CN102188694B - Application of osteogenesis inducible factor - Google Patents

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

The purposes of osteogenesis induction factor

Technical field

The present invention relates to protein application, particularly relate to a kind of purposes of osteogenesis induction factor.

Background technology

Osteogenesis induction factor (osteoinductive factor OIF), also known as osteoglycin or mimecan, 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 not clear.Multiple OIF obform body is formed in the montage organizing endogenous cause of ill different of cattle or the difference in PolyA site, but in the OIF gene obform body of these different lengths, the protein structure of its coding is identical, and this gene is between different plant species, structure is also high conservative, and this prompting OIF albumen may have important function in vivo.The amino acid whose secretory protein propetide of OIF gene code 298 of people, reach 86% with the OIF homology of cattle, its 1-19 position is signal peptide structure, 20-193 position is propetide, maturation zone, in 194-298 position, is made up of 105 amino acid residues, has 6 leucine rich regions of repeating in 124-260 position.

OIF research is the earliest thought that OIF may be relevant with the Metabolism regulation of bone, but finds in the mouse model of OIF gene knockout, the mice energy normal development after rejecting, do not affect its fertility; Compared with wild-type mice, its skeleton development Non Apparent Abnormality, illustrates that OIF may not play an important role in bone metabolism regulates.In the mice of OIF gene knockout, only find that the diameter of the collagen fiber of cornea and skin increases thick, the tension stress of skin declines.Meanwhile, the transcription regulation mechanism of some seminar to OIF gene conducts in-depth research in the world, find in OIF upstream region of gene 296bp, containing 3 initiator elements, an E box (E-box) and Oct-1, NF-nB, metal responsive element (MRE) binding site, and in the First Intron of this gene, the enhancer containing this gene transcription regulation and silencer sequence.Further research finds, contain the binding site of P53 gene, and P53 can strengthen the transcriptional activity of OIF gene in OIF gene First Intron district.OIF may play an important role in the physiological process of human body as can be seen here.

Although OIF is a kind of important secretory protein, at present the function of OIF gene be it be unclear that.Therefore, this area in the urgent need to understanding the physiological action of OIF, and provides corresponding application.

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, provide osteogenesis induction factor (osteoinductive factor, the OIF) purposes in the medicine preparing appetite-suppressing.

In another preference, described osteogenesis induction factor is osteoinductive factor OIF Osteoglycin Mimecan; Described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type human osteogenesis induction factor or recombined human osteogenesis induction factor; More preferably, described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragments or OIF maturation protein fragment.

Described osteoinductive factor OIF Osteoglycin Mimecan's core protein fragments is (AKYNKIKSRGIKANAFKKLNNLTFLYLDHNALESVPLNLPESLRVIHLQFNNIASI TDDTFCKANDTSYIRDRIEEIRLEGNPIVLGKHPNSFICLKRLPIGSYF) as shown in SEQ ID NO:1.

Described osteoinductive factor OIF Osteoglycin Mimecan's maturation protein fragment is (PNEKSLQLQKDEAITPLPPKKENDEMPTCLLCVCLSGSVYCEEVDIDAVPPLPKES AYLYARFNKIKKLTAKDFADIPNLRRLDFTGNLIEDIEDGTFSKLSLLEELSLAEN QLLKLPVLPPKLTLFNAKYNKIKSRGIKANAFKKLNNLTFLYLDHNALESVPLNLP ESLRVIHLQFNNIASITDDTFCKANDTSYIRDRIEEIRLEGNPIVLGKHPNSFICL KRLPIGSYF) as shown in SEQ ID NO:2.

In a second aspect of the present invention, provide a kind of compositions, described compositions contains the osteogenesis induction factor of effective dose.

In another preference, described osteogenesis induction factor is osteoinductive factor OIF Osteoglycin Mimecan; Described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type human osteogenesis induction factor or recombined human osteogenesis induction factor; More preferably, described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragments or OIF maturation protein fragment.

Accordingly, the invention provides the application of OIF.

