CN106117370B - Hyperglycosylated Extendin-4, fusion protein of analogue thereof, and preparation method and application of fusion protein - Google Patents

Abstract

The invention discloses hyperglycosylated Extendin-4, fusion protein of analogue thereof, and a preparation method and the application of fusion protein. The fusion protein comprises Extendin-4, the analogue of the Extendin-4, a flexible peptide joint, at least one human chorionic gonadotropin beta carboxyl terminal peptide rigid unit and a human immunoglobulin Fc fragment. The invention also discloses the preparation method and the application of the fusion protein. The fusion protein has optimal biological activity, obviously prolonged circulation half-time, lowered immunogenicity and improved bioavailability. The fusion protein can be used for treating diabetes, obesity and other diseases benefited by lowering fasting plasma glucose, inhibiting stomach and/or bowel movement and inhibiting and/or bowel evacuation or inhibiting 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, Its preparation method is further related to it in treatment diabetes, obesity and by reducing plasma glucose, suppressing stomach and/or bowel movement With suppress stomach and/or intestines emptying or suppress food intake and the application in benefited other diseases.

Background technology

Exendin-4 is isolated from South Africa Monster saliva, and it has 53% with the GLP-1 amino acid sequences of mammal Homology, is effective GLP-1 receptor stimulating agents.The physiologically active of Exendin-4 is similar to GLP-1, but Exendin-4-NH2 End second instead of the Ala in GLP-1 by Gly, it is had certain resistant function to DPP-IV enzymes, be difficult by DPP-IV Degraded, the half-life period circulated in vivo is more long, can reach 60-90 minutes.Additionally ,-COOH the ends of Exendin-4 are by 9 ammonia The special Trp-cage structures that base acid (PSSGAPPPS) is formed, make it be significantly higher than GLP- with the binding affinity of GLP-1 acceptors 1 (Neidigh JW etc., Biochemistry, 2001,40:13188-13200), its BA is about GLP-1's 1000 times.In addition, Exendin-4 is also different from the metabolic pathway of GLP-1, Exendin-4 is mainly by renal metabolism, and GLP-1 is metabolized by peripheral tissues and kidney, and peripheral tissues' 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 amino acid, and molecular weight is small, individually recombinantly expresses Difficulty is very big, and expression quantity is low.Thus, be applied at present clinic Exendin-4 be synthetic (Exenatide, Chinese name：Exenatide).Clinical effectiveness shows that mean half-life of the Exenatide in human body only has 2.4 hours, needs daily Injection twice, greatly pain and inconvenience is brought to the life of patient.Further, since there was only 53% with the homology of people GLP-1, So that patient produces the antibody ratio to increase.In sum, the Exendin-4 deficiencies such as have half-life short, immunogenicity strong, but together When its special molecular structure assign its natural GLP-1 or other advantages to be reached of GLP-1 analogs again, such as and GLP- 1 acceptor combine affinity it is strong, bioactivity is high, clinical medicine dose is extremely low, Stability Analysis of Structures be difficult by DPP-IV enzymolysis and The low advantage of metabolic rate, makes it have bigger clinical value compared with other GLP-1 analogs.

In order to extend the Half-life in vivo of Exendin-4, Exendin-4 or other GLP-1 analogs are melted with Fc fragments The technical scheme of conjunction studies have reported that, such as Li Lai companies exploitation the weekly hypodermic length merged with human IgG 4Fc Effect GLP-1 analogs --- Du Lalu peptides (Dulaglutide) are listed in the U.S., and its average organism long half time is small up to 90 When (China Patent No.：CN1802386B).In addition, disclosing a kind of Exendin-4 and its class in Chinese patent CN101891823 Like the fusion protein that thing is formed by connecting by specific connection peptide with natural human IgG2Fc fragments.To being applied to the treatment of people and Speech, when GLP-1/Fc fusion proteins are incorporated into receptor in target cell, the Fc regions of fusion protein must will not mediate ill effect Subfunction and crack or remove these cells.Therefore, fusion part Fc regions must be non-cracking performance, that is, combining Fc γ Rs In terms of C1q and trigger effect subfunction, Fc must be inactive or low activity.However, institute is public in patent CN101891823 The CDCC and complement activation effect that the natural Fc fragments of the Exendin-4 fusion proteins opened are mediated may be to bodies Cause certain injury.

The carboxy terminal peptide (hereinafter referred to as CTP) of human chorionic gonadotrophin (hCG) β chains is also with some eggs of extension The effect of white matter Half-life in vivo, thus fusion protein disclosed in some patent documents include extension half-life period part can select Select using immunoglobulin Fc, CTP or other can extend the fusion part of half-life period.In addition, CTP can also be used as joint Different subunits in two reactive proteins of connection or for connecting protein.For example, Chinese patent CN103539860A, In fusion protein disclosed in CN103539861A, CN103539868A and CN103539869A, CTP as joint, positioned at promoting ovum Between the beta subunits and alpha subunits of bubble hormone；In fusion protein disclosed in patent WO2005058953A2, CTP is used as connecing Head, beta subunits and alpha subunits for connecting glycoprotein hormones.

The present inventor is surprisingly merged by long-term research in Exendin-4 and the like C-terminals CTP peptide fragments and Fc fragments, both can bring synergy, be used to resist the scavenging action of kidney, so as to extend albumen exist Internal half-life period.Especially, the inventors discovered that, the CTP in Fc N-terminals also have metastable three-dimensional conformation so that Promote Exendin-4 and the like and Fc sections of independent folding to form more preferably three-dimensional conformation, show CTP as joint peptide A part (and not all) is in action.The fusion protein of Exendin-4 of the invention and the like is compared with Du Lalu peptides not only With longer in vivo functionality half-life period and BA is higher, and more make us unexpected, it is in animal body Bioavilability it is higher.In addition, in the preferred embodiment of the present inventor, the IgG Fc parts of fusion protein are from human IgG2 It is not IgG4, so as to have smaller ill effect subfunction relative to Du Lalu peptides, and with longer body-internal-circulation half Decline the phase.

The content of the invention

The present invention is intended to provide a kind of fusion protein of high-glycosylation Exendin-4 and the like, its preparation method and Using to solve the defects such as Exendin-4 half-life shorts, immunogenicity be strong.

One aspect of the present invention, there is provided a kind of high-glycosylation Exendin-4 and the like fusion proteins (hereinafter referred to as melt Hop protein), the fusion protein connects containing Exendin-4 or its analog (being expressed as Ex4), flexible peptide successively from N-terminal to C-terminal The carboxy terminal peptide rigid element of head (being expressed as L) and at least one human chorion gonadotrophic hormone beta subunit is (hereinafter referred to as (CTP) n, n are 1,2,3,4, or 5) chimeric human immunoglobulin(HIg) Fc fragments (being expressed as Fc), wherein (CTP) n can be embedded in Fc N-terminal or C-terminal；Thus, the fusion protein can be expressed as Ex4-L- (CTP) n-Fc or Ex4-L-Fc- (CTP) n.

Wherein, the primary structure of the 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, are represented by Ex (1- 39)。

Wherein, the Exendin-4 analogs are homologous with the amino acid sequence at least 70% of natural Exendin-4；It is more excellent Selection of land, the amino acid sequence of the Exendin-4 analogs is homologous with natural Exendin-4 at least 80%；It is highly preferred that described The amino acid sequence of Exendin-4 analogs is homologous with natural Exendin-4 at least 90%.Most preferably, the Exendin-4 The amino acid sequence of analog is homologous with natural Exendin-4 at least 95%.Different, the Exendin-4 according to method of modifying Analog is divided into saltant type, truncated-type or extended pattern.

In some embodiments of the present invention, the Exendin-4 analogs are the non-conservative amino of saltant type, i.e. at least one Acid is substituted, and it includes 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, the non-conservative site Xaa of at least one is substituted：

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.

As in the preferred embodiments of the present invention, the Exendin-4 analogs contain 1 substitution of 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 invention, the Exendin-4 analogs are truncated-type.9 amino acid of C-terminal Residue be not Exendin-4 combined with acceptor and its bioactivity necessary to, Exendin-4 analogs of the present invention can be with Missing C-terminal 31-39 amino acids, are represented by Ex (1-30).

