CN106117370A - The fusion protein of high-glycosylation Exendin 4 and the like, Preparation Method And The Use - Google Patents

The fusion protein of high-glycosylation Exendin 4 and the like, Preparation Method And The Use Download PDF

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CN106117370A
CN106117370A CN201610692679.4A CN201610692679A CN106117370A CN 106117370 A CN106117370 A CN 106117370A CN 201610692679 A CN201610692679 A CN 201610692679A CN 106117370 A CN106117370 A CN 106117370A
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xaa
ser
fusion protein
lys
leu
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CN106117370B (en
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李强
董炤
王著
李媛丽
冯雄
李子瑞
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Anyuan Pharmaceutical Technology (shanghai) Co Ltd
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Anyuan Pharmaceutical Technology (shanghai) Co Ltd
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Priority to CN201610692679.4A priority Critical patent/CN106117370B/en
Priority to EP16913393.1A priority patent/EP3502143A4/en
Priority to PCT/CN2016/106011 priority patent/WO2018032638A1/en
Priority to US16/326,412 priority patent/US11123438B2/en
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Priority to US17/391,535 priority patent/US11833212B2/en
Priority to US18/492,958 priority patent/US20240108743A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention discloses the fusion protein of a kind of high-glycosylation Exendin 4 and the like, described fusion protein comprises Exendin 4 and the like, flexible peptide linker, at least 1 human chorion gonadotrophic hormone beta subunit carboxyl terminal peptide rigid element and human normal immunoglobulin's Fc fragment.The invention also discloses preparation method and the purposes of described fusion protein.Described fusion protein has preferably biologic activity, notable circulating half-life, the immunogenicity of reduction and the bioavailability of raising extended.Described fusion protein can be used for treating diabetes, obesity and the benefited other diseases by reduction plasma glucose, suppression stomach and/or bowel movement and suppression stomach and/or intestinal emptying or suppression food intake.

Description

The fusion protein of high-glycosylation Exendin-4 and the like, its preparation method and Purposes
Technical field
The present invention relates to long-acting GLP-1 analog, more particularly, to the fusion protein of Exendin-4 and the like, Further relate to its preparation method and it is in treatment diabetes, obesity and by reducing plasma glucose, suppressing stomach and/or bowel movement With suppression stomach and/or intestinal emptying or suppression food intake and application in benefited other diseases.
Background technology
Exendin-4 is isolatable from South Africa Monster saliva, and it has 53% with the GLP-1 aminoacid sequence of mammal Homology, is effective GLP-1 receptor stimulating agent.The physiologically active of Exendin-4 is similar to GLP-1, but Exendin-4-NH2 End second be instead of the Ala in GLP-1 by Gly so that it is DPP-IV enzyme is had certain resistant function, is difficult to by DPP-IV Degraded, the half-life circulated in vivo is longer, can reach 60-90 minute.Additionally ,-COOH the end of Exendin-4 is by 9 ammonia The special Trp-cage structure that base acid (PSSGAPPPS) is formed so that it is be significantly higher than GLP-with the binding affinity of GLP-1 receptor 1 (Neidigh JW etc., Biochemistry, 2001,40:13188 13200), its biologic activity is about GLP-1's 1000 times.It addition, the metabolic pathway of Exendin-4 from GLP-1 is the most different, Exendin-4 is mainly by renal metabolism, and GLP-1 carries out metabolism by peripheral tissues and kidney, and peripheral tissues's approach be main mode (Simonsen L. etc., Regulatory Peptides, 2013,181:17 21), thus the metabolic rate of Exendin-4 is lower, this is also that it partly declines Phase is considerably longer than the major reason of natural GLP-1.Exendin-4 contains 39 aminoacid, and molecular weight is little, the most recombinant expressed Difficulty is very big, and expression is low.Thus, be applied at present clinic Exendin-4 be synthetic (Exenatide, Chinese name: Exenatide).Clinical effectiveness shows, Exenatide mean half-life in human body only has 2.4 hours, and every day needs Inject twice, bring to the life of patient the most painful and inconvenient.Additionally, due to only have 53% with the homology of people GLP-1, Make patient produce antibody ratio to increase.In sum, Exendin-4 has the deficiencies such as the half-life is short, immunogenicity is strong, but same Time its special molecular structure give again its natural GLP-1 or advantage that other GLP-1 analog cannot be reached, as with GLP- The affinity that 1 receptor combines is strong, biological activity is high, clinical medicine dose is extremely low, Stability Analysis of Structures be difficult to by DPP-IV enzymolysis and The advantages such as metabolic rate is low so that it is there is bigger clinical value compared with other GLP-1 analog.
In order to extend the Half-life in vivo of Exendin-4, Exendin-4 or other GLP-1 analog are melted with Fc fragment The technical scheme closed studies have reported that, such as the weekly hypodermic length merged with human IgG 4Fc of Li Lai company exploitation Effect GLP-1 analog Du Lalu peptide (Dulaglutide) lists in the U.S., and its average organism half-life is little up to 90 Time (China Patent No.: CN1802386B).It addition, Chinese patent CN101891823 discloses a kind of Exendin-4 and class thereof The fusion protein being formed by connecting with natural human IgG2Fc fragment by specific connection peptides like thing.To be applied to people treatment and Speech, when GLP-1/Fc fusion protein is incorporated into receptor in target cell, the Fc region of fusion protein must will not mediate ill effect Subfunction and crack or remove these cells.Therefore, merging part Fc region must be non-cracking performance, is i.e. combining Fc γ Rs With C1q and trigger effect subfunction aspect, Fc must be inactive or SA.But, in patent CN101891823, institute is public Cytotoxicity and complement activation effect that the natural Fc fragment of the Exendin-4 fusion protein opened is mediated may be to bodies Cause certain injury.
The carboxyl terminal peptide (hereinafter referred to as CTP) of human chorionic gonadotropin (hCG) β chain also has some egg of prolongation The effect of white matter Half-life in vivo, the prolongation half-life part that therefore fusion protein disclosed in some patent documentations comprises can be selected Select use immunoglobulin Fc, CTP or other can extend the fusion part of half-life.It addition, CTP can also use as joint In connecting two reactive proteins or for connecting the different subunits of protein.Such as, Chinese patent CN103539860A, In fusion protein disclosed in CN103539861A, CN103539868A and CN103539869A, CTP, as joint, is positioned at rush ovum Between beta subunit and the alpha subunit of bubble hormone；In fusion protein disclosed in patent WO2005058953A2, CTP is as connecing Head, for connecting beta subunit and the alpha subunit of glycoprotein hormones.
The present inventor, through long-term research, is surprisingly merged at Exendin-4 and the like C-terminal CTP peptide fragment and Fc fragment, both can bring synergism, in order to resist the scavenging action of kidney, thus extend albumen and exist The internal half-life.Especially, the inventors discovered that, the CTP being in Fc N end also has metastable three-dimensional conformation, thus Promote Exendin-4 and the like and Fc section independently to fold and form more preferably three-dimensional conformation, show that CTP is as joint peptide A part (and not all) is in action.The fusion protein of the Exendin-4 of the present invention and the like relatively Du Lalu peptide is not only Having the longer in vivo functionality half-life and biologic activity is higher, and more make us unforeseeable, it is in animal body Bioavailability higher.It addition, in the preferred embodiment of the present inventor, the IgG Fc part of fusion protein be derived from human IgG2 and It not IgG4, thus relative to Du Lalu peptide, there is less ill effect subfunction, and there is longer body-internal-circulation half Decline the phase.
Summary of the invention
It is desirable to provide the fusion protein of a kind of high-glycosylation Exendin-4 and the like, its preparation method and Application, to solve the defects such as the Exendin-4 half-life is short, immunogenicity is strong.
One aspect of the present invention, it is provided that a kind of high-glycosylation Exendin-4 and the like fusion protein (hereinafter referred to as melts Hop protein), described fusion protein contains Exendin-4 or its analog (being expressed as Ex4) successively from N end to C end, flexible peptide connects The carboxyl terminal peptide rigid element of head (being expressed as L) and at least 1 human chorion gonadotrophic hormone beta subunit is (hereinafter referred to as (CTP) n, n are 1,2,3,4, or 5) chimeric human normal immunoglobulin's Fc fragment (being expressed as Fc), wherein (CTP) n can be embedded in Fc N end or C end；Thus, described fusion protein can be expressed as Ex4-L-(CTP) n-Fc or Ex4-L-Fc-(CTP) n.
Wherein, the primary structure of described natural Exendin-4 is: His1-Gly-Glu-Gly-Thr5-Phe-Thr-Ser- Asp-Leu10-Ser-Lys-Gln-Met-Glu15-Glu-Glu-Ala-Val-Arg20-Leu-Phe-Ile-Glu-Trp25- Leu-Lys-Asn-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-Pro-Pro-Ser39, is represented by Ex (1- 39)。
Wherein, described Exendin-4 analog and aminoacid sequence at least 70% homology of natural Exendin-4；More excellent Selection of land, the aminoacid sequence of described Exendin-4 analog and natural Exendin-4 at least 80% homology；It is highly preferred that it is described The aminoacid sequence of Exendin-4 analog and natural Exendin-4 at least 90% homology.Most preferably, described Exendin-4 The aminoacid sequence of analog and natural Exendin-4 at least 95% homology.Different according to method of modifying, described Exendin-4 Analog is divided into saltant type, truncated-type or extended pattern.