Accompanying drawing explanation

Fig. 1 shows the impact of OIF core protein on C57BL/6J mice appetite.

Fig. 2 shows the impact of OIF core protein on db/db mice appetite.

Fig. 3 shows the impact of OIF core protein on KKAy mice appetite.

Fig. 4 shows SD lateral ventricle of rat brain injection OIF core protein to the impact of appetite.

Fig. 5 shows OIF maturation protein (HIS-OIF) to the impact of C57BL/6J mice appetite.

Detailed description of the invention

Inventor, through extensive and deep research, finds OIF, especially two of people OIF different size protein fragments, and namely OIF maturation protein and OIF core protein have the effect suppressing animal appetite.On this basis, the present invention is completed.

Particularly, inventor builds and have expressed the people OIF albumen of two kinds of different sizes, and name is people OIF maturation protein (as shown in SEQ ID NO:2,26.4KD, 66-298 amino acids) or substantially identical with its aminoacid sequence respectively; With people OIF core protein (as shown in SEQ ID NO:1,12KD, 194-298 amino acids) or substantially identical with its aminoacid sequence.The GenBank accession number of people OIF full length cDNA sequence is AF100758.

Term " aminoacid sequence is substantially identical " refers to sequence difference that is identical or that caused by one or more amino acid change (disappearance, increase, replacement), but this change does not reduce its biologic activity substantially, namely can by the developmental biology function in conjunction with OIF receptor in target cell.Anyly meet the OIF that " substantially identical " require and include in the present invention, no matter it is glycosylated (namely derive from natural or derive from eukaryotic expression system) or nonglycosylated (namely derive from prokaryotic expression system or chemosynthesis).

Term " treatment " refer to based on cure, alleviate, improve, alleviate, affect treatment target disease, symptom, disease body constitution (predisposition) object and give need treatment object OIF of the present invention.

Term " treatment target " refers to Mus, people and other mammals.

Term " treatment effective dose " refers to the amount that can reach His-OIF or MBP-OIF of therapeutic purposes in treatment target body.Those of ordinary skill in the art should be understood that described " treatment effective dose " can with the route of administration of His-OIF or MBP-OIF, excipient substance used and different and different from 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 entirety.

The host cell that can be used for expressing or cloning His-OIF or MBP-OIF of the present invention comprises prokaryotic cell, yeast cells or higher eukaryotic cell.The prokaryotic host cell be suitable for includes, but are not limited to: G ⁺or G ^-bacterium, as E. coli., the E.coli. bacterial strain that can be obtained by public approach is comprised: K12 MM294 (ATCC31,446), X1776 (ATCC31,537), W3110 (ATCC27,325) and K5772 (ATCC53,635, JM109, DH5 α, B strain arranges, 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 it is often used as the fermentation host of recombinant dna product.

Except prokaryotic cell, eukaryotic cell such as filamentous fungi (filamentousfungi) or yeast (yeast) etc. are equally applicable to express or clone His-OIF or MBP-OIF of the present invention.Saccharomyces cerevisiae (Saccharomyces) is exactly a kind of conventional microorganism such as low eucaryon host such as grade, and other hosts are as pompe 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) as Neurospora, penicillium (Penicillium), Tolypocladium, aspergillosis (Aspergillus) is as A.nidulans; Tilburmetal.; Yeltonetal. with aspergillus niger (A.niger).Methylotrophic yeast bacterium (Methylotropicyeasts) can be used for expressing His-OIF or MBP-OIF of the present invention equally, include but not limited to that the various yeast that can grow in methanol is as Hansenula yeast bacterium (Hansenula), candidiasis (Candida), Kloeckera japonica bacterium (kloeckera), Pichia yeast (Pichia), saccharomyces cerevisiae (Saccharomyces), torulopsis bacterium (Torulopsis), Rhodotorula sp (Rhodotorula).

