Generation Of Recombinant Adenovirus Containing Double Suicide Gene Driven By KDR Promoter By Using Modified AdEasy System

ObjectiveTo search for a novel modified AdEasy system on fabricating replicating-defective recombinant adenovirus. While traditional AdEasy system is somehow complicated, less successful and may have special request on laboratory condition, the modified AdEasy system in which AdEasier-1 cells are constructed before homologous recombination in bacteria may simplify the procedure, shorten period of experiment, improve rate of success, lower request of condition and thus be used in more laboratory. And by using this system, we construct an recombinant adenovirus of CD/TK double suicide gene driven by KDR promoter which is applied in anti-angiogenesis of colorectal carcinoma. KDR promoter which has specific targeting effect on new bom endothelial cell of blood vessel drives CD/5-FC and HSV-TK/GCV to kill specifically new bom endothelial cells of tumor, which pave a sound foundation on antiangiogenesis therapy of tumor.Methods1. Foundation of modified AdEasy systemAdenovirus backbone plasmid AdEasey-1 was transformed into BJ5183 cells whose positive product is called AdEasier-1 cell. The Pine I-linearized resultant plasmid pAdtrack was transformed into competent AdEasier-1 cells prepared by method of CaC12. The DNA of identified recombinant plasmid was digested with PacI and tranfected into 293 cells to package adenovirus. The recombinant adenovirus was identified by the expression of GFP.2. Construction of recombinant adenovirus containing double suicide gene driven by KDR promoter by using modified AdEasy systemKDR promoter, CD and TK gene were amplified by PCR and then were cloned into shuttle vector pAdtrack. The Pme I-linearized resultant plasmid pAdtrackKDR-CDTK was transformed into competent AdEasier-1 cells prepared by method of CaCl2. The DNA of identified recombinant plasmid was digestedwith Paci and tranfected into 293 cells to package adenovirus. The recombinant adenovirus was identified by the expression of GFP and PCR technique. After titer of the adenovirus, there were used to infect endothelial cells in vitro for killing test.Results1. After restrictive digestion of Hindllland PstI enzyme and electrophoresis, there were 3 positive clone of cell which were called AdEasier-1 cells.2. Chemical transfortiom of linearized transfer plasmid into AdEasier-1 cells resulted in very high success rate of positive clone(20/20).3. KDR promoter (amplified from the template of human DNA by PCR), CD gene (amplified from the template of E. coli JM109 by PCR) and TK gene (amplified from the template of pREP8-TK plasmid by PCR) were identical with the sequences reported. KDR promoter is around 580bp,CD 1.3kb,TK 1.2kb. DNA sequencing analysis indicate that the size of CD/TK refusion gene is 2439bp, without any introns and coding 813 amino acids.4. PCR test indicated that the recombinant adenovirus contained KDR promoter,CD and TK fusion gene. The titer of adenovirus is 2 X 1011 pfu/ml.Conclusions1. The modified AdEasy System is more convenient and efficient in construction of recombinant adenovirus than the tradional AdEasy System.2. The recombinant adenovirus of CD/TK double suicide gene driven by KDR promoter was constructed successfully and KDR promoter drives CD/5-FC and HSV-TK/GCV to effectively kill new bom endothelial cells, which pave a sound foundation on antiangiogenesis therapy of tumor.