Construction Of Recombinant Adenovirus Containing Fusion Suicide Gene CDglyTK By A Simple Method Of Homologous Recombination In Bacteria

The simple herpes virus-thymidine kinase (HSV-TK) gene and Escherichia coli cytosine deaminase (EC-CD) are the two most widely studied and most extensive application suicide genes in the virus-directed enzyme prodrug therapy (VDEPT) system. Their gene sequences and the mechanism of the expression products have been already definite. To acquire a biology therapy preparation with more anti-tumor efficient than a single suicide gene, this experiment successfully construct a recombinant virus containing fusion gene CDglyTK by homologous recombination in bacteria and then transfection of the replication-deficient adenovirus vector into eukaryotic packing cells 293.This study first construct PMD18-T-CD by a ligation reaction of the CD-PCR product carrying enzyme identification sites from the plasmid pBlueScript-CD and the PMD18-T vector by the use of base complementarity between the single A base at the end of CD-PCR product and the single T base at the end of the PMD18-T vector. The EC-CD gene is then cut from PMD18-T-CD and connected with the shuttle plasmid pAdTrack-CMV containing the same blunt ends to construct pAdTrack-CD, which is identified by PCR and digestion reaction. HSV-TK gene is from pAdElCMV-TK by PCR. At the 5' end of the PCR product, there are oligonulcleoside chains coding glycin by designing the primers to obtain glyTK. The shuttle plasmid pAdTrack-CMV-CDglyTK containing the fusion suicide gene is constructed and identified by the digestion and ligation reaction of the glyTK gene and pAdTrack-CMV-CD, which is identified by PCR, digestion and sequencing.The recombinant adenovirus plasmid pAdEasy-GFP-CDglyTK is constructed by the method of homologous recombination in bacteria, in which the shuttle plasmid pAdTrack-CMV-CDglyTK and the adenoviral backbone plasmid pAdEasy-1 carrying the main genome of adenovirus is co-transformed into the BJ5183 bacteria with high homologous recombination ability. After screening withkanamycin and identifying with digestion and PCR , the recombinant adenovirus plasmid is then transformed into the DH5a bacteria to obtain high copy plasmid. The plasmid is transfected into 293 cells with the liposome and the recombinant adenovirus rAd-CDglyTK is packed and proliferated, which sets up a substantial basis for the further study of malignant tumor gene therapy in vitro and in vivo and the ultimate clinical application.Moreover, during the period of the construction of the recombinant adenovirus rAd-CDglyTK, a in-depth study is made in the methodology and the selected AdEasy system is improved to some extent. On the one hand, the system does not need to ask for the help of the expensive instrument again, that is, in the general laboratory can proceed the recombination of plasmids in bacteria, save the financial power, and expand the application. On the other hand, the successful rate of homologous recombination is increased to some extent.