Expression Of Mouse Glucocorticoid Induced Tumor Necrosis Factor Receptor-Related Protein And Its Function On Experimental Autoimmune Thyroiditis

ObjectiveThe aim of this study is to clone the full-length gene of mouse GITR (mGITR) and construct two prokaryotic expression plasmids, containing mGITR extracellular domain alone or mGITR extracellular domain-mIgGFc binary. After the two prokaryotic expression plasmids are expressed in E.coli and the expression products are purified, the biological activities of these two prokaryotic proteins will be detected in vitro. In addition, the function of mGITR-Fc fusion protein on experimental autoimmune thyroiditis (EAT) will be evaluated and the potential mechanism is analyzed. It also contributes to understand the role of GITR in immune related diseases.Methods(1) The full-length gene of mGITR was obtained from mouse spleen leukomonocytes by RT-PCR, cloned into pMD18-T vector, transformed into E.coli DH5a, and then identified by sequencing. In addition, the amino acid sequence of GITR was analyzed by bioinformatic methods using the network platforms and some bioinformatic softwares.(2) The extracellular domain of mGITR and mIgGFc genes were amplified by PCR and then they were cloned into pMD18-T vector, respectively. After sequenced, mGITR extracellular domain was sub-cloned into pET32a(+) vector to construct the plasmid pET32a-mGITR ectodomain. Another plasmid pET32a-mGITR ectodomain-mIgGFc was also constructed by the method of enzyme digestion and ligation.(3) The two plasmids, including pET32a-mGITR ectodomain and pET32a-mGITR ectodomain-mIgGFc were transformed into E.coli Rosetta, respectively, and the mGITR and mGITR-Fc proteins containing His tag were then obtained and purified by nickel column. The two proteins were identified by Western Blot.(4) The mouse CD4+T cells from spleen were sorted by magnetic beads, and cultured into the96-well cell culture plates with the same amount cells. After the same quantity of mGITRL was added into the plates, the two different prokaryotic proteins were added at the indicated concentrations, respectively. Then the blocking function of the two prokaryotic proteins on the proliferation of CD4+T cells stimulated by mGITRL was deteced by3H-TdR incorporation assay. In addition, the dendritic cells were treated with mGITR-Fc fusion protein in vitro, and the expression of mGITRL on the surface of the dendritic cells was detected.(5) EAT mouse models were constructed and interfered by mGITR-Fc fusion protein. The function of mGITR-Fc on the disease development of EAT was detected by the methods of hematoxylin and eosin staining, flow cytometric analysis and so on.Results(1) The full-length of mGITR gene was amplified and cloned into the cloning vector successfully. The recombinant plasmid was named as pMD18-T-mGITR. The results of bioinformatic analysis showed that the extracellular region of mouse GITR was the potential functional domain with the high hydropHilicity, high accessibility and high polarity. These characters could provide a basis for the cloning, expression and functional research of mouse GITR.(2) The two plasmids were constructed and identified successfully, including the plasmids pET32a-mGITR ectodomain and pET32a-mGITR ectodomain-mlgGFc.(3) mGITR protein and mGITR-Fc protein containing His tag were expressed in E.coli Rosetta and purified by nickel column successfully. They were identified by SDS and Western Blot. (4) The interaction between mGITRL and mGITR expressed on CD4+T cells could be blocked by the two prokaryotic proteins, and the proliferation of CD4+T cells induced by mGITRL was inhibited obviously by mGITR or mGITR-Fc fusion protein in a concentration-dependent manner. In addition, when the dendritic cells were treated with mGITR-Fc fusion protein, the expression of mGITRL on the surface of the dendritic cells was down-regulated.(5) EAT mouse models were constructed successfully. The mGITR-Fc fusion protein could delay the disease development of EAT. The results showed that the level of mTgAb was down-regulated in the serum from mGITR-Fc fusion protein-treated mice and the inflammatory cell infiltration was also decreased in the thyroids from mGITR-Fc fusion protein-treated mice.ConclusionThe full-length and the extracellular domain of mGITR gene were cloned successfully. The prokaryotic plasmid of pET32a-mGITR ectodomain and the prokaryotic plasmid of pET32a-mGITR ectodomain-mlgGFc were constructed successfully. mGITR protein and mGITR-Fc protein were expressed and purified successfully. The interaction between mGITRL and mGITR expressed on CD4+T cells could be blocked by mGITR protein or mGITR-Fc protein and the proliferation of CD4+T cells induced by mGITRL could be inhibited. The expression of mGITRL on the surface of the dendritic cells was down-regulated by mGITR-Fc protein. The function of mGITR protein was partly dependent on GITR/GITRL co-stimulation signaling and partly dependent on the reverse signaling. The mGITR-Fc fusion protein could delay the disease development of EAT.