In some embodiments of the present invention, described Exendin-4 analog is saltant type, i.e. at least 1 non-conservative amino Acid is replaced, and it comprises the sequence such as Formulas I: His1-Xaa2-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Xaa10-Ser- Xaa12-Xaa13-Xaa14-Glu15-Glu-Glu-Ala-Xaa19-Xaa20-Xaa21-Phe-Ile-Xaa24-Trp25- Leu-Xaa27-Xaa28-Gly-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38- Xaa39；
In Formulas I, at least 1 non-conservative site Xaa is replaced:
Xaa2 is selected from Gly, Thr, Ala, Ser, Leu, Ile or Lys；
Xaa10 is selected from Leu, Ala, Ser, Leu, Ile, Glu or Lys；
Xaa12 is selected from Lys, Leu, Thr, Ser, Leu, Ile or Cys；
Xaa13 is selected from Gln, Thr, Ala, Val, Leu, Ile or Lys；
Xaa14 is selected from Met, Tyr, Thr, Ala, Ser, Ile or Lys；
Xaa19 is selected from Val, Cys, Ala, Ser, Leu, Ile or Lys；
Xaa20 is selected from Arg, Thr, Tyr, Ser, Leu, Ile or Lys；
Xaa21 is selected from Leu, Thr, Ala, Asp, Glu, His or Lys；
Xaa24 is selected from Glu, Leu, Thr, Ala, Ser, Lys or Ile；
Xaa27 is selected from Lys, Ala, Ser, Leu, Thr, Ile or Lys；
Xaa28 is selected from Asp, Thr, Ala, Ser, Leu, Ile or Lys；
Xaa30 is selected from Gly, Thr, Ala, Ser, Leu, Ile or Arg；
Xaa31 is selected from Pro, Val, Ser, Ala, Leu, Ile or Lys；
Xaa32 is selected from Ser, Thr, Glu, Ser, Asp, Lys or Ile；
Xaa33 is selected from Thr, Ser, Ala, Met, Leu, Ile or Lys；
Xaa34 is selected from Gly, Thr, Met, Ser, Ile, Leu or Lys；
Xaa35 is selected from Ala, Thr, Ala, Glu, Leu, Ile or Phe；
Xaa36 is selected from Pro, Ala, Thr, Ser, Leu, Ile or Cys；
Xaa37 is selected from Pro, Thr, Ser, Ala, His, Lys or Ile；
Xaa38 is selected from Pro, Thr, Val, Ser, Leu, Lys or Ile；
Xaa39 is selected from Ser, Tyr, Ala, Leu, Ser, Ile or Lys.
In the preferred embodiments of the present invention, described Exendin-4 analog contains the replacement of 1 conserved amino acid, i.e. Xaa2 is Ser, is represented by G2S Ex (1-39)；In another embodiment, Xaa19 is Ala, is represented by V19A Ex (1- 39)；In another embodiment, Xaa24 is Thr, is represented by E24T Ex (1-39)；In another embodiment, Xaa34 is Leu, is represented by G34LEx (1-39).
And for example, in other embodiments of the present invention, described Exendin-4 analog is truncated-type.9 aminoacid of C end Residue is not that Exendin-4 is combined with receptor and necessary to its biological activity, and Exendin-4 analog of the present invention is permissible Disappearance C end 31-39 amino acids, is represented by Ex (1-30).
For another example, in other embodiments of the present invention, described Exendin-4 analog is extended pattern, i.e. increases ammonia at C end Base acid.In one embodiment of the present invention, the 39th Ser connects LysLysLysLysLysLys and (is represented by Ex (1- 45))；Present invention discover that the carboxyl terminal at Exendin-4 increases these 6 aminoacid of KKKKKK, it resists DPP-IV enzyme hydrolysis The ability of effect strengthens, i.e. stability strengthens.
Wherein, the preferred non-immunogenic of described flexible peptide linker, and produce enough between Exendin-4 and Fc Distance, makes steric effect each other be down to minimum.It is preferred that use containing 2 or the flexibility of more Amino acid profile Peptide linker, and selected from following several aminoacid: Gly (G), Ser (S), Ala (A) and Thr (T).
Preferably, described flexible peptide linker comprises G and S residue.The length of connection peptides is the heaviest to the activity of fusion protein Want.For the purpose of the present invention, it is preferable that the general structure of described flexible peptide linker aminoacid composition is (GS) a (GGS) b (GGGS) c (GGGGS) d, wherein a, b, c and d are greater than or equal to the integer of 0, and a+b+c+d >=1.
In some embodiments of the present invention, described peptide linker is selected from following sequence:
(i) L1:GGGGS；
(ii) L2:GSGGGSGGGGSGGGGS；
(iii) L3:GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(iv) L4:GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(v) L5:GGGSGGGSGGGSGGGSGGGS；
(vi) L6:GGSGGSGGSGGS.
Wherein, described CTP rigid element choosing free human chorion gonadotrophic hormone beta subunit carboxyl terminal the 113rd to 145 Total length that aminoacid is formed or the sequence of truncate, specifically, described CTP rigid element comprises SEQ ID NO:1 or its truncate Sequence.
Preferably, described CTP rigid element comprises at least 2 glycosylation sites；Such as, a preferred embodiment of the present invention In, described CTP rigid element comprises 2 glycosylation sites, and exemplarily, described CTP rigid element comprises SEQ ID NO:1N 10 aminoacid, i.e. SSSS*KAPPPS* of end；Or described CTP rigid element comprises 14 amino of SEQ ID NO:1C end Acid, i.e. S*RLPGPS*DTPILPQ；And for example, in another embodiment, described CTP rigid element comprises 3 glycosylation sites, example Property, described CTP rigid element comprises 16 aminoacid of SEQ ID NO:1N end, i.e. SSSS*KAPPPS*LPSPS*R；Again As, in more another embodiments, described CTP rigid element comprises 4 glycosylation sites, and exemplarily, described CTP rigid element contains 28,29,30,31,32 or 33 aminoacid and start from the 113rd of human chorion gonadotrophic hormone beta subunit the, 114,115,116, 117 or 118, terminate at the 145th.Specifically, described CTP rigid element comprises 28 amino of SEQ ID NO:1N end Acid, i.e. SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ.In this article, * represents glycosylation site.Every kind of probability all generations The standalone embodiment of the table present invention.
In further embodiments, the CTP rigid element that the present invention provides is same with natural CTP aminoacid sequence at least 70% Source；In further embodiments, CTP rigid element and natural CTP aminoacid sequence at least 80% homology that the present invention provides；? In other embodiments, the CTP rigid element that the present invention provides and natural CTP aminoacid sequence at least 90% homology；At another In a little embodiments, the CTP rigid element that the present invention provides and natural CTP aminoacid sequence at least 95% homology.
Exemplarily, CTP rigid element of the present invention preferably comprises following sequence:
(i) CTP1:SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii) CTP2:PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii) CTP3:SSSSKAPPPS；
(iv) CTP4:SRLPGPSDTPILPQ；
(v) CTP5:SSSSKAPPPSLPSPSR.
In some embodiments of the invention, described fusion protein comprises 1 above-mentioned CTP rigid element.
Fusion protein of the present invention also can comprise the above-mentioned CTP rigid element of more than 1, it is preferable that comprises 2,3,4 or 5 above-mentioned CTP rigid elements.In one embodiment of the invention, described fusion protein comprises 3 CTP3 rigid elements: SSSSKAPPPSSSSSKAPPPSSSSSKAPPPS (CTP3-CTP3-CTP3, or it is expressed as (CTP3) 3)；Such as another of the present invention In embodiment, described fusion protein comprises 2 CTP5 rigid element: SSSSKAPPPSLPSPSRSSSSKAPPPSLPSPSR (CTP5-CTP5, or it is expressed as (CTP5) 2).
Wherein, Fc fragment preferably is selected from the Fc fragment of human normal immunoglobulin IgG, IgM, IgA and variant thereof；More preferably from people The Fc fragment of IgG1, IgG2, IgG3 or IgG4 and variant thereof；Wherein, described human IgG Fc variant (being expressed as vFc) comprises and is positioned at At least one in wild type human IgG Fc is amino acid modified, and Fc variant is without cracking performance, and demonstrates what minimum Fc-mediated Adverse side effect (ADCC and CDC effect) and/or the binding affinity with FcRn receptor strengthen.
Further, human IgG Fc variant is selected from lower group:
(i) vFc γ 1: containing Leu234Val, Leu235Ala and Pro331Ser sudden change human IgG1 hinge region, CH2 and CH3 region (aminoacid sequence as shown in SEQ ID NO:2)；
(ii) vFc γ 2-1: human IgG2 hinge region containing Pro331Ser sudden change, CH2 and CH3 region is (such as SEQ ID Aminoacid sequence shown in NO:3)；
(iii) vFc γ 2-2: the human IgG2 hinge region containing Thr250Gln and Met428Leu sudden change, CH2 and CH3 region (aminoacid sequence as shown in SEQ ID NO:4)；
(iv) vFc γ 2-3: the human IgG2 hinge region containing Pro331Ser, Thr250Gln and Met428Leu sudden change, CH2 With CH3 region (aminoacid sequence as shown in SEQ ID NO:5).
