CN106397607A

CN106397607A - Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases

Info

Publication number: CN106397607A
Application number: CN201610819163.1A
Authority: CN
Inventors: 叶贤龙; 齐剑英; 吴云舟; 朱升龙
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2016-09-13
Filing date: 2016-09-13
Publication date: 2017-02-15

Abstract

The invention discloses recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases. A nucleotide sequence of coding genes of the recombinant human fibroblast growth factor 21 fusion protein and an expression vector are connected to obtain a recombinant expression vector; the recombinant expression vector converts host cells, high-expression positive host cells are screened out, the cells are cultured, the recombinant human fibroblast growth factor 21 fusion protein is induced to be expressed, thalli are collected, breaking, centrifuging, clarifying and purifying are carried out, and the target product recombinant human fibroblast growth factor 21 fusion protein is obtained. The invention further discloses the application of the recombinant human fibroblast growth factor 21 fusion protein in preparation of the medicine for treating metabolic diseases. The recombinant human fibroblast growth factor 21 fusion protein can well control blood sugar fluctuation and remarkably improve the level of glycosylated hemoglobin and triglyceride.

Description

Recombinant human fibroblast growth factor 21 fusion protein and its preparation treatment metabolism Application in disease medicament

Technical field

The present invention relates to recombinant protein field, more particularly, to a kind of recombinant human fibroblast growth factor 21 fusion protein And preparation method thereof, the invention still further relates to this recombinant human fibroblast growth factor 21 fusion protein is in preparation treatment metabolism disease Purposes in medicine, belongs to fibroblast growth factor technical field.

Background technology

Diabetes（Diabetes mellitus, DM）, it is the chronic metabolic class disease by pancreas function pathology for the class, with Hyperglycaemia is principal character, is a kind of life-long disease.Its pathogenesis is insulin resistance or insulin secretion reduces, and leads to machine Caused by body glucose -lipid metabolism disorder.With the aging of world population, diabetes have become a kind of common disease, frequently-occurring disease, have become For after cancer and cardiovascular and cerebrovascular diseases, the third-largest disease threatening human life（Nathan DM, et al. Medical management of hyperglycaemia in type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy. Diabetes Care 2009; 32: 193–203. Culy CR, Jarvis B. Repaglinide: a review of its therapeutic use in type 2 diabetes mellitus [J]. Drugs, 2001, 61(11): 1625）.

With economical growing, diabetic's quantity of China is also in rising trend, and result is estimated according to investigations, existing China about 100,000,000 diabetic.The medicine great majority for the treatment of diabetes are to be played a role with insulin for core at present, such as Insulin type preparation or its sensitizer medicine etc., but the evening to some diabetics particularly type ii diabetes for these medicines Phase, patient outcome was still not ideal enough.Due to there is no the medicine of alternative insulin and more advanced treatment means, although patient Resistant function is produced to insulin, leads to said medicine cannot prove effective, insulin remains the drug of first choice for the treatment of type ii diabetes Thing.With the continuous aggravation of Insulin Resistance, the curative effect of insulin gradually weakens, blood sugar uncontrollable in normal level. Because blood sugar maintains higher level for a long time, thus leading to much serious complication, as blind, renal failure, cardiovascular and cerebrovascular disease With the nervous system disease etc..Therefore insulin can be replaced in the urgent need to one kind always in the clinical treatment of type ii diabetes, solve The newtype drug of insulin resistance.

Fibroblast growth factor（FGF）21 belong to a newcomer in FGF family, and FGF21 gene is mainly in liver Express with fat, FGF21 can promote HepG2 cell and 3T3-L1 adipocyte consumption of glucose in vitro, in animal body Inside there is the reduction function such as blood sugar and triglycerides, and the side effect such as will not produce hypoglycemia and cause tumour to occur （Kharitonenkov A, et al. FGF-21 as a novel metabolic regulator. J Clin Invest 2005; 115:1627–35. Kharitonenkov A, et al. The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology 2007;148: 774–81）.FGF21 safely, effectively and is independent of the feature of the biological blood sugar level of insulin regulation so as to be expected to become The newtype drug for the treatment of type ii diabetes.However, with the biology it has been found that wild type FGF21 is understood in depth to FGF21 Learn function and clinical practice receives internal stability and the restriction of autoantigenic（Huang Z, et al. A better anti-diabetic recombinant human fibroblast growth factor 21 (rhFGF21) modified with polyethylene glycol. PLoS One 2011;6:e20669. Ye X, et al. Enhancement of the pharmacological efficacy of FGF-21 by genetic modification and PEGylation. Curr Pharm Biotechnol 2014;14:1287–98.）, therefore FGF21 is transformed and repaiies Decorations will be increasingly becoming the focus of Recent study.

