组织特异性启动子SM22α逆转录病毒载体的构建和效率

Construction and Identification of a Tissue-specific Promoter SM22alpha-regulatory Recombinant Retrovirus Vector

目的克隆小鼠SM22α基因的启动子,检测启动子特异性及效率。方法构建重组慢病毒载体,提纯病毒并感染细胞。结果序列分析表明,PCR扩增片段包含有完整的启动子元件和重要的功能框,成功克隆SM22α启动子。获得含特异SM22α启动子的重组慢病毒,滴度为1×108TU/mL。重组病毒分别感染血管平滑肌细胞(VSMCs)与非血管平滑肌细胞(NSMCs)。与NSMCs相比,在VSMCs中荧光强度明显增高(P<0.05);与巨细胞病毒(CMV)启动子相比,含SM22α启动子的病毒感染效率无明显差异(P>0.05),但诱导绿色荧光蛋白表达强度约为CMV启动子的80%。结论 SM22α启动子具有高度的平滑肌细胞特异性;诱导蛋白表达的能力较强。

Objective To clone SM22alpha gene promoter,and test its specificity and efficiency.Methods By using PCR technique,SM22alpha promoter was constructed.The viruses were purified and transfected into vascular smooth muscle cells（VSMCs）.Results Sequencing analysis showed that the amplified fragment contained the complete promoter elements and the important function of box,and SM22alpha promoter was successfully cloned.The titer of recombinant lentiviruses was 1×108 TU/mL.As compared with non-smooth muscle cells（NSMCs）,the fluorescence intensity in VSMCs was significantly increased（P0.05）.There was no significant difference in the transfection efficiency between the SM22alpha promoter and CMV promoter.But the fluorescence intensity induced by SM22alpha promoter was only 80% of that induced by CMV promoter.Conclusion SM22alpha promoter of SMCs owns a high degree of specificity and has a strong ability to induce protein expression.