Construction of recombinant adenovirus containing mIκBα gene by homogenous recombination in E.coli. and its expression in Hep G2 cells

Abstract

Abstract: Objective To develop a rapid and efficient method for preparing recombinant adenovirus containing human mIκBα gene by homogenous recombination in E.coli. and detect its expression in Hep G2 cells.Methods The mIκBα 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- mIκBα by Pme I digestion and subsequent cotransformation into E.coli BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-mIκBα 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- mIκBα 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 mIκBα gene and the titer of Ad-mIκBα was 2.7×109 PFU/ml. With a multiplicity of infection (MOI) of 10, Ad-mIκBα could be expressed stably and efficiently in Hep G2 cells and the infection efficiency was 57% at 24h and 100% at 48h after transfection.Conclusions Homogenous recombination in E.coli can efficiently and conveniently construct recombinant adenovirus containing mIκBα 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 mIκBα gene and therapy for hepatocarcinoma.

CLC Number:

R373.2
R-33

JIN Wu-dong¹, CHEN Long-hua¹, MU Feng². Construction of recombinant adenovirus containing mIκBα gene by homogenous recombination in E.coli. and its expression in Hep G2 cells[J]. Journal of Southern Medical University, 2005, 25(03): 308-312.

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