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


METHODS
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.


RESULTS
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.


CONCLUSIONS
(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.
Lu
Feng
F
Department of Plastic Surgery, Nanfang Hospital of the First Military Medical University, Guangzhou 510515, China.
Gao
Jian-hua
JH
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001419:Bacteria / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007627:Keloid / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor; D019014:fas Receptor
D004274:DNA, Recombinant; C491901:FAS protein, human; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D019014:fas Receptor