CN108404119A - The preparation of FGF-21 analogs and the application in thrombus treatment - Google Patents
The preparation of FGF-21 analogs and the application in thrombus treatment Download PDFInfo
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- CN108404119A CN108404119A CN201810427139.2A CN201810427139A CN108404119A CN 108404119 A CN108404119 A CN 108404119A CN 201810427139 A CN201810427139 A CN 201810427139A CN 108404119 A CN108404119 A CN 108404119A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses a kind of preparation of 1 (FGF 21) analog of fibroblast growth factor 2 and the applications in thrombus treatment.The present invention provides 1 analog of fibroblast growth factor 2 it is following it is any in application：(A) it prepares for treating and/or the product of pre- preventing thrombosis relevant disease；(B) treatment and/or pre- preventing thrombosis relevant disease.Present invention discover that 1 analog of fibroblast growth factor 2 (i.e. fusion protein made of fibroblast growth factor 21 and the fusion of class elastin laminin) can effectively inhibit the generation of thrombus, drug therapy and/or pre- preventing thrombosis are can be used as, there is substantial worth for the treatment and prevention of thrombus.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to a kind of fibroblast growth factor-21 (FGF-21) is similar
The preparation of object and the application in thrombus treatment.
Background technology
The formation of thrombus is under physiology or pathologic condition, and blood coagulation system is activated to varying degrees, and blood constituent exists
It is formed by a kind of semisolid grumeleuse after being agglomerated in blood circulation.Thrombus is by insoluble fibrin, the blood platelet of deposition, accumulation
Leucocyte and be absorbed in red blood cell composition, cause lumen of vessels partially or completely to block, to cause blood flow unsmooth, make phase
Position blood supply disorder is answered, respective organization or organ ischemia, anoxic, necrosis can be caused to influence organ dysfunction or even jeopardize life
Life.
Thrombus can be happened at the intravascular of any position, belong to cardiovascular and cerebrovascular disease.The annual cardiovascular patient in the whole world is more
Up to several ten million；The generation number of China's thrombotic diseases is also apparent in recent years to be risen, and recurrence rate is higher after treatment.It is made by thrombus
At the infraction death rate may be up to 30%.The Disease Spectrum of cardiovascular disease increasingly aggravates, it has also become great public health problem.
Thrombus is the prefered method for treating the diseases such as myocardial infarction, cerebral thrombus, pulmonary embolism.But current thrombolytics, as urokinase,
Streptokinase, rt-PA etc., there are half-life short, side effect is big, all kinds of disadvantages such as expensive, because
This finds safe, good effect, economic and practical thrombolytic drug has a very important significance.
Under normal circumstances, there are the blood coagulation systems of mutual antagonism and anticoagulation system to be in equilibrium state in blood.If solidifying
Occur insufficiency of accommodation or unsuitable activation in blood and anti-coagulation process, will result in thrombosis.In being divided into coagulation process
Source property and exogenous two links, ultimately generate fibrin ferment.Fibrinogen (Fib) is under thrombin action through hydrolyzing, assembling
Etc. processes formed fibrin, form blood clot with platelet aggregation.Inflammation is interacted with blood coagulation by multiple links, is made
Original blood coagulation and anti-freezing, fibrinolytic and anti-fibrinolytic, inflammation and it is anti-inflammatory between balance be broken, form a kind of grade amplified automatically
Join effect.CRP can cause human endothelial cells to synthesize inflammatory mediator (IL-6) and coagulation factor (FV II), so as to cause thrombus shape
At.On the other hand, blood flow velocity locally or systemically slow down, be partially formed vortex can promote thrombosis and blood coagulation；It is red
Cell number (RBC) and platelet count (PLT) increase and blood viscosity can be caused to increase, and so that blood flow is slowed down, and then lead to thrombosis.
Fibrinolytic is internal important anticoagulation process, it is a kind of protectiveness physiological reaction of body.Under normal circumstances, the fibre in blood plasma
Lyase be in the form of inactive plasminogen (PLG) existing for.PLG issues unboiled water solution in activator effect, takes off one section of peptide
Chain and activate into fibrinolysin.The fibrinolytic of body blood plasma inhibits with plasminogen activator (tPA) and plasminogen activator
The interactively of agent (PAI) is close, and under physiological status, tPA is difficult to activate PLG, but when fiber protein content reaches a certain level
When, tPA effects can be enhanced, PAI can not inactivate tPA activity, cause fibrinolytic system to activate, inhibition thrombosis.Therefore, enhancing is fine
The activity of molten system and inhibition inflammatory reaction are the key that treatment thrombus target spots.
Invention content
The object of the present invention is to provide a kind of preparation of fibroblast growth factor-21 (FGF-21) analog and in blood
Application in bolt treatment.
In a first aspect, claimed fibroblast growth factor-21 analog it is following it is any in application：
(A) it prepares for treating and/or the product of pre- preventing thrombosis relevant disease；
(B) treatment and/or pre- preventing thrombosis relevant disease.
Second aspect, claimed fibroblast growth factor-21 analog it is following it is any in application：
(a) product for inhibiting thrombus to occur is prepared, or inhibits thrombus；
(b) product for inhibiting thrombus degree is prepared, or inhibits thrombus degree；
(c) product for reducing fibrinogen level in blood plasma is prepared, or reduces fibrinogen level in blood plasma；
(d) it prepares for reducing red blood cell number in blood and/or the product of platelet count, or reduces red blood cell number in blood
And/or platelet count；
(e) product for inhibiting inflammatory reaction is prepared, or inhibits inflammatory reaction；
(f) product for enhancing molten fine ability, or the molten fine ability of enhancing are prepared.
The third aspect, claimed fibroblast growth factor-21 analog it is following it is any in application：
(g) prepare Activated partial thromboplastin time (APTT) for extending blood plasma and/or prothrombin time (PT) and/
Or the product of thrombin time (TT), or extend the Activated partial thromboplastin time (APTT) and/or prothrombin time of blood plasma
(PT) and/or thrombin time (TT)；
(h) it prepares for reducing the horizontal of c reactive protein in blood (CRP) and/or inflammatory factor and/or coagulation factor
Product, or reduce c reactive protein (CRP) and/or inflammatory factor and/or coagulation factor level in blood；
Further, the inflammatory factor concretely IL-6；The coagulation factor concretely FV II.
