Survivin启动子调控sea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

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

目的构建Survivin启动子调控的葡萄球菌肠毒素A(SEA)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。方法以金黄葡萄球菌(ATCC25923)基因组为模板,PCR扩增sea基因。用sea基因替代以Survivin启动子为调控序列的pGL-S-RED质粒中的Red基因,构建真核表达质粒pGL-S-SEA。酶切及测序鉴定后,用脂质体转染A549细胞和MRC-5人胚肺成纤维细胞,RT-PCR检测两种细胞sea基因的转录情况。结果Survivin启动子调控的、以超抗原SEA编码序列CDS为目的基因的真核表达质粒pGL-S-SEA构建正确,并在A549细胞中启动了sea基因的转录,其强度为内参基因(GAPDH)的65.96%,而在MRC-5细胞对照中未检测到sea基因的转录。结论已成功构建Survivin启动子调控的sea基因真核表达质粒,Survivin启动子可在转录水平特异性地调控sea基因在A549细胞内表达,为下一步以其作为基因疫苗治疗肺癌奠定了基础。

Objective To construct a eukaryotic expression vector for staphylococcal enterotoxin A （SEA） gene modulated by survivin promoter and identify its specific expression in human lung cancer A549 cells. Methods The sea gene was amplified by PCR from the genome of Staphylococcus aureus ATCC25923. The Red gene in plasmid pGL-S-RED containing survivin promoter as a modulating sequence was substituted with sea gene, based on which a eukaryotic expression vector pGL-S-SEA was constructed, identified by restriction analysis and sequencing, and transfected to A549 and MRC-5 cells in mediation of liposome. The transcription of sea gene was tested by RT-PCR. Results The eukaryotic expression vector pGL-S-SEA was constructed correctly, and sea gene was transcribed in A549 cells. The ratio of transcription level of sea gene to that of GAPDH gene as internal reference was 65. 96%. However, no transcription of sea gene was observed in MRC-5 cells. Conclusion A eukaryotic expression vector for sea gene modulated by survivin promoter was successfully constructed, and survivin promoter specifically modulated the expression of sea gene in A549 cells at transcription level, which laid a foundation of therapy of lung cancer with the constructed recombinant plasmid as a gene vaccine.