CN113666999B - Polypeptides for the treatment of type 2 immune disorders - Google Patents
Polypeptides for the treatment of type 2 immune disorders Download PDFInfo
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- CN113666999B CN113666999B CN202111008519.0A CN202111008519A CN113666999B CN 113666999 B CN113666999 B CN 113666999B CN 202111008519 A CN202111008519 A CN 202111008519A CN 113666999 B CN113666999 B CN 113666999B
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Abstract
The invention provides a polypeptide for treating type 2 immune diseases, which comprises a polypeptide of an amino acid sequence shown as SEQ ID NO. 3. In a specific embodiment, the invention constructs an in vitro model of CLC crystal induced activation of natural immune factors of human nasal mucosal epithelial cells, the polypeptide can effectively inhibit the natural immune response activated by the CLC crystal, and the result proves that the polypeptide can be used for preparing the medicine for preventing or treating the type 2 immune diseases. The invention also provides the application of the polypeptide in preparing products for diagnosing 2-type immune diseases.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a polypeptide for treating type 2 immune diseases.
Background
More than 3 billion people worldwide suffer from helminth infections or allergic diseases such as asthma, allergic rhinitis, food allergy and eczema. A common feature of such infections or inflammatory reactions is the so-called "allergy" or "type 2 immune response". Type 2 immune responses are on the one hand induced by and produce a protective immune response against the helminths; on the other hand, pathological changes are also caused, including promotion of acute or chronic allergic inflammatory reactions caused by various allergens. Type 2 immune responses are primarily characterized by the production of type 2 cytokines. Under the stimulation of antigen, Dendritic Cells (DC) are activated to promote the differentiation of T cells into Th2 cells, release type 2 cytokines, and then stimulate the production of IgE and the aggregation of eosinophilic granulocyte.
Charcot-Leyden crystals (CLCs) were found extracellularly deposited biconical hexagonal protein crystals in heart and spleen tissue of a deceased leukemia patient in 1853 by Charcot, a neurologist of france, and were also found in sputum of asthmatic patients in 1872 by Leyden, a german physician, hence the name Charcot-Leyden crystals. Similar crystallization is also observed in diseased sites in patients with chronic rhino-sinusitis, allergic rhinitis, helminth infection, and some cancers in the later population. The Charcot Leiden crystal is a protein crystal formed by galectin-10 released after eosinophil lysis, mainly exists in a lesion part with Th2 type immune cell infiltration diseases, and the content change of the protein crystal can be used as one index for evaluating the disease condition.
Polypeptide generally refers to a compound of less than 100 amino acids linked by peptide bonds, and has a relative molecular mass of less than 10000. The polypeptide medicine has obvious curative effect on treating tumor, diabetes, cardiovascular disease, acromegaly, osteoporosis, gastrointestinal tract disease, central nervous system disease, immunological disease, antiviral and antibacterial diseases, and has over 80 kinds of medicine approved to be on the market globally, including 17 kinds of antitumor medicine, 7 kinds of diabetes treating medicine, 16 kinds of infection and immunological treating medicine, 9 kinds of blood vessel and urinary treating medicine and 31 kinds of other medicine. There is a lack in the art of polypeptides that can be used to treat type 2 immune disorders.
Disclosure of Invention
The invention aims to provide a polypeptide which can be used for diagnosing, preventing or treating type 2 immune diseases. In order to realize the purpose, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a polypeptide comprising an amino acid sequence as set forth in SEQ ID No.3, wherein the 8 th amino acid at the N-terminus of the sequence is guanadine.
In a second aspect, the present invention provides a polypeptide derivative comprising a modified product of the polypeptide of the first aspect of the present invention, a variant of the polypeptide of the first aspect of the present invention obtained by addition and/or substitution of one or more amino acids, and a conjugate of the polypeptide of the first aspect of the present invention with another substance.
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.
Further, the polypeptide derivative is a modified product of the polypeptide of the first aspect of the present invention.
Wherein, the modification includes but is not limited to amination modification, methylation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, phosphorylation modification, sulfation modification, esterification modification, glycosylation modification, cyclization modification, biotinylation modification, fluorophore modification, polyethylene glycol (PEG) modification, myristoylation modification, nonmetal chemical element modification and immobilization modification.
In a specific embodiment of the invention, the modification is an amination modification.
In a third aspect, the invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention or a polypeptide derivative according to the second aspect of the invention.
Further, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, excipient or carrier.
