CN102188694A - Application of osteogenesis inducible factor - Google Patents
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- CN102188694A CN102188694A CN2010101215962A CN201010121596A CN102188694A CN 102188694 A CN102188694 A CN 102188694A CN 2010101215962 A CN2010101215962 A CN 2010101215962A CN 201010121596 A CN201010121596 A CN 201010121596A CN 102188694 A CN102188694 A CN 102188694A
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Abstract
The invention discloses an application of an osteogenesis inducible factor, and the osteogenesis inducible factor is used for restraining the appetite of mammals.
Description
Technical field
The present invention relates to the protein application, relate in particular to a kind of purposes of osteogenesis induction factor.
Background technology
Osteogenesis induction factor (osteoinductive factor OIF) claims osteoglycin or mimecan again, is a kind of secretory protein, is the protein of an isolated 12KDa from the bone matrix of cattle the earliest.Its biological function is still indeterminate.In the difference of organizing different montage of endogenous cause of ill or PolyA site of cattle and form multiple OIF obform body, but in the OIF gene obform body of these different lengths, its encoded protein matter structure is identical, and this gene is between different plant species, structure also is a high conservative, and this prompting OIF albumen may have important function in vivo.298 amino acid whose secretory protein propetides of people's OIF gene code reach 86% with the OIF homology of cattle, and its 1-19 position is the signal peptide structure, the 20-193 position is a propetide, the maturation zone is made up of 105 amino acid residues in the 194-298 position, and 6 multiple leucine enrichment regions are arranged in the 124-260 position.
Research the earliest thinks that OIF may be relevant with the metabolism adjusting of bone to OIF, but finds that in the mouse model of OIF gene knockout the mice energy normal development after the rejecting does not influence its fertility; Compare with wild-type mice, its skeleton development Non Apparent Abnormality illustrates that OIF may not play an important role in bone metabolism is regulated.In the mice of OIF gene knockout, only find that the increasing diameter of collagen fiber of cornea and skin is thick, the tension stress of skin descends.Meanwhile, some seminar have carried out deep research to OIF gene transcription regulatory mechanism in the world, discovery is in OIF upstream region of gene 296bp, contain 3 initiator elements, an E box (E-box) and Oct-1, NF-nB, metal reaction element (MRE) binding site, and in first intron of this gene, contain the enhancer and the silencer sequence of this gene transcription regulation.Discover that further first includes the binding site that the P53 gene is contained in the subarea at the OIF gene, and P53 can strengthen OIF gene transcription activity.This shows that OIF may bring into play important effect in the physiological process of human body.
Though OIF is a kind of important secretory protein, the function to the OIF gene it be unclear that at present.Therefore, this area presses for the physiological action of understanding OIF, and application corresponding is provided.
Summary of the invention
The present invention aims to provide the purposes of a kind of OIF.
In a first aspect of the present invention, osteogenesis induction factor (osteoinductive factor, OIF) purposes in the medicine of preparation appetite-suppressing are provided.
In another preference, described osteogenesis induction factor is the osteoinductive factor OIF Osteoglycin Mimecan; Described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type osteoinductive factor OIF Osteoglycin Mimecan or recombined human osteogenesis induction factor; More preferably, described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragment or OIF maturation protein fragment.
Described osteoinductive factor OIF Osteoglycin Mimecan's core protein fragment (AKYNKIKSRGIKANAFKKLNNLTFLYLDHNALESVPLNLPESLRVIHLQFNNIASI TDDTFCKANDTSYIRDRIEEIRLEGNPIVLGKHPNSFICLKRL shown in SEQ ID NO:1
Described osteoinductive factor OIF Osteoglycin Mimecan's maturation protein fragment is (PNEKSLQLQKDEAITPLPPKKENDEMPTCLLCVCLSGSVYCEEVDIDAVPPLPKES AYLYARFNKIKKLTAKDFADIPNLRRLDFTGNLIEDIEDGTFSKLSLLEELSLAEN QLLKLPVLPPKLTLFNAKYNKIKSRGIKANAFKKLNNLTFLYLDHNALESVPLNLP ESLRVIHLQFNNIASITDDTFCKANDTSYIRDRIEEIRLEGNPIVLGKHPNSFICL KRLPIGSYF) shown in SEQ ID NO:2.
In a second aspect of the present invention, a kind of compositions is provided, described compositions contains the osteogenesis induction factor of effective dose.
In another preference, described osteogenesis induction factor is the osteoinductive factor OIF Osteoglycin Mimecan; Described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type osteoinductive factor OIF Osteoglycin Mimecan or recombined human osteogenesis induction factor; More preferably, described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragment or OIF maturation protein fragment.
In view of the above, the invention provides the application of OIF.
Description of drawings
Fig. 1 has shown the influence of OIF core protein to C57BL/6J mice appetite.
Fig. 2 has shown the influence of OIF core protein to db/db mice appetite.
