WO2023030407A1 - Polypeptide targeting charcot-leyden crystal protein and use thereof - Google Patents

Abstract

The present invention relates to a polypeptide targeting a charcot-leyden crystal protein and the use thereof. The polypeptide contains at least one of an amino acid sequence as represented by SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, or a fragment, a variant, a fusion or a derivative thereof, or a fusion of the fragment, the variant or the derivative thereof. The present invention also relates to a nucleic acid encoding the polypeptide, a recombinant vector and a cell for expressing the polypeptide. Also provided are the use of the polypeptide in the preparation of a drug for preventing or treating a type 2 immune disease, and a method for detecting a Gal-10 protein for non-diagnostic purposes.

Description

Peptides Targeting Charcot-Leyden Crystalline Proteins and Their Applications technical field

The invention relates to the field of biomedicine, in particular to a polypeptide targeting Charcot-Leyden crystal protein and its application.

Background technique

Type 2 immunity is a special immune response that includes innate immunity and adaptive immunity and promotes the formation of an immune barrier on the mucosal surface to clear pathogens. Studies in recent years have shown that type 2 inflammatory pathways play 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. Under the stimulation of antigen, the activation of dendritic cells (DC) promotes the differentiation of T cells into Th2 cells, releases type 2 cytokines, and then stimulates the production of IgE and the accumulation of eosinophils.

Galectins are carbohydrate-binding proteins involved in many physiological functions such as inflammation, immune response, cell migration, autophagy, and signal transduction, and they are also associated with fibrosis, cancer diseases. So far, a total of 16 galectins have been discovered in mammals. Galectin-10 is a member of the Galectin superfamily, which exists in eosinophils, basophils, granulocytes and some T cells. Charcot Leyden crystals (CLC) are non-soluble forms of Gal-10 that are only formed during eosinophil extracellular trap generation, and CLC are released extracellularly during eosinophil cell membrane disruption and cell death . Staphylococcus aureus and its exotoxin are important pathogens of chronic rhinosinusitis nasal polyposis (CRSwNP), and colonization by Staphylococcus aureus induces the formation of a large number of CLCs, which can further promote natural immune responses and aggravate type 2 Immune response, and can cause the aggregation of neutrophils, screening of polypeptides that can target CLC or Gal-10 protein is expected to be applied to the diagnosis or treatment of CLC-induced diseases and/or type 2 immune diseases, for the diagnosis or treatment of CLC Induced diseases and/or type 2 immune diseases provide new approaches.

Contents of the invention

The purpose of the present invention is to provide a polypeptide that can be used for diagnosing or treating type 2 immune diseases.

The technical scheme adopted in the present invention is:

The first aspect of the present invention provides a polypeptide, said polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, or its fragment, variant, fusion or derivatives, or fusions of said fragments, variants or derivatives thereof.

Said fragment, variant, fusion or derivative thereof, or said fusion of said fragment, variant or derivative thereof retains the inhibitory CLCs of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 Activity of the induced immune response.

Further, the variant comprises at least 55%, 60%, 65%, 70%, 75%, 80% of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5. %, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to amino acid sequences.

The second aspect of the present invention provides a pharmaceutical composition for preventing or treating type 2 immune diseases, said pharmaceutical composition comprising the polypeptide described in the first aspect of the present invention.

Further, the type 2 immune diseases include allergic diseases and mite infections.

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 includes a pharmaceutically acceptable buffer, carrier or excipient.

The third aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention in the detection of Gal-10 protein for non-diagnostic purposes.

The fourth aspect of the present invention provides a nucleic acid encoding the polypeptide described in the first aspect of the present invention.

The fifth aspect of the present invention provides a recombinant vector comprising the nucleic acid described in the fourth aspect of the present invention.

The sixth aspect of the present invention provides a cell comprising the nucleic acid of the fourth aspect of the present invention or the recombinant vector of the fifth aspect of the present invention.

Further, the cells include prokaryotic cells and eukaryotic cells.

Further, said prokaryotic cells include bacterial cells.

Further, the eukaryotic cells include protozoan cells, animal cells, plant cells, and fungal cells.

Further, the animal cells include mammalian cells, poultry cells, and insect cells.

The seventh aspect of the present invention provides the polypeptide described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention or the nucleic acid described in the fourth aspect of the present invention or the recombinant vector described in the fifth aspect of the present invention or The use of the cells described in the sixth aspect of the present invention in the preparation of drugs for preventing or treating type 2 immune diseases.

Further, the type 2 immune diseases include allergic diseases and mite infections.

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.

The eighth aspect of the present invention provides a non-diagnostic method for detecting Gal-10 protein, the method comprising:

(1) contacting the sample with the polypeptide described in the first aspect of the present invention;

(2) detecting the formation of a complex comprising the polypeptide described in the first aspect of the present invention;

Further, the method for detecting the formation of the complex comprising the polypeptide according to the first aspect of the present invention includes gel electrophoresis, chromatography techniques, western blot analysis, immunohistochemistry, mass spectrometry and/or high pressure liquid chromatography.

The ninth aspect of the present invention provides the polypeptide described in the first aspect of the present invention or the nucleic acid described in the fourth aspect of the present invention or the recombinant vector described in the fifth aspect of the present invention or the cell described in the sixth aspect of the present invention. Application in products for the diagnosis of type 2 immune diseases.

Further, the type 2 immune diseases include allergic diseases and mite infections.

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.

Description of drawings

Figure 1 RT-qPCR detection results of gene expression changes in human nasal polyp mucosal epithelial cells induced by different concentrations of CLCs, in which Figure A is a statistical chart of IL-1β expression changes, and Figure B is a statistical chart of TNF-α expression changes. C is a statistical chart of IL-6 expression changes, Figure D is a statistical chart of GM-CSF expression changes, and Figure E is a statistical chart of IL-8 expression changes;

Fig. 2 Statistical graph of gene expression changes in human nasal polyp mucosal epithelial cells when CLCs (100 μg/mL) were induced for 24 hours, wherein, graph A is a statistical graph of IL-1β expression changes, graph B is a statistical graph of TNF-α expression changes, and graph C is Statistical chart of IL-6 expression change, Figure D is a statistical chart of IL-8 expression change, Figure E is a statistical chart of GM-CSF expression change;

Figure 3 is a diagram of the experimental results of the immune response induced by eight kinds of polypeptides at the cellular level, where Figure A is the statistical chart of IL-1β expression, Figure B is the statistical chart of IL-6 expression, and Figure C is the expression of TNF-α Quantity statistical diagram, Figure D is a statistical diagram of IL-8 expression;

Figure 4 is a graph of the experimental results of the #1 polypeptide (SEQ ID NO.4) involved in the present invention to inhibit the immune response induced by CLCs at the cellular level, wherein, Figure A is a statistical graph of IL-1β expression, and Figure B is a TNF- Statistical graph of α expression, and Figure C is a statistical graph of IL-6 expression;

