Jin Wu-Dong, Chen Long-Hua, Mu Feng
Department of Radiotherapy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2005 Mar;25(3):308-12.
To develop a rapid and efficient method for preparing recombinant adenovirus containing human mIkappaBalpha gene by homogenous recombination in E.coli. and detect its expression in Hep G2 cells.
The mIkappaBalpha gene was cloned into the shuttle plasmid pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene, followed by linearization of the resultant plasmid pAdTrack-CMV- mIkappaBalpha by Pme I digestion and subsequent cotransformation into E.coli BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd- mIkappaBalpha was selected for kanamycin resistance and confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into 293 cells, in which Ad- mIkappaBalpha were generated within 7 to 10 days. The virus titer in 293 cells and its infection efficiency in Hep G2 cells were detected and calculated with the aid of GFP expression.
PCR indicated that the recombinant adenovirus contained mIkappaBalpha gene and the titer of Ad- mIkappaBalpha was 2.7x10(9) PFU/ml. With a multiplicity of infection (MOI) of 10, Ad- mIkappaBalpha could be expressed stably and efficiently in Hep G2 cells and the infection efficiency was 57% at 24 h and 100% at 48 h after transfection.
Homogenous recombination in E.coli can efficiently and conveniently construct recombinant adenovirus containing mIkappaBalpha gene capable of amplification in 293 cells and efficient infection of Hep G2 cells. The recombinant adenovirus may serve as a good gene transfer vector for study the function of mIkappaBalpha gene and therapy for hepatocarcinoma.
建立一种在大肠杆菌中通过同源重组制备含人mIkappaBalpha基因的重组腺病毒的快速高效方法，并检测其在肝癌细胞株Hep G2中的表达。
将mIkappaBalpha基因克隆到含绿色荧光蛋白（GFP）报告基因的穿梭质粒pAdTrack-CMV中，用Pme I酶切线性化所得质粒pAdTrack-CMV-mIkappaBalpha，随后与腺病毒骨架质粒pAdEasy-1共转化到大肠杆菌BJ5183细胞中。筛选出对卡那霉素耐药的重组质粒pAd-mIkappaBalpha，并通过多种限制性内切酶分析进行鉴定。最后，将线性化的重组质粒转染到293细胞中，7至10天内产生Ad-mIkappaBalpha。借助GFP表达检测并计算293细胞中的病毒滴度及其在Hep G2细胞中的感染效率。
PCR结果表明重组腺病毒含有mIkappaBalpha基因，Ad-mIkappaBalpha的滴度为2.7×10⁹ PFU/ml。感染复数（MOI）为10时，Ad-mIkappaBalpha可在Hep G2细胞中稳定高效表达，转染后24小时感染效率为57%，48小时为100%。
在大肠杆菌中进行同源重组能够高效便捷地构建含mIkappaBalpha基因的重组腺病毒，该病毒能在293细胞中扩增并有效感染Hep G2细胞。该重组腺病毒可作为研究mIkappaBalpha基因功能及肝癌治疗的良好基因传递载体。
