CN116836233A - Anti-inflammatory active polypeptide and application thereof - Google Patents
Anti-inflammatory active polypeptide and application thereof Download PDFInfo
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- CN116836233A CN116836233A CN202311115254.3A CN202311115254A CN116836233A CN 116836233 A CN116836233 A CN 116836233A CN 202311115254 A CN202311115254 A CN 202311115254A CN 116836233 A CN116836233 A CN 116836233A
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Abstract
The invention provides a polypeptide with anti-inflammatory biological activity or pharmaceutically acceptable salt thereof and application thereof, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. The polypeptide can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory bioactivity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of LPS-induced macrophages. Therefore, the pharmaceutical composition prepared by the polypeptide has anti-inflammatory bioactivity, and can be further used for developing products such as medicines, cell cultures or cosmetics.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-inflammatory active polypeptide or pharmaceutically acceptable salt thereof and application thereof.
Background
Inflammation is a complex pathological process that results from the body being subjected to a certain stimulus. Inflammation is often beneficial, being an automatic defensive response of the human body. However, when the body's immune balance is broken, uncontrolled inflammation can lead to increased body temperature, redness, swelling, pain and dysfunction. Mediators involved in inflammatory reactions such as NO, cytokines, PEG2, etc. are released in large amounts, thereby affecting the occurrence and development of the disease. In recent years, inhibition of synthesis or activity of inflammatory mediators has become a major direction for the treatment of inflammation. Although the existing clinical medicines can treat various inflammations, side effects with different degrees exist. Therefore, finding anti-inflammatory drugs with good safety is an important point in the development of anti-inflammatory drugs.
Bioactive peptides are a special class of protein fragments that can positively affect the function and status of the human body. A large number of researches show that the bioactive peptide has the functions of resisting oxidation, inhibiting bacteria, resisting inflammation, resisting tumor, resisting virus, reducing blood sugar and the like. Compared with chemical synthesis medicines, the bioactive peptide has the advantages of wide sources, simple preparation process and higher safety. Thus, bioactive peptides with good safety and high anti-inflammatory performance are still to be developed.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the invention provides polypeptides having anti-inflammatory activity or pharmaceutically acceptable salts, conjugates, nucleic acid molecules, constructs, cells, pharmaceutical compositions and uses thereof. The polypeptide can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory bioactivity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of LPS-induced macrophages. Therefore, the pharmaceutical composition prepared by the polypeptide has anti-inflammatory bioactivity, and can be further used for developing products such as medicines, cell cultures or cosmetics.
The present invention has been made based on the findings and knowledge of the inventors regarding the following facts and problems:
in the long-term research of bioactive polypeptide, the inventor unexpectedly obtains a heptapeptide with an amino acid sequence of LLPFFGR. Further, the experimental result shows that the polypeptide can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory biological activity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of LPS-induced macrophages.
Thus, in a first aspect of the invention, the invention provides a polypeptide or a pharmaceutically acceptable salt thereof. According to an embodiment of the invention, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1.
LLPFFGR（SEQ ID NO：1）。
The polypeptide can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory biological activity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of LPS-induced macrophages. Therefore, the pharmaceutical composition prepared by the polypeptide or the pharmaceutically acceptable salt thereof has anti-inflammatory biological activity and can be further used for developing products such as medicines, cell cultures or cosmetics.
In a second aspect of the invention, the invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises:
the aforementioned polypeptide;
a conjugate moiety, said conjugate moiety being coupled to said polypeptide.
The polypeptide may be linked to a suitable coupling moiety in order to obtain desired properties of the polypeptide, such as ease of administration, reduced toxicity of the polypeptide to normal tissue, prolonged residence time in the blood circulation system in vivo, improved targeting, etc. Thus, conjugates comprising the polypeptides of the embodiments of the invention are obtained.
