The Improved Method For Constructing Recombinant Adenovirus Bsed On The The AdEasy System

Objective: Adenovirus AdEasy system is a good method for rapid constructing recombinant adenovirus by employing homologous recombination between recombinant shuttle plasmid carrying interesting gene and adenovirus backbone plasmid in prokaryocyte. In order to construct recombinant adenovirus more easily, we try to make the protocol improved and obtain a more efficient, convenient and rapid method.Method: The replication-defective adenovirus type 5 backbone plasmid pAdEasy-1 was transformed into competent E.coli BJ5183 by electroporation. The resultant E.coli BJ5183/p strain was selected with ampicillin and screened by restriction endonuclease digestion, and its stability was assessed by restriction endonuclease digestion. Then the E.coli BJ5183 / p competent cells were prepared by CaCl2 technique and the recombinant shuttle plasmid carrying GFP (pAdTrack-CMV) was transformed into them. Recombinants were selected with kanamycin and screened by restriction endonuclease digestion. The recombination efficiency between recombinant shuttle plasmid carrying interesting gene and adenovirus backbone plasmid was compared to the routine method of co-transformation. The obtained recombinant adenovirus was further transformed into competent E.coli DH 10B in order to obtain high-copy recombinant plasmid. The confirmed recombinant adenovirus DNA was linearlized withPac I and transfected into 293 cells line to produce the recombinant adenovirus (rAdv). We also investigated the infection capability of the rAdv to primary schistosoma culture cells.Result: By using the improved method, we obtained the E.coli BJ5183 / p strain with high stability and it can be used repeatedly. The competent E.coli BJ5183 / p prepared by CaCl2 technique was easily transfomed by recombinant shuttle plasmid, and recombinant adenovirus plasmid can be produced by homologous recombination in E.coli BJ5183 / p. The recombination efficiency has been improved compared to the routine method. After the recombinant adenovirus DNA was transfected into 293 cells line, and the CPE of 293 cells and the green fluorescence were observed. The rAdv can infect some kind of primary schistosoma culture cells.Conclusion: This improved method for constructing recombinant adenovirus is more convenient and more efficient than the routine method. It can be applied well in a poorly equipped laboratory in our country.