Figure 5 is a graph of the experimental results of the #3 polypeptide (SEQ ID NO.5) involved in the present invention to inhibit the immune response induced by CLCs at the cellular level, wherein, Figure A is a statistical graph of IL-1β expression, and Figure B is a TNF- Figure C is a statistical chart of IL-6 expression, Figure D is a statistical chart of IL-8 expression, Figure E is a statistical chart of GM-CSF expression;

Figure 6 is a graph of the experimental results of the #8 polypeptide (SEQ ID NO.3) involved in the present invention to inhibit the immune response induced by CLCs at the cellular level, wherein, Figure A is a statistical graph of IL-1β expression, and Figure B is a TNF- Figure C is a statistical chart of IL-6 expression, Figure D is a statistical chart of IL-8 expression, Figure E is a statistical chart of GM-CSF expression;

Fig. 7 utilizes biomembrane interference technology to verify the interaction ability of #8 polypeptide (SEQ ID NO.3) involved in the present invention and soluble Gal-10 protein;

Figure 8 uses FITC-labeled polypeptides to verify the affinity of #8 polypeptide (SEQ ID NO.3) involved in the present invention with CLC;

Fig. 9 is the dynamic process of dissolving CLC by polypeptides of different concentrations, in order to verify that #8 polypeptide (SEQ ID NO.3) involved in the present invention has the ability to dissolve CLC;

Figure 10 is a mouse lung injury model induced by CLC was established, and the inflammation-inducing ability of exogenous CLC was verified in mice;

Figure 11 verifies the detection results of inflammatory factors that #8 polypeptide (SEQ ID NO.3) involved in the present invention alleviates lung injury in mice in the CLC-induced acute lung injury model in mice;

FIG. 12 verifies that #8 polypeptide (SEQ ID NO.3) involved in the present invention alleviates the lung pathological results of mouse lung injury in the mouse CLC-induced acute lung injury model.

Detailed ways

polypeptide

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 (eg, α,α-disubstituted amino acids, N-hydrocarbyl amino acids, etc.) and chemically derivatized amino acids.

Where an amino acid is explicitly recited, such as "alanine" or "Ala" or "A," the term refers to both L-alanine and D-alanine, unless expressly stated otherwise. Other unconventional amino acids may also be suitable components of the polypeptides of the invention, so long as the polypeptide retains the desired functional properties. For the peptides shown, each encoded amino acid residue is (where appropriate) represented by a one-letter designation corresponding to the conventional amino acid common name.

"Variants" of polypeptides include insertions, deletions and substitutions, which are either conservative or non-conservative. For example, a conservative substitution refers to the substitution of an amino acid within the same general class (eg, acidic amino acid, basic amino acid, non-polar amino acid, polar amino acid, or aromatic amino acid) with another amino acid within the same class. As such, the significance of conservative and non-conservative amino acid substitutions is well known in the art. In particular, variants of polypeptides that exhibit activity that binds to CLCs and/or that inhibit immune responses induced by CLCs are included.

In some embodiments, the variant comprises at least 55%, 60%, 65%, 70% of the amino acid sequence shown in any of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5. , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence homology.

A "fusion" of a polypeptide includes an amino acid sequence corresponding to a reference sequence (eg, SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5, or a fragment or variant thereof) fused to any other polypeptide. For example, the polypeptide can 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 can be fused to an oligohistidine tag such as His6 or an epitope recognized by an antibody such as the well known Myc tag epitope. Additionally, fusions comprising hydrophobic oligopeptide end tags can be used. Fusions to any variant or derivative of said polypeptide are also included within the scope of the invention.

Fusions may comprise additional moieties that confer desired characteristics on the polypeptides of the invention; for example, such moieties may be used to detect or isolate the polypeptides, or to facilitate cellular uptake of the polypeptides. The moiety may for example be a biotin moiety, a streptavidin moiety, a radioactive moiety, a fluorescent moiety, eg a small fluorophore or a green fluorescent protein (GFP) fluorophore, as known to those skilled in the art. A module may be an immunogenic tag, such as a Myc tag, as known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain capable of facilitating cellular uptake of the polypeptide, as known to those skilled in the art.

Those skilled in the art will appreciate that a polypeptide of the invention may comprise one or more amino acids modified or derivatized, eg, by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

As appreciated in the art, PEGylated proteins can 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 affect the outcome are important and should be considered. PEG molecules can vary, and PEG variants that have been used for PEGylation of proteins include PEG and monomethoxy-PEG. Additionally, they can be either linear or branched.

Increasing the degree of PEGylation has been shown to result in increased half-life in vivo. However, those skilled in the art will appreciate that the PEGylation process will require optimization for a particular protein on an individual basis.

PEG can be coupled at naturally occurring disulfide bonds as described in WO 2005/007197. Disulfide bonds can be stabilized through the addition of chemical bridges that do not damage the structure of the polypeptide. This allows the creation of bridges for site-specific attachment of PEG using the conjugation thiol selectivity of the two sulfurs that make up the disulfide bond. Thus, the need to engineer residues into the peptide for attachment to the target molecule is circumvented.

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 combinations of these methods. Methods for preparing such polypeptides are well known in the art.

At present, the nucleic acid sequence encoding the polypeptide of the present invention can be obtained completely through chemical synthesis. The nucleic acid sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the sequence of the polypeptides of the invention by chemical synthesis.

The term "nucleic acid encoding a polypeptide" as used herein includes a nucleic acid comprising a sequence encoding a polypeptide of the present invention, especially a polypeptide having any amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 . The term also includes nucleic acids comprising a single contiguous region or multiple discontinuous regions encoding the polypeptide (e.g., due to integrating phage, integrating insert sequences, integrating vector sequences, integrating transposon sequences, or due to RNA editing or genomic DNA reconstruction) and additional regions, which may also comprise coding and/or non-coding sequences.

The vectors for constructing the recombinant vectors of the present invention include (but are not limited to) the MarEx expression vectors produced by Celltrion Inc. (Korea); pCDNA vectors widely available on the market; F, R1, RP1, Col, pBR322, ToL, Ti Vectors; cosmids; bacteriophages such as lambda phages, lambda phages, M13 phages, Mu phages, P1 phages, P22 phages, Qμ phages, T-even phages, T2 phages, T4 phages, T7 phages, etc.; plant viruses. Any of various vectors known to those skilled in the art can be used in the present invention, and the choice of the vector depends on the properties of the selected cells. The introduction of the vector into the cell can be achieved by (but not limited to) calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofection or electroporation, and anyone skilled in the art can Choose and use an introduction method appropriate for the vector and cells used. Preferably, the above-mentioned vectors contain one or more selection markers, but are not limited thereto, and vectors that do not contain selection markers can also be used. The choice of selectable marker may depend on the cells selected (as is well known to those skilled in the art), but is not critical to the invention.

