Eukaryotic Expression Of Human GITRL_aa52-177 Protein And Development Of BA-ELISA For HGITRL Protein

Objective:In this study,we first used Bac-to-Bac baculovirus expression system to express 6×His-hGITRL_aa52-177recombinant protein,then identified the recombinant protein by SDS-PAGE,Western blot and indirect immunofluorescence staining.We also constructed recombinant pDisplay-hGITRL_aa52-177vector and transfected HEK-293 cells to express secretory hGITRL_aa52-177protein.Finally,we tried to develop BA-ELISA method to detect GITRL protein concentration.Methods:(1)The hGITRL_aa52-177cDNA was obtained by PCR amplification and inserted into pFastBacHTa donor plasmid.After the pFastBacHTa -hGITRL_aa52-177vector constructed successfully,we transformed this recombinant plasmid into E.coli DH10Bac for transposition into the bacmid.The recombinant bacmid DNA was purified and transfected Tn cells by cationic lipid.When Tn cells appeared cytopathic effect,the culture supemantants containing virus was collected.We used the baculoviral stock to infect Tn cells and assayed for expression of hGITRL_aa52-177recombinant protein by Western blot and indirect immunofluorescence staining.(2)The hGITR_aa52-177cDNA was obtained by PCR amplification from the full-length pMD18- hGITRL-T vector,and subcloned into the pDisplay expression plasmid.The pDisplay-hGITR_aa52-177vector was transformed into HEK-293 cellls with cationic lipid,and the target protein was identified by Western blot.(3)In this test,Dh8/Dd9 mAb recognize the different hGITRL antigenic epitopes,Dd9 mAb was used as capture antibody,and biotin conjugated Dh8 mAb was used as detection antibody.There was six-step procedure:1)coated the microtiter plate wells with capture antibody;2) blocked all unbound sites to prevent false positive results;3)added antigen to the wells;4)added biotinylated antibodies detection antibody;5)added avidin-HRP;6)added substrate solution and read the optical density at 450nm.Results:(1)The recombinant pFastBacHTa-hGITRL_aa52-177vector and the recombinant Bacmid-hGITRL_aa52-177vector were constructed successfully. The recombinant hGITRL_aa52-177protein was about 18KD,which was revealed by SDS-PAGE assay,and confirmed to be target protein by Western blot and indirect immunofluorescence staining.(2)After constructing the recombinant pDisplay-hGITRL_aa52-177vector successfully,we transfected this recombinant vector into HEK-293 cells. After 48 hours post transfection,we collected the culture supernatants and assayed the expression of hGITRL_aa52-177protein by Western blot.(3)The Dh8 mAb was successfully labeled with BNHS.Using Dd9 mAb as capture antibody,Bio-Dh8 mAb as detection antibody,we have detected some linearly dependence between the absorbance and the GITRL concentration.Still this approach needs to enhance the stabilization.Condusion:In this study,we employed Bac-to-Bac baculovirus expression system and pDisplay vector,prepared our recombinant hGITRL_aa52-177protein in eukaryotic cells.We also started to grope how to develop BA-ELISA kit to detect GITRL protein.Our studies may lay a foundation for the further exploration of its biological function and clinical application.