showed that this recombinant plasmid could be transferred into HEK-293 T cells with TRPA1 gene stable expression.Conclusions A HEK-293 T cell line with stable expression of TRPA1 channel is successfully constructed,which lays the foundation for studying the physiological and pathological functions of TRPA1 in vitro and screening of relevant TRPA1 channel regulators.
引文
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