Human serum albumins（HSA）The single-stranded not glycosyafated globular protein matter being made up of 585 amino acid residues, be Content highest albumen in human serum, molecular weight is 66kDa, and the threonine of the 356th is to there may be O- glycosylation site （He, X.M. and D.C. Carter, Atomic structure and chemistry of human serum albumin. Nature, 1992. 358(6383): p. 209-15.）, it is adjusting colloidal osmotic pressure and is promoting wound to heal The aspect such as close to play an important role, and have that human compatibility is good, molecular weight big, long half time and non-enzymatic activity and immunity The advantages of originality, this makes albumin fusion technology receive much attention in development Long Life Recombinant Protein Drug field（Nel, M.R., Human albumin administration in critically ill patients. Critical analysis of original studies has to take place. BMJ, 1998. 317(7162): p. 882.）.Much have and control The protein for the treatment of functions, such as interferon, human growth hormone (HGH) and IL-2 etc., after the transformation of albumin fusion technology, can It is developed into that there is the recombinant protein medicine that drug effect more preferably reduces with frequency injection.The advantage of albumin fusion technology exists Do not need extra chemical modification, simple production process in it, product is homogeneous, and quality control is relatively easy, extend medicine half The effect of phase of declining may compare chemical modification（As PEG modification and acetylation etc.）More effectively（Muller, N., et al., Superior serum half life of albumin tagged TNF ligands. Biochemical and Biophysical Research Communications, 2010. 396(4): p. 793-799.）.But merge in white egg With degraded and polymerism during the expression of albumen and storage, when the difficulty and the clinical application that increased purifying, immunity occurs The risk of reaction（Yao, X.Q., et al., Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast. J Biotechnol, 2009. 139(2): p. 131-6. Cordes, A.A., et al., Selective domain stabilization as a strategy to reduce fusion protein aggregation. J Pharm Sci, 2012. 101(4): p. 1400-9. Cordes, A.A., J.F. Carpenter, and T.W. Randolph, Selective domain stabilization as a strategy to reduce human serum albumin-human granulocyte colony stimulating factor aggregation rate. J Pharm Sci, 2012. 101(6): p. 2009-2016.）.Recently research finds, HSA 381-585 amino acids residue is constituted The 3rd domain（3DHSA）It is HSA and neonatal Fc receptor（FcRn）In conjunction with key position, receptor-mediated endocytosis protect The degraded of the lower tolerance protein enzyme of shield effect, thus ensure that HSA has the longer half-life（Andersen, J.T., J.D. Qian, and I. Sandlie, The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin. Eur J Immunol, 2006. 36(11): p. 3044-3051.）.Based on the depth to albumin permanent mechanism Enter understanding, Kenanova etc.（Kenanova, V.E., et al., Tuning the serum persistence of human serum albumin domain III:diabody fusion proteins. Protein Eng Des Sel, 2010. 23(10): p. 789-98.）Using 3DHSA as fusion partner, construct a series of anti-carcinoembryonic antigen double antibodies with The fusion protein of 3DHSA is it was demonstrated that 3DHSA extends the feasibility of protein drug half-life as fusion partner.

The height of protein stability directly affects its expression in host cell, in order to improve protein yield and stablize Property, and do not affect proteinogen activated on the basis of, need for FGF21 to carry out suitable transformation.The present invention passes through FGF21 The form connecting into fusion protein with HSA or 3DHSA to improve the stability of FGF21, and the industrialization for later FGF21 provides Important technology helps.

Content of the invention

Present invention solves the technical problem that there is provided a kind of recombinant human fibroblast growth factor 21 fusion protein and Its preparation method, this recombinant human fibroblast growth factor 21 fusion protein can be used in preparation treatment diabetes or obesity Medicine or pharmaceutical composition.

The present invention is to solve above-mentioned technical problem to adopt the following technical scheme that, recombinant human fibroblast growth factor 21 melts Hop protein is it is characterised in that be that human fibroblastic growth factor 21 recombinant protein hFGF21 and HSA or 3DHSA is formed by connecting Recombination fusion protein hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21, wherein restructuring merge egg SEQ ID NO in the amino acid sequence such as sequence table of white hFGF21-HSA:Shown in 5, the ammonia of recombination fusion protein HSA-hFGF21 SEQ ID NO in base acid sequence such as sequence table:Shown in 6, the amino acid sequence such as sequence of recombination fusion protein hFGF21-3DHSA SEQ ID NO in table:Shown in 7, SEQ ID NO in the amino acid sequence such as sequence table of recombination fusion protein 3DHSA-hFGF21:8 Shown.

Recombinant human fibroblast growth factor 21 fusion protein encoding gene of the present invention it is characterised in that：Institute SEQ ID NO in the nucleotide sequence of the recombination fusion protein hFGF21-HSA encoding gene stated such as sequence table:Shown in 1, restructuring SEQ ID NO in the nucleotide sequence of fusion protein HSA-hFGF21 encoding gene such as sequence table:Shown in 2, recombination fusion protein SEQ ID NO in the nucleotide sequence of hFGF21-3DHSA encoding gene such as sequence table:Shown in 3, recombination fusion protein 3DHSA- SEQ ID NO in the nucleotide sequence of hFGF21 encoding gene such as sequence table:Shown in 4.

The expression vector of recombinant human fibroblast growth factor 21 fusion protein encoding gene of the present invention and containing Have the host cell of this expression vector it is characterised in that：Expression vector used is preferably pET30a（+）, host cell is preferred For Rossetta（DE3）.

The preparation method of recombinant human fibroblast growth factor 21 fusion protein of the present invention is it is characterised in that have Body step is：By the nucleotide sequence of recombinant human fibroblast growth factor 21 fusion protein encoding gene and expression vector phase Connect and obtain recombinant expression carrier；Again by this recombinant expression carrier transformed host cell, then screen the positive host of high expression thin Born of the same parents, cultured cells abduction delivering recombinant human fibroblast growth factor 21 fusion protein, collects thalline, broken, centrifugation, clear Clearly, purify, obtain target product recombinant human fibroblast growth factor 21 fusion protein.

Recombinant human fibroblast growth factor 21 fusion protein of the present invention is in preparation treatment metabolic disease medicine In application, wherein metabolic disease includes diabetes, obesity and metabolic syndrome.

Pharmaceutical composition for treating diabetes or obesity of the present invention has it is characterised in that including treating Recombinant human fibroblast growth factor 21 fusion protein of effect dosage and pharmaceutically acceptable carrier or auxiliary material.

The test cell line of the present invention and zoology test result show, recombinant human fibroblast growth factor 21 merges egg Blood glucose fluctuation can preferably be controlled in vain, stably maintain 24 hours blood glucoses to be in normal level.Particularly hFGF21 and 3DHSA is even The recombination fusion protein connecing, its hypoglycemic effect is more preferably.The recombination fusion protein of the present invention can be used as drug treatment of diabetic, obesity The metabolic disease such as disease or metabolic syndrome.

