Molecular Design And Biological Functions Of SCF-TPO Fusion Protein In High Efficiently Promoting Thrombopoiesis.

Thrombocytopenia is not only a common medical problem during the induction, intensive, and consoledation therapy of patients with hematologic malignancies including leukemia, lymphoma, and mutiple myeloma, but also a common problem encounted with conventional radio and chemotherapy regimen usted to treat solid tumors and other diseases inflecting hematopoiesis.Thrombocytopenia increases the risk of mortality and morbidity caused by bleeding and infectious diseases, and limits the maximal uses of cytotoxic chemotherapy medicines. Thrombocytopenia associated with the use of new powerful chemotherapy agents such as gemcitabine may limit their use in patients with lung, ovarian, or breast cancer. Thrombocytopenia is also a frequent problem in the treatment of nonchemotherapy patients with myelodysplasia syndrome(MDS), idiopathic thrombocytopenic prurua(ITP),chronic liver disease, and acquired immunodeficiency syndrome(AIDs). The chronic thrombocytopenia during these diseases results from defective or diminished platlete production or enhanced immunologic and nonimmunologic platelet destruction and may be associated with abnormal platelet function. Additionally, patients undergoing liver transplantation, cardiovascular often experience severe thrombocytopenia that results in increased mortality. The treatment of thrombocytopenia related to chemotherapy is not resolved and remains a unsettled obstacles worldwide.Platelet transfusion is currently the only treatment for severe thrombocytopenia. Although temporarily effective in controlling severe thrombocytopenia, but many times of platelet transfusion is also associated with severe medical problem, including transmission of infectious disease, febril reactions, alloimmunization, and a graft-versus-host diseases, and contributes to increased health care costs and patient inconvenience. The limited supply of blood products can also limit the platelet transfusion. Furthermore, the more and more use of dose-intensive chemotherapy regimens and hematopoietic stem celltransplantation aimed to elimate the leukemia and other cancer cells has resulted in an increasing demand for platelet problems. The limitations of platelet transfusions and the increased costs with the complications of such transfusions have prompted a search for growth factors that stimulated platelet production, thereby reducing or eliminating the need for platelet products. The successful clone of tpo and scf gene gives us the hope to resulve the medical problem of platelet transfusion completely.Cytokine is a kind of proteins or peptides with a high activity which is produced by hematopoietic cells and immune cells. Once binding its receptor on the cell surface, cytokine can mediate the proliferation, differentiation of its target cells, and also mediate some physical and pathological processes, such as hematopoiesis regulation and immunoresponsiveness. Based on the network effects of the cytokines, researchers have designed and constructed by genetic engineering and protein engineering techniques some novel cytokines comprising dimeric cytokine proteins, which exhibited the biololgical effects of both its component cytokine, and better bioactivities by either complementation or synergy effects of single cytokine. These fusion proteins could be less side effects and more active, and could be used for the researches on the immune regulation as well as the clinical applications. Up to now, many kinds of cytokines fusion proteins have been constructed with the efforts of many researchers worldwide, and many of these fusion proteins ,included GM-CSF/EPO fusion protein, referred to as MEN11303, GM-CSF/IL-3, referred to as PIXY321, and IL-3/EPO, have completed phase I/II trial, which are well tolerated and merits consideration as a potential therapy. It can be prophesied that cytokine fusion protein could be one of topics of intense study in the fields of today immune reaseach.TPO gene localized to human chromosome 2q26 or 3q27, with a total 70KD molecular mass. The cloned human TPO cDNA encodes a polypeptide of 353 amino acids, including a 21 amino acids secretory leading signal sequence. TPO gene has 5 exons which enconded a protein of 332 amino acids. The structure of thrombopoietin suggests two distinct domains of the protein: an amino-terminal receptor binding domain, and a carboxyl-terminal region lacking obvious secondary structure termed the thrombopoietin glycan domain. The amino terminal 154 residues of TPO share 23% sequence identity to human EPO and lesser homology to the interferon. This domain is also most highly conserved between the homologues of TPO and its function is adequate for receptor binding, signaling and supporting cellular proliferation. Nearly half of the 70KD molecular mass, the