CN108404119B - Preparation of FGF-21 analogue and application thereof in thrombus treatment - Google Patents
Preparation of FGF-21 analogue and application thereof in thrombus treatment Download PDFInfo
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- CN108404119B CN108404119B CN201810427139.2A CN201810427139A CN108404119B CN 108404119 B CN108404119 B CN 108404119B CN 201810427139 A CN201810427139 A CN 201810427139A CN 108404119 B CN108404119 B CN 108404119B
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Abstract
The invention discloses a preparation method of a fibroblast growth factor-21 (FGF-21) analogue and application thereof in thrombus treatment. The invention provides an application of fibroblast growth factor-21 analogue in any one of the following applications: (A) preparing a product for treating and/or preventing diseases related to thrombus; (B) treating and/or preventing thrombosis related diseases. The invention discovers that the fibroblast growth factor-21 analogue (namely the fusion protein formed by fusing the fibroblast growth factor-21 and the elastin) can effectively inhibit the generation of thrombus, can be used as a medicament for treating and/or preventing thrombus, and has great value for treating and preventing the thrombus.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to preparation of a fibroblast growth factor-21 (FGF-21) analogue and application thereof in thrombus treatment.
Background
Thrombosis is a semi-solid clot formed by coagulation of blood components in circulating blood, which is activated to varying degrees by the coagulation system under physiological or pathological conditions. The thrombus consists of insoluble fibrin, deposited platelets, accumulated white blood cells and entrapped red blood cells, and partially or completely blocks a blood vessel cavity, so that blood flow is not smooth, the blood supply of corresponding parts is obstructed, and ischemia, hypoxia and necrosis of corresponding tissues or organs can be caused, so that the functions of the organs are influenced, and even the life is threatened.
Thrombus can occur in blood vessels of any part, and belongs to cardiovascular and cerebrovascular diseases. Cardiovascular patients are as many as several tens of millions worldwide each year; the number of people who suffer from thrombotic diseases in China also obviously increases in recent years, and the recurrence rate after treatment is high. Mortality from infarction by thrombus can be as high as 30%. The disease burden of cardiovascular diseases is increasing day by day and has become a major public health problem. Thrombolysis is the first choice for treating myocardial infarction, cerebral thrombosis, pulmonary embolism and other diseases. However, the existing thrombolytic agents, such as urokinase, streptokinase, recombinant tissue plasminogen activator, and the like, have various defects of short half-life, large side effect, high price and the like, so that the finding of thrombolytic drugs with high safety, good curative effect, economy and applicability is of great significance.
Normally, the mutually antagonistic coagulation system and anticoagulation system are present in the blood in an equilibrium state. If dysregulation or inappropriate activation occurs during coagulation and anticoagulation, thrombosis can result. The coagulation process is divided into an endogenous link and an exogenous link, and thrombin is finally generated. Fibrinogen (Fib) forms fibrin through hydrolysis, aggregation and other processes under the action of thrombin, and aggregates with platelets to form blood clots. Inflammation and blood coagulation interact through a plurality of links, so that the balance between original blood coagulation and anticoagulation, original fibrinolysis and anti-fibrinolysis, and original inflammation and inflammation resistance is broken, and an automatically amplified cascade effect is formed. CRP causes human endothelial cells to synthesize inflammatory mediators (IL-6) and blood coagulation Factors (FVI), thereby causing thrombosis. On the other hand, the blood flow speed is reduced locally or systemically, and the local eddy can promote the thrombosis and the blood coagulation; an increase in the number of Red Blood Cells (RBC) and Platelets (PLT) causes an increase in blood viscosity, slowing blood flow, and thus thrombosis. Fibrinolysis is an important anticoagulant process in the body, which is a protective physiological response of the body. Normally, plasmin in plasma is present in the inactive Plasminogen (PLG) form. PLG is hydrolyzed under the action of activator to remove a peptide chain and activate plasmin. The fibrinolytic activity of the body plasma is closely related to the action of plasminogen activator (tPA) and Plasminogen Activator Inhibitor (PAI), under the physiological state, the tPA is difficult to activate the PLG, but when the fibrin content reaches a certain degree, the action of the tPA can be enhanced, and the PAI can not inactivate the activity of the tPA, so that the activation of a fibrinolytic system is caused, and the formation of thrombus is inhibited. Therefore, enhancing the activity of the fibrinolytic system and inhibiting the inflammatory response are key targets for the treatment of thrombosis.
Disclosure of Invention
The invention aims to provide a preparation method of a fibroblast growth factor-21 (FGF-21) analogue and an application of the analogue in thrombus treatment.
In a first aspect, the invention claims the use of a fibroblast growth factor-21 analogue in any one of:
(A) preparing a product for treating and/or preventing diseases related to thrombus;
(B) treating and/or preventing thrombosis related diseases.
In a second aspect, the invention claims the use of a fibroblast growth factor-21 analogue in any one of:
(a) preparing a product for inhibiting thrombosis or inhibiting thrombosis;
(b) preparing a product for inhibiting the extent of thrombosis, or inhibiting the extent of thrombosis;
(c) preparing a product for reducing fibrinogen levels in plasma, or reducing fibrinogen levels in plasma;
(d) preparing a product for reducing the number of red blood cells and/or the number of platelets in blood, or reducing the number of red blood cells and/or the number of platelets in blood;
(e) preparing a product for inhibiting an inflammatory response, or inhibiting an inflammatory response;
(f) preparing a product for enhancing the fiber dissolving capacity, or enhancing the fiber dissolving capacity.