For another example, in other embodiments of the invention, the Exendin-4 analogs are extended pattern, i.e., increase ammonia in C-terminal Base acid.In one embodiment of the present invention, connection LysLysLysLysLysLys (is represented by Ex (1- on the 39th Ser 45))；Present invention discover that the carboxyl terminal in Exendin-4 increases this 6 amino acid of KKKKKK, its resistance DPP-IV enzyme hydrolysis The ability enhancing of effect, i.e. stability enhancing.

Wherein, the preferred non-immunogenic of the flexible peptide linker, and produced between Exendin-4 and Fc enough Distance, minimizes steric effect each other.It is preferred that using containing 2 or more the flexibilities of Amino acid profile Peptide linker, and selected from following several amino acid：Gly (G), Ser (S), Ala (A) and Thr (T).

Preferably, the flexible peptide linker includes G and S residues.The length for connecting peptide is weighed very much to the activity of fusion protein Will.For the purpose of the present invention, it is preferable that the general structure of the flexible peptide linker amino acid composition is (GS) a (GGS) b (GGGS) c (GGGGS) d, wherein a, b, c and d are greater than or equal to 0 integer, and a+b+c+d >=1.

In some embodiments of the present invention, the 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, the CTP rigid elements are selected from by human chorion gonadotrophic hormone beta subunit carboxyl terminal the 113rd to 145 Total length or the sequence of truncation that amino acid is constituted, specifically, the CTP rigid elements include SEQ ID NO：1 or its truncation Sequence.

Preferably, the CTP rigid elements include at least 2 glycosylation sites；For example, a preferred embodiment of the present invention In, the CTP rigid elements include 2 glycosylation sites, and exemplarily, the CTP rigid elements include SEQ ID NO：1N 10 amino acid at end, i.e. SSSS*KAPPPS*；Or the CTP rigid elements include SEQ ID NO：14 amino at 1C ends Acid, i.e. S*RLPGPS*DTPILPQ；And for example, in another embodiment, the CTP rigid elements include 3 glycosylation sites, example Property, the CTP rigid elements include SEQ ID NO：16 amino acid at 1N ends, i.e. SSSS*KAPPPS*LPSPS*R；Again Such as, in more another embodiment, the CTP rigid elements include 4 glycosylation sites, and exemplarily, the CTP rigid elements contain 28th, 29,30,31,32 or 33 amino acid and start from human chorion gonadotrophic hormone beta subunit the 113rd, 114,115,116, 117 or 118, terminate at the 145th.Specifically, the CTP rigid elements include SEQ ID NO：28 amino at 1N ends Acid, i.e. SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ.Herein, * represents glycosylation site.Every kind of possibility all generations Table standalone embodiment of the invention.

In further embodiments, the CTP rigid elements that the present invention is provided are same with natural CTP amino acid sequences at least 70% Source；In further embodiments, the CTP rigid elements that the present invention is provided are homologous with natural CTP amino acid sequences at least 80%； In other embodiments, the CTP rigid elements that the present invention is provided are homologous with natural CTP amino acid sequences at least 90%；Another In a little embodiments, the CTP rigid elements that the present invention is provided are homologous with natural CTP amino acid sequences at least 95%.

Exemplarily, CTP rigid elements of the present invention preferably include following sequence：

(i)CTP1：SSSSKAPPPSLPSPSRLPGPSDTPILPQ；

(ii)CTP2：PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；

(iii)CTP3：SSSSKAPPPS；

(iv)CTP4：SRLPGPSDTPILPQ；

(v)CTP5：SSSSKAPPPSLPSPSR.

In some embodiments of the invention, the fusion protein includes 1 above-mentioned CTP rigid element.

Fusion protein of the present invention can also include the above-mentioned CTP rigid elements of more than 1, it is preferable that comprising 2,3,4 or 5 above-mentioned CTP rigid elements.As in one embodiment of the invention, the fusion protein includes 3 CTP3 rigid elements： SSSSKAPPPSSSSSKAPPPSSSSSKAPPPS (CTP3-CTP3-CTP3, or it is expressed as (CTP3) 3)；It is such as of the invention another In embodiment, the fusion protein includes 2 CTP5 rigid elements：SSSSKAPPPSLPSPSRSSSSKAPPPSLPSPSR (CTP5-CTP5, or it is expressed as (CTP5) 2).

Wherein, Fc fragments preferably are selected from the Fc fragments of human immunoglobulin(HIg) IgG, IgM, IgA and its variant；More preferably from people The Fc fragments of IgG1, IgG2, IgG3 or IgG4 and its variant；Wherein, the human IgG Fc variants (being expressed as vFc) include and are located at It is at least one amino acid modified in wild type human IgG Fc, and Fc variants are without cracking performance, and show minimum Fc- mediations Adverse side effect (ADCC and CDC effects) and/or the binding affinity enhancing with FcRn acceptors.

Further, human IgG Fc variants may be selected from the following group：

(i)vFcγ1：Containing Leu234Val, Leu235Ala and Pro331Ser mutation human IgG1's hinge region, CH2 and CH3 regions (such as SEQ ID NO：Amino acid sequence shown in 2)；

(ii)vFcγ2-1：Human IgG2's hinge region, CH2 and CH3 regions (such as SEQ ID containing Pro331Ser mutation NO：Amino acid sequence shown in 3)；

(iii)vFcγ2-2：Containing human IgG2's hinge region, CH2 and CH3 regions that Thr250Gln and Met428Leu is mutated (such as SEQ ID NO：Amino acid sequence shown in 4)；

(iv)vFcγ2-3：Human IgG2's hinge region, CH2 containing Pro331Ser, Thr250Gln and Met428Leu mutation With CH3 regions (such as SEQ ID NO：Amino acid sequence shown in 5).

(v)vFcγ4：The hinge region of human IgG 4, CH2 and CH3 regions containing Ser228Pro and Leu235Ala mutation are (such as SEQ ID NO：Amino acid sequence shown in 6).

IgG Fc variants provided by the present invention can also be IgG including but not limited to 5 kinds of variants described in (i)~(v) The combination or superposition in two class functional variety mutational sites between isotype subclass, as described above variant described in (iv) be by (ii) and (iii) combinatory variants of superimposed the obtained new IgG2Fc in mutational site in.

Fc variants (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 regions.Contain amino acid at 228,234,235 and 331 (being determined by EU number systems) in this CH2 regions Mutation.It is believed that these amino acid mutations can reduce the effector function of Fc.Human IgG2 does not combine Fc γ R, but shows extremely weak Complement activity.The variants of Fc γ 2 with Pro331Ser mutation should be lower than the complement activity of natural Fc γ 2, and remains Fc γ R azygosperms.IgG4Fc is defective in activating complement cascade, and it is lower than IgG1 by about one with the binding affinity of Fc γ R The individual order of magnitude.Compared with natural Fc γ 4, the variants of Fc γ 4 being mutated with Ser228Pro and Leu235Ala should show minimum Effector function.Fc γ 1 with Leu234Val, Leu235Ala and Pro331Ser mutation are also shown than natural Fc γ 1 The effector function of reduction.These Fc variants are all more suitable for preparing Exendin-4 and the like fusions than natural human IgG Fc Albumen.And 250 and 428 (by EU number systems defined locations) contain amino acid mutation so that Fc areas and neonatal receptor The binding affinity of FcRn increases, so that further extension half-life period (Paul R etc., J Biol Chem, 2004,279:6213– 6216)；The variant of above-mentioned two classes function is mutually combined or is superimposed, and obtains new combinatory variants, makes the reduction of its effector function Simultaneously and extend its half-life period.Fc variants of the present invention include the mutation for being but not limited to above-mentioned several sites, can also draw The replacement for entering other sites causes that Fc has the effector function and/or the adhesion enhancing with FcRn acceptors for reducing, while also Fc will not be caused to become body function/activity reduction or cause bad conformation change, common mutational site may refer to Shields RL etc., J Biol Chem, 2001,276 (9):6591-604.

In a preferred embodiment of the present invention, CTP rigid elements are located at the N-terminal of vFc, constitute the amino acid of fusion protein Sequence such as SEQ ID NO：Shown in 8；In another preferred embodiment of the invention, CTP rigid elements are located at the C-terminal of vFc, are constituted The amino acid sequence of fusion protein such as SEQ ID NO：Shown in 10.

