反向前列腺特异性膜抗原增强子启动子调控重组质粒的构建及增强子调控活性的比较

Constructing Recombinant Plasmid with Prostate-specific Membrane Antigen Promoter and Reverse Enhancer and Comparing the Transcription Activity of Enhancers in Different Directions

目的探讨前列腺特异性膜抗原(PSM A)启动子和增强子调控目的基因表达的前列腺细胞特异性,了解增强子增强转录效率的能力。方法采用PCR方法从前列腺癌细胞DNA中分别扩增PSM A增强子序列,将其按不同方向亚克隆到含有报告基因——绿色荧光蛋白(GFP)的重组质粒载体pEGFP-PSM AP ro,脂质体介导基因转染不同癌细胞并观察GFP在所转染的细胞的表达情况。结果成功构建质粒pEGFP-PSM AE-P和pEGFP-PSM AE(r)-P,质粒转染结果显示PSM A表达阳性的LNC aP细胞中存在GFP的特异有效表达,增强子序列能使基因转录效率提高30倍,不同方向的增强子序列在增强转录效率方面具有同样效应。结论反向PSM A增强子启动子调控GFP表达的重组质粒的构建证明该调控序列具有前列腺细胞特异性,增强子具有增强启动子转录效率的功能且没有方向性。

Objective To assess the specificity of prostate-specific membrane antigen（PSMA） promoter and enhancer in controlling gene expression and to compare the activity of enhancers in different directions for choosing the most suitable prostate-specific PSMA controlling elements. Methods PSMA enhancer gene was amplified with PCR, then the enhancer gene was subcloned into the expressing vector pEGFP-PSMAPro reversely to construct the recombinant plasmid pEGFP-PSMAE（r）-p, which was transfected into different cell lines such as LNCaP, PC-3,MCF-7,A549. Green fluorescent protein（GFP） expression was observed and compared to other recombinants constructed previously. Results The recombinant plasmid with reverse enhancer was successfully constructed, the PSMA promoter and enhancer showed modulating activity in PSMA-expressed cell line uniquely. PSMA enhancer could increase 30-fold transcriptional activity over the basal level achieved by PSMA promoter alone, and no impact of the direction on the activity of enhancer was noted. Conclusion PSMA promoter/enhancer is specific to PSMA-expressed cells. The transcriptional activity of reverse enhancer is similar to that of enhancer. PSMA promoter/enhancer has the potential for use in targeted gene therapy of prostate adenocarcinoma.