肿瘤特异的sea基因真核表达质粒的构建及在肺癌细胞中的功能研究

Construction of Tumor-specific Eukaryotic Expression Vector of sea Gene and Its Function in Human Lung Cancer A549 Cells

构建Survivin启动子调控的葡萄球菌肠毒素A(SEA)的真核表达质粒,检测其在人肺腺癌A549细胞中的特异性表达以及表达产物的超抗原活性。以PCR法扩增sea基因,以含有Survivin启动子为调控序列的pGL-S-RED质粒为基础,构建pGL-S-SEA真核表达质粒。用脂质体转染A549细胞,以RT-PCR法检测sea基因表达水平。制备健康献血者PBMC,用pGL-S-SEA质粒转染A549细胞的上清液和细胞裂解液进行PBMC刺激试验,以MTT法检测其促细胞增殖效应。结果显示,成功构建Survivin启动子调控的真核表达质粒pGL-S-SEA。重组质粒在A549细胞中启动了sea基因的表达,其转录强度为内参(GADPH基因)的65.96%,在对照MRC-5细胞中无明显表达。该质粒转化A549细胞的裂解液具有促进人PBMC增殖活性、上清液无明显促PBMC增殖作用,裂解液组、上清液组和PHA阳性对照组的刺激指数分别为1.29、0.95和1.58。结论成功构建pGL-S-SEA质粒,其在A549细胞的表达产物具有诱导人PBMC增殖的超抗原活性,为下一步将其作为基因疫苗治疗肺癌奠定了基础。

The research aimed to construct eukaryotic expression vector of sea gene modulated by survivin promoter, to identify the expression of the plasmid in A549 ceils, and the superantigen effect of recombinant SEA. The sea gene was amplified by PCR, then the pGL-S-SEA was constructed based on the pGL-S-RED plasmid which contained surviving promoter as modulating sequence. The pGL-S-SEA plasmid was transfected into A549 and MRC-5 cells by liposome and RT-PCR was used to detect the expression of sea gene in A549 cells and MRC-5 cells. Also,the stimulation test of PBMC involved in to find out study the superantigen effect of recombinant SEA expressed in A549 cells. Results showed that,the eukaryotic expression vector was successfully constructed and the sea gene was expressed in lung cancer A549 cells,with the transcription strength rate of 65.96% , but no expression in normal control MRC-5 cells. The lysate of A549 cells can stimulate the PBMC activity,but without this activity of the supernatant. Therefore,the expression product of A549 cells that can induce the PBMC activation,may be useful in transcriptional targeting gene therapy of human lung cancer.