According to another aspect of the present invention, there is provided a kind of DNA for encoding above-mentioned fusion protein.Of the invention one is preferred real In applying example, the DNA sequence dna such as SEQ ID NO of the fusion protein：Shown in 7.It is described to melt in another preferred embodiment of the invention The DNA sequence dna of hop protein such as SEQ ID NO：Shown in 9.

According to a further aspect of the invention, there is provided a kind of carrier.The carrier includes above-mentioned DNA.

According to a further aspect of the invention, there is provided a kind of host cell.The host cell includes above-mentioned carrier, Huo Zhezhuan Above-mentioned carrier is contaminated.

In specific embodiment of the invention, host cell is the derived cell strain DXB-11 of CHO.

According to a further aspect of the invention, there is provided a kind of pharmaceutical composition.The pharmaceutical composition is included and can pharmaceutically connect Carrier, excipient or the diluent received, and effective dose above-mentioned fusion protein.

According to a further aspect of the invention, there is provided the fusion protein is preparing the II types for the treatment of non-insulin-dependent Purposes in the medicine of diabetes and the other diseases benefited by reducing plasma glucose.

According to a further aspect of the invention, there is provided the fusion protein is preparing treatment or prevention obesity and passing through Suppress stomach and/or bowel movement and suppress stomach and/or intestines emptying or suppress food intake and in the medicine of benefited other diseases Using.

Provide according to another aspect of the present invention a kind of from mammal cell line (cell line as derived from CHO) preparation Or the method for producing the fusion protein, comprise the steps of：

A DNA that () will encode above-mentioned fusion protein introduces mammalian cell；

It is interior during every 24 hours in its growth medium in (b) screening step (a), express more than 50 μ g/106 cells High yield cell line；

C cell line that () incubation step (b) screening is obtained；

D () harvests the zymotic fluid that step (c) is obtained, purified fusion protein；

Preferably, the mammalian cell in the step (a) is Chinese hamster ovary celI；More preferably CHO derived cells system DXB- 11。

Compared with existing product, the inventors discovered that, the Exendin-4 of the invention or its analog fusion have There are following outstanding advantages：

1st, there is longer circulating half-life in vivo relative to the Exendin-4Fc fusion proteins without CTP rigid elements, 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, so as to improve the quality of life of patient；On the other hand reduce because of the wind of the potential antibody tormation that frequent drug administration by injection triggers The reduction of danger, i.e. immunogenicity.

2nd, with the in vivo functionality half-life period of extension, damaged to db/db spontaneous types diabetic mice and STZ inducing pancreatics In the evaluating drug effect experiment of random blood sugar value observation after diabetic mice single-dose, respectively to give FP-A the 168th small When and after 144 hours still have significant hypoglycemic effect；And FP-B has longer body in two kinds of diabetes animal models Interior drug effect, its blood glucose value of 240h and 216h still has significant difference compared with model group after administration.The relative drawings of shutting out of FP-A and FP-B Shandong peptide more can for a long time, effectively control mouse blood sugar level.

3rd, bioavilability higher, single-dose pharmacokinetic data display, in dosage identical feelings Under condition, it is exhausted in rat body that FP-A and FP-B drug exposures (AUC0~∞) are above Du Lalu peptides, i.e. FP-A and FP-B It is higher to bioavilability, it is contemplated that its clinical medicine dose will also decrease.

4th, the content of the HbA1c of db/db diabetic mices can be effectively reduced, but FP-A groups do not occur after long term administration HbA1c significantly reduced situations compared with normal group, point out FP-A not increase the risk that hypoglycemia occurs.

Brief description of the drawings

Fig. 1, show the FP-A of SpeI/EcoRI fragments in PCDNA3.1 expression vectors according to embodiments of the present invention Nucleotide sequence and derivation amino acid sequence, by the microglobulin leader peptides of α 1 (1-19, with _ _ mark), Ex (1-39) (20- 58), flexible peptide linker (59-85, withMark), CTP rigid elements (86-113, withMark) and vFc (114-336) structure Into.

Fig. 2, show the FP-B of SpeI/EcoRI fragments in PCDNA3.1 expression vectors according to embodiments of the present invention Nucleotide sequence and derivation amino acid sequence, by the microglobulin leader peptides of α 1 (1-19, with _ _ mark), Ex (1-39) (20- 58), flexible peptide linker (59-74, withMark), vFc (75-297) and CTP rigid elements (298-330, withMark) structure Into.

RBG value changes curves between Fig. 3-1,0~6h of db/db diabetic mice single injection FP-A；Significant difference is marked Annotation：FP-A groups compared with model group,^*P ＜ 0.05,^**P ＜ 0.01；Du Lalu peptide groups compared with model group,^#P ＜ 0.05,^##P ＜ 0.01.

RBG value changes curves between Fig. 3-2,0~216h of db/db diabetic mice single injection FP-A；Significant difference mark Note annotation：FP-A groups compared with model group,^*P ＜ 0.05,^**P ＜ 0.01；Du Lalu peptide groups compared with model group,^#P ＜ 0.05,^## P ＜ 0.01.

Fig. 4, STZ induced diabetes mouse single injection FP-A and FP-B RBG value changes curve between 0~240h；Statistics Learn difference marker annotations：FP-A groups compared with model group,^*P ＜ 0.05,^**P ＜ 0.01；FP-B groups compared with model group,^#P ＜ 0.05,^##P ＜ 0.01；Du Lalu peptide groups compared with model group,^ΔP ＜ 0.05,^ΔΔP ＜ 0.01.

The influence of Fig. 5, various dose group FP-A to db/db diabetic mices HbA1c (%)；Significant difference mark note Release：FP-A groups compared with model group,^*P ＜ 0.05,^**P ＜ 0.01.

Fig. 6, FP-A feed the influence that Mouse Weight increases to high lipid food.

Fig. 7, FP-A feed the influence (means ± SD, n=8) of glucose tolerance in mice to high lipid food.Note：With normal group phase Than,^#P ＜ 0.05,^##P ＜ 0.01；Compared with fat group high, * P ＜ 0.05, * * P ＜ 0.01.

Fig. 8, FP-A feed the influence (means ± SD, n=8) of mice serum insulin content to high lipid food.Note：With Normal group is compared,^#P ＜ 0.05,^##P ＜ 0.01；Compared with fat group high, * P ＜ 0.05, * * P ＜ 0.01.

Fig. 9, FP-A feed the influence (means ± SD, n=8) of mouse islets element tolerance index to high lipid food.Note：With Normal group is compared,^#P ＜ 0.05,^##P ＜ 0.01；Compared with fat group high, * P ＜ 0.05, * * P ＜ 0.01.

Figure 10, FP-A feed the influence of fat cell cross-sectional area to high lipid food.Note：A：Normal group；B：Fat group high；C： FP-A groups.

Figure 11-1a, C57BL/6J mouse give carbohydrate tolerance test curve after FP-B 1d.

Influence (means ± SD, n=s of the 1d FP-B to 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 mouse give carbohydrate tolerance test curve after FP-B 4d.

Influence (means ± SD, n=s of the 4d FP-B to 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 mouse give carbohydrate tolerance test curve after FP-B 7d.

Influence (means ± SD, n=s of the 7d FP-B to 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 mouse give carbohydrate tolerance test curve after FP-B 10d.

Influence (means ± SD, n=s of the 10d FP-B to C57BL/6J glucose tolerance in mice (iAUC) after Figure 11-4b, administration 8).Note：Compared with normal group, * * P ＜ 0.01, * P ＜ 0.05.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation 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) described in condition, or according to the condition proposed by manufacturer.

Fusion protein of the present invention is generally prepared by the method for biosynthesis.According to nucleotide sequence of the present invention, this Technical field personnel easily can be obtained code nucleic acid of the invention with various known methods.These methods are such as, but not limited to： PCR, DNA are artificial synthesized etc., and specific method can be found in J. Pehanorm Brookers,《Molecular Cloning:A Laboratory guide》.As the present invention A kind of implementation method, the method that Overlap extension PCR can be again carried out by subsection synthesis nucleotide sequence is of the invention to build Nucleic acid sequence encoding.

