uPA基因重组GFP-腺相关病毒载体质粒的构建及其表达

Construction and Expression of Recombinant Plasmid of Adeno-associated Virus Vectors with GFP Carrying uPA Gene in Renal Tubular Epithelial Cells

利用基因重组技术,用RT-PCR法从幼鼠肾脏获得uPA cDNA,再克隆到质粒pAAV-IRES-hrGFP的多克隆位点,构建重组质粒pAAV-hrGFP-uPA,通过酶切和DNA测序鉴定重组质粒的正确性.采用磷酸钙共沉淀法,以重组质粒pAAV-hrGFP-uPA和pAAV-RC、pHelper共转染AAV-293细胞,产生具有传染性的病毒颗粒;用斑点杂交法测定重组病毒颗粒的滴度,再将此病毒颗粒体外转染到培养的肾小管细胞中,倒置荧光显微镜观察GFP的表达,用免疫组化法检测转染的uPA蛋白表达.结果表明:成功地构建uPA基因GFP-腺相关病毒重组质粒,病毒滴度达每mL 4×1013病毒颗粒,60%～70%肾小管细胞感染了病毒颗粒,感染的肾小管细胞能稳定、高效表达外源uPA蛋白,为今后建立AAV-uPA基因治疗肾纤维化的模型奠定了良好的基础.

To construct the recombinant plasmids adeno-associated virus （pAAV-IRES-hrGFP） with GFP carrying uPA gene, uPA cDNA were obtained from immature rat kidney by reverse transcription polymerase chain reaction method. Then the uPA cDNA were cloned into multiple cloning sites of plasmid pAAV-IRES-hrGFP to construct the recombinant pAAV-hrGFP-uPA plasmids. The recombinant plasmids were identified by restriction endonuclease digesting and DNA sequencing. The recombinant plasmids were transfected into the AAV-293 cells together with the control plasmids pHelper and pAAV-RC by phosphate-calcium deposit method. Recombinant AAV viral particles were prepared from infected AAV-293 cells and the titer was determined by dot hybridization. Then the viral particles were used to infect renal tubular epithelial cells in vitro. The expression of GFP and uPA in renal tubular epithelial cells were detected by using the inverted fluorescence microscope and immunocytochemical staining respectively. The results suggested the recombinant AAV vectors carrying uPA gene were constructed successfully. The virus titer was 4×10^13 viral particles per mL and the renal tubular epithelial cells could express exogenous uPA effectively and stably. These results indicate that AAV can deliver uPA gene to renal cells in vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.