AdMax系统构建腺病毒载体介导SCL基因转染Cajal样间质细胞及其表达

目的 构建含有人干细胞白血病(stem cell leukemia,SCL)基因的重组腺病毒载体,并观察其对豚鼠膀胱Cajal样间质细胞的转染及其介导SCL基因的表达.方法 采用PCR方法从含人SCL基因质粒中扩增SCL基因,连接到腺病毒穿梭质粒的多克隆位点上,构建重组穿梭质粒pDC315-EGFP/SCL,在脂质体介导下与腺病毒辅助大质粒pBHGlox(delta)E1,3Cre共转染293细胞,包装产生复制缺陷型重组腺病毒pDC315-SCL经HEK293细胞扩增,纯化后测定病毒滴度.通过观察绿色荧光蛋白的表达评估重组腺病毒对Cajal样间质细胞的转染率,RT-PCR法分析转染Cajal样间质细胞后SCL mRNA的表达.结果 PCR结果和Western blot法检测证实pDC315-SCL重组腺病毒载体构建成功,滴度达到1×1010 PFU/ml,对膀胱Cajal样间质细胞的转染率高达98%,RT-PCR法检测转染Cajal样间质细胞后SCL mRNA有表达.结论 成功构建pDC315-SCL重组腺病毒载体对Cajal样间质细胞有很强的转染能力,可介导SCL基因在Cajal样间质细胞的表达.

Construction of recombinant adenovirus vector with SCL gene by AdMax system and transfecting ICC-like cells and expression of adenovirus vector

Purpose To construct recombinant adenovirus vector containing human stem cell leukemia(hSCL) gene and to observe its ability in transfecting Cajal-like interstitial cells and mediating SCL gene expression.Methods SCL gene was amplified from plasmid with SCL gene by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pDC315-EGFP.HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC315-EGFP/SCL and large adenovirus helper plasmid pBHGlox(delta)E1,3Cre in mediation of liposome.The obtained replication-defective recombinant adenovirus pDC315-SCL was propagated in HEK293 cells,purified pDC315-SCL plasmid and determined for virus titer.The distribution and efficiency of recombinant adenovirus mediated hSCL was observed by the expression of green fluorescence protein(GFP) under the fluorescent microscope.The expression of hSCL mRNA transfected with Cajal-like interstitial cells was measured by RT-PCR method.Results Western blot inspection and PCR analysis confirmed that the hSCL gene was successfully inserted into the adenovirus vector,with titer of the recombinant adenovirus to 1.0×1010 PFU/ml.The adenovirus had a high transfection efficiency up to 98%.RT-PCR analysis showed expression of hSCL mRNA in Cajal interstitial cells transfected by hSCL.Conclusions The recombinant adenovirus containing human hSCL gene is successfully constructed by homogenous recombination in bacteria and it has a high transfection efficiency and can mediate expression of Cajal-like interstitial cells.