Polypeptide targeting summer-leden crystallin and application thereof
阅读说明：本技术 靶向夏科-莱登结晶蛋白的多肽及其应用 (Polypeptide targeting summer-leden crystallin and application thereof ) 是由 王晨轩 莫珊珊 张罗 赵妍 于兰兰 王向东 李小璐 张文博 郝蕴 于 2021-08-31 设计创作，主要内容包括：本发明涉及靶向夏科-莱登结晶蛋白的多肽及其应用,所述的多肽包含如SEQ ID NO.3所示的氨基酸序列,或其片段、变体、融合物或衍生物、或所述其片段、变体或衍生物的融合物。本发明还涉及编码所述多肽的核酸,表达该多肽的重组载体及细胞。本发明提供了所述多肽在制备用于预防或治疗2型免疫疾病的药物中的应用。本发明还提供了一种非诊断目的的检测Gal-10蛋白的方法。(The invention relates to a polypeptide targeting a Charcot-Laden crystal protein and application thereof, wherein the polypeptide comprises an amino acid sequence shown as SEQ ID NO.3, or a fragment, variant, fusion or derivative thereof, or a fusion of the fragment, variant or derivative thereof. The invention also relates to nucleic acids encoding the polypeptides, recombinant vectors and cells expressing the polypeptides. The invention provides application of the polypeptide in preparing a medicament for preventing or treating type 2 immune diseases. The invention also provides a method for detecting the Gal-10 protein for non-diagnostic purposes.)
1. A polypeptide comprising the amino acid sequence shown as SEQ ID No.3, or a fragment, variant, fusion or derivative thereof, or a fusion of a fragment, variant or derivative thereof, preferably wherein the variant comprises an amino acid sequence which is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown as SEQ ID No. 3.
2. A pharmaceutical composition for preventing or treating type 2 immune diseases, wherein the pharmaceutical composition comprises the polypeptide of claim 1, preferably, the type 2 immune diseases comprise allergic diseases and mite infection, preferably, the type 2 immune diseases are allergic diseases, preferably, the allergic diseases comprise chronic nasosinusitis, asthma, allergic rhinitis, allergic dermatitis and food allergy, and preferably, the allergic diseases are chronic nasosinusitis.
3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable buffer, carrier or excipient,
preferably, the buffer comprises Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPSO, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES,
preferably, the carrier comprises an antimicrobial agent, an isotonic agent, an antioxidant, a local anesthetic, a suspending agent, a dispersing agent, an emulsifier, a chelating agent, a thickening agent or a solubilizing agent,
preferably, the excipient comprises a carbohydrate, a polymer, a lipid or a mineral.
4. Use of the polypeptide of claim 1 for the detection of a Gal-10 protein for non-diagnostic purposes.
5. A nucleic acid encoding the polypeptide of claim 1.
6. A recombinant vector comprising the nucleic acid of claim 5.
7. A cell comprising the nucleic acid of claim 5 or the recombinant vector of claim 6, preferably said cell comprises a prokaryotic cell, preferably said prokaryotic cell comprises a bacterial cell, preferably said eukaryotic cell comprises a protist cell, an animal cell, a plant cell, a fungal cell, preferably said animal cell comprises a mammalian cell, an avian cell, an insect cell.
8. Use of the polypeptide of claim 1 or the pharmaceutical composition of claim 2 or the nucleic acid of claim 5 or the recombinant vector of claim 6 or the cell of claim 7 for the preparation of a medicament for the prevention or treatment of immune disease type 2, preferably, the immune disease type 2 comprises allergic disease, mite infection, preferably, the immune disease type 2 is allergic disease, preferably, the allergic disease comprises chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, food allergy, preferably, the allergic disease is chronic sinusitis.
9. A method for detecting a Gal-10 protein for non-diagnostic purposes, said method comprising:
(1) contacting a sample with the polypeptide of claim 1;
(2) detecting the formation of a complex comprising the polypeptide of claim 1;
preferably, the method of detecting the formation of a complex comprising a polypeptide according to the first aspect of the invention comprises gel electrophoresis, chromatographic techniques, immunoblot analysis, immunohistochemistry, mass spectrometry and/or high pressure liquid chromatography.
10. Use of the polypeptide of claim 1 or the nucleic acid of claim 5 or the recombinant vector of claim 6 or the cell of claim 7 for the preparation of a product useful for diagnosing type 2 immune disease, preferably, the type 2 immune disease comprises allergic disease, mite infection, preferably, the type 2 immune disease is allergic disease, preferably, the allergic disease comprises chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, food allergy, preferably, the allergic disease is chronic sinusitis.
Technical Field
The invention relates to the field of biomedicine, in particular to a polypeptide targeting Charcot-Laeden crystallin and application thereof.
Background
Type 2 immunity is a specific immune response that involves innate immunity and adaptive immunity and promotes the formation of an immune barrier at the mucosal surface to clear pathogens. Recent studies have shown that the type 2 inflammatory pathway plays an important role in the development of allergic diseases. Th2 cells play a key role in the type 2 inflammatory pathway by secreting type 2 cytokines. Activation of Dendritic Cells (DCs) under antigen stimulation promotes differentiation of T cells into Th2 cells, releasing type 2 cytokines, which in turn stimulate IgE production and eosinophil aggregation, etc.
Galectins are carbohydrate-binding proteins involved in many physiological functions, such as inflammation, immune response, cell migration, autophagy and signaling, and they are also associated with fibrosis, cancer diseases. To date, a total of 16 galectins have been found in mammals, Galectin 10(Galectin-10) being a member of the Galectin superfamily, also known as Charcot Leyden Crystal (CLC) proteins. Galectin-10 is actively present in various eosinophilic diseases. Staphylococcus aureus and exotoxin thereof can play an important role in type 2 immunity of chronic rhino-sinusitis nasal polyp (CRSwNP), staphylococcus aureus can induce a large amount of CLC to form after colonization, CLC formation can further promote type 2 immune response and can cause the aggregation of neutrophils, and Galectin-10(Gal-10) is closely related to the mature differentiation and function of eosinophils. The polypeptide capable of targeting the Gal-10 protein is expected to be applied to diagnosis or treatment of type 2 immune diseases by screening, and a novel method is provided for diagnosis or treatment of type 2 immune diseases.
