[Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage].

Objective: To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage.
Methods: PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni ²⁺ -beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin ^- Scal-1 ⁺ c-Kit ⁺ cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10.
Results: The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury.
Conclusions: A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.