Molecular Cloning, Expression And Preliminary Research In The Fuction Of Human Glucocorticoid-induced Tumor Necrosis Factor Receptor

Objective: Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a typeâ… transmembrane protein with homology to TNF receptor family members. GITR is expressed at low levels on resting CD4⁺ and CD8⁺ T cells and constitutively at high levels on CD4⁺CD25⁺ regulatory T cells (Tregs). Ligation of GITR provides a costimulatory signal that enhances both CD4⁺ and CD8⁺ T cell proliferation and effector functions, and neutralization signal that reverses the suppressive activity of Tregs. In this study, the full-length of human GITR(hGITR) cDNA was cloned, the extracellular region of hGITR (hGITR_aa27-165 ) fusion protein was expressed and purified, and polyclonal antibody (PAb). against fusion protein was prepared. Subsequently, the effects of anti-hGITR_aa27-165 PAb on the proliferation of CD4⁺T cells was investigated.Methods:1) Total cellular RNA was extracted from the peripheral blood mononuclear cells of normal adult, and the full-length of hGITR cDNA obtained by RT-PCR was cloned into pGEM-T vector. The pGEM-T-hGITR vector was transformed into B. coli DH5αand identified by restriction digestion and DNA sequencing.2) The hGITR_aa27-165 cDNA was obtained by PCR amplification and inserted into expression vector pQE30 plasmid. The pQE30-hGITR_aa27-165 vector was transformed into B. coli JM109 and identified by restriction digestion and PCR amplification.3) The pQE30-hGITR_aa27-165 expression vector was transformed into E. coli M15．The His-hGITR_aa27-165 fusion protein was expressed in E. coli M15 with Isopropylβ-D-1-thiogalacto-pyranoside (IPTG), and identified by SDS-PAGE. The target fusion protein was purified by IMAC column, and then identified by Western Blot.4) The rabbit was immunized by the target fusion protein combined with the Freud' s complete adjuvant. Anti-hGITR_aa27-165 PAb was obtained from the rabbit, and purified by Protein A Sepharose column. The titer of purified PAb was detected by enzyme-linked immunosorbent assay (ELISA), and the PAb specificity was analyzed by Western Blot and flow cytometry (FCM). The proliferation reaction of CD4T cell stimulated by anti-hGITR_aa27-165 PAb was detected by incorporating ³H-TdR.Results:1) The pGEM-T-hGITR plasmid was successfully constructed and the inserted target gene shared 100% homology with the sequence of mRNA for human GITR in Genbank.2) The prokaryotic expression vector pQE30-hGITR_aa27-165 was efficiently transformed into E.coli M15．The fusion preotein was expressed in E. coli M15 at 30℃after 0．5mmol/L IPTG induction for 6 hours. The hGITR_aa27-165 fusion protein was effectively expressed in E. coli as inclusion bodies and denaturation and refolding procedure was performed to acquire soluble protein. The fusion protein was conveniently purified using Profinity IMAC Ni-Charged Resin affinity column with above 90% purity.3) The anti-hGITR_aa27-165 PAb was prepared by immunization rabbit with purified target fusion protein. The titer of PAb was 1: 1．6×10⁵by ELISA method, meanwhile, the specificity and reactivity of anti-hGITR_aa27-165 PAb was satisfactory by Western Blot and FCM. The proliferation ability of CD4⁺T cells increased during being stimulated by anti_hGITR_aa27-165 PAb.Conclusion:The fusion protein hGITR_aa27-165 was obtained from E. coli, and anti-hGITR_aa27-165 PAb was successfully prepared. The anti-hGITR_aa27-165 PAb could increase the proliferation ability of CD4T cell in vitro. The fusion protein hGITR_aa27-165 and anti-hGITR_aa27-165 PAb laid a foundation for better understanding of the molecular mechanism of interaction between GITR and GITRL.