HDAC1重组腺病毒载体的构建及鉴定

Construction and Identification of Adenoviral Vector Containing Human HDAC1 Gene

利用Ad-Easy腺病毒表达系统构建含有人源性组蛋白去乙酰化酶1(HDAC1)基因的重组腺病毒,并检测其在脐带间充质干细胞(h UC-MSCs)中的表达及对h UC-MSCs增殖的影响.RT-PCR扩增人HDAC1 ORF序列,构建p Ad Track-CMV-HDAC1重组腺病毒穿梭质粒,Pme I单切后将其转化进入E.coli BJ5183与腺病毒骨架质粒同源重组,Pac I单切线性化后转染至HEK293细胞中扩增和包装p Ad-HDAC1,采用空斑法鉴定病毒滴度;p Ad-HDAC1感染h UC-MSCs,倒置荧光显微镜下观察荧光表达,qRT-PCR和Western Blot分别检测细胞中HDAC1在mRNA水平和蛋白水平的表达,CCK-8实验检测HDAC1高表达对h UC-MSCs增殖的影响.结果显示成功扩增人HDAC1 ORF序列并构建p Ad-HDAC1重组腺病毒;病毒感染后h UC-MSCs中HDAC1 mRNA和蛋白表达水平明显增高,并且促进h UC-MSCs的增殖.成功构建HDAC1高表达的腺病毒,可有效提高h UC-MSCs中HDAC1的表达并促进细胞增殖.

It was aimed to construct a recombinant adenovirus containing human histone deacetylase 1 （HDAC1） gene by using Ad-Easy system and detect its effect on the proliferation of hUC-MSCs. Human HDAC10RF was generated by RT-PCR and inserted into the shuttle adenovirus plasmid to construct re- combinant shuttle adenovirus plasmid （ pAdTrack-CMV-HDAC1 ）. The pAdTrack-CMV-HDAC1 was line- arized by PmeI and transformed into BJ5183 to homologously recombine with pAdEasyl. After linearized by PacI, pAd-HDAC1 was transfected into HEK293 to package and amplify. The viral titer was deter- mined by air-dilution method. After transfected into hUC-MSCs, the green fluorescence was observed by using fluorescent microscope, qRT-PCR and Western Blot were used to analyze the mRNA and protein expression of HDAC1 and CCK-8 assay was performed to detect the proliferation of hUC-MSCs. Recombi- nant adenovirus pAd-HDAC1 containing HDAC1 gene was successfully constructed. After transfeeted into hUC-MSCs, the mRNA and protein expression of HDAC1 in hUC-MSCs were increased significantly, and HDAC1 could promote the proliferation of hUC-MSCs, pAd-HDAC1 was successfully constructed, which can effectively improve expression of HDAC1 in hUC-MSCs and promote hUC-MSCs proliferation.