Preparation of Catalase-contained recombinant adenovirus vector by the homologous recombination in bacteria

Objective To prepare Catalase-contained recombinant adenovirus vector by the homologous recombination in bacteria. Methods Catalase gene was digested from pZeoSV2-Cat, subcloned into plasmid of pBluescriptⅡsk(+) and formed plasmid of pBluescriptⅡsk(+)-Cat. Then Catalase gene was digested from plasmid of pBluescriptⅡsk(+)-Cat, subcloned into shuttle plasmid of pShuttle-CMV and formed transfer plasmid of pShuttle-CMV-Cat.Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdEasy-1. Results The linealinzed pShuttle-CMV-Cat was transformed into ultracompletent BJ5183 containing pAdEasy-1.There were 30% positive recombinant plasmid. PCR test indicated that the recombinant adenovirus plasmid pAdEasy-1-Cat contained Catalase gene. Conclusion The homologous recombination in bacteria is a convenient and efficient method to prepare recombinant adenovirus plasmid pAdEasy-1-Cat. This offers a good gene transfer vector for the gene therapy in restenosis.