(v) vFc γ 4: containing Ser228Pro and Leu235Ala sudden change human IgG 4 hinge region, CH2 and CH3 region (as Aminoacid sequence shown in SEQ ID NO:6).
IgG Fc variant provided by the present invention is including but not limited to described in (i)～(v) 5 kinds of variants, it is also possible to be IgG The combination in two class functional variety mutational sites or superposition between isotype subclass, as described in above-mentioned (iv) variant be i.e. by (ii) and (iii) combinatory variants of superimposed the obtained new IgG2Fc in mutational site in.
Fc variant (vFc) in fusion protein of the present invention, it contains the strand of human IgG such as human IgG1, IgG2 and IgG4 Sequence, CH2 and CH3 region.Aminoacid is contained at 228,234,235 and 331 (being determined by EU number system) in this CH2 region Sudden change.It is believed that these amino acid mutations can reduce the effector function of Fc.Human IgG2 does not combine Fc γ R, but demonstrates the most weak Complement activity.Have Pro331Ser sudden change Fc γ 2 variant should complement activity than natural Fc γ 2 lower, and remain Fc γ R azygosperm.IgG4Fc is defective in activating complement cascades, and it is lower by about one than IgG1 with the binding affinity of Fc γ R The individual order of magnitude.Compared with natural Fc γ 4, Fc γ 4 variant with Ser228Pro and Leu235Ala sudden change should show minimum Effector function.The Fc γ 1 with Leu234Val, Leu235Ala and Pro331Ser sudden change also shows than natural Fc γ 1 The effector function reduced.These Fc variants are all more suitable for preparing Exendin-4 and the like than natural human IgG Fc and merge Albumen.And 250 and 428 (by EU number system defined location) is containing amino acid mutation so that Fc district and neonatal receptor The binding affinity of FcRn increases, thus extends half-life (Paul R etc., J Biol Chem, 2004,279:6213 further 6216)；The variant of above-mentioned two class functions is mutually combined or superposition, it is thus achieved that new combinatory variants so that it is effector function reduces Simultaneously and extend its half-life.Fc variant of the present invention comprises the sudden change being but not limited to above-mentioned several site, it is possible to draw Entering the replacement in other site makes Fc have the effector function of reduction and/or strengthen, also with the adhesion of FcRn receptor simultaneously Fc variant function/activity will not be caused to reduce or cause bad conformation change, common mutational site to may refer to Shields RL etc., J Biol Chem, 2001,276 (9): 6591-604.
In a preferred embodiment of the present invention, CTP rigid element is positioned at the N end of vFc, the aminoacid of formed fusion protein Sequence is as shown in SEQ ID NO:8；In another preferred embodiment of the present invention, CTP rigid element is positioned at the C end of vFc, is formed The aminoacid sequence of fusion protein is as shown in SEQ ID NO:10.
According to another aspect of the present invention, it is provided that a kind of DNA encoding above-mentioned fusion protein.The one of the present invention is the most real Executing in example, the DNA sequence of described fusion protein is as shown in SEQ ID NO:7.In another preferred embodiment of the present invention, described in melt The DNA sequence of hop protein is as shown in SEQ ID NO:9.
According to a further aspect of the invention, it is provided that a kind of carrier.This carrier comprises above-mentioned DNA.
According to a further aspect of the invention, it is provided that a kind of host cell.This host cell comprises above-mentioned carrier, or turns Contaminate above-mentioned carrier.
In the detailed description of the invention of the present invention, host cell is the derived cell strain DXB-11 of CHO.
According to a further aspect of the invention, it is provided that a kind of pharmaceutical composition.This pharmaceutical composition comprises and pharmaceutically can connect Carrier, excipient or the diluent being subject to, and the above-mentioned fusion protein of effective dose.
According to a further aspect of the invention, it is provided that described fusion protein is in the II type of preparation treatment non-insulin-dependent Diabetes and by reducing the purposes in the medicine of plasma glucose and benefited other diseases.
According to a further aspect of the invention, it is provided that described fusion protein is in preparation treatment or prevention obesity and passes through In the medicine of suppression stomach and/or bowel movement and suppression stomach and/or intestinal emptying or suppression food intake and benefited other diseases Application.
Provide one according to a further aspect in the invention to prepare from mammal cell line (cell line as derivative in CHO) Or the method producing described fusion protein, comprise the steps of
A the DNA encoding above-mentioned fusion protein is introduced mammalian cell by ()；
In (b) screening step (a) in its growth medium in every 24 hours periods, express more than g/106 cell of 50 μ High yield cell strain；
C cell strain that () incubation step (b) screening obtains；
D fermentation liquid that () results step (c) obtains, purified fusion protein；
Preferably, the mammalian cell in described step (a) is Chinese hamster ovary celI；More preferably CHO derived cell system DXB- 11。
Compared with existing product, the inventors discovered that, the described Exendin-4 of the present invention or its analog fusion tool There is a following outstanding advantages:
1, relative to the Exendin-4Fc fusion protein without CTP rigid element, there is longer circulating half-life in vivo, Circulating half-life in rat body is up to about 22 hours, on the one hand can reduce the fluctuation of drug in blood serum concentration, reduces injection Frequency, thus improve the quality of life of patient；On the other hand the wind of the potential antibody tormation that reduction causes because of frequent drug administration by injection Danger, i.e. immunogenicity reduce.
2, there is the in vivo functionality half-life of prolongation, db/db spontaneous type diabetic mice and STZ inducing pancreatic are being damaged In the evaluating drug effect test of random blood sugar value observation after diabetic mice single-dose, respectively to give FP-A the 168th little Time and after 144 hours, still there is significant hypoglycemic activity；And FP-B has longer body in two kinds of diabetes animal models Interior drug effect, after administration, its blood glucose value of 240h with 216h still has significant difference compared with model group.The relative Du of FP-A with FP-B is drawn Shandong peptide more can for a long time, effectively control mouse blood sugar level.
3, higher bioavailability, single-dose pharmacokinetic data show, in the feelings that dosage is identical Under condition, FP-A and FP-B drug exposure (AUC0～∞) is above exhausted in rat body of Du Lalu peptide, i.e. FP-A and FP-B Higher to bioavailability, it is contemplated that its clinical medicine dose will also decrease.
4, can effectively reduce the content of the HbA1c of db/db diabetic mice, but after long term administration, FP-A group does not occurs HbA1c is significantly reduced situation compared with normal group, and prompting FP-A will not increase the hypoglycemic risk of generation.
Accompanying drawing explanation
Fig. 1, show the FP-A of SpeI/EcoRI fragment in PCDNA3.1 expression vector according to embodiments of the present invention Nucleotide sequence and the aminoacid sequence of derivation, by α 1 microglobulin leader peptide (1-19, with _ _ mark), Ex (1-39) (20- 58), flexible peptide linker (59-85, withMark), CTP rigid element (86-113, withMark) and vFc (114-336) structure Become.
Fig. 2, show the FP-B of SpeI/EcoRI fragment in PCDNA3.1 expression vector according to embodiments of the present invention Nucleotide sequence and the aminoacid sequence of derivation, by α 1 microglobulin leader peptide (1-19, with _ _ mark), Ex (1-39) (20- 58), flexible peptide linker (59-74, withMark), vFc (75-297) and CTP rigid element (298-330, withMark) structure Become.
RBG value change curve between Fig. 3-1, db/db diabetic mice single injection FP-A 0～6h；Significant difference labelling Annotation: FP-A group compared with model group,*P ＜ 0.05,**P ＜ 0.01；Du Lalu peptide group compared with model group,#P ＜ 0.05,##P ＜ 0.01.
RBG value change curve between Fig. 3-2, db/db diabetic mice single injection FP-A 0～216h；Significant difference mark Note annotation: FP-A group compared with model group,*P ＜ 0.05,**P ＜ 0.01；Du Lalu peptide group compared with model group,#P ＜ 0.05,## P ＜ 0.01.
Fig. 4, STZ induced diabetes mice single injection FP-A and FP-B RBG value change curve between 0～240h；Statistics Learn difference marker annotations: FP-A group compared with model group,*P ＜ 0.05,**P ＜ 0.01；FP-B group compared with model group,#P ＜ 0.05,##P ＜ 0.01；Du Lalu peptide group compared with model group,ΔP ＜ 0.05,ΔΔP ＜ 0.01.
The impact on db/db diabetic mice HbA1c (%) of Fig. 5, various dose group FP-A；Significant difference labelling is noted Release: FP-A group compared with model group,*P ＜ 0.05,**P ＜ 0.01.
High lipid food is fed the impact that Mouse Weight increases by Fig. 6, FP-A.
High lipid food is fed the impact (means ± SD, n=8) of glucose tolerance in mice by Fig. 7, FP-A.Note: with normal group phase Ratio,#P ＜ 0.05,##P ＜ 0.01；Compared with high fat group, * P ＜ 0.05, * * P ＜ 0.01.
High lipid food is fed the impact (means ± SD, n=8) of mice serum insulin content by Fig. 8, FP-A.Note: with Normal group is compared,#P ＜ 0.05,##P ＜ 0.01；Compared with high fat group, * P ＜ 0.05, * * P ＜ 0.01.
