WO2018196743A1 - Oligopeptide fonctionnel amyloïde a1 sérique humain, son procédé de préparation et son application - Google Patents

Oligopeptide fonctionnel amyloïde a1 sérique humain, son procédé de préparation et son application Download PDF

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WO2018196743A1
WO2018196743A1 PCT/CN2018/084256 CN2018084256W WO2018196743A1 WO 2018196743 A1 WO2018196743 A1 WO 2018196743A1 CN 2018084256 W CN2018084256 W CN 2018084256W WO 2018196743 A1 WO2018196743 A1 WO 2018196743A1
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polypeptide fragment
human serum
serum amyloid
fragment
polypeptide
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叶R·德全
陈明杰
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上海交通大学
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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of bioengineering, and particularly relates to a functional fragment of human serum amyloid A1 and a preparation method and application thereof.
  • Serum Amyloid A is a major acute phase protein secreted by the body when infected and damaged.
  • SAA is a general term for a polymorphic protein consisting of related proteins (SAA1-SAA4) with independent genes.
  • human serum amyloid A1 (SAA1) is composed of 104 amino acids, mainly synthesized by hepatocytes.
  • Human SAA2 and SAA1 have 7 amino acid differences, and they are all acute phase proteins.
  • SAA3 is a pseudogene, and SAA4 does not change much in the acute phase reaction and continues to be produced in small amounts in the body.
  • SAA has a large increase in serum levels in patients with acute and chronic inflammation, and is therefore an important biomarker for reflecting infectious and non-infectious inflammatory diseases.
  • SAA is not only a biomarker for the acute exacerbation of chronic obstructive pulmonary disease, but also an expected factor for heart rupture in patients with acute myocardial infarction after emergency coronary intervention, which provides an important reference for early detection of cardiac rupture.
  • a number of other studies have also shown that in patients with pancreatitis, hepatitis, coronary heart disease, diabetes, chronic kidney disease, rheumatoid arthritis, inflammatory bowel disease, acute and chronic pneumonia, and obesity, SAA elevation and disease activity and disease progression have Very close relationship.
  • SAA is not only a simple marker of inflammation, but also actively involved in the process of disease.
  • the level of serum SAA can be increased by a factor of 1000.
  • High levels of SAA will cause amyloidosis, and a combination of low-density lipoprotein and SAA (LDL-SAA) may increase the risk of cardiovascular disease; at the same time, SAA also has a pro-inflammatory response that promotes multiple cytokines.
  • TLRs Toll-like receptors
  • SRB1 a class B scavenger receptor
  • FPR2 G-protein coupled receptor foryl peptide receptor 2
  • RAGE receptor For advanced glycation endproduct
  • P2X7 receptor P2X7 receptor
  • SAA promotes the metabolism of cholesterol; SAA binds to SRB1 to produce inflammation-inducing factors, leading to diseases such as atherosclerosis; SAA binds to TLR4 to induce NO production.
  • SAA1 exerts a variety of biological activities in the body.
  • this patent designs a yeast expression system to express recombinant human SAA1 and its structurally similar polypeptide without lipopolysaccharide interference.
  • This yeast-host expression not only rids the problem of lipopolysaccharide that has been encountered in SAA1 research for many years, but also realizes the biological effects of SAA1 which is not interfered by lipopolysaccharide in vitro and in vivo, and can also express it by yeast expression system.
  • a variety of SAA1 structurally similar polypeptides were functionally studied to discover the domains and domains in which SAA functions.
  • detailed bioassay results of different peptides of human SAA1 can be used to design corresponding blocking agents to provide potential drug candidates for clinical treatment.
  • a human serum amyloid A1 polypeptide fragment or a pharmaceutically acceptable salt thereof, is provided, the polypeptide fragment having the following characteristics:
  • sequence of the polypeptide fragment is derived from the amino acid sequence of human serum amyloid A1;
  • the polypeptide fragment is from position M to position N of the full-length amino acid sequence (104 amino acids) of human serum amyloid A1 (SAA1), and is 42-67 amino acids in length, wherein M is any of 9-14 A positive integer, N is any positive integer from 55-75.
