Preparation of gfp-bcl-X_L-contained recombinant adenovirus vector by the homologous recombination in bacteria / 中国病理生理杂志

ABSTRACT

AIM: To prepare gfp-bcl-X L-contained recombinant adenovirus(rAd-gfp-bcl-X L).METHODS: Bcl-X L gene was amplified from pEGFP-C 3-bcl-X L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X L. Then pAdTrack-CMV-bcl-X L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X L gene. The titer of the purified rAd-gfp-bcl-X L was 6 5?10 12 PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X L. This affords a good gene transfer vector for the gene therapy in human's diseases.