CN116903721A - Polypeptide PDBPE-001, pharmaceutical composition and application thereof - Google Patents
Polypeptide PDBPE-001, pharmaceutical composition and application thereof Download PDFInfo
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- CN116903721A CN116903721A CN202310717677.6A CN202310717677A CN116903721A CN 116903721 A CN116903721 A CN 116903721A CN 202310717677 A CN202310717677 A CN 202310717677A CN 116903721 A CN116903721 A CN 116903721A
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- OTQCKZUSUGYWBD-BRHMIFOHSA-N lepirudin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)[C@@H](C)O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)[C@@H](C)O)C1=CC=C(O)C=C1 OTQCKZUSUGYWBD-BRHMIFOHSA-N 0.000 description 1
- 229960004408 lepirudin Drugs 0.000 description 1
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- 239000012460 protein solution Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
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Classifications
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- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- 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
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The application relates to the technical field of polypeptide drug research and development. Specifically disclosed are polypeptide PDBPE-001, pharmaceutical compositions and uses thereof. The application discovers that the polypeptide PDBPE-001 derived from the dactylogyrus spider for the first time, and the polypeptide PDBPE-001 provided by the application comprises the following polypeptides of A1 or A2: a polypeptide with an A1 amino acid sequence of SEQ ID NO. 1; polypeptide with the identity of 90% or more with SEQ ID NO.1 and the same function as A1, which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues into the amino acid sequence of the A2 amino acid sequence of SEQ ID NO. 1; the application has obvious inhibition effect on the coagulation Factor Xa through carrying out different thrombin resisting researches and animal tail thrombus model researches on the polypeptide, and the research shows that the polypeptide can be used as a candidate molecule of a substitute drug or an auxiliary drug of the existing anticoagulants.
Description
Technical Field
The application belongs to the technical field of development of polypeptide medicaments, and particularly relates to a polypeptide PDBPE-001, a pharmaceutical composition and application thereof.
Background
Animal toxin is derived from venom produced by the animal evolving from the natural animal gland, contains active polypeptide with various structures and rich functions, can act on blood systems, complement systems, membrane receptors and ion channels respectively, and has wide pharmacological effects. In recent years, more various animal toxin-derived polypeptide drugs have been approved by the FDA and are in clinical laboratory stages, such as "Captopril" (Captopril) derived from agkistrodon halys, a conventional drug for treating hypertension; hirudin (Lepirudin) derived from Hirudo, a conventional drug for treating venous thromboembolism, exenatide (Exenatide) derived from lizard, and a conventional drug for treating type 2 diabetes. Thus, animal toxin polypeptides are highly valued as a natural treasury for drug development.
It is estimated that at least 220000 toxic animals live on earth, of which spiders are the most representative are toxic arthropods, and more than 50751 spiders have been reported (24 th edition of world spider catalog), and only toxin polypeptides of less than 600 spiders have been studied early on due to technical singleness and resource limitation, etc., and are a highly undeveloped edition. The inventor finds that the dactylogyrus spider Draconarius digitusiformis has not been developed and utilized in systematic research, and because of the fact, the dactylogyrus spider is subjected to functional activity and unreported toxin polypeptide excavation, and a new thought and a new source are provided for the development of polypeptide medicaments in the future.
Disclosure of Invention
The present application aims to overcome the above-mentioned disadvantages of the prior art and to provide a polypeptide PDBPE-001, a pharmaceutical composition and use thereof.
In order to achieve the above purpose, the present application adopts the following technical scheme:
it is a first object of the present application to provide a polypeptide PDBPE-001, said polypeptide PDBPE-001 comprising a polypeptide of A1 or A2 as follows: a polypeptide with an A1 amino acid sequence of SEQ ID NO. 1; the amino acid sequence A2 is polypeptide which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues in the amino acid sequence of SEQ ID NO.1, has the identity of 90% or more with the SEQ ID NO.1 and has the same function with A1.
It is a second object of the present application to provide a nucleic acid molecule encoding the polypeptide PDBPE-001.
