Preparation Of Genetic Recombinant Human FasAD Peptide And The Primary Study Of Its Immunosuppression In Graft-Versus-Host Reaction

Studies of GVHD (graft versus host diease) focus on the molecular immune mechanism recently. Evidence suggested that the pathway of Fas/ Fas ligand(FasL), specially the increasing FasL takes a important role in the process of GVHD in animal model. Our study in 20 patients undergoing HSCT support it. Our studies shows that the expression of FasL in patients undergoing allo-HSCT increased significantly compare to that of patients undergoing auto-HSCT (P <0.05) , and the expression of FasL in patients suffered II degrees acute GVI-ID increased significantly compare to that of patients sufferd 0?I degree acute GVHD (P<0.0l) , and the expression of FasL on T lymphocytes has positive correlation with the acute GVHD. Therefore, it may be a new strategy to blockade the pathway of Fas/ FasL to relieve the GVHD. Fas activation domain is the important domain mediated the recognization and activation to Fas by FasL. FasAD shared by Fas soluble variants can competitively bind to the FasL to prevent cells death in vitro. Thus we have the plan of preparation of the lower molecular weight peptide according to the FasAD by the recombinant technology used as blockage to apply to the biological immune therapy of GVHD. We designed two half of primers according to the sequence of Fas cDNA searched from the Genebank. Then, we cloned the FasAD cDNA by the method of seminested RT-PCR and genetic recombination. DNA sequence analysis verified that the base pairs were identical with that of Fas. And then we constituted the recombinant plasmid expressed intein protein of FasADN-pTYB2 which allow the fusion of target gene to the N-terminal of intein and have constituted the recombinant plasmid of FasAD~-pTYB 12 which allow the fusion of target gene to the C-terminal of intein. These two recombinant plasmids were transformed into E.coli ER2566 strain. Two clones of correct target gene insert were identified by the PCR and digestion of restriction enzymes. The recombinant plasmids successfully expressed the soluble fusion protein induced by IPTG which the molecular weight is estimated as 6OKD. The expression of recombinant protein by FasADc-pTYB 12 plasmid is higher than that of by the Fa5ADN-pTYB2 plasmid. We used a self-cleavable affinity tag to complete one-step purification of FasAD~ and FasAD~ peptides through the low pressure liquid chromatograph. The recombinant proteins and FasAD peptides were identified by the western-blot with the rabbit anti-human Fas antibody. It indicats that the IMPACT system can be used to preparation of low molecular peptide . The C-terminal fusion is more suitable for large scale production of recombinant proteins. We investigated the basic biologic activities of FasAD peptides in vitro. When apoptosis was induced in Jurkat cells by the rhFasL, the apoptosis inhibition could be occur in presence of either the Fa5ADN or FasAD~ peptide(P<0.0l) . It suggests that either the Fa5ADN or FasAD~ peptide can competitively bind to the FasL so as to prevent the apoptosis induced by FasL. The apoptosis could be induced in Jurkat cells by the FasL+ lymphocytes, which we used it as the model of GVH reaction mediated by the FasIFasL. The apoptosis inhibition could aslo be occur in presence of either the Fa5ADN or FasAD peptide(P<0.05) . It suggests that the FasADN or FasAD peptide has the activity of immunosuppression to GVH reaction in vitro. There is no significant dissimilarity between the Fa5ADN peptide and...