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 (polysulphonates), 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) 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 composition may also contain one or more mono-or disaccharides such as xylitol, sorbitol, mannitol, lactitol, isomalt (isomalt), maltitol or xyloside, and/or monoacylglycerols such as monolaurin (monolaurin).
The characteristics of the carrier depend on the route of administration. One route of administration is topical. For example, for topical application, one preferred carrier is an emulsified emulsion containing the active peptide, but other common carriers such as certain petrolatum/mineral and plant based ointments, as well as polymeric gels, liquid crystalline phases and microemulsions may be used.
The pharmaceutical compositions of the invention may also be in the form of liposomes, in which the polypeptide is combined with an amphiphile (amphetathic agent) such as a lipid, which is present in aggregated form as micelles, insoluble monolayers and liquid crystals, in addition to other pharmaceutically acceptable carriers. Lipids suitable for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponins, bile acids, and the like.
The pharmaceutical compositions of the present invention may also be in the form of biodegradable microspheres. Aliphatic polyesters such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly (caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. The preparation of such microspheres can be found in US 5,851,451 and EP 213303.
The pharmaceutical composition of the present invention may also be in the form of a polymer gel wherein polymers such as starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, chitosan, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinyl imidazole, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate and polyvinyl pyrrolidone, which are hydrolyzed to varying degrees, are used to thicken a peptide-containing solution. The polymer may also comprise gelatin or collagen.
Alternatively, the polypeptide of the present invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum and/or various buffers.
It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention may be administered topically or systemically. Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, vaginal, and rectal. Administration from an implant is also possible. Suitable dosage forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, microemulsions (which are defined as optically isotropic thermodynamically stable systems composed of water, oil and surfactant), liquid crystalline phases (which are defined as systems characterized by long-range order but short-range disorder (examples include lamellar, hexagonal and cubic phases, or are water or oil continuous), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, microdrops or ampoules, and also injectable solutions with prolonged release of the active compound, in which preparations excipients, diluents or carriers are generally used, as described above.
In a preferred embodiment, the pharmaceutical composition is suitable for pulmonary or nasal administration.
For example, the pharmaceutical compositions of the invention may be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray which is provided from a pressurised container, pump, spray or nebuliser, using a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1, 2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3, 3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas, hi the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered dose, the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and propellant as the solvent, it may additionally contain a lubricant, for example sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a polypeptide of the invention and a suitable powder base such as lactose or starch.
The pharmaceutical composition will be administered to the patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose sufficient to produce the desired effect with respect to the condition to which it is administered. The precise dosage will depend on the activity of the compound, the mode of administration, the nature and severity of the condition, the age and weight of the patient and may require different dosages. Dosage administration can be carried out by a single administration in the form of an individual dosage unit (otherwise, several smaller dosage units) and also by multiple administrations of finely divided doses at specific time intervals.
In a fourth aspect, the invention provides the use of a pharmaceutical composition according to the third aspect of the invention in the manufacture 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.
Furthermore, 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 type 2 immune disease is chronic sinusitis.
In a fifth aspect, the invention provides a use of a polypeptide according to the first aspect of the invention, said use comprising use of any one of:
1) use in the detection of CLC crystals for non-diagnostic purposes;
2) use in the preparation of a polypeptide derivative according to the second aspect of the invention;
3) use in the manufacture of a pharmaceutical composition according to the third aspect of the invention;
4) the use in the manufacture of a medicament for the prevention or treatment of type 2 immune disorders;
5) use in the manufacture of a product useful in the diagnosis of type 2 immune disorders.
The terms "charcot-leyden crystals", "CLCs", "CLC crystals" are used interchangeably herein and refer to crystals formed from galectin-10. The crystals formed from galectin-10 are typically biconical hexagonal crystals, approximately 20-40 μm in length and approximately 2-4 μm in width.
"galectin-10" as used herein, the term "galectin-10" (or Gal10 or Gal-10) refers to small hydrophobic glycan-binding proteins that self-crystallize to form charcot-leyden crystals (CLC 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.
Further, the type 2 immune diseases comprise allergic diseases and mite infections.
Furthermore, 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 type 2 immune disease is chronic sinusitis.
In a sixth aspect, the invention provides a use of a polypeptide derivative according to the second aspect of the invention, the use comprising any one of the following:
1) use in the detection of CLC crystals for non-diagnostic purposes;
2) use in the manufacture of a pharmaceutical composition according to the third aspect of the invention;
3) the use in the manufacture of a medicament for the prevention or treatment of type 2 immune disorders;
4) use in the manufacture of a product useful in the diagnosis of type 2 immune disorders.