Further, the nucleic acid molecules include the nucleic acid molecules shown in a1 or a2 or a3 as follows:
the a1 coding region comprises a nucleic acid molecule of SEQ ID NO. 2;
a2 nucleic acid molecule having the nucleotide sequence of SEQ ID NO. 2;
a3 has 90% or more identity to the nucleotide sequence defined in a1 or a2 and encodes the nucleic acid molecule of claim 2.
A third object of the present application is to provide a recombinant vector comprising the above-mentioned nucleic acid molecule.
It is a fourth object of the present application to provide a recombinant expression cell as described above comprising the recombinant vector as described above.
A fifth object of the present application is to provide a method for preparing the above polypeptide PDBPE-001, comprising the steps of: linearizing a recombinant expression vector of the polypeptide PDBPE-001 through an enzyme cleavage site; introducing the linearized recombinant expression vector into a host cell to obtain a recombinant expression cell, culturing the recombinant expression cell, and obtaining the polypeptide PDBPE-001 from the culture.
A sixth object of the present application is to provide a pharmaceutical composition comprising said polypeptide PDBPE-001 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
In the pharmaceutical composition, the polypeptide PDBPE-001 or pharmaceutically acceptable salt thereof can be used in an amount which is effective for treatment.
The pharmaceutical excipients can be widely used in the field of pharmaceutical production. Adjuvants are used primarily to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.
The pharmaceutical compositions of the present application may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes, and the like.
The pharmaceutical compositions of the present application may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the application may also be in controlled or delayed release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry formulations which may be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.
The seventh object of the application is to provide an application of the polypeptide PDBPE-001 or pharmaceutically acceptable salt thereof in preparing FXa inhibitor.
An eighth object of the present application is to provide an application of the polypeptide PDBPE-001 or its pharmaceutically acceptable salt in preparing a medicament for treating FXa-related diseases.
The FXa-related diseases of the application include deep vein thrombosis, thromboembolism, pulmonary embolism and acute coronary syndrome.
The ninth object of the present application is the use of the polypeptide PDBPE-001 or a pharmaceutically acceptable salt thereof for preparing a medicament, wherein the medicament is used for treating cardiovascular and cerebrovascular diseases, preferably deep vein thrombosis, thromboembolic diseases, pulmonary embolism diseases or acute coronary syndromes.
Further, the medicine is an antithrombotic medicine.
The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" refers to a salt-forming reaction conventional in the art, such as: salts are formed from chemical reactions between bases and acids, such as: NH (NH) 3 +H 2 SO 4 →(NH 4 ) 2 SO 4 。
The salt may be a basic salt, an acidic salt, or a neutral salt. The basic salt generates hydroxide ions in the water and the acidic salt generates hydronium ions.
The polypeptide PDBPE-001 may form a salt of the polypeptide PDBPE-001 of the application with a cation or anion between anionic groups or cationic groups, respectively. These groups may be located in the peptide portion of the polypeptide PDBPE-001 of the application.
The anionic group of the polypeptide PDBPE-001 of the application may comprise the free carboxyl group of the peptide moiety. The peptide moiety typically includes a free carboxylic acid group at the C-terminus.
The cationic group of the peptide moiety is not limited in the present application and includes the free amino group at the N-terminus (if present) as well as any free amino groups of internal basic amino acid residues (e.g., arg and Lys).
In a specific embodiment, the analog of the polypeptide PDBPE-001 of the application is an alkaline salt. These salts may be formed, for example, between the anionic groups of the peptide moiety and sodium or potassium cations.
In another specific embodiment, the analog of the polypeptide PDBPE-001 of the application is an acidic salt. These salts may be formed, for example, between the cationic groups of the peptide moiety and chloride or acetate anions.
The free carboxylic acid groups may also be reacted with alcohols or phenols to form esters of the derivatives of the application, which may involve free carboxylic groups at the C-terminus of the peptide and/or any free carboxylic groups in the side chains.