Polypeptides of the invention can be prepared by any of a variety of techniques. Generally, polypeptides can be produced by cell culture techniques, including production of polypeptides by conventional techniques, or by transfection of nucleic acid molecules of the polypeptides into suitable bacterial or mammalian cell hosts to allow production of the polypeptides, which can be recombinant of. The various forms of the term "transfection" are intended to include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the polypeptides of the invention may be expressed in prokaryotic or eukaryotic cells, expression of the polypeptides in eukaryotic cells is preferred, and most preferably in mammalian cells, since such eukaryotic cells (especially mammalian cells) are more likely to Assemble and secrete correctly folded polypeptides than prokaryotic cells. When a recombinant expression vector of a nucleic acid molecule encoding a polypeptide is introduced into a mammalian cell, the polypeptide is secreted by culturing the cell for a period of time sufficient to allow expression of the polypeptide in the cell, or more preferably, the medium in which the cell is cultured. Polypeptides can be recovered from the culture medium using standard protein purification methods.

pharmaceutical composition

"Pharmaceutically acceptable" means a non-toxic material that does not reduce the active ingredient. Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A.R Gennaro, Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A ed. Kibbe, Pharmaceutical Press (2000)).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose 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, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC , 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.

Excipients can 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 compositions, eg, to facilitate lyophilization. Examples of polymers are various degrees of hydrolyzed starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, ethylcellulose , methylcellulose, propylcellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulphonate, polyethylene glycol/ Polyethylene oxide, polyethylene oxide/polypropylene oxide copolymer, polyvinyl alcohol/polyvinyl acetate, poly(lactic acid), poly(glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone ( They all have different molecular weights), which are added to the composition, for example, to control viscosity, to achieve bioadhesion, 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 degrees of saturation), egg lecithin, soybean lecithin, hydrogenated lecithin and soy lecithin, which are added to the composition for similar reasons as the polymer. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to compositions for benefits such as reduced liquid build-up or favorable pigment properties.

The pharmaceutical compositions of the 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 yield active ingredients from previously sterile-filtered solutions of the active ingredient and other desired ingredients. ingredients and powders of other desired ingredients. Alternatively, the compositions of the invention may be in solution, and suitable pharmaceutically acceptable excipients may be added and/or mixed before or at the time of delivery to provide an injectable unit dosage form. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain proper fluidity, and delay absorption if necessary.

The selection of the optimal route of administration of the pharmaceutical composition of the present invention will be affected by several factors, including the physicochemical properties of the active molecule in the composition, the urgency of the clinical manifestation and the relationship between the plasma concentration of the active molecule and the desired therapeutic effect. relationship between. For example, polypeptides of the invention can be prepared with carriers that will protect them against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used in the present invention, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Further, the polypeptide may be coated with, or administered simultaneously with, a material or compound that prevents the inactivation of the polypeptide. For example, polypeptides can be administered with a suitable carrier such as liposomes or diluents.

The route of administration of the pharmaceutical composition of the present invention can be divided into oral administration and parenteral administration.

Oral dosage forms can be formulated as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels or ointment. These formulations may contain pharmaceutical excipients, which include but are not limited to: granulating and disintegrating agents, binders, lubricants, preservatives, coloring, flavoring or sweetening agents, vegetable oils or minerals Oils, humectants, and thickeners.

Preparations for parenteral administration may be in the form of aqueous or nonaqueous isotonic sterile nontoxic injection or perfusion solutions or suspensions. The solutions or suspensions may include agents such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic solutions, such as 1,3-butanediol, Ringer's solution, Hank's solution, etc. sodium chloride solutions, oils, fatty acids, local anesthetics, preservatives, buffers, viscosity or solubility increasing agents, water-soluble antioxidants, oil-soluble antioxidants, and metal chelating agents.

other

Unless otherwise defined, 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 to refer to crystals formed from Galectin-10. Crystals formed from galectin-10 are generally bipyramidal hexagonal crystals, about 20-40 μm in length and about 2-4 μm in width.

The term "galectin-10" (or Gal10 or Gal-10) refers to a small hydrophobic glycan-binding protein that self-crystallizes to form Charcot-Leyden crystals. Galectin-10 is also known as Charcot-Leyden crystal protein (CLCP), eosinophil lysophospholipase, and lysophosphatidyl hydrolase. The term "galectin-10" is broad enough to encompass the human protein and any species homologue.

The term "specificity" refers to the ability to bind a given target (eg CLC crystals). 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 natural state. If an "isolated" composition or substance exists in nature, it has been altered from its original environment, or has been removed from its original environment, or both. As this term is used herein, for example, a polypeptide naturally occurring in a living animal is not "isolated", but said polypeptide is "isolated" when it is separated from the coexisting substances of its natural state.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Experimental Materials:

1. Plasmid: After the Gal-10 protein sequence is humanized and codon-optimized, a solubilizing fragment is added to its N-terminus, and cloned into the pET-28a vector plasmid through the NcoI/XhoI double restriction site to form pET-28a -Gal10-TEV-6XHis. The Gal-10 protein sequence is shown in Table 1, and the Gal-10 protein sequence with a solubilizing fragment is shown in Table 2.

Table 1 Gal-10 protein sequence

Table 2 Gal-10 sequences with solubilizing fragments

2. Reagent materials and equipment

1. Chemical reagents: Kanamycin and Ampicillin were purchased from Tiangen Biotechnology Co., Ltd.; Isopropyl-beta-D-thiogalactoside (IPTG ) and Escherichia coli BL21 (DE3) were purchased from Beijing Quanshijin Company; SDS, Trizol and imidazole were purchased from Sigma Company; TEV enzyme was purchased from Beijing Yiqiao Shenzhou Co., Ltd.; Coomassie Brilliant Blue staining solution (self-made); cDNA reverse transcription reagent Purchased from Takara Company; real-time fluorescent quantitative PCR reagents were purchased from Aibotec Biotechnology; the peptide was synthesized in Guoping Pharmaceutical Co., Ltd., the sequence is shown in Table 3, wherein the first amino acid at the C-terminal of #3 polypeptide (SEQ ID NO.5) An amino group is connected, and the 8th amino acid at the N-terminal is citrulline.