Further, the type 2 immune diseases comprise allergic diseases and mite infections.
Furthermore, 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 type 2 immune disease is chronic sinusitis.
The polypeptides useful in the present invention may be prepared using any suitable means known in the art. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means and methods for preparing such polypeptides are well known in the art.
In a seventh aspect, the invention provides a nucleic acid encoding a polypeptide according to the first aspect of the invention or a polypeptide derivative according to the second aspect of the invention or a recombinant vector comprising the same.
As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising an intact replicon, such that when placed in a permissive cell, the vector can be replicated, e.g., by a transformation process. Vectors can replicate in one cell type (e.g., bacteria), but have a limited ability to replicate in another cell (e.g., mammalian cells). The vector may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers (e.g., heterogeneous polylysines, fixed length oligopeptides, and polyethylene imines) are, in some cases, also contained in liposomes.
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 pCDNA vector which is widely available on the market; F. r1, RP1, Col, pBR322, ToL, 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. Selection of a selectable marker may depend on the cell selected (as is well known to those skilled in the art), but is not critical to the present invention.
According to an eighth aspect of the invention there is provided a cell comprising a nucleic acid according to the seventh aspect of the invention or a recombinant vector comprising the same.
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 ninth aspect the invention provides a method of detecting a CLC crystal for non-diagnostic purposes, the method comprising:
(1) contacting the sample with a polypeptide according to the first aspect of the invention or a polypeptide derivative according to the second aspect of the invention;
(2) detecting the formation of a complex comprising a polypeptide according to the first aspect of the invention or a polypeptide derivative according to the second 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 or a polypeptide derivative according to the second aspect of the invention comprises gel electrophoresis, chromatographic techniques, immunoblot analysis, immunohistochemistry, mass spectrometry and/or high pressure liquid chromatography.
The invention has the following advantages and beneficial effects:
the invention discloses a polypeptide for preventing or treating type 2 immune diseases, provides a new method for preventing or treating type 2 immune diseases, and has potential clinical value.
The invention also discloses the application of the polypeptide in preparing products for diagnosing 2-type immune diseases.
The invention also discloses a method for detecting the CLC crystal for non-diagnosis purposes.
Drawings
FIG. 1 is a graph of the results of RT-qPCR testing the gene expression changes of human nasal mucosal epithelial cells induced by CLCs of different concentrations, wherein, 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 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 confirmed at the cellular level to inhibit 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
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 its N-terminus, and cloned into pET-28a vector plasmid via NcoI/XhoI double cleavage site 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 solubilizing 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); the cDNA reverse transcription reagent was purchased from Takara; the real-time fluorescent quantitative PCR reagent is purchased from Ebola tektes; the polypeptide is synthesized by the national peace pharmaceutical industry limited company, the sequence is shown in a table 2, and the first amino acid at the C terminal of the sequence is connected with an amino group.
TABLE 2 polypeptide sequences
2. Consumables and instrumentation: 10cm cell culture dishes, 12 well cell culture plates were 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 layerThe columns were purchased 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 (SHH W21600); micro uv spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); electronic analytical balance (Sartorius, BS 110S); an optical inverted microscope (XDS-1B); micropipettes (eppendorf research plus); 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;
pure 25 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:
using Kanamycin (25mg/mL) as a selection marker, the positive monoclonal BL21(DE3)/pET-28a-gal10-TEV-6His on the plate of the above step was selected, inoculated into 20mL LB liquid medium resistant to Kanamycin, and shake-cultured at 37 ℃ at 210r/min for 12 hours. Inoculating the bacterial liquid into 1L LB liquid culture medium containing kanamycin at the ratio of 1: 100, and culturing at 37 ℃ under the condition of 210r/min oscillation.
(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, volume about 75-100mL, 1: PMSF (1 mM final concentration) is added according to the proportion of 100 to protect the target protein;
C. ultrasonic disruption of bacteria: 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.
pure 25 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; 1.5 of Alarm delta column compressed enabled; alarmpit columnresursure 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 sample is continuously operated until the sample passes through 20mL, sterile water is replaced for cleaning, 10mL operation is performed, then 20mL operation is performed by 20% ethanol solution, and shutdown can be performed;
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 stood still and slowly melted.
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 for three times to obtain the pure CLCs.
Bca assay detects concentration of CLCs.
