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WO2018196743A1 - 人血清淀粉样蛋白a1功能性短肽及其制备方法和应用 - Google Patents

人血清淀粉样蛋白a1功能性短肽及其制备方法和应用 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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PCT/CN2018/084256
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English (en)
French (fr)
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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.

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Abstract

本发明公开了一种人血清淀粉样蛋白A1功能性短肽及其制备方法和应用。具体地，本发明提供一种人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐，所述的多肽片段具有以下特征：(i)该多肽片段的序列来源于人血清淀粉样蛋白A1的氨基酸序列；和(ii)该多肽片段为人血清淀粉样蛋白A1(SAA1)全长氨基酸序列(104氨基酸)的第M位到第N位，且长度为42-67个氨基酸，其中M为9-14的任一正整数，N为55-75的任一正整数。本发明的人血清淀粉样蛋白功能性短肽具有抑制致炎细胞因子表达、促进抗炎细胞因子表达的能力，可用于制备治疗炎症疾病的药物。

Description

人血清淀粉样蛋白A1功能性短肽及其制备方法和应用 技术领域
本发明属于生物工程技术领域，具体涉及一种人血清淀粉样蛋白A1功能性片段及其制备方法和应用。
背景技术
血清淀粉样蛋白A(Serum Amyloid A,SAA)是机体受到感染和损伤时分泌的一种主要的急性期蛋白。SAA是一个多态性蛋白的总称，由相关但各具独立基因的蛋白(SAA1-SAA4)组成。其中，人血清淀粉样蛋白A1(SAA1)由104个氨基酸组成，主要由肝细胞合成，人SAA2与SAA1有7个氨基酸的差别，它们均为急性时相蛋白。SAA3是一个假基因，SAA4在急性时相反应中变化不大，在机体中持续少量产生。
研究表明，SAA在急性和慢性炎症患者血清内含量均有大量上升，因此是一个反映感染性和非感染性炎症疾病的重要生物检测指标。SAA不仅是慢性阻塞性肺病急性加重期的生物标志物，也是急性心梗患者在接受急诊冠状动脉介入手术后发生心脏破裂的预期因子，这为早期发现心脏破裂患者提供了重要参考指标。其他大量研究也表明，在胰腺炎、肝炎、冠心病、糖尿病、慢性肾脏疾病、类风湿性关节炎、炎性肠炎、急性和慢性肺炎及肥胖病例中，SAA升高与疾病活动和病程发展有非常密切的关系。目前临床研究还认为，所有急性期反应蛋白中，SAA对急性炎症反应最敏感，升值幅度最大。同时研究还表明SAA与甲胎蛋白等肿瘤相关蛋白相似，其在血清中的浓度与癌症发生呈正相关。因此，在临床上对SAA在病人血清中含量的检测具有辅助诊断价值。
同时，SAA不仅是一种简单的炎症标志物，还积极介入疾病的相关过程。在急性感染或者炎症发生时，血清内SAA的水平可提升1000倍。高水平的SAA将导致淀粉样病变，而且低密度脂蛋白与SAA的结合物(LDL-SAA)可提高心血管疾病的风险；同时，SAA还具有促炎反应的特性，促进多种细胞因子的分泌，如IL-1β，IL-6，MMPs，CCL20等，这些细胞因子都参与炎症导致的疾病的发病过程，如，风湿性关节炎，肌肉萎缩，痛风，动脉粥样硬化等。由于SAA与多种疾病的发生相关，针对SAA的治疗方法已越来越受到重视。
目前，已知SAA与六种受体结合，Toll-like receptor(TLR)包括TLR2和TLR4，SRB1(a class B scavenger receptor)，及其G蛋白偶联受体formyl peptide receptor 2(FPR2)，receptor for advanced glycation endproduct(RAGE)，P2X7 receptor)。SAA与不同的受体结合所发挥的生物学活性不一样，例如，SAA与FPR2结合可诱导滑液超常增生及其内皮细胞的增殖，从而导致类风湿性关节炎的发生；SAA与CD36结合可促进胆固醇的代谢；SAA与SRB1结合可产生炎症诱发因子，导致动脉粥样硬化等疾病；SAA与TLR4结合可诱导NO的产生。由此可见，SAA1在机体中发挥多种生物学活性。