(i) it prepares in the product or elevating blood for tissue-type plasminogen activator (tPA) level in elevating blood
Tissue-type plasminogen activator (tPA) is horizontal；
(j) product for reducing plasminogen activator inhibitor in blood (PAI) level is prepared, or is reduced in blood
Plasminogen activator inhibitor (PAI) is horizontal.
In first aspect, second aspect and the third aspect, the fibroblast growth factor-21 analog is served as reasons
Fusion protein made of fibroblast growth factor-21 and the fusion of class elastin laminin.
Further, the fibroblast growth factor-21 is located at N-terminal；The class elastin laminin is located at C-terminal.
Further, the class elastin laminin can be protein shown in following (A1)-(A3) is any：
(A1) amino acid sequence is protein shown in 184-283 of SEQ ID No.1；
(A2) amino acid sequence shown in 184-283 of SEQ ID No.1 is residual by one or several amino acid
It the substitution of base and/or lacks and ors add and protein with the same function；
(A3) with (A1)-(A2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% with
Upper, 85% or more or 80% or more homology and protein with the same function.
The fibroblast growth factor-21 can be protein shown in following (B1)-(B3) is any：
(B1) amino acid sequence is protein shown in 1-183 of SEQ ID No.1；
(B2) amino acid sequence shown in 1-183 of SEQ ID No.1 is passed through into one or several amino acid residues
Substitution and/or lack and or add and protein with the same function；
(B3) with (B1)-(B2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% with
Upper, 85% or more or 80% or more homology and protein with the same function.
More specifically, the fibroblast growth factor-21 analog can be egg shown in following (C1)-(C4) is any
White matter：
(C1) amino acid sequence is protein shown in SEQ ID No.1；
(C2) by amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues substitution and/or
It lacks and ors add and protein with the same function；
(C3) with (C1)-(C2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% with
Upper, 85% or more or 80% or more homology and protein with the same function；
(C4) fusion obtained after the N-terminal of protein defined by any in (C1)-(C3) and/or C-terminal connection label
Albumen.
In first aspect, second aspect and the third aspect, the fibroblast growth factor-21 analog can be according to
The method included the following steps is prepared：
(a1) encoding gene of the fibroblast growth factor-21 analog is cloned into prokaryotic expression carrier, so
Expression obtains the fusion protein afterwards；
(a2) fusion protein is collected, the Desmocyte growth factor is obtained after carrying out temperature Reversible Cycle three times
Sub -21 analogs；
The realization method of " the temperature Reversible Cycle three times " is as follows：1) fusion protein is taken, 3-5 times is carried out with PBS
Volume dilution is placed in and is set as in 37 DEG C of thermostat, and high speed freezing centrifuge is preheated to 37 DEG C, after temperature stabilization, such as
19309g centrifuges 15min；2) it discards supernatant, centrifugation gained precipitation is resuspended with isometric PBS, is blown and beaten repeatedly to solution with suction pipe
Uniform, then ultrasonic, setting condition is power 1200w, working time 1s, interval time 3s, and work times 60 times surpass repeatedly
During which sound keeps bath temperature to be less than 20 DEG C until solution is clarified completely；3) 4 DEG C of refrigerators place 30min, and then high speed freezes
Centrifuge low-temperature centrifugation, setting condition are that 4 DEG C of 19309g centrifuge 20min, discard precipitation；4) step is substituted with supernatant obtained by step 3)
It is rapid 1) in the fusion protein repeat step 1) -3) twice.
Further, the PBS is PBS buffer solution (pH 7.2).Specifically, the solvent of the PBS be water, solute and its
Concentration is as follows：NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L。
In step (a1), the prokaryotic expression carrier is specially pET30a carriers.In the present invention, specially by it is described at
The encoding gene of -21 analog of fibroblast growth factor be cloned into pET30a carriers restriction enzyme site Nde I and BamH I it
Between.In the present invention, the host strain for being used for prokaryotic expression is Escherichia coli, specially e. coli bl21 (DE3).It is more specific
, induce the condition of prokaryotic expression to be：25 DEG C of induction 10h of the final concentration of 0.25mmol/L of IPTG.
Fourth aspect, a kind of claimed method preparing fibroblast growth factor-21 analog.
The method provided by the present invention for preparing fibroblast growth factor-21 analog, before specific steps can be found in
Described in text.
In previously described several respects, the encoding gene of the fibroblast growth factor-21 analog is by institute
It states and merges base made of the encoding gene fusion of the encoding gene and the class elastin laminin of fibroblast growth factor-21
Cause.
Further, the encoding gene of the class elastin laminin can be (D1)-(D3) in it is any shown in DNA molecular：
(D1) DNA molecular shown in 550-849 of SEQ ID No.2；
(D2) hybridize under strict conditions with (D1) DNA molecular limited and encode the DNA molecular of the class elastin laminin；
(D3) DNA sequence dna limited with (D1) or (D2) has 99% or more, 95% or more, 90% or more, 85% or more
Or 80% or more homology and the coding class elastin laminin DNA molecular.
The encoding gene of the fibroblast growth factor-21 can be (E1)-(E3) in it is any shown in DNA molecular：
(E1) DNA molecular shown in 1-549 of SEQ ID No.2；
(E2) hybridize under strict conditions with (E1) DNA molecular limited and encode the fibroblast growth factor-
21 DNA molecular；
(E3) DNA sequence dna limited with (E1) or (E2) has 99% or more, 95% or more, 90% or more, 85% or more
Or 80% or more homology and the coding fibroblast growth factor-21 DNA molecular；
Further, the encoding gene of the fibroblast growth factor-21 analog concretely (F1)-(F3)
In it is any shown in DNA molecular：
(F1) DNA molecular shown in SEQ ID No.2；
(F2) hybridize under strict conditions with (F1) DNA molecular limited and encode the fibroblast growth factor-
The DNA molecular of 21 analogs；
(F3) DNA sequence dna limited with (F1) or (F2) has 99% or more, 95% or more, 90% or more, 85% or more
Or 80% or more homology and the coding fibroblast growth factor-21 analog DNA molecular.
