Construction and expression of recombinant adenovirus vector carrying transforming growth factor-β3 gene

Abstract:

Objective：To construct the recombinant adenovirus vector containing transforming growth factor-β3（TGF-β3） and en－hanced green fluorescent protein（EGFP） in order to transfect rabbit bone marrow mesenchymal stem cells（MSCs）. Methods：The cD－NA TGF-β3 was integrated into the shuttle vector of pAdxsi-CMV with EGFP，then the product was transfected into package cell HEK 293 and the recombinant adenovirus vector expressing TGF-β3 gene was generated. The expression of EGFP was identified by PCR and fluorescent microscopy. The titer of pAdxsi-EGFP-TGF-β3 was tested with end-paint dilution assay and its tranfection ef－ficacy was examed. The expression of TGF-β3 was detected by Western blot. Results：The recombinant adenovirus vector pAdxsi-EGFP-TGF-β3 containing TGF-β3 and EGFP was constructed successfully. PCR analysis showed that TGF-β3 cDNA was inserted into the recombinant adenoviral plasmid. The recombinant virus vector was collected by packaging HEK293 cells. The titer of pAdxsi-EGFP-TGF-β3 was 2×1010 pfu/ml. Western blot demonstrated that TGF-β3 was expressed in rabbit bone marrow MSCs. Conclusions：EGFP-TGF-β3 recombinant adenovirus vector is successful constructed and TGF-β3 protein is effectively expressed in rabbit bone marrow MSCs，which provides the basis for research of gene therapy in preventing hypertrophic scar after wound healing.