Fig. 3 has shown the influence of OIF core protein to KKAy mice appetite.
Fig. 4 has shown the influence of SD rat intracerebroventricular injection OIF core protein to appetite.
Fig. 5 has shown the influence of OIF maturation protein (HIS-OIF) to C57BL/6J mice appetite.
The specific embodiment
The inventor finds OIF, especially two of people OIF big or small protein fragments of difference through extensive and deep research, and promptly OIF maturation protein and OIF core protein have the effect that suppresses animal appetite.On this basis, finished the present invention.
Particularly, the inventor has made up and has expressed the people OIF albumen of two kinds of different sizes, respectively name be people OIF maturation protein (shown in SEQ ID NO:2,26.4KD, the 66-298 amino acids) or basic identical with its aminoacid sequence; With people OIF core protein (shown in SEQ ID NO:1,12KD, the 194-298 amino acids) or basic identical with its aminoacid sequence.The GenBank accession number of people OIF full length cDNA sequence is AF100758.
Term " aminoacid sequence is basic identical " is meant sequence difference identical or that caused by one or more amino acid changes (disappearance, increase, replace), but this change does not reduce its biologic activity basically, promptly can be by the developmental biology function in conjunction with the OIF receptor in target cell.The OIF that requires any meeting " basic identical " includes in the present invention, and no matter it is glycosylated (promptly derive from natural or derive from eukaryotic expression system) or nonglycosylated (promptly deriving from prokaryotic expression system or chemosynthesis).
Term " treatment " is meant the object OIF of the present invention that needs treatment based on the purpose of curing, alleviating, improving, alleviating, influencing treatment target disease, symptom, disease body constitution (predisposition).
Term " treatment target " is meant Mus, people and other mammals.
Term " treatment effective dose " is meant the amount that can reach the His-OIF or the MBP-OIF of therapeutic purposes in the treatment target body.Those of ordinary skill in the art should be understood that described " treatment effective dose " can be with the route of administration of His-OIF or MBP-OIF, used excipient substance and different and different with other drug drug combination situation.
The function of the function representative OIF maturation protein of His-OIF; The function of the function representative OIF core protein of MBP-OIF.
The technology contents of institute of the present invention incorporated by reference data is incorporated herein by reference in the lump with regard to its integral body.
The host cell that can be used for expressing or clone His-OIF of the present invention or MBP-OIF comprises prokaryotic cell, yeast cells or higher eukaryotic cell.The prokaryotic host cell that is suitable for includes, but are not limited to: G
+Or G
-Bacterium as escherichia coli E.coli., can comprise by the E.coli. bacterial strain that public approach obtains: K12 MM294 (ATCC31,446), X1776 (ATCC31,537), W3110 (ATCC27,325) and K5772 (ATCC53,635, JM109, DH5 α, B strain series, B834, BL21, BLR etc.Other available prokaryotic cells include but not limited to: European bacillus (Erwinia), klebsiella spp (Klebsiella), Bacillus proteus (Proteus), salmonella (Salmonella) is as bacillus typhi murium (Salmonellatyphimurium), Serratia (Serratia) is as serratia marcesens (Serratiamarcescans), Shigella (Shigella), bacillus subtilis. (B.subtilis), bacillus licheniformis (B.licheniformis), Rhodopseudomonas (Pseudomonas) is as bacillus pyocyaneus (P.aeruginosa), streptococcus (Streptomyces).E.coli.W3110 is proposed as preferably because of the fermentation host that it is often used as recombinant dna product.
Except prokaryotic cell, eukaryotic cell such as filamentous fungi (filamentousfungi) or yeast (yeast) etc. is applicable to equally expresses or clones His-OIF of the present invention or MBP-OIF.Saccharomyces cerevisiae (Saccharomyces) is exactly a kind of microorganism such as low eucaryon host such as grade commonly used, other hosts such as pombe fission yeast bacterium (Schizosaccharomycespombe); Kluyveromyces sp (Kluyveromyceshosts); Pichia pastoris bacterium (PichiaPastoris); Candidiasis (Candida); Trichodermareesia; Arteries and veins born of the same parents bacterium (Neurosporacrassa); Execute ten thousand peculiar mycetes (schwanniomyces) as Schwanniomycesoccidentalis; Filamentous fungi (filamentousfungi) is as Neurospora, penicillium (Penicillium), and Tolypocladium, aspergillosis (Aspergillus) is as A.nidulans; Tilburmetal.; Yeltonetal. and aspergillus niger (A.niger).Methylotrophy yeast (Methylotropicyeasts) can be used for expressing His-OIF of the present invention or MBP-OIF equally, include but not limited to the various yeast that can in methanol, grow such as Hansenula yeast bacterium (Hansenula), candidiasis (Candida), Kloeckera japonica bacterium (kloeckera), Pichia yeast (Pichia), saccharomyces cerevisiae (Saccharomyces), torulopsis bacterium (Torulopsis), Rhodothece glutinis bacterium (Rhodotorula).
