| BackgroundWith the development of recombination technology, the completion of human genome project (HGP), gradual understand of target of many diseases and mechanism of immunology, bifunctional molecules, such as bispecific antibodies, immunocytokines and bifunctional cytokine fusion proteins, have attracted interest as immunotherapeutics in more and more diseases (tumor, autoimmune disease, viral infection) since they are specifically valuable reagents for establishing close cell contacts, for example between T cells and tumor cells, bispecific monoclonal antibodies (Bi-mAbs) which could bridge T cells to tumors cells may facilitate interaction within molecules that costimulate host immune response. So bifunctional molecules can open new ways to the treatment of cancer and viral infective disease.T cells are commonly recognized as the most potent effector cells to generate antitumor cytotoxicity. T cell recognition and activation is the core of immunologic reaction. Activation of T cells needs double signals offered by the antigen presentation cells, such as dendritic cells (DCs), as the most efficient APCs and vaccines delivery vehicles, pulsed with tumor-derived peptide, proteins, have been shown to activate tumor-specific CD8+ T cells and ruduce tumor load.The cytokines, interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF), have very strong regulation on T cells, antigen presentation cells and immunity response. So we manufacture a functional versatile fusion protein, hIL-2/GM-CSF,which may conjugate T cells to peptide-pulsed macrophages (M) or DCs by the corresponding surface receptor and form a "bridge" to facilitate interaction between T cells and APC, increase the efficiency of antigen presentation. To study this new probable biological function and the mechanisms involved in the "bridge", we carried out studies on the base of previous studies in order to be convenient for the further research.The expression system pLY-hL-2/GM-CSF,.Coil7 DH5 is not suitable for studies on the bifunctional cytokine fusion protein, hIL-2/GM-CSF's unknown functional assembly, such as its efforts on hemopoietic stem cell, dendritic cell, T cell and immunoloregulation, when output and purification were involved in. So we carried out the studies on the construction, expression and purification of hIL-2/GM-CSF in a superior expression and purification system in order to get the homogeneous bifunctional molecule free of endotoxin with high purity and specific activity and perform preliminary biological function studies on this bifunctional protein, for instance, the effect on antigen presentation efficiency and the T cell subpopulation.MethodsAccording to PHD prediction, hIL-2/GM-CSF is a globulin that composes of 268 amino acids. In this study, The fragments of hIL-2/GM-CSF gene amplified by PCR were cloned to these expression vector pET-22b(+),pET-28a(+),pET-llc. Then three constructs recombiaint strains that induced for expression of hIL-2/GM-CSF were screened for the best expression condition. After fermentation at optimal expression condition, the crude inclusion bodies was got by disrupted the cells four times at 4癈 using a high-pressure homogenizer, then the inclusion bodies was washed, denatured, refolded, the hIL-2/GM-CSF were purified by combination of anion exchange and affinity chrbmatography. After twostep purification, the purified and refolded protein was polished for endotoxin removal by being subjected to a SP Sepharose Fast Flow column. The evaluation of the recombinant fusion protein was carried out by SDS-PAGE, immunoblot, isoelectric focusing electrophoresis, lowery method, limulus lysate test and cell proliferation assay. The effect of this bifunctional protein on antigen presentation was detected by proliferation of peripheral blood monoeuclear cell(PBMC), mixed lymphocyte reacion (MLR) and the T cell subpopulation by flow cytometry.ResultsWe found that it's more suitable for the recombiaint strains, pET-llc-hIL-2/GM-CSF/? coli BL21(DE3), to be induced to express...
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