Present invention also offers a kind of expression vector, operate comprising the sequence for encoding fusion protein of the invention and therewith Property connected expression regulation sequence.Described-be operatively connected " or-be operably coupled to " refer to such a situation, i.e. line Some parts of property DNA sequence dna can adjust or control the activity of same linear DNA molecule other parts.If for example, started The transcription of sub- control sequence, then it is exactly to be operably coupled to coded sequence.

Expression vector can be such as, but not limited to using commercially available：It is thin that pcDNA3, pIRES, pDR, 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 conventionally by restricted Enzyme is sheared and spliced, and the coded sequence of fusion protein of the invention is inserted into suitable restriction site, is obtained of the invention heavy Group expression vector.

Present invention also offers the host cell for expressing fusion protein of the present invention, wherein containing fusion protein of the invention Coded sequence.Described host cell is preferably eukaryotic, such as but not limited to CHO, and COS cells, 293 cells, RSF is thin Born of the same parents etc..Used as preferred embodiment of the invention, described cell is Chinese hamster ovary celI, and it can well express fusion protein of the invention, Binding activity can be obtained good, the fusion protein having good stability.

The present invention also provides 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) nucleotide sequence 1) is inserted into suitable expression vector, obtains recombinant expression carrier；

3) recombinant expression carrier 2) is imported into suitable host cell；

4) culture converts host cell under conditions suitable for the expression；

5) supernatant, and purified fusion protein product are collected.

The coded sequence is imported into host cell can use various known technologies of this area, such as but not limited to：Phosphorus Sour calcium precipitate, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkali metal from Sub- method.

Culture and expression about host cell can be found in Olander RM Dev Biol Stand 1996；86：338. Clear liquid can be collected by the way that cell and residue in removal suspension is centrifuged.Can be identified by agarose gel electrophoresis technology.

Can be substantially uniform property by the above-mentioned fusion protein purification for preparing, for example, be on SDS-PAGE electrophoresis Single band.For example, when recombinant protein is secreting, expressing, the albumen, example can be separated using the milipore filter of commercialization Such as Millipore, Pellicon Products, first concentrate expression supernatant.Concentrate can be using the method for gel chromatography Further purified, or using the method purifying of ion-exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or sun from Sub- displacement chromatography.Gel-type vehicle can be the matrix that agarose, glucan, polyamide etc. are usually used in protein purification.Q- or SP- groups It is ideal ion-exchange group.Finally, hydroxylapatite adsorption is also can use to chromatograph, metal chelate chromatography is hydrophobic mutual The method such as effect chromatography and RPLC (RP-HPLC) is to the further polishing purification of above-mentioned purified product.Above-mentioned institute Having purification step can reach finally purity of protein substantially uniform using different combinations.

Fusion using the affinity column of specific antibody, acceptor or part containing the fusion protein to expressing Albumen is purified.According to the characteristic of the affinity column for being used, using conventional method, such as high-salt buffer, change pH Amalgamation polypeptide of the method elution of bound on affinity column.Selectively, the aminoterminal or c-terminus of described fusion protein be also One or more polypeptide fragments can be contained, as protein tag.Any suitable label may be used to the present invention.For example, institute The label stated can be FLAG, HA, HA1, c-Myc, 6-His or 8-His etc..These labels can be used to carrying out fusion protein pure Change.

Embodiment 1, the expression plasmid for building encoding fusion protein

The microglobulin of coding for alpha 1 (microglobulin of α 1) leader peptide and maturation Exendin-4 and the like, flexibility The gene order of peptide linker, CTP rigid elements and human IgG Fc variants is all the Chinese hamster ovary celI preference codon that artificial optimization crosses, Full length sequence is obtained through chemical synthesis process.For the ease of purpose fragment to be inserted the specific site of expression vector, synthesized Fragment 5 ' and 3 ' end respectively have a restriction enzyme site, respectively SpeI and EcoRI.Exendin-4 after checking and its SpeI the and EcoRI digestions of analog fusion gene, are then inserted into through the improved plasmid PXY1A1 phases of PCDNA3.1 Between answering restriction enzyme site, fusion gene expression plasmid has been obtained.Containing cytomegalovirus early promoter, it is mammal to the plasmid Enhancer needed for cell high level expression foreign gene.The plasmid also contains selected marker thing, so as to can be with bacterium With kalamycin resistance, and there can be G418 resistances in mammalian cell.In addition, when host cell is DHFR genes During expression deficiency, PXY1A1 expression vectors contain dihyrofolate reductase (DHFR) gene of mouse, so as to there is ammonia first Can coamplification fusion and DHFR genes during pterin (MTX) (referring to United States Patent (USP) US 4,399,216).

As shown in table 1, the present invention constructs a series of fusion protein of Exendin-4 and the like, and they are not comprising Constituted with the flexible peptide linker (Linker) of length and IgG Fc variant (vFc) element of several different subtypes, and CTP rigidity Cell position and length are also different.In order to verify containing at least one, and different length CTP rigid elements be respectively provided with it is higher BA, 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 elements.The nucleotide sequence of wherein FP-A and FP-B and the amino acid sequence of translation point Not as depicted in figs. 1 and 2.The amino acid composition of fusion protein is shown in the content of the invention or sequence table.

The composition of several fusion proteins that table 1. builds

Expression of the embodiment 2, fusion protein in transfectional cell series

Recombinant expression carrier plasmid transfection is entered into mammalian host cell line, to express Exendin-4 and the like Fusion protein.In order to stablize high-caliber expression, preferred host cell line is DHFR deficient CHO- cells (referring to U.S. State patent US 4,818,679), host cell chooses CHO derived cells strain DXB11 in the present embodiment.A kind of preferred transfection side Method is electroporation, it is also possible to use other methods, including calcium phosphate co-sedimentation, fat transfection and Protoplast fusion.In electroporation, Be set to 300V electric fields and 1050 μ Fd electric capacity Gene Pulser electroporation apparatus (Bio-Rad Laboratories, Hercules, CA), in cuvette 5 × 10⁷The expression plasmid of 50 μ g high-purities is added in individual cell.In transfection two days later, Make culture medium into growth medium containing 0.6mg/mL G418.With the elisa assay method of anti-human igg Fc, screening is to choosing Select the resistant transfectant of medication.Also can use the ELISA of anti-Exendin-4 carries out quantifying for fusion protein expression.Pass through The well culture plate of Method of Limited Dilution 96, subclone produces the hole of high-level fusion protein.

In order to realize the expression of fusion protein higher level, preferably coamplification is carried out with the DHFR genes by MTX Drug inhibitions. In the growth medium containing progressive concentration MTX, the antigen-4 fusion protein gene transfected with DHFR genes coamplification.Method of Limited Dilution The positive subclone of DHFR expression, progressively pressurizes and filters out the transfectant that can be grown in up to 6 μM MTX culture mediums, determines Its secretion rate, filters out the cell line of expression foreign protein high.Secretion rate is exceeded into about 30 (being preferably about 50) μ g/10⁶(i.e. hundred Ten thousand) cell line of individual cell/24 hour carries out adaptability suspension culture using serum free medium, and CMC model is then used again Base purified fusion protein.

The purifying of embodiment 3, fusion protein with it is qualitative

The conditioned medium containing fusion protein is titrated to pH 7~8 with 1N NaOH, then with 0.45 micron of nitric acid Cellulose filter is filtered.Filtrate is loaded onto on the Protein A posts of phosphate buffer saline (PBS) balance.It is to be fused Protein binding discards the component of outflow in after Protein A posts.The post is washed with PBS, the OD values at 280nm are less than 0.01.Then with the fusion protein of the citrate buffer solution elution of bound that 0.1M pH are 3.75.The eluent 1M of 0.4 volume K₂HPO₄Neutralize, merge the component containing purifying protein, and use PBS.Then with 0.22 micron of nitrocellulose filter Filtering, and store at 4 DEG C.Under non-reduced and reducing condition, protein product is identified by SDS-PAGE, is analyzed.Use BSA As standard, by the quantitative fusion protein of BCA protein analyses.

