OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.