In a third aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes a polypeptide as described above. According to the embodiment of the invention, the polypeptide coded by the nucleic acid can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory biological activity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of the LPS-induced macrophages. Therefore, the prepared pharmaceutical composition can be further used for developing products such as medicines, cell cultures or cosmetics by utilizing the anti-inflammatory biological activity of the polypeptide encoded by the nucleic acid.
In a fourth aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises the aforementioned nucleic acid molecule. Thus, the above-described polypeptide can be efficiently expressed by using the constructed construct such as a vector or a transformant.
In a fifth aspect of the invention, the invention provides a cell. According to an embodiment of the invention, the cell carries the aforementioned nucleic acid molecule or the aforementioned construct; or expressing the aforementioned polypeptide. According to the embodiment of the invention, the cell can efficiently express the polypeptide under proper conditions, and further, the obtained polypeptide has anti-inflammatory bioactivity on reducing the amount of NO released by LPS-induced macrophages, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory reactions of the LPS-induced macrophages.
In a sixth aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned conjugate, the aforementioned nucleic acid molecule, the aforementioned construct, or the aforementioned cell. The pharmaceutical composition provided by the embodiment of the invention has anti-inflammatory bioactivity, and can be further used for developing products such as medicines, cell cultures or cosmetics.
In a seventh aspect, the invention provides the use of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned conjugate, the aforementioned nucleic acid molecule, the aforementioned construct, the aforementioned cell or the aforementioned pharmaceutical composition for the preparation of a product for inhibiting LPS-induced release of NO by macrophages, or for the prevention and/or treatment of inflammatory diseases.
Those skilled in the art will appreciate that the features and advantages described above for the polypeptide or a pharmaceutically acceptable salt, conjugate, nucleic acid molecule, construct, cell, inhibitor or pharmaceutical composition thereof are equally applicable to the use of the prepared product and will not be described in detail herein.
The beneficial effects are that:
(1) The invention provides a heptapeptide with an amino acid sequence of Leu-Leu-Pro-Phe-Phe-Gly-Arg (LLPFFGR), which has small molecular weight and easy absorption, has no obvious toxicity in a cell level experiment, and has good drug development value and clinical application prospect.
(2) The heptapeptide can obviously inhibit inflammatory response of LPS-induced macrophages (such as RAW264.7 macrophages), reduce the release amount of NO, has anti-inflammatory biological activity, can be further used for developing products such as medicines, cell cultures or cosmetics, and has wide application prospect.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a high performance liquid chromatography result diagram of a heptapeptide LLPFFGR according to an embodiment of the present invention;
FIG. 2 is a graph showing the results of the primary mass spectrum of the heptapeptide LLPFFGR according to an embodiment of the present invention;
FIG. 3 is a graph showing the effect of different concentrations of heptapeptide on RAW264.7 macrophage growth proliferation according to an embodiment of the invention, with different letters indicating significant differences in mean values (P < 0.05);
FIG. 4 is a graph showing the effect of different concentrations of heptapeptide on LPS-induced RAW264.7 macrophage release of NO according to an embodiment of the invention, with different letters indicating significant differences in average values (P < 0.05).
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
Terms and definitions
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
Herein, the term "conservatively modified form of an amino acid sequence such as" refers to an amino acid modification that does not significantly affect or alter the biological properties of a polypeptide comprising the amino acid sequence of the amino acid, including amino acid substitutions, additions and deletions. Modifications may be introduced into the polypeptides of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
In this context, the term "vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
The invention provides a polypeptide or pharmaceutically acceptable salt, conjugate, nucleic acid molecule, construct, cell, pharmaceutical composition and application thereof, which are respectively described in detail below.
Polypeptides
The invention provides a polypeptide. Or a pharmaceutically acceptable salt thereof. According to an embodiment of the invention, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1.
LLPFFGR（SEQ ID NO：1）。
It should be noted that the amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.
In this context, a "polypeptide" is sometimes referred to as a "heptapeptide" or a "bioactive peptide" or a "bioactive polypeptide".