为了更明确地对SAA1进行结构及其生物学活性的研究与分析，本专利设计了酵母表达系统，表达没有脂多糖干扰的重组人SAA1及其结构类似的多肽。这种以酵母为宿主的表达方式不仅摆脱了多年来SAA1研究中所碰到的脂多糖困扰的问题，实现体内外研究不受脂多糖干扰的SAA1的生物学作用，还可利用酵母表达系统表达多种SAA1结构类似的多肽进行功能研究，从而发现SAA发挥活性的结构域和功能域。同时还可利用详尽的人SAA1不同肽段所发挥的生物学活性的研究结果，设计相应的阻断剂，为临床治疗提供潜在的候选药物。
然而，本领域尚缺乏令人满意的能够有效抑制致炎细胞因子，从而可有效治疗炎症或炎症相关疾病的药物。
发明内容
本发明的目的在于提供一种可有效抑制致炎细胞因子，可促进抗炎因子表达，从而可有效治疗炎症或炎症相关疾病的药物。
本发明的第一方面，提供一种人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐，所述的多肽片段具有以下特征：
(i)该多肽片段的序列来源于人血清淀粉样蛋白A1的氨基酸序列；和
(ii)该多肽片段为人血清淀粉样蛋白A1(SAA1)全长氨基酸序列(104氨基酸)的第M位到第N位，且长度为42-67个氨基酸，其中M为9-14的任一正整数，N为55-75的任一正整数。
在另一优选例中，所述的多肽片段还具有选自下组的一个或多个(或全部)特征：
(iii)该多肽片段具有抑制革兰氏阴性细菌内毒素LPS诱导巨噬细胞炎性 细胞因子表达的能力；
(iv)所述炎性细胞因子选自：IL-6,IL-1β,TNF-α；
(v)该多肽片段具有抑制革兰氏阴性细菌内毒素LPS诱导败血症致死的能力；
(vi)该多肽片段具备诱导巨噬细胞表达抗炎细胞因子的能力；
(vii)该多肽片段具备诱导巨噬细胞中p38MAPK磷酸化的能力。
在另一优选例中，所述抗炎细胞因子选自：IL-10；所述诱导抗炎细胞因子IL-10的能力依赖于Toll样受体2(TLR2)。
在另一优选例中，P M-N表示从血清淀粉样蛋白A1氨基酸序列第M位至第N位所构成的多肽。
在另一优选例中，M为9、10、或11。
在另一优选例中，N为58、59、60、61、62、63、64、65、66、67、68、69、70、71、或72。
在另一优选例中，所述的多肽选自下组：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、或其组合。
在另一优选例中，所述多肽具有野生型人血清淀粉样蛋白A1氨基酸序列的第11-58位、第11-68位、第11-72位或其组合。
在另一优选例中，野生型人血清淀粉样蛋白A1的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述多肽片段的N端和/或C端还带有用于纯化分离等目的的标签序列(如6His、或HIS6-SUMO-TEV片段)。
在另一优选例中，所述多肽片段为以酵母为宿主进行表达获得的多肽片段。
在另一优选例中，所述的人血清淀粉样蛋白A1的氨基酸序列为野生型序列。
在另一优选例中，所述人血清淀粉样蛋白A1的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述致炎细胞因子选自下组：IL-6、IL-1β、TNF-α、或其组合。
在另一优选例中，所述多肽片段具有促进抗炎细胞因子表达的能力。
在另一优选例中，所述抗炎因子为IL-10。
在另一优选例中，所述多肽片段是重组的。
在另一优选例中，所述的多肽是在酵母中表达的。
在本发明的第二方面，提供了一种融合蛋白，所述融合蛋白包括本发明的第一方面所述的人血清淀粉样蛋白A1多肽片段以及与所述片段融合在一起的标签序列。
在另一优选例中，所述标签序列融合于所述多肽片段的N端和/或C端。
在另一优选例中，所述的标签序列选自下组：His6、His6-SUMO、His6-SUMO-TEV片段。
在本发明的第三方面，提供了一种多核苷酸序列，所述多核苷酸序列编码本发明的第一方面所述的人血清淀粉样蛋白A1多肽片段、或本发明的第二方面所述的融合蛋白。
在本发明的第四方面，提供了一种酵母表达载体，该载体含有编码本发明的第一方面所述的多肽片段的多核苷酸。
在另一优选例中，所述载体为含有编码本发明的第一方面所述的多肽片段的多核苷酸的酵母表达载体pPIC9K。
在本发明的第五方面，提供了一种宿主细胞，所述宿主细胞选自下组：
(a)含有本发明的第四方面所述的载体的宿主细胞；
(b)染色体中整合有编码本发明的第一方面所述的多肽片段的多核苷酸的宿主细胞。
在另一优选例中，所述的宿主细胞选自下组：酵母细胞、用于表达SAA1蛋白的大肠杆菌。
在本发明的第六方面，提供了一种制备本发明的第一方面所述的多肽片段的方法，包括步骤：