High lipid food is fed the impact (means ± SD, n=8) of mouse islets element tolerance index by Fig. 9, FP-A.Note: with Normal group is compared,#P ＜ 0.05,##P ＜ 0.01；Compared with high fat group, * P ＜ 0.05, * * P ＜ 0.01.
High lipid food is fed the impact of adipose cell cross-sectional area by Figure 10, FP-A.Note: A: normal group；B: high fat group；C: FP-A group.
Figure 11-1a, C57BL/6J mice give carbohydrate tolerance test curve after FP-B 1d.
1d FP-B impact (means ± SD, n=on C57BL/6J glucose tolerance in mice (iAUC) after Figure 11-1b, administration 8).Note: compared with normal group, * * P ＜ 0.01, * P ＜ 0.05.
Figure 11-2a, C57BL/6J mice give carbohydrate tolerance test curve after FP-B 4d.
4d FP-B impact (means ± SD, n=on C57BL/6J glucose tolerance in mice (iAUC) after Figure 11-2b, administration 8).Note: compared with normal group, * * P ＜ 0.01, * P ＜ 0.05.
Figure 11-3a, C57BL/6J mice give carbohydrate tolerance test curve after FP-B 7d.
7d FP-B impact (means ± SD, n=on C57BL/6J glucose tolerance in mice (iAUC) after Figure 11-3b, administration 8).Note: compared with normal group, * * P ＜ 0.01, * P ＜ 0.05.
Figure 11-4a, C57BL/6J mice give carbohydrate tolerance test curve after FP-B 10d.
10d FP-B impact (means ± SD, n=on C57BL/6J glucose tolerance in mice (iAUC) after Figure 11-4b, administration 8).Note: compared with normal group, * * P ＜ 0.01, * P ＜ 0.05.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Fusion protein of the present invention is generally prepared by biosynthetic method.According to nucleotide sequence of the present invention, this Technical field personnel can prepare the code nucleic acid of the present invention easily with various known methods.These methods such as but not limited to: PCR, DNA synthetic etc., concrete method can be found in J. Pehanorm Brooker, " Molecular Cloning: A Laboratory guide ".As the present invention A kind of embodiment, the method that can be carried out Overlap extension PCR by salvage nucleotide sequence again builds the present invention's Nucleic acid sequence encoding.
Present invention also offers a kind of expression vector, the sequence of the fusion protein comprising code book invention and operating therewith Property be connected expression regulation sequence.Described-be operatively connected " or-be operably coupled to " refer to such a situation, i.e. line Some part of property DNA sequence can regulate or control the activity of same linear DNA molecule other parts.Such as, if started Son controls transcribing of sequence, then it is operably coupled to coded sequence exactly.
Expression vector can use commercially available such as but not limited to: pcDNA3, pIRES, pDR, it is thin that pUC18 etc. can be used for eucaryon The carrier of born of the same parents' system expression.Those skilled in the art can select suitable expression vector according to host cell.
According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can be conventionally by restricted Enzyme is sheared and splicing, and the coded sequence of the fusion protein of the present invention is inserted suitable restriction site, prepares the weight of the present invention Group expression vector.
Present invention also offers the host cell expressing fusion protein of the present invention, wherein contain the fusion protein of the present invention Coded sequence.Described host cell preferably eukaryotic cell, such as but not limited to CHO, COS cell, 293 cells, RSF is thin Born of the same parents etc..As the optimal way of the present invention, described cell is Chinese hamster ovary celI, and it can express the fusion protein of the present invention well, Combination activity can be obtained good, the fusion protein having good stability.
The present invention also provides for a kind of method that recombinant DNA prepares fusion protein of the present invention, and its step includes:
1) nucleotide sequence of encoding fusion protein is provided；
2) by 1) nucleotide sequence be inserted into suitable expression vector, it is thus achieved that recombinant expression carrier；
3) by 2) recombinant expression carrier import suitable host cell；
4) transformed host cell is cultivated under conditions suitable for the expression；
5) supernatant is collected, and purified fusion protein product.
Described coded sequence importing host cell can be used the multiple known technology of this area, such as but not limited to: phosphorus Acid calcium deposit, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkali metal from Sub-method.
Cultivation and expression about host cell can be found in Olander RM Dev Biol Stand 1996；86:338. Clear liquid can be collected by the cell in centrifugal segregation suspension and residue.Can be identified by agarose gel electrophoresis technology.
Can be substantially uniform character by the above-mentioned fusion protein purification prepared, such as on SDS-PAGE electrophoresis in Single band.Such as, when recombiant protein is secreting, expressing, the ultrafilter membrane of commercialization can be used to separate described albumen, example Such as Products such as Millipore, Pellicon, first will express supernatant and concentrate.Concentrated solution can use the method for gel chromatography The most in addition purification, or use the method purification of ion-exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or sun from Sub-displacement chromatography.Gel-type vehicle can be the substrate that agarose, glucosan, polyamide etc. are usually used in protein purification.Q-or SP-group It it is ideal ion-exchange group.Finally, can also be used with hydroxylapatite adsorption chromatography, metal chelate chromatography, hydrophobic mutually The method polishing purification further to above-mentioned purified product such as effect chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC).Above-mentioned institute There is purification step to may utilize different combinations, finally make purity of protein reach substantially uniform.
The fusion to expressing of the available affinity column containing the specific antibody of described fusion protein, receptor or part Albumen is purified.According to the characteristic of the affinity column used, available conventional method, such as high-salt buffer, change pH etc. Method elution of bound amalgamation polypeptide on affinity column.Selectively, the aminoterminal of described fusion protein or c-terminus are also One or more polypeptide fragments can be contained, as protein tag.Any suitable label may be used to the present invention.Such as, institute The label stated can be FLAG, HA, HA1, c-Myc, 6-His or 8-His etc..These labels can be used for carrying out pure to fusion protein Change.
Embodiment 1, the expression plasmid of structure encoding fusion protein
Coding for alpha 1 microglobulin (α 1 microglobulin) leader peptide and ripe Exendin-4 and the like, flexibility The gene order of peptide linker, CTP rigid element and human IgG Fc variant is all the Chinese hamster ovary celI preference codon that artificial optimization crosses, Full length sequence obtains through chemical synthesis process.For the ease of the specific site by purpose fragment inserting expressioning carrier, synthesized Fragment 5 ' and 3 ' end be respectively arranged with restriction enzyme site, respectively SpeI and EcoRI.Exendin-4 after checking and Analog fusion gene SpeI and EcoRI enzyme action, be then inserted into through PCDNA3.1 improved plasmid PXY1A1 phase Answer between restriction enzyme site, obtained fusion gene expression plasmid.This plasmid is containing cytomegalovirus early promoter, and it is mammal Enhancer needed for cell high level expression exogenous gene.This plasmid is possibly together with selected marker thing, thus permissible in antibacterial There is kalamycin resistance, and can have G418 resistance in mammalian cell.It addition, when host cell is DHFR gene When expressing deficiency, PXY1A1 expression vector contains dihydrofolate reductase (DHFR) gene of mice, thus there is ammonia first Can coamplification fusion gene and DHFR gene (seeing United States Patent (USP) US 4,399,216) during pterin (MTX).
As shown in table 1, the present invention constructs the fusion protein of a series of Exendin-4 and the like, and they comprise not With flexible peptide linker (Linker) and IgG Fc variant (vFc) the element composition of several different subtype of length, and CTP rigidity Cell position and length are the most different.In order to verify containing at least 1, and the CTP rigid element of different length is respectively provided with higher Biologic activity, we construct fusion protein F P-A, FP-B, FP-C, FP-D, FP-E, FP-F, FP-G and FP-H；Simultaneously Also construct the FP-I without CTP rigid element.Wherein the nucleotide sequence of FP-A and FP-B and the aminoacid sequence of translation divide Not as depicted in figs. 1 and 2.The aminoacid composition of fusion protein is shown in summary of the invention or sequence table.
The composition of several fusion protein that table 1. builds
The expression in transfectional cell series of embodiment 2, fusion protein
Recombinant expression carrier plasmid is transfected into mammalian host cell line, to express Exendin-4 and the like Fusion protein.In order to stablize high-caliber expression, preferred host cell system is that DHFR deficient CHO-cell (sees U.S. State's patent US 4,818,679), in the present embodiment, host cell chooses CHO derived cell strain DXB11.The one preferably side of transfection Method is electroporation, it is possible to use other method, including calcium phosphate cosedimentation, fat transfection and Protoplast fusion.In electroporation, Be set to 300V electric field and 1050 μ Fd electric capacity Gene Pulser electroporation apparatus (Bio-Rad Laboratories, Hercules, CA), 5 × 10 in cuvette7Individual cell adds the 50 highly purified expression plasmids of μ g.In transfection two days later, Culture medium is made into the growth medium containing 0.6mg/mL G418.By the elisa assay method of anti-human igg Fc, screening is to choosing Select medication and there is the transfectant of resistance.Also the quantitative of fusion protein expression can be carried out with the ELISA of anti-Exendin-4.Pass through Method of Limited Dilution 96 well culture plate, sub-clone produces the hole of high level fusion protein.