Embodiment 4, In vitro biological activity is determined

The diabetes medicament screening cell model with GLP-1 signal paths as target spot is set up, is swashed for screening GLP-1 acceptors The New-type long-acting GLP-1 analogs of dynamic agent class.According to document (Zlokarnik G etc., Science, 1998,279 (5347):84- 88) methods described is determined.By band someone GLP-1R expression plasmids and the expression plasmid PGL-4.29 of CRE-Luc reporter genes (Luc2P/CRE/Hygro) the cotransfection CHO-K1 cells (purchased from Promega companies), pressurizeing to screen by antibiotic obtains table altogether Up to two kinds of stable cell lines of plasmid.When external activity is analyzed, it is inoculated into 96 hole cells with 16000 cells/well/200 μ l and trains Support in plate, with the DMEM medium cultures 16~24 hours containing 10%FBS, treat cell growth to the area of cover plate bottom more than 90% When, by with DMEM culture medium gradient dilutions fusion protein F P-A, FP-B, FP-C, FP-D, FP-E, FP-F, FP- containing 10%FBS G, FP-H and FP-I, then added by the μ l of every hole 10, concentration gradient is set to 0.010,0.020,0.039,0.078,0.156, 0.313rd, 0.625,1.25 and 2.5nM, while setting Du Lalu peptides positive controls (the Eli Lilly Company of isoconcentration Production, article No.：9301897).In 37 DEG C, 5%CO₂Under the conditions of be incubated 5-6 hours after, suck supernatant, be slowly added to 300 μ l PBS washed cells, then suck PBS, add 40 μ l lysates, shake 15min, and 40 μ l luciferase substrates are then added per hole (luciferase (Luciferase) reporter gene detection kit, article No.：GM-040501B, the full biology Co., Ltd of Ji produces Product), react 2 minutes, with multi-function microplate reader (SpectraMax M5system, Molecular Device companies) in wavelength Fluorescent value is determined under 560nm, and dose-effect curve is drawn 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 peptides₅₀Value be about 0.02987nM, illustrate FP-A, FP-B is suitable with the BA of Du Lalu peptides；The EC of FP-C, FP-D, FP-E, FP-F, FP-G, FP-H and FP-I₅₀Value is referred to Table 2；It can be seen that, each fusion protein has certain activation to GLP-1 acceptors.

The external activity EC of each fusion protein of table 2.₅₀Value compares

The change of blood sugar situation of embodiment 5, db/db diabetic mice single injections FP-A

Female diabetes db/db mouse (being purchased from Shanghai Slac Experimental Animal Co., Ltd.), 8 week old, body weight 42 ± 2g, 3 groups, every group 6 are randomly divided into by body weight.Reagent group presses 3mg/kg dose subcutaneous injection FP-A, positive group injection (Eli Lilly and Company are produced the Du Lalu peptides of 3mg/kg, article No.：9301897), model group injects isometric dosage The PBS of (10mL/kg).Each group animal respectively at (0h) before administration, 1h after administration, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h and 216h carry out tail vein blood, determine random with 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 is represented, using SPSS18.0 statistical softwares analyze data.Normal distribution, multigroup mean difference is using single factor test variance point Analysis, homogeneity of variance is checked using LSD, and heterogeneity of variance uses Dunnett ' T3 to check；Non-normality distribution uses non-parametric test, P<0.05 represents there is notable statistics difference.

Under same dose, FP-A and positive control drug Du Lalu peptides have hypoglycemic effect, such as Fig. 3-1 and Fig. 3-2 institutes Show.Checked through t-test, in 0h~6h, FP-A groups occur showing with the random blood sugar value relative model group of Du Lalu peptide group animals Work property is reduced, 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；In addition from Du Lalu peptide hypoglycemic effects are can be seen that after administration in 9 days in mouse RBG value changes curve map (Fig. 3-2) to be only capable of maintaining extremely 4th day, the 120th hour upon administration, its blood glucose value did not had a significant difference compared with model group, and FP-A can maintain to 168 hours after administration, i.e., the 7th day blood sugar level of mouse compared with model group, still with 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.

The change of blood sugar situation of embodiment 6, db/db diabetic mice single injections FP-B

SPF grades of selection, the male db/db mouse of 7 week old and C57BL/6J male mices (are bought in Shanghai Si Laike experiments Company of Animals Ltd., animal productiong licensing SCXK (Shanghai)：2012-0002).Feeding environment：22-25 DEG C of temperature, it is relatively wet Degree 45-65%, lighting hours 12h/d.After adaptability is raised 1 week, 30 db/db mouse are pressed into random blood sugar value and body weight is random It is divided into 5 groups：Model control group；Du Lalu peptide group (dosages：3mg/kg)；FP-B：Set 0.75,1.5 and 3mg/kg it is low, Middle and high three dosage groups, every group 6；C57BL/6J mouse are used as Normal group (n=6).Each administration group hypodermic injection gives Relative medicine solution, model control group and Normal group hypodermic injection PBS, administered volume are 10ml/kg.Each group Animal respectively at (0h) before administration, 1h after administration, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h and 240h tail vein bloods, determine the random blood sugar value RBG of each group animal, and remember with blood glucose meter (quasi- blood glucose meter is pacified in three promises) Record data.Data are poor with mean ± markForm is represented, using the statistical software analyze datas of SPSS 18.0.Normal distribution, Multigroup mean difference uses one-way analysis of variance, and homogeneity of variance is checked using LSD, and heterogeneity of variance is examined using Dunnet T3 Test；Non-normality distribution uses non-parametric test, P<0.05 represents there is notable statistics difference.

As can be known from Table 3, after single-dose, the middle and high dosage groups of FP-B show more longlasting compared with Du Lalu peptide groups Hypoglycemic effect.144h is compared Du Lalu peptides group upon administration, and there was no significant difference with the RBG values of model group, and this When FP-B middle dose groups RBG values compared with model group, still with significant difference (P<0.05), FP-B high doses group still has Significant difference (P<0.01).On the other hand, the blood sugar reducing function of FP-B is presented dose dependent, and compared with model group, FP-B is high The blood sugar reducing function of dosage group continues to administration 240h (P<0.05)；The blood sugar reducing function of FP-B middle dose groups continues to administration 196h (P<0.05)；And the blood sugar reducing function of FP-B low dose groups only continues the 120h (P to administration<0.01), with Du Lalu The peptide hypoglycemic duration is similar.

Influences (means ± SD, n=7) of the single subcutaneous injection FP-B of table 3. to db/db mouse random blood sugars

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 groups,^ΔP ＜ 0.05,^ΔΔP ＜ 0.01.

Influences (means ± SD, n=7) of the single subcutaneous injection FP-B of continued 3. to db/db mouse random blood sugars

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 groups,^ΔP ＜ 0.05,^ΔΔP ＜ 0.01.

Influences (means ± SD, n=7) of the single subcutaneous injection FP-B of continued 3. to db/db mouse random blood sugars

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 groups,^ΔP ＜ 0.05,^ΔΔP ＜ 0.01.

The pharmacodynamic study of embodiment 7, STZ induced diabetes mouse single injections FP-A and FP-B

Choose SPF grades of male mouse of kunming (being purchased from Shanghai Slac Experimental Animal Co., Ltd.), 25 ± 2g of body weight, by body Diabetes group and normal group are randomly divided into again.Adapt to raise the kunming mice of 1 week, fasting 18h is weighed, and diabetes group is pressed 150mg/kg intraperitoneal injections give 1%STZ solution, and pH=4.4 (is purchased from Sigma companies, article No. S0130)；Normal group mouse (n =8) intraperitoneal injection give isometric citric acid-sodium citrate buffer solution (purchased from Chemical Reagent Co., Ltd., Sinopharm Group). Detection diabetes group mouse random blood sugar value (Random blood glucose, RBG) in 10th day after injection, wherein RBG >= 16.7mmol/L is the successful diabetic mice of modeling.To observe the hypoglycemic of the diabetic mice that test medicine is induced STZ Effect, chooses STZ- induced diabetes mouse 32, is randomly divided into 4 groups, every group 8,3mg/kg is given respectively at hypodermic injection FP-A, 3mg/kg FP-B and 3mg/kg Du Lalu peptides.Model group and normal group give isometric dosage (10ml/ respectively Kg PBS).Respectively at (0h) before administration, 1h after administration, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h and 240h tail vein blood, the random blood sugar of each group animal is determined with blood glucose meter (quasi- blood glucose meter is pacified in three promises) Value RBG, and record data.Data are poor with mean ± markForm is represented, using the statistical software analyze datas of SPSS 18.0. Normal distribution, multigroup mean difference uses one-way analysis of variance, and homogeneity of variance is checked using LSD, and heterogeneity of variance is used Dunnet T3 are checked；Non-normality distribution uses non-parametric test, P<0.05 represents there is notable statistics difference.

