CN102464712A - Deletion human fibroblast growth factor 21 variant and conjugate thereof - Google Patents
Deletion human fibroblast growth factor 21 variant and conjugate thereof Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- C07K14/501—Fibroblast growth factors [FGF] acidic FGF [aFGF]
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- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to a natural wild type N-terminal deletion variant human Fibroblast Growth Factor 21 (FGF-21), characterized in that 9 amino acid sequences (delta N9FGF-21) are deleted from the N-terminal. The deletion variant has strong stability in vitro and high biological activity. Simultaneously, the invention further provides a conjugate of the deletion variant, characterized in that: deletion variant human FGF-21 is chemically modified by polyethylene glycol having a molecular weight of 5-40 KD to form the delta N9FGF-21 conjugate. The conjugate keeps the activity in vivio of the delta N9FGF-21 , and improves the pharmacokinetic properties in vivio of the delta N9FGF-21. The deletion variant and the conjugate are suitable for being used as a medicine composition for treating or preventing obesity, diabete, hyperglycemia or hyperlipidemia.
Description
Technical field
The present invention relates to DNA recombinant technology and pharmaceutical field.More specifically, the present invention relates to a kind of varient of human fibroblastic growth factor 21 (FGF-21) of N-terminal brachymemma, the covalently bound and purposes of corresponding D NA molecule, expression vector, this varient and polyoxyethylene glycol.
Background of invention
Fibroblast growth factor (Fibroblast Growth Factor; FGF) be one type of protein by the structurally associated of FGF gene family member coding; Cell to multiple mesoderm such as endotheliocyte, inoblast, smooth muscle cell and neuroderm source has the synthetic and cell fission effect of the DNA of promotion; Mainly by inoblast, endotheliocyte, osteocyte, polyblast secretion, relative molecular weight is generally at 17～34kD.FGF family member's central zone amino acid has the homology of 30%-70%.FGF-21 is a up-to-date found FGFs member, belongs to the FGF-19 subfamily.The FGF-19 subfamily comprises FGF-15, FGF-19, FGF-21 and FGF-23, and different with other FGF members is that they play regulating and controlling effect with endocrine form to metabolism, and do not have and heparin-bounding function.FGF-21 mainly expresses in ripe liver and thymus gland; The earliest by (Nishimura T etc. such as Nishimura; Biochim Biophys Acta; 2000,1492:203-206) in mice embryonic, to separate first in 2000 and obtain, the homology of its aminoacid sequence and FGF-19 and FGF-23 is respectively 35% and 24%.MRNA (Inagaki T etc., Cell Metab, 2007,5 (6): 415-425) of FGF-21 have been found subsequently equally in pancreas, fatty tissue and muscle tissue.People source FGF-21 gene is positioned at karyomit(e) No. 19, and total length FGF-21 albumen is made up of 181 amino acid whose ripe FGF-21 albumen and 28 amino acid whose signal peptides that are positioned at N end, and it is 75% identical that its aminoacid sequence and mouse source FGF-21 have approximately.