Brief description

Fig. 1 is recombination fusion protein hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 big The SDS-PAGE electrophoretic analysis figure of expression in enterobacteria；

Fig. 2 is recombination fusion protein hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 after purification SDS-PAGE electrophoretic analysis figure；

Fig. 3 is the Half-life in vivo comparison diagram of 5 kinds of albumen；

Fig. 4 is the cell in vitro Activity determination figure of 5 kinds of albumen；

Fig. 5 be STZ induction 5 kinds of albumen of type i diabetes mouse long term injections after blood sugar level weekly change curve；

Fig. 6 is type i diabetes injected in mice difference albumen blood sugar, glycosylated hemoglobin and triglyceride water after 8 weeks of STZ induction Flat change curve；

Fig. 7 is the change curve of blood sugar level weekly after type ii diabetes 5 kinds of albumen of db/db mouse long term injections；

Fig. 8 is type ii diabetes db/db injected in mice difference albumen blood sugar, glycosylated hemoglobin and triglyceride level after 8 weeks Change curve.

Specific embodiment

To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and Apparent.But these embodiments are only exemplary, any restriction is not constituted to the scope of the present invention.People in the art Member should be understood that can be to enter to the details of technical solution of the present invention and form under without departing from the spirit and scope of the present invention Row modification or replacement, but these modifications and replacement each fall within protection scope of the present invention.

Explanation：The design of the gene being related in the present invention, synthesis and clone, the structure of expression vector, nucleic acid extraction, sequencing And identification, and the operating procedure such as the separation of expression product and purifying, can carry out according to techniques known in the art（Referring to CURRENT PROTOCOLS IN MOLECULAR BIOLOGY）.If not specializing, in embodiment, technological means used is Conventional meanses well-known to those skilled in the art.

Embodiment 1

The structure of hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 expression vector

According to e. coli codon Preference, design 4 kinds of genes, its nucleotide sequence is respectively as SEQ ID in sequence table NO:1（hFGF21-HSA）、SEQ ID NO:2（HSA-hFGF21）、SEQ ID NO:3（hFGF21-3DHSA）With SEQ ID NO:4（3DHSA-hFGF21）Shown.This 4 kinds of genes are delivered to the synthesis of Shanghai JaRa biotech firm, sets at each gene two ends simultaneously Meter NdeI and BamHI two restriction enzyme site.

By the carrier containing respective genes of interest fragment of 4 kinds of synthesis and pET30a（+）Use NdeI and Bam HI double respectively Digestion, after digestion finishes, the target fragment that glue reclaim each needs.Using T4 DNA ligase by 4 kinds of purpose fragments respectively with Prokaryotic expression carrier pET30a（+）Connect, coupled reaction system be 10 L, mix, 4 DEG C connect overnight, then each inverting extremely In bacillus coli DH 5 alpha.Picking positive colony, after digestion identification, builds respectively and obtains 4 kinds of recombinant plasmid pET30a- HFGF21-HSA, pET30a-HSA-hFGF21, pET30a-hFGF21-3DHSA and pET30a-3DHSA-hFGF21.

Embodiment 2

The expression of tetra- kinds of fusion proteins of hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 and purifying

（1）Conversion, culture abduction delivering

By 4 kinds of recombinant plasmids pET30a-hFGF21-HSA, pET30a-HSA-hFGF21, pET30a- containing correct sequence HFGF21-3DHSA and pET30a-3DHSA-hFGF21 converts respectively to expression bacterial strain Rosseta（DE3）（The full Shi Jinsheng in Beijing Thing Technology Co., Ltd., catalog number (Cat.No.)：CD801）.Single bacterium colony after conversion is seeded to 20mL respectively and contains Kan（50µg/mL）LB training In foster base, 37 DEG C of culture 8h, with volume ratio for 1:100 are inoculated in another 20mL contains Kan（50µg/mL）LB culture medium in, 37 DEG C culture, work as A₆₀₀When 0.35 about, IPTG to final concentration of 0.25mmol/L is added to be induced, inducing temperature is 30 DEG C, Harvest thalline after 5h, use Binding buffer（20mmol/L Na₃PO₄, pH 7.0）Resuspended thalline, is centrifuged after broken thalline, Supernatant precipitation is taken to carry out 12wt% SDS-PAGE electrophoretic analysis respectively.After result display hFGF21 is connected with HSA or 3DHSA Fusion protein is most of to be expressed with soluble form, as shown in figure 1, A figure swimming lane 1：Protein standard molecular weight Marker；2、3、4： HFGF21-HSA whole bacterial protein, thalline supernatant, bacterial sediment；5、6、7：HSA-hFGF21 whole bacterial protein, thalline supernatant, thalline sink Form sediment；B figure swimming lane 1：Protein standard molecular weight Marker；2：Do not induce thalline；3、4、5：3DHSA-hFGF21 whole bacterial protein, thalline Supernatant, bacterial sediment；6、7、8：HFGF21-3DHSA whole bacterial protein, thalline supernatant, bacterial sediment.

（2）Protein purification

Finite concentration lysozyme is added in thalline（1mg/mL）, place 30min, ultrasonication somatic cells on ice（Work 1s, Interval 1s, 4min/ time, totally 3 circulations）.After crushing thoroughly, 12000rpm, 4 DEG C of centrifugation 15min, collect supernatant.Supernatant mistake After 0.22 μm of filter membrane clarification, AKTA purifier 100 system is entered by pump, with 2-3 times of column volume Binding buffer The Blue Sepharose 6FF post having balanced（It is loaded on XK16/20 void column, pillar height 10cm, flow velocity 100cm/h）After being completely combined, Rinse foreign protein with the binding buffer of 4-5 times of column volume, when ultraviolet curve reaches stable baseline, then with 2-3 times Column volume Elution buffer（20mmol/L Na₃PO₄, 2 mol/L NaCl, pH 7.0）Wash-out destination protein, exists combination Fusion protein on filler elutes and collects in test tube.

