| Midkine (MK) is one of the heparin-binding growth factors that strongly expressed during embryogenesis, while the level and distribution is restricted in adults. Multiple kinds of MK receptors attribute to its various functions via different downstream signaling pathways. MK has an anti-apoptotic activity and is known to promote the growth, survival, differentiation and migration of many cell types. It is vigorously involved in development and regeneration of normal tissues, as well as in tumorigenesis. Now Midkine has been the gene target in the treatment of many diseases including malignant tumors.According to the gene expression profile analysis during murine bone marrow regeneration after chemotherapy, the expression pattern of Midkine during chemotherapy induced BM regeneration was found to be related to the recovery of the damaged BM. The purpose of this project was to produce human Midkine recombinant protein (rhMidkine, rhMK) and to investigate its effects on hematopoiesis.We used a bacteria expression system to produce non-tagged rhMK. The coding sequence for hMK was cloned into the pET-30a (+) vector and transformed into Escherichia coli (E. coli) BL21 (DE3). Expression of rhMK was induced by IPTG. Bacteria inclusion bodies containing the protein were isolated and washed to remove contaminated bacteria proteins, and then resolved in guanidine hydrochloride (GdnHCl) buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using Sp-Sepharose cation exchange chromatography. The identity of the purified protein was confirmed through Western Blotting. The biologic activity and endotoxin level of this purified recombinant protein were measured. Then we injected this protein into wild type mice to investigate its regulatory roles in BM hematopoiesis.The main results and conclusions of our work are as follows: we got the hMidkine gene fragment from human bone marrow cDNA library. After agarose gel electrophoresis, we saw the gene fragment 380 base pairs. The gene fragment was cloned into pET30a (+) to construct recombinant plasmid, which was transformed to non-expressing bacteria DH5α. PCR method and double enzyme digestion were used to select the right clone. Sequencing result showed that it was identical to hMidkine gene encoding the mature protein.The recombinant plasmid pET30a (+)-hMidkine was transformed into E. coli BL21 (DE3). Then we optimized the expression of rhMK, and decided that the induction of rhMK expression was performed at 42℃for 3 hours, with 1mmol/L IPTG and 100ug/mL Kana in the LB culture medium. After induced by IPTG, the protein was found mainly in inclusion bodies, and its molecular weight was about 16 kDa. The collected bacteria went through sonication and lysozyme digestion. Then we used 6M GdnHCl to resolve the inclusion bodies, the denatured proteins were renatured with renaturing solution (pH 7.4). At last, the refolded protein was purified using Sp-Sepharose cation exchange chromatography. The final preparation of the purified protein was 0.8mg/mL measured by BSA protein quantify method. Silver staining and SDS-PAGE showed the protein purity was above 99%. The endotoxin content was less than 0.2EU/ug as measured by Quantitative Chromogenic End-point TAL. The rhMK protein promoted the proliferation of NIH3T3 cells in a concentration-dependent way measured by methylthiazoleterazolium (MTT) assays.Then we injected the rhMK protein into wild type mice to study its effects on BM hematopoiesis. After continuous administration of rhMK to mice, the number of White Blood Cells (WBC) and Platelets (PLT) significantly increased, while the number of Bone Marrow cells (BM) significantly decreased. From these results, we came to a conclusion that the exogenous rhMK might promote the migration of BM cells to peripheral blood (PB), and rhMK might mobilize BM hematopoietic stem cells (HSC) to peripheral blood. The results of this study have laid solid foundation for the future study of regulatory roles of Midkine on bone marrow hematopoiesis. |
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