Amides of the derivatives of the application may also be formed by reacting free carboxylic acid groups with amines or substituted amines, or by reacting free or substituted amino groups with carboxylic acids. Amide formation may involve free carboxyl groups at the C-terminus of the peptide, any free carboxyl groups in the side chains, free amino groups at the N-terminus of the peptide and/or any free or substituted amino groups of the peptide and/or the peptide in the side chains.
In particular embodiments, the polypeptide PDBPE-001 is in the form of a pharmaceutically acceptable salt. In another specific embodiment, the polypeptide PDBPE-001 is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C-terminal end of the peptide. In yet another specific embodiment, the polypeptide PDBPE-001 is in the form of a pharmaceutically acceptable ester.
Compared with the prior art, the technical scheme provided by the application has the beneficial effects that:
(1) According to the application, the dactylogyrus spider Draconarius digitusiformis is taken as a research object, and the polypeptide PDBPE-001 derived from the dactylogyrus spider and the encoding gene thereof are discovered for the first time through sequencing a transcriptome of a poison gland sample, analyzing data and selecting the polypeptide, and the polypeptide has a remarkable inhibition effect on a coagulation Factor Xa.
(2) The application efficiently prepares the polypeptide PDBPE-001 with the amino acid sequence of SEQ ID NO.1 by constructing recombinant plasmids and expressing recombinant cells, has the molecular weight of 9889.82Da, consists of 92 amino acid residues and has 4 pairs of disulfide bonds.
(3) According to the application, by carrying out different thrombin resistance researches on the polypeptide PDBPE-001 and animal tail thrombus model researches, the research results show that the polypeptide PDBPE-001 has obvious concentration-dependent inhibition on Factor Xa, and the half inhibition concentration is about 0.807 mu M+/-0.14 mu M. Through the verification of a tail thrombus model, 30mg/kg of PDBPE-001 can play a good role in resisting thrombus.
(4) The application obtains a novel Factor Xa inhibitor, provides a brand new lead polypeptide molecule for the development of novel anticoagulants, provides a method reference for other toxic animal resources which do not develop active polypeptides, and can be used as candidate molecules of alternative drugs or auxiliary drugs of the existing anticoagulants.
Drawings
FIG. 1A is a schematic diagram of the structure of a recombinant expression plasmid PDBPE-001-PET-32a (+) constructed in the application;
FIG. 1B is a diagram showing the identification result of the recombinant expression plasmid PDBPE-001-PET-32a (+) constructed in the present application;
FIG. 2A is a graph showing the results of HPLC analysis of the polypeptide PDBPE-001 of the present application;
FIG. 2B is a diagram showing the SDS-PAGE identification of the polypeptide PDBPE-001 of the present application; swimming band 1: bacterial liquid supernatant; swimming band 2: fusion protein PDBPE-001, band 3: degradation of the fusion protein PDBPE-001;
FIG. 2C is a graph showing the mass spectrum identification result of the polypeptide PDBPE-001 of the application;
FIGS. 3A and 3B are graphs showing the results of evaluation of the activity of the polypeptide PDBPE-001 of the present application;
FIG. 4A is a graph comparing black tail images of different treatment groups;
FIG. 4B is a graph showing the comparison of tail vein plug darkening area length for different treatment groups;
fig. 4C is a graph showing the tail black tail rate variation trend for different treatment groups.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the following detailed description of the specific embodiments of the present application will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
SPF grade ICR Male mice (20+ -2 g) 40, 4 week old, laboratory animals ethical examination number PA23041501-1, purchased from Sichuan Tonglihua animal laboratory center.
RNA stable preservation solution is purchased from Thermo company, polypeptide expression vector is purchased from Nanjing gold Style company, thrombin, kallikrein, factor XIa, factor Xa blood coagulation Factor is purchased from French Enzyme research, TEV Enzyme is purchased from Soilebao company, BL21 (DE 3) is purchased from full gold company, XB-C18 reverse phase chromatographic column is purchased from Nami micro-tech Co., ltd., carrageenan is purchased from Sigma company, IPTG, ampicillin, LB medium, imidazole, sodium chloride and the like are all analytically pure reagents purchased from Shanghai company, S2222 (Xa Factor chromogenic substrate) is purchased from Italy Chromogenix.