For expressing the host cell resources of glycosylated His-OIF or MBP-OIF of the present invention in multi-cell organism.The example of invertebral zooblast comprises insect cell as DrosophilaS2 and SpodopteraSf9, plant cell.The example of the mammalian host cell be suitable for comprises Chinese hamster ovary cell (CHO), COS cell.Particularly, through monkey kidney CV1 cell strain (COS-7, ATCCCRL1651) of SV40 conversion; Human embryonic kidney cell's strain 293; CHO/-DHFR (UrlaubandChasin); Mouse testis trophocyte (TM4); 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 suitable host cell.

Above-mentioned host cell is through His-OIF or MBP-OIF expression vector or cloning vehicle transfection or can cultivate in traditional Nutrient medium (nutrientmedia) 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 Nutrient medium is modified.The selection of condition of culture 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 operating technology can see 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, such as CaCl ₂method, calcium phosphate precipitation, liposome mediator method or electroporation.According to the difference of the host cell used, those of ordinary skill in the art can select corresponding standard transformation techniques, such as CaCl ₂method (Sambrooketal., supra.) or electroporation are 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 not having cell wall, then can use calcium phosphate precipitation (GrahamandvanderEb, Virology, 52:456-457 (1978)); About comprehensive description of mammalian host cell transfection can see U.S.Pat.N..4,399,216.Relevant how transformed yeast cell can see Van Solingenetal., J.Bact., 130:946 (1977) and Hsiaoetal., Proc.Natl.Acad.Sci. (USA), 76:3829 (1979)..DNA is introduced the method for cell by other, if nucleic acid micro-injection, electroporation, whole bacterial cells protoplast fusion (bacterialprotoplastfusion with intactcells) or polycation method (polycations) are as 1,5-dimethyl-1,5-phenodiazine 11 methylene gathers Methobromide (polybrene), poly ornithine (polyornithine) etc. and all can be used in the present invention.About the description of various transforming mammalian cells technology can see Keownetal., MethodsinEnzymology, 185:527-537 (1990) and Mansouretal., Nature, 336:348-352 (1988).

The nucleotide sequence (i.e. 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 carrier such as plasmid, cosmid (cosmid, coemid), virion or phage etc. all obtain by public approach.Use the known technology of this area, can by restriction endonuclease site suitable on the coding nucleotide sequence of OIF of the present invention routinely step insertion replicable vector.A replicable vector generally includes but is not limited to lower component: 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 of transcriptional pause sequence (transcriptiontermination sequence).Those of ordinary skill in the art can use the interconnection technique of this area standard (ligation techniques) to build the suitable replicable vector containing one or more above-mentioned parts.

His-OIF or MBP-OIF of the present invention not only directly can be expressed by gene recombinaton, also can be produced by the mode forming warm polypeptide with polypeptide, the latter can be one section of signal sequence being positioned at mature protein or polypeptide N end, also can be other polypeptide fragments with specific cleavage site being positioned at mature protein or polypeptide N end.Under normal circumstances, this segment signal sequence is a part for above-mentioned replicable vector, or it also can be a part for His-OIF or the MBP-OIF coding nucleotide sequence of the present invention inserting replicable vector.Described signal sequence can be prokaryotic signal sequence, such as alkali phosphatase, penicillinase, lpp, or Thermostable α-amylase II targeting sequencing.In yeast secretion systems (yeast secretion), this signal sequence can be yeast invertase leader (yeast invertase leader), alpha factor targeting sequencing (comprises saccharomyces cerevisiae and Kluyveromyces sp alpha factor targeting sequencing, see U.S.Pat.No.5,, or acid phosphatase leader 010,182), C.albicans glucoamylase leader (EP362,179).In mammalian expression systems, mammalian signal sequences can be directly used in secretion target protein, and this type of sequence comprises the signal sequence and viral secretory leaders that derive from identical or close species mammal secretes albumen.

Expression vector and cloning vehicle are all containing one section of nucleotide sequence, and this sequence enables carrier copy in one or more corresponding host cell.It is known that the nucleotides sequence corresponding with various antibacterial, yeast or virus host cells is classified as those of ordinary skill in the art.Such as, the origin of replication of pBR322 plasmid is applicable to most of G ^-antibacterial, 2.mu. Plasmid replication origins is applicable to yeast cells, and various virus origin of replication (SV40, polyoma virus, adenovirus, VSV or BPV) is then applicable to the cloning vehicle in mammalian cell.