In a third aspect, the invention claims the use of a fibroblast growth factor-21 analogue in any one of:
(g) preparing a product for prolonging the Activated Partial Thrombin Time (APTT) and/or the Prothrombin Time (PT) and/or the Thrombin Time (TT) of plasma, or prolonging the Activated Partial Thrombin Time (APTT) and/or the Prothrombin Time (PT) and/or the Thrombin Time (TT) of plasma;
(h) preparing a product for reducing the level of C-reactive protein (CRP) and/or inflammatory and/or coagulation factors in blood, or reducing the level of C-reactive protein (CRP) and/or inflammatory and/or coagulation factors in blood;
further, the inflammatory factor may specifically be IL-6; the coagulation factor may specifically be FVI.
(i) Preparing a product for increasing the level of tissue plasminogen activator (tPA) in blood, or increasing the level of tissue plasminogen activator (tPA) in blood;
(j) for the manufacture of a product for use in reducing the level of or for reducing the level of a Plasminogen Activator Inhibitor (PAI) in blood.
In the first, second and third aspects, the fibroblast growth factor-21 analog is a fusion protein formed by fusing fibroblast growth factor-21 and elastin-like protein.
Further, the fibroblast growth factor-21 is located at the N-terminal; the elastin-like protein is located at the C-terminus.
Further, the elastin-like protein may be a protein as shown in any one of (a1) - (A3):
(A1) protein with the amino acid sequence shown as position 184-283 of SEQ ID No. 1;
(A2) a protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 184-283 position of SEQ ID No. 1;
(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence defined in any one of (A1) to (A2) and having the same function.
The fibroblast growth factor-21 may be a protein as shown in any one of (B1) - (B3):
(B1) protein with amino acid sequence shown in 1-183 of SEQ ID No. 1;
(B2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the 1 st to 183 th positions of SEQ ID No.1 and has the same function;
(B3) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in any one of (B1) to (B2) and having the same function.
More specifically, the fibroblast growth factor-21 analog may be a protein as shown in any one of (C1) to (C4):
(C1) protein with an amino acid sequence shown as SEQ ID No. 1;
(C2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;
(C3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (C1) to (C2) and having the same function;
(C4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (C1) to (C3).
In the first, second and third aspects, the fibroblast growth factor-21 analog may be prepared according to a method comprising the steps of:
(a1) cloning the coding gene of the fibroblast growth factor-21 analogue into a prokaryotic expression vector, and then expressing to obtain the fusion protein;
(a2) collecting the fusion protein, and performing reversible temperature cycle for three times to obtain the fibroblast growth factor-21 analogue;
the implementation mode of the three-time temperature reversible cycle is as follows: 1) taking the fusion protein, diluting with PBS by 3-5 times, placing in a thermostatic bath set at 37 ℃, preheating to 37 ℃ by a high-speed refrigerated centrifuge, and centrifuging for 15min at 19309g after the temperature is stable; 2) discarding the supernatant, resuspending and centrifuging the obtained precipitate with PBS of the same volume, repeatedly blowing and beating the precipitate with a straw until the solution is uniform, and then performing ultrasonic treatment under the conditions of 1200w of power, 1s of working time, 3s of interval time and 60 working times, repeatedly performing ultrasonic treatment until the solution is completely clarified, wherein the water bath temperature is kept lower than 20 ℃; 3) standing in a refrigerator at 4 deg.C for 30min, centrifuging at low temperature with a high-speed refrigerated centrifuge under the condition of 19309g at 4 deg.C for 20min, and discarding precipitate; 4) repeating steps 1) -3) twice with the supernatant obtained in step 3) instead of the fusion protein in step 1).
Further, the PBS was PBS buffer (pH 7.2). Specifically, the solvent of the PBS is water, and the solutes and the concentrations thereof are as follows: NaCl 137mmol/L, KCl2.7mmol/L, Na2HPO410mmol/L，KH2PO42mmol/L。
In the step (a1), the prokaryotic expression vector is specifically pET30a vector. In the invention, the coding gene of the fibroblast growth factor-21 analogue is specifically cloned between enzyme cutting sites Nde I and BamH I of pET30a vector. In the present invention, the host bacterium used for prokaryotic expression is Escherichia coli, specifically Escherichia coli BL21(DE 3). More specifically, the conditions for inducing prokaryotic expression are as follows: the final concentration of IPTG was 0.25mmol/L and induction was carried out at 25 ℃ for 10 h.
In a fourth aspect, the invention claims a method for preparing fibroblast growth factor-21 analogs.
The specific steps of the method for preparing the fibroblast growth factor-21 analogue provided by the invention can be seen in the above.
In the aspects described above, the gene encoding the fibroblast growth factor-21 analog is a fusion gene obtained by fusing the gene encoding the fibroblast growth factor-21 and the gene encoding the elastin-like protein.
Further, the encoding gene of the elastin-like protein can be any one of DNA molecules shown in (D1) - (D3):
(D1) a DNA molecule shown in position 550-849 of SEQ ID No. 2;
(D2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (D1) and encodes said elastin-like protein;
(D3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology to the DNA sequence defined in (D1) or (D2) and encoding the elastin-like protein.
The coding gene of the fibroblast growth factor-21 can be a DNA molecule shown in any one of (E1) to (E3):
(E1) a DNA molecule shown in the 1 st to 549 th sites of SEQ ID No. 2;
(E2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (E1) and encodes said fibroblast growth factor-21;
(E3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology to the DNA sequence defined in (E1) or (E2) and encoding the fibroblast growth factor-21;
further, the gene encoding the fibroblast growth factor-21 analog may specifically be a DNA molecule represented by any one of (F1) to (F3):
(F1) DNA molecule shown in SEQ ID No. 2;
(F2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (F1) and encodes said fibroblast growth factor-21 analog;
(F3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in (F1) or (F2) and encodes the fibroblast growth factor-21 analogue.