Construction of a polypeptide PDBPE-001 expression vector: the polypeptide PDBPE-001 fragment is sent to Nanjing Jinsrui company for expression vector construction, PET-32a (+) prokaryotic expression plasmid is adopted, a protease cleavage site is positioned at the N end of mature peptide, TEV enzyme cleavage (-ENLYFQG-) is adopted, HIS tag is used for purification, escherichia coli preferrably optimizes the target fragment nucleotide sequence, and the target fragment nucleotide sequence is inserted into the expression vector through EcoRI/HindIII.
LB medium containing ampicillin resistance: the medium components contained tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, glycerol 4mL/L, and ampicillin resistance 100. Mu.g/mL.
0.04mM S2222 mixture: weighing substrate S2222.0 mg, adding lml FXa buffer solution, and uniformly mixing.
FXa buffer: 2.42g Tris (100 mM), 2.34g NaCl (200 mM), 0.1% BSA, pH adjusted to 7.4, dissolved to 200ML with 1 XPBS and then sub-packed into 50ML centrifuge tubes for further use.
The positive control drug Fondaparinux (Fondaparinux sodium) was purchased from MedChemExpress.
Definition and description:
the term "identity" as used herein refers to identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
The term "protein having the same function" as used in the present application has the same or similar meaning as conventionally understood by those skilled in the art, and means that the amino acid sequence of a fragment is a part of the amino acid sequence of an intact protein or polypeptide, and has the same or similar function or activity as the intact protein or polypeptide. It will be appreciated by those of ordinary skill in the art that altering a minority of amino acid residues in certain regions of a polypeptide, e.g., non-important regions, does not substantially alter biological activity, e.g., the sequence resulting from appropriate substitution of certain amino acids does not affect its activity (see Watson et al Molecular Biologyof The Gene, fourth edition, 1987,The Benjamin/Cummings pub. Co. P224). Thus, one of ordinary skill in the art would be able to perform such substitutions and ensure that the resulting molecule still has the desired biological activity.
The discovery process of the polypeptide PDBPE-001 comprises the following steps: is selected from the constructed virtual library of natural polypeptides from Pitot peaceful biological medicine Co.
Example 1
1. Preparation of the polypeptide PDBPE-001:
(1) Recombinant vector construction
The polypeptide PDBPE-001 fragment sequence (the amino acid sequence is shown as SEQ ID NO. 1) is sent to Nanjing Jinsrui company for expression vector construction. The PET-32a (+) prokaryotic expression plasmid is adopted, double enzyme cutting sites are EcoRI/HindIII, protease cutting sites are positioned at the N end of mature peptide, TEV enzyme cutting (-ENLYFQG-) is adopted, and HIS tag is purified.
The results are shown in FIG. 1A and FIG. 1B, and the vector is identified by MluI and Hind III enzyme digestion and gel electrophoresis, and the results show that the vector construction is successful. The recombinant fusion protein is composed of linker protein (molecular weight about 18 kDa) and target polypeptide band (molecular weight about 9 kDa) at about 22-27kDa, and the target polypeptide can be obtained after TEV enzyme digestion.