Recent study finds that FGF-21 plays a very important role to the metabolism and the weight management of blood sugar, blood fat.In these mechanisms; Its N end regions is being brought into play different biological functions with the C end regions; Be the interaction of participation of N terminal amino acid and FGF acceptor, the C terminal amino acid is participated in and a kind of proteic interaction (Micanovic R etc., J CellPhys that is called β-Klotho; 2009,219 (2): 227-234).Kharitonenkov A etc. reported first in 2005 recombinant human FGF-21 albumen can stimulate glucose uptake (the Kharitonenkov A etc. of adipocyte before mouse 3T3-L1 cell and the people of differentiation; J ClinInvest; 2005,115 (6): 1627-1635).Research subsequently further shows; FGF-21 is (the Amer P etc. that increase noninsulin dependent glucose uptake through the expression of glucose transporter-1 (GLUT-1) in the adipocyte before increase 3T3-L1 cell and the people; FEBS Lett, 2008,582 (12): 1725-1730).With insulin-mediated utilize glucose different fast, the sustainable several hrs of the effect of FGF-21 aspect glucose uptake.The more important thing is that the glucose uptake under the FGF-21 effect is a non-insulin-dependent, and when existing jointly, can increase effect (Alexei Kharitonenkov etc., Biodrugs, 2008,22 (1): 37-44) of ingestion of glucose with Regular Insulin.FGF-21 can improve the function of beta Cell of islet.The db/db mouse postprandial blood sugar that FGF-21 continue to handle for 8 weeks is normal, glucose clearance be improved significantly, the transcribing, express and secrete increase of Regular Insulin, but islet cells quantity does not change.In addition; In isolating mouse islets and INS-1E cell, FGF-21 can stop glycolipid toxicity and cytokine mediated apoptosis, the quantity and the function of protection beta Cell of islet; Release (the Wente W etc. that suppress the hyperglycemic-glycogenolytic factor of glucose mediation; Diabetes, 2006,55:2470-2478).Aspect the non-human primates diabetes animal model, injection recombinant human FGF-21 can reduce the LDL cholesterol level, increases the HDL cholesterol level; Reduce risk (the Kharitonenkov A etc. that suffer from cardiovascular aspect disease; Endocrinology, 2007,148:774-781).In the recombinant human FGF-21 animal body that carries out at present, in the pharmacodynamic experiment, find that the side reaction that has other mellitus such as hypoglycemia, oedema and obesity increase often to occur occurs, and has demonstrated good prospects for application and security.
Lilly company discloses the aminoacid sequence and reorganization preparation, purposes (US7259248 of people FGF-21; WO/2009/020802); The fusion rotein patent (US20070237768) of people FGF-21 albumen and IgG4 or HSA; 4 amino acid whose two mutants of disappearance and 153 Leu of the N end of a kind of people FGF21 have been announced simultaneously, the two mutants of 167 Ala and 118 and 134 Cys.But biological effect reduces in various degree, active 90% (US 7622445) that keep of wherein simple N end disappearance 4 amino acids varients.Researchist of the present invention finds, the poor stability of recombinant human wild-type FGF-21 shows as the N-terminal fracture.With behind 9 sequential amino acid deletions of N-terminal of people FGF21 protein molecular (code name is Δ N9FGF-21), have higher stability and biological activity.Through adopting genetically engineered recombinant protein technology preparation absence type Δ N9FGF21; The three big expression systems that comprise intestinal bacteria (E.coli), yeast (Pichia Pastoris) and mammalian cell (CHO) have all proved the above characteristic of recombination deficient type people FGF21.
Simultaneously, researchist of the present invention finds, the conjugate through to the polyethyleneglycol modified back gained of this absence type varient has kept this absence type varient activity in vivo, improved this absence type varient in vivo pharmacokinetics character.This absence type varient and conjugate thereof are more suitable for being used for treatment or prevention of obesity, mellitus, high sugar or hyperlipidaemia as a kind of drug regimen.
Summary of the invention
One of the object of the invention provides a kind of deletion human fibroblast growth factor 21 (Δ N9FGF-21) varient.It has the aminoacid sequence characteristic of seq ID No:1, the sequence of this characteristic sequence and wild-type human fibroblastic growth factor 21 (FGF-21) relatively, in the N-terminal brachymemma HPIPDSSPL totally 9 amino acid, form by 172 amino acid.When recombinant expressed, its N-terminal can have methionine(Met) (Met) residue, is expressed as Met-Δ N9FGF-21 in prokaryotic organism such as intestinal bacteria.The N-terminal that relates to according to the invention lacks its N end of 9 amino acids human fibroblastss 21 (Δ N9FGF-21) and whether has Met, does not influence its biological nature.
