前列腺特异性膜抗原启动子增强子调控重组质粒的构建及鉴定

Construction and identification of recombinant plasmid with prostate-specific membrane antigen promoter and enhancer in controlling gene expression

目的探讨前列腺特异性膜抗原(PSMA)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PSMA启动子和增强子序列,先后克隆到含有报告基因绿色荧光蛋白(GFP)的质粒载体,脂质体介导基因转染不同癌细胞并观察GFP在不同细胞的表达情况。结果成功构建质粒pEGFPPSMAPro和pEGFPPSMAEP,转染结果显示PSMA表达阳性的LNCaP细胞中存在GFP的有效表达,且pEGFPPSMAEP的调控转录能力较pEGFPPSMAPro强20倍。结论PSMA启动子增强子调控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,增强子能够明显增强启动子的转录效率,增加了目的基因的表达水平。PSMA启动子和增强子的共调控可以保证基因表达的强度和细胞特异性,为前列腺癌基因靶向性治疗研究提供实验依据。

Objective To study the specificity of prostate-specific membrane antigen (PSMA)promoter and enhancer in controlling gene expression. Methods PSMA promoter and enhancer genes were amplified using PCR method,then the promoter gene and enhancer gene were separately cloned into expressing vector pEGFP-1 to construct the recombinant plasmids (pEGFP-PSMA_ Pro and pEGFP-PSMA_ EP),which were transfected into different cell lines such as LNCaP,PC-3,MCF-7 and A549.The green fluorescent protein (GFP) expression was observed. Results The construction of the recombinant plasmids was successful and PSMA promoter and enhancer uniquely showed modulating activity in PSMA positive cell line.Modulating activity of PSMA promoter and enhancer showed 20 times stronger than that of PSMA promoter. Conclusions PSMA promoter possesses PSMA positive cell specificity,as well as prostatic tissue.PSMA enhancer can increase the activity of promoter obviously.PSMA promoter and enhancer are potential to targeted gene therapy of prostate adenocarcinoma.