| PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and containing the immunoreceptor tyrosine-based inhbitory motif (ITIM). PD-1, like CD28, CTLA-4 and ICOS, is a member of the CD28 superfamily. It has 24% homology to CTLA-4 at the extracelluar region, and it also delivers an inhibitory signal to T cells. The PD-l receptor is a 55KD type I transmembrane protein, it is mainly expressed on activated CD4+, CD8+T cells and B cells as a monomer. Its ligand PD-L1(B7-H1) and PD-L2(B7-DC) are expressed broadly on lymphoid cells and non-lymphoid cells.In vitro studies,they have shown that after the engagement of PD-L by PD-L1/PD-L2,PD-1 can inhibit the proliferation of activated T cells and reduce cytokine production,especially suppress the function of effector CD8+ T cell.PD-1-PD-Ll pathway can antagonize a stronger CD28-B7 signal after the antigen exposure.Thus,it suggests that the negative role of PD-1-PD-L 1 pathway in regulating Tcell responses.The expression of PD-L1/PD-L2 in non-lymphoid tissues indicate they can down-regulate the response of auto-reactive T,B cells in peripheral tissues.PD-1-/-mice on the C57BL/6 genetic background cause a variety of autoimmune diseases such as rheumatosis, glomerulonephritis, dilated cardiomyopathy and SLE.This opinion summarizes the loss of PD-l can lead the autoreactive T cells to activate.Studies show that engagement of PD-l can downregulate the immune response of activated T,B cells, and its biological significance in sustaining peripheral tolerance.Now PD-l becomes the study focus on immunology research for its key rolein negative signal and its effects on T cell function and memory. To further explore its regulatory mechanisms and other biological functions, it is very necessary to get souble PD-1-Fc and PD-Ll-Fc(B7-Hl-Fc) protein.It means much:on one hand,if souble PD-L1-Fc engages in its receptor ,it can enhance the negative regulatory effect,thus we can apply it to avoid transplant rejection.as the time is mature,this souble protein can be designed as a immunosuppressive drug;on the other hand,the acquirement of souble PD-l-Fc provides us a basis to study its role in immune regulation ,kinetics and signal transduction. To get this goal, we conducted the following works:1. The construction of hPD-1 eukaryotic expression plasmids(1) The total length human PD-1 cDNA was cloned from a normal human activated T cells cDNA library phAD.CAD with PCR, confirming its open reading frame is the same as the public sequence of PD-1 in Genebank by DNA sequencing.(2) To recombinant espression human PD-1 on the surface of mammal cells,PD-l cDNA was subcloned into the eukaryotic expression plasmids: pcDNA3.1(+) at HindIII and XbaI site.2. The construction of human PD-l-Fc and B7-Hl-Fc eukaryotic expression plasmidsPD-1 and B7-H1 extramembrane domain encoding region were cloned with PCR, and inserted into pcDNA3.1(+) and pCI-neo eukaryotic expression plasmids,respectively, together with human IgG1 Fc cDNA, confirmed by DNA sequencing.3. The expression and purification of hPD-l-Fc and hB7-Hl-Fc fusion protein(1) Plasmids wre transfected into mammal cells CHO by liposome transfection, proving that the hPD-l-Fc and hB7-H1-Fc fusion protein insupernatantwre detected with sandwich-ELISA(2) The hPD-1-Fc and hB7-H1-Fc fusion protein were purified by recombinated protein A affinity chromatography column,then examining its molecular weight and purity by SDS-PAGE,we found those fusion protein's MW coincided with the expected.(3) The Immunologic competence of hPD-l-Fc and hB7-H1-Fc fusion protein were proved by using Western blot.
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