Expression And Purification Of Human SDF-1α In Prokaryotic Cells And Its Regulative Role In Murine Bone Marrow Hematopoiesis

Cancer is still the leading cause of human death. Radiation and chemotherapy is still the most used therapy to treat cancer. However, their hematopoietic and immune suppression are the most life threatening side-effects. Treating and managing these side-effects of radiochemotherapy have becoming the active field for cancer therapy since the introduction of the therapies.Hematopoietic stem cell transplantation has been used to treat the chemotherapy induced bone marrow suppression. However, patients who receive stem cell transplant need long time to recover their hematopoietic and immune Function. Anti-infection drugs and blood transfusion to stop bleeding are frequently used during transplantation. Most of the patients could not afford the high cost of the transplantation.Chemokines and their receptors have been revealed to regulate hematopoiesis. We have identified one of the chemokines, stromal derived factor-1 (SDF-1), as a potential regulator for BM regeneration. The expression pattern of SDF-1 during chemotherapy induced BM regeneration was found to be related to the recovery of the damaged BM, which is in agreement to the results of SDF-1 and its receptor CXCR4 knock-out mouse. The knock-out mouse showed suppressed lymphopoiesis and myelopoiesis. In my thesis, I plan to produce human SDF-1 recombinant protein (rhSDF-1) and to investigate its effects on hematopoiesis and its possible therapeutic value in treatment of chemotherapy induced BM suppression.I cloned the human SDF-1 (hSDF-1) full length cDNA, and constructed the gens into the prokaryotic expessing plasmid pET-28a. The plasmid was induced to express the rhSDF-1 in E. coli. After denaturation and renaturation, the recombinant protein was purified by ion-change chromatography. The biologic activity and endotoxin level of this recombinant protein were measured. I injected this protein into wild type mice and mice treated with 5-fluorouracil (5-Fu) to investigate its regulatory roles of BM regeneration. The main results and conclusion of our work is as follows: First, we got the hSDF-1αgene fragment from human bone marrow cDNA library. After agarose gel electrophoresis, we saw the gene fragment 213 base pairs. The gene fragment was cloned into pET28a(+) to construct recombinant plasmid, that was transformed to non-expressing bacterial DH5α. PCR method was used to select the right clone, sequencing result showed that it was indentical to hSDF-1αgene encoding the mature protein.The recombinant plasmid pET28a(+)-hSDF-1αwas transformed into E.coli BL21(DE3). The protein expressing condition was optimized, that was induced overnight in 37℃, and the LB culture medium contained 100ug/mL Kana, IPTG concentration was 1mmol/L. After induced by IPTG, the protein was found mainly in inclusion bodies, and its molecular weight was 8 kDa. The E. coli were centrifuged, sonicated and washed for three times. We used 8M urea (pH 11.0) to denature the inclusion bodies, then the proteins were renatured with renature solution (pH 11.0). At last, the protein solution was obtained after anion exchange chromatogram. The protein concentration was 0.67mg/mL measured by BSA protein quantify method, and the protein purity was more than 95% analyzed by SDS-PAGE. The endotoxin and biologic activity of this recombinant protein were also measured. The endotoxin content was less than 1EU/ug by endotoxin measure kit. The protein has chemotaxis Function to T cell of mouse spleen. We injected the protein into wild type and 5-Fu treated mice to study their effects on BM regeneration. In wild type mice model, rhSDF-1 significantly increased the number of BM cells. Pretrement of mice with rhSDF-1 before 5-Fu injection led to increased mortality, which indicated the activation of BM hematopoiesis by rhSDF-1; injection of rhSDF-1 after 5-Fu treatment delayed the peripheral blood cell recovery, suggesting that the exogenous rhSDF-1 played a negative role in the repair of BM damage. The results of this study have layed foundation by providing valubel recombinant proteins and functional analysis for the future study of regulatory roles of SDF-1 during BM regeneration.