Construction and Identification of Recombinant Adenovirus Vector of Small Interfering RNA Targeting Basic Fibroblast Growth Factor

Objective To construct a recombinant adenovirus vector carrying small interfering RNA(siRNA) which could efficiently silence the basic fibroblast growth factor(bFGF), and provide relevant study for further transgene technology. Methods Preferred specifc siRNA sequences targeting bFGF was designed and synthesized. First, by the method of restriction endonuclease digestion and T4 DNA ligase, the bFGF-siRNA sequence was cloned to the shuttle plasmid pGStrack, then LR site-specific recombination was performed in vitro between recombinant shuttle plasmid pGStrack-bFGF-siRNA and adenovirus backbone plasmid pGSadeno. Finally, after correct identification of the recombinant backbone plasmid pGSadeno-bFGF-siRNA, it was digested by PacⅠand transfected into HEK 293 cells and packaged as recombinant adenovirus rAd5-bFGF-siRNA. Further, it was amplified in HEK 293 cells and identified by PCR analysis. The titer was detected by tracing total cytopathic effect. Results PCR identification showed the construction of recombinant adenovirus rAd5-bFGF-siRNA was successful. The titer was about 5×10⁸ PFU/ml after amplifcation. Western blot showed bFGF protein silencing effciency was greater than 90%. Conclusion The recombinant adenovirus vector carrying bFGF-siRNA was constructed successfully via LR recombination. The vector showed a high effciency of silencing target protein in U251 cell line, which laid a foundation for further relevant study.