The aminoacid sequence of people's Δ N9FGF-21 (SEQ ID NO:1):
Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp
1 5 10 15
Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val
20 25 30
Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala
35 40 45
Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe
50 55 60
Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp
65 70 75 80
Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn
85 90 95
Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn
100 105 110
Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu
115 120 125
Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu
130 135 140
Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val
145 150 155 160
Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
165 170
Another object of the present invention provides the dna molecular of the sequence (SEQ IDNO:1) of coding deletion human fibroblast growth factor 21 varients, and the recombinant expression vector that contains this dna molecular.Its expression vector can be to come from prokaryotic organism or eukaryote system; Except that the gene order that contains coded delta N9FGF-21, also comprise regulating and controlling sequence that target protein expresses such as promotor and selection markers such as penbritin, tsiklomitsin, Xin Meisu, G418, Zeocin, DHFR etc.
Another object of the present invention provides a kind of above-mentioned expression vector transformed host cells that is used for.This host cell can be intestinal bacteria, yeast or mammalian cell.
Another object of the present invention provides a kind of through cultivating above-mentioned transformed host cell and from nutrient solution, separating and the method for purification of Recombinant deletion human FGF-21 varient.
Another object of the present invention has provided the analogue of the human fibroblastic growth factor 21 that a kind of stability and biological activity all obtain to improve, i.e. people's Δ N9FGF-21.
Another object of the present invention provides the N-terminal absence type varient and the conjugate thereof of human fibroblastic growth factor 21.This conjugate is through the N-terminal amino of activated polyglycol molecule and people's Δ N9FGF-21 or amino covalently bound the forming of Lys; Wherein in the main and SEQ ID NO:1 sequence of polyoxyethylene glycol the 47th; The 50th; The 60th amino covalence with the 113rd Lys is connected, and wherein peg molecule can be straight chain or branched chain or star-like shape, and its molecular weight is 5KD-40KD.
The N-terminal absence type varient of this human fibroblastic growth factor 21 and conjugate albumen thereof have more and are used to treat or the using value of prevention of obesity, mellitus, high fat and hyperglycemia.For realizing that its medicinal application is worth, under effective dosage ranges, can be prepared into the medicine or the drug regimen of corresponding treatment or prevention usefulness through technology known in the art.According to the difference of the administering mode or the approach of medicine, can be made into injection liquid, freeze-dried, sustained release dosage, tablet or capsule with known open formulation method.
Description of drawings
Accompanying drawing 1 recombinant human Δ N9FGF-21 protein expression and purifying SDS-PAGE electrophoretogram.Wherein, M is the protein standard, and 1 for not inducing, 2 be abduction delivering after, 3 is behind the Phenyl purifying, 4 for behind the anti-phase purifying.
The PEG of accompanying drawing 2 recombinant human Δ N9FGF-21 modifies and purifying SDS-PAGE electrophoretogram.Wherein, M is the protein standard, and 1 is sample before modifying, and 2 for modifying the back sample, and 3 is PEG-Δ N9FGF21 behind the purifying.
(ordinate zou is glucose content (mMol/L) to the effect of 3T3-L1 grape cell Sugar intake rate for accompanying drawing 3 recombinant human Δ N9FGF21 and PEG-Δ N9FGF21; X-coordinate be the time (hour); Wherein ▲ be control group, ● be Δ N9FGF21, ■ is PEG-Δ N9FGF21.
Embodiment
The inventor is through extensive and deep research; The expression of carrying out recombination deficient type human fibroblastic growth factor 21 varients with preferred expression vector pET-3c and host cell BL21 (DE3) PlysS with separate preparation, adopting molecular weight is that the straight chain of 20KD is modified deletion human fibroblast growth factor 21 varients by aldehyde radical activatory mono methoxy polyethylene glycol molecule (mPEG-ButylALD-20KD).Said instance is merely illustration purpose, should not be construed as limitation of the present invention.
