摘要
目的通过引入内部核糖体进入位点序列,构建并鉴定smad7和uPA基因共表达的腺病毒载体。方法PCR扩增uPA和smad7全长cDNA的片段,先后亚克隆入pIRES质粒;更换酶切位点后,将smad7IRESuPA片段克隆至穿梭质粒pAdTrackCMV,再与pAdEasy1质粒在BJ5183菌中同源重组产生腺病毒载体质粒。酶切鉴定后在293细胞中包装成重组腺病毒Adsmad7/uPA。RTPCR检测Adsmad7/uPA在L02肝细胞中的表达。结果该重组腺病毒质粒经测序、酶切鉴定,均与预期结果一致;转染AD293细胞后,2天可观察到GFP明显表达;RTPCR检测到转染后的L02细胞中smad7和uPA表达增强。结论成功构建了smad7和uPA双基因共表达重组腺病毒载体,为研究mad7和uPA双基因共表达对大鼠肝纤维化的预防、治疗作用奠定基础。
Objective To construct adenoviral vector co-expressing rat smad7 and urokinase-type plasminogen activator by utilizing an internal ribosome entry site（IRES） sequences to link the two cDNAs. Methods The uPA and smad7 sequences were amplified by PCR from rat kidney total cDNAs and a T-vector containing rat smad7 cDNAs, respectively. The DNA fragments were inserted into T vector and sequenced, then subcloned into plRES. After changing the restriction enzyme site by PCR amplification, the smadT-IRES-uPA fragment was cloned into the shuttle plasmid pAdTrack-CMV, then the linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1. Further the recombinant plasmid was packaged in AD-293 cells after Pac I digestion to get adenovirn （Adsmad7/uPA）, smad7 and uPA expression in the Adsmad7/uPA transfected L02 liver cell were also assessed. Results The recombinant plasmid were confor reed by sequencing and restriction endonucleases digestion. GFP expression was observed on the second day after packaging the pAdsmadT/uPA in AD-293 cells. The smad7 and uPA mRNA significantly enhance in AdsmadT/uPA transfected L02 cell. Conclusion the IRES containing adenoviral vector allows functional coexpression of smad7 and uPA cDNAs.
出处
《胃肠病学和肝病学杂志》
CAS
2005年第5期448-451,共4页
Chinese Journal of Gastroenterology and Hepatology