Table 3 Polypeptide Sequence

pure 25蛋白质纯化系统(superdex 75,GE Healthcare)；SDS-PAGE电泳仪(Bio-Rad)；凝胶成像仪(Molecular Imager Gel Doc XR,Bio-Rad)；ABI PCR仪器(ABI 7500)。 2. Consumables and equipment: 10cm cell culture dishes and 12-well cell culture plates were purchased from CORNING Company; BEGM medium for cell culture was purchased from Lonza Company; DMEM high-glucose medium, fetal bovine serum FBS, digestive solution (2.5g/ L trypsin, 0.02g/L EDTA, pH=8.0, 0.22μm filter), penicillin (20mg/mL), streptomycin (20000 U/mL) were purchased from GIBCO; Ni-NTA affinity chromatography column was purchased from GE Company; 10kDa concentration tube was purchased from Millipore Company. Electric constant temperature incubator (XMTD HH.B11-600); PCR instrument (Biometra Tgradient); desktop centrifuge (eppendorf, Centrifuge 5415D); electric constant temperature water tank (SHH W21 600); trace ultraviolet spectrophotometer (NanoDrop 2000); Balance (Sartorius 2000S); electronic analytical balance (Sartorius, BS110S); optical inverted microscope (XDS-1B); micropipette (eppendorf Research plus); cell _CO2 incubator (SANYO); , MDF-382E); pH meter (Thermo Orion 868); magnetic stirrer (IKA RH-KT/C); microplate reader (Bio-Rad, 680); ultrasonic breaker;

pure 25 protein purification system (superdex 75, GE Healthcare); SDS-PAGE electrophoresis instrument (Bio-Rad); gel imager (Molecular Imager Gel Doc XR, Bio-Rad); ABI PCR instrument (ABI 7500).

Expression and purification of embodiment 1Gal-10 recombinant protein and generation of CLCs

1. Experimental method:

(1) Conversion:

A. Take a BL21(DE3) competent cell and thaw it on ice;

B. Add 0.5 μL of pET-28a-gal10-TEV-6His recombinant plasmid, and incubate on ice for 15-20 minutes;

C. Heat shock: heat shock in 42°C water bath for 90s;

D. Quickly place on ice, ice bath for 5 minutes;

E. Add 500 μL of non-resistant LB, and incubate in a constant temperature incubator at 37°C for 40-50 minutes;

F. Take 40 μL of the bacterial liquid, inoculate it on the LB solid medium of Kanamycin (25 mg/mL), and cultivate it overnight in a constant temperature incubator at 37°C.

(2) Bacterial culture:

Using Kanamycin (25mg/mL) as a selection marker, pick the positive monoclonal BL21(DE3)/pET-28a-gal10-TEV-6His on the plate in the previous step, and inoculate it in 20mL of Kanamycin-resistant LB liquid medium , at 37°C, 210r/min shaker shaking culture for 12h. The bacterial liquid was inoculated into 1L LB liquid medium containing kanamycin at a ratio of 1:100, and cultured at 37°C and 210r/min shaking.

(3) Induced expression and acquisition of target protein:

A. When the optical density at 600nm (OD 600 ) of Escherichia coli is 0.6-0.8h, add IPTG with a final concentration of 1mM, and culture on a shaker at 28°C overnight to induce the expression of the target protein;

B. Enrich the overnight expressed bacterial culture, centrifuge at 6 000×g, 4°C for 20min, and discard the supernatant. Resuspend with Lysis Buffer, concentrate it in a beaker with a volume of about 75-100mL, then add PMSF (final concentration: 1mM) at a ratio of 1:100 to protect the target protein;

C. Ultrasonic disruption of bacteria: power 25%, working time 25min, ultrasonic on time 3s, ultrasonic off time 9s; centrifuge (4°C, 13,000×g, 30min) to remove cell debris, and at the same time break up the nucleic acid released after cell disruption, Centrifugal sedimentation makes the cell lysate less viscous and facilitates subsequent processing. Collect the supernatant, and the soluble protein of interest exists in the supernatant.

(4) Purification of Gal-10 protein:

A. Ni-NTA affinity column chromatography

a. Equilibrate the Ni column: use Lysis Buffer to penetrate the Ni column for about 2-3 column volumes, and then start to elute the protein;

b. Load the supernatant obtained by centrifugation, and pass through the Ni column 2-3 times;

c. Wash the column with Lysis Buffer containing 20mM imidazole and 0.1% Empigen detergent to elute the impurity protein;

d. Wash the column with Lysis Buffer containing 500mM imidazole to remove the target protein from the nickel column;

e. Concentration: Put the eluted and collected target protein into a 10kDa concentrator tube, centrifuge at 2000×g for 10 min at 4°C, add a small amount of Lysis Buffer or PBS to dilute the imidazole during the concentration process to prevent the target protein from aggregation and precipitation.

pure 25蛋白纯化 b.

pure 25 protein purification

a. Clean the chromatography column: Use sterile water to clean the chromatography column, about 8mL. Then put the pump head in the PBS solution, repeat the above steps and execute, and clean about 36mL;

b. Parameters: System flow: 0.5mL/min; Column position: 3; Alarm delta column pressure enabled: 1.5; Alarm pre column pressure enabled: 5;

c. Sample loading: Rinse the sample loop two or three times with PBS first, then inject 2mL of a sample with a concentration of about 6.35mg/mL into the sample loop, and finally absorb PBS slightly before injecting into the sample loop to avoid sample residue. Arrange the collection tube and select inject valve:inject;

d. Collection: When the UV 280 marking line representing the protein content rises, use the automatic sample collector to collect the target protein, and set the collection volume of each tube to 0.3mL;

e. Cleaning the chromatographic column: After collecting the samples, put the pump head in PBS, continue to run until 20mL, replace with sterile water for cleaning, run 10ml, and then replace with 20% ethanol solution to run 20mL, that is can shut down;

f. Protein treatment: Measure the concentration of each tube of the collected protein and mark it, place it in liquid nitrogen for quick freezing, and store it in a -80°C refrigerator. Let stand at room temperature and melt slowly before the experiment.

C. SDS-PAGE identification of expression products

a. Sample preparation: Whole bacteria sample, centrifuged supernatant sample, protein supernatant passed through the chromatographic column sample, 20mM imidazole passed through the sample, 500mM imidazole passed through the sample, collected at the peak head, peak peak and peak tail in the molecular sieve protein samples;

b. Gel preparation: 5% stacking gel; according to the size of the protein, choose 15% separating gel;

c. Sample preparation: 2×Loading Buffer mixed with sample 1:1;

d. Sample loading: 5 μL of whole bacteria sample, 3 μL of marker, and 10 μL of other samples;

e. Staining: put protein glue in Coomassie brilliant blue staining solution, microwave at medium-high heat, 2min;

f. Decolorization: Put the dyed protein glue in tap water, microwave on high heat, 20-40min.

(5) Generation and concentration determination of CLCs:

A. TEV enzyme and pET-28a-gal10-TEV-6His recombinant protein were mixed at a mass ratio of 1:10, and incubated overnight at 4°C for enzyme digestion on a shaker;

B. Centrifuge at 600×g and 4°C for 10 minutes. After the centrifugation, remove the supernatant and add an appropriate amount of PBS to resuspend. Repeat three times to obtain pure CLCs.