Superdex 75 solvent resistant column（It is loaded in Column XK26/70 void column, column volume 340mL, flow velocity 2mL/ min）It is connected in AKTA purifier 100 system, first replace it with the distilled water of 2 times of column volumes and protect liquid（Volume fraction is 20% ethanol）, then the Desalting buffer with 2 times of column volumes（20mmol/L Na₃PO₄, 150mmol/L NaCl, pH7.0） Then affinity chromatography eluent is passed through Superloop sample introduction by balance pillar.Collect each eluting peak, and carry out 15wt% SDS- PAGE electrophoretic analysis, result show purified after, four kinds of fusion protein purity are more than 95%, as shown in Fig. 2 A figure swimming lane 1： Protein standard molecular weight Marker；2：HFGF21-HSA fusion protein after purification；3：HSA-hFGF21 after purification merges egg In vain；B figure swimming lane 1：Protein standard molecular weight Marker；2：HFGF21-3DHSA fusion protein after purification；3：After purification 3DHSA-hFGF21 fusion protein.

Embodiment 3

The Half-life in vivo detection of 4 kinds of albumen hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21

Choose the rabbit 18 of body weight about 2kg, be randomly divided into 6 groups.Every group of difference hypodermic injection 6 kinds of albumen hFGF21, hFGF21- HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21, dosage 30nmol/kg, 0h upon administration, 1h, 3h, 5h, 7h, 24 h, in ear edge vein exploitating blood 800 μ L about.12000r/m be centrifuged 10min, take supernatant be stored in -20 DEG C standby.

The Half-life in vivo of 6 kinds of albumen of ELISA indirect Determination：With having diluted hFGF21, hFGF21- of variable concentrations HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 albumen（2μg/mL、0.2μg/mL、200ng/mL、20ng/ ML and 2ng/mL）Set up the calibration curve of protein concentration content respectively, by the standard protein after dilution and serum coated elisa plate, The content of target protein in the application each serum of ELISA indirect Determination, statistical analysis simultaneously calculate partly declining in vivo of 6 kinds of albumen Phase.Half-life in vivo t_1/2=0.301*（t₂-t₁）/log（OD₁/OD₂）, wherein OD₁And OD₂Blood is taken out when representing t1 and t2 respectively Average light absorption value on corresponding clearly ELISA Plate.

Result as shown in figure 3, through formula calculate fusion protein hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA, The Half-life in vivo of 3DHSA-hFGF21 and wild-type protein hFGF21 respectively may be about 396min, 402min, 318min, 331min And 36min, after illustrating the fused expression of hFGF21 albumen, Half-life in vivo dramatically increases.

Embodiment 4

The sugar in vitro of 4 kinds of albumen hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 absorbs activity inspection Survey

HepG2 cell culture：HepG2 cell（Basis institute of Chinese Academy of Medical Sciences cell bank）For human hepatoma cell strain, its growth Condition is DMEM in high glucose culture medium, wherein adds 10wt% NBCS NCS（Invitrogen Corporation）, mould Plain 100 μ g/mL, streptomysin 100 μ g/mL, in 37 DEG C, volume fraction 5% CO₂, cultivate under the conditions of saturated humidity.Work as cell growth During high density, should be passed on, after the digestion of 0.25wt% trypsin solution, with volume ratio for 1:3-1:5 ratio is by cell It is inoculated in new culture culture in glassware, the cell in growth period of taking the logarithm is used for testing.

The inoculation of HepG2 cell and process：It is that 0.25% tryptic digestive juice digestion growth conditions are good with mass concentration HepG2 cell, is collected by centrifugation cell, according to 2.5 × 10⁴Density by cell be inoculated in 96 orifice plates continue culture, in every hole Nutrient solution volume is 200 μ L.When cell growth is to uniform monolayers, discards culture supernatants and add fresh serum free medium After continuing culture 12h, you can add testing protein detection activity.

After sample-adding HepG2 cell starvation 12h, use variable concentrations respectively（10nmol/L、100nmol/L、1000nmol/L） 5 kinds of albumen of hFGF21, hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 stimulate cell 24h, Detect the glucose content of residual in culture medium with GOD-POD method.Medium supernatant 2 μ L is taken to be added to 200 μ L glucose inspections Survey in liquid, every hole glucose at least duplicate detection 3 times, after 37 DEG C of reaction 5-10min, survey OD value under 500nm wavelength.Calculate Portugal Grape sugar consumption rate, and use statistical analysis experimental result.

In nutrient solution, the concentration of glucose of residual and the computing formula of grape cell sugar consumption rate are as follows：

Concentration of glucose（mmol/L）=OD_Sample/OD_Standard×5.55mmol/L

Grape cell sugar consumption rate（%）=[(C_{Blank glucose}-C_{Administration glucose}) / C_{Blank glucose}] ×100%

Data results as shown in figure 4, result shows, with respect to hFGF21 albumen, hFGF21-HSA, HSA-hFGF21, Tetra- kinds of fusion proteins of hFGF21-3DHSA and 3DHSA-hFGF21 cell in vitro sugar absorb activity also dramatically increase, and low, Under middle and high Three doses, difference is all extremely notable（^##p<0.01）, illustrate that the external activity of 4 kinds of fusion proteins will be significantly better than HFGF21 albumen.The external activity of wherein 3DHSA-hFGF21 fusion protein is optimal.

Embodiment 6

The internal Composition analyzed of 4 kinds of albumen hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21

First, Activity determination on I patients with type Ⅰ DM animal model for the 4 kinds of fusion proteins of the present invention

Prepared by I patients with type Ⅰ DM animal model：Male C57BL/6 mouse（Purchase public in Shanghai Si Laike animal used as test Limited Liability Department, animal quality quality certification number SCXK（Shanghai）2012-0005）Adaptability feed to body weight reach 35g about when start test.Suitable Answering property is fed 2 weeks, chooses body weight 25-30g mouse 60 and is only randomly divided into 6 Normal group mouse and 52 modeling group mouse, Prepare to start modeling.Before modeling, mouse needs fasting to can't help water 12h, next day, and modeling group presses weight ratio by lumbar injection mode 40mg/kg injects STZ parenteral solution, recovers normal diet, and gives the G/W of 1wt% and drink, and after the 1d of interval, fasting is not again After prohibiting water 12h, weight ratio 30mg/kg is pressed by lumbar injection mode and injects STZ parenteral solution, after the 1d of interval, pass through abdominal cavity the 3rd time Injection system presses weight ratio 20mg/kg injection STZ parenteral solution；Control group only injects citric acid-sodium citrate（pH 4.4）Buffering Liquid.During lumbar injection, note depth and the angle of syringe needle insertion, it is to avoid hurt Viscera in Mice.STZ injects Afterwards, the change of a mouse fasting blood-glucose and body weight is detected every 7d.Fasting plasma glucose concentration after 4 weeks will be injected>16.65mmol/L Mouse be judged to type i diabetes animal model.