The host cell that is used to express glycosylated His-OIF of the present invention or MBP-OIF derives from multi-cell organism.The example of invertebral zooblast comprises insect cell such as DrosophilaS2 and SpodopteraSf9, plant cell.The example of the mammalian host cell that is suitable for comprises Chinese hamster ovary cell (CHO), COS cell.Particularly, and the monkey kidney CV1 cell strain that transforms through SV40 (COS-7, ATCCCRL1651); Human embryonic kidney cell's strain 293; CHO/-DHFR (UrlaubandChasin); Mouse testis trophocyte (TM4); The human pneumonocyte (WI38, ATCCCCL75); Human liver cell (HepG2, HB8065); Mouse mastopathy cell (MMT060562, ATCCCCL51).Those of ordinary skill in the art should know how to select proper host cell.
Above-mentioned host cell can be cultivated in traditional nutrition base (nutrientmedia) through His-OIF or MBP-OIF expression vector or cloning vehicle transfection or after transforming, and is suitable for evoked promoter (promoter), selective conversion body (selectingtransformant) or amplification His-OIF or MBP-OIF coding gene sequence after described nutrition base is modified.The selection of condition of culture such as culture medium, temperature, pH etc. then should be known to those skilled in the art.How to make the maximized rule of culturing and propagating power, scheme and the operating technology can be referring to Mammalian Cell Biotechnology:a PracticalApproach, M.Butler, ed. (IRLPress, 1991) and Sambrooketal., supra..Those of ordinary skill in the art should know the method for eukaryotic cell transfection and prokaryotic cell conversion, for example CaCl
2Method, calcium phosphate precipitation method, liposome mediator method or electroporation.According to the difference of the host cell that uses, those of ordinary skill in the art can select corresponding standard transformation technology, for example CaCl
2Method (Sambrooketal., supra.) or electroporation be generally used for prokaryotic cell; Agrobacterium tumefaciems (Agrobacteriumtumefaciens) infects and is mainly used in certain plants transformation (Shawetal., Gene, 23:315 (1983) and WO89/05859); For the mammalian cell that does not have cell wall, then can use calcium phosphate precipitation method (GrahamandvanderEb, Virology, 52:456-457 (1978)); Comprehensive description of relevant mammalian host cell transfection can be referring to U.S.Pat.N..4, and 399,216.Relevant how transformed yeast cell can be referring to Van Solingenetal., J.Bact., 130:946 (1977) and Hsiaoetal., Proc.Natl.Acad.Sci. (USA), 76:3829 (1979)..Other introduce DNA the method for cell, merge (bacterialprotoplastfusion with intactcells) or polycation method (polycations) as 1 as nucleic acid micro-injection, electroporation, intact bacterial cell protoplast, 5-dimethyl-1,5-phenodiazine 11 methylene gather Methobromide (polybrene), poly ornithine (polyornithine) etc. and all can be used among the present invention.The description of relevant various mammalian cell transformation technologies can be referring to Keownetal., MethodsinEnzymology, 185:527-537 (1990) and Mansouretal., Nature, 336:348-352 (1988).
The nucleotide sequence (being cDNA or genomic DNA) of OIF of the present invention of encoding can be inserted into a replicable vector (replicable vector) to carry out gene clone (DNA cloning) or expresses.Various carriers such as plasmid, cosmid (cosmid, coemid), virion or phage etc. all can obtain by public approach.The known technology of utilization this area, can with the coding nucleotide sequence of OIF of the present invention routinely step insert restriction endonuclease site suitable on the replicable vector.Replicable vector generally includes but is not limited to lower member: one or more signal sequence (signal sequence), an origin of replication (origin ofreplication), one or more marker gene (marker gene), an enhancer element (enhancerelement), a promoter (promoter), and one section transcription pausing sequence (transcriptiontermination sequence).Those of ordinary skill in the art can use the interconnection technique (ligation techniques) of this area standard to make up the suitable replicable vector that contains one or more above-mentioned parts.
His-OIF of the present invention or MBP-OIF not only can directly express by gene recombinaton, also can produce by the mode that forms warm polypeptide with the xenogenesis polypeptide, the latter can be one section signal sequence that is positioned at mature protein or polypeptide N end, also can be other polypeptide fragments with specificity cleavage site that are positioned at mature protein or polypeptide N end.Generally, this segment signal sequence is the part of above-mentioned replicable vector, and perhaps it also can be to insert the His-OIF of the present invention of replicable vector or the part of MBP-OIF coding nucleotide sequence.Described signal sequence can be a prokaryotic signal sequence, alkali phosphatase for example, penicillinase, lpp, perhaps thermally-stabilised enterotoxin 1 I targeting sequencing.In yeast secretary system (yeast secretion), this signal sequence can be yeast invertase targeting sequencing (yeast invertase leader), the alpha factor targeting sequencing (comprises saccharomyces cerevisiae and Kluyveromyces sp alpha factor targeting sequencing, referring to U.S.Pat.No.5,010,182), or the acid phosphatase targeting sequencing, C.albicans glucoamylase targeting sequencing (EP362,179).In mammalian expression systems, the mammalian signal sequence can be directly used in the secretion target protein, and this type of sequence comprises signal sequence and the viral secretory targeting sequencing that derives from identical or close species mammal secretory protein.