Disclosure of Invention
The invention aims to provide a polypeptide which can be applied to diagnosis or treatment of type 2 immune diseases.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a polypeptide comprising an amino acid sequence as shown in SEQ ID No.3, or a fragment, variant, fusion or derivative thereof, or a fusion of a fragment, variant or derivative thereof.
Said fragment, variant, fusion or derivative thereof, or said fusion of a fragment, variant or derivative thereof, retains the activity of SEQ ID No.3 of inhibiting an immune response induced by CLCs.
Further, the variant comprises an amino acid sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence shown in SEQ ID No. 3.
In a second aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of type 2 immune disorders, said pharmaceutical composition comprising a polypeptide according to the first aspect of the invention.
Further, the type 2 immune diseases comprise allergic diseases and mite infection.
Further, the type 2 immune disease is an allergic disease.
Further, the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy.
Further, the allergic disease is chronic sinusitis.
Further, the pharmaceutical composition also comprises a pharmaceutically acceptable buffer, carrier or excipient.
In a third aspect, the invention provides the use of a polypeptide according to the first aspect of the invention for the detection of a Gal-10 protein for non-diagnostic purposes.
In a fourth aspect, the invention provides a nucleic acid encoding a polypeptide according to the first aspect of the invention.
In a fifth aspect, the invention provides a recombinant vector comprising a nucleic acid according to the fourth aspect of the invention.
In a sixth aspect, the invention provides a cell comprising a nucleic acid according to the fourth aspect of the invention or a recombinant vector according to the fifth aspect of the invention.
Furthermore, the cell comprises a prokaryotic cell and a eukaryotic cell.
Further, the prokaryotic cell includes a bacterial cell.
Further, the eukaryotic cell includes a protist cell, an animal cell, a plant cell, a fungal cell.
Further, the animal cells include mammalian cells, avian cells, insect cells.
In a seventh aspect, the invention provides a polypeptide according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention or a nucleic acid according to the fourth aspect of the invention or a recombinant vector according to the fifth aspect of the invention or a cell according to the sixth aspect of the invention for use in the preparation of a medicament for the prevention or treatment of a type 2 immune disorder.
Further, the type 2 immune diseases comprise allergic diseases and mite infection.
Further, the type 2 immune disease is an allergic disease.
Further, the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy.
Further, the allergic disease is chronic sinusitis.
In an eighth aspect, the invention provides a method for detecting a Gal-10 protein for non-diagnostic purposes, said method comprising:
(1) contacting the sample with a polypeptide according to the first aspect of the invention;
(2) detecting the formation of a complex comprising a polypeptide according to the first aspect of the invention;
further, the method for detecting the formation of a complex comprising a polypeptide according to the first aspect of the invention comprises gel electrophoresis, chromatographic techniques, immunoblot analysis, immunohistochemistry, mass spectrometry and/or high pressure liquid chromatography.
In a ninth aspect, the invention provides the use of a polypeptide according to the first aspect of the invention or a nucleic acid according to the fourth aspect of the invention or a recombinant vector according to the fifth aspect of the invention or a cell according to the sixth aspect of the invention in the manufacture of a product for use in the diagnosis of an immune disease type 2.
Further, the type 2 immune diseases comprise allergic diseases and mite infection.
Further, the type 2 immune disease is an allergic disease.
Further, the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy.
Further, the allergic disease is chronic sinusitis.
Drawings
FIG. 1 is a graph showing the results of RT-qPCR experiments for detecting the gene expression changes of mucosal epithelial cells of human nasal polyps induced by CLCs of different concentrations, in which graph A is a statistical graph of IL-1. beta. expression changes, graph B is a statistical graph of TNF-. alpha. expression changes, graph C is a statistical graph of IL-6 expression changes, graph D is a statistical graph of GM-CSF expression changes, and graph E is a statistical graph of IL-8 expression changes;
FIG. 2 is a statistical graph of gene expression changes in human nasal polyp mucosal epithelial cells induced by CLCs (100. mu.g/mL) for 24h, wherein Panel A is a statistical graph of IL-1. beta. expression changes, Panel B is a statistical graph of TNF-. alpha. expression changes, Panel C is a statistical graph of IL-6 expression changes, Panel D is a statistical graph of IL-8 expression changes, and Panel E is a statistical graph of GM-CSF expression changes;
FIG. 3 is a graph showing the results of experiments in which 8 polypeptides were tested at the cellular level for inhibiting the immune response induced by CLCs, wherein Panel A is a statistical graph of the expression level of IL-1. beta., Panel B is a statistical graph of the expression level of IL-6, Panel C is a statistical graph of the expression level of TNF-. alpha., and Panel D is a statistical graph of the expression level of IL-8;
FIG. 4 is a graph showing the results of an experiment in which the polypeptide of the present invention was confirmed to inhibit an immune response induced by CLCs at the cellular level, wherein Panel A is a statistical graph of the expression level of IL-1. beta., Panel B is a statistical graph of the expression level of TNF-. alpha., Panel C is a statistical graph of the expression level of IL-6, Panel D is a statistical graph of the expression level of IL-8, and Panel E is a statistical graph of the expression level of GM-CSF.
Detailed Description
Polypeptides
The term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers in the "D" form (as compared to the natural "L" form), omega-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., alpha-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids.
In specific lists of amino acids, such as "alanine" or "Ala" or "A", the term refers to both L-alanine and D-alanine, unless specifically stated otherwise. Other unconventional amino acids may also be suitable components for the polypeptides of the present invention, as long as the polypeptide retains the desired functional properties. For the peptides shown, each encoded amino acid residue (where appropriate) is represented by a single letter name corresponding to the common amino acid name.
