Construction And Identification Of Three Strains Of Recombinant Replication-defective Adenovirus Expressing HFGF2, HVEGF165 And HVEGF121 Respectively

Objective To apply gene therapy to ischemic flap in plastic surgery, we obtained three strains of replication-defective recombinant adenoviruses expressing hFGF2 , hVEGF165 and hVEGF121 respectively. Methods Based on the AdEasy system, the replication-defective recombinant adenoviruses can be constructed by employing homologous recombination in E. coli. First, three cDNA segments encoding hVEGF165, hVEGF121 and hFGF2 were obtained by RT-PCR from Hep-2 cells. Second, they were recombined into pGEM-T Easy vector respectively, and sequenced by automatic sequence analyzer. Third, they are subcloned into pShuttle-CMV respectively. After digesting with restriction endonuclease Pme I, the recombinant pShuttle-CMVs were linerized respectively. Subsequently, they are transformed into competent E.coli BJ5183 , p and recombinants were selected with kanamycin and screened by restriction endonuclease digestion. Finally the recombinant adenoviral plasmids were digested with restriction endonuclease Pac I, and transfected into adenovirus packaging cell lines (293 cells) to produce recombinant adenovirues. These recombinant adenoviruses were identified further by PCR and RT-PCR. Results We obtained three cDNA segments of hFGF2, hVEGF165, hVEGF121. Then we constructed recombinant clone vectors and recombinant shuttle vectors carrying these genes successfully. Three recombinant adenoviral plasmids were produced and identified respectively. By transfecting 293 cells, we obtained three strains recombinant adenoviruses, and they were identified by PCR and RT-PCR. Conclusion Three strains of replication-defective adenovirus expressing hFGF2, hVEGF165, hVEGF121 respectively have been constructed and identified successfully, which makes it possible that we can further our research on gene therapy to ischemic flap.