Aim: To express the mouse glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) protein with Bac-to-Bac baculovirus expression system.
Methods: GITRL gene was obtained by double digestion using EcoR I and Sal I and cloned into the baculovirus transfer vector pFastBacHTA. Then the pFastBacHTA was transformed into competent 10BacTM E.coli cells. The transposition occurred between pFastBacHTA and bacmid and a recombinant bacmid was obtained. The positive clones were picked out and the recombinant bacmid was isolated, and then complete transfected into Tn cells for producing complete recombinant baculovirus. The baculoviral stock was amplified and the GITRL protein was expressed.
Results: The presence of GITRL gene containing the recombinant bacmid was verified by PCR and gene sequencing. The cytopathic effect (CPE) displayed in the transfected Tn cells assumed that the transfection was successful. Western blot analysis showed that the molecular weight of mGITRL protein was about 20 KD.
Conclusion: The GITRL protein is expressed successfully with Bac-to-Bac baculovirus expression system, which may lay the foundation for further study on biological activity and function of GITRL protein.