Expression vector and cloning vehicle all contain one section nucleotide sequence, and this sequence can be duplicated carrier in one or more corresponding host cells.With various antibacterials, yeast or virus host cell corresponding nucleotide sequences be that those of ordinary skills are known.For example, the origin of replication of plasmid pBR322 is applicable to most of G
-Antibacterial, 2.mu. plasmid replication starting point is applicable to yeast cells, and various virus replication starting point (SV40, polyoma virus, adenovirus, VSV or BPV) then is applicable to the cloning vehicle in the mammalian cell.
Expression vector and cloning vehicle contain one section usually selects gene, has another name called " selected marker ".Typical select protein (a) that gene code produces to some antibiotic or toxin such as ampicillin, neomycin, methotrexate, tetracyclines etc. have resistance; (b) can remedy auxotroph and lack (auxotrophicdeficiencies); (c) replenish the encoding gene of the D-alanine racemase that crucial nutrient substance that compound culture medium can not provide such as bacillus host cell need.
The selection gene that is applicable to mammalian host cell should receive the host cell of His-OIF of the present invention or MBP-OIF encoding gene to distinguish having the ability, as DHFR or breast (gland nuclear pyrimidine) glycosides kinases.What be suitable for serves as that to select the host cell of gene be the active Chinese hamster ovary celI strain of no DHFR with wild type DHFR, and the preparation of this cell strain and propagation method can be referring to Urlaubetal., Proc.Natl.Acad.Sci.USA, 77:4216 (1980).The selection gene that is applicable to yeast cells is trpl gene (Stinchcombet al., Nature, the 282:39 (1979) that expresses in yeast plasmid Yrp7; Kingsmanetal., Gene, 7:141 (1979); Tschemperetal., Gene, 10:157 (1980)).The trpl gene can be used for screening yeast mutant such as the ATCCNo.44047 or the PEP4-1 (Jones, Genetics, 85:12 (1977)) that can not grow in tryptophan.
But expression vector and cloning vehicle are connected to the promoter on His-OIF of the present invention or the MBP-OIF coding nucleotide sequence with containing a hand control usually, and be synthetic to instruct mRNA's.The promoter corresponding with various host cells is that those of ordinary skills are known.The promoter that is applicable to prokaryotic host cell comprises beta-lactamase and lactose promoter systems (Changetal., Nature, 275:615 (1978); Goeddeletal., Nature, 281:544 (1979)), alkali phosphatase, tryptophan (trp) promoter systems (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP36,776), hybrid promoter such as tac promoter (deBoeretal., Proc.Natl.Acad.Sci.USA, 80:21-25 (1983)).But the bacterial host cell promoter is connected to Shine-Dalgarno (S.D.) sequence of His-OIF of the present invention or MBP-OIF coding gene sequence with comprising one section hand control equally.
The promoter sequence that is applicable to yeast host cell comprises 3-phoshoglyceric acid kinase promoter (Hitzemanet al., J.Biol.Chem., 255:2073 (1980)) or the promoter of other glycolytic ferments (Hessetal., J.Adv.EnzvmeReg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)), as Enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phoshoglyceric acid mutase, pyruvate kinase, triose-phosphate isomerase, phosphoglucoisomerase, and glucokinase.
Also have some derivable Yeast promoters, they can be regulated according to growing state better and transcribe, and comprise alcoholdehydrogenase 2, different cell pigment C, acid phosphatase, nitrogen metabolism related degradation enzyme, metallothionein, glyceraldehyde-3-phosphate, the promoter of maltose and galactose metabolic enzyme etc.Further describe referring to EP73 657 about the carrier that is applicable to yeast expression system and promoter.
Promoter can be controlled in the mammalian host cell transcribing of His-OIF of the present invention on the replicable vector or MBP-OIF coding gene sequence.Described promoter comprises and comes from polyoma virus, fowlpox virus (UK2,211,504), adenovirus, cow teats (shape) tumor virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, or simian virus 40 virus genomic promoteres such as (SV40), come from the mammiferous promoter of xenogenesis such as actin promoter or immunoglobulin promoter, and the promoter that comes from heat shock protein, prerequisite is that these promoteres are compatible with the host cell expression system.