The polypeptide can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory biological activity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of LPS-induced macrophages. Therefore, the pharmaceutical composition prepared by the polypeptide or the pharmaceutically acceptable salt thereof has anti-inflammatory biological activity and can be further used for developing products such as medicines, cell cultures or cosmetics.
According to an embodiment of the invention, the polypeptide has the sequence as set forth in SEQ ID NO:1, and a conservatively modified form of the amino acid sequence shown in 1.
It is noted that one or more amino acid residues of a polypeptide of the invention may be replaced with other amino acid residues from the same side chain family without substantially affecting the anti-inflammatory properties (retaining at least 90% of the activity) of the polypeptide of the invention, and that altered retention functions of the polypeptide may be tested using the functional assays described herein. Preferably, the conservative modifications do not exceed 1 or 2 in number.
Conjugate(s)
The invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises:
the aforementioned polypeptide;
a conjugate moiety, said conjugate moiety being coupled to said polypeptide.
The term "conjugate" is sometimes referred to herein as a "conjugate" or "conjugate" and refers to a protein or polypeptide fragment conjugated to a coupling moiety such as a carrier substance, drug, toxin, cytokine, protein tag, modification, therapeutic agent, chemotherapeutic agent, or the like, using any covalent or non-covalent bioconjugation strategy.
The polypeptide may be linked to a suitable coupling moiety in order to obtain desired properties of the polypeptide, such as ease of administration, reduced toxicity of the polypeptide to normal tissue, prolonged residence time in the blood circulation system in vivo, improved targeting, etc. Thus, conjugates comprising the polypeptides of the embodiments of the invention are obtained.
According to an embodiment of the invention, the coupling moiety comprises at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modification, a therapeutic agent, and a chemotherapeutic agent.
Nucleic acid molecules
The invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes a polypeptide as described above. According to an embodiment of the invention, the nucleic acid molecule encodes a polypeptide as described above. According to the embodiment of the invention, the polypeptide coded by the nucleic acid molecule can reduce the amount of NO released by LPS-induced macrophages, has anti-inflammatory biological activity, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory response of LPS-induced macrophages. Thus, the prepared pharmaceutical composition can be further used for developing products such as medicines, cell cultures or cosmetics by utilizing the anti-inflammatory biological activity of the polypeptide encoded by the nucleic acid molecules.
It is noted that, for the nucleic acid molecules mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, although only one strand is shown in most cases herein, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosure of one of them means that the other is also disclosed.
Constructs
The invention provides a construct. According to an embodiment of the invention, the construct comprises the nucleic acid molecule described above. Thus, the above-described polypeptide can be efficiently expressed by using the constructed construct such as a vector or a transformant.
According to an embodiment of the invention, the construct may be a vector or a transformant.
According to embodiments of the invention, the construct (e.g., vector or transformant) may include an optional control sequence operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The resulting construct (e.g., vector or transformant) thus constructed can efficiently express the above polypeptide.
In the case of the above-described nucleic acid molecules being linked to the construct (e.g., vector or transformant), such as an expression vector, the nucleic acid molecule may be linked directly or indirectly to control elements on the expression vector, so long as these control elements are capable of controlling translation and expression of the nucleic acid molecule, etc. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. The nucleic acid molecule may be operably linked to a control element.
According to embodiments of the present invention, the above-described vector may refer to a cloning vector, or may refer to an expression vector, which may be obtained by operably linking the nucleic acid to a commercially available vector (e.g., a plasmid or viral vector). The vector of the present invention is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, pMH3, etc. can be used.
As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the nucleic acid molecule can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the polypeptide encoded by the nucleic acid molecule.
According to an embodiment of the invention, the vector is a eukaryotic vector or a prokaryotic vector.