As shown in figure 4,0-240 hours random blood sugar value after diabetic mice the single injection FP-A and FP-B of STZ inductions Change curve shows that FP-A and FP-B can be effectively reduced the random blood sugar value of the diabetic mice of STZ inductions.FP-A exists The 24th hours blood glucose level is minimized after administration, thereafter slow rise, in the 96th hour its random blood sugar value and model group phase Than still with significant difference (P ＜ 0.05)；And the hypoglycemic effect of FP-B groups is more preferably, the 24th hours blood glucose level upon administration Also continue to reduce, minimized in 48 hours after administration, upon administration the 216th hour random blood sugar value compared with model group, still With significant difference (P ＜ 0.01).

The continuous random blood sugar for giving FP-A for 10 weeks of embodiment 8, db/db diabetic mices and HbA1c changes of contents

SPF grades of female db/db mouse (being purchased from Shanghai Slac Experimental Animal Co., Ltd.), 8 week old, adaptability raises 1 24 db/db mouse 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), (3mg/kg) dosage high administration.Each administration group hypodermic injection is given The drug solution of corresponding dosage is given, model group hypodermic injection PBS, administered volume is 10ml/kg.Each group animal is weekly It is administered once, successive administration 10 weeks, with blood glucose meter, (the quasi- blood glucose meter of peace, Changsha Sinocare Biosensing Co., Ltd produces respectively Product) detect different blood sampling time point each group mouse RBG values after administration, and record data.Blood sampling time point sets：It is administered for the first time Before (0d), administration after 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d and 70d.70d, each group mouse fasting 14h Posterior orbit takes blood, and (the uncommon Lay perseverance medical electric in Shenzhen has to determine kit (immunoturbidimetry) with glycosylated hemoglobin immediately after Limit Products, equipment registration number：Guangdong food medicine supervises tool (standard) word 2013 the 2400025th) and its specific eggs of necessary instrument H700 Glycosylated hemoglobin (HbA1c) content, knot in white analyzer (Shenzhen Xi Laiheng medical electrics Co., Ltd product) detection whole blood The percentage (%) that fruit accounts for total hemoglobin with HbA1c is represented.

Data are represented in mean ± standard deviation (means ± SD) form, using SPSS18.0 statistical softwares analyze data.Just State is distributed, and multigroup mean difference uses one-way analysis of variance, and homogeneity of variance is checked using Dunnett t-, and heterogeneity of variance is adopted Checked with Dunnett ' T3；Non-normality distribution uses non-parametric test, P<0.05 represents there is notable statistics difference.

From table 4 knowable to 10 weeks variation tendencies of random blood sugar value of each group mouse successive administration, 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 is presented dose dependent.Carry Show that FP-A can effectively, constantly control the blood sugar level of db/db diabetic mices.And, first administration and last dose FP-A Hypoglycemic effect it is similar, illustrate not occur to be reacted due to the quick resistance to the action of a drug of acceptor, trigger the tolerance effect to FP-A.

Glycosylated hemoglobin (HbA1c) is the product that glucose is combined with the hemoglobin of red blood cell in blood, it and blood In the proportional relation of glucose level.Because life-span of the red blood cell in blood circulation is about 120 days, therefore HbAle egg Can reflect in vain and take the 4-12 weeks aggregate level of blood sugar before blood, compensate for the deficiency that fasting blood-glucose only reflects instantaneous blood sugar.Thus, HbA1c is to control the most important evaluation index of blood sugar for a long time, is also the clinical important evidence for deciding whether to change therapeutic scheme. In the present embodiment HbA1c inspection results can stablize, it is reliable reflect take 2~3 months glycemic control situations of mouse before blood. Successive administration after 10 weeks each group mouse HbA1c assays result as shown in figure 5, showing high, medium and low dosage group FP-A saccharification There is conspicuousness reduction (P ＜ 0.01) compared with model group in HbA1c contents, and dose dependent, wherein high dose group HbA1c is presented (%) is reduced most significantly (6.38 ± 1.63), but (normal value refers to model to remain above the HbA1c levels of normal C57BL/6J mouse Enclose：2.5%-3.5%), the generation of long-term hypoglycemic reaction will not also be caused even if illustrating the FP-A of 3mg/kg；Result above is carried Show FP-A can for a long time, effectively, smoothly control mouse blood sugar, and do not increase the risk for causing hypoglycemia, this and blood sugar in table 4 Variation tendency is consistent.

Influences (means ± SD, n=6) of the table 4.FP-A to db/db mouse random blood sugars

Note：Each dosage group is compared with model group^*P ＜ 0.05；^**P ＜ 0.01.

Influences (means ± SD, n=6) of the continued 4.FP-A to db/db mouse random blood sugars

Note：Each dosage group is compared with model group^*P ＜ 0.05；^**P ＜ 0.01.

The obesity mice that embodiment 9, FP-A is induced high lipid food prevents the experimental study of antiobesity action

First, model is set up and is administered with packet

7 week old C57BL/6J male mices 24 (from Shanghai Slac Experimental Animal Co., Ltd., permitted for purchase by animal productiong Can the number of card SCXK (Shanghai)：2012-0002).Feeding environment：22-25 DEG C of temperature, relative humidity 45-65%, lighting hours 12h/d. After C57BL/6J mouse adaptability is raised 1 week, 3 groups are randomly divided into by body weight：Normal group (NFD), fat group high (HFD), FP-A groups (HFD+FP-A 0.3mg/kg).Fat group high, FP-A groups give high lipid food (D12492 high lipid foods, U.S. Research Diets Products) feed, normal group mouse gives standard feed (normal fat diet, NFD) nursing.FP-A groups are pressed The every 6 days hypodermic injection relative medicines solution of 0.3mg/kg dosage once, give by normal group and model group hypodermic injection PBS Medicine body accumulates 10ml/kg.After 96 days, fasting blood sugar is weighed and detected in each group mouse fasting 16 hours, and eye socket takes blood, 400 × g Centrifugation 15min, separates to obtain serum.After taking blood, take off cervical vertebra and put to death mouse, determine mouse nose to anus length (body is long), calculate Lee ' s indexes.Bilateral epididymal peripheral adipose tissue is separated, claims weight in wet base.Take same position epididymal adipose and be stored in 10% good fortune In your Malin's solution, for Pathomorphology detection.

2nd, Indexs measure

2.1st, body weight and obese degree

Every 6 days mouse weights once, draw Mouse Weight growth curve, and calculate Mouse Weight increment.Mouse Weight Body weight during body weight-mice group when increment=mouse last is weighed.With Lee ' s index assessment mouse obese degrees.

2.2nd, fat weight and index

Weighed with assay balance (BSA223S electronic balances, Sai Duolisi scientific instrument (Beijing) Co., Ltd product) small Mouse both sides epididymis peripheral adipose tissue, claims weight in wet base.Calculate epididymal adipose tissues mass fraction (mg/g)：Epididymal adipose tissues mass fraction=attached Testis fat mass (mg)/empty body weight (g).

2.3rd, oral glucose tolerance experiment

After experiment is carried out 84 days, each group mouse fasting 16h (17:00am-9:00pm), with blood glucose meter (the quasi- blood glucose meter of peace, length Husky Sinocare Biosensing Co., Ltd's product) detection each group mouse fasting blood sugar (FBG), weigh.Orally (i.g) gives 2g/kg glucose solutions (the pure DEXTROSE ANHYDROUS of analysis, Chemical Reagent Co., Ltd., Sinopharm Group's product), after detection gavage 30min, 60min, 90min and 120min each group mouse blood sugar value, draw glucose tolerance curve, and trapezoidal method calculates amendment blood glucose curve Lower area value (iAUC).

