前列腺组织特异性启动子介导的TAT-EGFP的靶向表达

Targeting expression of TAT-EGFP driven by the prostate specific antigen promoter

目的构建由前列腺特异抗原(PSA)启动子介导、表达带有蛋白转导结构域序列(TAT)的增强型绿色荧光蛋白(EGFP)融合蛋白pPSA-TAT-EGFP表达载体,使TAT所携带的目的蛋白能够在靶组织中高效表达,提高其特异性及表达效率。方法以pEGFP-1载体为模板,设计包含TAT序列的引物,PCR技术扩增含有酶切位点的小片段SalI-TAT-EGFP-Not I,经胶回收纯化,连接到T克隆载体。双酶切及测序鉴定后,提取质粒、双酶切后胶回收得到目的小片段。含有PSA启动子的pPSA-EGFP载体经Sal I和Not I双酶切后,胶回收含有PSA启动子序列的大片段。过夜连接目的小片段与PSA启动子大片段,得到pPSA-TAT-EGFP表达载体。转化、筛选阳性克隆后酶切与测序鉴定。将构建载体分别转染前列腺癌细胞和大肠癌细胞检测其表达情况。结果成功构建pPSA-TAT-EGFP表达载体。转染结果显示其能够在前列腺癌细胞中特异性表达。结论引入了PSA组织特异性启动子的TAT所携带的目的蛋白能够在前列腺癌组织中表达。

Objective We constructed an expression vector containing EGFP fused to the viral TAT transduction domain and driven by the prostate-specific antigen promoter （pPSA-TAT-EGFP） by gene cloning technology. The targeting expression of pPSA-TAT-EGFP was examined in prostate cancer cells and colon ceils. Methods The vector pEGFP-1 served as the framework, and the fragment with the TAT sequence and EGFP was produced by using polymerase chain reaction. After digestion with Sall and Nod enzymes, the fragment was inserted into the T cloning vector. The EGFP expression plasmid driven by the PSA promoter （pPSA-EGFP） was digested overnight with SalI and Nod, and a large fragment containing the PSA promoter was purified by gel purification. The TAT-EGFP, cut with Sail and NotI from the T-vector, was linked with the large fragment with the PSA promoter sequence and generated the pPSA-TAT-EGFP construct. The construct was confirmed by restriction enzyme digestion and DNA sequencing, and tested by transfection assay. Result The expression vector pPSA-TAT-EGFP was constructed. It was expressed in prostate carcinoma cells, but was not detected in colon cells. Conclu^on A slightly higher expression was observed in cells transfected with EGFP fused to TAT compared to the EGFP control ptasmid in prostate cancer cells, but no detectable expression was shown in colon cells.