肿瘤特异性Survivin启动子驱动的FAK shRNA真核表达载体的构建

目的 构建肿瘤特异性Survivin启动子驱动的FAK shRNA真核表达载体.方法 PCR扩增Survivin启动子,构建重组载体pGEMT/pSurv并酶切、测序鉴定.体外化学合成编码FAK shRNA寡核苷酸链并退火互补成双链,构建真核表达载体pGenesil-FAK shRNA并酶切、测序鉴定.用Survivin启动子替换pGenesil-FAK shRNA中的U6启动子,重组为Survivin启动子驱动的FAK shRNA真核表达载体pGenesil-pSurv-FAK shRNA.将pGenesil-pSurvFAK shRNA转染至肝癌细胞系HepG2,观察转染效率.48h后应用RT-PCR法检测转染重组质粒后HepG2细胞中FAK mRNA的表达变化.结果 成功构建了肿瘤特异性Survivin启动子驱动的真核表达载体pGenesil-pSur-FAKshRNA,转染至肝癌细胞HepG2后可明显下调HepG2细胞中FAK mRNA表达水平.结论 以Survivin启动子驱动的FAK shRNA真核表达载体能够有效下调肝癌细胞FAK的表达,为进一步以靶向干扰FAK表达为主要手段的肝癌联合治疗提供实验依据.

Construction of FAK shRNA vector driven by the tumor-specific survivin promoter

Objective To construct a eukaryotic FAK shRNA expression vector driven by the tumor-specific survivin gene promoter and to evaluate its effect on HepG2 FAK mRNA expression.Methods The Survivin gene promoter was amplified by PCR,and then cloned to pGEMT vector and recombinated as pGEMT/pSurv,which was identified by double-enzyme digestion and sequencing.FAK shRNA sequences were designed and chemically synthesized,and then conjugated into the pGenesil-1 and recombined as pGenesil-FAK shRNA vectors,which were identified by double-enzyme digestion and sequencing.The U6 promoter in pGenesil-FAK shRNA vectors was then replaced by survivin gene promoter and recombined as pGenesil-pSurv-FAK shRNA vectors.The pGenesil-pSurv-FAK shRNA vectors were tranfected into HepG2 cell,and the transfection efficiency was observed by fluorescence microscope.After 48-hour transfection,the FAK mRNA expression level in HepG2 was evaluated by RT-RCR.Results The eukaryotic FAK shRNA expression vector pGenesil-pSurv-FAK shRNA driven by the tumor-specific survivin gene promoter was constructed successfully.The expression of FAK mRNA in HepG2 cell was down-regulated after transfection.Conclusion FAK shRNA expression vector driven by the tumor-specific Survivin gene promoter can downregulate the expression of FAK mRNA in HepG2,which may lay the foundation for further study of targeting FAK as an alternative treatment of HCC.