  • polypeptide fragment further has one or more (or all) characteristics selected from the group consisting of:
  • the polypeptide fragment has the ability to inhibit the expression of macrophage inflammatory cytokines by Gram-negative bacterial endotoxin LPS;
  • the inflammatory cytokine is selected from the group consisting of: IL-6, IL-1 ⁇ , TNF- ⁇ ;
  • the polypeptide fragment has the ability to inhibit death caused by Gram-negative bacterial endotoxin LPS-induced sepsis;
  • the polypeptide fragment is capable of inducing expression of an anti-inflammatory cytokine by a macrophage
  • the polypeptide fragment has the ability to induce phosphorylation of p38 MAPK in macrophages.
  • the anti-inflammatory cytokine is selected from the group consisting of: IL-10; the ability to induce the anti-inflammatory cytokine IL-10 is dependent on Toll-like receptor 2 (TLR2).
  • TLR2 Toll-like receptor 2
  • P MN represents a polypeptide consisting of the Mth to Nth positions of the amino acid sequence of serum amyloid A1.
  • M is 9, 10, or 11.
  • N is 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • the polypeptide is selected from the group consisting of: P 11-58, P 11-68, P 11-72, P 11-55, P 11-75, P 10-58, P 10-68 , P 10-72 , P 10-55 , P 10-75 , or a combination thereof.
  • polypeptide has positions 11-58, 11-68, 11-72, or a combination thereof of the amino acid sequence of wild-type human serum amyloid A1.
  • amino acid sequence of wild-type human serum amyloid A1 is set forth in SEQ ID NO.: 2.
  • the N-terminus and/or C-terminus of the polypeptide fragment also carries a tag sequence (such as a 6His, or HIS6-SUMO-TEV fragment) for purification isolation or the like.
  • a tag sequence such as a 6His, or HIS6-SUMO-TEV fragment
  • the polypeptide fragment is a polypeptide fragment obtained by expressing the yeast as a host.
  • amino acid sequence of the human serum amyloid A1 is a wild type sequence.
  • amino acid sequence of the human serum amyloid A1 is shown in SEQ ID NO.: 2.
  • the proinflammatory cytokine is selected from the group consisting of IL-6, IL-1 ⁇ , TNF- ⁇ , or a combination thereof.
  • the polypeptide fragment has the ability to promote expression of an anti-inflammatory cytokine.
  • the anti-inflammatory factor is IL-10.
  • polypeptide fragment is recombinant.
  • polypeptide is expressed in yeast.
  • a fusion protein comprising the human serum amyloid A1 polypeptide fragment of the first aspect of the invention and a tag sequence fused to the fragment is provided.
  • the tag sequence is fused to the N-terminus and/or C-terminus of the polypeptide fragment.
  • the tag sequence is selected from the group consisting of His6, His6-SUMO, His6-SUMO-TEV fragments.
  • a polynucleotide sequence encoding the human serum amyloid A1 polypeptide fragment of the first aspect of the invention, or the second aspect of the invention is provided The fusion protein described.
  • a yeast expression vector comprising a polynucleotide encoding the polypeptide fragment of the first aspect of the invention is provided.
  • the vector is a yeast expression vector pPIC9K comprising a polynucleotide encoding the polypeptide fragment of the first aspect of the invention.
  • a host cell is provided, the host cell being selected from the group consisting of:
  • the host cell is selected from the group consisting of a yeast cell, Escherichia coli for expressing the SAA1 protein.
  • a method of preparing a polypeptide fragment of the first aspect of the invention comprising the steps of:
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient; and the human serum amyloid A1 polypeptide fragment of the first aspect of the invention as an active ingredient.
  • the pharmaceutical composition is in the form of an injection preparation, a transdermal preparation, or a lyophilized powder.
  • the invention provides a human serum amyloid A1 polypeptide fragment of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, for use in the preparation of a medicament for inhibiting inflammation and/or treating inflammation The drug of the disease.
  • the disease caused by the inflammation is selected from the group consisting of sepsis, rheumatoid arthritis, atherosclerosis, acute and chronic pneumonia, chronic enteritis, pancreatitis, hepatitis, diabetes, chronic kidney disease. Obesity, muscle atrophy, gout.