(2) Recombinant expression of polypeptide PDBPE-001
Optimizing the nucleotide sequence of target polypeptide PDBPE-001 according to the preference of escherichia coli, wherein the nucleotide sequence is shown as SEQ ID NO.2, inserting the nucleotide sequence into a constructed Pet32a vector through EcoRI/HindIII, transforming E.coli BL21 (DE 3) escherichia coli, picking up monoclonal and inoculating the monoclonal into LB culture medium containing ampicillin resistance, culturing for 6-8 hours at 37 ℃ in 220pm shaking mode, sub-packaging 300 mu L, sequencing the strain in a carrier of Optimus in a Optimus in Qinkekoogin, and carrying out subsequent prokaryotic expression by using bacterial liquid with correct sequencing. Bacterial liquid with correct sequencing is prepared according to the following ratio of 1:100 is inoculated in LB culture medium containing ampicillin resistance for overnight culture, bacterial liquid is added into 1L of LB culture medium containing resistance for the next day, the bacterial liquid is cultured in a shaking table at 37 ℃ and 220rpm, when the OD600 value reaches 0.6-0.8, IPTG with the final concentration of 1mM is added, the shaking table is set at 28 ℃ at the moment, 100pm is subjected to induction expression, and the induction time is 12-16h. The bacterial liquid is collected centrifugally in the next day, washed twice by ultrapure water and centrifuged to remove the supernatant. And (3) re-suspending the thalli by using PBS until obvious thalli particles are eliminated, pouring the thalli liquid into a homogenizer for continuous crushing for 3 times, ensuring that no obvious precipitate exists in the thalli liquid, and centrifugally collecting the bacterial liquid supernatant. Loading a pretreated bacterial liquid supernatant sample into an equilibrated Ni column, eluting a hybrid protein by using 10mM and 30mM imidazole solution, eluting a target protein by using 300mM imidazole solution, and collecting the target protein; and (3) performing TEV enzyme digestion according to the mass ratio of 1:50, wherein the enzyme digestion condition is 16 ℃, and the enzyme digestion is performed for 12-14 hours to obtain enzyme digestion solution.
As shown in FIG. 2B, the target polypeptide PDBPE-001 is efficiently expressed in the escherichia coli expression bacterium BL21 by pET32a in a prokaryotic expression mode, the expression condition of the recombinant polypeptide is analyzed by SDS-PAGE electrophoresis after TEV digestion, and two bands of obvious fusion protein and target polypeptide can be observed in lane 3, so that the digestion is proved to be successful.
(3) Polypeptide PDBPE-001 separation and purification
Separating and purifying the enzyme-digested solution by preparative reversed phase high performance liquid chromatography (RP-HPLC, column type: XB-C18 reversed phase column), wherein the mobile phase is composed of ultrapure water and acetonitrile; detection wavelengths 215nm and 280nm; the flow rate is 10mL/min; wherein the mobile phase A phase is acetonitrile containing 0.1% TFA, and the mobile phase B phase is ultrapure water containing 0.1% TFA. The elution method is 15-45% of phase A, 85-55% of phase B, and the elution time is 40min. The elution peaks were collected and lyophilized to obtain the polypeptide PDBPE-001. Mass spectrometry detects molecular weight to identify whether the polypeptide is expressed successfully.
As shown in FIG. 2A, the target polypeptide PDBPE-001 has larger hydrophobicity than the fusion protein, so that the protein solution after enzyme digestion can be separated by high-efficiency reversed phase chromatography to finish the capture of high-purity polypeptide, different absorption peaks are respectively collected for mass spectrum identification, as shown in FIG. 2C, the molecular weight of an elution component with the retention time of 22.3min is matched with the theoretical molecular weight of the target polypeptide, the molecular weight of the polypeptide PDBPE-001 is 9889.82Da, and the polypeptide PDBPE-001 consists of 92 amino acid residues and contains 4 pairs of disulfide bonds.
2. Polypeptide PDBPE-001 has inhibitory effect on Thrombin, kallikrein, factor XIa and Factor Xa 4 thrombin activities
mu.L of the final concentration of 1mM polypeptide PDBPE-001 sample and 1. Mu.L of HFXa at a final concentration of 4.65 (mU/ml) were mixed in 58. Mu.L of buffer (100 mM NaCl, 50mM Tris-HCl, 5mM NaCl) in 96-well plates 2 pH 8.0). After gentle shaking and mixing, 5 minutes at room temperature, 35. Mu.L of buffer was added to a 5. Mu.L mixture of 0.04mM S2222, at a final volume of 100. Mu.L. Dynamic program (wave) is setLength 405nm, run for 30min, interval 40 s) and then put the 96-well plate into an enzyme label instrument for monitoring. Thrombin, kallikrein, factor XIa, activity assays were performed with enzyme concentrations of 10.02, 3.17 and 11.36 (mU/ml) and corresponding substrate substitutions.
