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目的构建deltex-1基因腺病毒载体并研究其在大鼠骨髓间充质干细胞(bone marrow mesenchymal stemcells,bMSCs)中的表达。方法采用高保真酶PCR扩增deltex-1基因并测序,亚克隆至pAdTrack-CMV腺病毒穿梭载体后在BJ5183细菌中进行重组,挑选正确的重组子在FT293细胞中包装成病毒。以CsCl2方法纯化病毒,并进行滴度测定。将目的基因病毒感染bMSCs细胞后收集总RNA及蛋白,用PCR及Western blot方法检测目的基因表达水平。结果①成功扩增了大小为1 883 bp的大鼠deltex-1,并成功构建了大小为4.5 kb的同源重组腺病毒穿梭载体,滴度为1.23×1011 IU/ml;②对培养的大鼠bMSCs进行流式检测,结果为CD90、CD44阳性表达,分别为99.57%和85.66%,而CD45表达阴性,阳性率为0.35%;③荧光显微镜观察deltex-1转染效率为60%～70%,RT-PCR及Western blot检测结果显示转染后deltex-1表达明显增强,说明转染成功。结论成功构建deltex-1基因腺病毒载体并能在bMSCs中高表达。
Objective To construct adenovirus vector of deltex-1 gene and study its expression in rat bone marrow mesenchymal stem cells (bMSCs). Methods The deltex-1 gene was amplified by high-fidelity PCR and sequenced. The recombinant plasmid was subcloned into pAdTrack-CMV adenovirus shuttle vector and then recombined in BJ5183 bacteria. The correct recombinant plasmid was selected and packaged into virus in FT293 cells. The virus was purified by CsCl2 method and the titer was determined. The total RNA and protein were collected after infecting bMSCs with virus of interest. The expression of target gene was detected by PCR and Western blot. Results ① The deltx-1 gene was successfully amplified in rat with a size of 1 883 bp. The recombinant adenovirus shuttle vector with a size of 4.5 kb was successfully constructed with a titer of 1.23 × 1011 IU / ml. Flow cytometry results showed that the expression of CD90 and CD44 were 99.57% and 85.66%, respectively, while the expression of CD45 was negative. The positive rate was 0.35%. ③The transfection efficiency of deltex-1 was 60% -70% The results of RT-PCR and Western blot showed that the expression of deltex-1 was significantly increased after transfection, indicating that the transfection was successful. Conclusion The deltex-1 gene adenovirus vector was successfully constructed and highly expressed in bMSCs.