  • the inflammation is inflammation caused by excessive SAA1.
  • the inflammation is characterized by an overexpression of SAA1.
  • the use of the human serum amyloid A1 polypeptide fragment of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, for the preparation of a composition or formulation is used to stimulate macrophage secretion of the anti-inflammatory factor IL-10.
  • a method of treating a disease caused by inflammation comprising administering a human serum amyloid A1 polypeptide fragment of the first aspect of the invention or a pharmaceutically acceptable salt thereof to a subject in need thereof Or protein complex.
  • Figure 1 is a gel electrophoresis analysis of the position of the SAA1 fragment relative to the full-length SAA1 and yeast expression.
  • Figure 2 shows a comparison of the ability of the SAA1 fragment to block the expression of the inflammatory cytokines IL-6, IL-1 ⁇ and TNF- ⁇ compared to full-length SAA1.
  • Figure 3 is a graph showing the relationship between the inflammatory cytokines IL-6, IL-1 ⁇ and TNF- ⁇ expression by the SAA1 fragment blocking LPS.
  • Figure 4 is a graph showing the dose relationship of the 11-58 peptide fragment blocking LPS-induced expression of the inflammatory cytokines IL-6, IL-1 ⁇ and TNF- ⁇ .
  • Figure 5 shows that the 11-58 peptide fragment effectively inhibits LPS-induced mouse death.
  • FIG. 6 shows that the 11-58 peptide fragment itself does not induce elevation of lung myeloperoxidase (MPO) in mice, but inhibits LPS-induced elevation of MPO in the lung; MPO is characteristic of neutrophils, Its elevation reflects the infiltration of neutrophils in the lungs.
  • MPO myeloperoxidase
  • Figure 7 shows that the 11-58 peptide fragment inhibits LPS-induced expression of the inflammatory cytokines IL-6, IL-1 ⁇ and TNF- ⁇ in mouse lung tissues.
  • Figure 8 is a section of lung tissue showing that mice receiving the 11-58 peptide fragment have some resistance to LPS-induced acute lung injury, maintaining intact lung tissue structure and less inflammatory cell infiltration.
  • Figure 9 shows that the ability of the 11-58 peptide fragment to induce MAP kinase activation by TLR2 differs from full-length SAA1.
  • Figure 10 shows that the 11-58 peptide fragment is capable of modulating LPS-induced MAP kinase activation in macrophages.
  • Figure 11 shows that the 11-58 peptide fragment induces the expression of mRNA and protein of the anti-inflammatory cytokine IL-10.
  • Figure 12 is a graph comparing the expression of the anti-inflammatory cytokine IL-10 after the 11-58 peptide fragment was used alone or in combination with LPS.
  • Figure 13 shows macrophages derived from mice lacking the TLR2 and TLR4 genes, which have different responses to 11-58 peptide fragments and full-length SAA1-induced IL-10 expression.
  • Figure 14 shows an RBL cell line expressing human FPR2, which is stimulated by the 11-58 peptide fragment and full-length SAA1 to produce different chemotactic migration reactions.
  • Figure 15 shows that the 11-58 peptide fragment and full length SAA1 have different ability to induce cellular calcium production.
  • Figure 16 shows the results of analysis of 11-58 and full-length SAA1 by exclusion chromatography using SEC-HPLC markers as a reference.
  • Figure 17 shows the results of comparing 11-58 stimulation of p38 MAPK phosphorylation in HeLa and TLR2-HeLa.
  • the anti-inflammatory polypeptide of the present invention is a human serum amyloid A1 functional short peptide having an anti-inflammatory action.
  • the short peptide of the present invention (exemplified by the short peptide 11-58) not only has a significant decrease in the inflammatory effect (even a substantial loss or complete loss of the inflammatory effect), but also can extremely effectively inhibit the inflammatory response caused by LPS, and is extremely efficient. Induces the expression of anti-inflammatory cytokines. On the basis of this, the present invention has been completed.