In order to realize the expression of fusion protein higher level, preferably carry out coamplification with the DHFR gene by MTX Drug inhibition. In the growth medium containing progressive concentration MTX, with the antigen-4 fusion protein gene of DHFR gene coamplification transfection.Method of Limited Dilution DHFR expresses positive sub-clone, progressively pressurizes and filters out the transfectant that can grow in up to 6 μMs of MTX culture medium, measures Its secretion rate, filters out the cell line of high expressed foreign protein.Secretion rate is exceeded about 30 (being preferably about 50) μ g/106(i.e. hundred Ten thousand) individual cell/24 hour cell line use serum-free medium carry out adaptability suspension culture, use CMC model the most again Base purified fusion protein.
Embodiment 3, fusion protein purification with qualitative
With 1N NaOH, the conditioned medium containing fusion protein is titrated to pH 7～8, then with the nitric acid of 0.45 micron Cellulose filter filters.Filtrate is loaded onto on the Protein A post that phosphate buffer saline (PBS) balances.To be fused Protein binding, after Protein A post, discards the component of outflow.This post is washed, until the OD value at 280nm is less than with PBS 0.01.Then with the fusion protein of the citrate buffer solution elution of bound that 0.1M pH is 3.75.The eluent 1M of 0.4 volume K2HPO4Neutralize, merge the component containing purifying protein, and use PBS.Then with the nitrocellulose filter of 0.22 micron Filter, and be stored in 4 DEG C.Non-reduced with under reducing condition, SDS-PAGE protein product identified, analyze.Use BSA As standard, by BCA protein analysis quantitatively this fusion protein.
Embodiment 4, In vitro biological activity measure
Set up the diabetes medicament screening cell model with GLP-1 signal path as target spot, be used for screening GLP-1 receptor and swash The New-type long-acting GLP-1 analog of dynamic agent class.According to document (Zlokarnik G etc., Science, 1998,279 (5347): 84- 88) described method measures.By the expression plasmid PGL-4.29 with people's GLP-1R expression plasmid and CRE-Luc reporter gene (Luc2P/CRE/Hygro) the cotransfection CHO-K1 cell (purchased from Promega company), obtains table altogether by antibiotic pressurization screening Reach the stable cell line of two kinds of plasmids.When external activity is analyzed, it is inoculated into 96 porocyte trainings with 16000 cells/well/200 μ l Support in plate, with the DMEM culture medium culturing 16 containing 10%FBS～24 hours, treat that cell grows to cover more than 90% area at the bottom of plate Time, by with DMEM culture medium gradient dilution fusion protein F P-A containing 10%FBS, FP-B, FP-C, FP-D, FP-E, FP-F, FP- G, FP-H and FP-I, then add by every hole 10 μ l, Concentraton gradient is set to 0.010,0.020,0.039,0.078,0.156, 0.313,0.625,1.25 and 2.5nM, isocyatic Du Lalu peptide positive controls (Eli Lilly Company is set simultaneously Produce, article No.: 9301897).At 37 DEG C, 5%CO2Under the conditions of hatch 5-6 hour after, suck supernatant, be slowly added to 300 μ l PBS washed cell, sucks PBS subsequently, adds 40 μ l lysates, shakes 15min, and every hole adds 40 μ l luciferase substrate subsequently (lucky full biological company limited produces for luciferase (Luciferase) reporter gene detection kit, article No.: GM-040501B Product), react 2 minutes, by multi-functional microplate reader (SpectraMax M5system, Molecular Device company) in wavelength Measure fluorescent value under 560nm, and draw dose-effect curve according to fluorescent value, calculate EC50Value, the results are shown in Table 2.FP-A and The EC of FP-B50Value about 0.03086nM and 0.02854, and the EC of Du Lalu peptide50Value be about 0.02987nM, illustrate FP-A, The biologic activity of FP-B with Du Lalu peptide is suitable；The EC of FP-C, FP-D, FP-E, FP-F, FP-G, FP-H and FP-I50Value refers to Table 2；Visible, each fusion protein all has certain activation to GLP-1 receptor.
The external activity EC of each fusion protein of table 2.50Value compares
Embodiment 5, the change of blood sugar situation of db/db diabetic mice single injection FP-A
Female diabetes db/db mice (purchased from Shanghai Slac Experimental Animal Co., Ltd.), 8 week old, body weight 42 ± 2g, is randomly divided into 3 groups by body weight, often group 6.3mg/kg injected sc FP-A, positive group injection is pressed by reagent group The Du Lalu peptide (Eli Lilly and Company produces, article No.: 9301897) of 3mg/kg, model group injection equal-volume dosage (10mL/kg) PBS.Each treated animal respectively at be administered before (0h), be administered after 1h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h and 216h carry out tail vein blood, measure random by blood glucose meter (quasi-blood glucose meter is pacified in three promises) Blood glucose value (Random blood glucose, RBG), and record data.Blood glucose level data is with mean ± standard deviation (means ± SD) Form represents, uses SPSS18.0 statistical software analytical data.Normal distribution, between many groups, mean difference uses single factor test variance to divide Analysis, homogeneity of variance uses LSD inspection, and heterogeneity of variance uses Dunnett ' T3 inspection；Non-normality distribution uses non parametric tests, P < 0.05 represents have notable statistics difference.
Under same dose, FP-A and positive control drug Du Lalu peptide all have hypoglycemic activity, such as Fig. 3-1 and Fig. 3-2 institute Show.Checking through t-test, showing all occurs in the random blood sugar value relative model group in 0h～6h, FP-A group with Du Lalu peptide treated animal Work reduces, P < 0.01 (Fig. 3-1), illustrates that the two onset time in vivo is close, and hypoglycemic effect is similar in a short time；Additionally from In being administered latter 9 days in mice RBG value change curve (Fig. 3-2) it can be seen that Du Lalu peptide hypoglycemic activity be only capable of maintaining to 4th day, the 120th hour upon administration, its blood glucose value did not the most have significant difference compared with model group, and FP-A can maintain extremely Being administered latter 168 hours, i.e. the blood sugar level of the 7th day mice is compared with model group, still has significant difference (P < 0.05).Cause And, it is expected to be developed into weekly or longer cycle administration potential long-acting GLP-1 receptor agonists once.
Embodiment 6, the change of blood sugar situation of db/db diabetic mice single injection FP-B
Choose SPF level, the male db/db mice of 7 week old and C57BL/6J male mice (to buy and test in Shanghai Si Laike Company of Animals Ltd., animal productiong credit number SCXK (Shanghai): 2012-0002).Feeding environment: temperature 22-25 DEG C is the wettest Degree 45-65%, lighting hours 12h/d.After adaptability raises 1 week, 30 db/db mices are pressed random blood sugar value and body weight is random It is divided into 5 groups: model control group；Du Lalu peptide group (dosage: 3mg/kg)；FP-B: arrange 0.75,1.5 and 3mg/kg is low, Middle and high three dosage groups, often group 6；C57BL/6J mice is as Normal group (n=6).Each administration group subcutaneous injection gives Relative medicine solution, model control group and Normal group subcutaneous injection PBS, be administered volume and be 10ml/kg.Each group Animal respectively at be administered before (0h), be administered after 1h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h and 240h tail vein blood, measures random blood sugar value RBG of each treated animal, and remembers by blood glucose meter (quasi-blood glucose meter is pacified in three promises) Record data.Data are poor with mean ± markForm represents, uses SPSS 18.0 statistical software analytical data.Normal distribution, Between many groups, mean difference uses one factor analysis of variance, and homogeneity of variance uses LSD inspection, and heterogeneity of variance uses Dunnet T3 inspection Test；Non-normality distribution uses non parametric tests, and P < 0.05 represents have notable statistics difference.
As can be known from Table 3, after single-dose, FP-B middle and high dosage group, compared with Du Lalu peptide group, all shows more longlasting Hypoglycemic activity.Du Lalu peptide group 144h upon administration compares, and there was no significant difference with the RBG value of model group, and this Time FP-B in the RBG value of dosage group compared with model group, still there is significant difference (P < 0.05), FP-B high dose group still has Significant difference (P < 0.01).On the other hand, the blood sugar reducing function of FP-B presents dose dependent, and compared with model group, FP-B is high The blood sugar reducing function of dosage group persistently to be administered after 240h (P < 0.05)；After in FP-B, the blood sugar reducing function of dosage group is persistently extremely administered 196h (P < 0.05)；And the blood sugar reducing function of FP-B low dose group only continues 120h (P < 0.01) to administration, with Du Lalu The peptide blood sugar lowering persistent period is similar.
The impact (means ± SD, n=7) on db/db mice random blood sugar of table 3. single subcutaneous injection FP-B
Note: compared with Normal group,#P ＜ 0.05,##P ＜ 0.01；Compared with model comparison,*P ＜ 0.05,**P ＜ 0.01；Compared with Du Lalu peptide group,ΔP ＜ 0.05,ΔΔP ＜ 0.01.
The impact (means ± SD, n=7) on db/db mice random blood sugar of continued 3. single subcutaneous injection FP-B
Note: compared with Normal group,#P ＜ 0.05,##P ＜ 0.01；Compared with model comparison,*P ＜ 0.05,**P ＜ 0.01；Compared with Du Lalu peptide group,ΔP ＜ 0.05,ΔΔP ＜ 0.01.