Above-mentioned stringent condition can be with 6 × SSC, and the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC,
It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
5th aspect, the claimed product at least one of following function, active constituent are above
The fibroblast growth factor-21 analog；
(A) treatment and/or pre- preventing thrombosis relevant disease；
(a) inhibit thrombus；
(b) inhibit thrombus degree；
(c) fibrinogen level in blood plasma is reduced；
(d) red blood cell number and/or platelet count in blood are reduced；
(e) inhibit inflammatory reaction；
(f) enhance molten fine ability；
(g) Activated partial thromboplastin time (APTT) of extension blood plasma and/or prothrombin time (PT) and/or fibrin ferment
Time (TT)；
(h) level of c reactive protein (CRP) and/or inflammatory factor and/or coagulation factor in blood is reduced；
Further, the inflammatory factor concretely IL-6；The coagulation factor concretely FV II.
(i) tissue-type plasminogen activator (tPA) is horizontal in elevating blood；
(j) it is horizontal to reduce plasminogen activator inhibitor (PAI) in blood.
In previously described several respects, the product can be drug.
The drug further includes other pharmaceutically acceptable carriers or auxiliary material.In the drug, in addition to it is described activity at
Exceptionally, the excipient pharmaceutically allowed, filler, sorbefacient, surfactant, absorption carrier, synergist can be added
With additive etc..The administration form of the drug can be injection (such as pulvis, aqua, finish).Ability all can be used in the preparation
Field technique personnel know common preparation method and obtain.The administration route of the drug can be subcutaneously injected, intravenous injection or
Intramuscular injection.
Following biomaterial and its application is also claimed in 6th aspect, the present invention.
The biomaterial is following any：(b1) volume of previously described fibroblast growth factor-21 analog
Code gene；(b2) contain recombinant vector, recombinant bacterium or the expression cassette of the encoding gene.
The application be the biomaterial it is following it is any in application：
(A) it prepares for treating and/or the product of pre- preventing thrombosis relevant disease；
(a) product for inhibiting thrombus to occur is prepared；
(b) product for inhibiting thrombus degree is prepared；
(c) product for reducing fibrinogen level in blood plasma is prepared；
(d) it prepares for reducing red blood cell number in blood and/or the product of platelet count；
(e) product for inhibiting inflammatory reaction is prepared；
(f) product for enhancing molten fine ability is prepared；
(g) prepare Activated partial thromboplastin time (APTT) for extending blood plasma and/or prothrombin time (PT) and/
Or the product of thrombin time (TT)；
(h) it prepares for reducing the horizontal of c reactive protein in blood (CRP) and/or inflammatory factor and/or coagulation factor
Product；
Further, the inflammatory factor concretely IL-6；The coagulation factor concretely FV II.
(i) product for tissue-type plasminogen activator (tPA) level in elevating blood is prepared；
(j) product for reducing plasminogen activator inhibitor in blood (PAI) level is prepared.
In previously described several respects, the product can be drug.
In the specific implementation mode of the present invention, previously described fibroblast growth factor-21 analog is specially
DNA fragmentation shown in sequence SEQ ID No.2 is replaced to the small fragment between restriction enzyme site Nde I and the BamH I of pET30a carriers
The protein containing amino acid sequence shown in SEQ ID No.1 expressed afterwards.
Present invention discover that fibroblast growth factor-21 analog (i.e. fibroblast growth factor-21 and class elasticity
Fusion protein made of protein fusion) generation that can effectively inhibit thrombus, drug therapy and/or pre- preventing thrombosis are can be used as, it is right
There is substantial worth in the treatment and prevention of thrombus.
Description of the drawings
Fig. 1 is the polyacrylate hydrogel electrophoretogram of FGF-21 analog solution.
HE coloration results of the Fig. 2 by the tail tissue slice taken after Ca/LPS joint modelings.A is Mouse tail artery, and B is
Mouse tail vein.
Fig. 3 is APTT, PT and TT testing result of blood plasma.1：Normal group；2：Model control group；3：Low dose of FGF21
Amount group；4：FGF21 high dose groups.
Fig. 4 is Fib content detection results.
Fig. 5 is RBC and PLT count detection results.1：Normal group；2：Model control group；3：FGF21 low dose groups；
4：FGF21 high dose groups.
Fig. 6 is the expression of II gene of CRP, IL-6 and FV, the expression of II albumen of CRP, IL-6 and FV.A is
The result of Real-time PCR detection CRP genes.B is the result that Real-time PCR detect IL-6 genes.C is Real-
The result of time PCR detection II genes of FV.D is the result that ELISA detects CRP albumen.E is the knot that ELISA detects IL-6 albumen
Fruit.F is the result that ELISA detects II albumen of FV.1：Normal group；2：Model control group；3：FGF21 low dose groups；4：
FGF21 high dose groups.
Fig. 7 is the expression of rat tPA genes, the expression of tPA albumen.A is that Real-time PCR detect rat
The result of tPA genes.B is the result that ELISA detects rat tPA protein contents.1：Normal group；2：Model control group；3：
FGF21 low dose groups；4：FGF21 high dose groups.
Fig. 8 is the expression of mouse tPA genes, the expression of tPA albumen.A is that Real-time PCR detect mouse
The result of tPA genes.B is the result that ELISA detects mouse tPA protein contents.1：Normal group；2：Model control group；3：
FGF21 low dose groups；4：FGF21 high dose groups.
Fig. 9 is the expression of P of Rats AI genes, the expression of PAI albumen.A is that Real-time PCR detect rat
The result of PAI genes.B is the result that ELISA detects P of Rats AI protein contents.1：Normal group；2：Model control group；3：
FGF21 low dose groups；4：FGF21 high dose groups.
Figure 10 is the expression of mouse PAI genes, the expression of PAI albumen.A is that Real-time PCR detections are small
The result of mouse PAI genes.B is the result that ELISA detects mouse PAI protein contents.1：Normal group；2：Model control group；
3：FGF21 low dose groups；4：FGF21 high dose groups.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Prokaryotic expression carrier is pET30a：Novagen products
Escherichia coli Rosetta (DE3)：Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.) CD801.
Male Wistar rat：Harbin Veterinary Medicine Inst., China Academy of Agriculture's experimental animal center.
Male mouse of kunming：Harbin Veterinary Medicine Inst., China Academy of Agriculture's experimental animal center.