C. BCA method to detect the concentration of CLCs.

D. Coomassie brilliant blue staining and Western blot verification

2. Experimental results:

Through the above experimental methods, we obtained CLC crystals and soluble Gal-1 protein.

Example 2 Screening of polypeptide sequences that can bind to CLC crystals or to Gal-10 protein

1. Experimental method

1. Use the peptide chip to screen the peptides that can bind to CLCs:

The laboratory self-made polypeptide chip (containing 400 different polypeptide sequences), by respectively incubating the CLCs labeled with the control group-EGFP fluorescent protein and the experimental group-EGFP protein with the chip, to screen out the polypeptide sequences that can interact with CLCs.

(1) Incubate the peptide chip with 5% BSA on a shaker for 1 h.

(2) Discard BSA and wash 3 times with TBST.

(3) Add EGFP fluorescent protein-labeled Gal-10 and EGFP protein to two identical polypeptide arrays respectively, and incubate at room temperature for 1 hour on a shaker.

(4) Wash 4 times with TBST, and shake on the shaker for 10 minutes each time.

(5) Use Fluorescence Detection FLA9500 for fluorescence detection.

2. Use phage display technology to screen peptides that bind to Gal-10 protein:

(1) Washing:

A. Dilute the target protein (Gal10 protein with solubilization tag) to 100 μg/mL with 0.1M NaHCO ₃ (PH 8.6), spread 1.5 mL on a 6 cm plastic petri dish, place in a humid box, and shake overnight at 4°C.

B. Flip the protein-coated plate upside down on a clean paper towel, add 1.5mL Blocking buffer (prepared now) for at least 1 hour for the target protein. Remove the Blocking buffer and wash quickly 6 times with TBST (TBS+0.1% Tween-20). Repeated rotation ensures that the bottom of the well and the sides of the well are washed. Buckle it upside down on a clean paper towel, and wash it quickly to avoid drying the plate.

C. Dilute the phage (10μL phage+1mL TBS) with 1mL TBS, add to the coated plate, shake gently at room temperature for 10-60min. Wash the plate 10 times with TBST, changing to a clean paper towel each time to avoid cross-contamination.

D. 1mL elution buffer (contains BSA, ready-to-use) to elute the bound phage, shake gently for no more than 10min, transfer the eluate into a microcentrifuge tube, and dilute with 150μL 1M Tris-HCL (PH 9.1) neutralize.

E. Measure the titer to determine the dilution factor for the next round of elutriation.

(2) Amplify phage:

A. Add the eluate to 20 mL of ER2738 culture (in the early logarithmic phase, dilute E. coli overnight at a ratio of 1:100), and incubate at 37°C for 4.5 hours with vigorous shaking.

B. Transfer the culture to a microcentrifuge tube, 4°C, 12000g, 10min, transfer the supernatant to a new centrifuge tube, and centrifuge again.

C. Transfer 80% of the supernatant to a new centrifuge tube and add 1/6 volume of 20% PEG/NaCl. Phage were precipitated overnight at 4°C.

D. Centrifuge the supernatant at 12000g for 15 minutes at 4°C, pour off the supernatant, centrifuge again, and suck off the excess supernatant.

E. Suspend the precipitate with 1mL TBS and transfer it to a microcentrifuge tube, centrifuge at 14000rpm at 4°C for 5min to precipitate the remaining cells, and discard the precipitate.

F. Transfer the supernatant to a new centrifuge tube, use 1/6 volume of PEG/NaCl to precipitate again, and incubate on ice for 15-60min. Centrifuge at 14,000 rpm for 10 min at 4°C, discard the supernatant, centrifuge briefly again, and suck off the remaining supernatant.

G. Use 200μL TBS to suspend the precipitate, and transfer the supernatant to a new tube, which is the amplified eluate. (The amplified eluate is used for the next round of elutriation)

(3) Titer determination:

A. Melt the upper layer of agar in a microwave oven, divide it into 3mL aliquots in sterilized test tubes, and store them in a 45°C water bath for later use. Pre-warm LB/IPTG/Xgal plates at 37°C.

B. Prepare serial dilutions of phage in LB.

C. Take ER2738 at OD600 0.5, divide it into 200 μL aliquots in microcentrifuge tubes, add 10 μL of different dilutions of phage to each tube, shake and mix quickly, and incubate at room temperature for 1-5 minutes.

D. Add the infected Escherichia coli to the upper agar culture tube pre-warmed at 45°C, mix quickly, and immediately pour it onto the LB/IPTG/Xgal plate pre-warmed at 37°C. Tilt the plate to evenly spread the upper layer of agar.

E. After the plate is cooled, place it upside down at 37°C and incubate overnight.

F. Count the number of plaques on the plate with 1-100 plaques on the next day. This number was then multiplied by the dilution factor to obtain the plaque forming unit (pfu) titer per 10 μL of phage. Or for picking single clones.

(4) Extract phage DNA:

A. Use a 200μL pipette tip to pierce a single blue and add it to the OD0.05 bacterial solution, shake for 4.5h.

B. Put the culture at 4°C, 14000rpm, for 30s, transfer the supernatant, and centrifuge again. Transfer 80% of the supernatant (can be stored at 4°C for several weeks).

C. Transfer 500 μL to a new microcentrifuge tube, add 200 μL of PEG, and let stand at room temperature for 10-20 minutes.

D. 4°C, 14 000rpm, 10min, discard the supernatant, and centrifuge again.

E. Add 100 μL of iodide solution to dissolve the precipitate completely, add 250 μL of ethanol, and keep at room temperature for 10-20 minutes.

F. 4°C, 14 000rpm, 10min, discard the supernatant, add 70% ethanol, centrifuge again, discard the supernatant, vacuum dry, use 30μL of PCR-grade pure water to dissolve the DNA, and then send it for sequencing.

G. According to the sequencing results, the polypeptides that may interact with the Gal-10 with the solubilizing tag can be obtained.

3. Verify that the peptide inhibits the immune response induced by CLCs at the cellular level

The cells cultured in step (1) of step 3 in Example 1 were co-stimulated with polypeptide (50 μM) and CLCs (100 μg/ml), and cellular RNA was collected after 24 hours for reverse transcription and qPCR.