  • pPIC9K is a secreted vector for the efficient expression of foreign proteins with a signal peptide ⁇ -factor, which can secrete SAA1 and its fragments into the medium for subsequent purification.
  • the restriction enzyme sites XhoI and EcoRI were used to link SAA1 to the downstream of the ⁇ -factor of the vector pPIC9K, and indirectly have a Kex2 signal peptide cleavage sequence, which can excise the ⁇ -factor during secretion expression and release the N-terminus of SAA1.
  • the expressed SAA1 fragment was stained with Coomassie brilliant blue after SDS gel electrophoresis and showed the expected length (Fig. 1).
  • SAA1 fragment sequences include, but are not limited to, the following fragments (numbers represent the starting and ending amino acid positions): 11-58, 11-68, 11-72, 27-72, 27-90, 29-104.
  • These polypeptide fragments were also expressed using the yeast expression vector pPIC9K and yeast strain GS115.
  • fragment 11-58 was found to have the most significant inhibitory effect on LPS-induced inflammatory response.
  • the 11-58 fragment with the most amino acid removal at the C-terminus and the deletion of 10 amino acids at the N-terminus showed the strongest inhibition of the expression of inflammatory cytokines IL-6, TNF- ⁇ and IL-1 ⁇ induced by LPS (Fig. 3).
  • the fragment showed concentration-dependent inhibition (Fig. 4).
  • SAA1 fragments expressed by yeast as a host are more helpful in obtaining the ability to inhibit proinflammatory cytokines.
  • mice were subsequently examined.
  • C57BL/6 mice were selected, which were the same strain as the acute lung injury test.
  • the mortality at 48 hours at a dose of 20 mg/kg LPS was 90%.
  • the 11-58 fragment dose of 5 mg/kg was pre-administered in the abdominal cavity, and the administration of LPS after half an hour reduced the mortality rate to 40%, indicating that the 11-58 fragment was effective in reducing the mortality caused by LPS (Fig. 5).
  • Interleukin 10 is a major anti-inflammatory factor. It was found that mouse bone marrow macrophages were stimulated by 11-58 fragment, and there was a large amount of IL-10 mRNA expression within 2-8 hours, and IL-10 protein level also increased, especially in the interval of 4-12 hours ( Figure 11). In contrast, LPS induced little IL-10 expression, whereas LPS shared no significant effect on IL-10 production when shared with 11-58 fragments ( Figure 12).
  • bone marrow macrophages were isolated from wild-type C57BL/6 hours and TLR2 knockout mice of the same genetic background, using full-length SAA1 and 11-58 fragment stimulation, to investigate the expression level of IL-10.
  • the 11-58 fragment was found to stimulate wild-type mouse bone marrow macrophages to produce more (about 6-fold) IL-10 compared to full-length SAA1.
  • the ability of the full-length SAA1 and 11-58 fragments to stimulate IL-10 production was significantly reduced (Fig. 13). Therefore, it is speculated that the 11-58 fragment mainly stimulates macrophages to produce IL-10 by TLR2.
  • polypeptide fragment of the invention As used herein, the terms “polypeptide fragment of the invention”, “polypeptide fragment of human serum amyloid A1 of the invention”, “fragment of the invention” or “short peptide of the invention” are used interchangeably and refer to the first aspect of the invention A polypeptide fragment derived from serum amyloid A1, or a pharmaceutically acceptable salt thereof, as defined in the aspects. Furthermore, it is to be understood that although the preferred serum amyloid A1 is derived from humans, it may also be derived from other mammals (e.g., other non-human primates).
  • the present invention mainly provides a human serum amyloid A1 polypeptide fragment having the following characteristics:
  • sequence of the polypeptide fragment is derived from the amino acid sequence of human serum amyloid A1;
  • the polypeptide fragment is from position M to position N of the full-length amino acid sequence (104 amino acids) of human serum amyloid A1 (SAA1), and is 42-67 amino acids in length, wherein M is any of 9-14 A positive integer, N is any positive integer from 55-75.
  • Representative polypeptide fragments of the present invention including, but not limited to, P 11-58, P 11-68, P 11-72 , or a combination thereof.