The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In a fifth aspect, the invention claims a product having at least one of the following functions, as an active ingredient, a fibroblast growth factor-21 analog as described hereinbefore;
(A) treating and/or preventing thrombosis related diseases;
(a) inhibiting thrombosis;
(b) inhibiting the extent of thrombosis;
(c) reducing fibrinogen levels in plasma;
(d) reducing the number of red blood cells and/or the number of platelets in the blood;
(e) inhibiting inflammatory response;
(f) the fiber dissolving capacity is enhanced;
(g) prolonging the Activated Partial Thrombin Time (APTT) and/or Prothrombin Time (PT) and/or Thrombin Time (TT) of plasma;
(h) reducing the level of C-reactive protein (CRP) and/or inflammatory and/or coagulation factors in the blood;
further, the inflammatory factor may specifically be IL-6; the coagulation factor may specifically be FVI.
(i) Increasing tissue plasminogen activator (tPA) levels in blood;
(j) reduces the level of Plasminogen Activator Inhibitor (PAI) in the blood.
In several of the aspects described above, the product may be a medicament.
The medicine also comprises other pharmaceutically acceptable carriers or auxiliary materials. In addition to the active ingredient, pharmaceutically acceptable excipients, fillers, absorption enhancers, surfactants, adsorption carriers, synergists, additives and the like may be added to the drug. The administration form of the medicine can be injection (such as powder, aqua and oil). The formulations can be obtained by conventional methods well known to those skilled in the art. The route of administration of the drug may be subcutaneous, intravenous or intramuscular.
In a sixth aspect, the invention also claims the following biomaterials and their uses.
The biological material is any one of the following materials: (b1) a gene encoding a fibroblast growth factor-21 analog as described above; (b2) a recombinant vector, a recombinant bacterium or an expression cassette containing the coding gene.
The application is the application of the biological material in any one of the following steps:
(A) preparing a product for treating and/or preventing diseases related to thrombus;
(a) preparing a product for inhibiting thrombosis;
(b) preparing a product for inhibiting the extent of thrombosis;
(c) preparing a product for reducing fibrinogen levels in plasma;
(d) preparing a product for reducing the number of red blood cells and/or the number of platelets in blood;
(e) preparing a product for inhibiting inflammatory reactions;
(f) preparing a product for enhancing the dissolving capacity of the fiber;
(g) preparing a product for prolonging the Activated Partial Thrombin Time (APTT) and/or the Prothrombin Time (PT) and/or the Thrombin Time (TT) of plasma;
(h) preparing a product for reducing the level of C-reactive protein (CRP) and/or inflammatory and/or coagulation factors in blood;
further, the inflammatory factor may specifically be IL-6; the coagulation factor may specifically be FVI.
(i) Preparing a product for increasing the level of tissue plasminogen activator (tPA) in blood;
(j) the preparation of a product for reducing the level of Plasminogen Activator Inhibitor (PAI) in the blood.
In several of the aspects described above, the product may be a medicament.
In a specific embodiment of the present invention, the fibroblast growth factor-21 analog described above is specifically a protein comprising the amino acid sequence shown in SEQ ID No.1, which is expressed by replacing a small fragment between the enzyme cutting sites Nde I and BamH I of the pET30a vector with a DNA fragment shown in SEQ ID No. 2.
The invention discovers that the fibroblast growth factor-21 analogue (namely the fusion protein formed by fusing the fibroblast growth factor-21 and the elastin) can effectively inhibit the generation of thrombus, can be used as a medicament for treating and/or preventing thrombus, and has great value for treating and preventing the thrombus.
Drawings
FIG. 1 is a diagram of electrophoresis on a polypropylene gel of FGF-21 analogue solution.
FIG. 2 shows the HE staining results of tail tissue sections taken after Ca/LPS combined modeling. A is the mouse tail artery, B is the mouse tail vein.
FIG. 3 shows the results of plasma APTT, PT and TT assays. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
FIG. 4 shows the results of Fib content measurement.
Fig. 5 shows RBC and PLT count detection results. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
FIG. 6 shows the expression levels of CRP, IL-6 and FVI genes, and the expression levels of CRP, IL-6 and FVI proteins. A is the result of CRP gene detection by Real-time PCR. B is the result of Real-time PCR detection of IL-6 gene. C is the result of Real-time PCR detection of FVI gene. D is the result of detecting CRP protein by ELISA. E is the result of detecting IL-6 protein by ELISA. F is the result of detecting FVI protein by ELISA. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
FIG. 7 shows the expression level of rat tPA gene and the expression level of tPA protein. A is the result of Real-time PCR detection of rat tPA gene. B is the result of ELISA to detect the content of rat tPA protein. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
FIG. 8 shows the expression level of mouse tPA gene and the expression level of tPA protein. A is the result of detecting mouse tPA gene by Real-time PCR. And B is the result of detecting the content of the mouse tPA protein by ELISA. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
FIG. 9 shows the expression level of rat PAI gene and the expression level of PAI protein. A is the result of Real-time PCR detection of rat PAI gene. B is the result of measuring the PAI protein content of rats by ELISA. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
FIG. 10 shows the expression level of mouse PAI gene and the expression level of PAI protein. A is the result of Real-time PCR detection of mouse PAI gene. B is the result of measuring the PAI protein content of the mice by ELISA. 1: a normal control group; 2: a model control group; 3: FGF21 low dose group; 4: FGF21 high dose group.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The prokaryotic expression vector is pET30 a: novagen products
Escherichia coli Rosetta (DE 3): beijing Quanjin Biotechnology Ltd, catalog number CD 801.