(a)在适合表达的条件下，培养本发明的第五方面所述的宿主细胞，从而表达本发明的第一方面所述的多肽片段；和
(b)从培养产物中分离出本发明的第一方面所述的多肽片段。
在本发明的第七方面，提供了一种药物组合物，它含有药学上可接受的载体或赋形剂；以及本发明的第一方面所述人血清淀粉样蛋白A1多肽片段作为活性成分。
在另一优选例中，所述的药物组合物的剂型为注射制剂、透皮制剂、冻干粉剂。
在本发明的第八方面，提供了一种本发明的第一方面所述人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐的用途，用于制备抑制炎症和/或治疗炎症相关疾病的药物。
在另一优选例中，所述的炎症导致的疾病选自下组：败血症、类风湿性关节炎、动脉粥样硬化、急性和慢性肺炎、慢性肠炎、胰腺炎、肝炎、糖尿病、慢性肾脏疾病、肥胖病、肌肉萎缩、痛风。
在另一优选例中，所述炎症为SAA1过高所造成的炎症。
在另一优选例中，所述炎症的特征为SAA1表达过高。
在本发明的第九方面，提供了一种本发明的第一方面所述的人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐的用途，用于制备一组合物或制剂，所述组合物或制剂用于刺激巨噬细胞分泌抗炎因子IL-10。
在本发明的第十方面，提供了一种治疗炎症导致的疾病的方法，包括给需要的对象施用本发明的第一方面所述人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐或蛋白复合体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为SAA1片段的相对于全长SAA1的位置和酵母表达后的凝胶电泳分析图。
图2显示SAA1片段阻断与全长SAA1相比，诱导炎性细胞因子IL-6,IL-1β和TNF-α表达的能力对比。
图3显示SAA1片段阻断LPS导致的炎性细胞因子IL-6,IL-1β和TNF-α表达的关系图。
图4为11-58肽片段阻断LPS诱导炎性细胞因子IL-6,IL-1β和TNF-α表达的剂量关系图。
图5显示11-58肽片段有效地抑制LPS导致的小鼠死亡。
图6显示11-58肽片段本身不能诱导小鼠肺部髓过氧化酶(MPO)的升高，但能够抑制LPS诱导的MPO在肺部的升高；MPO为嗜中性粒细胞所特有，其升高反映肺部嗜中性粒细胞的浸润。
图7显示11-58肽片段抑制LPS诱导的小鼠肺组织中炎性细胞因子IL-6,IL-1β和TNF-α的表达。
图8为肺组织切片图，显示接受11-58肽片段的小鼠，对LPS诱导的急性肺损伤有一定的抵御，保持肺部组织结构完整和较少的炎症细胞浸润。
图9显示11-58肽片段通过TLR2诱导MAP激酶活化的能力与全长SAA1有所不同。
图10显示11-58肽片段在巨噬细胞中能够调控LPS导致的MAP激酶活化。
图11显示11-58肽片段诱导抗炎细胞因子IL-10的mRNA和蛋白的表达。
图12为11-58肽片段与LPS单独使用或合用后诱导抗炎细胞因子IL-10表达的对比图。
图13显示来源于TLR2及TLR4基因缺失的小鼠的巨噬细胞，对11-58肽片段和全长SAA1诱导的IL-10表达有不同的反应。
图14显示表达人FPR2的RBL细胞株，受11-58肽片段和全长SAA1刺激后，产生不同的趋化迁移反应。
图15显示11-58肽片段和全长SAA1具备不同的诱导细胞产生钙流的能力。
图16显示通过排阻色谱法分析11-58和全长SAA1的结果，使用SEC-HPLC标记物作为参考。
图17显示在HeLa和TLR2-HeLa中比较11-58刺激p38MAPK磷酸化的结果。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次意外地开发了一类结构新颖的抗炎多肽。本发明的抗炎多肽为具有抗炎作用的人血清淀粉样蛋白A1功能性短肽。本发明的短肽(以短肽11-58为例)，不仅致炎作用显著下降(甚至基本丧失或完全丧失致炎作用)，并且能够极其高效地抑制LPS导致的炎症反应，并且可极其高效地诱导抗炎细胞因子的表达。在此基础上，完成了本发明。
本发明人的研究还表明，SAA1C末端对其致炎作用十分重要，C末端缺失的越多，导致本发明短肽的致炎作用越弱。
本发明人的研究亦表明，去除SAA1N端数个氨基酸能够改变SAA1的功能包括减弱其诱导炎症功能。
具体地，本发明人采用了酵母表达载体pPIC9K，酵母菌株为GS115，表达重组人SAA1，及其源于人SAA1的片段。pPIC9K为一个高效表达外源蛋白质的分泌型载体，其上带有信号肽α-因子，能将SAA1和其片段分泌到培养基中，方便后续的纯化工作。利用酶切位点XhoI和EcoRI将SAA1连接到载体pPIC9K的α-因子下游，中间接有Kex2信号肽切割序列，可在分泌表达的过程中将α-因子切除，释放SAA1的N末端。表达后的SAA1片段在SDS凝胶电泳分离后，经考马氏亮蓝染色，显示预计的长度(图1)。