By in replicable vector, inserting coding nucleotide sequence the transcribing in the higher eucaryote expression system that enhancer can strengthen His-OIF of the present invention or MBP-OIF.Enhancer is a kind of cis acting element of dna molecular, is generally 10 to 300 bp, strengthens transcribing of dna molecular by acting on promoter.Known a lot of enhancer sequence come from mammalian genes (globin, elastoser, albumin, α-fetoprotein, insulin) at present.And use more is the enhancer that comes from the eucaryon virocyte, as be positioned at the SV40 enhancer (100-270bp) in origin of replication downstream (lateside), the sub-enhancer of cytomegalovirus early promoter is positioned at the polyoma virus enhancer in origin of replication downstream, adenovirus enhancer.Enhancer can be sheared the 5 ' end or the 3 ' end of the coding nucleotide sequence that inserts the His-OIF of the present invention be positioned on the replicable vector or MBP-OIF, is preferably placed at 5 ' end of promoter.
Expression vector in the eukaryotic host cell (yeast cells, fungal cell, insect cell, plant cell, zooblast, human cell perhaps derive from the nucleated cell of other multi-cell organisms) contains equally ends to transcribe the nucleotide sequence required with stable mRNA.This type of sequence is taken from 5 ' end of eucaryon or viral DNA or cDNA untranslated region usually, also can take from 3 ' end sometimes.The nucleotide fragments that described " untranslated region " comprises can be transcribed and be produced the poly-adenylylation fragment that is positioned at His-OIF of the present invention or MBP-OIF mRNA untranslated region.
Other method, carrier and host cells that can be used for synthetic His-OIF of the present invention or MBP-OIF in the recombinant vertebrate cultivating system can be referring to Gethingetal., Nature, 293:620-625 (1981); Manteietal., Nature, 281:40-46 (1979); EP117,060; And EP117,058.
The coding nucleotide sequence of OIF of the present invention can be applied in the gene therapy.In the gene therapy process, gene is imported in the cell in the hope of synthesizing the gene expression product with therapeutic effect in vivo, such as alternative original dcc gene." gene therapy " comprises traditional gene therapy, promptly by seance and permanently effective; Comprise also giving gene therapy medicament that the latter comprises once or repeatedly gives effective DNA or mRNA.Antisense RNA and DNA also can be used as gene therapy medicament with some expression of gene in the blocking-up body.Confirmed already, although antisense oligonucleotide short-movie section is absorbed limited by cell membrane, cause its intracellular concentration to reduce, it still can bring into play the effect (Zamecniketal. of inhibitor in cell, Proc.Natl.Acad.Sci.USA83:4143-4146[1986])) described oligonucleotide fragment is modified for example replace its electronegative phosphodiester group with uncharged group after, can improve its cell membrane trap.
His-OIF of the present invention or MBP-OIF can be used as medicine.Those of ordinary skill in the art can be prepared into various pharmaceutically effective preparations (formulation) with it by the conventional method of this area, wherein comprises His-OIF or MBP-OIF and pharmaceutically suitable carrier of effective dose.
When making lyophilized formulations or solution, also need in the pharmaceutical composition of the present invention to add some other physiologically acceptable carrier, excipient, stabilizing agent etc. so that store (Remington ' sPharmaceutical Sciences16
ThEdition, Osol, A.Ed. (1980)).Its pharmaceutical dosage such as the carrier of described " can accept on the physiology ", excipient, stabilizing agent and concentration reply administration object (people, Mus and other mammals) are nontoxic, comprise buffer agent such as phosphate, citrate and other organic acid; Antioxidant such as ascorbic acid (VitC); Low-molecular-weight (being less than 10 amino acid residues) polypeptide; Protein such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone (PVP), aminoacid such as glycine, glutamate, Glu, agedoite acid, arginine or lysine; Monosaccharide, disaccharide and other carbohydrates such as glucose, mannose or dextrin; Intercalating agent such as EDTA; Sugar alcohol (sugaralcohols) is as mannitol, sorbitol; Salify counter ion counterionsl gegenions (counterions) are as sodium ion; And/or nonionic surfactant such as TWEEN.TM., PLURONICS.TM. or PEG etc.
The preparation that contains His-OIF of the present invention or MBP-OIF must process be sterilized before the administration in vivo.This step can be before or after lyophilizing and recomposition (reconstitution) be filtered by sterilising filtration film (sterile filtration membranes) and is finished.