Cells
The invention provides a cell. According to an embodiment of the invention, the cell carries the aforementioned nucleic acid molecule or the aforementioned construct; or expressing the aforementioned polypeptide. According to the embodiment of the invention, the cell can efficiently express the polypeptide under proper conditions, and further, the obtained polypeptide has anti-inflammatory bioactivity on reducing the amount of NO released by LPS-induced macrophages, has low cytotoxicity and good safety, and can be used for preventing and/or treating inflammatory diseases or inhibiting inflammatory reactions of the LPS-induced macrophages.
In some embodiments of the invention, the cell is a prokaryotic cell, a eukaryotic cell, or a phage.
In some embodiments of the invention, the prokaryotic cell is E.coli, B.subtilis, streptomyces, or Proteus mirabilis.
In some embodiments of the invention, the eukaryotic cell is a fungus, an insect cell, a plant cell, or a mammalian cell.
In some embodiments of the invention, the fungus is pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, or trichoderma.
In some embodiments of the invention, the insect cell is a myxoplasma gondii cell; according to an embodiment of the invention, the plant cell is a tobacco plant cell; according to an embodiment of the invention, the mammalian cell is a BHK cell, CHO cell, COS cell, myeloma cell or human embryonic kidney 293 cell; and does not include animal germ cells, fertilized eggs, or embryonic stem cells.
In some embodiments of the invention, the cell is a mammalian cell.
In some embodiments of the invention, the cell is a BHK cell, CHO cell, COS cell, or NSO cell.
The term "suitable conditions" as used herein refers to conditions suitable for expression of the polypeptide of the present invention. Those skilled in the art will readily appreciate that conditions suitable for polypeptides include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for forming the optimum expression of the polypeptide according to the specific environment of the laboratory.
Pharmaceutical composition
The invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned conjugate, the aforementioned nucleic acid molecule, the aforementioned construct, or the aforementioned cell. The pharmaceutical composition provided by the embodiment of the invention has anti-inflammatory bioactivity, and can be further used for developing products such as medicines, cell cultures or cosmetics.
According to an embodiment of the invention, further comprising pharmaceutically acceptable excipients.
According to an embodiment of the present invention, the pharmaceutical composition is a pharmaceutical injection.
The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, solid carriers, or both.
As used herein, the term "pharmaceutically acceptable ingredients" is a substance suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the polypeptides of the invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.
Use of the same
The present invention provides the use of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned conjugate, the aforementioned nucleic acid molecule, the aforementioned construct, the aforementioned cell or the aforementioned pharmaceutical composition for the preparation of a product for inhibiting the release of NO by LPS-induced macrophages, or for the prevention and/or treatment of inflammatory diseases.
Those skilled in the art will appreciate that the features and advantages described above for the polypeptide or a pharmaceutically acceptable salt, conjugate, nucleic acid molecule, construct, cell, inhibitor or pharmaceutical composition thereof are equally applicable to the use of the prepared product and will not be described in detail herein.
According to an embodiment of the invention, the product comprises a medicament, a cell culture or a cosmetic;
when the product is a medicament, the product is for use in the prevention and/or treatment of inflammatory diseases;
when the product is a cell culture or a cosmetic, the product is used to inhibit LPS-induced macrophage release of NO.
According to an embodiment of the invention, the inflammatory disease is at least one of hyperacute inflammation, acute inflammation, chronic inflammation or subacute inflammation.
According to an embodiment of the invention, the inflammatory disease is at least one of degenerative, exudative, serous, cellulosic or proliferative inflammation.
Method for treating disease
The present invention provides a method for preventing and/or treating inflammatory diseases. According to an embodiment of the invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned conjugate, the aforementioned nucleic acid molecule, the aforementioned construct, the aforementioned cell, or the aforementioned pharmaceutical composition.
It is noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and/or being treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, and the like. The subject may be a human, but also includes other mammals, particularly mammals that may be used as laboratory models of human diseases, such as mice, rats, and the like.
As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The polypeptide, conjugate, nucleic acid molecule, construct, cell, inhibitor or pharmaceutical composition of the invention may be administered by any common route, provided that it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these illustrated modes of administration. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.
In this context, the term "treatment" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.