  • the numbers represent the amino acid positions at the beginning and end of the amino acid sequence of human serum amyloid A1.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • isolated polypeptide fragment of the invention means that the polypeptide fragment of the invention is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify the polypeptide fragments of the invention using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide fragments of the invention can be analyzed by amino acid sequence.
  • the polypeptide fragment of the invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide fragments of the invention may be naturally purified products including products hydrolyzed by proteases, or products of chemical synthesis, or recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals). Produced in cells).
  • the polypeptide of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the polypeptide fragments of the invention.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of a native polypeptide fragment of the invention of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment).
  • a polypeptide having one or more conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It
  • short peptide of the present invention refers to a polypeptide having the sequence of SEQ ID NO: 2 which is active in the polypeptide fragment of the present invention.
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 2 that have the same function as the polypeptide fragments of the invention. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the polypeptide fragments of the invention.
  • the invention also provides analogs of the polypeptide fragments of the invention.
  • the difference between these analogs and the natural short peptide of the present invention may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • polypeptide fragment conservative variant polypeptide of the present invention means having up to 8, preferably up to 5, more preferably up to 3, optimally compared to the amino acid sequence of SEQ ID NO: 2. Up to 2 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1 or may be a degenerate variant.
  • a "degenerate variant" in the present invention refers to a polypeptide fragment encoding a sequence having the MN position in SEQ ID NO: 2, but differs from the coding region sequence shown in SEQ ID NO: 1. Nucleic acid sequence.
  • a polynucleotide encoding a polypeptide of position MN of SEQ ID NO: 2 comprises: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding) Sequence) and non-coding sequences.
  • polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the full length nucleotide sequence of the short peptide of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant polypeptide fragments of the present invention by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:
  • a polynucleotide sequence encoding a polypeptide of the present invention can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to, T7-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells, etc.
  • a preferred host cell is a yeast cell.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the recombinant human serum amyloid A1 polypeptide fragment of the present invention can be used for detecting the effects of these products on the secretion of cytokines such as IL-6, TNF- ⁇ , IL-1 ⁇ , IL-10.
  • the recombinant SAA1 polypeptide fragment of the present invention has the ability to inhibit the expression of proinflammatory cytokines and stimulate the expression of the anti-inflammatory factor IL-10, and can be used for preparing a medicament for treating an inflammatory disease; the inflammatory disease is selected from the group consisting of rheumatism. Arthritis, acute and chronic lung injury, chronic enteritis, atherosclerosis, cardiovascular disease, pancreatitis, hepatitis, diabetes, chronic kidney disease, obesity, muscle atrophy, gout.
  • the recombinant human serum amyloid A1 polypeptide fragment of the present invention has the property of reducing the lethality of LPS, and can be used for treating sepsis and tissue damage caused by accumulation of a large amount of endotoxin LPS after Gram-negative bacterial infection.
  • the recombinant human serum amyloid A1 polypeptide fragment of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually from about 5 to 8, preferably from about 6 to about 8. , although the pH may vary depending on the nature of the substance being formulated and the condition being treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • the recombinant human serum amyloid A1 polypeptide fragment of the present invention can be directly used for the treatment of diseases caused by inflammation, for example, for the treatment of rheumatoid arthritis diseases.
  • other therapeutic agents may be used simultaneously or in combination with other therapies, for example, sepsis and tissue damage caused by accumulation of endotoxin LPS after Gram-negative bacterial infection. Treatment of (eg acute lung injury).
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of a recombinant human serum amyloid A1 polypeptide fragment of the invention, or a combination thereof; and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition can be prepared by a conventional method.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the recombinant human serum amyloid A1 polypeptide fragments of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of a recombinant human serum amyloid A1 polypeptide fragment of the invention, or a combination thereof is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases Not more than about 8 mg/kg body weight, preferably about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the recombinant human serum amyloid A1 polypeptide fragment of the present invention has a significantly reduced activity of inducing expression of an inflammatory cytokine, and a significantly increased induction, as compared with the full-length recombinant human serum amyloid A1.