为了研究SAA1与受体结合的结构域和功能域，本发明人又设计了多种SAA1的片段序列，并在表达后进行了生物学活性分析。这些SAA1片段包括但不限于以下这些片段(数字代表始端和末端氨基酸位置)：11-58，11-68，11-72，27-72，27-90，29-104。这些多肽片段也使用酵母表达载体pPIC9K以及酵母菌株GS115进行表达。这些SAA1片段对骨髓巨噬细胞(BMDM)进行孵育后，收集上清液分析各种细胞因子的表达水平，发现SAA1C末端缺失越多，其片段的致炎作用越弱(图2)。上述片段在N末端均缺失10至28个氨基酸。
同样实验条件下，发现片段11-58对LPS诱导的炎症反应有最明显的抑制作用。其中又以C端去除氨基酸最多、N端缺失10个氨基酸的11-58片段，显示最强的抑制LPS诱导炎性细胞因子IL-6，TNF-α，IL-1β表达的作用(图3)。在使用不同浓度的11-58片段的情况下，该片段显示与浓度相关的抑制作用(图4)。此外，试验还表明，由酵母作为宿主表达的SAA1片段，更有助于获得抑制致炎细胞因子的能力。
随后检测了11-58片段在小鼠体内的抗炎作用。在小鼠致死率检测中，选择了C57BL/6小鼠，与急性肺损伤实验为同一品系。在20mg/kg LPS剂量下48小时死亡率为90％。腹腔预先给药11-58片段剂量5mg/kg，半小时后给药LPS可将致死率降低至40％，说明11-58片段能有效地降低LPS所导致的死亡率(图5)。
较低剂量的LPS(15mg/kg)，给予C57BL/6小鼠腹腔注射，造成全身炎症反应，包括急性肺损伤。按上述方法给予11-58片段，结果显示能够有效地抑制LPS导致的肺内MPO水平的升高(图6)。11-58片段还能够有效地抑制LPS导致的肺内炎症因子水平的上调，如TNF-α，IL-6，IL-1β(图7)。根据肺切片苏木精-伊红染色观察，发现11-58片段能够有效地抑制肺部嗜中性粒细胞的浸润(图8)。
之前的研究发现，全长SAA1能够通过TLR2产生信号转导，刺激细胞内MAP激酶的活化，具体表现为激酶的磷酸化水平上升。为了对比11-58片段与全长SAA1在信号转导功能上的差别，TLR2-HeLa稳转细胞株分别给予0.5μM全长SAA1或11-58片段。经过不同时间的刺激，用识别磷酸化激酶的特异抗体检测三种不同MAP激酶的磷酸化水平。图9显示11-58片段较全长SAA1更能刺激p38MAPK的磷酸化，而丧失了刺激JNK磷酸化的能力。
为了进一步研究11-58片段抑制LPS所导致的炎症反应的信号传导途径及其作用机理。利用不同浓度的11-58片段与LPS共刺激骨髓巨噬细胞，其中，LPS先于短肽半小时加入到骨髓巨噬细胞中，然后采用western-blot检测ERK，JNK，p38MAPK的磷酸化水平，发现11-58片段可抑制LPS导致的ERK,JNK磷酸化水平的上升，而11-58片段本身具有刺激p38磷酸化水平上升的能力，如果与LPS合用，则该片段进一步加强了LPS诱导的p38MAPK磷酸化(图10)。
白介素10(IL-10)是一个主要的抗炎因子。发现小鼠骨髓巨噬细胞经11-58片段刺激后，在2-8小时内有大量的IL-10mRNA表达，而IL-10蛋白水平也有升高，在4-12小时的区间内尤为明显(图11)。相比之下，LPS诱导很少的IL-10表达，而LPS与11-58片段共用时，对其刺激IL-10产生的功能并没有显著的影响(图12)。
为检测11-58片段通过哪一个受体刺激IL-10的产生，从野生型C57BL/6小时和同样遗传背景的TLR2基因敲除小鼠中，分离骨髓巨噬细胞，分别用全长SAA1和11-58片段刺激，考察IL-10的表达水平。发现同全长SAA1相比， 11-58片段能够刺激野生型的小鼠骨髓巨噬细胞产生更多(约6倍)的IL-10。但是在TLR2 -/-小鼠中，全长SAA1和11-58片段刺激IL-10产生的能力大幅下降(图13)。因此，推测11-58片段主要通过TLR2刺激巨噬细胞产生IL-10。
进一步对比了11-58片段与全长SAA1通过TLR4刺激IL-10的差别，发现SAA1和11-58片段刺激IL-10的产生与TLR4基因是否敲除没有依赖关系，而无论是在mRNA还是在蛋白水平上，11-58片段都较全长SAA1诱导更多的IL-10产生(图13)。
已知全长SAA1能够通过FPR2诱导细胞迁移。相比之下，11-58片段不能诱导表达有FPR2的大鼠粒细胞株(FPR2-RBL)产生有意义的迁移(图14)，说明N端和C端的去除导致该SAA1片段丧失了诱导细胞趋化迁移的功能。