Pharmaceutical composition of the present invention is placed in one usually and is equipped with in " sterilization inlet " container (sterileaccessport), for example, the intravenous solution bottle that bottle stopper is arranged, described stopper can be by the subcutaneous injection needle penetration.Pharmaceutical composition of the present invention can include but not limited to by the conventional route administration of this area: intravenous injection or infusion, lumbar injection, intracerebral injection, intramuscular injection, intraocular injection, intra-arterial injection or infusion, topical or by slow-released system (sustainedreleasesystems) administration.The dosage of pharmaceutical composition of the present invention can change because of the different of actual operating position with concentration range.Those of ordinary skill in the art should know and goes to select proper dosage and route of administration how according to the actual requirements.The zoopery that the inventor did provides a believable guidance for His-OIF or MBP-OIF in the amount ranges of human body.For system not of the same race, the adjustment principle of drug dose scope between people and the Mus for example, can be referring to Mordenti, J.and Chappell, W. " The use of interspecies scaling intoxicokinetics " In Toxicokinetics and New Drug Development, Yacobietal.; PergamonPress, NewYork 1989, pp.42-96..When His-OIF of the present invention or MBP-OIF vivo medicine-feeding, its common dosage range is 0.01uM/kg/d-0.1uM/kg/d weight of mammal every day, and preferable range 0.025uM/kg/d-0.05uM/kg/d is according to the difference of route of administration and different.Predictably, different His-OIF or MBP-OIF preparations will be effective to appetite-suppressing; When drug target (organ or tissue) when changing, administering mode also needs to do corresponding adjustment.
The microcapsule that contains His-OIF of the present invention or MBP-OIF can be used for the sustained-release administration of His-OIF of the present invention or MBP-OIF.The microcapsule controlled-release medicine-feeding technology of recombiant protein has been successfully applied to recombinant human somatropin (rhGH), recombinant human interferon alpha 2 (rhIFN), interleukin-2 and MNrgp120 (Johnsonetal., Nat.Med., 2:795-799 (1996); Yasuda, Biomed.Ther27:1221-1223 (1993); WO97/03692, WO96/40072, WO96/07399; U.S.Pat.No.5654010.The slow releasing preparation of His-OIF of the present invention or MBP-OIF can prepare with having good biological lactic acid hydroxyacetic acid high polymer (PLGA) compatible and wide in range biodegradability.The catabolite of PLGA, lactic acid and hydroxyacetic acid can be by the very fast removings of human body.And, the degradation capability of this high polymer can be with its molecular weight and composition different, from the some months (Lewis that extends to several years, " Controlled release of bioactiveagentsformlactide/glycolide polymer; " in:M.ChasinandR.Langer (Eds.), Biodegradable Polymersas Drug Delivery Systems (MarcelDekker:NewYork, 1990), pp.1-41)).
The also available various activatory molecular weight of His-OIF of the present invention or MBP-OIF are 5,000-100, and 000 Polyethylene Glycol (PEG) is modified to make it producing high-molecular, prolongs its half-life.Concrete operations can be referring to GreenWaldetal., Bioorg.Med.Chem.Lett.1994,4,2465; Calicetietal., ILFarmaco, 1993,48,919; ZalipskyandLee, " Polyethylene Glycol chemistry: biotechnology and biomedical applications ", J.M.Harris compiles, PlenumPress, N.Y., 1992.The preferred active PEG (CNZL02101672.0, WO9932139, PCT/US95/0755, PCT/US94/13013, USPat:4,640,835,4,496,689,4,301,144,4,670,417,4,791,192,4,179,337) of multi-arm fork type that uses.
His-OIF of the present invention or MBP-OIF can also make " chimeric molecule " (ChimericMolecule) or fusion rotein (Fusion Protein), thereby strengthen its biologic activity or prolong its intravital biological half-life.Express such as linking to each other with partial immunity globulin gene (Fc) with the whole of people.The part of His-OIF or MBP-OIF can be with whole or section H is-OIF or MBP-OIF cDNA sequence in " chimeric molecule ".Produce and express the visible USPat:5 of Fc fusion rotein, 428,130.His-OIF of the present invention or MBP-OIF gene can be placed on the N-end of Fc, and the C-end that also can be placed on Fc is expressed.
The His-OIF of covalent modification or MBP-OIF protein are also included within the present invention.The chemistry covalent modification comprises that changing N-end or C-end or other aminoacid adds a chemical molecular.This modification also comprises the aminoacid sequence that changes His-OIF or MBP-OIF.Change the glycosylation state (Glycosalytion) of His-OIF or MBP-OIF itself, comprise increasing glycosylation or reducing glycosylation, perhaps directly change the glycosylation state by chemical reaction.
Other preparation techniques such as nanometer formulation, spray, inhalant etc. are included within protection scope of the present invention equally.In a preference of the present invention, the inventor adopts the pET-28a of Novagen company (+) carrier to merge people OIF maturation protein sequence, makes up and give expression to the former nucleoprotein of His-OIF (about 31KD).The inventor adopts the pMAL-c2x of NEB company carrier to merge people OIF core protein sequence simultaneously, makes up and give expression to the former nucleoprotein of MBP-OIF (about 55KD).By optimizing abduction delivering and the various chromatographic techniques of integrated application (affine, ion exchange, molecular sieve, displacement buffer, take off endotoxin etc.), obtained low residual His-OIF and the former nucleoprotein of MBP-OIF of toxin of a large amount of high-purities.
