The construction of recombinant β-galactosidase adenovirus and its expression in transduced rat aortic smooth muscle cells

Objective To construct a replication-defective recombinant adenovirus expressing β-galactosidase for providing a control vector in future study of gene therapy for restenosis, and of safety, feasibility, and efficiency of gene transfer mediated by adenovirus. Methods The shuttle vector pAd-β-gal was constructed by recombinant DNA technology and characterized by endonuclease digestion and agarose gel electrophoresis. Ad-β-gal was generated by homologous recombination and characterized by staining the transfected 293 cells with X-gal. The virus titer was determined by measuring the absorbance at 260 nm.Rat aortic vascular smooth muscle cells (VSMCs) were cultured by explant methods and characterized by specific smooth muscle α-actin immunohistochemical staining. VSMCs were transfected with Ad-β-gal at 100 moi and stained with X-gal to observe the expression of β-galactosidase. Results (1)pAd-β-gal shuttle vector and Ad-β-gal were constructed successfully;(2)The transfected 293 cells and VSMCs were stained blue with X-gal;(3)The virus titer is 9×10 11 pfu/ml;(4)The efficiency of gene transfer in VSMCs at 100 moi was nearly 100%. Conclusions (1)The recombinant adenovirus expressing β-galactosidase, Ad-β-gal, was constructed, successfully;(2) This investigation provides an excellent control vector for future study of gene therapy in restenosis;(3)Ad-β-gal is a useful tool in the research of safety, feasibility and the efficiency of gene transfer mediated by adenovirus.