Ferric trichloride：Sigma-Aldrich companies；CAS 7705-08-0.
Carrageenan(Ca)：Sigma-Aldrich companies；CAS 9000-07-1.
Lipopolysaccharides(LPS)：Sigma-Aldrich companies.
PBS buffer solution (pH 7.2)：Solvent is water, and solute and its concentration are as follows：NaCl 137mmol/L, KCl
2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L。
The preparation of embodiment 1, FGF-21 analogs
One, the sequence design of FGF-21 analogs
By NCBI retrieval income earner FGF21 sequences and class elastin laminin sequence assembly, class elastin laminin amino acid sequence exists
Article (Urry DW etc., Biochim Biophys Acta.1974Dec 18；371(2):It is carried out on the basis of 597-602)
Optimization, particular sequence are that 8 VPGAG pentapeptide repetitive sequences add 12 VPGVG pentapeptide repetitive sequences.Final design obtains FGF-21
Analog (abbreviation FGF-21 analogs) sequence of albumen, (1-183 are FGF-21 albumen sequences as shown in SEQ ID No.1
Row；184-283 are class elastin laminin sequence)；The encoding gene of FGF-21 analogs (1- as shown in SEQ ID No.2
549 are FGF-21 protein coding gene sequences；The 550-849 coding gene sequences for class elastin laminin).
Two, the structure of recombinant plasmid
1, gene order (" the catatg+SEQ ID No.2+ that step 1 of the synthesis both ends with restriction enzyme site designs
Ggatcc "), by restriction enzyme NdeI and the BamHI digestion of obtained synthetic plasmid, recycle digestion products.Expression by
Fusion protein made of fibroblast growth factor -21 is merged with class elastin laminin.
2, with restriction enzyme NdeI and BamHI double digestion prokaryotic expression carrier pET30a, digestion products are recycled.
3, the digestion products of step 1 are connected with the carrier framework of step 2, obtains recombinant plasmid pET30a-FGF-21-
ELP.The structure of recombinant plasmid pET30a-FGF-21-ELP is described as：In the restriction enzyme site NdeI of prokaryotic expression carrier pET30a
The recombinant plasmid of DNA fragmentation shown in SEQ ID No.2 is inserted between BamHI.
4, in recombinant plasmid pET30a-FGF-21-ELP, the encoding gene and class elastin gene of FGF-21 mature peptides
Fusion forms fusion, and (fusion protein is followed successively by FGF-21 mature peptides and class elasticity egg to expressed fusion protein from N-terminal to C-terminal
In vain).
The expection molecular weight of FGF-21- class elastin laminins is that (reckoning molecular weight is 32KD, SDS-PAGE electrophoresis showeds to 32KD
Molecular weight is 35KD or so).
Three, the preparation and purification of FGF-21- classes elastin laminin
1, recombinant plasmid pET30a-FGF-21-ELP is imported into e. coli bl21 (DE3), obtains recombinant bacterium.
2, the single bacterium colony for the recombinant bacterium that step 1 obtains is seeded in 5mL LB culture mediums, 37 DEG C, 120rpm shaken cultivations
Then 10h takes bacterium solution, with 1:100 volume ratio be inoculated in 500mL cards containing 50mg/mL receive mycin LB culture mediums in, 37 DEG C,
120rpm shaken cultivation 2h, at this time OD600nmIt is 0.5 or so.
3, in the bacterium solution obtained to step 2 be added IPTG and make its a concentration of 0.25mmol/L with induced (25 DEG C,
60rpm shaken cultivation 10h), then 4 DEG C, 4000rpm centrifugation 30min collect thalline.
4, the thalline for taking step 3 to obtain, carries out ultrasonication (8-12min, work 1s stop 1s), 4 DEG C, 12000rpm from
The heart collects supernatant and precipitation respectively.
Supernatant and precipitation are subjected to 12%SDS-PAGE electrophoretic analysis respectively.
5, the supernatant for taking step 4 to obtain carries out temperature Reversible Cycle three times, specific as follows：
(1) fusion protein is taken, four times is diluted with PBS, is placed in and is set as in 37 DEG C of thermostat.High speed refrigerated centrifuge
Machine is preheated to 37 DEG C, is centrifuged after temperature stabilization, and setting condition is 19309g 15min.
(2) discard supernatant, centrifugation gained precipitation be resuspended with isometric PBS, blown and beaten repeatedly with suction pipe it is uniform to solution, so
Ultrasonic afterwards, setting condition is power 1200w, working time 1s, interval time 3s, during which work times 60 times keep water-bath temperature
Degree is less than 20 DEG C, repeatedly ultrasonic, until solution is clarified completely.
(3) 4 DEG C of refrigerators of solution that step (2) obtains are placed into 30min.Item is arranged in high speed freezing centrifuge low-temperature centrifugation
Part is 4 DEG C, 19309g, 20min.Discard precipitation.
(4) supernatant for obtaining step (3) again repeat step (1) to step (3) twice, obtained supernatant solution is
FGF-21- class elastin laminin solution.
The polyacrylate hydrogel electrophoretogram of FGF-21- class elastin laminin solution is shown in that (swimming lane 1 is molecular weight marker, swimming lane 2 to Fig. 1
For FGF-21- class elastin laminins solution).Recycling purpose band (35KD or so) simultaneously carries out N-terminal sequencing, the results showed that, 15 before N-terminal
Amino acid residue such as SEQ ID No.1 are from shown in the 1st to 15 amino acids residue of N-terminal.
The therapeutic effect of embodiment 2, FGF-21- classes elastin laminin to thrombus
Experimental animal is six week old detergent male Wistar rats, six week old cleaning grade male mouse of kunming.
The FGF-21- class elastin laminin solution prepared using embodiment 1 is tested.