As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.
The effective amount of the polypeptide or pharmaceutically acceptable salt, conjugate, nucleic acid molecule, construct, cell or pharmaceutical composition of the invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.
According to an embodiment of the invention, the inflammatory disease is at least one of hyperacute inflammation, acute inflammation, chronic inflammation or subacute inflammation.
According to an embodiment of the invention, the inflammatory disease is at least one of degenerative, exudative, serous, cellulosic or proliferative inflammation.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. 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.
Example 1: solid-phase chemical synthesis, separation and identification of polypeptides
The chemical synthesis of the polypeptide (Leu-Leu-Pro-Phe-Phe-Gly-Arg) adopts the Fmoc solid-phase synthesis technology of the polypeptide, and the reverse synthesis is carried out from the C end to the N end of the polypeptide sequence.
The carboxyl of the first amino acid is covalently linked to the carrier resin, and then the amino of the first amino acid is taken as a reaction starting point to carry out acylation reaction with the carboxyl of the adjacent amino acid to form peptide bond. This process is repeated until the target polypeptide is synthesized, the Fmoc protecting group is removed and the resin is drained. With 6 times the resin volume of the cleavage liquid (97.50% TFA+2.50% H) 2 O) cutting, washing the precipitate 3 times with anhydrous diethyl ether, and obtaining the crude polypeptide.
The polypeptide with the purity of more than 95 percent is obtained through high performance liquid chromatography separation and purification. The results are shown in FIG. 1.
The molecular weight of the polypeptides was confirmed using mass spectrometry techniques. The results are shown in FIG. 2.
The above experimental results show that the polypeptide of the present invention (amino acid sequence LLPFFGR (SEQ ID NO: 1)) can be obtained by chemical synthesis.
Further, the polypeptide was formulated into a 10mg/mL polypeptide stock with PBS solution to examine its effect on growth and proliferation of different tumor cells.
Example 2: effect of different concentrations of polypeptide on RAW264.7 macrophage growth proliferation
Adherent cells, preparation of reagents: cell counting of RAW264.7 macrophages in logarithmic growth phase was performed according to 1X 10 4 The wells were inoculated into 96-well plates, a 37℃incubator (5% CO) 2 ) Culturing until the cells adhere to the wall. 10mg/mL of the polypeptide stock was diluted in whole medium in gradient to a concentration of 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 400. Mu.g/mL, 800. Mu.g/mL, 1000. Mu.g/mL of polypeptide dilution.
Polypeptide intervention and cell viability detection: the 96-well plate was removed, old medium was aspirated, and 100. Mu.L of polypeptide dilutions of different concentrations (3 wells per polypeptide dilution concentration) were added, respectively, for incubation for 24 hours. Subsequently, 20. Mu.L of 5mg/mL MTT solution was added to each well and incubation was continued for 4 hours, and absorbance was measured at 490 nm. Taking a complete culture medium without adding polypeptide solution as a blank control, and calculating the cell survival rate according to the formula (1):
wherein: a1 is the absorbance value of the sample group and A2 is the absorbance value of the blank group.
The results are shown in FIG. 3. When the polypeptide solution concentration is 50, 100, 200, 400, 800 and 1000 mug/mL, the survival rate of RAW264.7 macrophages is 101.96%, 96.99%, 93.91%, 94.90%, 92.87% and 93.64%, respectively, and the polypeptide has no significant effect on the survival rate of RAW264.7 macrophages (P < 0.05).
The experimental results show that the polypeptide has low toxicity and good safety.
Example 3: effect of different concentrations of polypeptide on LPS-induced RAW264.7 macrophage NO production
Cell counting of RAW264.7 macrophages in logarithmic growth phase was performed according to 1X 10 4 The wells were inoculated into 96-well plates, a 37℃incubator (5% CO) 2 ) Culturing until the cells adhere to the wall. The polypeptide solution was diluted with complete medium, and after adding 100. Mu.L of the above dilution to each well for incubation for 8 hours, 2. Mu.g/mL of LPS solution was added thereto for incubation for 24 hours. The amount of NO released was determined using a kit (bi yun tian corporation), i.e. 50 μl of supernatant was mixed with 50 μl of Griess reagent i, griess reagent ii, OD values were detected at 540nm, and the sample NO concentration was calculated according to a standard curve.