Male Wistar rats: the animal center of the Harbin veterinary institute, national academy of agricultural sciences.
Male Kunming mice: the animal center of the Harbin veterinary institute, national academy of agricultural sciences.
Ferric chloride: Sigma-Aldrich; CAS number 7705-08-0.
Carrageenanan (ca): Sigma-Aldrich; CAS number 9000-07-1.
Lipopolysacchrides (LPS): Sigma-Aldrich.
PBS buffer (pH 7.2): the solvent is water, and the solutes and their concentrations are as follows: NaCl 137mmol/L, KCl2.7mmol/L, Na2HPO410mmol/L，KH2PO42mmol/L。
EXAMPLE 1 preparation of FGF-21 analogues
Sequence design of FGF-21 analogues
The human FGF21 sequence obtained from the NCBI search was spliced to an elastin-like amino acid sequence optimized based on the article (Urry DW et al, Biochim Biophys acta.1974Dec 18; 371(2): 597-. Finally, an analogue (FGF-21 analogue for short) sequence of the FGF-21 protein is designed and obtained, as shown in SEQ ID No.1 (the 1 st to 183 th sites are FGF-21 protein sequences and the 184 th and 283 th sites are elastin-like protein sequences); the coding gene of the FGF-21 analogue is shown as SEQ ID No.2 (the 1 st-549 th site is the coding gene sequence of FGF-21 protein; the 550 th-849 th site is the coding gene sequence of elastin-like protein).
Second, construction of recombinant plasmid
1. The gene sequence (catatg + SEQ ID No.2+ ggatcc) designed in step 1 with cleavage sites at both ends was synthesized, and the resulting synthetic plasmid was digested with restriction enzymes NdeI and BamHI to recover the digested product. Expressing the fusion protein formed by fusing fibroblast growth factor-21 and elastin-like protein.
2. The prokaryotic expression vector pET30a was digested with NdeI and BamHI restriction enzymes, and the digested product was recovered.
3. And (3) connecting the enzyme digestion product in the step (1) with the vector skeleton in the step (2) to obtain a recombinant plasmid pET30 a-FGF-21-ELP. The structure of the recombinant plasmid pET30a-FGF-21-ELP is described as follows: the recombinant plasmid of the DNA fragment shown in SEQ ID No.2 is inserted between the enzyme cutting sites NdeI and BamHI of the prokaryotic expression vector pET30 a.
4. In the recombinant plasmid pET30a-FGF-21-ELP, the coding gene of FGF-21 mature peptide is fused with the elastin-like gene to form a fusion gene, and the fusion protein is expressed (the fusion protein is FGF-21 mature peptide and elastin-like protein from N end to C end in sequence).
The expected molecular weight of FGF-21-elastin-like protein is 32KD (estimated molecular weight is 32KD, and SDS-PAGE electrophoresis shows that its molecular weight is about 35 KD).
Preparation and purification of FGF-21-elastin-like proteins
1. The recombinant plasmid pET30a-FGF-21-ELP is introduced into Escherichia coli BL21(DE3) to obtain a recombinant strain.
2. Inoculating the single colony of the recombinant bacteria obtained in the step 1 into 5mL LB culture medium, carrying out shaking culture at 37 ℃ and 120rpm for 10h, then taking the bacterial liquid, and inoculating the bacterial liquid according to the volume ratio of 1:100In 500mL of LB medium containing 50mg/mL kanamycin, the medium was cultured at 37 ℃ for 2 hours with shaking at 120rpm, at which time OD600nmAbout 0.5.
3. IPTG was added to the bacterial solution obtained in step 2 to give a concentration of 0.25mmol/L for induction (shaking culture at 25 ℃ C. and 60rpm for 10 hours), and then centrifugation was carried out at 4 ℃ C. and 4000rpm for 30 minutes to collect the cells.
4. And (3) taking the thalli obtained in the step (3), carrying out ultrasonic disruption (8-12min, stopping working for 1s and 1s), centrifuging at 4 ℃ and 12000rpm, and respectively collecting supernatant and precipitate.
The supernatant and the pellet were analyzed by 12% SDS-PAGE electrophoresis, respectively.
5. Taking the supernatant obtained in the step 4, and performing reversible temperature cycle for three times, wherein the reversible temperature cycle specifically comprises the following steps:
(1) the fusion protein was taken, diluted four-fold with PBS, and placed in a constant temperature bath set at 37 ℃. Preheating a high-speed refrigerated centrifuge to 37 ℃, and centrifuging after the temperature is stable, wherein the setting condition is 19309g for 15 min.
(2) Discarding the supernatant, resuspending and centrifuging the obtained precipitate with PBS of the same volume, repeatedly blowing with a pipette until the solution is uniform, and performing ultrasonic treatment under the conditions of 1200w of power, 1s of working time, 3s of interval time and 60 working times, wherein the temperature of the water bath is kept lower than 20 ℃ during the ultrasonic treatment, and the ultrasonic treatment is repeated until the solution is completely clarified.
(3) And (3) placing the solution obtained in the step (2) in a refrigerator at 4 ℃ for 30 min. Centrifuging at low temperature with a high-speed refrigerated centrifuge under the set conditions of 4 deg.C, 19309g for 20 min. The precipitate was discarded.