The impact (means ± SD, n=7) on db/db mice random blood sugar of continued 3. single subcutaneous injection FP-B
Note: compared with Normal group,#P ＜ 0.05,##P ＜ 0.01；Compared with model comparison,*P ＜ 0.05,**P ＜ 0.01；Compared with Du Lalu peptide group,ΔP ＜ 0.05,ΔΔP ＜ 0.01.
Embodiment 7, the pharmacodynamic study of STZ induced diabetes mice single injection FP-A and FP-B
Choosing SPF level male mouse of kunming (purchased from Shanghai Slac Experimental Animal Co., Ltd.), body weight 25 ± 2g, by body Heavily it is randomly divided into diabetic groups and normal group.Adapting to raise the kunming mice of 1 week, fasting 18h, weigh, diabetic groups is pressed 150mg/kg lumbar injection gives 1%STZ solution, pH=4.4 (purchased from Sigma company, article No. S0130)；Normal group mice (n =8) lumbar injection gives isopyknic citric acid-sodium citrate buffer (purchased from Chemical Reagent Co., Ltd., Sinopharm Group). After injection the 10th day detection diabetic groups mice random blood sugar value (Random blood glucose, RBG), wherein RBG >= 16.7mmol/L is the successful diabetic mice of modeling.For observing the test medicine blood sugar lowering to the diabetic mice that STZ induces Effect, chooses STZ-induced diabetes mice 32, is randomly divided into 4 groups, and often group 8, gives 3mg/kg respectively at subcutaneous injection The Du Lalu peptide of FP-B and 3mg/kg of FP-A, 3mg/kg.Model group and normal group give equal-volume dosage (10ml/ respectively Kg) PBS.Respectively at be administered before (0h), be administered after 1h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h and 240h tail vein blood, measures the random blood sugar of each treated animal by blood glucose meter (quasi-blood glucose meter is pacified in three promises) Value RBG, and record data.Data are poor with mean ± markForm represents, uses SPSS 18.0 statistical software analytical data. Normal distribution, between many groups, mean difference uses one factor analysis of variance, and homogeneity of variance uses LSD inspection, and heterogeneity of variance uses Dunnet T3 checks；Non-normality distribution uses non parametric tests, and P < 0.05 represents have notable statistics difference.
As shown in Figure 4,0-240 hour random blood sugar value after diabetic mice single injection FP-A and FP-B of STZ induction Change curve shows, FP-A and FP-B all can be effectively reduced the random blood sugar value of the diabetic mice of STZ induction.FP-A exists After administration, the 24th hours blood glucose level is down to minimum, the most slowly gos up, in the 96th hour its random blood sugar value and model group phase Ratio, still has significant difference (P ＜ 0.05)；And the hypoglycemic effect of FP-B group is more preferably, the 24th hours blood glucose level upon administration Also continuing to reduce, within latter 48 hours, be down to minimum in being administered, the 216th hour random blood sugar value is compared with model group upon administration, still There is significant difference (P ＜ 0.01).
The continuous random blood sugar giving FP-A in 10 weeks of embodiment 8, db/db diabetic mice and HbA1c changes of contents
SPF level female db/db mice (purchased from Shanghai Slac Experimental Animal Co., Ltd.), 8 week old, adaptability raises 1 24 db/db mices are pressed random blood sugar value (random blood glucose, RBG) and are randomly divided into 4 groups (n=6) by Zhou Hou: Model group, FP-A by low (0.75mg/kg), in (1.5mg/kg), high (3mg/kg) dosed administration.Each administration group subcutaneous injection is given Give the drug solution of corresponding dosage, model group subcutaneous injection PBS, be administered volume and be 10ml/kg.Each treated animal is weekly Being administered once, successive administration 10 weeks, by blood glucose meter, (pacifying quasi-blood glucose meter, Changsha Sinocare Biosensing Co., Ltd produces respectively Product) detection after being administered different blood sampling time points respectively organize mice RBG value, and record data.Blood sampling time point sets: be administered for the first time Before (0d), be administered after 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d and 70d.70d, each group mice fasting 14h Posterior orbit takes blood, and (Lay perseverance medical electric is wished in Shenzhen to be had to measure test kit (immunoturbidimetry) with glycolated hemoglobin immediately after Limit Products, equipment registration number: Guangdong food medicine prison tool (accurate) word 2013 the 2400025th) and the specific egg of necessary instrument H700 Glycolated hemoglobin (HbA1c) content in white analyser (Shenzhen Xi Laiheng medical electric company limited product) detection whole blood, knot The percentage ratio (%) that fruit accounts for total hemoglobin with HbA1c represents.
Data represent with mean ± standard deviation (means ± SD) form, use SPSS18.0 statistical software analytical data.Just State is distributed, and between many groups, mean difference uses one factor analysis of variance, and homogeneity of variance uses Dunnett t-inspection, and heterogeneity of variance is adopted Check with Dunnett ' T3；Non-normality distribution uses non parametric tests, and P < 0.05 represents have notable statistics difference.
From table 4, the variation tendency of each group 10 weeks random blood sugar values of mice successive administration understands, the high, medium and low dosage of FP-A Mouse blood sugar value relative model group all decreases to some degree of group, and its hypoglycemic activity presents dose dependent.Carry Show that FP-A can control the blood sugar level of db/db diabetic mice effectively, constantly.And, first administration and last are administered FP-A Hypoglycemic effect similar, illustrate not occur, owing to the Fast Anti property of medicine of receptor is reacted, to cause the tolerance effect to FP-A.
Glycolated hemoglobin (HbA1c) is the product that in blood, glucose combines with erythrocytic hemoglobin, it and blood In the proportional relation of glucose level.Owing to the erythrocyte life-span in blood circulation is about 120 days, therefore HbAle egg Can reflect in vain and take the aggregate level of 4-12 week blood glucose before blood, compensate for fasting glucose and only reflect the deficiency of instantaneous blood glucose.Thus, HbA1c is the most important evaluation index of long-term control blood glucose, be also clinic decide whether to change therapeutic scheme important evidence. In the present embodiment, HbA1c checks that result can be stablized, reflect reliably and take before blood 2～the glycemic control situation of 3 months mices. Successive administration respectively organizes mice HbA1c assay result as it is shown in figure 5, show high, medium and low dosage group FP-A saccharifying after 10 weeks HbA1c content relatively model group all occurs significance to reduce (P ＜ 0.01), and presents dose dependent, wherein high dose group HbA1c (%) reduce the most notable (6.38 ± 1.63), but remain above HbA1c level (the normal value reference model of normal C57BL/6J mice Enclose: 2.5%-3.5%), even if illustrating that the FP-A of 3mg/kg also will not cause the generation of long-term hypoglycemic reaction；Result above carries Show that FP-A can for a long time, effectively, smoothly control mouse blood sugar, and do not increase and cause hypoglycemic risk, this and blood glucose in table 4 Variation tendency is consistent.
The table 4.FP-A impact (means ± SD, n=6) on db/db mice random blood sugar
Note: each dosage group is compared with model group*P ＜ 0.05；**P ＜ 0.01.
The impact (means ± SD, n=6) on db/db mice random blood sugar of continued 4.FP-A
Note: each dosage group is compared with model group*P ＜ 0.05；**P ＜ 0.01.
The experimentation of the obesity mice prevention antiobesity action that high lipid food is induced by embodiment 9, FP-A
One, model is set up and is administered with packet
7 week old C57BL/6J male mices 24 (buying from Shanghai Slac Experimental Animal Co., Ltd., permitted by animal productiong Can the number of card SCXK (Shanghai): 2012-0002).Feeding environment: temperature 22-25 DEG C, relative humidity 45-65%, lighting hours 12h/d. After C57BL/6J mice adaptability raises 1 week, it is randomly divided into 3 groups by body weight: normal group (NFD), high fat group (HFD), FP-A group (HFD+FP-A 0.3mg/kg).High fat group, FP-A group give high lipid food (D12492 high lipid food, U.S. Research Diets Products) feed, normal group mice gives standard feed (normal fat diet, NFD) and feeds.FP-A group is pressed Every 6 days subcutaneous injection relative medicine solution of 0.3mg/kg dosage once, normal group and model group subcutaneous injection PBS, give Medicine volume 10ml/kg.After 96 days, each group mice fasting 16 hours, to weigh and detect fasting blood sugar, eye socket takes blood, 400 × g Centrifugal 15min, separates to obtain serum.After taking blood, de-cervical vertebra puts to death mice, measures mice nose to anus length (body length), calculating Lee ' s index.Separate bilateral epididymal peripheral adipose tissue, claim weight in wet base.Take same position epididymal adipose and be stored in 10% good fortune In your Malin's solution, detect for pathomorphology.
Two, Indexs measure
2.1, body weight and obese degree
Every 6 days mouse weights once, are drawn Mouse Weight growth curve, and are calculated Mouse Weight increment.Mouse Weight Body weight during body weight-mice group when increment=mice last weighs.With Lee ' s index assessment mice obese degree.
2.2, fat weight and index
Weigh little with analytical balance (BSA223S electronic balance, Sai Duolisi scientific instrument (Beijing) company limited product) Mus both sides epididymis peripheral adipose tissue, claims weight in wet base.Calculating epididymal adipose tissues mass fraction (mg/g): epididymal adipose tissues mass fraction=attached Testis fat mass (mg)/empty body weight (g).