OIF provided by the invention can be used for the appetite of non-therapeutic purposes and the adjusting of energy metabolism.As training athlete, satisfy performance worker or ordinary people's plastotype requirement etc.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can combination in any.All features that this case description is disclosed can with any composition forms and usefulness, each feature that is disclosed in the description can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is: core protein (MBP-OIF) fragment in finder's osteogenesis induction factor and maturation protein (His-OIF) fragment have the effect that suppresses mammal appetite first.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio and umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
OIF core protein (MBP-OIF) suppresses the research of laboratory animal appetite
At first the inventor tests normal C57 mice.(8W n=8) spends the night after the hunger, and the abdominal cavity is injected OIF albumen (MBP-OIF, 0.05 μ M/kg) and reference protein (MBP, 0.05 μ M/kg) and PBS respectively for three groups of C57BL/6J mices.* P＜0.05 expression MBP-OIF group has significant difference with MBP group, MBP-OIF group with PBS group accumulative total food-intake.And the MBP group is almost completely consistent with PBS group accumulative total food-intake, does not have significant difference (Fig. 1).
The inventor buys 10 of db/db male mices (5W) from Nanjing University's model animal institute simultaneously, gives being divided into two groups at random after adaptability is fed a week.One group of injection MBP-OIF (dosage is 0.05 μ M/kg), another group is contrast, injection MBP (dosage is 0.05 μ M/kg).Continuous three days lumbar injections, find that MBP-OIF group food-intake obviously reduces * * P＜0.001 (Fig. 2) at every day twice.
The inventor buys the male KK of cleaning level from Chinese Academy of Sciences's Shanghai Experimental Animal Center again immediately
Ay10 of mices, ad lib experimentizes after adapting to a week.10 mices are divided into two groups of row lumbar injections at random, and one group is MBP-OIF (dosage is 4mg/kg), and another group is contrast, injection MBP (dosage is 3.2mg/kg).The feed of per hour observing two groups of mices changes.Find that food-intake has significant difference between two groups, * P＜0.05 expression MBP-OIF group has significant difference with MBP group accumulative total food-intake.(Fig. 3)
In order further to observe OIF to the appetite regulation effect, the appetite that the inventor observes rat at SD rat intracerebroventricular injection OIF albumen changes.The rat ventricles of the brain are put pipe back and are recovered a week, treat that index such as appetite recovers the normal hungry 24h in back, then from indwelling tube give two groups of rats inject respectively MBP-OIF (24 μ g/ only, n=7) and MBP (24 μ g/ only, n=7), the observation food-intake.* P＜0.05 expression MBP group accumulative total feed is organized more than MBP-OIF, and significant difference (Fig. 4) is arranged.
OIF maturation protein (His-OIF) suppresses the research of laboratory animal appetite
The inventor tests normal C57BL/6J mice.After two groups of C57 mices (8 week) hunger of spending the night, OIF albumen is injected in the abdominal cavity respectively, and (HIS-OIF, 0.05 μ M/kg n=5) and PBS (n=4), observe appetite.* P＜0.05 expression HIS-OIF group accumulative total food-intake is obviously lacked and the PBS group, and two groups of differences have statistical significance (Fig. 5).
Above experimental result shows, no matter be the OIF core protein (shown in SEQ ID NO:1,12KD, the 194-298 amino acids), still be that maturation protein is (shown in SEQ ID NO:2,26.4KD, the 66-298 amino acids), can both suppress mammiferous appetite significantly.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Sequence table
＜110〉Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
＜120〉purposes of osteogenesis induction factor
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<170>PatentIn?version?3.3
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＜213〉homo sapiens
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Ala?Lys?Tyr?Asn?Lys?Ile?Lys?Ser?Arg?Gly?Ile?Lys?Ala?Asn?Ala?Phe
