Recombinant Adenovirus Plasmid Constructed by Simplied and High Performance Two-step Homologous Recombination in Bacteria

Objective To construct a recombinant adenovirus plasmid by a simplied and efficient two step homologous recombination in bacteria.Methods First,pAdEasy 1 was transformed into E.coli BJ5183 to generate the so called BJ5183pAdEasy 1 which was selected,amplified and preserved in the glycerol at -70℃.Second,TK gene was liberated from the pRep8.TK (HindⅢ and XbaⅠ) and subcloned into the shuttle plasmid pAdtrack CMV to make pAdtrack CMV TK which was then linearized by PmeⅠand transferred into BJ5183pAdEasy 1 bacteria.Transformants were selected on LB agar plates containing 12 5 μg/mL kanamycin.Plasmid DNA was prepared from individual colonies,amplied and purified.Results The pAdtrack CMV TK(about 10 kb)and pAdEasy CMV TK(about 34 kb),both selected by kanamycin resistance,could be clearly identified by size after fractionating DNA in agarose gel.The simplied two step homologous recombination in bacteria generated recombinant adenovirus plasmids at a frequency of 90%(9/10).Conclusions The simplied two step homologous recombination in bacteria is a convenient and effective way to construct the recombinant adenovirus plasmid which is the most important to construct the recombinant adenovirus. China J Cancer Prev Treat,2003,10(5):473-475