2. Experimental results

Based on the above method, the present invention has screened 8 polypeptides that may bind to Gal10 protein, and the polypeptides (#1, #3, #8) involved in the present invention can bind to CLCs. The experimental results of these eight polypeptides inhibiting the immune response induced by CLCs were verified at the cellular level, as shown in Figure 3 (in the figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, ** ** indicates P<0.0001). As shown in Figure 4, the polypeptide (#1) involved in the present invention can effectively inhibit the increased expression of human nasal mucosal epithelial cells IL-1β, TNF-α, and IL-6 caused by CLCs (100 μg/mL); 5, the polypeptide (#3) involved in the present invention can effectively inhibit the human nasal mucosal epithelial cells IL-1β, TNF-α, IL-6, IL-8 and GM-CSF induced by CLC crystals (100 μg/mL). As shown in Figure 6, the polypeptide (#8) involved in the present invention can effectively inhibit the human nasal mucosal epithelial cells IL-1β, TNF-α, IL-6, Expression of IL-8 and GM-CSF was increased. The above experimental results prove that the polypeptide involved in the present invention can effectively inhibit the natural immune response activated by CLC crystals.

Example 3 CLC-induced nasal polyp-derived human nasal mucosal epithelial cell innate immune response activation model

1. Experimental method

1. Acquisition of human nasal polyp tissue epithelial cells

(1) Enzymatic digestion of tissue:

A. Put the surgically removed mucosal specimen into sterile saline.

B. Take out the specimen and soak it in 5 mL of tissue washing solution (physiological saline containing 200 μg/mL of penicillin and streptomycin) at 4°C for 1 hour.

C. Put the soaked specimen on a 6cm petri dish, wash it repeatedly with PBS + double antibody (can be washed several times, generally ≥10 times), wash the blood clots and dirt on the specimen, and place it under a dissecting microscope The mucus and blood vessels on the specimen were removed.

D. Put the cleaned specimen into a 15mL centrifuge tube and add 10mL human protease solution (collagenase type 2 and DNase I).

E. Take human placental collagen (Collagen I) and coat a 10cm culture dish, 2mL/well, overnight at 37°C.

(2) Nasal epithelial cell inoculation:

A. Add 2mL whole serum culture solution to the digested tissue tube and shake repeatedly for 20s.

B. Take out the supernatant, add 5mL whole serum culture solution again, shake repeatedly for 20s, and take out the supernatant again.

C. Centrifuge the supernatant taken out at 800r for 5min and remove the supernatant.

D. Add 1mL of whole serum culture solution and put it into a 6cm culture dish for pre-attachment for 1 hour.

E. Aspirate the human placental collagen from the coated culture dish (recovery after coating ≤3 times), rinse with distilled water ≥10 times (more or less). Disinfect by ultraviolet light for 20 minutes.

F. Aspirate the supernatant from a 6cm culture dish, add 5mL whole serum culture solution, and centrifuge at 800r for 5min. Discard the supernatant (aspirate as clean as possible).

G. Add 3mL red blood cell lysate to mix the cells, lyse at room temperature for 5min, centrifuge at 800r for 5min, and discard the supernatant.

H. After washing with 3mL sterile PBS, centrifuge at 800r for 5min, discard the supernatant, and resuspend with BEGM.

I. Take 1 μL, add 9 μL PBS, mix well and add to a cell counting plate for counting.

J. Cell counting: (number of cells in cell suspension)/mL=(number of n cells/n)×16×10 ⁴

K. After counting, add 2×10 ⁶ to the coated 10cm culture dish, and add 12mL BEGM epithelial medium.

2. CLCs stimulate human nasal mucosal epithelial cells

(1) Nasal epithelial cell inoculation:

A. The primary nasal mucosal epithelial cells of nasal polyps grow to about 85-90%, discard the medium, wash with 2mL of sterile PBS, digest with 2mL of 0.25% trypsin at 37°C for 4-5min, and then add 1mL of FBS-containing medium Complete medium was terminated.

B. After blowing the cells to a single-cell suspension, centrifuge at 800r for 5min and discard the supernatant.

C. After cell counting, spread 2×10 ⁵ cells to a 12-well plate/well and culture with 1 mL BEGM medium.

(2) CLCs stimulate nasal epithelial cell model:

A. The 12-well plate was cultured for about 2 days. When the primary nasal mucosal epithelial cells grew to about 80%, the medium was discarded, and the culture medium was continued for 24 hours with BEGE medium containing different concentrations of CLC.

B. Collect the cell supernatant, centrifuge at 800r at 4°C for 5 minutes, and take the upper layer and freeze it in a -80°C refrigerator for later use.

C. Wash the cells with 1mL sterile PBS and add 1mL Trizol/well to lyse the cells for RNA extraction.

3. RNA extraction and cDNA reverse transcription

(1) RNA extraction:

A. Thaw the lysate of cells or tissues frozen in a -80°C refrigerator in Trizol and slowly thaw on ice.

B. After the RNA sample is completely thawed, take the sample to room temperature for 10 minutes to fully dissolve the RNA.

C. Add chloroform according to the volume ratio of 200 μL chloroform/mL Trizol, shake vigorously to make it fully mixed, and place it at room temperature for 15 minutes.

D. Centrifuge at 12000r·min ^-1 for 15min at 4°C.

E. Transfer the upper aqueous phase to a new RNase-free 1.5 mL centrifuge tube.

F. Add pre-cooled isopropanol according to the volume ratio of 800μL isopropanol/mL water phase, mix thoroughly, and place at room temperature for 10-15min.

G. Centrifuge at 12000r·min ^-1 at 4°C for 10min, discard the supernatant, and there is RNA precipitation at the bottom of the centrifuge tube.

H. Add 1 mL of 750 mL/L ethanol prepared with DEPC-treated H ₂ O, and shake the centrifuge tube gently to resuspend the pellet.

I. Centrifuge at 12000r·min ^-1 at 4°C for 5min, and discard the supernatant as much as possible.

J. Repeat steps 8-9.

K. Dry at room temperature for 5-10 minutes, and wait for the 750mL/L ethanol to evaporate.

L. Dissolve the RNA pellet with 25-100 μL DEPC-treated H ₂ O depending on the size of the RNA pellet.

M. Take 1 μL RNA sample and measure the RNA concentration and purity with a micro-volume ultraviolet spectrophotometer NanoDrop2000 (Thermo Scientific). And take 1 μL (just greater than 100ng) mixed with 7 μL of DEPC-treated H ₂ O and 2 μL of 5×RNA Loading Buffer, and use 1% agarose gel electrophoresis to detect the integrity of the RNA. If the integrity of the RNA is good, the brightness ratio of 28S to 18S is about 2:1.

(2) RNA reverse transcription into cDNA:

Reverse transcription of cDNA was performed according to the instructions of cDNA Reverse Transcription Kits (cat.no RR036A) of Takara Company.

1) 5×PrimeScript RT Master Mix (Perfect Real Time): 2μL

1×Total RNA: 0.5μg

RNase Free dH2O up to 10μL

2) Perform reverse transcription reaction after gentle mixing, the conditions are as follows:

37°C 15min (reverse transcription reaction)

85°C 5sec (reverse transcriptase inactivation reaction)

Store at 4°C for short-term storage.

