阿片受体μ-Myc-pIRES2-EGFP的构建及其在CHO细胞中的表达

Construction of μ-Myc-pIRES2-EGFP and its expression in CHO

目的:构建带Myc标签的大鼠μ型阿片受体的pIRES2-EGFP表达质粒(μ-Myc-pIRES2-EGFP),并实现其在CHO细胞中的表达。方法:以含大鼠全长μ阿片受体基因的μ-pMD20T载体为模板,通过引物设计和扩增,在μ基因C端引入Myc标签,AT克隆至pMD20-T载体中,测序鉴定,经酶切连接克隆入pIRES2-EGFP中,将获得的μ-Myc-pIRES2-EGFP转染入CHO细胞中,应用荧光显微镜观察EGFP表达情况,并用细胞免疫荧光和蛋白印迹检测μ-Myc的表达。结果:测序及酶切结果表明获得带Myc标签的μ基因,构建入pIRES2-EGFP表达质粒中,在荧光显微镜下,转染细胞可以观察到绿色荧光,应用免疫荧光和蛋白印迹观察到目的基因表达。结论:成功构建了μ-Myc-pIRES2-EGFP表达质粒,并在CHO细胞中实现了高效表达。

Objective：To construct the μ-Myc-pIRES2-EGFP plasmid and investigate its expression in CHO. Methods：μ gene was tagged with a 10-residue Myc epitope through PCR. Then the μ gene with Myc epitope was inserted into pIRES2-EGFP to form μ-Myc-pIRES2-EGFP recombinant eukaryotic plasmid. The μ-Myc-pIRES2-EGFP plasmid was transfected into CHO using Lipofectamine2000. The expression of μ-Myc-pIRES2-EGFP was examined by the fluorescence microscopy and Western blot. Results：The sequence of μ opioid receptor with Myc epitode was inserted into pIRES2-EGFP and confirmed by sequencing. High expression of EGFP and μ gene with Myc epitope was detected in CHO after μ-Myc-pIRES2-EGFP plasmid transfection. Conclusions：μ-Myc-pIRES2-EGFP was cloned correctly and expressed in high level in CHO cells.