1, rat experimental design
Rat is randomly divided into 4 groups, every group 15, carries out following parallel processing respectively after adaptability is raised 1 week：
Model control group：It tests the 1-7 days, every morning 8:00 or so is subcutaneously injected PBS buffer solution, single injection volume
For 0.2mL；Test the morning 9 on the 7th:00 or so 10% chloral hydrate anesthesia of intraperitoneal injection；Detach left common carotid
2cm, small pieces plastic film (3 × 1.5cm) is set in lower section, and for protecting tissues surrounding vascular, suction is had 2.16mol/L FeCl3It is molten
The small filter paper (1 × 1cm) of liquid is spread in exposed artery surface, and filter paper will be close to vascular wall；
FGF21 low dose groups：It tests the 1-7 days, every morning 8:00 or so intraperitoneal injection FGF-21 maturations peptide solution (is used
PBS buffer solution adjusts albumen concentration), bolus doses are 1mg FGF-21 mature peptides (in terms of total protein)/kg weight, single
Volume injected is 0.2mL；Test the morning 9 on the 7th:00 or so 10% chloral hydrate anesthesia of intraperitoneal injection；Separation left side neck
Total artery 2cm, small pieces plastic film (3 × 1.5cm) is set in lower section, and for protecting tissues surrounding vascular, suction is had 2.16mol/L
FeCl3The small filter paper (1 × 1cm) of solution is spread in exposed artery surface, and filter paper will be close to vascular wall；
FGF21 high dose groups：It tests the 1-7 days, every morning 8:00 or so intraperitoneal injection FGF-21 maturations peptide solution (is used
PBS buffer solution adjusts albumen concentration), bolus doses are 2mg FGF-21 mature peptides (in terms of total protein)/kg weight, single
Volume injected is 0.2mL；Test the morning 9 on the 7th:00 or so 10% chloral hydrate anesthesia of intraperitoneal injection；Separation left side neck
Total artery 2cm, small pieces plastic film (3 × 1.5cm) is set in lower section, and for protecting tissues surrounding vascular, suction is had 2.16mol/L
FeCl3The small filter paper (1 × 1cm) of solution is spread in exposed artery surface, and filter paper will be close to vascular wall；
Normal group：It tests the 1-7 days, every morning 8:00 or so is subcutaneously injected PBS buffer solution, single injection volume
The morning 9 on the 7th is tested for 0.2mL:00 or so 10% chloral hydrate anesthesia of intraperitoneal injection；Detach left common carotid
2cm, small pieces plastic film (3 × 1.5cm) is set in lower section, for protecting tissues surrounding vascular, will inhale the small filter paper for having PBS solution
(1 × 1cm) is spread in exposed artery surface, and filter paper will be close to vascular wall；
Remove filter paper (timing since applying filter paper) after effect 90s, cut the blood vessel of thrombus happening part, then from the right side
Side arteria carotis communis takes blood, 4 DEG C of centrifugation 15min of 3000r/min to take supernatant blood plasma for detecting；
2, mouse experiment designs
Mouse is randomly divided into 4 groups, every group 15, carries out following parallel processing respectively after adaptability is raised 1 week：
Model control group：It tests the 1-7 days, one morning 8:00 or so intraperitoneal injection Ca solution, bolus doses
For 3mg/kg weight, single injection volume is 0.3mL；Tail vein injection LPS after 24 hours, bolus doses 50ug/kg
Weight；
FGF21 low dose groups：It tests the 1-7 days, one morning 8:00 or so intraperitoneal injection Ca solution, single injection agent
Amount is 3mg/kg weight, and single injection volume is 0.3mL；Tail vein injection LPS after 24 hours, bolus doses 50ug/
Kg weight；It tests the 1-7 days, every morning 9:00 or so is subcutaneously injected FGF-21 maturations peptide solution (adjusts egg with PBS buffer solution
White concentration), 1mg FGF-21 mature peptides (in terms of total protein)/kg weight, single injection volume is 0.2mL；
FGF21 high dose groups：It tests the 1-7 days, one morning 8:00 or so intraperitoneal injection Ca solution, single injection agent
Amount is 3mg/kg weight, and single injection volume is 0.3mL；Tail vein injection LPS after 24 hours, bolus doses 50ug/
Kg weight；It tests the 1-7 days, every morning 9:00 or so is subcutaneously injected FGF-21 maturations peptide solution (adjusts egg with PBS buffer solution
White concentration), 2mg FGF-21 mature peptides (in terms of total protein)/kg weight, single injection volume is 0.2mL；
Normal group：It tests the 1-7 days, one morning 8:00 or so is subcutaneously injected PBS buffer solution, single injection body
Product is 0.3mL, and tail vein injection PBS buffer solution after 24 hours, bolus doses are 50ug/kg weight；It tests the 1-7 days,
Every morning 9:00 or so is subcutaneously injected PBS buffer solution, and single injection volume is 0.2mL；
It tests the 7th day, mouse is put to death in anesthesia, takes Mouse whole blood and tail tissue respectively.
3, detection and result
Microexamination is carried out to the tail tissue slice taken after Ca/LPS joint modelings, as a result sees that (A is mouse tail to Fig. 2
Artery, B are mouse tail vein).As seen from the figure：Visible thrombosis in model control group Mouse tail artery and tail vein；With
Model control group is compared, FGF21 low dose group mouse thrombus degree significantly reduce, FGF21 high dose group mouse reached with just
It is horizontal similar in normal control group mice.The result shows that FGF-21 mature peptides can significantly inhibit the formation of thrombus.
Coagulation process is a series of enzymatic reaction, including endogenous blood coagulation system, external source blood coagulation system and common pathway.It is living
Change the activity of partial prothrombinase time (APTT) reaction intrinsic coagulation pathway, prothrombin time (PT) reacts extrinsic coagulation
The activity of approach；And thrombin time (TT) reaction is the two common pathway i.e. activity of fibrin ferment.Neck always moves on the right side of rat
Arteries and veins takes blood, CS2000i full automatic blood-coagulation instrument to measure APTT, PT and TT, as a result see Fig. 3.As seen from the figure：Model control group rat
APTT, PT and TT time shorten, i.e. blood coagulation system excessive activation；Compared with model control group, FGF21 low dose groups and FGF21
High dose group rat APTT, PT and TT time lengthening, i.e., play the role of directly inhibiting to Coagulation test.The result shows that FGF-21
Mature peptide can significantly inhibit the formation of thrombus.
The horizontal reverse of fibrinogen (Fib) has answered thrombus degree.Rat right carotid takes blood, CS2000i complete certainly
Dynamic Blood coagulation instrument measures fibrinogen, as a result sees Fig. 4.As seen from the figure：Model control group rat annulus proteinogen level is high, i.e. blood
Bolt degree is high；Compared with model control group, FGF21 low dose groups and FGF21 high dose group rat annulus proteinogen levels are low.Knot
Fruit shows that FGF-21 mature peptides can significantly inhibit thrombus degree.