已知全长SAA1能够通过FPR2刺激细胞内钙流的产生。这一功能同样在FPR2-RBL细胞上进行了检测，结果发现11-58片段基本丧失了诱导钙流产生的功能(图15)。
通过排阻色谱法分析各个表达的片段，结果表明它们(例如aa 11-58片段)以单体形式(monomers)存在，而全长SAA1似乎是是多聚化(multimerized)的(图16)。p38MAPK的磷酸化早在aa 11-58刺激后2分钟出现，但在没有稳定表达TLR2的HeLa细胞中未检测到p38MAPK的磷酸化。未转染的HeLa细胞对具有增强p38MAPK磷酸化的aa 11-58片段无反应，这一现象支持该反应是TLR2依赖性的观点(图17)。具体参见J Immunol 2017；199:1105-1112。
术语
如本文所用，术语“本发明多肽片段”、“本发明的人血清淀粉样蛋白A1的多肽片段”、“本发明片段”或“本发明短肽”，可互换使用，指本发明第一方面中所定义的来源于血清淀粉样蛋白A1的多肽片段或其药学上可接受的盐。此外，应理解，虽然优选的血清淀粉样蛋白A1是来自于人的，但是也可来自其他哺乳动物(如其他非人灵长动物)。
人血清淀粉样蛋白A1的多肽片段
本发明主要提供了一类人血清淀粉样蛋白A1多肽片段，其具有以下特征：
(i)该多肽片段的序列来源于人血清淀粉样蛋白A1的氨基酸序列；和
(ii)该多肽片段为人血清淀粉样蛋白A1(SAA1)全长氨基酸序列(104氨基 酸)的第M位到第N位，且长度为42-67个氨基酸，其中M为9-14的任一正整数，N为55-75的任一正整数。
本发明的代表性多肽片段，包括但不限于P 11-58、P 11-68、P 11-72、或其组合。其中数字代表在人血清淀粉样蛋白A1的氨基酸序列中始端和末端氨基酸位置。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的本发明多肽片段”是指本发明多肽片段基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化本发明多肽片段。基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。本发明多肽片段的纯度能用氨基酸序列分析。
本发明的多肽片段可以是重组多肽、天然多肽、合成多肽，优选重组多肽。本发明的多肽片段可以是天然纯化的产物包括经由蛋白酶水解后的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括本发明多肽片段的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然本发明多肽片段相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中，术语“本发明短肽”指具有本发明多肽片段活性的SEQ ID NO:2序列的多肽。该术语还包括具有与本发明多肽片段相同功能的、SEQ ID NO:2序列的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50 个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明多肽片段的活性片段和活性衍生物。
发明还提供本发明多肽片段的类似物。这些类似物与天然本发明短肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，“本发明多肽片段保守性变异多肽”指与SEQ ID NO:2的氨基酸序列相比，有至多8个，较佳地至多5个，更佳地至多3个，最佳地至多2个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:2中第M-N位所示序列的多肽片段，但与SEQ ID NO:1所示的编码区序列有差别的核酸序列。
编码SEQ ID NO:2第M-N位所示多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
本发明中的多肽和多核苷酸优选以分离的形式提供，更佳地被纯化至均质。
本发明短肽的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中 分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本发明的多聚核苷酸序列可用来表达或生产重组的本发明多肽片段。一般来说有以下步骤：