Embodiment 1: the structure of recombinant human Δ N9FGF-21 protein expression engineering bacteria
Aminoacid sequence (deriving from Genebank) according to wild-type people FGF-21; Behind 9 His Pro of N-terminal Ile ProAsp Ser Ser Pro Leu sequence deletion; Aminoacid sequence (SEQ ID No:1) as Δ N9FGF-21; And according to the codon of intestinal bacteria preferences, the encode eDNA (SEQ ID NO:2) of corresponding people's Δ N9FGF-21 of optimization design.Entrusting precious biological (Dalian) ltd to carry out full gene synthesizes; Wherein introduce Nde I restriction enzyme site (CATATG) at cDNA 5 ' end; 3 ' end is introduced terminator codon (TCA) and BamH I restriction enzyme site (GGATCC), behind the insertion pMD-19-T simplevector, changes host bacterium DH5 α over to; Positive colony after cDNA sequence verification and implementation sequence are in full accord, called after pMD-Δ N9FGF21.
The cDNA sequence of people's Δ N9FGF-21 (SEQ ID NO:2):
5′-CTG CAG TTT GGC GGC CAG GTG CGT CAG CGT TAT CTG TAT ACC GAT GAT
GCG CAG CAG ACC GAA GCG CAT CTG GAA ATT CGT GAA GAT GGC ACC GTG
GGC GGC GCG GCG GAT CAG AGC CCG GAA AGC CTG CTG CAG CTG AAA GCG
CTG AAA CCG GGC GTG ATT CAG ATT CTG GGC GTG AAA ACC AGC CGT TTT
CTG TGC CAG CGT CCG GAT GGC GCG CTG TAT GGC AGC CTG CAT TTT GAT
CCG GAA GCG TGC AGC TTT CGT GAA CTG CTG CTG GAA GAT GGC TAT AAT
GTG TAT CAG AGC GAA GCG CAT GGC CTG CCG CTG CAT CTG CCG GGC AAT
AAA AGC CCG CAT CGT GAT CCG GCG CCG CGT GGC CCG GCG CGT TTT CTG
CCG CTG CCG GGC CTG CCG CCG GCG CTG CCG GAA CCG CCG GGC ATT CTG
GCG CCG CAG CCG CCG GAT GTG GGC AGC AGC GAT CCG CTG AGC ATG GTG
GGC CCG AGC CAG GGC CGT AGC CCG AGC TAT GCG AGC-3′
Extract pMD-Δ N9FGF21 recombinant plasmid, behind Nde I and BamH I double digestion, reclaim 540bp left and right sides purpose fragment, be connected with the expression plasmid pET-3c (Invitrogen) that the switchback of BamH I enzyme is received through Nde I with same with the T4DNA ligase enzyme.Adopt CaCl2 method transformed into escherichia coli host bacterium DH5 α, select positive colony, cut and the correct recombinant plasmid called after pET-3C-Δ N9FGF21 of PCR checking with enzyme.This plasmid is through CaCl2 method transformed into escherichia coli expressive host bacterium BL21 (DE3) PlysS (Novagen) competence, and screening is also cloned the proteic bacterial strain of efficiently expressing recombinant human Δ N9FGF-21, and in-80 ℃ of preservations.
Embodiment 2: the recombinant expressed and preparation of recombinant human Δ N9FGF-21 in intestinal bacteria.