"variants" of a polypeptide include insertions, deletions, and substitutions, either conservative or non-conservative. For example, a conservative substitution refers to the replacement of an amino acid within the same general class (e.g., acidic amino acid, basic amino acid, non-polar amino acid, or aromatic amino acid) with another amino acid within the same class. Thus, the meaning of conservative amino acid substitutions and non-conservative amino acid substitutions are well known in the art. In particular, variants of the polypeptides that exhibit activity that can inhibit CLCs-induced immune responses are included.
In some embodiments, the variant comprises an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence set forth in SEQ ID No. 3.
"fusions" of polypeptides include amino acid sequences corresponding to a reference sequence (e.g., SEQ ID No.3, or a fragment or variant thereof) fused to any other polypeptide. For example, the polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A to facilitate purification of the polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the polypeptide may be fused to an oligohistidine tag such as His6 or an epitope recognized by an antibody such as the well-known Myc tag epitope. In addition, fusions comprising hydrophobic oligopeptide terminal tags may be used. Also included within the scope of the invention are fusions to any variant or derivative of the polypeptide.
The fusion may comprise further portions conferring desired characteristics to said polypeptide of the invention; for example, the moiety may be used to detect or isolate the polypeptide, or to facilitate cellular uptake of the polypeptide. The moiety may for example be a biotin moiety, a streptavidin moiety, a radioactive moiety, a fluorescent moiety, e.g. a small fluorophore or a Green Fluorescent Protein (GFP) fluorophore, as known to the person skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.
It will be appreciated by those skilled in the art that the polypeptides of the invention may comprise one or more amino acids modified or derivatized, for example, by pegylation, amidation, esterification, acylation, acetylation, and/or alkylation.
As appreciated in the art, pegylated proteins may exhibit reduced renal clearance and proteolysis, reduced toxicity, reduced immunogenicity, and increased solubility.
In order to obtain a successfully pegylated protein with maximally extended half-life and retained biological activity, several parameters that can influence the outcome are important and should be considered. PEG molecules may vary, and PEG variants that have been used for protein pegylation include PEG and monomethoxy-PEG. In addition, they may be either linear or branched.
It has been shown that increasing the degree of pegylation results in an increased half-life in vivo. However, it will be appreciated by those skilled in the art that the pegylation process will require optimization of a particular protein on an individual basis.
PEG may be coupled at a naturally occurring disulfide bond as described in WO 2005/007197. Disulfide bonds can be stabilized via the addition of chemical bridges that do not damage the structure of the polypeptide. This allows the selective creation of bridges for site-specific attachment of PEG with the conjugated thiols of the two sulfur that make up the disulfide bond. Thus, the need to engineer residues into peptides to attach to target molecules is circumvented.
The polypeptides for use in the invention may be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for making such polypeptides are well known in the art.
At present, nucleic acid sequences encoding the polypeptides of the invention can be obtained entirely by chemical synthesis. The nucleic acid sequence can then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the sequence of the polypeptide of the present invention by chemical synthesis.
The term "nucleic acid encoding a polypeptide" as used herein includes nucleic acids comprising a sequence encoding a polypeptide of the present invention, in particular a polypeptide having the amino acid sequence shown in SEQ ID NO. 3. The term also includes nucleic acids that: the nucleic acid comprises a single continuous region or multiple discontinuous regions encoding the polypeptide (e.g., polynucleotides interrupted by integrating phage, integrating insert sequences, integrating vector sequences, integrating transposon sequences, or by RNA editing or genomic DNA reconstitution) and additional regions, which may also comprise coding and/or non-coding sequences.
Vectors for constructing the recombinant vector of the present invention include, but are not limited to, MarEx expression vectors produced by Celltrion inc. (korea); a commercially available pCDNA vector; F. r1, RP1, Col, pBR322, ToL and Ti vector; sticking particles; phages such as lambda phage, lambda-shaped phage, M13 phage, Mu phage, P1 phage, P22 phage, Q μ phage, T-even phage, T2 phage, T4 phage, T7 phage, and the like; a plant virus. Any of a variety of vectors known to those of skill in the art can be used in the present invention, and the choice of vector depends on the nature of the cell selected. Introduction of the vector into the cell can be achieved by, but is not limited to, calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection, or electroporation, and any person skilled in the art can select and use an introduction method suitable for the vector and cell used. Preferably, the above-mentioned vector contains one or more selection markers, but is not limited thereto, and a vector not containing a selection marker may also be used. The selection of the selectable marker may depend on the cell chosen (as is well known to those skilled in the art), but is not critical to the invention.
The polypeptides of the invention can be prepared by any of a variety of techniques. In general, the polypeptide may be produced by cell culture techniques, including producing the polypeptide by conventional techniques, or by transfecting the nucleic acid molecule of the polypeptide into a suitable bacterial or mammalian cell host, to allow for production of the polypeptide, wherein the polypeptide may be recombinant. The term "transfection" of various forms is intended to include usually used to introduce exogenous DNA into prokaryotic or eukaryotic cells in various techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although the polypeptides of the invention may be expressed in prokaryotic or eukaryotic cells, it is preferred to express the polypeptides in eukaryotic cells, and most preferably in mammalian cells, since such eukaryotic cells (particularly mammalian cells) are more likely to assemble and secrete correctly folded polypeptides than prokaryotic cells. When a recombinant expression vector encoding a nucleic acid molecule for a polypeptide is introduced into a mammalian cell, the polypeptide is secreted into the culture medium in which the cell is cultured, by culturing the cell for a period of time sufficient to allow the polypeptide to be expressed in the cell, or more preferably. The polypeptide can be recovered from the culture medium using standard protein purification methods.
Pharmaceutical composition
By "pharmaceutically acceptable" is meant a non-toxic material that does not detract from the active ingredient. Such pharmaceutically acceptable buffers, carriers or Excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, A.R Gennaro, eds., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3 rd edition, A.Kibbe eds., Pharmaceutical Press (2000)).
The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the aim of stabilizing the pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPSO, imidazole lactate, PIPES, SSPE, POPSO, TAPS, TABS, TAPSO and TES.
