High efficient generation of recombinant adenovirus containing human Fas gene using a method of homologous recombination in bacteria

Abstract

To generate recombinant adenovirus with human Fas gene and transfect the Fas gene into keloid-derived fibroblasts to take place of the dysfunctional Fas gene, reconstruct the blocked Fas signal. We used a new bacterial homologous recombination system (Ad-Easy system) to construct recombinant adenovirus vector. Fas gene was cut down from the PMD-T-Fas plasmid and then transferred to track plasmid. Recombination was successfully completed in bacteria BJ5183 and recombinant adenovirus was produced in 293 cells. Then we infected keloid derived fibroblasts and compared the expression of Fas protein. Finally, we detected the function of newly produced Fas protein. We successfully constructed the recombinant adenovirus with Fas gene and detected its highly expressed Fas protein in infected keloid derived fibroblasts. Obvious apoptosis was also detected in infected keloid derived fibroblasts exposed to FasMcab. (1) The recombinant adenovirus with Fas gene can transfect the Fas gene into keloid-derived fibroblasts and highly improved the expression of Fas protein. The newly expressed Fas gene can reconstruct the blocked Fas signal. (2) The correlation between keloid and Fas gene was further proved and it may paves a sound foundation for further gene therapy in keloid.