| Part I Study on receptor blocking by IgECH2-3 and mast cells apoptosis by rat lgECH2-3-FasLObjective To obtain spleen and peripheral blood of rat asthma model. To study biological activity of the rat IgE Fc CH2-3 ; To determine the surface expression of Fas Ag on rat mast cells and its function. To show whether the Fas Ligand gene induces mast cells apoptosis. To obtain rat IgECH2-3-FasL fusion protein in eukaryotic expressing plasmid. To research rat lgECH2-3-FasL fusion protein inducing apoptosis and blocking degranulation on RBL-2H3.Methods Rat was sensitized i.p with OVA and Al(OH).To transfer the pBAD/gIILA/CH2-3 which is constructed by ourselves into E. Coil ToplO. Rat IgE Fc CH2-3 was produed in periplasmic space of E. Coil ToplO by inducing it with arabinose. Biological activity was detected on RBL-2H3 and animals. RT-PCR and Western blot were used to detect the tansfection and expression of Fas in RBL-2H3. Surface expression of Fas Ag was studied by immunoichemistry .Apoptosis changes following treatment with anti-Fas antibody were analyzed using flow cytometic analysis of Annexin V. RT-PCR was used to amplify the gene of rat Fas Ligand ex trace]]uar domain and transmembrane domain, which is cloned into eukaryotic expression plasmid pcDNAS. 1. After transcently transfecting RBL-2H3, the expression of Fas ligand in RBL-2H3 was detected by RT-PCR. Western blot. The Annexin V FCM was used to detect the RBL-2H3 apoptosis after the transfection of Fas Ligand. Designing and synthesing primer was used to amplify the gene of rat IgECH2-3 and FasL then cloned it into pcDNAS. 1 one by one. Eukaryotic expression plasmid pcCDNA3. 1/IgECH2-3-FasL transcently transfect RBL-2H3, the expression of IgECH2-3-FasL in RBL-2H3 was detected by RT-PCR, Western blot. The Annexin VFCM was used to detect the RBL-2H3 apoptosis after the transfection of Fas Ligand. The realease rate of histamin was detecated to analysis the blocking role of fusion protein.Results The asthma rat was obtained. It is successful in amplifying gene of rat IgE Fc CH2-3 and cloning it into pBAD/gHIA and obtaining it by expressing, it can inhibit passive sensitization of skin oast cells in vivo and sensitized RBL-2H3 to degranulation by anti-OVA-IgE in vitro. We can see the Fas Ag expression on the surface of RBL-2H3 by immunolchemistry. RBL-2H3 exhibit apoptosis in response to anti-Fas treatment. It is successful to obtain the gene of rat Fas Ligand extracellular domain and transmeabrane segment, cloning it into pcDNAS. 1, FasL was expressed on the surface of RBL-2H3 and it' s supernatant after the transfection of pcDNAS. 1/FasL. The cell show apoptosis It is successful in amplif ing gene of IgECH2-3 and FasL , then cloning them into pcDNAS.1, obtain rat IgECH2-3-FasL fusion gene eukaryotic expressing plasmid. IgECH2-3-FasL was expressed on the surface of RBL-2H3 after the transfection of pcDNAS. 1/FasL. The cell start to he apoptosis and degranulation was blocked after the transfection of pcI)NA3.1/ IgECH2-3-FasL. Conclusions Rat IgE Fc CH2-3 can block IgE high affinity receptor-Fc t RI , inhibit the allergic reactions. Induction of rat mast cell apoptosis by activation of the Fas pathway provide an mechanism to regulate the number of the mast cells. Our study reveals that Fas Ligand gene transfection in RBL-2H3 can effectively induced apoptosis. IgECH2-3-FasL have double roles in reducing the cell apoptosis and blocking degranulation. It is a promising strategy to be used in the therapy of allergic disease. [Key words] IgE;Constant domain; Prokaryoric expression;Allergic reaction; RBL-2H3;FasL;apoptosis;transfection IgECH2-3; FasL;fusion protein eukaryotic expression; mast cellsPartllCloning and expressing extracellular domain of human Fc e R I a subunitand preparation and detecting function of the anti-serum.Objective To obtain extraceller domain of Fc ?R I a chain with biologicalactivity and its antisurm, research its function.Methods Construct the extraceller domain of Fc c R I a chain prokaryoticexpression plasmid, pBAD/... |
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