2.4th, biochemical index

With automatic clinical chemistry analyzer (XL-200 automatic clinical chemistry analyzers are seized by force in Europe, and Products are seized by force in German Europe) and supporting TG (triglycerides detection kit, Meikang Biotech Co., Ltd., Ningbo's product) and TC (total courages in kit detection serum Sterol detection kit, Meikang Biotech Co., Ltd., Ningbo's product) content, concrete operations enter according to instrument specification OK.

2.5th, insulin and insulin resistant index

With ELISA method (mouse islets element ELISA detection kit, U.S. ALPCO Products) detection mice serum pancreas Island cellulose content, and calculate insulin resistant index.

2.6th, adipose tissue pathology detection

The same side epididymal adipose is taken, adipose tissue is observed with hematoxylin eosin staining method (abbreviation HE dyeing) Morphology.

3rd, statistics and analysis

Data are represented in mean ± standard deviation (means ± SD) form, using SPSS18.0 statistical softwares analyze data.Just State is distributed, and multigroup mean difference uses one-way analysis of variance, and homogeneity of variance is checked using LSD, and heterogeneity of variance is used Dunnet T3 are checked；Non-normality distribution uses non-parametric test, P<0.05 represents there is notable statistics difference.

4th, result

4.1st, FP-A feeds the influence of Mouse Weight and obese degree to high lipid food

Compared with normal group, fat group Mouse Weight high, body weight increase amount and Lee ' s indexes significantly raise (P ＜ 0.01). FP-A can significantly reduce the body weight that high lipid food feeds mouse, body weight increase amount and Lee ' s indexes (P ＜ 0.01), the results are shown in Table 5 And Fig. 6.

Table 5.FP-A feeds the influence (means ± SD, n=8) of Mouse Weight and Lee ' s indexes to high lipid food

Note：Compared with normal group,^#P ＜ 0.05,^##P ＜ 0.01；Compared with fat group high, * P ＜ 0.05, * * P ＜ 0.01.

4.2nd, influences of the FP-A to epididymal adipose tissues quality and mass fraction

As shown in table 6, compared with normal group, fat group mouse epididymis fat mass high and mass fraction significantly raise (P ＜ 0.01).Compared with fat group high, FP-A group mouse epididymis fat masses and mass fraction are significantly reduced (P ＜ 0.05).

Influences (means ± SD, n=8) of the table 6.FP-A to epididymal adipose tissues quality and mass fraction

Note：Compared with normal group, #P ＜ 0.05, ##P ＜ 0.01；Compared with fat group high, * P ＜ 0.05, * * P ＜ 0.01.

4.3rd, influences of the FP-A to mice serum TG and TC content

Compared with normal group, fat group mice serum TC and TG content high significantly raises (P ＜ 0.01).With fat group phase high Than FP-A group mice serum TC contents are significantly reduced (P ＜ 0.01), and TG contents are also significantly reduced (P ＜ 0.01).Result is shown in Table 7.

Influences (means ± SD, n=8) of the table 7.FP-A to mice serum TG and TC content

Note：Compared with normal group, #P ＜ 0.05, ##P ＜ 0.01；Compared with fat group high, * P ＜ 0.05, * * P ＜ 0.01.

4.4th, FP-A feeds the influence of glucose tolerance in mice to high lipid food

As shown in fig. 7, fat group iAUC high is significantly higher than normal group (P ＜ 0.05).Compared with fat group high, the iAUC of FP-A groups Significantly reduce (P ＜ 0.05).

4.5th, FP-A feeds the influence of mice serum insulin and insulin resistant index to high lipid food

Compared with normal group, the serum insulin concentration (P ＜ 0.01) and insulin resistant index (P ＜ of fat group mouse high 0.05) conspicuousness is raised, i.e., mouse has occurred obvious Insulin resistance, thus understands the hypersecretion pancreas islet of compensatory Element produces hyperinsulinemia.Compared with fat group high, FP-A can significantly reduce mice serum insulin (INS) content (P ＜ 0.05) mouse islets element tolerance index (HOMA-IR) (P ＜ 0.05), are improved.Result is shown in Fig. 8 and Fig. 9 respectively.

4.6th, Pathomorphology inspection

HE coloration results are shown, compared with normal group, fat group mouse epididymis fat cell cross-sectional area high is dramatically increased.With Fat group high is compared, and FP-A group mouse epididymis fat cell cross-sectional areas are reduced significantly, and as a result see Figure 10.

In summary result of study, it was demonstrated that FP-A can efficiently control the body weight of the obesity mice of high lipid food induction, With antiobesity action.

Influence of the embodiment 10, FP-B to C57BL/6J glucose tolerance in mice

8 week old SPF ranks 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：22-25 DEG C of temperature, relative humidity 45-65%, lighting hours 12h/ d.24 SPF grades of male C 57 BL/6 J mouses, after adaptability is raised, are randomly divided into normal group and FP-B groups, respectively hypodermic injection Give PBS or 3mg/kg FP-B solution.1d, 4d, 7d and 10d carry out sugar tolerance experiment respectively after administration.Specific method It is as follows：Each group mouse fasting 16h (17:30pm-9:30am), each group mouse blood sugar value is detected, is weighed, i.p gives 2g/kg grapes Sugar juice, administered volume 10mL/kg.15min, 30min, 60min, 90min and 120min each group mouse blood sugar after detection injection Value, draws glucose tolerance curve, and trapezoidal method calculates Area under the curve of blood glucose (iAUC) after amendment.

Data are represented in mean ± standard deviation (means ± SD) form, using SPSS18.0 statistical softwares analyze data.Just State is distributed, and multigroup mean comparison in difference uses one-way analysis of variance, homogeneity of variance selection LSD inspections, heterogeneity of variance selection Dunnet T3 are checked；Non-normality distribution uses non-parametric test, P<0.05 represents there is notable statistics difference.

C57BL/6J mouse give 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 groups mouse under the sugar tolerance empirical curve of normal group relative to increasing face Product is significantly reduced, and 10d still has the sugared tolerance effect for significantly increasing after single-dose.As Figure 11-1b, 11-2b, 11-3b and Shown in 11-4b, after C57BL/6J mouse give FP-B 1d, 4d, 7d and 10d, the iAUC values of FP-B groups show compared with normal group Writing reduces (P ＜ 0.01).This experiment confirms that FP-B can promote islet β cell insulin, suppresses exogenous glucose intake Caused temporary blood glucose rise, promotes glucose utilization, and the long-acting hypoglycemic effects of FP-B are explained from mechanism of action.

Embodiment 11, FP-A, FP-B and FP-I is determined in single-dose pharmacokinetics in rat body

Male SPF grades of SD rat (being purchased from Shanghai Bi Kai experimental animals Co., Ltd), single subcutaneous injection after raising a week in advance (sc) FP-A, FP-B and FP-I of 0.5mg/kg, every group 4, respectively at 0h before administration, 2h after administration, 8h, 24h, 32h, 48h, 56h, 72h, 96h, 120h and 144h eye socket take blood, and about 0.3ml or so, is denoted as respectively every time：T₀、T₂、T₈、T₂₄、T₃₂、T₄₈、T₅₆、 T₇₂、T₉₆、T₁₂₀And T₁₄₄.Stood after taking blood, then serum is separated with 5000rpm centrifugations 10min, in merging after -70 DEG C of freezen protectives Inspection.When being determined with double crush syndrome, resisted with self-control or commercially available anti-Exendin-4 or the NH2 terminal monoclonals of GLP-1 Body (such as Santa Cruz companies production, article No. SC-65389) coating, making by oneself or commercially available horseradish peroxidase-labeled Mouse anti-human igg Fc monoclonal antibodies (such as Sino Biological Inc., article No.：10702-MM01E-50) detected. After enter data into analysis software PKSOLVER, draw medicine to be measured T in blood_1/2, C_maxAnd AUC_0~48hIn the medicines such as~∞ generation, is dynamic Mechanics parameter.