The carriers of the present invention include antimicrobial agents, isotonic agents, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifying agents, chelating agents, thickening agents, or solubilizing agents.
The excipient may be one or more of the following: carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates (alginates), carrageenans (carageenans), hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates (polysulfonates), polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate, poly (lactic acid), poly (glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone (all of different molecular weights) which are added to the composition, for example to control viscosity, to achieve bio-adhesion, or to protect the active ingredient from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-and triglycerides, ceramides, sphingolipids and glycolipids (all with different acyl chain lengths and saturations), lecithin (eg lecithin), soy lecithin, hydrogenated lecithin and soy lecithin, which are added to the composition for similar reasons as the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or favorable pigment properties.
The pharmaceutical compositions of the present invention must be sterile and stable under the conditions of manufacture and storage. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus other desired ingredient from a previously sterile-filtered solution thereof. Alternatively, the compositions of the present invention may be in solution, and suitable pharmaceutically acceptable excipients may be added and/or mixed prior to or at the time of delivery to provide injectable unit dosage forms. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain adequate flowability, and delay absorption if necessary.
The choice of the optimal route of administration of the pharmaceutical composition of the invention will be influenced by several factors, including the physicochemical properties of the active molecule in the composition, the urgency of clinical presentation, and the relationship between the plasma concentration of the active molecule and the desired therapeutic effect. For example, the polypeptides of the invention may be prepared with a carrier that will protect them from rapid release (such as a controlled release formulation), including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used in the present invention. Further, the polypeptide may be coated with, or co-administered with, a material or compound that prevents inactivation of the polypeptide. For example, the polypeptide may be administered with a suitable carrier (e.g., liposomes or diluents).
The administration route of the pharmaceutical composition of the present invention can be divided into oral administration and parenteral administration.
Oral dosage forms may be formulated as tablets, troches, lozenges, aqueous or oily suspensions, dispersed powders or granules, emulsions, hard gelatin capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels or pastes. These formulations may contain pharmaceutical excipients, including but not limited to: granulating and disintegrating agents, binding agents, lubricants, preservatives, coloring agents, flavoring agents or sweetening agents, vegetable or mineral oils, wetting agents, and thickening agents.
Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solutions or suspensions. The solution or suspension may include agents that are non-toxic to the recipient, such as 1, 3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oils, fatty acids, topical anesthetics, preservatives, buffers, agents that increase viscosity or solubility, water-soluble antioxidants, oil-soluble antioxidants, and metal chelators, in the amounts and concentrations employed.
Others
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The terms "charcot-leyden crystals", "CLCs", "CLC crystals", "CLC" are used interchangeably herein and refer to crystals formed from galectin-10. The crystals formed from galectin-10 are generally biconical hexagonal crystals, approximately 20-40 μm in length and approximately 2-4 μm in width.
The term "galectin-10" (or Gal10 or Gal-10) refers to small hydrophobic glycan binding proteins that self-crystallize to form charcot-leyden crystals. Galectin-10 is also known as charcot-leyden crystallin (CLCP), eosinophil lysophospholipase and lysoegg phosphatidyl hydrolase. The term "galectin-10" is broad enough to cover human proteins and any species homologue.
The term "specificity" refers to the ability to bind to a given target (e.g., a CLC crystal). A polypeptide may be monospecific and contain one or more binding sites that specifically bind a target, or a polypeptide may be multispecific and contain two or more binding sites that specifically bind the same or different targets.
The term "isolated" refers to an "artificial" alteration of the native state. If an "isolated" composition or substance exists in nature, its original environment has been altered, or it has been removed from its original environment, or both. As used herein, a polypeptide that occurs naturally, for example, in a living animal, is not "isolated," but the polypeptide is "isolated" from materials with which it coexists in its natural state.
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
Experimental materials:
firstly, plasmid: after the Gal-10 protein sequence was subjected to humanized codon optimization, a solubilizing fragment was added to the N-terminus thereof, and cloned into pET-28a vector plasmid via NcoI/XhoI double cleavage sites to form pET-28a-Gal10-TEV-6 XHis. The Gal-10 protein sequence is shown in Table 1, and the Gal-10 protein sequence with the lysogenic fragment is shown in Table 2.
TABLE 1 Gal-10 protein sequences
TABLE 2 Gal-10 sequences with lysogenic fragments
Reagent material and instrument
1. Chemical reagents: kanamycin (Kanamycin) and Ampicillin (Ampicillin) were purchased from Tiangen biotechnology limited; Isopropyl-beta-D-thiogalactoside (IPTG) and E.coli BL21(DE3) were purchased from Beijing Quanjin; SDS, Trizol and imidazole were purchased from Sigma; TEV enzyme was purchased from Beijing Yiqiao Shenzhou, Inc.; coomassie brilliant blue dye liquor (self-made); cDNA reverse transcription reagents were purchased from Takara; the real-time fluorescent quantitative PCR reagent is purchased from Ebola tektes; the polypeptide was synthesized by guoping pharmaceutical limited and the sequence is shown in table 2.
TABLE 2 polypeptide sequences
2. Consumables and instrumentation: 10cm cell culture dishes, 12 well cell culture plates purchased from CORNING; BEGM medium for cell culture was purchased from Lonza; DMEM high-glucose medium, fetal bovine serum FBS, digest (2.5g/L pancreatin, 0.02g/L EDTA, pH 8.0, 0.22 μm filtration), penicillin (20mg/mL), streptomycin (20000U/mL) were purchased from GIBCO; Ni-NTA affinity chromatography column from GE; 10kDa concentration tubes were purchased from Millipore. An electric heating constant temperature incubator (XMTD HH.B 11-600); PCR instrument (Biometra tgradent); desktop centrifuges (eppendorf, Centrifuge 5415D); an electric constant temperature water tank (SHHW 21600); a micro ultraviolet spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); electronic analytical balance (Sartorius, BS 110S); an optical inverted microscope (XDS-1B); micropipette (eppendorf research plus)) (ii) a Cell CO2 incubator (SANYO); ultra low temperature refrigerator at-80 deg.C (SANYO, MDF-382E); a pH meter (Thermo Orion 868); magnetic stirrer (IKARH-KT/C); microplate reader (Bio-Rad, 680); an ultrasonic crusher;pure25 protein purification system (superdex 75, GE Healthcare); SDS-PAGE electrophoresis apparatus (Bio-Rad); gel Imager (Molecular Imager Gel Doc XR, Bio-Rad); ABI PCR instrument (ABI 7500).