(4) And (4) repeating the steps (1) to (3) twice on the supernatant obtained in the step (3), and obtaining a supernatant solution, namely the FGF-21-elastin-like solution.
The polypropylene gel electrophoresis of the FGF-21-elastin-like solution is shown in FIG. 1 (lane 1 is the molecular weight marker, lane 2 is the FGF-21-elastin-like solution). The target band (about 35 KD) is recovered and the N-terminal sequencing is carried out, and the result shows that the first 15 amino acid residues of the N-terminal are shown as the 1 st to 15 th amino acid residues from the N-terminal of SEQ ID No. 1.
EXAMPLE 2 therapeutic Effect of FGF-21-Elastin-like on thrombosis
The experimental animals are male Wistar rats with six-week-old detergent and male Kunming mice with six-week-old detergent.
The FGF-21-elastin-like solution prepared in example 1 was used for the experiment.
1. Experimental design in rats
After 1 week of acclimatization, the rats were randomly divided into 4 groups of 15 animals each, and were treated in parallel as follows:
model control group, on test days 1-7, injecting PBS buffer solution subcutaneously at about 8:00 every morning with a single injection volume of 0.2mL, on test day seven, injecting 10% chloral hydrate anesthesia intraperitoneally at about 9:00 every morning, separating left common carotid artery 2cm, placing small piece of plastic film (3 × 1.5.5 cm) below to protect perivascular tissue, and adsorbing 2.16mol/L FeCl3A small piece of filter paper (1 × 1cm) of the solution was applied to the exposed arterial surface, the piece of filter paper was to be held against the vessel wall;
FGF21 Low dose group, i.e., FGF-21 mature peptide solution (protein concentration adjusted by PBS buffer) is intraperitoneally injected about 8:00 a day on days 1-7 a.m., single injection dose is 1mg FGF-21 mature peptide (total protein)/kg body weight, single injection volume is 0.2mL, 10% chloral hydrate anesthesia is performed once about 9:00 a.m. on day seven, left common carotid artery is separated by 2cm, small plastic film (3 × 1.5.5 cm) is arranged below the left common carotid artery for protecting perivascular tissue, and 2.16mol/LFeCl is absorbed3A small piece of filter paper (1 × 1cm) of the solution was applied to the exposed arterial surface, the piece of filter paper was to be held against the vessel wall;
FGF21 high dose group, on test days 1-7, FGF-21 mature peptide solution (protein concentration adjusted by PBS buffer) was intraperitoneally injected at 8: 00/day around morning, single injection dose was 2mg FGF-21 mature peptide (total protein)/kg body weight, single injection volume was 0.2mL, on test day seven, 10% chloral hydrate anesthesia was performed at one time around 9: 00/day around morning, left common carotid artery was isolated by 2cm, small piece of plastic film (3 × 1.5.5 cm) was placed below to protect perivascular tissue, 2.16mol/LFeCl was inhaled3A small piece of filter paper (1 × 1cm) of the solution was applied to the exposed arterial surface, the piece of filter paper was to be held against the vessel wall;
normal control group: on the 1 st to 7 th days of the experiment, PBS buffer solution is injected subcutaneously at about 8:00 a day in the morning, and the volume of single injection is 0.2mL, and 10% chloral hydrate is injected intraperitoneally at about 9:00 a day in the seventh day of the experiment for anesthesia; separating left common carotid artery by 2cm, placing a small piece of plastic film (3 × 1.5cm) below the left common carotid artery for protecting perivascular tissue, applying a small filter paper sheet (1 × 1cm) absorbed with PBS solution on the exposed surface of the artery, wherein the filter paper sheet is required to be tightly attached to the vessel wall;
after 90s of action, removing filter paper (timing from the application of the filter paper sheet), cutting off blood vessels at the position where thrombus occurs, collecting blood from the right common carotid artery, centrifuging at 3000r/min at 4 ℃ for 15min, and taking supernatant blood plasma for detection;
2. mouse experimental design
After 1 week of adaptive feeding, the mice were randomly divided into 4 groups of 15 mice each, and were treated in parallel as follows:
model control group: on the 1 st to 7 th days of the experiment, the abdominal cavity is injected with Ca solution about 8:00 am on the first day, the single injection dose is 3mg/kg body weight, and the single injection volume is 0.3 mL; LPS is injected into tail vein after 24 hours, and the single injection dosage is 50ug/kg body weight;
FGF21 low dose group: on the 1 st to 7 th days of the experiment, the abdominal cavity is injected with Ca solution about 8:00 am on the first day, the single injection dose is 3mg/kg body weight, and the single injection volume is 0.3 mL; LPS is injected into tail vein after 24 hours, and the single injection dosage is 50ug/kg body weight; on days 1-7 of the experiment, the FGF-21 mature peptide solution (protein concentration adjusted by PBS buffer solution) is injected subcutaneously about 9:00 a day, 1mg of FGF-21 mature peptide (calculated by total protein) is per kg of body weight, and the volume of single injection is 0.2 mL;
FGF21 high dose group: on the 1 st to 7 th days of the experiment, the abdominal cavity is injected with Ca solution about 8:00 am on the first day, the single injection dose is 3mg/kg body weight, and the single injection volume is 0.3 mL; LPS is injected into tail vein after 24 hours, and the single injection dosage is 50ug/kg body weight; on days 1-7 of the experiment, the FGF-21 mature peptide solution (protein concentration adjusted by PBS buffer solution) is injected subcutaneously about 9:00 a day, 2mg of FGF-21 mature peptide (calculated by total protein) is per kg of body weight, and the volume of single injection is 0.2 mL;
normal control group: on the 1 st to 7 th days of the test, the PBS buffer solution is injected subcutaneously at about 8:00 am in the first day, the volume of single injection is 0.3mL, the PBS buffer solution is injected into the tail vein after 24 hours, and the dose of single injection is 50ug/kg body weight; on the 1 st to 7 th days of the test, PBS buffer solution is injected subcutaneously about 9:00 a day, and the volume of single injection is 0.2 mL;
on day 7 of the experiment, the mice were sacrificed under anesthesia and whole blood and tail tissue were taken from the mice, respectively.
