Human GITRL_(aa52-177) protein expressed in Bac-to-Bac baculovirus expression system

Objective To produce the extracellular region of hGITRL in eukaryotic expression system.Methods Bac-to-Bac baculovirus expression system was employed in the present study.The hGITRL extracellular region gene was amplified by PCR from the full-length pMD18-hGITRL-T vector and subcloned into the pFastBacHTa plasmid,then the latter was transformed into E.coli DH10Bac competent cells to construct a recombinant baculovirus and to infect Tn cells.The interest protein was identified by SDS-PAGE,Western blot and indirect immunofluorescence staining.Results The recombinant pFastBacHTa-hGITRLaa52-177 plasmid and the recombinant Bacmid-hGITRLaa52-177 vector were constructed successfully.And the recombinant hGITRLaa52-177 protein was roughly 18KD,which was revealed by SDS-PAGE assay,and confirmed to be hGITRLaa52-177 protein by Western blot and indirect immunofluorescence staining.Conclusion Bac-to-Bac baculovirus expression system can effectively prepare hGITRLaa52-177 protein with bioactivity in vitro.