Example 1 construction of in vitro model for inducing activation of innate immune factors of human nasal mucosal epithelial cells by CLCs
The first experiment method comprises the following steps:
1. expression and purification of Gal-10 recombinant proteins and production of CLCs
(1) And (3) transformation:
A. taking a BL21(DE3) competent cell and thawing the cell on ice;
B. adding 0.5 μ L of pET-28a-gal10-TEV-6His recombinant plasmid, and incubating on ice for 15-20 min; C. heat shock: carrying out water bath heat shock at 42 ℃ for 90 s;
D. rapidly placing on ice, and ice-cooling for 5 min;
E. adding 500 μ L of nonresistant LB, and culturing at 37 deg.C for 40-50 min;
F. mu.L of the bacterial suspension was inoculated onto LB solid medium of Kanamycin (25mg/mL) and cultured overnight in a 37 ℃ incubator.
(2) And (3) bacterial culture:
the positive monoclonal BL21(DE3)/pET-28a-gal10-TEV-6His on the plate of the above step was picked up using Kanamycin (25mg/mL) as a selection marker, inoculated into 20mL of LB liquid medium resistant to Kanamycin, and shake-cultured at 37 ℃ and 210r/min for 12 hours. Inoculating the bacterial liquid into 1LLB liquid culture medium containing kanamycin at a ratio of 1: 100, and culturing at 37 ℃ under the condition of oscillation at 210 r/min.
(3) Induced expression and acquisition of the protein of interest:
A. when the optical density of Escherichia coli at 600nm (OD 600) is 0.6-0.8h, adding IPTG with final concentration of 1mM, shake culturing at 28 deg.C overnight, and inducing the expression of target protein;
B. the overnight expressed bacterial culture was enriched, centrifuged at 6000 Xg at 4 ℃ for 20min and the supernatant discarded. Resuspend with Buffer Lysis Buffer, concentrate it in a beaker, about 75-100mL in volume, that is, 1: PMSF (1 mM final concentration) is added according to the proportion of 100 to protect the target protein;
C. ultrasonic bacteria disruption: the power is 25%, the working time is 25min, the ultrasonic on time is 3s, and the ultrasonic off time is 9 s; centrifuging (4 deg.C, 13,000 Xg, 30min) to remove cell debris, breaking nucleic acid released after cell disruption, centrifuging to precipitate to make cell lysate not viscous, and facilitating subsequent treatment. The supernatant is collected and the soluble protein of interest is present in the supernatant.
(4) Purification of Gal-10 protein:
A.Ni-NTA affinity column chromatography
a. Balancing Ni columns: the elution of the protein can be started by penetrating the lysine Buffer out of the Ni column by the capacity of about 2-3 column volumes;
b. centrifuging to obtain a supernatant sample, and passing through the Ni column for 2-3 times;
c. the column was washed with Lysis Buffer containing 20mM imidazole and 0.1% Empigen detergent to elute the heteroprotein;
d. washing the column with Lysis Buffer containing 500mM imidazole to allow the target protein to be unbound to the nickel column;
e. concentration: and (3) putting the target protein collected by elution into a 10kDa concentration tube, centrifuging at 4 ℃ at 2000 Xg for 10min, and adding a small amount of lysine Buffer or PBS to dilute imidazole in the concentration process so as to prevent the target protein from aggregating and precipitating.
B.pure25 protein purification
a. Cleaning a chromatographic column: the chromatographic column was washed with sterile water, about 8 mL. Placing the pump head in a PBS solution, repeating the steps and performing to wash about 36 mL;
b. parameters are as follows: system flow 0.5 mL/min; column position 3; alarm delta columnresure enabled: 1.5; alarmpit columnresurserving enabled: 5;
c. loading: the sample loop was washed twice and three times with PBS, 2mL of sample with concentration of about 6.35mg/mL was injected into the sample loop, and PBS was slightly aspirated and injected into the sample loop to avoid sample residue. Placing the collecting pipe, and selecting inject valid as inject;
d. collecting: when the UV 280 marking line representing the protein content rises, collecting the target protein by using an automatic sample collector, and setting the collection amount of each tube to be 0.3 mL;
e. cleaning a chromatographic column: after the sample is collected, the pump head is placed in PBS, the pump head is continuously operated until the sample passes through 20mL, the pump head is replaced by sterile water for cleaning, the operation is carried out for 10mL, and then the pump head is replaced by 20% ethanol solution for operating for 20mL, and then the machine can be shut down;
f. protein treatment: measuring the concentration of each tube of the collected protein, marking, quickly freezing in liquid nitrogen, and freezing and storing in a refrigerator at-80 ℃. The experimental antechamber is statically placed at a room temperature and slowly melts.
C. SDS-PAGE identification of expression products
a. Sample preparation: a whole bacteria sample, a supernatant sample after centrifugation, a protein supernatant sample after passing through a chromatographic column, a 20mM imidazole sample, a 500mM imidazole sample, and protein samples collected at three positions of a peak head, a peak tip and a peak tail in a molecular sieve;
b. preparing glue: 5% concentrated gum; selecting 15% of separation gel according to the size of the protein;
c. preparing a sample: mixing 2 XLoading Buffer with sample 1: 1;
d. loading: 5 mu L of whole bacteria sample, 3 mu L of marker and 10 mu L of other samples;
e. dyeing: placing the protein gel in Coomassie brilliant blue dye solution, and heating with microwave for 2 min;
f. and (3) decoloring: placing the dyed protein gel in tap water, and heating with microwave for 20-40 min.