Expression vector and cloning vehicle containing one section of Select gene, have another name called " selected marker " usually.The protein (a) that typical Select gene coding produces to some antibiotic or toxin as ampicillin, neomycin, methotrexate, tetracyclines etc. have resistance; B () can make up auxotroph and lack (auxotrophicdeficiencies); The encoding gene of the D-alanine racemase that the critical nutrients that c () supplements compound culture medium can not provide needs as bacillus host cell.

The Select gene being applicable to mammalian host cell should receive the host cell of His-OIF or MBP-OIF encoding gene of the present invention to distinguish by having the ability, as DHFR or breast (gland nuclear pyrimidine) glycosides kinases.What be suitable for is Chinese hamster ovary celI strain without DHFR activity with the wild type DHFR host cell that is Select gene, and the preparation of this cell strain and propagation method can see Urlaubetal., Proc.Natl.Acad.Sci.USA, 77:4216 (1980).The Select gene being applicable to yeast cells is the trpl gene (Stinchcombet al., Nature, the 282:39 (1979) that express in yeast plasmid Yrp7; Kingsmanetal., Gene, 7:141 (1979); Tschemperetal., Gene, 10:157 (1980)).Trpl gene can be used for screening the yeast mutant that can not grow in tryptophan as ATCCNo.44047 or PEP4-1 (Jones, Genetics, 85:12 (1977)).

Expression vector and cloning vehicle contain a promoter that can be connected to hand control on His-OIF or MBP-OIF coding nucleotide sequence of the present invention usually, to instruct the synthesis of mRNA.The promoter corresponding with various host cell is that those of ordinary skill in the art are known.The promoter being applicable to prokaryotic host cell comprises beta-lactamase and lactose promoter system (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 is as tac promoter (deBoeretal., Proc.Natl.Acad.Sci.USA, 80:21-25 (1983)).Bacterial host cell promoter comprises Shine-Dalgarno (S.D.) sequence that a section can be connected to hand control His-OIF or MBP-OIF coding gene sequence of the present invention equally.

The promoter sequence being applicable to yeast host cell comprises glycerol 3-phosphate kinase promoter (Hitzemanet al., J.Biol.Chem., 255:2073 (1980)) or the promoter (Hessetal. of other glycolytic ferments, 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 regulate 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 metabolism enzyme etc.Further describe see EP73 about what be applicable to the carrier of yeast expression system and promoter, 657.

Promoter can to control in mammalian host cell transcribing of His-OIF or MBP-OIF coding gene sequence of the present invention on replicable vector.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 the virus genomic promoter such as simian virus 40 (SV40), come from the promoter of heterologous mammalian as actin promoter or immunoglobulin promoter, and come from the promoter of heat shock protein, prerequisite is that these promoteres are compatible with host cell expression systems.

Coding nucleotide sequence the transcribing in higher eucaryote expression system of His-OIF or MBP-OIF of the present invention can be strengthened by inserting enhancer in replicable vector.Enhancer is a kind of cis acting element of DNA molecular, being generally 10 to 300 bp, strengthening transcribing of DNA molecular by acting on promoter.Current known a lot of enhancer sequence comes from mammalian genes (globin, elastoser, albumin, α-fetoprotein, insulin).And to use more be the enhancer coming from eukaryotic viral cell, as being positioned at the SV40 enhancer (100-270bp) in origin of replication downstream (lateside), the sub-enhancer of cytomegalovirus early promoter, be positioned at the polyoma virus enhancer in origin of replication downstream, adenovirus cancers.Enhancer can be sheared 5 ' end or the 3 ' end of the coding nucleotide sequence inserting His-OIF or MBP-OIF of the present invention be positioned on replicable vector, is preferably placed at 5 ' end of promoter.