The absorbance value at the time point of 30min is taken for inhibition rate calculation,
wherein A is the absorbance value.
And further according to the concentration relation of PDBPE-001 in hFXa enzyme dynamics, nonlinear fitting is performed in GraphPad Prism 9 software, and half inhibition concentration of PDBPE-001 on hFXa is calculated.
The results are shown in Table 1, the polypeptide PDBPE-001 has different inhibition effects on 4 enzymes, wherein the polypeptide PDBPE-001 has obvious inhibition effect on Factor Xa, has good selectivity and has no obvious inhibition effect on other enzymes.
Table 1.
| Project | Thrombin | Kallikrein | Factor Xa | Factor XIa |
| PDBPE-001 | NI a | NI a | 0.807 | NI a |
Note that: NI (NI) a No inhibition of the polypeptide was observed at inhibitor concentrations up to 1 mM.
Further, the IC50 values of 1mM polypeptide PDBPE-001 sample solution were measured at 4 concentrations of 0.24, 0.49, 0.97 and 1.94. Mu.M, respectively, and as a result, as shown in FIGS. 3A-3B, the IC50 value of PDBPE-001 to Factor Xa was about 0.807. Mu.M.+ -. 0.14. Mu.M, and the inhibition effect was remarkably enhanced with the increase of the concentration.
3. Effect of polypeptide PDBPE-001 in animal thrombus model
Mouse tail thrombus model, reference: the model reported in the model comparison of the thrombus model of the tail of the mice prepared by combining two administration modes of carrageenan in the 7 th stage of the 11 th volume of the journal of cardiovascular and cerebrovascular diseases 2013 of traditional Chinese medicine and Western medicine is molded.
SPF ICR mice were housed in SPF animal houses, allowing free access to water and food, 12h light/dark cycle. The number of mice per cage is not more than 5. The experimental animals were euthanized after the end of the experiment. During molding, the ambient temperature was 20.+ -. 2 ℃. Animals were randomly divided into 6 groups (n=5) according to body weight after one week of adaptation, respectively, normal group, model group, positive drug group 6mg/kg Fondaparinux and polypeptide PDBPE-00130mg/kg, PDBPE-001 mg/kg, PDBPE-0017.5mg/kg. Modeling was performed according to the above model, and 200 μl of carrageenan solution (6 mg/mL) was intraperitoneally injected after 10 minutes of drug of different concentrations was injected into the tail vein, and after 24 hours, the length of the blackening region of the blood vessel plug at the tail of the mouse was observed and recorded, and the blacktail rate was calculated. Black tail rate calculation formula: black tail rate% = number of black tail mice in group/number of mice in group x 100%.
The results are shown in FIGS. 4A-4C, and the application adopts a tail thrombus model to evaluate the antithrombotic efficacy of the polypeptide PDBPE-001 in vivo. After the thrombus model is established, compared with a control group, the model group has 80% of black tail rate, and the tail thrombus length is 2.28cm, so that the modeling is proved to be successful. Compared with a model group, 6mg/kg Fondaparinux and polypeptide PDBPE-00130mg/kg of a positive group are administered for 24 hours, the black tail rate of mice is reduced to 20%, and meanwhile, the length of tail thrombus is obviously reduced by 86.4% and 88.5% (P < 0.05), so that the anti-thrombus effect is obvious.
Example 2
Taking 0.096 g-1.8 g of the polypeptide PDBPE-001 prepared in the embodiment 1, adding proper auxiliary materials of injection (including freeze-dried powder injection and sterile split-charging dry powder injection), and preparing the antithrombotic injection according to the injection (including freeze-dried powder injection and sterile split-charging dry powder injection) process.
