前列腺特异性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 plasmids with prostate specific antigen enhancer and promoter in gene expression regulation

目的:利用前列腺特异性抗原(pros-tate-specific antigen,PSA)组织特异性表达的特点,克隆出其上游增强子(prostate-specific antigen enhancer,PSAE)和启动子(prostate-specificantigen promoter,PSAP)片断,并对其表达效率和组织特异性表达进行初步研究。方法:从前列腺癌组织提取基因组DNA,并采用PCR方法分别扩增PSA增强子和启动子序列;利用报告基因pEGFP-1,分别构建含不同调控序列的表达载体;脂质体介导基因转染不同细胞并观察绿色荧光蛋白(GFP)的表达情况。结果:成功构建质粒pPSAE-EGFP、pPSAP-EGFP和pPSAE-PS-AP-EGFP;转染结果显示,pPSAE-PSAP-EGFP在前列腺癌细胞PC-3中的荧光强度明显高于pPSAP-EGFP,pPSAE-EGFP同样观察到荧光表达,说明PSA增强子能显著提高PSA启动子的转录能力,并具有单独调控基因表达的能力。结论:PSA增强子和启动子的共同调控可以提高基因表达的有效性和细胞特异性,为前列腺癌的临床基因治疗提供实验依据。

OBJECTIVE： To study the expression efficiency and specificity of prostate-specific antigen （PSA） enhancer and promoter in a possible targeted gene therapy scheme for prostate cancer. METHODS： Genomic DNA was obtained from prostate cancer tissues. PSA enhancer and pro motet sequences were amplified by PCR method, then the two fragments were cloned into plasmid pEGFP-1 to construct the expression vectors. After transfecting into different cell lines, the expression status was observed by the fluorecent microscopic examination method. RESULTS： The recombinant plasraids （pPSAE-EGFP, pPSAP-EGFP and pPSAE-PSAP EG- FP） were successfully constructed. In prostate cancer cell PC-3, pPSAE-PSAP EGFP expressed more GFP than that in pPSAP EGFP and the green fluorescence was also observed in pPSAE-EGFP, which showed that PSA enhancer not only no tably increased the transcription efficiency of PSA promoter but also had modulating ability singly used. CONCLUSION： The expression efficiency and specificity of gene can be inreased by PSA enhancer/promoter combination which should provide trial evidences for clinical gene therapy of prostate cancer.