The Expression Of Notch Ligand Delta-like1, Delta-like4 And The Detection Of Protein Activity

The Notch signaling pathway, which is conserved during evolution from worm to man , has multiple and essential roles in embryogenesis, hematopoiesis and cancers. During mammalian hematopoiesis and lymphopoiesis, Notch signaling has multiple functions. Recently, it is discovered that Notch receptors and its ligands 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 can not only inhibits HSCs differentiation but also promotes their expansion. These include regulation of T-cell commitment from a common lymphoid precursor and restrain the development of B-cell, regulation of T-cell development and marginal zone B-cell development. However, Notch signaling must be precisely regulated, as constitutive Notch signaling leads to T-cell malignancies. In the study of cell proliferation, differentiation and apoptosis in vitro, researchers always activate Notch signaling by Notch ligand. And Notch ligands expression system can classify into eukaryotic and prokaryotic expression systems. The eukaryotic expression system is mostly mammalian cell lines, such as CHO, COS7. However, the mammalian expression system could not be applied extensively because of the expensive cost, strict manipulation and low expression. The prokaryotic expression system has no post-translational modification system and the exogenous proteins always emerge in the inclusion body, complicating the course of renaturation and purification. Pichia pastoris expression system has the advantages of mammalian and prokaryotic expression systems: post- translation system, low cost, high production, etc. Therefore, we choose the Pichia pastoris expression system firstly, then the prokaryotic expression system which comes to a soluble expression.Several experiments were included as follows:1. Expression recombinant human Delta-like1ext-Fc (hDll1ext-Fc) in Pichia pastoris. Got hDll1ext fragments by PCR, the Fc fragment from pET32a -Fc. Constructed expression vectors pPIC9K- hDll1ext -Fc and pPIC9K-Fc. Screened the transformants by the histidine-deficiency and G418 media, and induced the expression of positive transformants with methanol.2. Expression recombinant hDll4 in E.coli BL21. Constructed expression vectors pET32a-hdll4ext-26-217 and pET32a-hdll4ext -93-217. Induced expression with IPTG at 25℃.3. Detected the recombinant hDll4 bioactivity though reporter assay in HeLa and COS7. Results: We got recombinant hDll1ext-Fc and human IgG1 Fc proteins in Pichia pastoris; obtained and purified a soluble recombinant protein His- hdll4ext -93-217 in E.coli BL21 at 25℃; proved His- hdll4ext -93-217 bioactivity by reporter assay.