Hybrid promoter and recombinant vector comprising the same
The present invention relates to a hybrid promoter, in which a whole or a part of a CMV enhancer, a whole or a part of a β-actin promoter, a whole or a part of a CMV promoter, and a whole or a part of a β-actin intron are operably linked to each other, a recombinant vector comprising the same, a transformant transformed with the recombinant vector, a pharmaceutical composition comprising the recombinant vector or the transformant, and a method for preparing a target protein using the recombinant vector or the transformant. The hybrid promoter of the present invention is able to induce high expression of a target protein in a eukaryotic cell. Therefore, the hybrid promoter of the present invention can be effectively used for the development of an antibody or the production of a DNA vaccine.
1. A hybrid promoter comprising a cytomegalovirus (CMV) enhancer having the nucleotide sequence of SEQ ID NO:13, a β-actin promoter having the nucleotide sequence of SEO ID NO:9 or SEQ ID NO:10, a TATA box of a CMV promoter having the nucleotide sequence of SEQ ID NO:11, and a β-actin intron having the nucleotide sequence of SEQ ID NO:12, wherein the CMV enhancer, β-actin promoter, CMV promoter TATA box and β-actin intron are operably linked to each other in a 5′ to 3′ direction.
2. A recombinant vector comprising the hybrid promoter of claim 1 and a target protein-encoding gene operably linked thereto.
3. The recombinant vector according to claim 2 , further comprising one or more expression regulatory elements selected from the group consisting of a replication origin, a selectable marker, a reporter gene, a terminator and combinations thereof.
4. The recombinant vector according to claim 3 , wherein the selectable marker is a drug resistance gene.
5. The recombinant vector according to claim 4 , wherein the drug resistance gene is a gene resistant to antibiotics selected from the group consisting of ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, tetracycline, chloramphenicol and neomycin.
6. The recombinant vector according to claim 3 , wherein the reporter gene is a gene encoding a protein selected from the group consisting of green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (dsRFP), luciferase (Luc), chloramphenicol acetyltransferase (CAT), 13-galactosidase (LacZ) and 13-glucuronidase (Gus).
7. An isolated transformant in which the recombinant vector of claim 2 is introduced into a host cell.
8. The transformant according to claim 7 , wherein the host cell is an animal cell.
9. The transformant according to claim 8 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell.
10. A composition comprising the recombinant vector of claim 2 and a pharmaceutically acceptable carrier.
11. An in vitro method for preparing a target protein, comprising the steps of:
1) culturing the transformant of claim 7 ;
2) inducing the expression of a target protein from the transformant; and
3) harvesting the expressed target protein from the transformant or the culture solution thereof.
12. The method according to claim 11 , wherein the target protein is selected from the group consisting of human growth hormones, growth hormone release hormones, growth hormone release peptides, interferons, interferon receptors, colony stimulating factors, glucagons-like peptides, G-protein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukin- or cytokine-binding proteins, macrophage activating factors, macrophage peptides, B cell factors, T cell factors, protein A, allergy inhibitors, cell necrosis glycoprotein, immune toxins, lymph toxins, tumor necrosis factors, tumor suppressing factors, transitional growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptides, thrombomodulin, blood factor VII, Vila, VIII, IX, and XIII, plasminogen activating factor, fibrin-binding peptides, urokinases, streptokinases, hirudin, protein C, C-reactive proteins, rennin inhibitors, collagenase inhibitors, superoxide dismutases, leptin, platelet-originated growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, myelopoiesis growth factor, myelopoiesis stimulating factor, calcitonin, insulin, atriopeptin, cartilage inducer, elcatonin, joint tissue activating factor, tissue factor pathway inhibitor, follicle stimulating hormone, progesterone forming hormone, progesterone forming hormone releasing hormone, nerve growth factors, parathormone, relaxin, cycretin, somatomedine, insulin-like growth factor, adrenocortical hormones, glucagons, cholecystokynine, pancreatic polypeptides, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, virus-originated vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.