3. Detection and results
Microscopic observation of the tail tissue sections obtained after the Ca/LPS combined molding was carried out, and the results are shown in FIG. 2(A is mouse tail artery, B is mouse tail vein). As can be seen from the figure: thrombosis can be seen in the tail artery and tail vein of the mouse in the model control group; compared with a model control group, the thrombus degree of mice in an FGF21 low-dose group is obviously reduced, and the level of mice in an FGF21 high-dose group is similar to that of the mice in a normal control group. The result shows that the FGF-21 mature peptide can obviously inhibit the formation of thrombus.
The coagulation process is a series of enzymatic reactions, including the intrinsic coagulation system, the extrinsic coagulation system, and the common pathway. Activated Partial Thrombin Time (APTT) reflects the activity of the intrinsic coagulation pathway and Prothrombin Time (PT) reflects the activity of the extrinsic coagulation pathway; the Thrombin Time (TT) response is the activity of thrombin, which is a common pathway between the two. The rat right carotid artery was bled and APTT, PT and TT were measured by a CS2000i fully automatic coagulometer, the results are shown in FIG. 3. As can be seen from the figure: the APTT, PT and TT time of the model control group rat is shortened, namely the blood coagulation system is over activated; compared with a model control group, rats APTT, PT and TT time of an FGF21 low-dose group and an FGF21 high-dose group are prolonged, namely, the rats have a direct inhibition effect on blood coagulation response. The result shows that the FGF-21 mature peptide can obviously inhibit the formation of thrombus.
The level of fibrinogen (Fib) reflects the extent of thrombosis. The rat right carotid artery was bled and fibrinogen was measured by a CS2000i fully automatic coagulometer, and the results are shown in fig. 4. As can be seen from the figure: the fibrinogen level of the rats in the model control group is high, namely the thrombus degree is high; rats with low fibrinogen levels in the FGF21 low dose group and FGF21 high dose group were low compared to the model control group. The result shows that the FGF-21 mature peptide can obviously inhibit the thrombus degree.
The number of Red Blood Cells (RBC) and the small blood count (PLT) are closely related to both blood viscosity and thrombosis. The rat right carotid artery was bled and RBC and PLT were measured by an XT1800i hematology analyzer, the results of which are shown in fig. 5. As can be seen from the figure: the model control group rats had high RBC and PLT values, i.e., high blood viscosity, and the FGF21 low dose group and FGF21 high dose group rats had significantly reduced RBC and PLT values compared to the model control group. The result shows that the FGF-21 mature peptide can obviously reduce the viscosity of whole blood and reduce thrombosis.
There is a network relationship between inflammation and thrombosis, and inflammation promotes thrombosis, and products in thrombosis also cause inflammation. C-reactive protein (CRP) as an acute-phase protein can cause endothelial cells of human bodies to synthesize inflammatory mediators and coagulation factors, thereby causing thrombosis. The rat right side carotid artery was bled, total RNA was extracted and reverse-transcribed into cDNA, and the expression levels of CRP gene, inflammatory factor (IL-6) and coagulation Factor (FVI) gene were detected by Real-time PCR (using GAPDH gene as reference gene). The primer pairs for detecting the CRP gene are as follows: 5'-CCCCAATTCTCCCTCGAAT-3', respectively; 5'-GGATGGATGGATGGATGGA-3' are provided. The primer pair for detecting the IL-6 gene is as follows: 5'-TAGTCCTTCCTACCCCAACTTCC-3', respectively; 5'-TTGGTCCTTAGCCACTCCTTC-3' are provided. The primer pair for detecting FVI gene is as follows: 5'-CGCAACTAAGGCAGTTCTATGT-3', respectively; 5'-GCTAGATCGTGGCTTTTCTTTCT-3' are provided. The primer pair for detecting the reference gene is as follows: 5'-ATGATTCTACCCACGGCAAG-3', respectively; 5'-CTGGAAGATGGTGATGGGTT-3' are provided. The rat right side carotid artery is blood-taken, centrifuged at 3000R/min at 4 ℃ for 15min, supernatant plasma is taken, and ELISA is used for detecting CRP, IL-6 and FVI contents (an ELISA detection kit for detecting rat CRP, an ELISA detection kit for detecting rat IL-6 and an ELISA detection kit for detecting rat FVI are all products of R & D company, and the operation is carried out according to the kit specifications). The results of the CRP gene detection by Real-time PCR are shown in A in FIG. 6. The results of the Real-time PCR detection of the IL-6 gene are shown in FIG. 6B. The results of Real-time PCR detection of FV II gene are shown in FIG. 6C. The results of ELISA detection of CRP protein are shown in FIG. 6D. The results of ELISA detection of IL-6 protein are shown in FIG. 6E. The results of ELISA detection of FVI protein are shown in FIG. 6F. As can be seen from fig. 6: the CRP content, IL-6 content and FVI content of the model control group rat are very high, namely the inflammatory reaction is activated to promote the release of the blood coagulation factor; compared with a model control group, the CRP content, the IL-6 content and the FVI content of rats in the FGF21 low-dose group and the FGF21 high-dose group are obviously reduced. The result shows that the FGF-21 mature peptide can obviously inhibit inflammatory reaction and thrombosis.