  • Activity of anti-inflammatory cytokine IL-10 expression is a significantly reduced activity of inducing expression of an inflammatory cytokine, and a significantly increased induction, as compared with the full-length recombinant human serum amyloid A1.
  • the present inventors have unexpectedly found that recombinant human serum amyloid A1 polypeptide fragments have an inhibitory effect on the expression of various proinflammatory cytokines induced by LPS.
  • the inventors have unexpectedly discovered that recombinant human serum amyloid A1 polypeptide fragments are capable of protecting mice from acute lung damage caused by LPS.
  • the present invention determines that these polypeptide fragments can reduce the lethality of LPS in mice by preliminary animal experiments.
  • the first step design primers, clone hSAA1 gene and its polypeptide fragments (11-58, 11-68, 11-72, 27-72, 27-90, 29-104 and other different peptides), both ends of the fragment With the enzyme cleavage site XhoI/EcoRI.
  • the target fragment was obtained by PCR, digested with XhoI/EcoRI, and the product was ligated with pPIC9k XhoI/EcoRI double-digested product to obtain an expression vector.
  • the second step the expression vector was transfected into yeast GS115 by electroporation, the yeast monoclonal was screened by histidine auxotrophic plate, and the obtained monoclonal coated G418 screening plate was used, and the concentration of G418 was 0.5 mg/mL, 1 mg, respectively. /mL, 2mg/mL, cultured at 30 °C for 3-4 days, the monoclonal culture grown on the G418 concentration 2mg/mL plate was selected, and the target protein was induced by methanol. A large number of single clones with higher yields were selected for further fermentation and purification.
  • the yeast fermentation broth supernatant was concentrated by ultrafiltration, mixed with Ni Sepharose HP at 4 ° C for 1-2 hours, and packed.
  • the target protein was eluted by washing the column with 20 mM sodium phosphate, 25 mM imidazole, 100 mM NaCl, pH 7.4, 20 mM sodium phosphate, 500 mM imidazole, 100 mM NaCl, pH 7.4, and detected by SDS-PAGE. Then, the His6-TEV enzyme was added to digest at 4 ° C overnight, and the digested product was again passed through a Ni 2+ affinity chromatography column to remove the enzyme-cut His6-SUMO and His6-TEV enzymes.
  • the purified SAA1 or fragment was stored in 20 mM phosphate buffer (pH 7.4) and stored at -80 °C for a long period of time.
  • the molecular weight and purity of SAA were determined by SDS-PAGE, the protein concentration was determined by BCA method, and the endotoxin content was detected by sputum reagent method.
  • hSAA1 and its polypeptide fragment were added to the serum-free cultured BMDM cell culture medium at a final concentration of 0.5 ⁇ M. After 5 hours, the cells were collected, TRIzol was lysed, RNA was extracted, reverse transcribed into cDNA, and cytokine was detected by real-time PCR. Expression status. For experiments with LPS co-stimulation, short peptides were added and half an hour later, 100 ng/ml LPS was added. The cells were collected after 5 hours, and the expression of cytokines was detected by real-time PCR.
  • Figure 3 shows the effect of different peptides blocking inflammatory cytokines by LPS. the result shows,
  • polypeptide fragments (11-58, 11-68, 11-72, 27-72, 27-90, 29-104, 11-45) showed some inhibitory effects, among which peptides 27-72, 27 The inhibition effects of -90, 29-104, and 11-45 are low.
  • the polypeptide fragments 11-58, 11-68 and 11-72 exhibited a surprisingly high inhibitory effect, for example, a higher inhibitory activity than peptides such as peptides 27-72 or 27-90.
  • the inhibitory activities of P11-58 and P11-68 were increased by about 5-10 times as compared with the inhibitory activity of P27-72.
  • P11-58 ie, fragment 11-58
  • P11-58 had the most significant inhibitory effect on LPS-induced inflammatory response, indicating the strongest inhibition of LPS-induced inflammatory cytokines IL-6, TNF- ⁇ , IL- The role of 1 ⁇ expression.
  • the 11-58 polypeptide fragment inhibits LPS-induced release of proinflammatory cytokines and induces higher levels of IL-10 secretion by itself.
