Preparation of gfp-bcl-X_L-contained recombinant adenovirus vector by the homologous recombination in bacteria
- VernacularTitle:细菌内同源重组高效制备含绿色荧光蛋白和抗凋亡基因bcl-X_L的重组腺病毒载体
- Author:
Yan LUO
;
Shibo TANG
;
Xianghong LI
;
Shuangxi LIU
- Publication Type:Journal Article
- Keywords:
Recombination;
Adenovirus;
Apoptosis;
Genes, bcl-X L;
Gene therapy
- From:
Chinese Journal of Pathophysiology
2000;0(12):-
- CountryChina
- Language:Chinese
-
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.