4. Detection of gene expression changes by real-time fluorescent quantitative PCR (RT-qPCR)

The experimental steps of real-time fluorescent quantitative PCR were carried out according to the instructions of ABclonal SYBGreen Mix.

(1) The components added in each reaction system are as follows:

Table 4 RT-qPCR reaction system

(2) The reaction system was added to a 384-well plate, and the sealing film was evenly pasted on the 384-well plate, and centrifuged at 1500 r·min ⁻¹ for 2 min.

(3) Put the 384-well plate on the reaction bracket according to the instructions of the instrument, and set the following reaction program.

e: Melting point curve reaction program

f: cooling reaction

(4) After the reaction, set the sample sequence and the detected gene and other information, and use the system software to analyze the data. The system program will automatically calculate the relative expression value of the detected gene according to the Ct value of the target gene and the internal reference gene.

(5) The algorithm used is: relative gene expression value = 2 ^{(-(Ct target gene-Ct internal reference))} , and finally set the control expression value to 1 to calculate the relative expression value of the gene in other samples.

2. Experimental results

As shown in Figure 1, different concentrations of CLC crystals can increase the expression of IL-1β, TNF-α, IL-6, GM-CSF and IL-8 in human nasal mucosal epithelial cells in a concentration-gradient manner. As shown in Figure 2, the expressions of IL-1β, TNF-α, IL-6, GM-CSF and IL-8 in human nasal mucosal epithelial cells were significantly increased after induction of CLCs (100 μg/mL) for 24 h, and the difference was statistically significant . The experimental results prove that the in vitro model of CLCs-induced activation of innate immune factors in human nasal mucosal epithelial cells was successfully constructed. And as shown in Figures 4-6, all the three polypeptides in the present invention can effectively inhibit the natural immune response of human nasal mucosa epithelial cells induced by CLC.

Example 4 Verifying the Affinity of the Polypeptide and Gal-10 or CLC and the Dissolving Effect of the Polypeptide on the Crystal

1. Experimental method

1. Biofilm interference

(1) Dissolving the polypeptide in PBS with a final concentration of 2 μM;

(2) Dilute His-tagged gal-10 protein with PBS to 300nM, 150nM, 75nM, 37.5nM, 18.75nM respectively;

(3) Add PBS, 2 μM polypeptide, PBS, and His-tagged gal-10 protein in four columns in sequence in a black 96-well plate, and the concentration of His-tagged gal-10 protein in the last well of the His-tagged gal-10 protein is 0;

(4) The affinity between the peptide and His-tagged gal-10 protein was detected using the Fortibio OctetRed96 molecular interaction instrument.

2. Binding of FITC-labeled peptides to CLC

(1) Dissolving the FITC-labeled polypeptide in PBS to a final concentration of 1 mM after dissolution;

(2) Mix CLC (100 μg/mL) and FITC-labeled polypeptide solution for a period of time, and the final concentration of the polypeptide is 10 μM;

(3) Wash several times with PBS to avoid background influence as much as possible;

(4) Use a microscope to take fluorescent photos and white light photos.

3. In vitro characterization of the dissolution effect of peptides on crystals

(1) Use PBS to dissolve the polypeptide, and prepare the peptide concentrations as 12mM, 16mM, 20mM, and 24mM;

(2) Dilute CLC with PBS to a concentration of 0.3 mg/mL;

(3) Mix the peptide solution and CLC at a volume ratio of 1:1, drop it on a crystal plate, observe it with an optical microscope, and take pictures at regular intervals;

2. Experimental results

As shown in Figure 7 and Figure 8, taking polypeptide No. 8 as an example, it can be directly combined with Gal-10 protein and CLC crystals, which proves that the polypeptide involved in the present invention can be used as a diagnostic agent for the diagnosis of CLC-induced diseases and/or type 2 immunity Reagents of disease. As shown in Figure 9, taking polypeptide No. 8 as an example, it can directly dissolve CLC crystals, which proves that the polypeptide involved in the present invention can target and dissolve CLC, and provide a basis for the treatment of CLC-induced diseases and/or type 2 immune diseases .

Example 5 The polypeptide can alleviate the inflammatory response of CLC-induced lung injury in mice

1. Experimental method

1. Intratracheal injection

1) Before the experiment, the mice were randomly divided into five groups, 6 in each group;

2) Medicines for tracheal injection: PBS (negative control), 0.15 mg/mL CLC, 300 μM polypeptide, 0.15 mg/mL CLC+300 μM polypeptide, 0.15 mg/mL CLC+0.5 mg/kg dexamethasone (positive control);

3) The mice were intraperitoneally injected with tribromoethanol anesthesia, the anesthesia dose was: mouse body weight (g) × 15 μL/mouse, and the mice were fully anesthetized (foot squeeze reflex negative) and then immobilized;

4) Keep the airway of the mouse vertical, expose a longitudinal incision with a length of 0.5-0.8 cm at the center of the neck skin 1.5 cm away from the mandible, and use ophthalmic curved forceps to bluntly separate the subcutaneous tissue and connective tissue along the midline , neck muscles until the trachea is fully exposed;

5) Tilt the mouse fixture at about 60°, suck 150 μL of air into the syringe first, and then inhale 70 μL of the pre-prepared drug to ensure that the drug can fully enter the lungs. Insert the needle parallel to the trachea under the thyroid cartilage in the middle of the trachea. 1cm, quickly inject the air and medicine in the syringe into the mouse trachea;

6) After the drug successfully enters the lungs, a sudden increase in the respiratory rate of the mouse can be observed, and the mouse neck tissue is clamped layer by layer according to the anatomy, and the fixing device is placed vertically on the operating table for about 1 minute to help the drug disperse;

7) Release the immobilization of the mouse, put the mouse back into the cage in a lateral position, and observe its state until it returns to autonomous activities.

1.2 Collection of mouse alveolar lavage fluid and lung tissue

alveolar lavage fluid

1) Collect samples 12 hours after tracheal injection;

2) The mice were intraperitoneally injected with tribromoethanol anesthesia, the anesthesia dose was: mouse body weight (g) × 15 μL/mouse, and the mice were fully anesthetized (foot squeeze reflex negative) and then immobilized;

3) Expose the trachea again along the trachea injection incision, cut the trachea transversely under the thyroid cartilage, be careful not to cut the trachea, and perform tracheal intubation, pay attention to the gentle operation to avoid disconnecting the mouse trachea;

4) Use the method of using a large tip with a small tip. A 1000 μL tip is covered with a 200 μL tip to obtain alveolar lavage fluid from mice. First, absorb 600 μL of sterile PBS, and place the pipette parallel to the trachea. Stretch into the incision until it is completely attached to the inner wall of the mouse trachea, slowly and evenly inject PBS into the mouse alveoli through the mouse trachea, and then slowly and evenly suck back. After the operation is successful, after repeating the injection-backup process three times, put the lavage solution into a 1.5mL microcentrifuge tube;

5) Repeat step 4), put the lavage solution into the same 1.5mL microcentrifuge tube, freeze it at -196°C, and store it in a -80°C refrigerator;

lung tissue

1) The thorax of the mouse was opened, the fascia and connective tissue between the lung tissue and the thorax were cut off, the lung tissue was taken out, rinsed with sterile PBS, quick-frozen at -196°C, and stored in a -80°C refrigerator.