2.3, oral glucose tolerance test
After test carries out 84 days, each group mice fasting 16h (17:00am-9:00pm), (pacify quasi-blood glucose meter, length by blood glucose meter Husky Sinocare Biosensing Co., Ltd product) detect each group of mice fasting blood sugar (FBG), weigh.Oral (i.g) gives 2g/kg glucose solution (analytical pure anhydrous glucose, Chemical Reagent Co., Ltd., Sinopharm Group's product), after detection gavage 30min, 60min, 90min and 120min respectively organize mouse blood sugar value, draw glucose tolerance curve, and trapezoidal method calculates revises blood glucose curve Lower area value (iAUC).
2.4, biochemical index
And supporting with automatic clinical chemistry analyzer (Europe despot's XL-200 automatic clinical chemistry analyzer, Germany's Europe despot's Products) TG (triglyceride detection kit, Meikang Biotech Co., Ltd., Ningbo's product) and TC (total gallbladder in test kit detection serum Sterin detection kit, Meikang Biotech Co., Ltd., Ningbo's product) content, concrete operations are entered according to instrument description OK.
2.5, insulin and insulin resistant index
Mice serum pancreas is detected by ELISA method (mouse islets element ELISA detection kit, U.S.'s ALPCO Products) Island cellulose content, and calculate insulin resistant index.
2.6, fatty tissue pathology detection
Take the same side epididymal adipose, observe adipose tissue with hematoxylin-eosin staining method (being called for short HE dyeing) Morphology.
Three, add up and analyze
Data represent with mean ± standard deviation (means ± SD) form, use SPSS18.0 statistical software analytical data.Just State is distributed, and between many groups, mean difference uses one factor analysis of variance, and homogeneity of variance uses LSD inspection, and heterogeneity of variance uses Dunnet T3 checks；Non-normality distribution uses non parametric tests, and P < 0.05 represents have notable statistics difference.
Four, result
4.1, FP-A high lipid food is fed Mouse Weight and obese degree impact
Compared with normal group, high fat group Mouse Weight, body weight increment and Lee ' s index significantly raise (P ＜ 0.01). FP-A can significantly reduce high lipid food and feed the body weight of mice, body weight increment and Lee ' s index (P ＜ 0.01), the results are shown in Table 5 And Fig. 6.
High lipid food is fed Mouse Weight and the impact (means ± SD, n=8) of Lee ' s index by table 5.FP-A
Note: compared with normal group,#P ＜ 0.05,##P ＜ 0.01；Compared with high fat group, * P ＜ 0.05, * * P ＜ 0.01.
4.2, FP-A is on epididymal adipose tissues quality and the impact of mass fraction
As shown in table 6, compared with normal group, high fat group mouse epididymis fat mass and mass fraction significantly raise (P ＜ 0.01).Compared with high fat group, FP-A group mouse epididymis fat mass and mass fraction all significantly reduce (P ＜ 0.05).
The table 6.FP-A impact (means ± SD, n=8) on epididymal adipose tissues quality and mass fraction
Note: compared with normal group, #P ＜ 0.05, ##P ＜ 0.01；Compared with high fat group, * P ＜ 0.05, * * P ＜ 0.01.
4.3, FP-A is on mice serum TG and the impact of TC content
Compared with normal group, high fat group mice serum TC and TG content significantly raise (P ＜ 0.01).With high fat group phase Ratio, FP-A group mice serum TC content significantly reduces (P ＜ 0.01), and TG content also significantly reduces (P ＜ 0.01).Result is shown in Table 7.
The table 7.FP-A impact (means ± SD, n=8) on mice serum TG and TC content
Note: compared with normal group, #P ＜ 0.05, ##P ＜ 0.01；Compared with high fat group, * P ＜ 0.05, * * P ＜ 0.01.
4.4, high lipid food is fed the impact of glucose tolerance in mice by FP-A
As it is shown in fig. 7, high fat group iAUC is significantly higher than normal group (P ＜ 0.05).Compared with high fat group, the iAUC of FP-A group Significantly reduce (P ＜ 0.05).
4.5, high lipid food is fed mice serum insulin and the impact of insulin resistant index by FP-A
Compared with normal group, the serum insulin concentration (P ＜ 0.01) of high fat group mice and insulin resistant index (P ＜ 0.05) all significance raises, i.e. mice has occurred obvious Insulin resistance, thus understands the supersecretion islets of langerhans of compensatory Element produces hyperinsulinemia.Compared with high fat group, FP-A can significantly reduce mice serum insulin (INS) content (P ＜ 0.05) mouse islets element tolerance index (HOMA-IR) (P ＜ 0.05), is improved.Result is shown in Fig. 8 and Fig. 9 respectively.
4.6, pathomorphology inspection
HE coloration result shows, compared with normal group, high fat group mouse epididymis adipose cell cross-sectional area dramatically increases.With High fat group is compared, and FP-A group mouse epididymis adipose cell cross-sectional area is reduced significantly, and result is shown in Figure 10.
Comprehensive above result of study, it was demonstrated that FP-A can efficiently control the body weight of the obesity mice of high lipid food induction, There is antiobesity action.
Embodiment 10, the FP-B impact on C57BL/6J glucose tolerance in mice
8 week old SPF rank male C 57 BL/6 J mouses are (purchased from Shanghai Slac Experimental Animal Co., Ltd., animal productiong Credit number SCXK (Shanghai): 2012-0002).Feeding environment: temperature 22-25 DEG C, relative humidity 45-65%, lighting hours 12h/ d.24 SPF level male C 57 BL/6 J mouses, after adaptability is raised, are randomly divided into normal group and FP-B group, respectively subcutaneous injection Give PBS or 3mg/kg FP-B solution.After administration, 1d, 4d, 7d and 10d carry out carbohydrate tolerance experiment respectively.Concrete grammar As follows: each group mice fasting 16h (17:30pm-9:30am), detect each group of mouse blood sugar value, weigh, i.p gives 2g/kg Fructus Vitis viniferae Sugar juice, is administered volume 10mL/kg.After detection injection, 15min, 30min, 60min, 90min and 120min respectively organize mouse blood sugar Value, draws glucose tolerance curve, and trapezoidal method calculates Area under the curve of blood glucose (iAUC) after revising.
Data represent with mean ± standard deviation (means ± SD) form, use SPSS18.0 statistical software analytical data.Just State is distributed, and between many groups, mean comparison in difference uses one factor analysis of variance, and homogeneity of variance selects LSD inspection, and heterogeneity of variance selects Dunnet T3 checks；Non-normality distribution uses non parametric tests, and P < 0.05 represents have notable statistics difference.
C57BL/6J mice gives carbohydrate tolerance test blood glucose curve after FP-B 1d, 4d, 7d and 10d, respectively as Figure 11-1a, Shown in 11-2a, 11-3a and 11-4a, display FP-B administration group mice is relative to increasing face under the carbohydrate tolerance empirical curve of normal group Amassing and significantly reduce, after single-dose, 10d still has the sugared tolerance effect being obviously enhanced.As Figure 11-1b, 11-2b, 11-3b and Shown in 11-4b, after C57BL/6J mice gives FP-B 1d, 4d, 7d and 10d, the iAUC value of FP-B group is the most aobvious compared with normal group Write and reduce (P ＜ 0.01).This test confirms that FP-B can promote that islet β cell insulin, suppression exogenous glucose are taken in Caused temporary blood glucose raises, and promotes glucose utilization, explains the long-acting hypoglycemic activity of FP-B from mechanism of action.
Embodiment 11, FP-A, FP-B and FP-I single-dose pharmacokinetics in rat body measures
Male SPF level SD rat (purchased from Shanghai Bi Kai laboratory animal company limited), raises single subcutaneous injection after a week in advance (sc) FP-A, FP-B and FP-I of 0.5mg/kg, often group 4, before being administered 0h, 2h after administration, 8h, 24h, 32h, 48h, 56h, 72h, 96h, 120h and 144h eye socket takes blood, the most about about 0.3ml, is denoted as respectively: T0、T2、T8、T24、T32、T48、T56、 T72、T96、T120And T144.Stand after taking blood, then be centrifuged 10min separation serum with 5000rpm, merge after-70 DEG C of freezen protective Inspection.When measuring with double crush syndrome, resist with the NH2 terminal monoclonal of self-control or commercially available anti-Exendin-4 or GLP-1 Body (as Santa Cruz company produces, article No. SC-65389) is coated, with self-control or commercially available horseradish peroxidase-labeled Mouse-anti human IgG Fc monoclonal antibody (such as Sino Biological Inc., article No.: 10702-MM01E-50) detects. After enter data into analysis software PKSOLVER, draw medicine to be measured T in blood1/2, CmaxAnd AUC0～48h～in the medicine such as ∞ generation, is dynamic Mechanics parameter.
As in table 8, result shows, the FP-I of 0.5mg/kg circulating half-life T in rat body1/2It is 14.1 ± 1.67 little Time, and the T that FP-A and FP-B of 0.5mg/kg is in rat body1/2It is respectively 21.4 ± 2.51 and 22.6 ± 3.6 hours.FP-A With FP-B maximum plasma concentration CmaxValue is all remarkably higher than FP-I.It addition, recorded by blood sampling time points different in comparison sheet 8 AUC0～t(t=2h, 5h, 8h, 24h, 28h, 32h or 48h) can learn, in the case of dosage is identical, FP-A's and FP-B Drug exposure is all higher than FP-I, i.e. FP-A and the FP-B absolute bioavailability in rat body relatively without CTP rigid element FP-I higher, it is contemplated that its clinical administration dosage will also decrease.