(5) Production and concentration determination of CLCs:
mixing TEV enzyme and pET-28a-gal10-TEV-6His recombinant protein according to the mass ratio of 1:10, and carrying out enzyme digestion incubation on a shaker at 4 ℃ overnight;
and B.600 Xg, centrifuging at 4 ℃ for 10min, sucking out the supernatant after the centrifugation is finished, adding a proper amount of PBS (phosphate buffer solution) for heavy suspension, and repeating the process for three times to obtain the pure CLCs.
Bca assay detects the concentration of CLCs.
2. Acquisition of human nasal polyp tissue epithelial cells
(1) And (3) carrying out enzyme digestion on the tissue:
A. and (4) placing the mucosa specimen taken out by the operation into sterile normal saline.
B. The specimen was taken out and immersed in 5mL of a tissue rinse (physiological saline containing 200. mu.g/mL of penicillin) at 4 ℃ for 1 hour.
C. And (3) putting the soaked specimen on a culture dish of 6cm, repeatedly washing with PBS and double antibodies (for several times, generally more than or equal to 10 times), cleaning blood clots and dirt on the specimen, and removing mucus and blood vessels on the specimen by placing under a dissecting microscope.
D. The cleaned specimen was placed in a 15mL centrifuge tube and 10mL of human zymogen solution (collaagenase type 2 and Dnase I) was added.
E. Human placental Collagen (Collagen I) was coated onto 10cm dishes at 2 mL/well overnight at 37 ℃.
(2) Nasal mucosal epithelial cell inoculation:
A. 2mL of whole serum culture medium was added to the digested tissue tube and the tube was shaken repeatedly for 20 seconds.
B. The supernatant was removed, 5mL of whole serum culture medium was added again, shaking was repeated for 20 seconds, and the supernatant was removed again.
C. The supernatant was centrifuged at 800r for 5min and removed.
D. Adding 1mL of whole serum culture solution, and placing in a 6cm culture dish for pre-adherence for 1 h.
E. Sucking out the human placenta collagen of the coated culture dish (recovering the coating for less than or equal to 3 times), and washing with distilled water for more than or equal to 10 times (more and more). Sterilizing with ultraviolet lamp for 20 min.
The supernatant was aspirated from an F.6cm petri dish, 5mL of whole serum culture medium was added, and centrifugation was carried out at 800r for 5 min. Discard the supernatant (suck clean as much as possible).
G. Adding 3mL of erythrocyte lysate, mixing the cells evenly, lysing the cells for 5min at room temperature, centrifuging the cells at 800r for 5min, and removing the supernatant.
H.3mL sterile PBS after washing, 800r 5min centrifugal, abandon the supernatant, use BEGM heavy suspension.
I. And adding 9 mu LPBS into 1 mu L of the mixture, uniformly mixing, and adding a cell counting plate for counting.
J. Cell counting: (cell suspension cell number)/mL ═ n grid cell number/n). times.16X 104
K. After counting, according to 2X 106Adding into a coated 10cm culture dish, and adding 12mLBEGM epithelial culture medium.
3. CLCs stimulate human nasal mucosal epithelial cells
(1) Nasal mucosal epithelial cell inoculation:
A. primary nasal mucosal epithelial cells of nasal polyps grow to about 85-90%, the culture medium is discarded, the cells are cleared by 2mL of sterile PBS, and then digested for 4-5min by 2mL of 0.25% trypsin at 37 ℃, and then 1mL of complete culture medium containing FBS is added for termination.
B. After the cells were blown into a single cell suspension, centrifugation was carried out at 800r for 5min, and the supernatant was discarded.
C. After cell counting 2X 105Individual cells were plated in 12-well plates/well and cultured in 1mLBEGM medium.
(2) CLCs stimulated nasal mucosal epithelial cell model:
a.12 well plate culture for about 2 days, when primary nasal mucosa epithelial cells grow to about 80%, discarding the culture medium, and continuously culturing for 24h by BEGE culture medium containing different CLC concentrations.
B. Collecting cell supernatant, centrifuging at 4 deg.C for 5min at 800r, and freezing the supernatant in-80 deg.C refrigerator.
C. After washing the cells with 1mL sterile PBS, 1mL of Trizol/well was added to lyse the cells for RNA extraction.
4. RNA extraction and cDNA reverse transcription
(1) RNA extraction:
A. lysates of cells or tissues frozen in a-80 ℃ freezer stored in Trizol were thawed slowly on ice.
B. After the RNA sample is completely thawed, the sample is taken to room temperature and placed for 10min, so that the RNA is fully dissolved.
C. Chloroform was added in a volume ratio of 200. mu.L of chloroform/ml of trizol, and the mixture was thoroughly mixed by vigorous shaking and allowed to stand at room temperature for 15 min.
D.4℃12000r·min-1Centrifuge for 15 min.
E. The upper aqueous phase was transferred to a new RNase-free 1.5mL centrifuge tube.
F. Adding precooled isopropanol according to the volume ratio of 800 mu L of isopropanol to mL of water phase, fully and uniformly mixing, and standing for 10-15min at room temperature.
G.4℃12000r·min-1Centrifuging for 10min, and removing the supernatant to see that RNA precipitates at the bottom of the centrifuge tube.
H. 1mL of 750mL/L of DEPC-treated H was added2Ethanol in O-configuration, gently shake the centrifuge tube to resuspend the pellet.
I.4℃12000r·min-1Centrifuge for 5min, discard the supernatant as much as possible.
J. And 8-9 are repeated.
K. Air drying at room temperature for 5-10min, and evaporating 750mL/L ethanol.
L. H treated with 25-100 μ LDEPC depending on the size of the RNA pellet2O to dissolve the RNA pellet.
M. 1. mu.L RNA samples were taken and RNA concentration and purity determined using a micro-UV spectrophotometer NanoDrop2000(Thermo Scientific). Mixing 1 μ L (more than 100 ng) with 7 μ L of H treated with LDEPC2O and 2. mu.L of 5 × RNALoading Buffer, the integrity of the RNA was checked by electrophoresis on a 1% agarose gel. If RNA integrity is good, the brightness ratio of 28S to 18S is about 2: 1.