To express best engineering strain streak inoculation in LA (adding 100 μ g/ml Amp in the LB substratum) agar plate, 37 ℃ of overnight cultures.The picking lawn is inoculated in and contains LB liquid nutrient medium (Tryptones 10g, yeast extract 5g, NaCl 10g from the LA flat board of incubated overnight; Adding water dissolves to 1000mL surely; 121 ℃, autoclaving 30min gets final product) in the test tube, 37 ℃ of activation 12 hours; Be transferred in the 1000mL triangular flask that contains 200mL LB nutrient solution in 1% ratio then, 37 ℃ of overnight cultures promptly become and go up a jar seed liquor.Last jar seed liquor is inoculated in the 30L fermentor tank that contains 20L M9+YT nutrient solution in 5% ratio; 37 ℃ of cultivations; Have suffered in the fermenting process, keep dissolved oxygen＞30% through adjusting rotating speed, blowing air amount, logical pure oxygen amount, the ammoniacal liquor with 28% is regulated pH and is also remained on 7.0.Culture bacteria liquid OD600=4.0～6.0 o'clock reduces to 28 ℃ with culture temperature, to bacterium liquid OD
600=8.0～10.0 o'clock, the adding final concentration was 0.5mmol/L IPTG, continued to cultivate to stop fermentation after 6 hours, collected bacterium liquid, and centrifugal 10 minutes of 8000rpm abandons supernatant, and the collection thalline is put into-20 ℃ of refrigerators and preserved subsequent use.
Fermented liquid at 4 ℃ of centrifugal 10min of following 8000rpm, is collected thalline with high-speed refrigerated centrifuge, put-20 ℃ frozen 24 hours.The broken bacterium damping fluid of every 1g thalline adding 10ml (50mmol/LTris, 5mmol/L EDTA, pH10.3); 30 ℃ of stirring in water bath are after 1 hour, and (model: AH-BASIC) 500Pa homogenate twice, uses microscopic examination with the pressure ramp pulp grinder of ATS company; Thalline is broken fully, is cotton-shaped.4 ℃ of centrifugal 10min of following 10000rpm collect supernatant then.
Mend 10% saturated ammonium sulphate solution in the supernatant, behind 4 ℃ of placement 30min, 4 ℃ of centrifugal 10min of following 8000rpm, disgorging.Phenyl Sepharose (FF) column chromatography method subsequently; Solution A is 10mmol/L PB, 15% saturated ammonium sulphate solution (pH8.0); Solution B is 10mmol/L PB (pH8.0).After the appearance,, contain Δ N9FGF-21 elution peak with the collection of B liquid wash-out again on the supernatant with A liquid multiple equilibria.Q-Srpharose (FF) column chromatography method: solution A is 10mmol/L/L PB (PH8.0), and solution B is 10mmol/L/L (PH8.0) and 0.5M NaCl.After the mercapto water layer analysed on the elution peak appearance,, collect the target protein peak (about 50% gradient) that contains Δ N9FGF-21 with the ion gradient wash-out of solution A to solution B.C18 reversed phase chromatography method: separating medium is WelchXB-C18, and solution A is 5% acetonitrile and 0.1%TFA, and solution B is 75% acetonitrile (containing 0.1%TFA).Q post elution peak transfers PH to 2.0～3.0 backs to go up appearance with TFA, carries out wash-out (10 column volumes) with the gradient of solution A to solution B, collects Δ N9FGF-21 elution peak (about 35% gradient).After lyophilize, become the sample of recombinant human Δ N9FGF-21 again.
Sample is through the above-mentioned steps purifying, promptly saltouts, behind the hydrophobic chromatography, ion exchange chromatography, reversed phase chromatography, and purity reaches more than 95%.Through technical study, recombinant human Δ N9FGF-21 is a solubility expression, but every 100g thalline purifying prepares about recombinant human Δ N9FGF-21 protein 10 0mg
Embodiment 3:mPEG-ButylALD-20KD is to modification and the purifying of Δ N9FGF-21
Δ N9FGF-21 solution is by instance 2 preparations, and (relative molecular mass is 20 * 10 to mPEG-ButylALD-20KD
3), sodium cyanoborohydride (CH3BNNa) solution is as α-NH
2Acvator.Δ N9FGF-21 solution is after dialysing to 100mM PB pH5.0, and adding the CH3BNNa final concentration is 20mM.Got Δ N9FGF-21 and mPEG-ButylALD-20KD mass ratio 1: 2, under 4 ℃ of conditions, 100r/min stirs, reaction 24hr, and SDS-PAGE electrophoresis (Fig. 3) is carried out in sampling, confirms the modification ratio of PEG-Δ N9FGF21, and its modification rate reaches 75%.Modify back sample P EG-20-Δ N9FGF21, after 5-10 times of DDW dilution, transfer pH to 8.0.Adopt the further separation and purification of Source 15-Q.After balance liquid (50mM Tris-HClpH8.0) balance, last appearance reequilibrate.With the NaCl linear elution of 0-500mM, collect the PEG-Δ N9FGF21 elution peak of the Δ N9FGF-21 that does not contain unmodified.
