Survivin启动子调控的EGFP基因真核表达质粒的构建及其在肺癌细胞中的特异性表达

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。

Construction of Eukaryotic Expression Vector for EGFP Gene Modulated by Survivin Promoter and Its Specific Expression in Human Lung Cancer Cells

Objective To construct a eukaryotic expression vector for enhanced green fluorescent protein(EGFP)gene modulated by survivin promoter and identify its specific expression in human lung cancer A549 cells. Methods Survivin promoter was amplified by PCR using the genomic DNA of A549 cells as a template,with which the CMV promoter in pEGFP-C1 vector was substituted. The constructed recombinant plasmid pEGFP-C1 / Surp was transfected to A549 and MRC-5 cells,and the expression of EGFP was observed by fluorescent microscopy. Results Restriction analysis and sequencing proved that recombinant plasmid pEGFP-C1 / Surp was constructed correctly. Strong green fluorescence was observed by microscopy in the A549 cells transfected with recombinant plasmid pEGFP-C1 / Surp. However,no green fluorescence was observed in transfected MRC-5 cells. Conclusion A eukaryotic expression vector for EGFP gene modulated by survivin promoter was successfully constructed,and survivin showed strong tumor-specific promoting activity. It laid a foundation of further development of vector for gene therapy targeting tumors.