Erythrocyte number (RBC) and blood smallest number (PLT) have substantial connection with blood viscosity and thrombosis.Rat Right
Side arteria carotis communis takes blood, XT1800i cellanalyzers to measure RBC and PLT, as a result see Fig. 5.As seen from the figure：Model control group
Rat RBC and PLT value are high, i.e., blood viscosity is high, and compared with model control group, FGF21 low dose groups and FGF21 high dose groups are big
Mouse RBC and PLT value significantly reduces.The result shows that FGF-21 mature peptides can significantly reduce whole blood viscosity, thrombosis is reduced.
There are cyberrelationship between inflammation and thrombosis, inflammation promotes thrombosis, while the product in thrombosis
Cause inflammation.C reactive protein (CRP) is used as a kind of acute phase protein, can cause human endothelial cells synthesis inflammatory mediator and coagulate
Blood factor, so as to cause thrombosis.Rat right carotid takes blood, and simultaneously reverse transcription is cDNA to extraction total serum IgE, is passed through
The expression of Real-time PCR detection CRP genes, inflammatory factor (IL-6) and coagulation factor (FV II) gene (uses
GAPDH genes are reference gene).Primer pair for detecting CRP genes is：5'-CCCCAATTCTCCCTCGAAT-3'；5'-
GGATGGATGGATGGATGGA-3'.Primer pair for detecting IL-6 genes is：5'-TAGTCCTTCCTACCCCAACTTCC-
3'；5'-TTGGTCCTTAGCCACTCCTTC-3'.Primer pair for detecting II genes of FV is：5'-
CGCAACTAAGGCAGTTCTATGT-3'；5'-GCTAGATCGTGGCTTTTCTTTCT-3'.For detecting drawing for reference gene
Object to for：5'-ATGATTCTACCCACGGCAAG-3'；5'-CTGGAAGATGGTGATGGGTT-3'.Rat right carotid
Blood, 4 DEG C of centrifugation 15min of 3000r/min is taken to take supernatant blood plasma, ELISA II contents of detection CRP, IL-6 and FV (big for detecting
The ELISA detection kit of mouse CRP, the ELISA detection kit for detecting rat IL-6 and for detecting rat FV's II
ELISA detection kit is R＆D Products, is operated by kit specification).Real-time PCR detection CRP genes
As a result see A in Fig. 6.The result of Real-time PCR detection IL-6 genes is shown in B in Fig. 6.Real-time PCR detect II bases of FV
The result of cause is shown in C in Fig. 6.The result of ELISA detection CRP albumen is shown in D in Fig. 6.The result of ELISA detection IL-6 albumen is shown in Fig. 6
Middle E.The result of ELISA detection II albumen of FV is shown in F in Fig. 6.By Fig. 6 it can be seen that：Model control group rats and mice CRP contents, IL-
6 and FV, II contents are very high, i.e., inflammatory reaction, which is activated, promotes the release of coagulation factor；Compared with model control group, FGF21 is low
Dosage group and FGF21 high dose group rat CRP contents, IL-6 and II contents of FV significantly reduce.The result shows that FGF-21 mature peptides
Inflammatory reaction, inhibition thrombosis can be significantly inhibited.
Tissue-type plasminogen activator (tPA) can promote plasminogen to be changed into fibrinolysin, promote thrombolysis.Rat
It is cDNA that right carotid, which takes blood, extraction total serum IgE and reverse transcription, and the expression water of tPA genes is detected by Real-time PCR
Flat (using GAPDH genes for reference gene).Primer pair for detecting rat tPA genes is：5'-
AGAGGGAGTGACAGTCTTTAGGC-3'；5'-GAGGATTGTGGGAGGATGG-3'.Real-time PCR detection rats tPA
The result of gene is shown in A in Fig. 7.Rat right carotid takes blood, 3000r/min4 DEG C of centrifugation 15min to take supernatant blood plasma,
(ELISA detection kit for detecting rat tPA is R＆D Products to ELISA detection tPA contents, by kit specification
Operation).As a result see B in Fig. 7.By Fig. 7 it can be seen that：Model control group rat tPA contents are low, i.e., fibrinolytic capacity is low；With model
Control group is compared, and FGF21 low dose groups and FGF21 high dose group rat tPA contents significantly increase.Mouse takes blood, extracts total serum IgE
And reverse transcription is cDNA, the expression that tPA genes are detected by Real-time PCR (uses GAPDH genes for internal reference base
Cause).Primer pair for detecting mouse tPA genes is：5'-TGACCAGGGAATACATGGGAG-3'；5'-
GTCTGCGTTGGCTCATCTCTG-3'.The result of Real-time PCR detection mouse tPA genes is shown in A in Fig. 8.Mouse takes blood,
4 DEG C of centrifugation 15min of 3000r/min take supernatant blood plasma, and (ELISA for detecting mouse tPA is detected ELISA detection tPA contents
Kit is R＆D Products, is operated by kit specification).As a result see B in Fig. 8.By Fig. 8 it can be seen that：Model control group
Mouse tPA contents are low, i.e., fibrinolytic capacity is low；Compared with model control group, FGF21 low dose groups and FGF21 high dose group mouse
TPA contents significantly increase.The result shows that FGF-21 mature peptides can significantly increase fibrinolytic capacity, anti thrombotic action is shown.