Expression vector in eukaryotic host cell (yeast cells, fungal cell, insect cell, plant cell, zooblast, human cell, or the nucleated cell deriving from other multi-cell organisms) is transcribed and the nucleotide sequence needed for stable mRNA containing stopping equally.This type of sequence takes from 5 ' end of eucaryon or viral DNA or cDNA untranslated region usually, sometimes also can take from 3 ' end.The transcribed generation of nucleotide fragments that described " untranslated region " comprises is positioned at the poly-adenylylation fragment of His-OIF or MBP-OIF mRNA untranslated region of the present invention.

Other can see Gethingetal., Nature, 293:620-625 (1981) for the synthesis of the method for His-OIF or MBP-OIF of the present invention, carrier and host cell in recombinant vertebrate cultivating system; 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 gene therapy.In gene therapy process, gene is imported into synthesizing the gene expression product with therapeutic effect in vivo in cell, such as alternative original dcc gene." gene therapy " comprises traditional gene therapy, namely permanently effective by seance; Also comprise and give gene therapy medicament, the latter comprises once or gives effective DNA or mRNA in multiple times.Antisense RNA and DNA also can be used as gene therapy medicament to block the expression of some gene in body.Already confirmed, although antisense oligonucleotide short-movie section is absorbed limited by cell membrane, its intracellular concentration is caused to reduce, it still can play the effect (Zamecniketal. of inhibitor in cell, Proc.Natl.Acad.Sci.USA83:4143-4146 [1986])) described oligonucleotide fragment is modified such as replace its electronegative phosphodiester group with uncharged group after, its cell membrane trap can be improved.

His-OIF or MBP-OIF of the present invention can be used as medicine.Those of ordinary skill in the art can be prepared into various pharmaceutically effective preparation (formulation) by the conventional method of this area, wherein includes His-OIF or MBP-OIF and pharmaceutically suitable carrier of effective amount.

When making lyophilized formulations or solution, also need in 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)).It is nontoxic that its pharmaceutical dosage such as carrier, excipient, stabilizing agent of described " physiology can accept " and concentration tackle administration object (people, Mus and other mammals), comprises buffer agent as phosphate, citrate and other organic acid; Antioxidant is as ascorbic acid (VitC); Low-molecular-weight (being less than 10 amino acid residues) polypeptide; Protein as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer is as polyvinylpyrrolidone (PVP), and aminoacid is as glycine, glutamate, Glu, agedoite acid, arginine or lysine; Monosaccharide, disaccharide and other carbohydrates are as glucose, mannose or dextrin; Intercalating agent is as EDTA; Sugar alcohol (sugaralcohols) is as mannitol, sorbitol; Salt-forming counterion (counterions) is as sodium ion; And/or nonionic surfactant is as TWEEN.TM., PLURONICS.TM. or PEG etc.

Preparation containing His-OIF or MBP-OIF of the present invention has to pass through sterilizing before administration in vivo.This step can have been filtered by sterilising filtration film (sterile filtration membranes) before or after lyophilizing and recomposition (reconstitution).

Pharmaceutical composition of the present invention is usually placed in one and is equipped with in the container of " sterile access port " (sterileaccessport), and such as, an intravenous solution bottle having a bottle stopper, described stopper can be penetrated by hypodermic needle.Pharmaceutical composition of the present invention by the conventional route administration of this area, can include but not limited to: 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 and concentration range can change because of the difference of actual service condition.Those of ordinary skill in the art should know and goes how according to the actual requirements to select suitable dosage and route of administration.The zoopery that the present inventor does provides a believable guidance for His-OIF or MBP-OIF in the amount ranges of human body.For different germline, the such as Adjustment principle of drug dose scope between people and Mus, can see 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 or MBP-OIF vivo medicine-feeding of the present invention, its usual dosage range is 0.01uM/kg/d-0.1uM/kg/d weight of mammal every day, preferable range 0.025uM/kg/d-0.05uM/kg/d, different according to the difference of route of administration.Predictably, different His-OIF or MBP-OIF preparations will be effective to appetite-suppressing; When drug target (organ or tissue) changes, administering mode also needs to do corresponding adjustment.

