Construction of Recombinant Adenovirus by An Efficient Method of Chemical Transformation and Homologous Recombination in Bacteria

Objective The AdEasy system to construct recombinant adenoviruses by homologous recombination in bacteria was modified to establish the basis for preparing a biological preparation with more anti-tumor efficiency. Methods The suicide genes of CD and HSV-TK were inserted into the shuttle plasmid pAdTrack-CMV and the resultant plasmid was digested by Pme I and then purified by phenol chloroform extraction and ethanol precipitation or gelextraction. The linearized shuttle plasmid pAdTrackCMV-CDglyTK and the backbone plasmid pAdeasy-1 were co-tranformed into bacteria by electro-transformation or chemical transformation to attain homologous recombination. The recombinant plasmid rpAdEasyGFP-CDglyTK was tranformed into 293 cells by liposome to package and proliferate adenovirused. Results The chemical transformation with gel extraction got the highest positive rate of recombination 100%. The fusion suicide genes were successfully inserted into adenoviruses identified by PCR amplification digestion and sequencin. Conclusion The method of homologous recombination in bacteria to consturct recombinant adenoviruses was improved to decrease the cost of experiments as well as to obtain the highest positive recombinant rate. The replication-deficient recombinant adenovirus rAd-CDglyTK was successfully constructed.