Embodiment 4: recombinant human Δ N9FGF-21 biological characteristics detects
1, the N-terminal aminoacid sequence detects
The Δ N9FGF-21 albumen of reorganization preparation is analyzed (Edman edman degradation Edman) automatically through the N terminal amino acid sequence; 15 aminoacid sequences of N-terminal are: Met-Leu-Gln-Phe-Gly-Gly-Gln-Val-Arg-Gln-Arg-Tyr-Leu-Tyr-Thr is Met-Δ N9FGF-21
2, molecular weight and purity
The recombinant human Δ N9FGF-21 of 15%SDS-PAGE reduction electrophoresis showed preparation is molecular weight 19,000 daltonian single electrophoresis bands, and performance liquid chromatography (HPLC) C18 reversed-phase column detected result shows that its purity is greater than 98.0%.
Embodiment 5: active determination in vitro
Cell cultures and induce differentiation: at 37 ℃, 5%CO
2Condition under; Adipocyte (Shanghai cell biological institute cell bank) is cultivated in containing the high sugared DMEM of 10%FBS before the 3T3-Ll; Add the high sugared DMEM that contains 0.5mmol/Lol/L IBMX, 1 μ mol/L DEXAMETHASONE BP98,10mg/L insulin human after 2 days and cultivate 48hr; Change the high sugared DMEM that contains the 10mg/L insulin human again and cultivate 48hr, induce 8～12 days 3T3-L1 cell about 90% of differentiation to be the adipocyte phenotype, be used for following experiment.
1) glucose transport analysis: 3T3-L1 adipocyte in 24 orifice plates; Add the substratum (the high sugared DMEM of 0.2%BSA) that contains recombinant human Δ N9FGF-21, PEG-Δ N9FGF21 and recombinant human FGF-21 (rhFGF-21) (Beijing Ai Dibo biotechnology company) simultaneously respectively; Make final concentration be respectively 0,0.032,0.16,0.8,4,20 and 100nM, cultivated 24 hours down for 37 ℃.Glucose is absorbed detection: with KRP damping fluid (NaCl 131.2mmol/Lol/L, KCl 4.7mmol/Lol/L, the MgSO of heating (37 ℃)
41.2mmol/Lol/L, CaCl
22.5mmol/Lol/L, NaH
2PO
42.5mmol/Lol/L pH 7.4) after the washed twice; KRP damping fluid with containing 1%BSA, 0.2 μ ci/ hole 2-deoxidation-(14C)-glucose (Institute for Atomic Research, Beijing) (100 μ l) is hatched 1hr, adds the picked-up that the 10umol/l cytochalasin B stops glucose.Other establishes one group of non-special uptake ratio that adds 10 μ mol/L cytochalasin Bs (cytochalasin B) as 2-deoxidation-(14C)-glucose, and all data deduct this value, as the glucose uptake rate (CPM, PM liquid scintillation instrument count value) of each group cell.The result shows that recombinant human Δ N9FGF-21 extracts the active high by about 45% of glucose than people FGF-21 at the external promotion adipocyte of measuring under the same experimental conditions, and the activity that the external promotion adipocyte that PEG-Δ N9FGF21 measures under same experimental conditions extracts glucose keeps about 70%.