As result shows in table 8, the circulating half-life T of the FP-I of 0.5mg/kg in rat body_1/2For 14.1 ± 1.67 small When, and Ts of the FP-A of 0.5mg/kg and FP-B in rat body_1/2Respectively 21.4 ± 2.51 and 22.6 ± 3.6 hours.FP-A With FP-B maximum plasma concentrations C_maxValue is all remarkably higher than FP-I.In addition, measured by different blood sampling time points in comparison sheet 8 AUC_0~t(t=2h, 5h, 8h, 24h, 28h, 32h or 48h) can learn, in the case of dosage identical, FP-A's and FP-B Drug exposure is all relatively free of CTP rigid elements higher than the absolute bioavailability of FP-I, i.e. FP-A and FP-B in rat body FP-I it is higher, it is contemplated that its clinical administration dosage will also decrease.

The pharmacokinetic parameter of FP-A, FP-B and FP-I of the male SD rat single subcutaneous injection 0.5mg/kg of table 8.

The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, those skilled in the art can Made various changes or modifications with to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims (30)

1. The fusion protein of 1.Exendin-4 or its analog, the fusion protein from N-terminal to C-terminal successively comprising Exendin-4 or Its analog, flexible peptide linker, at least one human chorion gonadotrophic hormone beta subunit carboxy terminal peptide rigid element and people are immunized Immunoglobulin Fc fragment；Or, the fusion protein connects comprising Exendin-4 or its analog, flexible peptide successively from N-terminal to C-terminal Head, human immunoglobulin(HIg) Fc fragments and at least one human chorion gonadotrophic hormone beta subunit carboxy terminal peptide rigid element, wherein,

   The amino acid sequence that the Exendin-4 analogs are included is

   His¹-Xaa²-Glu-Gly-Thr⁵-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Xaa¹³-Xaa¹⁴-Glu¹⁵-Glu-G lu-Ala-Xaa¹⁹-Xaa²⁰-Xaa²¹-Phe-Ile-Xaa²⁴-Trp²⁵-Leu-Xaa²⁷-Xaa²⁸-Gly-Xaa³⁰-Xaa³¹-Xa a³²- 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,

   Or, the amino acid sequence of the Exendin-4 analogs 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-Gly³⁰Or 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。
2. 2. fusion protein as claimed in claim 1, it is characterised in that the fusion protein is glycosylated.
3. 3. fusion protein as claimed in claim 2, it is characterised in that the fusion protein is by mammalian cell Expression and it is glycosylated.
4. 4. fusion protein as claimed in claim 3, it is characterised in that the fusion protein is by thin in Chinese hamster ovary In born of the same parents expression and it is glycosylated.
5. 5. fusion protein as claimed in claim 1, it is characterised in that the amino acid sequence of the 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。
6. 6. fusion protein as claimed in claim 1, it is characterised in that the fusion protein is included successively from N-terminal to C-terminal Exendin-4 or its analog, flexible peptide linker, at least one human chorion gonadotrophic hormone beta subunit carboxy terminal peptide rigidity are single Unit and human immunoglobulin(HIg) Fc fragments.
7. 7. fusion protein as claimed in claim 1, it is characterised in that the fusion protein is included successively from N-terminal to C-terminal Exendin-4 or its analog, flexible peptide linker, at least one human chorion gonadotrophic hormone beta subunit carboxy terminal peptide rigidity are single Unit and human immunoglobulin(HIg) Fc fragments.
8. 8. fusion protein as claimed in claim 1, it is characterised in that the amino acid sequence of the Exendin-4 analogs 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。
9. 9. fusion protein as claimed in claim 1, it is characterised in that the amino acid sequence of the Exendin-4 analogs 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。
10. 10. fusion protein as claimed in claim 1, it is characterised in that the flexible peptide linker contains 2 or more and is selected from The amino acid of G, S, A and T.
11. 11. fusion proteins as claimed in claim 10, it is characterised in that the structure of the flexible peptide linker amino acid composition is led to Formula is (GS)_a(GGS)_b(GGGS)_c(GGGGS)_d, wherein a, b, c and d are greater than or equal to 0 integer, and a+b+c+d >=1.
12. 12. fusion proteins as claimed in claim 11, it is characterised in that the amino acid of the flexible peptide linker is selected from following sequence Row：

    (i)GGGGS；

    (ii)GSGGGSGGGGSGGGGS；

    (iii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；

    (iv)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；

    (v)GGGSGGGSGGGSGGGSGGGS；

    (vi)GGSGGSGGSGGS。
13. 13. fusion protein as claimed in claim 1, it is characterised in that the carboxyl of the human chorion gonadotrophic hormone beta subunit Terminal peptide rigid element includes SEQ ID NO：The sequence of 1 or its truncation, wherein the sequence of the truncation includes at least 2 glycosyls Change site,
14. 14. fusion proteins as claimed in claim 13, it is characterised in that the carboxylic of the human chorion gonadotrophic hormone beta subunit Base terminal peptide rigid element includes following amino acid sequence：

    (i)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；

    (ii)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；

    (iii)SSSSKAPPPS；

    (iv)SRLPGPSDTPILPQ；Or

    (v)SSSSKAPPPSLPSPSR。
15. 15. fusion proteins as claimed in claim 14, it is characterised in that the carboxylic of the human chorion gonadotrophic hormone beta subunit The amino acid sequence of base terminal peptide rigid element be SSSSKAPPPSLPSPSRLPGPSDTPILPQ or PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ。
16. 16. fusion proteins as claimed in claim 1, it is characterised in that the fusion protein includes 1,2,3,4 or 5 people's suedes The carboxy terminal peptide rigid element of chorionic gonadotropin beta subunit.
17. 17. fusion proteins as claimed in claim 1, it is characterised in that the human immunoglobulin(HIg) Fc fragments are with reduction ADCC effects and/or CDC effects and/or variant enhanced with the binding affinity of FcRn acceptors.
18. 18. fusion proteins as claimed in claim 17, it is characterised in that the Fc fragments are selected from human IgG Fc variants.
19. 19. fusion proteins as claimed in claim 18, it is characterised in that the human IgG Fc variants are selected from：

    I human IgG1's hinge region, CH2 and CH3 regions that () is mutated containing Leu234Val, Leu235Ala and Pro331Ser；

    (ii) human IgG2's hinge region, CH2 the and CH3 regions of Pro331Ser mutation are contained；

    (iii) human IgG2's hinge region, CH2 the and CH3 regions being mutated containing Thr250Gln and Met428Leu；

    (iv) human IgG2's hinge region, CH2 the and CH3 regions of Pro331Ser, Thr250Gln and Met428Leu mutation are contained；

    The v hinge region of human IgG 4, CH2 and CH3 regions that () is mutated containing Ser228Pro and Leu235Ala.
20. 20. fusion protein as described in right wants 1, it is characterised in that the amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 8 or 10.
21. The DNA molecular of 21. coding fusion proteins as any one of claim 1-20.
22. 22. DNA moleculars as claimed in claim 21, it is characterised in that comprising such as SEQ ID NO：Sequence shown in 7 or 9.
23. 23. a kind of carriers, it is characterised in that comprising the DNA molecular as described in claim 21 or 22.
24. 24. a kind of host cells, it is characterised in that comprising carrier as claimed in claim 23, or transfected claim Carrier described in 23.
25. 25. a kind of pharmaceutical compositions, it is characterised in that including pharmaceutically acceptable carrier, excipient or diluent, Yi Jiyou Imitate the fusion protein as any one of claim 1-20 of dosage.
26. A kind of 26. methods for preparing the fusion protein as any one of claim 1-20, methods described includes：

    A the DNA sequence dna of encoding fusion protein described in claim 21 or 22 is introduced mammalian cell by ()；

    It is interior during every 24 hours in its growth medium in (b) screening step (a), express more than 50 μ g/10⁶(million) it is individual thin The high yielding cell sarain of born of the same parents；

    C cell line that () incubation step (b) is screened, expressed fusion protein；

    D () harvests the zymotic fluid obtained in step (c), purified fusion protein.
27. 27. methods as claimed in claim 26, it is characterised in that the mammalian cell in the step (a) is that CHO is thin Born of the same parents.
28. 28. methods as claimed in claim 27, it is characterised in that the mammalian cell in the step (a) is CHO derivatives Cell line DXB-11.
29. 29. fusion protein as any one of claim 1-20 is preparing the II types for treating non-insulin-dependent Purposes in diabetes medicament.
30. Use of 30. fusion protein as any one of claim 1-20 in the medicine for preparing treatment or prevention obesity On the way.