摘要
目的利用AdMax腺病毒载体系统构建大鼠血管紧张素转换酶-短发夹RNA(ACE-shRNA)腺病毒载体并在人胚胎肾-293细胞中扩增制备重组病毒。方法自先期构建的pGenesil-1-ACE-shRNA真核表达载体中用反转录聚合酶链反应(RT-PCR)法扩增出ACE-shRNA片段,并克隆进入穿梭质粒pDC316中,将构建好的穿梭质粒pDC316-ACE-shRNA载体和骨架病毒pBHGlox-E1,3Cre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,聚合酶链反应(PCR)检测和绿色荧光蛋白(FGFP)表达证实成功地构建了携带ACE-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ACE-shRNA片段的重组腺病毒载体,为进一步利用基因沉默技术抗高血压的研究奠定了基础。
Objective To construct an adenovirus vector expressing shRNA of rat angiotensin-converting enzyme （ACE） and amplify the adenovirus vector in HEK-293 cells. Methods The rat ACE-shRNA segments was obtained from plasmid pGenesil-1-ACE-shRNA that was constructed at an earlier date by RT-PCR and then was cloned into the shuttle plasmid pDC316 to form the pDC316-ACE-shRNA vector. The pDC316-ACE-shRNA plasmid was cotransfected with genomic plasmid pBHGlox-E1, 3Cre into HEK-293 cells to package the recombinant adenovirus. The recombinant adenovirus was transfeeted into rat vascular endothelial cells （ECs）, and the green fluorescence protein expression was detected. Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by restriction enzyme digestion, PCR and GFP expression. Conclusion The recombinant adenoviral vector carrying ACE-shRNA was successfully constructed, which would become a basis for gene silence in antihypertensive treatment.
出处
《中国药物与临床》
CAS
2009年第2期111-115,161,共6页
Chinese Remedies & Clinics
基金
山西省归国留学人员基金(2007005)