The pharmacokinetic parameter of FP-A, FP-B and FP-I of table 8. male SD rat single subcutaneous injection 0.5mg/kg
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally Enclose.

Claims (17)

1.Exendin-4 or the fusion protein of its analog, described fusion protein from N end to C end comprise successively Exendin-4 or Its analog, flexible peptide linker, at least 1 human chorion gonadotrophic hormone beta subunit carboxyl terminal peptide rigid element and people's immunity Immunoglobulin Fc fragment, or, described fusion protein comprises Exendin-4 or its analog successively from N end to C end, flexible peptide connects Head, human normal immunoglobulin's Fc fragment and at least 1 human chorion gonadotrophic hormone beta subunit carboxyl terminal peptide rigid element.
2. fusion protein as claimed in claim 1, it is characterised in that described fusion protein is glycosylated, preferably through Glycosylated middle expression of mammalian cell (e.g., Chinese hamster ovary cell).
3. fusion protein as claimed in claim 1, it is characterised in that the aminoacid sequence of described Exendin-4 is His1- Gly-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Leu10-Ser-Lys-Gln-Met-Glu15-Glu-Glu-Ala-Val- Arg20-Leu-Phe-Ile-Glu-Trp25-Leu-Lys-Asn-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-Pro- Pro-Ser39。
4. fusion protein as claimed in claim 1, it is characterised in that described in described Exendin-4 analog and claim 3 The aminoacid sequence of the Exendin-4 in fusion protein has at least 70%, the homogeneity of 80%, 90% or 95%；Preferably, The aminoacid sequence that described Exendin-4 analog comprises is His1-Xaa2-Glu-Gly-Thr5-Phe-Thr-Ser-Asp- Xaa10-Ser-Xaa12-Xaa13-Xaa14-Glu15-Glu-Glu-Ala-Xaa19-Xaa20-Xaa21-Phe-Ile-Xaa24-Trp25- Leu-Xaa27-Xaa28-Gly-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39；
Wherein:
Xaa2Selected from Gly, Thr, Ala, Ser, Leu, Ile or Lys；
Xaa10Selected from Leu, Ala, Ser, Leu, Ile, Glu or Lys；
Xaa12Selected from Lys, Leu, Thr, Ser, Leu, Ile or Cys；
Xaa13Selected from Gln, Thr, Ala, Val, Leu, Ile or Lys；
Xaa14Selected from Met, Tyr, Thr, Ala, Ser, Ile or Lys；
Xaa19Selected from Val, Cys, Ala, Ser, Leu, Ile or Lys；
Xaa20Selected from Arg, Thr, Tyr, Ser, Leu, Ile or Lys；
Xaa21Selected from Leu, Thr, Ala, Asp, Glu, His or Lys；
Xaa24Selected from Glu, Leu, Thr, Ala, Ser, Lys or Ile；
Xaa27Selected from Lys, Ala, Ser, Leu, Thr, Ile or Lys；
Xaa28Selected from Asp, Thr, Ala, Ser, Leu, Ile or Lys；
Xaa30Selected from Gly, Thr, Ala, Ser, Leu, Ile or Arg；
Xaa31Selected from Pro, Val, Ser, Ala, Leu, Ile or Lys；
Xaa32Selected from Ser, Thr, Glu, Ser, Asp, Lys or Ile；
Xaa33Selected from Thr, Ser, Ala, Met, Leu, Ile or Lys；
Xaa34Selected from Gly, Thr, Met, Ser, Ile, Leu or Lys；
Xaa35Selected from Ala, Thr, Ala, Glu, Leu, Ile or Phe；
Xaa36Selected from Pro, Ala, Thr, Ser, Leu, Ile or Cys；
Xaa37Selected from Pro, Thr, Ser, Ala, His, Lys or Ile；
Xaa38Selected from Pro, Thr, Val, Ser, Leu, Lys or Ile；
Xaa39Selected from Ser, Tyr, Ala, Leu, Ser, Ile or Lys,
Preferably, the aminoacid sequence of described Exendin-4 analog is His1-Gly-Glu-Gly-Thr5-Phe-Thr-Ser- Asp-Leu10-Ser-Lys-Gln-Met-Glu15-Glu-Glu-Ala-Val-Arg20-Leu-Phe-Ile-Glu-Trp25-Leu- Lys-Asn-Gly-Gly30；The aminoacid sequence of the most described Exendin-4 analog is His1-Gly-Glu-Gly- Thr5-Phe-Thr-Ser-Asp-Leu10-Ser-Lys-Gln-Met-Glu15-Glu-Glu-Ala-Val-Arg20-Leu-Phe- Ile-Glu-Trp25-Leu-Lys-Asn-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-Pro-Pro-Ser39- Lys40-Lys-Lys-Lys-Lys-Lys45。
5. fusion protein as claimed in claim 1, it is characterised in that described flexible peptide linker contains 2 or more is selected from The aminoacid of G, S, A and T, it is preferable that the general structure of described flexible peptide linker aminoacid composition is (GS)a(GGS)b(GGGS)c (GGGGS)d, wherein a, b, c and d are greater than or equal to the integer of 0, and a+b+c+d >=1,
It is highly preferred that the aminoacid of described flexible peptide linker is selected from following sequence:
(i)GGGGS；
(ii)GSGGGSGGGGSGGGGS；
(iii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(iv)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(v)GGGSGGGSGGGSGGGSGGGS；
(vi)GGSGGSGGSGGS。
6. fusion protein as claimed in claim 1, it is characterised in that the carboxyl of described human chorion gonadotrophic hormone beta subunit Terminal peptide rigid element comprises SEQ ID NO:1 or the sequence of its truncate, and the sequence of wherein said truncate comprises at least 2 glycosyls Change site,
Preferably, the carboxyl terminal peptide rigid element of described human chorion gonadotrophic hormone beta subunit comprises following aminoacid sequence:
(i)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)SSSSKAPPPS；
(iv)SRLPGPSDTPILPQ；
(v)SSSSKAPPPSLPSPSR。
7. fusion protein as claimed in claim 1, it is characterised in that the carboxyl of described human chorion gonadotrophic hormone beta subunit Terminal peptide rigid element at least has 70%, 80%, 90% with the CTP aminoacid sequence in fusion protein described in claim 6 Or the homogeneity of 95%.
8. fusion protein as claimed in claim 1, it is characterised in that described fusion protein comprises 1,2,3,4 or 5 human chorionics The carboxyl terminal peptide rigid element of film gonadotrophin beta subunit.
9. fusion protein as claimed in claim 1, it is characterised in that described human normal immunoglobulin's Fc fragment is to have reduction The mutant that ADCC effect and/or CDC effect and/or the binding affinity with FcRn receptor strengthen；Preferably, described Fc fragment Selected from human IgG variant；
It is highly preferred that described human IgG Fc variant is selected from:
I () contains the human IgG1 hinge region of Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region；
(ii) the human IgG2 hinge region of Pro331Ser sudden change, CH2 and CH3 region are contained；
(iii) the human IgG2 hinge region of Thr250Gln and Met428Leu sudden change, CH2 and CH3 region are contained；
(iv) the human IgG2 hinge region of Pro331Ser, Thr250Gln and Met428Leu sudden change, CH2 and CH3 region are contained；
V () contains human IgG 4 hinge region of Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region.
10. want the fusion protein as described in 1 such as right, it is characterised in that the aminoacid sequence of described fusion protein such as SEQ ID Shown in NO:8 or 10.
11. coding DNA of fusion protein, the most described DNA sequence such as SEQ ID as according to any one of claim 1-10 Shown in NO:7 or 9.
12. 1 kinds of carriers, it is characterised in that comprise DNA as claimed in claim 11.
13. 1 kinds of host cells, it is characterised in that comprise carrier as claimed in claim 12, or transfected claim Carrier described in 12.
14. 1 kinds of pharmaceutical compositions, it is characterised in that include pharmaceutically acceptable carrier, excipient or diluent, Yi Jiyou The fusion protein as according to any one of claim 1-10 of effect dosage.
Preparing the method for fusion protein as according to any one of claim 1-10 for 15. 1 kinds, described method includes:
A the DNA sequence of encoding fusion protein described in claim 11 is introduced mammalian cell by ()；
In (b) screening step (a) in its growth medium in every 24 hours periods, express more than 50 μ g/106(million) individual carefully The high yielding cell sarain of born of the same parents；
C cell strain that () incubation step (b) screens, expressed fusion protein；
The fermentation liquid obtained in (d) results step (c), purified fusion protein.
Preferably, the mammalian cell in described step (a) is Chinese hamster ovary celI；More preferably CHO derived cell system DXB-11.
16. fusion protein as according to any one of claim 1-10 are used for treating the II type of non-insulin-dependent in preparation Purposes in diabetes medicament.
17. fusion protein as according to any one of claim 1-10 use in the medicine of preparation treatment or prevention obesity On the way.
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