(1).用本发明的编码本发明短肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码本发明多肽的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。在本发明中适用的载体包括但不限于：在细菌中表达的基于T7的表达载体(Rosenberg,et al.Gene,1987,56：125)；在哺乳动物细胞中表达的pMSXND表达载体(Lee and Nathans,J Bio Chem.263：3521,1988)和在昆虫细胞中表达的来源于杆状病毒的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。优选的宿主细胞是酵母细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用 CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
用途
(1)本发明的重组人血清淀粉样蛋白A1多肽片段，能用于检测这些产物对细胞因子(如IL-6，TNF-α，IL-1β，IL-10)分泌的影响。
(2)本发明的重组SAA1多肽片段具有抑制致炎细胞因子表达和刺激抗炎因子IL-10表达的能力，能用于制备治疗炎症疾病的药物；所述的炎症疾病选自下组：风湿性关节炎、急性和慢性肺损伤、慢性肠炎、动脉粥样硬化、心血管疾病、胰腺炎、肝炎、糖尿病、慢性肾脏疾病、肥胖病、肌肉萎缩、痛风。
(3)本发明的重组人血清淀粉样蛋白A1多肽片段，具备降低LPS致死率的特性，能用于治疗革兰氏阴性细菌感染后大量内毒素LPS积累所引起的败血症及组织损伤。
药物组合物和施用方式
本发明重组人血清淀粉样蛋白A1多肽片段，可配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：肌内、腹膜内、静脉内、皮下、皮内、或局部给药。
本发明重组人血清淀粉样蛋白A1多肽片段可直接用于炎症导致的疾病的治疗，例如，用于类风湿关节炎疾病的治疗。在使用本发明重组人血清淀粉样蛋白A1多肽片段时，还可同时使用其他治疗剂或与其他疗法联用，例如用于革兰氏阴性细菌感染后内毒素LPS积累所引起的败血症及组织损伤(例如急性肺损伤)的治疗。
本发明还提供了一种药物组合物，它含有安全有效量的本发明重组人血清淀粉样蛋白A1多肽片段或其组合；以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明重组人血清淀粉样蛋白A1多肽片段还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的本发明重组人血清淀粉样蛋白A1多肽片段或其组合施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点在于：
(1)本发明中所述的重组人血清淀粉样蛋白A1多肽片段，与全长重组人血清淀粉样蛋白A1相比，具有明显降低的诱导致炎细胞因子表达的活性，显著升高的诱导抗炎细胞因子IL-10表达的活性。
(2)本发明人出乎意料地发现重组人血清淀粉样蛋白A1多肽片段对LPS诱导的多种致炎细胞因子的表达具有抑制作用。
(3)本发明人出乎意料地发现重组人血清淀粉样蛋白A1多肽片段能够保护小鼠免受LPS引起的急性肺部损伤。
(4)本发明通过初步的动物实验确定这些多肽片段可降低LPS的小鼠致死率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
hSAA1及其多肽片段的酵母表达载体的构建，及其目的蛋白质的表达
第一步：设计引物，克隆hSAA1基因及其多肽片段(11-58，11-68，11-72，27-72，27-90，29-104等多种不同的肽段)，片段两端带有酶切位点XhoI/EcoRI。PCR获得目的片段，XhoI/EcoRI双酶切，产物回收与pPIC9k XhoI/EcoRI双酶切产物连接，获得表达载体。
第二步：利用电转化方法将表达载体转染酵母GS115，组氨酸营养缺陷型平板筛选酵母单克隆，将所获得的单克隆涂布G418筛选平板，G418浓度分别为0.5mg/mL，1mg/mL，2mg/mL，30℃培养3-4天，选取G418浓度2mg/mL平板上所生长的单克隆培养，甲醇诱导表达目的蛋白质。选取表达产量较高单克隆进行下一步的大量发酵及其纯化。
实施例2
hSAA1及其多肽片段的纯化
将酵母发酵液上清超滤浓缩，与Ni Sepharose HP 4℃混合1-2小时，装柱。20mM sodium phosphate,25mM imidazole,100mM NaCl,pH 7.4洗涤柱子，20mM sodium phosphate,500mM imidazole,100mM NaCl,pH 7.4洗脱目的蛋白质，SDS-PAGE检测。然后加入His6-TEV酶4℃过夜酶切，酶切产物再次过Ni 2+亲和层析柱，将酶切下来的His6-SUMO和His6-TEV酶除去。纯化的SAA1或片段存放于20mM磷酸缓冲液中(pH 7.4)，可放在-80℃长期保存。采用SDS-PAGE确定SAA分子量和纯度，BCA法检测蛋白浓度，鲎试剂法检测内毒素含量。