Packet, administration and Indexs measure：The blood glucose value being selected to mould is less than the type i diabetes mouse 36 of 25mmol/L, It is randomly divided into model control group, hFGF21 group, hFGF21-HSA group, HSA-hFGF21 group, hFGF21-3DHSA group and 3DHSA- HFGF21 group, every group 6.Give the corresponding tested material of experimental group once about thirty every morning 8, hypodermic injection, dosage 50nmol/kg, model control group injects the physiological saline of same volume, successive administration 8 weeks.Free diet, drink in experimentation Water.

In weekly the one morning 8 points about tail vein bloods measure each experimental mice blood sugar levels, after administration 8 weeks, Each experimental mice is put to death（Eve fasting）, eyeball takes hematometry experiment mice blood sugar（BG）, GH（GHb）With Triglyceride（TG）Level.Obtained experimental data carries out statistical analysis.

As shown in Figure 5,6, Fig. 5 result shows experimental test data, with respect to model control group, 5 kinds of administration group mouse blood Sugar declines significantly after being administered 1 week, and change of blood sugar trend is consistent weekly afterwards, but compared with hFGF21 group, hFGF21-HSA, 4 groups of mouse blood sugar levels of HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 decline rapid.hFGF21-HSA、HSA- Long-acting hypoglycemic effect on I patients with type Ⅰ DM mouse for the 4 kinds of fusion proteins of hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 Fruit is substantially better than hFGF21, and acting duration is long, and the wherein long-term hypoglycemic effect of 3DHSA-hFGF21 group mouse is optimal.

GH（GHb）Test generally can reflect the glycemic control situation in patient nearly 8-12 week, therefore this reality After testing detection administration 8 weeks, each experimental mice blood sugar and glycated hemoglobin level control blood glucose fluctuations comparing 6 kinds of albumen Ability.As shown in fig. 6, this result further illustrates hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA- 4 kinds of fusion proteins of hFGF21 have the regulating and controlling effect of long duration to the blood sugar level of I patients with type Ⅰ DM mouse, and effect shows Write and be better than hFGF21.

After being administered 8 weeks, each experimental mice serum levels of triglyceride level result as shown in fig. 6, with respect to physiological saline group, 5 kinds of albumen can significantly reduce TG content.And compared with hFGF21, hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and Tetra- kinds of albumen of 3DHSA-hFGF21 show more preferably improvement on the blood lipid level of type i diabetes mouse.

2nd, Activity determination on II patients with type Ⅰ DM animal model for the 4 kinds of fusion proteins of the present invention

Packet, administration and Indexs measure：Take SPF level 9-11 week old male db/db mouse（Have purchased from Shanghai Si Laike animal used as test Limit responsible company, animal quality quality certification number SCXK（Shanghai）2012-0005）50, pre- raising was weighed after 1 week, and next day fasting is not Prohibit water 6h, tail vein takes the fasting blood-glucose of hematometry mouse, reject body weight extremely, screening blood glucose value is relatively close to the Cheng Mo of average Mouse 36, is randomly divided into model control group, hFGF21 group, hFGF21-HSA group, HSA-hFGF21 group, hFGF21-3DHSA group With 3DHSA-hFGF21 group, every group 6.Give the corresponding tested material of experimental group once about thirty every morning 8, subcutaneous note Penetrate, dosage 50nmol/kg, model control group injects the physiological saline of same volume, successive administration 8 weeks.In experimentation freely Diet, drinking-water.

As shown in Figure 7,8, Fig. 7 result shows experimental test data, with respect to model control group, 5 kinds of administration group mouse blood Sugar declines significantly after being administered 1 week, and change of blood sugar trend is consistent weekly afterwards, but compared with hFGF21 group, hFGF21- 4 groups of mouse blood sugar levels of HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 decline rapid.hFGF21-HSA、 Long-acting hypoglycemic on type ii diabetes mouse for the 4 kinds of fusion proteins of HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 Effect is substantially better than hFGF21, and acting duration is long, and the wherein long-term hypoglycemic effect of 3DHSA-hFGF21 group mouse is optimal.

GH（GHb）Test generally can reflect the glycemic control situation in patient nearly 8-12 week, therefore this reality After testing detection administration 8 weeks, each experimental mice blood sugar and glycated hemoglobin level control blood glucose fluctuations comparing 5 kinds of albumen Ability.As shown in figure 8, result further illustrates hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA- 4 kinds of fusion proteins of hFGF21 have the regulating and controlling effect of long duration to the blood sugar level of type ii diabetes mouse, and effect is significant Better than hFGF21.

After being administered 8 weeks, each experimental mice serum levels of triglyceride level result as shown in figure 8, with respect to physiological saline group, 5 kinds of albumen can significantly reduce TG content.And compared with hFGF21, hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 4 kinds of fusion proteins of 3DHSA-hFGF21 show more preferably improvement on the blood lipid level of type ii diabetes mouse.

