| The inducible costimulator (ICOS) and its ligand GL50 are a couple of the newest member of the CD28/B7 family involved in regulating T cell activation. Ulike the constitutively expressed CD28, ICOS has to be de novo induced on the T-cell surface, while GL50 is expressed on B cells and macrophages or induced on fibroblasts etc. Present studies have demonstrated that GL50-ICOS plays a key role in inflammation regulation, B cell reaction and transplantation immunity ,which discerns a broad perspective of clinic application. However, the molecular mechanism by which GL50-ICOS involves in immune-regulation is not clearly known yet. Furthermore, studies so far on GL50-ICOS are mainly focused on mice . In view of these points, we first cloned human ICOS/GL50 gene, constructed ICOS/GL50-transfectant cells, and expressed ICOS/GL50 recombinant protein in E.coli. Then we purified ICOS/GL50 recombinant proteins with bioactivity, and used them for further biological study. Due to a kinship between the expression pattern and function of co-stimulatory molecules, the expression characteristics of ICOS and GL50 molecules on cells were studied. On the basis we focused on the role of this couple of co-stimulatory molecules on immunopathogensis of Graves'disease as well as underlying mechanism. Several of our findings are first reported. 1. ICOS/GL50 gene cloning, expression, purification of recombinant protein and biological activity in vitro1.1 ICOSThe published cDNA sequence for ICOS was used for designing primer. Full-length of ICOS was amplified through RT-PCR from activated human T cells and the cDNA fragment was cloned into T-vector plasmid. The derived plasmid(named as T-ICOS) was verified by DNA sequence analysis, and then used as template for PCR to obtain the cDNA fragment encoding extra-cellular domain of ICOS . The production of PCR was cloned into expression vector pET-28a to construct recombinant expression vector pET-ICOS. After confirmed by DNA sequencing, the recombinant expression vector pET-ICOS was transformed into BL-21 E. coli strain. Induced by l-5mmol/L IPTG, ICOS recombinant protein was expressed in BL-21 E. coli in forms of inclusion bodies. ICOS recombinant protein with bioactivity was obtained through denaturation, renaturation and purification by FPLC .1.2 GL50A specific primer was designed according to published cDNA sequence of GL50. Full-length of GL50 was amplified through RT-PCR from activated human Tonsil B cells and cloned into T-vector plasmid. The derived plasmid was verified by DNA sequencing and named as T-GL50. The ' cDNA fragment encoding extra-cellular domain of GL50 was then amplified through PCR and cloned into GST fusion protein expression vector (pGEX-5x-3). The derived recombinant expression vector (named as pGEX-5x-3-GL50) was verified by DNA sequencing and then transformed into BL-21 E. coli strain. Induced by O.lmmol/L IPTG, the target protein (GST-GL50) was expressed in soluble form. The fusion protein GST-GL50 was purified step by step through cation-exchange chromatography and affinity-chromatography according to manufactory's protocol.1.3 The biological function of ICOS/GLSO recombinant protein in vitro According to the previous reports, soluble GL50-Ig could up-regulate thesecretion of IL-10 by activated CD4+ T cells, which could be blocked by adding soluble ICOS-Ig. Therefore, the bioactivity of recombinant protein ICOS/GST-GL50 could be assayed by its effect on the induction of IL-10 by activated CD4+ T cells invitro. Our findings showed that GST-GL50 recombinant protein could strikinglyup-regulate the expression of IL-10 by activated CD4"1" T cells in vitro, while the level of IL-10 is decreased when soluble ICOS recombinant protein was added. It demonstrated that the ICOS and GST-GL50 recombinant protein obtained were of biological function. We also found that the expression level of IL-10 could further elevated by adding IL-2 or anti-CD28 agonist monoclonal antibody but down-regulated by anti-IL-2 neutralization antibody, which suggested that the f...
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