Construction of rat Smad7 recombinant adenovirus vector

Objective To construct a recombinant adenovirus vector to express rat Smad7. Methods Total RNA was extracted from the rat liver to clone Smad7 cDNA. The shuttle plasmid pAdTrack-CMV-Smad7 in which the Smad7 cDNA was inserted into the downstream of CMV promoter was constructed by digestion and ligation. Then the linearized shuttle plasmid was cotransformed into bacteria with backbone vector AdEasy-1 to obtain the recombinant adenoviral plasmid-pAdSmad7 by homologous recombination. After identifying the desired recombinant adenovirus was packaged in HEK293 cells. The expression of Smad7 in HEK293 cells was detected by RT-PCR. Results The recombinant plamid pAdSmad7 was constucted by homologous recombination and confirmed by restriction endo-nucleases digestion, sequencing and PCR. GFP expression was observed on the third day after packing the linearized pAdSmad7 in HEK293 cells. After the HEK293 cells were infected, the expression of Smad7 increased significantly. Conclusion Using the AdEasy system the rat Smad7 recombinant adenovirus can be rapidly generated and their formal the basis for further research in the signal transduction pathway of TGF-βand gene therapy of hepatic fibrosis.