(2) Reverse transcription of RNA into cDNA:
reverse Transcription of cDNA was performed according to the instruction of cDNA Reverse Transcription kit (cat. no RR036A) of Takara.
1)5×PrimeScript RTMaster Mix(Perfect Real Time):2μL
1×Total RNA:0.5μg
RNase Free dH2O up to 10μL
2) After gentle and uniform mixing, carrying out reverse transcription reaction under the following conditions:
15min at 37 ℃ (reverse transcription)
5sec at 85 ℃ (inactivation reaction of reverse transcriptase)
Short-term storage at 4 ℃.
5. Real-time fluorescent quantitative PCR (RT-qPCR) detection of gene expression changes
Real-time fluorescent quantitative PCR assay procedures were performed according to the instructions of ABClonal SYBGreen Mix.
(1) The components added in each reaction system were as follows:
TABLE 3 RT-qPCR reaction System
(2) Adding the reaction system into a 384-well plate, and flatly sticking a sealing film on the 384-well plate at 1500 r.min-1Centrifuge for 2 min.
(3) The 384 well plate was placed in the reaction tray as specified in the instrument and set up for the following reaction protocol.
(4) After the reaction is finished, information such as sample sequence, detected gene and the like is set, and system software is used for analyzing data. The system program will automatically calculate the relative expression value of the gene of the detection gene according to the Ct values of the target gene and the reference gene.
(5) The algorithm used is: relative gene expression value of 2((Ct target gene-Ct internal reference))Finally, the control quantity is set as
1, the relative expression value of the gene in other samples can be calculated.
Second, experimental results
As shown in FIG. 1, CLC crystals at different concentrations can cause increased expression of IL-1 beta, TNF-alpha, IL-6, GM-CSF and IL-8 in human nasal mucosal epithelial cells, and are concentration gradient dependent. As shown in figure 2, after CLCs (100 mu g/mL) are induced for 24 hours, the expression of IL-1 beta, TNF-alpha, IL-6, GM-CSF and IL-8 of human nasal mucosal epithelial cells is remarkably increased, and the difference has statistical significance. The experimental result proves that the in vitro model for inducing the natural immune factor activation of the human nasal mucosa epithelial cells by the CLCs is successfully constructed.
Example 2 screening of polypeptide sequences that can bind to CLC crystals or to Gal-10 proteins
First, experiment method
1. The polypeptide chip is used for screening the polypeptides capable of being combined with the CLCs:
polypeptide chips (containing 400 different polypeptide sequences) are self-prepared in laboratories, and polypeptide sequences capable of acting with CLCs are screened out by incubating the CLCs marked by a control group-EGFP fluorescent protein and an experimental group-EGFP protein with the chips respectively.
(1) The polypeptide chips were incubated for 1h with a 5% BSA shaker.
(2) BSA was discarded and washed 3 times with TBST.
(3) And adding Gal-10 marked by EGFP fluorescent protein and EGFP protein to the two same polypeptide arrays respectively, and incubating for 1h at room temperature by using a shaker.
(4) Washing with TBST for 4 times, shaking for 10min each time.
(5) Fluorescence Detection was performed using the Fluorescence Detection FLA 9500.
2. Screening polypeptides binding to Gal-10 protein using phage display technology:
(1) elutriation:
A. with 0.1M NaHCO3(pH 8.6) the target protein (Gal 10 protein with a lysotropic tag) was diluted to 100. mu.g/mL, plated in 1.5mL plastic plates of 6cm, placed in wet boxes, and shaken overnight at 4 ℃.
B. The protein coated plate was back-buckled on a clean paper towel, and 1.5 mLBpacking buffer (ready-to-use) was added to the target protein for at least 1 h. Blocking buffer was removed and washed rapidly 6 times with TBST (TBS + 0.1% Tween-20). Repeated rotation ensures that both the bottom and the sides of the well are washed. The washing liquid is reversely buckled on a clean paper towel, the washing is rapid, and the plate is prevented from being dried.
C. Phage (10. mu.L phase +1mL TBS) were diluted with 1mL TBS and added to the coated plate and gently shaken at room temperature for 10-60 min. The plates were washed 10 times with TBST, each time with a clean paper towel to avoid cross contamination.
D. (4)1mL elution buffer (containing BSA, ready to use) eluted the bound phage with gentle shaking for no more than 10min, and the eluate was transferred to a microcentrifuge tube and neutralized with 150. mu.L of 1M Tris-HCl (pH 9.1).
E. Titers were measured to determine dilution fold for the next round of panning.
(2) And (3) amplifying the phage:
A. the eluate was added to 20mL of ER2738 culture (early log, overnight cultured E.coli diluted 1: 100) and incubated at 37 ℃ with vigorous shaking for 4.5 h.
B. The culture was transferred to a microcentrifuge tube at 4 ℃ at 12000g for 10min, and the supernatant was transferred to a new microcentrifuge tube and centrifuged again.
C. 80% of the supernatant was transferred to a new centrifuge tube and 1/6 volumes of 20% PEG/NaCl were added. Phage were precipitated overnight at 4 ℃.
The supernatant was centrifuged at 12000g for 15min at D.4 ℃ and decanted, centrifuged again and excess supernatant aspirated.
E. The pellet was suspended with 1ml of LTBS and transferred to a microcentrifuge tube, and the remaining cells were pelleted by centrifugation at 14000rpm for 5min at 4 ℃ and the pellet was discarded.
F. The supernatant was transferred to a new centrifuge tube, reprecipitated with 1/6 volumes of PEG/NaCl and incubated on ice for 15-60 min. Centrifugation was carried out at 14000rpm for 10min at 4 ℃ and the supernatant was discarded and centrifuged again briefly to remove the residual supernatant.
G. The precipitate was suspended with 200 μ LTBS and the supernatant was transferred to a new tube, the amplified eluate. (the amplified eluate was used for the next round of panning)
(3) And (3) titer determination:
A. the upper agar was melted in a microwave oven, divided into 3mL portions, and stored in a 45 ℃ water bath for further use in sterile test tubes. The LB/IPTG/Xgal plates were pre-warmed at 37 ℃.