Plasminogen activator inhibitor (PAI) is main negative regulation substance in fibrinolytic system.Neck always moves on the right side of rat
Arteries and veins is taken a blood sample, and simultaneously reverse transcription is cDNA to extraction total serum IgE, and the expression that PAI genes are detected by Real-time PCR (uses
GAPDH genes are reference gene).Primer pair for detecting P of Rats AI genes is：5'-GTTCGCTTCACCCCTTCCAGA-
3'；5'-GAAATAGAGGGCGTTCACCAG-3'.The result of Real-time PCR detection PAI genes is shown in A in Fig. 9.Rat Right
Side arteria carotis communis blood sampling, 4 DEG C of centrifugation 15min of 3000r/min, takes supernatant blood plasma, ELISA detection PAI contents (big for detecting
The ELISA detection kit of mouse PAI is R＆D Products, is operated by kit specification).As a result see B in Fig. 9.It can by Fig. 9
To see：Model control group P of Rats AI contents are high, i.e., fibrinolytic capacity is low；Compared with model control group, FGF21 low dose groups and
FGF21 high dose group P of Rats AI contents significantly reduce.Mouse is taken a blood sample, and simultaneously reverse transcription is cDNA to extraction total serum IgE, passes through Real-
The expression (using GAPDH genes for reference gene) of time PCR detection PAI genes.For detecting mouse PAI genes
Primer pair is：5'-TTCAGCCCTTGCTTGCCTC-3'；5'-ACACTTTTACTCCGAAGTCGGT-3'.Real-time PCR
Detect A in the result is shown in Figure 10 of PAI genes.Mouse is taken a blood sample, and 4 DEG C of centrifugation 15min of 3000r/min take supernatant blood plasma, ELISA inspections
Survey PAI contents (ELISA detection kit for detecting mouse PAI is R＆D Products, is operated by kit specification).
B in the result is shown in Figure 10.By Figure 10 it can be seen that：Model control group mouse PAI contents are high, i.e., fibrinolytic capacity is low；With model comparison
Group is compared, and FGF21 low dose groups and FGF21 high dose group mouse PAI contents significantly reduce.The result shows that FGF-21 mature peptides
Fibrinolytic function can be significantly increased, anti thrombotic action is shown.
<110>Harbin Bo'ao Biopharmaceutical Technology Development Co., Ltd.
<120>The preparation of FGF-21 analogs and the application in thrombus treatment
<130> GNCLN180967
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 283
<212> PRT
<213> Artificial sequence
<400> 1
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Ala Ser Arg Ser Val Pro Gly Ala Gly Val Pro Gly Ala
180 185 190
Gly Val Pro Gly Ala Gly Val Pro Gly Ala Gly Val Pro Gly Ala Gly
195 200 205
Val Pro Gly Ala Gly Val Pro Gly Ala Gly Val Pro Gly Ala Gly Val
210 215 220
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
225 230 235 240
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
245 250 255
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
260 265 270
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
275 280
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<213> Artificial sequence
<400> 2
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120
gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180
ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240
gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300
gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360
aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 420
ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 480
ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 540
tccagatctg tcccaggcgc cggcgtccca ggcgccggcg tcccaggcgc cggcgtccca 600
ggcgccggcg tcccaggcgc cggcgtccca ggcgccggcg tcccaggcgc cggcgtccca 660
ggcgccggcg tcccaggcgt cggcgtccca ggcgtcggcg tcccaggcgt cggcgtccca 720
ggcgtcggcg tcccaggcgt cggcgtccca ggcgtcggcg tcccaggcgt cggcgtccca 780
ggcgtcggcg tcccaggcgt cggcgtccca ggcgtcggcg tcccaggcgt cggcgtccca 840
ggcgtcggct aa 852
Claims (10)
1. fibroblast growth factor-21 analog it is following it is any in application：
(A) it prepares for treating and/or the product of pre- preventing thrombosis relevant disease；
(B) treatment and/or pre- preventing thrombosis relevant disease.
2. fibroblast growth factor-21 analog it is following it is any in application：
(a) product for inhibiting thrombus to occur is prepared, or inhibits thrombus；
(b) product for inhibiting thrombus degree is prepared, or inhibits thrombus degree；
(c) product for reducing fibrinogen level in blood plasma is prepared, or reduces fibrinogen level in blood plasma；
(d) prepare for reducing red blood cell number in blood and/or the product of platelet count, or reduce in blood red blood cell number and/
Or platelet count；
(e) product for inhibiting inflammatory reaction is prepared, or inhibits inflammatory reaction；
(f) product for enhancing molten fine ability, or the molten fine ability of enhancing are prepared.
3. application according to claim 2, it is characterised in that：Fibroblast growth factor-21 analog is being appointed as follows
Application in one：
(g) Activated partial thromboplastin time for extending blood plasma and/or prothrombin time and/or thrombin time are prepared
Product, or the Activated partial thromboplastin time of extension blood plasma and/or prothrombin time and/or thrombin time；
(h) the horizontal product for reducing c reactive protein in blood and/or inflammatory factor and/or coagulation factor, or drop are prepared
C reactive protein and/or inflammatory factor and/or coagulation factor are horizontal in low blood；
Further, the inflammatory factor is IL-6；The coagulation factor is FV II；
(i) it is fine to prepare tectotype in product or elevating blood for tissue-type plasminogen activator's level in elevating blood
Dissolved preferment activator is horizontal；
(j) product for reducing plasminogen activator inhibitor level in blood is prepared, or reduces plasminogen in blood
Activator inhibitor is horizontal.
4. according to any application in claim 1-3, it is characterised in that：The fibroblast growth factor-21 class
It is the fusion protein made of fibroblast growth factor-21 and the fusion of class elastin laminin like object.
5. application according to claim 4, it is characterised in that：The class elastin laminin is following (A1)-(A3) any institute
Show protein：
(A1) amino acid sequence is protein shown in 184-283 of SEQ ID No.1；
(A2) by amino acid sequence shown in 184-283 of SEQ ID No.1 by one or several amino acid residues
Replace and/or lacks and ors add and protein with the same function；
(A3) with (A1)-(A2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% or more,
85% or more or 80% or more homology and protein with the same function；And/or
The fibroblast growth factor-21 is protein shown in following (B1)-(B3) is any：
(B1) amino acid sequence is protein shown in 1-183 of SEQ ID No.1；
(B2) the taking by one or several amino acid residues by amino acid sequence shown in 1-183 of SEQ ID No.1
It generation and/or lacks and ors add and protein with the same function；
(B3) with (B1)-(B2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% or more,
85% or more or 80% or more homology and protein with the same function.
6. application according to claim 5, it is characterised in that：The fibroblast growth factor-21 analog is such as
Under (C1)-(C4) it is any shown in protein：
(C1) amino acid sequence is protein shown in SEQ ID No.1；
(C2) substitution by amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues and/or missing
And/or addition and protein with the same function；
(C3) with (C1)-(C2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% or more,
85% or more or 80% or more homology and protein with the same function；
(C4) fusion protein obtained after the N-terminal of protein defined by any in (C1)-(C3) and/or C-terminal connection label.