  • the BMDM cells were isolated, and the 11-58 short peptide was added to the serum-free cultured BMDM cell culture medium at a final concentration of 0.5 ⁇ M. After half an hour, 100 ng/ml LPS was added, and the cells were collected 5 hours later (or cells were collected at different time points). ), TRIzol cleavage, RNA extraction, reverse transcription into cDNA, SYBR Green dye was added for Real-time PCR detection, detection of proinflammatory cytokines IL-6, IL-1 ⁇ , TNF- ⁇ , anti-inflammatory cytokine IL-10 mRNA expression level. Finally, the experimental Ct values were analyzed to calculate the level of transcriptional regulation of specific genes between different samples.
  • the polypeptide fragment 11-58 inhibited the production of IL-6, TNF ⁇ and IL-1 ⁇ by LPS in a dose-dependent manner.
  • the results showed that the 11-58 polypeptide fragment alone stimulated bone marrow macrophages to produce large amounts of IL-10.
  • the ability to stimulate IL-10 production was not significantly affected when used in combination with LPS.
  • the 11-58 polypeptide fragment stimulated bone marrow macrophages to produce more IL-10 (Fig. 13).
  • the 11-58 short peptide exerts an anti-inflammatory effect mainly by inhibiting the elevation of inflammatory factors caused by LPS and simultaneously stimulating the production of the anti-inflammatory factor IL-10 by bone marrow macrophages.
  • 11-58 short peptide inhibits LPS-induced ERK and JNK phosphorylation levels up-regulated
  • Mouse bone marrow macrophages were isolated, and different concentrations of 11-58 polypeptide fragments were added. After one hour, LPS was added to a final concentration of 500 ng/ml, mixed, and cultured at 37 °C. One hour later, the cells were lysed, and the phosphorylation level of MAPK was detected by Western blot. The results are shown in Fig. 10. LPS can up-regulate the phosphorylation levels of ERK, p38 and JNK in mouse bone marrow macrophages. The peptide fragments can block the increase of phosphorylation of ERK and JNK by LPS, but can cause the increase of p38 phosphorylation level by itself. .
  • the present inventors isolated wild-type mouse bone marrow macrophages and TLR2 ⁇ / ⁇ mouse bone marrow macrophages, and stimulated with apoSAA and 11-58 polypeptide fragments respectively, and examined the expression levels of IL-10 at different time points. 6.11-58 polypeptide fragment is capable of stimulating the production of IL-10 by wild-type mouse bone marrow macrophages, but this ability is substantially lost in TLR2 ⁇ / ⁇ mice, therefore, the inventors speculate that 11-58 polypeptide Fragments stimulate macrophages to produce IL-10 which is TLR2-dependent.
  • the 11-58 short peptide activates TLR2 to produce a signaling pathway for IL-10.
  • the TLR2-HeLa stable cell line was cultured to detect the activation of NF- ⁇ B and the phosphorylation level of MAPK by 11-58 polypeptide.
  • the results are shown in Figure 9.
  • the full-length SAA1 is a positive control.
  • the 11-58 polypeptide is different from the full-length SAA1 in the activation of MAPK phosphorylation.
  • SAA1 can activate ERK, p38, and JNK phosphorylation.
  • the 11-58 polypeptide does not activate JNK phosphorylation but activates p38 phosphorylation and ERK phosphorylation; the 11-58 polypeptide fragment activates p38MAPK phosphorylation more efficiently than full-length SAA1. Moreover, this activation was significantly reduced in TLR2 -/- macrophages, suggesting a TLR2-dependent.
  • 11-58 peptide fragments reduce the mortality caused by LPS and reduce acute lung injury caused by LPS
  • mice C57BL/6 mice were used to detect the mortality of mice induced by LSA by SAA1 short peptide.
  • the specific experimental method was divided into four groups, 10 mice in each group, short peptide group: short injection of 5 mg/kg intraperitoneally Peptide; PBS group: intraperitoneal injection of the corresponding dose of PBS; LPS group: intraperitoneal injection of 20 mg / kg LPS; short peptide + LPS group: intraperitoneal injection of 5 mg / kg of short peptide, half an hour after intraperitoneal injection of 20 mg / kg of LPS . Mice mortality was observed within 72 hours and a survival rate curve was plotted.