B. Gradient diluted phages were prepared in LB.
C. And dividing ER2738 with OD6000.5 into 200 mu L equal parts, adding 10 mu L phage with different dilutions into each tube, rapidly shaking and mixing uniformly, and incubating at room temperature for 1-5 min.
D. The infected colibacillus is added into an upper agar culture tube pre-warmed at 45 ℃, quickly mixed evenly and immediately poured on an LB/IPTG/Xgal plate pre-warmed at 37 ℃. The plate was tilted to spread the upper agar evenly.
E. After the plate was cooled, it was inverted and incubated at 37 ℃ overnight.
F. The following day the number of plaques on the plates with 1-100 plaques was counted. This number was then multiplied by a dilution factor to give a plaque forming unit (pfu) titer per 10. mu.L phage. Or for picking single clones.
(4) Extracting phage DNA:
A. a single blue chip was pricked with a 200. mu.L tip and added to OD0.05 of the broth, followed by shaking for 4.5 h.
B. The culture was centrifuged again at 14000rpm for 30s and the supernatant was transferred. 80% of the supernatant was transferred (it could be stored at 4 ℃ for several weeks).
C. Transferring 500. mu.L into a new microcentrifuge tube, adding 200. mu.L of PEG, and standing at room temperature for 10-20 min.
D.4 ℃, 14000rpm, 10min, abandoning the supernatant and centrifuging again.
E. Add 100. mu.L of iodide solution to dissolve the precipitate completely, add 250. mu.L of ethanol, and room temperature 10-20 min.
F.4 ℃, 14000rpm, 10min, discarding the supernatant, adding 70% ethanol, centrifuging again, discarding the supernatant, vacuum drying, using 30 μ LPCR grade pure water to dissolve DNA, and sending to sequencing.
G. According to the sequencing result, the polypeptide which can have the interaction with the Gal-10 with the lysotropic tag can be obtained.
3. Verification of polypeptide inhibition of CLCs-induced immune response at cellular level
The cells cultured in step (1) of step 3 of example 1 were stimulated with the polypeptide (50. mu.M) and CLCs (100. mu.g/ml), and after 24 hours, the cellular RNA was collected and subjected to reverse transcription and qPCR.
Second, experimental results
Based on the above method, 8 polypeptides that can be combined with Gal10 protein were screened, and the polypeptide (#8) related to the present invention can be combined with CLCs. The results of experiments in which these 8 polypeptides inhibited immune responses induced by CLCs were verified at the cellular level, as shown in figure 3 (in the figure, P <0.05, P <0.01, P <0.001, P < 0.0001). As shown in FIG. 4, the polypeptide (#8) involved in the invention can effectively inhibit the increase of the expression of human nasal mucosal epithelial cells IL-1 beta, TNF-alpha, IL-6, IL-8 and GM-CSF caused by CLC crystals (100 mu g/mL). The experimental results prove that the polypeptide can effectively inhibit the natural immune response activated by the CLC crystal.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> institute of basic medicine of Chinese academy of medical sciences
BEIJING TONGREN HOSPITAL, CAPITAL MEDICAL University
<120> polypeptide targeting Charcot-Laeden crystallin and application thereof
<141> 2021-08-31
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 142
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ser Leu Leu Pro Val Pro Tyr Thr Glu Ala Ala Ser Leu Ser Thr
1 5 10 15
Gly Ser Thr Val Thr Ile Lys Gly Arg Pro Leu Ala Cys Phe Leu Asn
20 25 30
Glu Pro Tyr Leu Gln Val Asp Phe His Thr Glu Met Lys Glu Glu Ser
35 40 45
Asp Ile Val Phe His Phe Gln Val Cys Phe Gly Arg Arg Val Val Met
50 55 60
Asn Ser Arg Glu Tyr Gly Ala Trp Lys Gln Gln Val Glu Ser Lys Asn
65 70 75 80
Met Pro Phe Gln Asp Gly Gln Glu Phe Glu Leu Ser Ile Ser Val Leu
85 90 95
Pro Asp Lys Tyr Gln Val Met Val Asn Gly Gln Ser Ser Tyr Thr Phe
100 105 110
Asp His Arg Ile Lys Pro Glu Ala Val Lys Met Val Gln Val Trp Arg
115 120 125
Asp Ile Ser Leu Thr Lys Phe Asn Val Ser Tyr Leu Lys Arg
130 135 140
<210> 2
<211> 181
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Ala Ser Thr Thr His His His His His His Asp Thr Asp Ile Pro
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Thr Thr Gly Gly Gly Ser Arg Pro Asp Asp Asp Asp Asp Lys Glu Asn
20 25 30
Leu Tyr Phe Gln Gly His Met Met Ser Leu Leu Pro Val Pro Tyr Thr
35 40 45
Glu Ala Ala Ser Leu Ser Thr Gly Ser Thr Val Thr Ile Lys Gly Arg
50 55 60
Pro Leu Ala Cys Phe Leu Asn Glu Pro Tyr Leu Gln Val Asp Phe His
65 70 75 80
Thr Glu Met Lys Glu Glu Ser Asp Ile Val Phe His Phe Gln Val Cys
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Phe Gly Arg Arg Val Val Met Asn Ser Arg Glu Tyr Gly Ala Trp Lys
100 105 110
Gln Gln Val Glu Ser Lys Asn Met Pro Phe Gln Asp Gly Gln Glu Phe
115 120 125
Glu Leu Ser Ile Ser Val Leu Pro Asp Lys Tyr Gln Val Met Val Asn
130 135 140
Gly Gln Ser Ser Tyr Thr Phe Asp His Arg Ile Lys Pro Glu Ala Val
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Lys Met Val Gln Val Trp Arg Asp Ile Ser Leu Thr Lys Phe Asn Val
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Ser Tyr Leu Lys Arg
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ile Ser Gln Phe Pro Gln Thr Pro Arg Leu Tyr Glu
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