Aim: To construct recombinant adenovirus vector pAdEasy-GFP-GITRL and detect the viral titer.
Methods: GITRL gene was obtained by double digestion using Bgl II and Sal I, and cloned into the baculovirus transfer vector(pAdtrack-CMV), then the recombinant adenovirus vector (pAdtrack-CMV-GITRL) was digested by restrictive endoenzyme Pme I. The linear recombinant adenorirus vector and pAdEasy-1 were cotransfected into HEK293 cells by co-precipitate of calcium phosphate. Recombinant adenovirus was packaged and purified in HEK293A cells.
Results: Recombinant adenovirus vector pAdEasy-GFP-GITRL was constructed successfully and high titer of recombinant adenovirus was obtained (2.0 x 10⁹ pfu/mL). Western blotting analysis also revealed the expression of GITRL by recombinant adenovirus vector.
Conclusion: The construction of recombinant adenovirus vector pAdEasy-GFP-GITRL and recombinant adenovirus will facilitate the potential GITRL gene therapy.