前列腺特异性启动子rPB的改造及其载体质粒的构建和活性测定

Construction and activity determination of modified prostatic specific promoters rPB-DRs

目的 构建一个前列腺特异性的、可启动针对雄激素非依赖性前列腺癌的基因治疗的改良启动子并测定其活性。方法 选择前列腺特异性启动子rPB ,通过两次PCR对其进行改造,用维甲酸反应元件(RARE)替代雄激素反应元件(ARE)或直接插入RARE ,构建成为不同拷贝条件的rPB DR ,并与质粒连接。转染细胞后,进行荧光素酶活性测定。结果 通过电泳鉴定及基因测序,证实构建成功。结论 根据实验设计,构建出的rPB DR应具有在维甲酸的调控下,针对雄激素非依赖性前列腺癌特异性启动基因治疗的功能,现构建成功不同拷贝条件的rPB DR ,证实实验设计构想,同时通过活性测定比较各种拷贝条件的rPB DR启动子活性的优劣,为下一步与治疗基因的连接及功能测定奠定基础。

Objective To construct the prostatic specific promoter used to induce the gene therapy aiming at androgen independent prostatic carcinoma and evaluate the activity. Methods Through two steps of polymerase chain reaction (PCR), androgen response elements (AREs) in rPB were replaced by synthetic retinoic acid response elements (RAREs). Then the modified rPBs were connected with the plasmid. The activity was evaluated. Results Constructions were confirmed to be successful by electrophoresis and gene sequencing. Conclusion Modified rPB should be the prostatic specific promoter which can induce the gene therapy aiming at androgen-independent prostatic carcinoma controlled by retinoic acid. After being constructed, the modified rPBs were used to confirm our ideas about the experiment design and the capability inducing the gene expression of different modified rPBs was compared.