carboxyl terminal domain bears no homology with otherknown proteins, but is rich in serine, threonine, and proline residues. The TPO glycan domain also functions to increase the circulatory survival of the protein. Two disulfide bonds are formed in TPO between Cys7 and Cys151, and between Cys29 and Cys85. Both of the disulfide bonds of thrombopoietin are essential for its function. The physiologic functions of thrombopoietin is the principal regulator of megakaryocyte maturation, platelet production, and also a potent inducer of proliferation of megakaryocytic progenitor cells. In addition to its effects on megakaryocytes and their immediate precursors, TPO can support the survival of hematopoietic stem cells and acts in synergy with IL-3 and stem cell factor to incuce these cells into the cell cycle and increase their output of primitive and committed hematopoietic progenitor cells of all lineages.Stem cell factor(SCF), also called c-kit ligand, is a hematopoietic growth factor that acts on both primitive and mature progenitor cells. Human SCF is encoded by a gene located on chromosome 12q22-24,. The SCF gene is more than 40kb in length and consists of eight exons. The gene is alternatively spliced at exon6, encoding two isoforms of SCF: SCF248 and SCF220. SCF248 contain a specific proteolytic cleavage site at Ala165, which generates a soluble isoform, 164 amino acids in length. SCF220 lacks the proteolytic cleavage site and so forms transmembrane protein that consists of a 157 amino acid extra cellular domain, a 27 amino acids transmembrane domain and a 36 amino acids cytoplasmic tail. Both soluble and transmembrane forms of SCF are biologically active. In synergy with other hematopoietic growth factor EPO,GM-CSF, IL-3 so on ,SCF supports the colony growth of BFU-E, CFU-GEMM in semisolid media. There are three clinical areas where SCF has been used today: in vivo and ex vivo expansion of stem cells; stem-cell mobilization; and aplastic aneamia.In the present study, we constructed expression vector encoding SCF-TPO fusion protein. By collecting and purifying these expressed fusion protein, we investigated its hematopoiesis.First, two primers were designed according to known sequence of TPO an SCF to amplify the functional amino acid domain of TPO and SCF by RT-PCR, respectively from fetus hepatic cells. The coloned TPO and SCF functional segment was fused to the sall-Notl site in expression plasmid pET32a by VOE gene fusion technique. The cells hosting recombinant plasmid was induced for 4h with 0.5mM IPTG ar 37°C after the OD600 reaching 0.6. Induction in this method, the fusion protein is nearly expressed in inclusion body and takes more than 30 percents of the totally expressed protein. SDS-PAGE results showed that there existed an additional 60KD fusion protein compared with the uninduced and mock BL21 host cells. By anti-SCF and anti-TPO multiplecolony antibody, the western blot assay showed positive band at 60KD. A remarkable SCF-TPO fusion protein was obtained successfully.Then, We successively set up an effective downstream purification line named metal chelating chromatography method in which the aimed fusion protein can be purified in high speed and high efficiency. It is interesting that we have selected pET32a as an expression vector because it has a 6 histidines in the N terminal of the aimed fusion protein, which allows us to purify the fusion protein by metal chelating chromatography easily. By this method we obtained the more than 90% expressed fusion protein with a high total recovery rate.Finally, we investigated the biological functions of SCF-TPO fusion protein in vitro. The molecular biology characteristics of SCF-TPO fusion protein, such as flexibility, antigenicity, hydrophilicity, and secondary structure, were predicted by using a computer software. The results showed the fusion protein reserved both SCF and TPO amino domain biology property. Otherwise, the linker inserted in the fusion protein had low antigenicity, low epitope, and high flexibility. As a rhGM-CSF cytokine-dependent human erythrolekemic cell line, MO-7e cells were employed to determine the activity of SCF-TPO fusion protein by MTT method. Like GM-CSF, the fusion protein could stimulate the proliferation of M0-7e cell. Further study revealed that rhSCF-TPO fusion protein could maintained CFU-MK for 3 weeks with 20%FCS present in semi-solid culture.In conclusion, we have successfully constructed expression vector encoding SCF-TPO fusion gene, and obtained the expressed fusion protein effectively.By the hematopoiesis assay, we concluded that the fusion SCF-TPO protein have similar biological function of SCF and TPO. Like GM-CSF, SCF-TPO fusion protein can improve proliferation of hematopoietic cells. Our results demonstrated that the SCF-TPO fusion protein is a functional cytokine which can be used to promote hematopoiesis.