Aim: To construct a eukaryotic expression vector containing the extracellular domain of human Jagged1 and Fc fragment of human IgG1 fusion gene and to express the hJagged1(ext)-Fc fusion protein in mammalian cells.
Methods: The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. In order to obtain hJagged1(ext)-Fc fusion gene, the extracellular domain of Jagged1 gene was inserted into pBluescript-sk II-hc gamma 1, and then the fusion gene was inserted into pEF-BOSneo to get the eukaryotic expression vector pEF-BOSneo-hJagged1(ext)-Fc. The recombination plasmid was transiently transfected into COS7 cells and the expression of the fusion protein was identified by RT-PCR, immunofluorescent assay and sandwich ELISA.
Results: The extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in mammalian cells.
Conclusion: The hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, for example, the Jagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in-vitro.