SEQUENCE LISTING

<110>He'nan Normal University

<120>Recombinant human fibroblast growth factor 21 fusion protein and its answering in preparation treatment metabolic disease medicine With

<160> 10

<170> PatentIn version 3.3

<210> SEQ ID NO:1

<211> 2352

<212> DNA

<213>Artificial sequence

<400> SEQ ID NO:1

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gctgaccagt ctccggaatc tctgctgcag ctgaaagctc tgaaaccggg tgttatccag 180

atcctgggtg ttaaaacctc tcgtttcctg tgccagcgtc cggacggtgc tctgtacggt 240

tctctgcact tcgacccgga agcttgctct ttccgtgaac tgctgctgga agacggttac 300

aacgtttacc agtctgaagc tcacggtctg ccgctgcacc tgccgggtaa caaatctccg 360

caccgtgacc cggctccgcg tggtccggct cgtttcctgc cgctgccggg tctgccgccg 420

gctctgccgg aaccgccggg tatcctggct ccgcagccgc cggacgttgg ttcttctgac 480

ccgctgtcta tggttggtcc gtctcagggt cgttctccgt cttacacctc tggtggtggt 540

ggttctggtg gtggtggttc tggtggtggt ggttctagag gtgttttcag aagagacgct 600

cacaagtctg aagttgctca cagattcaag gacttgggtg aagaaaactt caaggctttg 660

gttttgatcg ctttcgctca atacttgcaa caatgtccat tcgaagacca cgttaagttg 720

gttaacgaag ttactgaatt tgctaagact tgtgttgctg acgaatctgc tgaaaactgt 780

gacaagtctt tgcacacttt gttcggtgac aagttgtgta ctgttgctac tttgagagaa 840

acttacggtg aaatggctga ctgttgtgct aagcaagaac cagaaagaaa cgaatgtttc 900

ttgcaacaca aggacgacaa cccaaacttg ccaagattgg ttagaccaga agttgacgtt 960

atgtgtactg ctttccacga caacgaagaa actttcttga agaagtactt gtacgaaatc 1020

gctagaagac acccatactt ctacgctcca gaattgttgt tcttcgctaa gagatacaag 1080

gctgctttca ctgaatgttg tcaagctgct gacaaggctg cttgtttgtt gccaaagttg 1140

gacgaattga gagacgaagg taaggcttct tctgctaagc aaagattgaa gtgtgcttct 1200

ttgcaaaagt tcggtgaaag agctttcaag gcttgggctg ttgctagatt gtctcaaaga 1260

ttcccaaagg ctgaatttgc tgaagtttct aagttggtta ctgacttgac taaggttcac 1320

actgaatgtt gtcacggtga cttgttggaa tgtgctgacg acagagctga cttggctaag 1380

tacatctgtg aaaaccaaga ctctatctct tctaagttga aggaatgttg tgaaaagcca 1440

ttgttggaaa agtctcactg tatcgctgaa gttgaaaacg acgaaatgcc agctgacttg 1500

ccatctttgg ctgctgactt cgttgaatct aaggacgttt gtaagaacta cgctgaagct 1560

aaggacgttt tcttgggtat gttcttgtac gaatacgcta gaagacaccc agactactct 1620

gttgttttgt tgttgagatt ggctaagact tacgaaacta ctttggaaaa gtgttgtgct 1680

gctgctgacc cacacgaatg ttacgctaag gttttcgacg aatttaagcc attggttgaa 1740

gaaccacaaa acttgatcaa gcaaaactgt gaattgttcg aacaattggg tgaatacaag 1800

ttccaaaacg ctttgttggt tagatacact aagaaggttc cacaagtttc tactccaact 1860

ttggttgaag tttctagaaa cttgggtaag gttggttcta agtgttgtaa gcacccagaa 1920

gctaagagaa tgccatgtgc tgaagactac ttgtctgttg ttttgaacca attgtgtgtt 1980

ttgcacgaaa agactccagt ttctgacaga gttactaagt gttgtactga atctttggtt 2040

aacagaagac catgtttctc tgctttggaa gttgacgaaa cttacgttcc aaaggaattt 2100

aacgctgaaa ctttcacttt ccacgctgac atctgtactt tgtctgaaaa ggaaagacaa 2160

atcaagaagc aaactgcttt ggttgaattg gttaagcaca agccaaaggc tactaaggaa 2220

caattgaagg ctgttatgga cgacttcgct gctttcgttg aaaagtgttg taaggctgac 2280

gacaaggaaa cttgtttcgc tgaagaaggt aagaagttgg ttgctgcttc tcaagctgct 2340

ttgggtttgt aa 2352

<210> SEQ ID NO:2

<211> 2352

<212> DNA

<213>Artificial sequence

<400> SEQ ID NO:2

agaggtgttt tcagaagaga cgctcacaag tctgaagttg ctcacagatt caaggacttg 60

ggtgaagaaa acttcaaggc tttggttttg atcgctttcg ctcaatactt gcaacaatgt 120

ccattcgaag accacgttaa gttggttaac gaagttactg aatttgctaa gacttgtgtt 180

gctgacgaat ctgctgaaaa ctgtgacaag tctttgcaca ctttgttcgg tgacaagttg 240

tgtactgttg ctactttgag agaaacttac ggtgaaatgg ctgactgttg tgctaagcaa 300

gaaccagaaa gaaacgaatg tttcttgcaa cacaaggacg acaacccaaa cttgccaaga 360

ttggttagac cagaagttga cgttatgtgt actgctttcc acgacaacga agaaactttc 420

ttgaagaagt acttgtacga aatcgctaga agacacccat acttctacgc tccagaattg 480

ttgttcttcg ctaagagata caaggctgct ttcactgaat gttgtcaagc tgctgacaag 540

gctgcttgtt tgttgccaaa gttggacgaa ttgagagacg aaggtaaggc ttcttctgct 600

aagcaaagat tgaagtgtgc ttctttgcaa aagttcggtg aaagagcttt caaggcttgg 660

gctgttgcta gattgtctca aagattccca aaggctgaat ttgctgaagt ttctaagttg 720

gttactgact tgactaaggt tcacactgaa tgttgtcacg gtgacttgtt ggaatgtgct 780

gacgacagag ctgacttggc taagtacatc tgtgaaaacc aagactctat ctcttctaag 840

ttgaaggaat gttgtgaaaa gccattgttg gaaaagtctc actgtatcgc tgaagttgaa 900

aacgacgaaa tgccagctga cttgccatct ttggctgctg acttcgttga atctaaggac 960

gtttgtaaga actacgctga agctaaggac gttttcttgg gtatgttctt gtacgaatac 1020

gctagaagac acccagacta ctctgttgtt ttgttgttga gattggctaa gacttacgaa 1080

actactttgg aaaagtgttg tgctgctgct gacccacacg aatgttacgc taaggttttc 1140

gacgaattta agccattggt tgaagaacca caaaacttga tcaagcaaaa ctgtgaattg 1200

ttcgaacaat tgggtgaata caagttccaa aacgctttgt tggttagata cactaagaag 1260

gttccacaag tttctactcc aactttggtt gaagtttcta gaaacttggg taaggttggt 1320

tctaagtgtt gtaagcaccc agaagctaag agaatgccat gtgctgaaga ctacttgtct 1380

gttgttttga accaattgtg tgttttgcac gaaaagactc cagtttctga cagagttact 1440

aagtgttgta ctgaatcttt ggttaacaga agaccatgtt tctctgcttt ggaagttgac 1500

gaaacttacg ttccaaagga atttaacgct gaaactttca ctttccacgc tgacatctgt 1560

actttgtctg aaaaggaaag acaaatcaag aagcaaactg ctttggttga attggttaag 1620

cacaagccaa aggctactaa ggaacaattg aaggctgtta tggacgactt cgctgctttc 1680

gttgaaaagt gttgtaaggc tgacgacaag gaaacttgtt tcgctgaaga aggtaagaag 1740

ttggttgctg cttctcaagc tgctttgggt ttgggtggtg gtggttctgg tggtggtggt 1800

tctggtggtg gtggttctga ctcttctccg ctgctgcagt tcggtggtca ggttcgtcag 1860

cgttacctgt acaccgacga cgctcagcag accgaagctc acctggaaat ccgtgaagac 1920

ggtaccgttg gtggtgctgc tgaccagtct ccggaatctc tgctgcagct gaaagctctg 1980

aaaccgggtg ttatccagat cctgggtgtt aaaacctctc gtttcctgtg ccagcgtccg 2040

gacggtgctc tgtacggttc tctgcacttc gacccggaag cttgctcttt ccgtgaactg 2100

ctgctggaag acggttacaa cgtttaccag tctgaagctc acggtctgcc gctgcacctg 2160

ccgggtaaca aatctccgca ccgtgacccg gctccgcgtg gtccggctcg tttcctgccg 2220

ctgccgggtc tgccgccggc tctgccggaa ccgccgggta tcctggctcc gcagccgccg 2280

gacgttggtt cttctgaccc gctgtctatg gttggtccgt ctcagggtcg ttctccgtct 2340

tacacctctt aa 2352

<210> SEQ ID NO:3

<211>1194

<212> DNA

<213>Artificial sequence

<400> SEQ ID NO:3

gactcttctc cgctgctgca gttcggtggt caggttcgtc agcgttacct gtacaccgac 60

gacgctcagc agaccgaagc tcacctggaa atccgtgaag acggtaccgt tggtggtgct 120

gctgaccagt ctccggaatc tctgctgcag ctgaaagctc tgaaaccggg tgttatccag 180

atcctgggtg ttaaaacctc tcgtttcctg tgccagcgtc cggacggtgc tctgtacggt 240

