Construction Of Conditionally Replicating Adenoviruses Driven By MK Promoter

Gene therapy, by definition, utilizes directed gene transfer to treat disease. The first clinical gene therapy study for cancer emerged 10 years ago. Early results on the clinical efficacy of gene therapies were somewhat disappointing. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanism of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes that are available for gene therapy. The current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of viruses, increase in stability and control of transgene expression. By using AdEasy system, which is based on the homologous recombination in bacteria, an AP(alkaline phosphatase)-labled recombinant adenovirus vector containing adenovirus E1A driven by MK promoter was constructed.By endonuclease digestion, fragment harboring of MK promoter- AP-IRES-E1A coding sequence was subcloned into shuttle plasmid., then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. After positive kanamycin resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with Pac-I and transfected into HEK293 cells to assembly conditionally replicating adenovirus Ad-MK- AP-IRES- E1A, the further restriction analysis was performed for testing the recombinant adenovirus. The results of restriction analysis showed that the successful insert of MK promoter-AP-IRES-E1A coding sequence and the successful construction of recombinant adenovirus vector.It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the successful construction of conditionally replicating adenoviruses make the further in vivo experiments possible.