The sustained-release administration of His-OIF or MBP-OIF used in the present invention of the microcapsule containing His-OIF or MBP-OIF of the present invention.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 or MBP-OIF of the present invention can be prepared with having good biological lactic-glycolic acid high polymer (PLGA) that is compatible and wide in range biodegradability.The catabolite of PLGA, lactic acid and hydroxyacetic acid can be removed very soon by human body.And, the degradation capability of this high polymer can with the difference of its molecular weight and composition, several years (Lewis are extended to from some months, " Controlled release of bioactiveagentsformlactide/glycolide polymer; " in:M.ChasinandR.Langer (Eds.), Biodegradable Polymersas Drug Delivery Systems (MarcelDekker:NewYork, 1990), pp.1-41)).

The molecular weight of His-OIF or MBP-OIF of the present invention also available various activation is 5,000-100, and the Polyethylene Glycol (PEG) of 000 is modified to make it producing high-molecular, extends its half-life.Concrete operations can see GreenWaldetal., Bioorg.Med.Chem.Lett.1994, and 4,2465; Calicetietal., ILFarmaco, 1993,48,919; ZalipskyandLee, " Polyethylene Glycol chemistry: biotechnology and biomedical applications ", J.M.Harris compiles, PlenumPress, N.Y., 1992.The 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 preferred use multi-arm fork type.

His-OIF or MBP-OIF of the present invention can also make " chimeric molecule " (ChimericMolecule) or fusion rotein (Fusion Protein), thus the biological half-life strengthening its biologic activity or extend in its body.Such as be connected with partial immunity globulin gene (Fc) with the whole of people and express.In " chimeric molecule ", the part of His-OIF or MBP-OIF can use His-OIF or MBP-OIF cDNA sequence all or in part.Produce and express the visible USPat:5 of Fc fusion rotein, 428,130.His-OIF or MBP-OIF gene of the present invention can be placed on the N-end of Fc, and the C-end that also can be placed on Fc is expressed.

His-OIF or the MBP-OIF protein of covalent modification is also included within the present invention.Chemical covalent modification comprises change N-end or C-end or other aminoacid and adds a chemical molecular.This modification also comprises the aminoacid sequence changing His-OIF or MBP-OIF.Change the glycosylation state (Glycosalytion) of His-OIF or MBP-OIF itself, comprise and increase glycosylation or reduce glycosylation, or directly change 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, inventor adopts Novagen company pET-28a (+) carrier to merge people OIF maturation protein sequence, builds and give expression to His-OIF protokaryon albumen (about 31KD).Inventor adopts NEB company pMAL-c2x carrier to merge people OIF core protein sequence simultaneously, builds and give expression to MBP-OIF protokaryon albumen (about 55KD).By optimizing abduction delivering and the various chromatographic technique of integrated application (affine, ion exchange, molecular sieve, displacement buffer, de-endotoxin etc.), obtain His-OIF and the MBP-OIF protokaryon albumen of the low the toxin remains of a large amount of high-purity.

OIF provided by the invention can be used for the appetite of non-treatment object and the adjustment of energy metabolism.As training athlete, the plastotype requirement etc. meeting performance worker or ordinary people.

The above-mentioned feature that the present invention mentions, or the feature that embodiment is mentioned can combination in any.All features that this case description discloses can with any composition forms and use, each feature disclosed in description, anyly can provide identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.

Major advantage of the present invention is: core protein (MBP-OIF) fragment in Late Cambrian osteoinductive factor OIF Osteoglycin Mimecan and maturation protein (His-OIF) fragment have the effect suppressing mammalian appetite.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise all percentage ratio and number by weight.

Unit in percent weight in volume in the present invention is well-known to those skilled in the art, such as, refer to the weight of solute in the solution of 100 milliliters.

Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.