Tissue plasminogen activator (tPA) promotes the conversion of plasminogen to plasmin, which promotes thrombolysis. The rat right side neck total artery is bled, total RNA is extracted and is reversely transcribed into cDNA, and the expression level of tPA gene is detected by Real-time PCR (GAPDH gene is adopted as an internal reference gene). The primer pair for detecting rat tPA gene is as follows: 5'-AGAGGGAGTGACAGTCTTTAGGC-3', respectively; 5'-GAGGATTGTGGGAGGATGG-3' are provided. The results of Real-time PCR detection of rat tPA gene are shown in FIG. 7A. The rat right side neck common artery is taken blood, centrifuged for 15min at 3000R/min4 ℃, supernatant plasma is taken, and the content of tPA is detected by ELISA (the ELISA detection kit for detecting rat tPA is a product of R & D company, and is operated according to the kit instruction). The results are shown in FIG. 7B. As can be seen from fig. 7: the tPA content of the rat of the model control group is low, namely the fibrinolytic capacity is low; compared with a model control group, the content of tPA in rats in an FGF21 low-dose group and an FGF21 high-dose group is obviously increased. Mice were bled, total RNA was extracted and reverse-transcribed into cDNA, and the expression level of tPA gene was detected by Real-time PCR (using the GAPDH gene as the reference gene). The primer pairs for detecting mouse tPA gene are: 5'-TGACCAGGGAATACATGGGAG-3', respectively; 5'-GTCTGCGTTGGCTCATCTCTG-3' are provided. The results of Real-time PCR detection of mouse tPA gene are shown in FIG. 8A. The mouse is bled, centrifuged at 3000R/min and 4 ℃ for 15min, supernatant plasma is taken, and the content of tPA is detected by ELISA (the ELISA detection kit for detecting the mouse tPA is a product of R & D company, and is operated according to the kit specification). The results are shown in FIG. 8B. As can be seen from fig. 8: the tPA content of the mouse of the model control group is low, namely the fibrinolytic capacity is low; compared with a model control group, the content of tPA in mice in an FGF21 low-dose group and an FGF21 high-dose group is obviously increased. The result shows that the FGF-21 mature peptide can obviously enhance the fibrinolytic capacity and show the antithrombotic effect.
Plasminogen Activator Inhibitor (PAI) is a major negative regulator in the fibrinolytic system. Blood is collected from the right common carotid artery of a rat, total RNA is extracted and is reversely transcribed into cDNA, and the expression level of PAI gene (GAPDH gene is adopted as an internal reference gene) is detected by Real-time PCR. The primer pairs for detecting rat PAI gene are as follows: 5'-GTTCGCTTCACCCCTTCCAGA-3', respectively; 5'-GAAATAGAGGGCGTTCACCAG-3' are provided. The results of Real-time PCR detection of the PAI gene are shown in A of FIG. 9. Collecting blood from the right common carotid artery of a rat, centrifuging at 3000R/min and 4 ℃ for 15min, taking supernatant plasma, and detecting PAI content by ELISA (an ELISA detection kit for detecting PAI of the rat is a product of R & D company and is operated according to the kit specification). The results are shown in FIG. 9B. As can be seen from fig. 9: the PAI content of the rats in the model control group is high, namely the fibrinolytic capacity is low; rats with the low dose group of FGF21 and the high dose group of FGF21 had significantly reduced PAI levels compared to the model control group. Blood was collected from mice, total RNA was extracted and reverse-transcribed into cDNA, and the expression level of PAI gene was detected by Real-time PCR (using GAPDH gene as reference gene). The primer pairs for detecting the mouse PAI gene are as follows: 5'-TTCAGCCCTTGCTTGCCTC-3', respectively; 5'-ACACTTTTACTCCGAAGTCGGT-3' are provided. The results of Real-time PCR detection of the PAI gene are shown in FIG. 10, panel A. Blood is collected from mice, centrifugation is carried out for 15min at 3000R/min and 4 ℃, supernatant plasma is taken, and PAI content is detected by ELISA (an ELISA detection kit for detecting PAI of mice is a product of R & D company, and the operation is carried out according to the kit specification). The results are shown in FIG. 10, panel B. As can be seen from fig. 10: the PAI content of the mouse in the model control group is high, namely the fibrinolytic capacity is low; compared with a model control group, PAI content of mice in an FGF21 low-dose group and an FGF21 high-dose group is remarkably reduced. The result shows that the FGF-21 mature peptide can obviously enhance the fibrinolysis function and show the antithrombotic effect.
<110> Haerbin soaring biomedical technology development Co., Ltd
<120> preparation of FGF-21 analogue and 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 9095
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 250255
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
<210>2
<211>852
<212>DNA
<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 (8)
1. Use of a fibroblast growth factor-21 analogue in the manufacture of a product for the treatment and/or prevention of thrombosis;
the fibroblast growth factor-21 analogue is a fusion protein formed by fusing fibroblast growth factor-21 and elastin-like protein;
the elastin-like protein is the protein with the amino acid sequence shown as position 184-283 of SEQ ID No. 1;
the fibroblast growth factor-21 is a protein shown by the 1 st to 183 th sites of an amino acid sequence SEQ ID No. 1.
