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目的构建由Survivin启动子调控的EGFP基因真核表达质粒,并检测其在人肺腺癌A549细胞中的特异性表达。方法以A549细胞基因组DNA为模板,PCR扩增Survivin启动子,替换pEGFP-C1载体中的CMV启动子,构建携带Survivin启动子的pEGFP-C1真核表达质粒pEGFP-C1/Surp,转染A549细胞及人胚肺成纤维细胞MRC-5,在荧光显微镜下观察EGFP的表达。结果重组表达质粒pEGFP-C1/Surp经双酶切和测序鉴定,证明构建正确。转染A549细胞和MRC-5细胞后,在荧光显微镜下可见A549细胞有较强的绿色荧光,而MRC-5细胞则未见绿色荧光。结论已成功构建以Survivin启动子为调控序列、EGFP为标示蛋白的真核表达质粒,且具有较强的肿瘤特异性启动活性,为进一步开发肿瘤靶向性基因治疗载体奠定了基础。
Objective To construct the eukaryotic expression plasmid of EGFP gene regulated by Survivin promoter and detect its specific expression in human lung adenocarcinoma A549 cells. Methods The Survivin promoter was amplified by PCR using the genomic DNA of A549 as a template. The CMV promoter in pEGFP-C1 vector was replaced by the recombinant plasmid pEGFP-C1 / Surp carrying Survivin promoter. The transfected A549 cells And human embryonic lung fibroblasts MRC-5, observed under a fluorescence microscope EGFP expression. Results The recombinant plasmid pEGFP-C1 / Surp was confirmed by double enzyme digestion and sequencing. After transfected A549 cells and MRC-5 cells, A549 cells showed strong green fluorescence under fluorescence microscope, while no fluorescence was observed in MRC-5 cells. Conclusion The eukaryotic expression plasmid with Survivin promoter as regulatory sequence and EGFP as marker protein has been successfully constructed and has strong tumor-specific promoter activity, which lays the foundation for further development of tumor-targeted gene therapy vector.