7. according to any application in claim 4-6, it is characterised in that：The fibroblast growth factor-21 class
It is prepared by a method comprising the following steps like object：
(a1) encoding gene of the fibroblast growth factor-21 analog is cloned into prokaryotic expression carrier, then table
Reach the fusion protein；
(a2) fusion protein is collected, the fibroblast growth factor-21 is obtained after carrying out temperature Reversible Cycle three times
Analog；
The realization method of " the temperature Reversible Cycle three times " is as follows：1) fusion protein is taken, 3-5 times of volume is carried out with PBS
Dilution, 37 DEG C of 19309g centrifuge 15min；2) centrifugation gained precipitation is resuspended with isometric PBS, then ultrasonic, setting condition is
During which power 1200w, working time 1s, interval time 3s keep bath temperature to be less than 20 DEG C until solution is clarified completely；3)4
DEG C 30min is placed, then 4 DEG C of 19309g centrifuge 20min；4) gained supernatant alternative steps 1 are centrifuged with step 3)) in described melt
Hop protein repeats step 1) -3) twice.
8. a kind of method preparing fibroblast growth factor-21 analog, includes the following steps：
(a1) by claim 4-6 it is any described in the encoding gene of fibroblast growth factor-21 analog be cloned into
Prokaryotic expression carrier, then expression obtain the fusion protein；
(a2) fusion protein is collected, the fibroblast growth factor-21 is obtained after carrying out temperature Reversible Cycle three times
Analog；
The realization method of " the temperature Reversible Cycle three times " is as follows：1) fusion protein is taken, 3-5 times of volume is carried out with PBS
Dilution, 37 DEG C of 19309g centrifuge 15min；2) centrifugation gained precipitation is resuspended with isometric PBS, then ultrasonic, setting condition is
During which power 1200w, working time 1s, interval time 3s keep bath temperature to be less than 20 DEG C until solution is clarified completely；3)4
DEG C 30min is placed, then 4 DEG C of 19309g centrifuge 20min；4) gained supernatant alternative steps 1 are centrifuged with step 3)) in described melt
Hop protein repeats step 1) -3) twice.
9. application according to claim 7 or method according to any one of claims 8, it is characterised in that：The fibroblast
The encoding gene of 1 analog of growth factor-2 is the encoding gene and the class bullet by the fibroblast growth factor-21
Property albumen encoding gene fusion made of fusion；
Further, the encoding gene of the class elastin laminin be (D1)-(D3) in it is any shown in DNA molecular：
(D1) DNA molecular shown in 550-849 of SEQ ID No.2；
(D2) hybridize under strict conditions with (D1) DNA molecular limited and encode the DNA molecular of the class elastin laminin；
(D3) with (D1) or (D2) limit DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or
The DNA molecular of 80% or more homology and the coding class elastin laminin；And/or
The encoding gene of the fibroblast growth factor-21 be (E1)-(E3) in it is any shown in DNA molecular：
(E1) DNA molecular shown in 1-549 of SEQ ID No.2；
(E2) hybridize under strict conditions with (E1) DNA molecular limited and encode the fibroblast growth factor-21
DNA molecular；
(E3) with (E1) or (E2) limit DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or
The DNA molecular of 80% or more homology and the coding fibroblast growth factor-21；
Further, the encoding gene of the fibroblast growth factor-21 analog is any shown in (F1)-(F3)
DNA molecular：
(F1) DNA molecular shown in SEQ ID No.2；
(F2) hybridize under strict conditions with (F1) DNA molecular limited and encode the fibroblast growth factor-21 class
Like the DNA molecular of object；
(F3) with (F1) or (F2) limit DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or
The DNA molecular of 80% or more homology and the coding fibroblast growth factor-21 analog.
10. biomaterial or its application, it is characterised in that：
The biomaterial is following any：
I the product) at least one of following function, active constituent are described in claim 1-8 is any into fiber
Growth factor-21 analog；
(A) treatment and/or pre- preventing thrombosis relevant disease；
(a) inhibit thrombus；
(b) inhibit thrombus degree；
(c) fibrinogen level in blood plasma is reduced；
(d) red blood cell number and/or platelet count in blood are reduced；
(e) inhibit inflammatory reaction；
(f) enhance molten fine ability；
(g) Activated partial thromboplastin time of extension blood plasma and/or prothrombin time and/or thrombin time；
(h) c reactive protein and/or inflammatory factor and/or the level of coagulation factor in blood are reduced；
(i) tissue-type plasminogen activator is horizontal in elevating blood；
(j) it is horizontal to reduce plasminogen activator inhibitor in blood；
II) claim 1-8 it is any described in fibroblast growth factor-21 analog encoding gene；
II) contain recombinant vector, recombinant bacterium or the expression cassette of the encoding gene；
The application is II) or III) described in biomaterial it is following it is any in application：
(A) it prepares for treating and/or the product of pre- preventing thrombosis relevant disease；
(a) product for inhibiting thrombus to occur is prepared；
(b) product for inhibiting thrombus degree is prepared；
(c) product for reducing fibrinogen level in blood plasma is prepared；
(d) it prepares for reducing red blood cell number in blood and/or the product of platelet count；
(e) product for inhibiting inflammatory reaction is prepared；
(f) product for enhancing molten fine ability is prepared；
(g) Activated partial thromboplastin time for extending blood plasma and/or prothrombin time and/or thrombin time are prepared
Product；
(h) the horizontal product for reducing c reactive protein in blood and/or inflammatory factor and/or coagulation factor is prepared；
(i) product for tissue-type plasminogen activator's level in elevating blood is prepared；
(j) product for reducing plasminogen activator inhibitor level in blood is prepared.
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Cited By (2)
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| CN113735977A (en) * | 2020-05-28 | 2021-12-03 | 江苏康缘瑞翱生物医药科技有限公司 | rhFGF21 fusion protein, polynucleotide for coding same, composition containing same and application thereof |
| WO2023077413A1 (en) * | 2021-11-02 | 2023-05-11 | 南京大学 | Use of protein sequence capable of binding to substrate in preparation of product for inhibiting fibrin assembly |
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