实施例3
hSAA1及其多肽片段对BMDM分泌细胞因子的影响
将hSAA1及其多肽片段以0.5μM的终浓度加入到无血清培养的BMDM细胞培养液中，5小时后收集细胞，TRIzol裂解，提取RNA，反转录为cDNA，real-time PCR检测细胞因子的表达状况。对于加入LPS共刺激的实验，先加入短肽，半小时后，加入100ng/ml LPS。5小时后收集细胞，real-time PCR检测细胞因子的表达状况。
图1和图2所示，随着hSAA1片段的缩短，致炎作用逐渐减弱，并且伴随着抑制LPS导致的炎症反应作用加强。这说明，SAA C末端的在炎症反应中发挥重要作用，C末端缺失的越多，致炎作用丧失的越多，其中短肽11-58完全丧失了致炎作用，并且具有高效的抑制LPS导致的炎症反应，本发明人进一步考察其抑炎的作用机制。
图3显示了不同肽段阻断LPS导致的炎性细胞因子的影响。结果表明，
多个多肽片段(11-58，11-68，11-72，27-72，27-90，29-104、11-45)均表现出一定的抑制效果，其中，肽段27-72，27-90，29-104、11-45的抑制效果较低。令人出乎意料的是，多肽片段11-58、11-68和11-72表现出出乎意的高抑制效果，例如，比肽段27-72或27-90等肽段的抑制活性高出数倍。其中，尤其是P11-58和P11-68的抑制活性与P27-72的抑制活性相比，提高了约5-10倍。
在众多肽段中，P11-58(即片段11-58)对LPS诱导的炎症反应有最明显的抑制作用，显示最强的抑制LPS诱导炎性细胞因子IL-6，TNF-α，IL-1β表达的作用。
实施例4
11-58多肽片段可抑制LPS诱导的致炎因子的释放，并且本身可诱导较高水平的IL-10的分泌
分离BMDM细胞，将11-58短肽以0.5μM的终浓度加入到无血清培养的BMDM细胞培养液中，半小时后，加入100ng/ml LPS，5小时后收集细胞(或者不同时间点收集细胞)，TRIzol裂解，提取RNA，反转录为cDNA，加入SYBR Green染料进行Real-time PCR检测，检测致炎细胞因子IL-6,IL-1β，TNF-α，抑炎细胞因子IL-10的mRNA表达水平。最后分析实验所得Ct值，计算不同样品之间特定基因转录调控水平。
如图4所示，多肽片段11-58抑制LPS产生的IL-6、TNFα和IL-1β呈现剂 量依赖性。如图11、12和13所示，结果统计发现，11-58多肽片段单独刺激骨髓巨噬细胞可产生大量IL-10。当于LPS联合使用时，刺激IL-10产生的能力未受显著影响。相比较全长SAA1，11-58多肽片段刺激骨髓巨噬细胞可产生更多的IL-10(图13)。
上述结果表明，11-58短肽发挥抗炎作用主要是通过抑制LPS导致的炎症因子的升高，同时刺激骨髓巨噬细胞产生抗炎因子IL-10来发挥作用的。
实施例5
11-58短肽可抑制LPS导致的ERK和JNK磷酸化水平上调
分离小鼠骨髓巨噬细胞，先加入不同浓度的11-58多肽片段，一小时后加入终浓度500ng/ml的LPS，混匀，37℃培养。一小时后，裂解细胞，Westernblot检测MAPK磷酸化水平，结果见图10。LPS可诱导小鼠骨髓巨噬细胞ERK、p38和JNK磷酸化水平的上调，多肽片段可以阻断LPS导致的ERK,JNK的磷酸化水平的升高，但是本身能够引起p38磷酸化水平的升高。
实施例6
SAA诱导IL-10的产生是TLR2依赖性的
本发明人分离野生型的小鼠骨髓巨噬细胞和TLR2 -/-小鼠骨髓巨噬细胞，分别用apoSAA和11-58多肽片段刺激，考察不同时间点IL-10的表达水平，结果见图6。11-58多肽片段能够刺激野生型的小鼠骨髓巨噬细胞产生IL-10，但是在TLR2 -/-小鼠中，这种能力基本丧失，因此，本发明人推测，11-58多肽片段刺激巨噬细胞产生IL-10是TLR2依赖性的。
本实施例中，考察11-58短肽激活TLR2产生IL-10的信号传导途径。培养TLR2-HeLa稳转细胞株，检测11-58多肽对NF-κB的激活及其MAPK磷酸化水平。结果见图9。全长SAA1为阳性对照，在激活MAPK磷酸化的方面，11-58多肽与全长SAA1有所不同，SAA1能够激活ERK，p38，JNK磷酸化。11-58多肽不能激活JNK磷酸化，但能激活p38磷酸化和ERK磷酸化；相比全长SAA1，11-58多肽片段能够更有效地激活p38MAPK的磷酸化。而且这种激活在TLR2 -/-巨噬细胞中显著降低，提示TLR2依赖性。