The experimental results are shown in FIG. 4.
(1) Compared with a blank group, the release rate of the cell NO in the control group induced by LPS reaches 255.22%, which shows that the LPS can obviously promote the generation of inflammatory mediator NO, and the modeling of a cell inflammation model is successful.
(2) Compared with a control group, the sample group added with the polypeptide solution has remarkably reduced cell NO release rate (P < 0.05) and dose dependency in the range of 100-1000 mug/mL. The sample group had a cell NO release rate of 158.55% when the concentration of the polypeptide solution was 1000. Mu.g/mL.
The experimental result shows that the polypeptide can inhibit the NO secretion of RAW264.7 cells induced by LPS and has stronger anti-inflammatory effect.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (13)
1. A polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is as set forth in SEQ ID NO: 1.
2. A conjugate, comprising:
the polypeptide of claim 1;
a conjugate moiety, said conjugate moiety being coupled to said polypeptide.
3. The conjugate of claim 2, wherein the coupling moiety comprises at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modification, a therapeutic agent, and a chemotherapeutic agent.
4. A nucleic acid molecule encoding the polypeptide of claim 1.
5. A construct comprising the nucleic acid molecule of claim 4.
6. A cell carrying the nucleic acid molecule of claim 4 or the construct of claim 5; or expressing the polypeptide of claim 1.
7. The cell of claim 6, wherein the cell is a prokaryotic cell, a eukaryotic cell, or a phage.
8. A pharmaceutical composition comprising:
the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 2 to 3, the nucleic acid molecule of claim 4, the construct of claim 5, or the cell of any one of claims 6 to 7.
9. The pharmaceutical composition of claim 8, further comprising a pharmaceutically acceptable adjuvant.
10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is a pharmaceutical injection.
11. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 2 to 3, the nucleic acid molecule of claim 4, the construct of claim 5, the cell of any one of claims 6 to 7 or the pharmaceutical composition of any one of claims 8 to 10 for the preparation of a product for inhibiting LPS-induced release of NO by macrophages, or for the prevention and/or treatment of inflammatory diseases.
12. Use according to claim 11, wherein the product comprises a medicament, a cell culture or a cosmetic;
when the product is a medicament, the product is for use in the prevention and/or treatment of inflammatory diseases;
when the product is a cell culture or a cosmetic, the product is used to inhibit LPS-induced macrophage release of NO.
13. The use according to claim 11, wherein the inflammatory disease is at least one of hyperacute inflammation, acute inflammation, chronic inflammation or subacute inflammation.
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| CN102639556A (en) * | 2009-09-22 | 2012-08-15 | X免疫股份公司 | Polypeptides and uses thereof |
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| WO2022094437A1 (en) * | 2020-11-02 | 2022-05-05 | Oneskin, Inc. | Polypeptides having anti-inflammatory effects and uses thereof |
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| CN102639556A (en) * | 2009-09-22 | 2012-08-15 | X免疫股份公司 | Polypeptides and uses thereof |
| CN108794588A (en) * | 2018-06-29 | 2018-11-13 | 上海铂辉生物科技有限公司 | A kind of biologically active polypeptide FDPTLHQ and its preparation method and application |
| CN108794590A (en) * | 2018-06-29 | 2018-11-13 | 上海铂辉生物科技有限公司 | A kind of biologically active polypeptide EPGIVNLD and its preparation method and application |
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| CN118005729A (en) * | 2024-02-27 | 2024-05-10 | 中国农业大学 | Polypeptide with tyrosinase inhibitory activity and application thereof |
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| CN116836233B (en) | 2023-11-14 |
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