人血红素氧合酶-1重组腺病毒的构建与鉴定

Construction and identification of recombinant adenovirus carrying human hHO-1 gene

目的构建人血红素氧合酶-1(hHO-1)重组腺病毒,用以对供体器官的预处理。方法将人hHO-1cDNA克隆于腺病毒穿梭质粒pSGCMV,得到重组质粒pSGCMV-hHO-1。采用位点特异性重组系统(Cre/Loxp)将pSGCMV-hHO-1与腺病毒骨架载体pBHGloxP△E3通过lipofectamine2000共转染至293细胞,生成带有hHO-1基因的重组腺病毒载体Ad-hHO-1。重组腺病毒质粒在293细胞中扩增,CsCl梯度离心纯化。结果经酶切鉴定和测序证实载体构建的正确性,病毒滴度为2.0×1010pfu/ml。结论成功构建带有hHO-1cDNA的重组腺病毒,用以器官移植时缺血再灌注损伤的基因治疗。

Objective The heme oxygenase-1 （HO-1） overexpression exerts cellular protection and immune regulatory functions by protecting grafts from ischemia/reperfusion and immunologic injury in organ transplantation. We constructed a recombinant adenovirus carrying human HO-1 gene for pretreatment donor organ. Methods Human HO-1 （hHO-1）cDNA was cloned into adenovirus shuttle vector pSGCMV by standard procedure. The recombinant adenoviral plasmid pSGCMV-HO-1 was identified and the correct clones containing target gene were selected, pSGCMV-hHO-1 and backbone vector pBHGloxPAE3, each bearing a loxP site and the latter containing a cre site,were cotransfered into the adenoviral packaging 293 cells by lipofectamine mediated gene transfer method to recombine and pack the virus（site-specific recombination）. The Ad-hHO-1 was then amplified in 293 cells and purified by Cscl. Results The cloned hHO-1cDNA was demonstrated correct by sequencing. The titer of Ad-hHO-1 was 2. 0 × 10^10 pfu/mL Conclusion The successful construction Ad-hHO-1 may provide an opportunity for the gene therapy of ischemia/reperfusion injury after transplantation.