Example 3
Adding proper auxiliary materials into tablets (including sustained-release tablets, skeleton tablets, coated tablets, dispersible tablets and the like) by taking and adding 0010.096 g-1.8 g of the polypeptide PDBPE prepared in the embodiment 1, and preparing the antithrombotic tablet by a tablet (including the sustained-release tablets, the skeleton tablets, the coated tablets, the dispersible tablets and the like) process.
Example 4
Taking 0.096 g-1.8 g of the polypeptide PDBPE-001 prepared in the example 1, adding proper auxiliary materials of the capsule, and preparing the antithrombotic capsule according to a capsule process.
Example 5
The polypeptide PDBPE-001 prepared in the embodiment 1 is taken to be 0.096 g-1.8 g, and proper auxiliary materials of emulsion (including microemulsion, nanoemulsion and the like) are added to prepare the antithrombotic emulsion according to the emulsion (including microemulsion, nanoemulsion and the like) process.
Example 6
Taking 0010.096 g-1.8 g of the polypeptide PDBPE prepared in the embodiment 1, adding proper auxiliary materials of the granule, and preparing the antithrombotic granule according to a granule process.
Example 7
Taking 0.096 g-1.8 g of the polypeptide PDBPE-001 prepared in the example 1, adding proper auxiliary materials of the slow-release and controlled-release agent, and preparing the antithrombotic slow-release and controlled-release agent according to a slow-release and controlled-release agent process.
Example 8
Taking 0.096 g-1.8 g of the polypeptide PDBPE-001 prepared in the example 1, adding proper auxiliary materials of the oral liquid, and preparing the antithrombotic oral liquid according to an oral liquid process.
Example 9
Taking 0.096 g-1.8 g of the polypeptide PDBPE-001 prepared in the example 1, adding proper auxiliary materials of liposome formulation, and preparing the antithrombotic liposome formulation according to a liposome process.
The embodiments described above and features of the embodiments herein may be combined with each other without conflict.
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.
Claims (10)
1. A polypeptide PDBPE-001, characterized in that: the polypeptide PDBPE-001 comprises the following polypeptides of A1 or A2: a polypeptide with an A1 amino acid sequence of SEQ ID NO. 1; the amino acid sequence A2 is polypeptide which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues in the amino acid sequence of SEQ ID NO.1, has the identity of 90% or more with the SEQ ID NO.1 and has the same function with A1.
2. A nucleic acid molecule encoding the polypeptide PDBPE-001 of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises a nucleic acid molecule as set forth in a1 or a2 or a 3:
the a1 coding region comprises a nucleic acid molecule of SEQ ID NO. 2;
a2 nucleic acid molecule having the nucleotide sequence of SEQ ID NO. 2;
a3 has 90% or more identity to the nucleotide sequence defined in a1 or a2 and encodes the nucleic acid molecule of claim 2.
4. A recombinant vector comprising the nucleic acid molecule of any one of claims 2-3.
5. A recombinant expression cell comprising the recombinant vector of claim 4.
6. A method of making the polypeptide PDBPE-001 as defined in claim 1, comprising the steps of: linearizing a recombinant expression vector of the polypeptide PDBPE-001 through an enzyme cleavage site; introducing the linearized recombinant expression vector into a host cell to obtain a recombinant expression cell, culturing the recombinant expression cell, and obtaining the polypeptide PDBPE-001 from the culture.
7. A pharmaceutical composition comprising the polypeptide PDBPE-001 or a pharmaceutically acceptable salt thereof as defined in claim 1, and a pharmaceutically acceptable adjuvant.
8. Use of the polypeptide PDBPE-001 as defined in claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of an FXa inhibitor.
9. Use of a polypeptide PDBPE-001 as defined in claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a FXa related disease.
10. Use of a polypeptide PDBPE-001 or a pharmaceutically acceptable salt thereof as claimed in claim 1, in the manufacture of a medicament for the treatment of cardiovascular and cerebrovascular diseases, preferably deep vein thrombosis, thromboembolic diseases, pulmonary embolism, or acute coronary syndromes; preferably, the drug is an antithrombotic drug.
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