  • mice used in the acute lung injury experiment were C57BL/6, 6-8 weeks old, and the rats were divided into four groups of six mice each at a time point of 4 hours and 24 hours.
  • Short peptide group intraperitoneal injection of 5 mg/kg short peptide
  • PBS group intraperitoneal injection of the corresponding dose of PBS
  • LPS group intraperitoneal injection of 15 mg/kg LPS
  • short peptide + LPS group intraperitoneal injection of 5 mg/kg of short peptide, After half an hour, 15 mg/kg of LPS was intraperitoneally injected again.
  • mice were anesthetized, the lungs were lavaged with PBS containing no calcium and magnesium, lung lavage fluid was collected, the total number of cells in the lung lavage fluid was calculated, and then the sepals were stained with hematoxylin-eosin. The number of neutrophils was counted under a microscope. Then, the largest lung formalin of the mouse was lavaged and fixed, and paraffin sections were prepared, and hematoxylin-eosin staining was performed to observe the condition of lung infiltration. The remaining lung tissue was used for real-time detection, neutrophil detection in lung tissue (detection of MPO activity).
  • the 11-58 polypeptide was effective in inhibiting the increase in intrapulmonary MPO levels caused by LPS. As shown in Figure 7, the 11-58 polypeptide is also effective in inhibiting the up-regulation of inflammatory factors in the lung caused by LPS, such as TNF ⁇ , IL-6, IL-1 ⁇ . According to the hematoxylin-eosin staining of the lung sections, as shown in Fig. 8, it was found that the 11-58 polypeptide was effective in inhibiting the infiltration of neutrophils in the lung.
  • the present invention develops a new class of polypeptide fragments, taking SAA1 functional polypeptide 11-58 (P 11-58 ) as an example, and its possible working mechanism is to stimulate the massive production of IL-10 by TLR2 and inhibit the LPS-induced inflammation. The production of factors, thereby playing a role in inhibiting the inflammatory response.
  • the 11-58 polypeptide fragment itself does not induce the large-scale production of proinflammatory cytokines, nor does it stimulate the chemotaxis and infiltration of leukocytes, and thus its anti-inflammatory effect becomes the main biological activity.
  • the discovery of this 11-58 anti-inflammatory short peptide may bring new ideas to the development of new anti-inflammatory drugs.

Abstract

L'invention concerne un oligopeptide fonctionnel amyloïde A1 sérique humain, son procédé de préparation et une application de celui-ci. La présente invention concerne particulièrement un fragment polypeptidique amyloïde A1 sérique humain, ou un sel pharmacologiquement acceptable de celui-ci. Le fragment polypeptidique comprend les caractéristiques suivantes : (i) la séquence du fragment polypeptidique est dérivée de la séquence d'acides aminés de l'amyloïde A1 sérique humain ; et (ii) le fragment polypeptidique est dérivé du Mème au Nème acides aminés d'une séquence d'acides aminés pleine longueur (104 acides aminés) de l'amyloïde A1 sérique humain (SAA1), et a une longueur de 42-67 acides aminés, M étant tout nombre entier positif de 9 à 14, et N étant tout nombre entier positif de 55 à 75. L'oligopeptide fonctionnel amyloïde sérique humain de la présente invention a la capacité d'inhiber l'expression de cytokines pro-inflammatoires et de favoriser l'expression de cytokines anti-inflammatoires, et peut être utilisé pour préparer des médicaments pour le traitement de maladies inflammatoires.
PCT/CN2018/084256 2017-04-24 2018-04-24 Oligopeptide fonctionnel amyloïde a1 sérique humain, son procédé de préparation et son application WO2018196743A1 (fr)

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CN105407972A (zh) * 2013-06-06 2016-03-16 法国诗华动物保健公司 使用急性期蛋白在非人类哺乳动物中控制感染的组合物和方法

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CN105407972A (zh) * 2013-06-06 2016-03-16 法国诗华动物保健公司 使用急性期蛋白在非人类哺乳动物中控制感染的组合物和方法

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