2. Use of a fibroblast growth factor-21 analogue in any one of:
(a) preparing a product for inhibiting thrombosis;
(b) preparing a product for inhibiting the extent of thrombosis;
(c) preparing a product for reducing fibrinogen levels in plasma;
(d) preparing a product for reducing the number of red blood cells and/or the number of platelets in blood;
(e) preparing a product for inhibiting inflammatory reactions;
(f) preparing a product for enhancing the dissolving capacity of the fiber;
the fibroblast growth factor-21 analogue is a fusion protein formed by fusing fibroblast growth factor-21 and elastin-like protein;
the elastin-like protein is the protein with the amino acid sequence shown as position 184-283 of SEQ ID No. 1;
the fibroblast growth factor-21 is a protein shown by the 1 st to 183 th sites of an amino acid sequence SEQ ID No. 1.
3. Use of a fibroblast growth factor-21 analogue in any one of:
(g) preparing a product for extending the activated partial thrombin time and/or prothrombin time and/or thrombin time of plasma;
(h) preparing a product for reducing the level of C-reactive protein and/or inflammatory and/or coagulation factors in blood;
the inflammatory factor is IL-6; the blood coagulation factor is FVI;
(i) preparing a product for increasing the level of tissue plasminogen activator in blood;
(j) preparing a product for reducing the level of a plasminogen activator inhibitor in blood;
the fibroblast growth factor-21 analogue is a fusion protein formed by fusing fibroblast growth factor-21 and elastin-like protein;
the elastin-like protein is the protein with the amino acid sequence shown as position 184-283 of SEQ ID No. 1;
the fibroblast growth factor-21 is a protein shown by the 1 st to 183 th sites of an amino acid sequence SEQ ID No. 1.
4. Use according to any one of claims 1 to 3, characterized in that: the fibroblast growth factor-21 analogue is a protein with an amino acid sequence shown as SEQ ID No. 1.
5. Use according to any one of claims 1 to 3, characterized in that: the fibroblast growth factor-21 analogue is prepared by a method comprising the following steps:
(a1) cloning the coding gene of the fibroblast growth factor-21 analogue into a prokaryotic expression vector, and then expressing to obtain the fusion protein;
(a2) collecting the fusion protein, and performing reversible temperature cycle for three times to obtain the fibroblast growth factor-21 analogue;
the implementation mode of the three-time temperature reversible cycle is as follows: 1) taking the fusion protein, diluting the fusion protein by 3-5 times of volume by PBS, and centrifuging the fusion protein for 15min at 37 ℃ at 19309 g; 2) resuspending and centrifuging the obtained precipitate with PBS (phosphate buffer solution) with the same volume, and then carrying out ultrasonic treatment under the conditions of 1200w of power, 1s of working time and 3s of interval time until the solution is completely clarified, wherein the temperature of a water bath is kept to be lower than 20 ℃; 3) standing at 4 deg.C for 30min, and centrifuging at 19309g at 4 deg.C for 20 min; 4) repeating the steps 1) to 3) twice by using the supernatant obtained by the centrifugation in the step 3) instead of the fusion protein in the step 1).
6. Use according to claim 5, characterized in that: the coding gene of the fibroblast growth factor-21 analogue is a fusion gene formed by fusing the coding gene of the fibroblast growth factor-21 and the coding gene of the elastin-like protein;
the encoding gene of the elastin-like protein is a DNA molecule shown in the 550 th and 849 th positions of SEQ ID No. 2; the coding gene of the fibroblast growth factor-21 is a DNA molecule shown in the 1 st-549 th site of SEQ ID No. 2.
7. Use according to claim 6, characterized in that: the coding gene of the fibroblast growth factor-21 analogue is a DNA molecule shown in SEQ ID No. 2.
8. Use of a biomaterial in any one of:
(A) preparing a product for treating and/or preventing thrombosis;
(a) preparing a product for inhibiting thrombosis;
(b) preparing a product for inhibiting the extent of thrombosis;
(c) preparing a product for reducing fibrinogen levels in plasma;
(d) preparing a product for reducing the number of red blood cells and/or the number of platelets in blood;
(e) preparing a product for inhibiting inflammatory reactions;
(f) preparing a product for enhancing the dissolving capacity of the fiber;
(g) preparing a product for extending the activated partial thrombin time and/or prothrombin time and/or thrombin time of plasma;
(h) preparing a product for reducing the level of C-reactive protein and/or inflammatory and/or coagulation factors in blood;
the inflammatory factor is IL-6; the blood coagulation factor is FVI;
(i) preparing a product for increasing the level of tissue plasminogen activator in blood;
(j) preparing a product for reducing the level of a plasminogen activator inhibitor in blood;
the biological material is the coding gene of the fibroblast growth factor-21 analogue in any one of claims 1-7 or a recombinant vector, a recombinant bacterium or an expression cassette containing the coding gene.
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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 |
| CN113968901A (en) * | 2021-11-02 | 2022-01-25 | 南京大学 | Use of protein sequences that bind to substrates for the preparation of products inhibiting fibrin assembly |
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| CN101955960A (en) * | 2010-08-03 | 2011-01-26 | 中山大学 | Method for purifying recombinant lactase by using elastin like protein tag |
| CN103191415A (en) * | 2013-04-03 | 2013-07-10 | 哈尔滨博翱生物医药技术开发有限公司 | Application of fibroblast growth factor-21 mature peptide in preparing medicine for treating hypertension |
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