Cloning, Expression And Purification Of Human Stem Cell Growth Factor-α And Its Study On Human Umbilical Cord Mesenchymal Stem Cells

Objective:1. To clone, express and purify gene encoding recombinant human Stem Cell Growth Factor-α.2. To construct and purify the cell penetrating peptides-TAT-SCGF.3. To study the possible role of rhSCGF-αand TAT-SCGF in proliferation of human Umbilical Cord Mesenchymal Stem Cells.4. To research the possible influence of rhSCGF-αon adipogenic differentiation of hUCMSCs.Methods:1. hSCGF-αgene was amplified from hUCMSCs cDNA by using two-step PCR according to the GC content and the amplified production was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E.coli BL21(DE3) was mainly existing in soluble form.The soluble protein was purified by using NI-NTA affinity chromatography. The biological activity of hSCGF-αwas assessed by the granulocyte/macrophage colony forming assay. MTT assay was used to test the proliferation of rhSCGF-αon hUCMSCs and the differention of standard control on hUCMSCs2. TAT-SCGF gene was amplified from hSCGF-αby using two-step PCR and the amplified production was inserted into pET-28a(+) plasmid vector.The fusion protein(TAT-SCGF), mainly existing in inclusion body form when induced with IPTG, was expressed in E.coli BL21(DE3).The inclusion body protein was purified by using NI-NTA affinity chromatography. We used the proliferation assay to reveal that TAT-SCGF had promoting activity for hUCMSCs.3. we added rhSCGF-αin adipogenic differentiation process of hUCMSCs.By detecting GPD, PPARγ2, marker genes of adipogenic differentiation,we researched the adipogenic differentiation influence of rhSCGF-αon hUCMSCs by Westernblot and Realtime PCR.Results:1. hSCGF-αgene was successfully amplified by using two-step PCR and was inserted into pET-28a(+) plasmid vector form to recombinant pET-28a-SCGF-α. The fusion protein, mainly existing in soluble form when induced with IPTG at 20 degrees Celsius for 24 h, was successfully expressed in E.coli BL21(DE3). hSCGF-αprotein, purified by using NI-NTA affinity chromatography, had the granulocyte/macrophage (GM) promoting activity for murine bone marrow GM progenitor with rmGM-CSF but it did not function when acting alone. The proliferation percent of rhSCGF-αon hUCMSCs was (10.61±1.09)%, and the standard was (10.18±0.96)%.2. Recombinant TAT-SCGF-28a of penetrating peptide was successfully constructed. The fusion protein(TAT-SCGF), mainly existing in inclusion body form when induced with IPTG, was expressed in E.coli BL21(DE3). The proliferation percent of TAT-SCGF on the proliferation hUCMSCs was (12.95±1.37)%.3. rhSCGF-α's influence on hUCMSCs in promoting adipogenic differentiation could be detected on the third day.Conclusion:1. TAT-SCGF and rhSCGF-α, obtained by genetic engineering technology, have biological activity. rhSCGF-α,compared to the standard, has no significant difference in biological activity.2. Both TAT-SCGF and rhSCGF-αhave proliferation effect on hUCMSCs.3. rhSCGF-αcan promote adipogenic differentiation on hUCMSCs.