| The stem cell factor (SCF), mainly produced by bone marrow stromal cells, is an important hematopoietic growth factor. Used with or without other cytokines, SCF acts directly on hematopoietic stem cells, mast cells, cancer cells, melanocytcs, germ cells and induces the survival, proliferation and differentiation of these cells. SCF/c-kit signaling pathway may constitute an attractive target for the treatment of cancers and play a protective role in neural injury and radiation damage.In the peculiar respect of the kind, the homology of SCF of people and mouse is up to 83%. Though people's SCF have a little effect on hyperplasic of mouse's hematopoietic stem cell, the mouse SCF function on people's hematopoietic stem cells is similar to people's SCF. Natural SCF is mainly produced by bone marrow stromal cells; there are few sources, difficult with the extensive purification. But the biological activation of SCF does not depend on glycosylation, so prokaryotic expression system is a kind of effective means to offer a large number of SCFIn this research, we used the mouse embryo which had been developed to ten days, and extracted the total RNA from it. We adopted RT-PCR method to get mouse cDNA of SCF from mouse embryo total RNA, and then cloned them into pGEM-T plasmid to become pGEM-T-SCF. Used BamHI and XhoI to cut mouse cDNA of SCF from pGF.M-T-SCF, and cloned them into the downstream of GST gene according to the right open reading frame in pGEX-4T-3 vector to become expressing plasmid pGEX-4T-3-SCF, and then the recombinant was transformed into host E.coil BL21 which were induced and expressed under 5mM IPTG and 35 ℃. The expression of rmSCF was identified by SDS-PAGE and Western-blot, and found a 53KD according to a GST fusion protein.A liter of bacterial culture was induced in this research, and been broken by means of ultrasonic process. After centrifuged, the supernatant of lysate was obtained. When the supernatant of lysate was loaded on affinity column, rmSCF was absorbed by Glutathione Sepharose-4B, and was digested directly on column using thrombin. In the end, the column was washed with PBS which was five times as much as column volume to get rmSCF. As a result, 80% of the purity of recombinant mSCF was obtained, which molecular weight is 27kP. Because of GST and impurity proteins in the purified rmSCF, the purified rmSCF was carried on Glutathione Sepharose-4B affinity column again in order to get fid of GST and the impurityproteins. The purity of rmSCF can be up to 85% after 5 times of Glutalhione Scpharose-4B affinity chromalography. For improving purity further, ion exchange chromatography was used, which is Q-Scpharosc column. Recombinant mSCF that had been up to 85% of the purity was carried on Q-Sepharose column. Balancing buffer is 10mmol/L Tris- HCL, pH 7. 2; eluting buffer was 10mmol/LTris- IICL pH 7. 2 and lmol/L NaCL Eluted by the gradient, recombinant mSCF was obtained as the ilensity of salt of eluting buffer was 0.36mol/L, and then separately with 10000Don molecular weight and 30000Don molecular weight membrane, rmSCF was further purified by ultra filtration. The purity of final product was over 90%.Used rmSCF with rhGM-CSF to stimulate the bone marrow cells clones and detect the activity of SCF. The result showed that, using50 ng/ml, 100 ng/ml, and 200 ng/ml of rmSCF separately, the number of clones stimulated by rmSCF with rhGM-CSF was two times more than the rhGM-CSF only.Conclusion: obtained rmSCF, then set up a high efficient expression and purification method of the soluble protein. The expressed recombinant protein can be used in cell culture.
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