靶向大鼠AT1-R基因的短发夹RNA重组腺病毒载体的构建及鉴定

Construction of recombinant adenovirus expressing the short hairpin RNA of rat angiotensin Ⅱreceptor subtype 1

目的利用AdMax腺病毒载体系统构建大鼠AT1-shRNA腺病毒载体并在293细胞中扩增制备重组病毒。方法自先期构建的pGenesil-1-AT1-shRNA真核表达载体中用RT-PCR法扩增出AT1-shRNA片段,将其克隆入PUC18质粒中。酶切构建好的pUC18-AT1-shRNA载体并克隆进入穿梭质粒pDC316中,将构建好的穿梭质粒pDC316-AT1-shRNA载体和骨架病毒pBHGloxΔ1,3Cre共转染293细胞,包装成重组的病毒颗粒,荧光显微镜观察绿色荧光表达。结果经限制性内切酶、PCR检测和GFP表达证实成功地构建了携带AT1-shRNA的重组腺病毒载体并制备出高滴度重组病毒。结论成功地构建了携带AT1-shRNA片段的重组腺病毒载体,为进一步抗血压研究奠定了基础。

Objective To construct the adenovirus vector expressing the shRNA of rat angiotensin Ⅱ receptor and amplify the adenovirus vector in 293 cells. Methods The rat AT1-shRNA segments was obtained from plasmid pGenesil-1-AT1-shRNA which was constructed at an earlier date by RT-PCR and then was inserted into linearized PUC18 plasmid. After having been screened, the constructed PUC18 ATI-shRNA plasmid was digested with restriction endonucleases and cloned into the shuttle plasmid pDC316 to form the pIX;316-AT1 shRNA vector. The pIX;316-ATI-shRNA plasmid was cotransfected with genomic plasmid pBHGlox△1, 3Cre into 293 cells to package the recombinant adenovirus. The recombinant adenovirus was transfected into rat glioma C6 cells, and the green fluorescence protein expression was detected. Results Recombinant adenoviral vector Ad-AT1-shRNA was constructed successfully, which was confirmed by restriction enzyme digestion, PCR and GFP exprexsion. Conclusion The recombinant adenoviral vector carrying AT1-shRNA is successfully constructed, and will lay the foundation of application of gene therapy to antihypertension.