Glucose utilization is analyzed: gets 3T3-L1 and induces (method is the same) behind the lipoblast, add PEG-Δ N9FGF21 respectively and people's Δ N9FGF-21 sample (with the DMEM+5%FCS of low sugar) is mixed with final concentration 4nM, and in 37 ℃, 5%CO
2Middle 12hr, 24hr and the 48hr time point cultivated got culture supernatant respectively; Content and mapping (Fig. 3) with glucose in the glucose assays kit measurement supernatant; Can know that by figure recombinant human Δ N9FGF-21 and PEG-Δ N9FGF21 can promote significantly that the 3T3-L1 cell utilizes glucose.
Instance 6: recombinant human Δ N9FGF-21 and PEG-Δ N9FGF21 are in the intravital pharmacokinetics of CD-1 mouse
Get 24 of CD-1 mouse, be divided into two groups (Δ N9FGF-21 group and PEG-Δ N9FGF21 groups) at random.Press the 0.4mg/kg subcutaneous injection for every group, between injection back 0 to 144hr, repeatedly get blood at interval.Adopt FGF-21ELISA Kit (health peptide biotech firm) to measure the Plasma Concentration of FGF-21 in the blood, calculate the transformation period.
| Title | t 1/2(h) |
| PEG-Δ N9FGF21 group | 39.5 |
| Δ N9FGF-21 group | 4.1 |
Experimental result shows that the transformation period of recombinant human Δ N9FGF-21 is 4.1hr; And the transformation period of PEG-Δ N9FGF21 reaches 39.5hr, transformation period significant prolongation in the body of PEGization Δ N9FGF-21.
Instance 7: the stability of recombinant human Δ N9FGF-21
People's Δ N9FGF-21 protein solution of reorganization preparation replaces with in 10mmol/L Mol/L phosphate-buffered (PH7.0) and the 0.15Mol/L sodium-chlor through dialysis treatment, and adjustment protein concentration (Lowry method mensuration) is 1.0mg/ml.Place respectively after the sterile filtration in 4 ℃ of refrigerators and 25 ℃ of constant temperature investigation casees and carry out examine stability, do external activity, SDS-PAGE electrophoresis and reverse hplc analysis in the different time points sampling.
Above result proves that recombinant human Δ N9FGF-21 is near having very stiff stability under the physiological condition.
Instance 8: recombinant human Δ N9FGF-21 expresses in pichia spp
The cDNA sequence of Δ N9FGF-21 coding is pressed Invitrogen company specification sheets insert in pIC9K (AOX promotor) or pPICZ α (GAP promotor) plasmid, make up the recombinant expression vector of corresponding secretion induction type (methanol induction) or composing type.After electricity transforms GS115 or X33 host bacterium, use G
418Or the recombinant expressed son of Zencin resistance screening.The Pichia yeast that contains Δ N9FGF-21 foreign gene is expressed target protein through fermentation culture, with centrifugal, ultrafiltration, concentrate, common stripping technique such as deposition, column chromatography can obtain the Δ N9FGF-21 that recombinates.Carry out recombinant expressed Δ N9FGF-21 albumen (N-terminal does not have Met) in the yeast saccharomyces cerevisiae expression plasmid of the same available GAL-1 of containing gene promoter.The recombinant human Δ N9FGF-21 albumen that obtains is analyzed with the method for instance 3, proves to have identical biological nature equally.
Instance 9: Δ N9FGF-21 is hypoglycemic and reducing blood lipid in the db/db mouse model
Get totally 30 of the db/db mouse in 8 ages in week, be divided into three groups (Δ N9FGF-21 group, Met-Δ N9FGF-21 group and model control group) at random, press 2mg/kg subcutaneous injection every day, successive administration 10 days.Model control group is by same procedure and volume injecting normal saline, and animal is routine feeding in SPF, when experiment finishes, handles animal measuring blood sugar of blood extracting and blood fat (triglyceride and SUV).The result shows, compares blood sugar and blood fat that Met-Δ N9FGF-21 and Δ N9FGF-21 have identical reduction diabetes model animal with model group.