实施例7
11-58肽片段可降低LPS导致的致死率，以及减少LPS导致的急性肺损伤
C57BL/6小鼠用于检测SAA1短肽抑制LPS所导致的小鼠死亡率，具体实验方法为，实验分为四组，每组10只小鼠，短肽组：腹腔注射5mg/kg的短肽；PBS组：腹腔注射相应剂量的PBS；LPS组：腹腔注射20mg/kg的LPS；短肽+LPS组：先腹腔注射5mg/kg的短肽，半小时后再次腹腔注射20mg/kg的LPS。72小时内观察小鼠死亡率，绘制生存率曲线。
在小鼠致死率检测中，在20mg/kg LPS剂量下48小时死亡率为90％。腹腔预先给药11-58多肽剂量5mg/kg，半小时后给药LPS可将致死率降低至40％，说明11-58短肽能有效的降低LPS所导致的死亡率。结果见图5。
急性肺损伤实验所用小鼠为C57BL/6，6-8周，雄鼠，实验分为四组，每组六只小鼠，时间点为4小时，和24小时。短肽组：腹腔注射5mg/kg的短肽；PBS组：腹腔注射相应剂量的PBS；LPS组：腹腔注射15mg/kg的LPS；短肽+LPS组：先腹腔注射5mg/kg的短肽，半小时后再次腹腔注射15mg/kg的LPS。4小时和24小时后，分别麻醉小鼠，用不含钙，镁的PBS灌洗肺，收集肺灌洗液，计算肺灌洗液中细胞总数，然后甩片，苏木精-伊红染色，显微镜下计数，计算中性粒细胞的数量。再将小鼠的一叶最大的肺福尔马林灌洗，固定，制作石蜡切片，苏木精-伊红染色，观察肺浸润的状况。其余肺组织用于real-time检测，肺组织内中性粒细胞检测(MPO活性检测)。如图6所示，11-58多肽能够有效地抑制LPS导致的肺内MPO水平的升高。如图7所示，11-58多肽还能够有效地抑制LPS导致的肺内炎症因子水平的上调，如TNFα，IL-6，IL-1β。根据肺切片苏木精-伊红染色观察，如图8所示，发现11-58多肽能够有效地抑制肺内中性粒细胞的浸润。
讨论
本发明开发了一类新的多肽片段，以SAA1功能性多肽11-58(P 11-58)为例，其可能的工作机制是通过TLR2刺激IL-10的大量产生，抑制LPS诱导的致炎因子的产生，从而发挥抑制炎症反应的作用。相对于LPS和全长SAA1来说，11-58多肽片段本身并不能诱导致炎因子的大量产生，也不能刺激白细胞的趋化和浸润，因而其抗炎作用成为主要的生物学活性。本11-58抗炎短肽的发现可能为新型抗炎药物研发带来了新的思路。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐，其特征在于，所述的多肽片段具有以下特征：
    (i)该多肽片段的序列来源于人血清淀粉样蛋白A1的氨基酸序列；和
    (ii)该多肽片段为人血清淀粉样蛋白A1全长氨基酸序列的第M位到第N位，且长度为42-67个氨基酸，其中M为9-14的任一正整数，N为55-75的任一正整数。
  2. 一种融合蛋白，其特征在于，所述融合蛋白包括权利要求1所述的人血清淀粉样蛋白A1多肽片段以及与所述片段融合在一起的标签序列。
  3. 一种多核苷酸序列，其特征在于，所述多核苷酸序列编码权利要求1所述的人血清淀粉样蛋白A1多肽片段、或权利要求2所述的融合蛋白。
  4. 一种酵母表达载体，其特征在于，该载体含有编码权利要求1所述的多肽片段的多核苷酸。
  5. 一种宿主细胞，其特征在于，所述宿主细胞选自下组：
    (a)含有权利要求4所述的载体的宿主细胞；
    (b)染色体中整合有编码权利要求1所述的多肽片段的多核苷酸的宿主细胞。
  6. 一种制备权利要求1所述的多肽片段的方法，其特征在于，包括步骤：
    (a)在适合表达的条件下，培养权利要求5所述的宿主细胞，从而表达权利要求1所述的多肽片段；和
    (b)从培养产物中分离出权利要求1所述的多肽片段。
  7. 一种药物组合物，其特征在于，它含有药学上可接受的载体或赋形剂；以及权利要求1所述人血清淀粉样蛋白A1多肽片段作为活性成分。
  8. 一种权利要求1所述人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐的用途，其特征在于，用于制备抑制炎症和/或治疗炎症相关疾病的药物。
  9. 一种权利要求1所述的人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐的用途，其特征在于，用于制备一组合物或制剂，所述组合物或制剂用于刺激巨噬细胞分泌抗炎因子IL-10。
  10. 一种治疗炎症导致的疾病的方法，其特征在于，包括给需要的对象施用权利要求1所述人血清淀粉样蛋白A1多肽片段或其药学上可接受的盐或蛋 白复合体。
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