Embodiment 1

The research of OIF core protein (MBP-OIF) Inhibition test animal appetite

First inventor tests normal C57 mice.After three groups of C57BL/6J mice (8W, n=8) starved overnight, abdominal cavity injects OIF albumen (MBP-OIF, 0.05 μM/kg) and reference protein (MBP, 0.05 μM/kg) and PBS respectively.* P < 0.05 represents that MBP-OIF group and the accumulative food-intake of MBP group, MBP-OIF group and PBS group have significant difference.And the accumulative food-intake of MBP group and PBS group is almost completely the same, there is no significant difference (Fig. 1).

Inventor buys db/db male mice 10 (5W) from Nanjing University's model animal institute simultaneously, gives after adaptability feeds one week and is divided into two groups at random.One group of injection MBP-OIF (dosage is 0.05 μM/kg), another group is contrast, injection MBP (dosage is 0.05 μM/kg).Continuous three days lumbar injections, twice daily, find that MBP-OIF group food-intake obviously reduces, * * P < 0.001 (Fig. 2).

Inventor buys the male KK of cleaning grade from Chinese Academy of Sciences's Shanghai Experimental Animal Center again immediately ^aymice 10, ad lib is tested after adapting to one 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).Observe the feed change of two groups of mices per hour.Find that between two groups, food-intake has significant difference, * P < 0.05 represents that MBP-OIF group and the accumulative food-intake of MBP group have significant difference.(Fig. 3)

In order to further observe the regulating action of OIF to appetite, inventor observes the appetite change of rat at SD lateral ventricle of rat brain injection OIF albumen.The rat ventricles of the brain put the rear recovery of pipe one week, recover hungry 24h normally until indexs such as appetite, then inject MBP-OIF (24 μ g/, n=7) and MBP (24 μ g/, n=7) respectively to two groups of rats from indwelling tube, observe food-intake.* P < 0.05 represents that the accumulative feed of MBP group is more than MBP-OIF group, and has significant difference (Fig. 4).

Embodiment 2

The research of OIF maturation protein (His-OIF) Inhibition test animal appetite

Inventor tests normal C57BL/6J mice.After two groups of C57 mice (8 weeks) starved overnight, abdominal cavity injects OIF albumen (HIS-OIF, 0.05 μM/kg, n=5) and PBS (n=4) respectively, observes appetite.* P < 0.05 represents the accumulative food-intake of HIS-OIF group obviously few and PBS group, and two groups of differences have statistical significance (Fig. 5).

Above experimental result shows, no matter be that OIF core protein is (as shown in SEQ ID NO:1,12KD, 194-298 amino acids) or maturation protein (as shown in SEQ ID NO:2,26.4KD, 66-298 amino acids), mammiferous appetite can be suppressed significantly.

The foregoing is only preferred embodiment of the present invention, and be not used to limit substantial technological context of the present invention, substantial technological content of the present invention is broadly defined in the right of application, any technology entities that other people complete or method, if with application right define identical, also or a kind of change of equivalence, be all covered by being regarded as among this right.

Sequence table

<110> Ruijin Hospital, Shanghai Jiao Tong University School of Medicine

 

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<170>PatentIn version 3.3

 

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Claims (4)

1. osteogenesis induction factor (osteoinductive factor, the OIF) purposes in the medicine preparing appetite-suppressing;

Wherein, described osteogenesis induction factor is osteoinductive factor OIF Osteoglycin Mimecan; And described osteoinductive factor OIF Osteoglycin Mimecan is OIF core protein fragments or OIF maturation protein fragment;

And described osteoinductive factor OIF Osteoglycin Mimecan's core protein fragments is as shown in SEQ ID NO:1;

And described osteoinductive factor OIF Osteoglycin Mimecan's maturation protein fragment is as shown in SEQ ID NO:2.

2. purposes as claimed in claim 1, it is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is selected from wild type human osteogenesis induction factor or recombined human osteogenesis induction factor.

3. purposes as claimed in claim 1, it is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is as shown in SEQ ID NO:1.

4. purposes as claimed in claim 1, it is characterized in that, described osteoinductive factor OIF Osteoglycin Mimecan is as shown in SEQ ID NO:2.