Claims (11)
1. the N-terminal absence type varient and the conjugate thereof of a human fibroblastic growth factor 21.
2. the N-terminal absence type varient of human fibroblastic growth factor 21 in the claim 1 is characterized by 9 aminoacid sequences of N-terminal disappearance of wild-type human fibroblastic growth factor 21.
3. deletion human fibroblast growth factor 21 varients have the sequence signature of Seq ID No.1 in the claim 1.
4. 9 aminoacid sequences of N-terminal of disappearance are His Pro Ile Pro Asp Ser Ser Pro Leu in the claim 1.
5. the corresponding dna molecular of deletion human inoblast factor 21 varient, this dna molecular contains inclined to one side code weight profit and requires 1,2,3 nucleotide sequence.
6. the dna molecular that the expression vector of the deletion human fibroblast growth factor 21 of a recombinant expressed claim 1, this expression vector contain claim 5 be used for the regulating and controlling sequence that this dna molecular is expressed.
7. the conjugate in the claim 1, it is characterized by it is to modify absence type varient human fibroblastic growth factor 21 and the △ N9FGF-21 conjugate that forms with the polyoxyethylene glycol chemistry.
8. the conjugate in the claim 1,7 is characterized in that amino covalently bound of N-terminal amino or the Lys of polyoxyethylene glycol and this absence type varient.
9. the polyoxyethylene glycol in the claim 7,8 is characterized by straight chain or branched chain or star-like shape, and molecular weight is 5KD-40KD.
10. claim 1,2,4,5,7, the fibroblast growth factor of deletion human described in 8 21 varients have stronger vitro stability and the biological activity of Geng Gao.
11. the biological property that claim 1 possessed is more suitable for being used for treatment or prevention of obesity, mellitus, high sugar or hyperlipidaemia as a kind of drug regimen.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105396740A CN102464712A (en) | 2010-11-11 | 2010-11-11 | Deletion human fibroblast growth factor 21 variant and conjugate thereof |
| PCT/CN2011/071948 WO2012062078A1 (en) | 2010-11-11 | 2011-03-18 | N-terminal deletion variant of human fibroblast growth factor 21 and conjugate thereof |
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| CN106220724A (en) * | 2016-09-13 | 2016-12-14 | 河南师范大学 | Human fibroblastic growth factor 21 recombiant protein and its preparation method and application |
| CN106397607A (en) * | 2016-09-13 | 2017-02-15 | 河南师范大学 | Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases |
| CN106432509A (en) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | RhFGF-21(recombinant human fibroblast growth factor-21) fusion protein capable of treating metabolic diseases as well as preparation method and application of rhFGF-21 fusion protein |
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| CN106220724A (en) * | 2016-09-13 | 2016-12-14 | 河南师范大学 | Human fibroblastic growth factor 21 recombiant protein and its preparation method and application |
| CN106397607A (en) * | 2016-09-13 | 2017-02-15 | 河南师范大学 | Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases |
| CN106432509A (en) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | RhFGF-21(recombinant human fibroblast growth factor-21) fusion protein capable of treating metabolic diseases as well as preparation method and application of rhFGF-21 fusion protein |
| CN106432509B (en) * | 2016-09-13 | 2019-05-21 | 河南师范大学 | A kind of 21 fusion protein of recombinant human fibroblast growth factor and its preparation method and application for treating metabolic disease |
| CN106220724B (en) * | 2016-09-13 | 2019-10-11 | 河南师范大学 | 21 recombinant protein of human fibroblastic growth factor and its preparation method and application |
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| WO2012062078A1 (en) | 2012-05-18 |
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