1 5 10 15
Lys?Lys?Leu?Asn?Asn?Leu?Thr?Phe?Leu?Tyr?Leu?Asp?His?Asn?Ala?Leu
20 25 30
Glu?Ser?Val?Pro?Leu?Asn?Leu?Pro?Glu?Ser?Leu?Arg?Val?Ile?His?Leu
35 40 45
Gln?Phe?Asn?Asn?Ile?Ala?Ser?Ile?Thr?Asp?Asp?Thr?Phe?Cys?Lys?Ala
50 55 60
Asn?Asp?Thr?Ser?Tyr?Ile?Arg?Asp?Arg?Ile?Glu?Glu?Ile?Arg?Leu?Glu
65 70 75 80
Gly?Asn?Pro?Ile?Val?Leu?Gly?Lys?His?Pro?Asn?Ser?Phe?Ile?Cys?Leu
85 90 95
Lys?Arg?Leu?Pro?Ile?Gly?Ser?Tyr?Phe
100 105
<210>2
<211>233
<212>PRT
＜213〉homo sapiens
<400>2
Pro?Asn?Glu?Lys?Ser?Leu?Gln?Leu?Gln?Lys?Asp?Glu?Ala?Ile?Thr?Pro
1 5 10 15
Leu?Pro?Pro?Lys?Lys?Glu?Asn?Asp?Glu?Met?Pro?Thr?Cys?Leu?Leu?Cys
20 25 30
Val?Cys?Leu?Ser?Gly?Ser?Val?Tyr?Cys?Glu?Glu?Val?Asp?Ile?Asp?Ala
35 40 45
Val?Pro?Pro?Leu?Pro?Lys?Glu?Ser?Ala?Tyr?Leu?Tyr?Ala?Arg?Phe?Asn
50 55 60
Lys?Ile?Lys?Lys?Leu?Thr?Ala?Lys?Asp?Phe?Ala?Asp?Ile?Pro?Asn?Leu
65 70 75 80
Arg?Arg?Leu?Asp?Phe?Thr?Gly?Asn?Leu?Ile?Glu?Asp?Ile?Glu?Asp?Gly
85 90 95
Thr?Phe?Ser?Lys?Leu?Ser?Leu?Leu?Glu?Glu?Leu?Ser?Leu?Ala?Glu?Asn
100 105 110
Gln?Leu?Leu?Lys?Leu?Pro?Val?Leu?Pro?Pro?Lys?Leu?Thr?Leu?Phe?Asn
115 120 125
Ala?Lys?Tyr?Asn?Lys?Ile?Lys?Ser?Arg?Gly?Ile?Lys?Ala?Asn?Ala?Phe
130 135 140
Lys?Lys?Leu?Asn?Asn?Leu?Thr?Phe?Leu?Tyr?Leu?Asp?His?Asn?Ala?Leu
145 150 155 160
Glu?Ser?Val?Pro?Leu?Asn?Leu?Pro?Glu?Ser?Leu?Arg?Val?Ile?His?Leu
165 170 175
Gln?Phe?Asn?Asn?Ile?Ala?Ser?Ile?Thr?Asp?Asp?Thr?Phe?Cys?Lys?Ala
180 185 190
Asn?Asp?Thr?Ser?Tyr?Ile?Arg?Asp?Arg?Ile?Glu?Glu?Ile?Arg?Leu?Glu
195 200 205
Gly?Asn?Pro?Ile?Val?Leu?Gly?Lys?His?Pro?Asn?Ser?Phe?Ile?Cys?Leu
210 215 220
Lys?Arg?Leu?Pro?Ile?Gly?Ser?Tyr?Phe
225 230
Claims (10)
- Osteogenesis induction factor (osteoinductive factor, OIF) the preparation appetite-suppressing medicine in purposes.
- 2. purposes as claimed in claim 1 is characterized in that, described osteogenesis induction factor is the osteoinductive factor OIF Osteoglycin Mimecan.
- 3. purposes as claimed in claim 2 is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type osteoinductive factor OIF Osteoglycin Mimecan or recombined human osteogenesis induction factor.
- 4. purposes as claimed in claim 3 is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragment or OIF maturation protein fragment.
- 5. purposes as claimed in claim 4 is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan's core protein fragment is shown in SEQ ID NO:1.
- 6. purposes as claimed in claim 4 is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan's maturation protein fragment is shown in SEQ ID NO:2.
- 7. a compositions is characterized in that, described compositions contains the osteogenesis induction factor of effective dose.
- 8. compositions as claimed in claim 7 is characterized in that, described osteogenesis induction factor is the osteoinductive factor OIF Osteoglycin Mimecan.
- 9. compositions as claimed in claim 8 is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type osteoinductive factor OIF Osteoglycin Mimecan or recombined human osteogenesis induction factor.
- 10. compositions as claimed in claim 9 is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragment or OIF maturation protein fragment.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004015060A2 (en) * | 2001-08-08 | 2004-02-19 | Curagen Corporation | Therapeutic polypeptides, nucleic acids encoding same, and methods of use |
| WO2004105784A1 (en) * | 2003-05-29 | 2004-12-09 | The University Of Manchester | Class iii slrp agonists for the reduction of blood vessel formation |
| CN1951967A (en) * | 2005-10-21 | 2007-04-25 | 上海第二医科大学附属瑞金医院 | Monoclonal antibody for resisting human osteogenesis induction factor and its preparation method and uses |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004015060A2 (en) * | 2001-08-08 | 2004-02-19 | Curagen Corporation | Therapeutic polypeptides, nucleic acids encoding same, and methods of use |
| WO2004105784A1 (en) * | 2003-05-29 | 2004-12-09 | The University Of Manchester | Class iii slrp agonists for the reduction of blood vessel formation |
| CN1951967A (en) * | 2005-10-21 | 2007-04-25 | 上海第二医科大学附属瑞金医院 | Monoclonal antibody for resisting human osteogenesis induction factor and its preparation method and uses |
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