AP2α重组腺病毒质粒的构建及其在大鼠骨髓间充质干细胞中的表达

Construction of recombinant adenovirus vector containing transcriptional factor activator protein 2α and its expression in rat mesenchymal stem cells

目的构建转录因子AP2α(Activator protein 2α)重组腺病毒质粒,感染大鼠骨髓间充质干细胞(Mesenchymalstem cells,MSCs),并检测其表达。方法从大鼠PC12细胞总RNA中PCR扩增AP2α基因,定向克隆至腺病毒穿梭质粒pAdtrace-TOX,构建重组质粒,与腺病毒骨架质粒pAd-Easy1在大肠杆菌BJ5183中同源重组,获得重组腺病毒质粒pAd-AP2α,转染HEK293细胞进行包装,获得重组腺病毒Ad-AP2α,感染大鼠骨髓MSCs,荧光显微镜下观察RFP的表达;Real-time PCR及Western blot检测AP2α的表达。结果 pAdtrace-AP2α质粒经PCR及双酶切鉴定,均可见约1 400 bp的目的基因条带,测序结果与GenBank报道一致,并正确克隆至腺病毒质粒中;重组腺病毒pAd-AP2α在HEK293细胞中包装,病毒滴度可达2.6×108pfu/ml;感染MSCs 48 h可观察到60%以上的RFP阳性细胞,经Real-time PCR及Western blot鉴定,感染后72 h,MSCs中AP2α的表达显著增高(P<0.01)。结论成功构建了携带转录因子AP2α的重组腺病毒质粒,其能有效感染MSCs,增强MSCs中AP2α的表达。

Objective To construct the recombinant adenovirus vector containing transcriptional factor activator protein （AP） 2α, infect rat mesenchymal stem cells （MSCs） and determine its expression. Methods AP2α gene was amplified from the total RNA of rat PC12 cells by PCR and cloned into adenovirus shuttle plasmid pAdtrace-TOX, and the constructed recombinant plasmid pAdtrace-AP2α was subjected to homologous combination with adenovirns skeleton plasmid pAd-Easyl in E. coli BJ5183. The obtained recombinant adenovirus vector pAd-AP2α was transfected to HEK293 cells for packaging. Rat MSCs were infected with the prepared recombinant adenovirus Ad-AP2α, then observed for expression of RFP by fluorescent microscopy, and determined for expression of AP2α by real-time PCR and Western blot. Results The target gene band at a length of about 1 400 bp was observed in recombinant plasmid pAdtrace-AP2ct by PCR and restriction analysis, of which the sequence was consistent with that reported in GenBank, and was cloned into adenovirus vector correctly. Recombinant adenovirus vector pAd-AP2ct was successfully packaged in HEK293 cells, and the obtained recombinant adenovirns Ad-AP2α reached a titer of 2. 6 x l0s pfu / ml. More than 60% of MSCs were positive for RFP 48 h after infection. Real-time PCR and Western blot showed that the expression level of AP2c~ increased significantly 72 h after infection （P 〈 0. 01 ）. Conclusion Recombinant adenovirus vector containing AP2ot was successfully constructed, which infected MSCs effectively and enhanced the expression of AP2α in MSCs.