Construction And Expression Of A Fusion Protein Containing The Extracellular Domain Of Human Jaggedl And Fc Fragment Of Human IgG1

Research background Hematopoietic stem cells transplantation is an efficacious therapy in patients with leukemia or other solid cancers, as well as some severe inherited immunodeficiency diseases and radiation diseases. For the limited resources of hematopoietic stem cells, More than 50% patients can not benefit from hematopoietic stem cells transplantation. With the development of stem cell biological project, stem cells are expanded ex vivo for clinical medicine. The classical method is to use several optimal cytokines combination for hematopoietic stem cells expansion in vitro. Experiments show that the self-renewal potential of hematopoietic stem cells in such culture systems decreases, but hematopoietic stem cells become lineage-committed. It is important to mimic hematopoietic microenvironment for inhibiting differentiation of hematopoietic stem cells in hematopoietic stemcells expansion in vitro.HSCs are those that can self-renew and generate differentiated progeny. The mechanisms that govern these cells fate decisions are unclear so far. Perhaps the key aspects of stem cell regulation are likely to be the interaction between adjacent cells as well as cytokines and their receptors. Notch signaling pathway is evolutionarily conserved from Caenorhabditis elegans to human. Notch is typically involved in binary cell fate decisions. Notch regulates development of many cells, tissues and organs. Jaggedl is one of Notch ligands. It comprises 1218 amino acids, and it is one of transmembrane protein. Its extracellular fragment has 3 distinct regions: 16 EGF-like repeated sequences, a cysteine-riched DSL(Delta-Serrate-Lag-2) motif, a cysteine-riched domain. The DSL motif plays key function in interaction between jaggedl and Notch. Recently, it is discovered that Notch receptors and its jaggedl ligand are expressed on the surface of the HSCs and bone marrow cells respectively, which denotes that Notch signaling plays a fundamental role in determining bone marrow hematopoietic precursor renewal and differentiation. Some researches show that Notch inhibits HSCs differentiation but promotes expansion.Experiment aim In order to study the function of jaggedl in HSCs proliferation in vitro, plasmids comprise human Jagged1ext-Fc fusion gene are to be constructed, and the fusion protein are to be expressed in eukaryotic cells.Experiment methods The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. In order to obtain Jagged1ext -Fc fusion gene, the extracellular domain of Jaggedl gene was inserted into pBluescript-sk II -hcyl, and then the fusion gene was inserted into pEF-BOSneo to get the eukaryotic expression vector pEF-BOSneo-hJagged1ext-Fc. The recombinationplasmids were transiently transfected into COS7 cells and the expression of the fusion protein was identified by RT-PCR, immunofluorescent assay and sandwich ELISA. The recombination plasmids were stablely transfected into X63 cells in order to obtain large amount of fusion protein.Experiment results The extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmids containing hJagged1ext-Fc fusion gene were the same as that designed. The fusion protein was successfully expressed in mammalian cells and protein-expressing cell lines were obtainedExperiment conclusions The extracellular domain of human Jaggedl gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, for example, the Jagged1ext-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.