tctctgcact tcgacccgga agcttgctct ttccgtgaac tgctgctgga agacggttac 300

aacgtttacc agtctgaagc tcacggtctg ccgctgcacc tgccgggtaa caaatctccg 360

caccgtgacc cggctccgcg tggtccggct cgtttcctgc cgctgccggg tctgccgccg 420

gctctgccgg aaccgccggg tatcctggct ccgcagccgc cggacgttgg ttcttctgac 480

ccgctgtcta tggttggtcc gtctcagggt cgttctccgt cttacacctc tggtggtggt 540

ggttctggtg gtggtggttc tggtggtggt ggttctgttg aagaaccaca aaacttgatc 600

aagcaaaact gtgaattgtt cgaacaattg ggtgaataca agttccaaaa cgctttgttg 660

gttagataca ctaagaaggt tccacaagtt tctactccaa ctttggttga agtttctaga 720

aacttgggta aggttggttc taagtgttgt aagcacccag aagctaagag aatgccatgt 780

gctgaagact acttgtctgt tgttttgaac caattgtgtg ttttgcacga aaagactcca 840

gtttctgaca gagttactaa gtgttgtact gaatctttgg ttaacagaag accatgtttc 900

tctgctttgg aagttgacga aacttacgtt ccaaaggaat tcaacgctga aactttcact 960

ttccacgctg acatctgtac tttgtctgaa aaggaaagac aaatcaagaa gcaaactgct 1020

ttggttgaat tggttaagca caagccaaag gctactaagg aacaattgaa ggctgttatg 1080

gacgacttcg ctgctttcgt tgaaaagtgt tgtaaggctg acgacaagga aacttgtttc 1140

gctgaagaag gtaagaagtt ggttgctgct tctcaagctg ctttgggttt gtaa 1194

<210> SEQ ID NO:4

<211> 1194

<212> DNA

<213>Artificial sequence

<400> SEQ ID NO:4

gttgaagaac cacaaaactt gatcaagcaa aactgtgaat tgttcgaaca attgggtgaa 60

tacaagttcc aaaacgcttt gttggttaga tacactaaga aggttccaca agtttctact 120

ccaactttgg ttgaagtttc tagaaacttg ggtaaggttg gttctaagtg ttgtaagcac 180

ccagaagcta agagaatgcc atgtgctgaa gactacttgt ctgttgtttt gaaccaattg 240

tgtgttttgc acgaaaagac tccagtttct gacagagtta ctaagtgttg tactgaatct 300

ttggttaaca gaagaccatg tttctctgct ttggaagttg acgaaactta cgttccaaag 360

gaattcaacg ctgaaacttt cactttccac gctgacatct gtactttgtc tgaaaaggaa 420

agacaaatca agaagcaaac tgctttggtt gaattggtta agcacaagcc aaaggctact 480

aaggaacaat tgaaggctgt tatggacgac ttcgctgctt tcgttgaaaa gtgttgtaag 540

gctgacgaca aggaaacttg tttcgctgaa gaaggtaaga agttggttgc tgcttctcaa 600

gctgctttgg gtttgggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct 660

gactcttctc cgctgctgca gttcggtggt caggttcgtc agcgttacct gtacaccgac 720

gacgctcagc agaccgaagc tcacctggaa atccgtgaag acggtaccgt tggtggtgct 780

gctgaccagt ctccggaatc tctgctgcag ctgaaagctc tgaaaccggg tgttatccag 840

atcctgggtg ttaaaacctc tcgtttcctg tgccagcgtc cggacggtgc tctgtacggt 900

tctctgcact tcgacccgga agcttgctct ttccgtgaac tgctgctgga agacggttac 960

aacgtttacc agtctgaagc tcacggtctg ccgctgcacc tgccgggtaa caaatctccg 1020

caccgtgacc cggctccgcg tggtccggct cgtttcctgc cgctgccggg tctgccgccg 1080

gctctgccgg aaccgccggg tatcctggct ccgcagccgc cggacgttgg ttcttctgac 1140

ccgctgtcta tggttggtcc gtctcagggt cgttctccgt cttacacctc ttaa 1194

<210> SEQ ID NO:5

<211> 783

<212> PRT

<213>Artificial sequence

<400> SEQ ID NO:5

Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp 20

Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala 40

Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly 80

Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr 100

Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro 120

His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 160

Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Thr Ser Gly Gly Gly 180

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gly Val Phe Arg Arg Asp Ala 200

His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu 220

Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 240

Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys 260

Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu 280

Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe 300

Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 320

Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile 340

Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys 360

Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu 380

Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 400

Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg 420

Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His 440

Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys 460

Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 480

Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu 500

Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala 520

Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser 540

Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 560

Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu 580

Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys 600

Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr 620

Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 640

Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val 660

Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val 680

Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe 700

Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 720

Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu 740

Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp 760

Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala 780

Leu Gly Leu *** 783

<210> SEQ ID NO:6

<211> 783

<212> PRT

<213>Artificial sequence

<400> SEQ ID NO:6

Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu 20

Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys 40

Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val 60

Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 80

Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln 100

Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg 120

Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe 140

Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 160

Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys 180

Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala 200

Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp 220

Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 240

Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala 260

Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys 280

Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu 300

Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 320

Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr 340

Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu 360

Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe 380

Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 400

Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys 420

Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly 440

Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser 460

Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 480

Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp 500

Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys 520

Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys 540

His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 560

Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys 580

Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly 600

Ser Gly Gly Gly Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 620

Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp 640

Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu 660

Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro 680

Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu 700

Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu 720

Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro 740

Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro 760

Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser 780

Tyr Thr Ser *** 783

<210> SEQ ID NO:7

<211> 397

<212> PRT

<213>Artificial sequence

<400> SEQ ID NO:7

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Glu Glu Pro Gln Asn Leu Ile 200

Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 220

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 240

Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 260

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro 280

Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 300

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr 320

Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 340

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met 360

Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 380

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu *** 397

<210> SEQ ID NO:8

<211> 397

<212> PRT

<213>Artificial sequence

<400> SEQ ID NO:8

Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 20

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr 40

Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 60

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu 80

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 100

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 120

Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 140

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 160

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 180

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 200

Ala Ala Leu Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 220

Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp 240

Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala 260

Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 280

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly 300

Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr 320

Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro 340

His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 360

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 380

Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Thr Ser *** 397

Claims

1. recombinant human fibroblast growth factor 21 fusion protein is it is characterised in that be by human fibroblastic growth factor 21 Recombination fusion protein hFGF21-HSA, HSA-hFGF21, hFGF21- that recombinant protein hFGF21 and HSA or 3DHSA is formed by connecting SEQ ID in 3DHSA and 3DHSA-hFGF21, the wherein amino acid sequence of recombination fusion protein hFGF21-HSA such as sequence table NO:Shown in 5, SEQ ID NO in the amino acid sequence such as sequence table of recombination fusion protein HSA-hFGF21:Shown in 6, restructuring is merged SEQ ID NO in the amino acid sequence of albumen hFGF21-3DHSA such as sequence table:Shown in 7, recombination fusion protein 3DHSA- SEQ ID NO in the amino acid sequence of hFGF21 such as sequence table:Shown in 8.

2. the recombinant human fibroblast growth factor 21 fusion protein encoding gene described in a kind of claim 1, its feature exists In：SEQ ID NO in the nucleotide sequence of described recombination fusion protein hFGF21-HSA encoding gene such as sequence table:Shown in 1, SEQ ID NO in the nucleotide sequence of recombination fusion protein HSA-hFGF21 encoding gene such as sequence table:Shown in 2, restructuring is merged SEQ ID NO in the nucleotide sequence of albumen hFGF21-3DHSA encoding gene such as sequence table:Shown in 3, recombination fusion protein SEQ ID NO in the nucleotide sequence of 3DHSA-hFGF21 encoding gene such as sequence table:Shown in 4.

3. a kind of expression containing the recombinant human fibroblast growth factor 21 fusion protein encoding gene described in claim 2 Carrier and the host cell containing this expression vector it is characterised in that：Expression vector used is preferably pET30a（+）, host Cell is preferably Rossetta（DE3）.

4. the preparation method of recombinant human fibroblast growth factor 21 fusion protein described in a kind of claim 1, its feature It is to concretely comprise the following steps：By the nucleotide sequence of recombinant human fibroblast growth factor 21 fusion protein encoding gene and expression Carrier is connected and obtains recombinant expression carrier；Again by this recombinant expression carrier transformed host cell, then screen high expression positive Host cell, cultured cells abduction delivering recombinant human fibroblast growth factor 21 fusion protein, collects thalline, broken, Centrifugation, clarification, purifying, obtain target product recombinant human fibroblast growth factor 21 fusion protein.

5. recombinant human fibroblast growth factor 21 fusion protein described in claim 1 is in preparation treatment metabolic disease medicine In application, wherein metabolic disease includes diabetes, obesity and metabolic syndrome.

6. a kind of pharmaceutical composition for treating diabetes or obesity is it is characterised in that include the power of the upper effective dose for the treatment of Profit requires recombinant human fibroblast growth factor 21 fusion protein and pharmaceutically acceptable carrier or auxiliary material described in 1.