2. Detection of protein changes by enzyme-linked immunosorbent assay (ELISA)

Coated ELISA 96-well plate

1) Dilute the capture antibody with sterile PBS according to the working concentration, add the diluted capture antibody at 100 μL per well into a high-binding 96-well microtiter plate, completely cover it with a parafilm, and incubate overnight at room temperature;

2.2 Detection process

1) Discard the capture antibody, add 200 μL washing solution to each well, shake on the shaker for 30 s, discard the washing solution and dry the residual liquid with filter paper, repeat 3 times;

2) Add 300 μL of diluent to each well and incubate at room temperature for at least 1 hour;

3) Discard the diluent and repeat step 1 to wash the plate;

4) Prepare the standard substance, dilute the standard substance according to the working concentration of the standard substance, and obtain the standard substance stock solution S7, gradiently dilute the standard substance to obtain S7-S0, a total of eight tubes (the standard substance concentration of S0 is 0), each well Add 100μL prepared standard respectively;

5) Add 100 μL of sample to each well, set up two secondary wells, shake gently, cover the plate, and incubate at 37°C for 2 hours;

6) Discard the standard and sample and repeat step 1) to wash the plate;

7) Dilute the detection antibody to the working concentration, add 100 μL detection antibody working solution to each well, cover the plate, and incubate at room temperature for 2 hours;

8) Discard the detection antibody and repeat step 1) to wash the plate;

9) Dilute Streptavidin-HRP to the working concentration, add 100 μL to each well, cover the plate, and incubate at room temperature in the dark for 20 minutes;

10) Discard Streptavidin-HRP and repeat step 1) to wash the plate;

11) Add 100 μL of TMB single-component chromogenic solution to each well, cover the plate, and incubate at room temperature in the dark for 10-20 minutes; when the first three wells of the standard have obvious discoloration, and the discoloration of the last four wells is not obvious, stop the reaction;

12) Add 50 μL of chromogenic stop solution to each well, shake and mix well, and detect the absorbance at 450 nm with a microplate reader.

2.3 Detection of gene expression changes by real-time fluorescent quantitative PCR (RT-qPCR)

1) extraction of total RNA;

2) RNA reversed to cDNA and real-time fluorescent quantitative PCR (RT-qPCR) to detect changes in gene expression.

3. H&E staining of pathological samples of mouse lung tissue

Dehydration, embedding, dewaxing, staining, dehydration and sealing (this step is completed by the company).

2. Experimental results

As shown in Figure 10, a CLC-induced mouse lung injury model was successfully established, and the inflammation-inducing ability of exogenous CLC was verified in mice. As shown in FIG. 11 , in the acute lung injury model induced by CLC in mice, the detection results of the inflammatory factors of #8 polypeptide (SEQ ID NO.3) involved in the present invention in alleviating lung injury in mice were verified. As shown in Figure 12, in the CLC-induced acute lung injury model in mice, it was verified that #8 polypeptide (SEQ ID NO.3) involved in the present invention alleviates the lung pathological results of lung injury in mice. The results of animal experiments prove the therapeutic effect of the polypeptide on CLC-induced diseases and/or type 2 immune diseases.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (23)

A polypeptide, characterized in that the polypeptide comprises at least one of the amino acid sequences shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, or fragments, variants, fusions thereof or derivatives thereof, or fusions of said fragments, variants or derivatives thereof, said variants comprising the amino acid sequence shown in any one of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 Amino acid sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, SEQ ID NO . The 8th amino acid at the N-terminal of the amino acid sequence shown in 4 is citrulline. A fusion, characterized in that the fusion comprises the polypeptide according to claim 1 or its fragment, variant or derivative, or a fusion of said fragment, variant or derivative thereof, or comprises a fluorescent marker , his-tag, CPP, connecting peptide. The polypeptide according to claim 1 or the fusion according to claim 2, wherein the polypeptide or the fusion can bind to CLC and/or Gal-10 protein; and/or Treatment of CLC-induced disease and/or type 2 immune disease. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polypeptide of claim 1 or the fusion of claim 2. The pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition can prevent or treat CLC-induced diseases and/or type 2 immune diseases. The pharmaceutical composition according to claim 5, wherein the type 2 immune diseases include allergic diseases and mite infections. The pharmaceutical composition according to claim 6, wherein the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy. The pharmaceutical composition according to claim 7, wherein the allergic disease is chronic sinusitis. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable buffer, carrier or excipient. Use of the polypeptide of claim 1 or the fusion of claim 2 in the detection of CLC protein for non-diagnostic purposes. A nucleic acid encoding the polypeptide of claim 1 or the fusion of claim 2. A recombinant vector comprising the nucleic acid of claim 11. A cell comprising the nucleic acid of claim 11 or the recombinant vector of claim 12. The polypeptide of claim 1 or the fusion of claim 2 or the pharmaceutical composition of any one of claims 4-9 or the nucleic acid of claim 11 or the recombinant vector of claim 12 or the claim Use of the cell described in 13 in the preparation of a medicament for preventing or treating CLC-induced diseases and/or type 2 immune diseases. The use according to claim 14, characterized in that the type 2 immune diseases include allergic diseases and mite infections. The use according to claim 15, characterized in that the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy. The use according to claim 16, characterized in that the allergic disease is chronic sinusitis. A method for detecting CLC protein for non-diagnostic purposes, characterized in that the method comprises: (1) contacting the sample with the polypeptide of claim 1 or the fusion of claim 2; (2) detecting the formation of a complex comprising the polypeptide of claim 1 or the fusion of claim 2. The method according to claim 18, wherein the method for detecting the formation of a complex comprising the polypeptide according to claim 1 or the fusion compound according to claim 2 comprises gel electrophoresis, chromatographic techniques, Western blot analysis, immunohistochemistry, mass spectrometry and/or high pressure liquid chromatography. The polypeptide according to claim 1 or the fusion according to claim 2 or the nucleic acid according to claim 11 or the recombinant vector according to claim 12 or the cell according to claim 13 can be used for the diagnosis of diseases induced by CLC And/or type 2 immune diseases or application in a kit for detecting CLC protein. The use according to claim 20, characterized in that the type 2 immune diseases include allergic diseases and mite infections. The application according to claim 21, characterized in that the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy. The use according to claim 22, characterized in that the allergic disease is chronic sinusitis.

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