2. Acquisition of human nasal polyp tissue epithelial cells
(1) And (3) carrying out enzyme digestion on the tissue:
A. the mucosa specimen removed by the operation is put 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 the 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 from 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 than or less). Sterilizing for 20min under ultraviolet irradiation.
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 as clean 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 taking 1 mu L, adding 9 mu LPBS, mixing uniformly, and adding a cell counting plate for counting.
J. Cell counting: (cell suspension cell number)/mL ═ n grid cell number/n). times.16X 10 4
K. After counting, according to 2X 10 6 Add to coated 10cm petri dishes and add 12ml of egm epithelial 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 10 5 Individual 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 LTrizol/well was added to lyse the cells for RNA extraction.
4. RNA extraction and cDNA reverse transcription
(1) RNA extraction:
A. the lysate of cells or tissues stored in Trizol frozen at-80 ℃ freezer was placed on ice and slowly thawed.
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 -1 Centrifuge 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 -1 Centrifuging for 10min, and removing the supernatant to see that RNA precipitates at the bottom of the centrifuge tube.
H. 1mL of 750mL/L of H treated with DEPC was added 2 Ethanol in O-configuration, gently shake the centrifuge tube to resuspend the pellet.
I.4℃12000r·min -1 Centrifuging for 5min, and discarding 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 pellet 2 O 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 LDEPC 2 O and 2. mu.L of 5 × RNALOADING Buffer, and 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 dH 2 O 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 deg.C.
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 -1 Centrifuge for 2 min.
(3) The 384 well plate was placed on the reaction tray as specified by the instrument and the following reaction protocol was set up.
a:95℃5min
Melting Point Curve reaction procedure
f cooling reaction
(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:
finally, setting the contrast expression quantity as 1, and calculating the relative expression value of the gene of other samples.
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 activating the natural immune factors of CLCs inducing the epithelial cells of the human nasal mucosa 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 NaHCO 3 (pH 8.6) dilution of the target protein (Gal 10 protein with a lysotropic tag) to 100. mu.Lg/mL, 1.5mL in a 6cm plastic petri dish placed in a wet box 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 well bottom and well sides 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, added to the coated plate and gently shaken at room temperature for 10-60 min. The plates were washed 10 times with TBST, and clean paper towels were used each time 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. The titer was measured to determine the 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 in 200. mu.L TBS 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 microwave oven melts the upper agar, divides into 3mL portions, and puts them into a sterilized test tube and keeps them in a water bath at 45 ℃ for standby. 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 of 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 at 4 ℃. 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.
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 possibly interacting with Gal-10 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 may interact with Gal10 protein were co-screened, and the polypeptide (#3) according to the present invention could bind to 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 (#3) 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> Polypeptides for the treatment of type 2 immune disorders
<160> 3
<170> SIPOSequenceListing 1.0
<210> 2
<211> 142
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
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> 3
<211> 181
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Ala Ser Thr Thr His His His His His His Asp Thr Asp Ile Pro
1 5 10 15
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
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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
145 150 155 160
Lys Met Val Gln Val Trp Arg Asp Ile Ser Leu Thr Lys Phe Asn Val
165 170 175
Ser Tyr Leu Lys Arg
180
<210> 3
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> UNSURE
<222> (8)..(8)
<223> Xaa=Cit
<400> 3
Glu Lys Gly Val Pro Leu Tyr Xaa His Ile Ala Asp Leu Ala Gly Lys
1 5 10 15
Claims (25)
1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO.3, and the 8 th amino acid at the N end of the sequence is citrulline.
2. A polypeptide derivative, wherein the polypeptide derivative is a modified product of the polypeptide of claim 1, and the modification comprises an amination modification, a methylation modification, an amidation modification, a hydroxylation modification, a carboxylation modification, a carbonylation modification, an alkylation modification, an acetylation modification, a phosphorylation modification, a sulfation modification, an esterification modification, a glycosylation modification, a cyclization modification, a biotinylation modification, a fluorophore modification, a polyethylene glycol (PEG) modification, a myristoylation modification, a non-metal chemical element modification, and an immobilization modification.
3. The polypeptide derivative of claim 2, wherein the modification is an amination modification.
4. A pharmaceutical composition comprising the polypeptide of claim 1, or the polypeptide derivative of claim 2 or 3.
5. The pharmaceutical composition of claim 4, further comprising a pharmaceutically acceptable buffer, excipient or carrier.
6. The pharmaceutical composition of claim 5, wherein 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, cacoate (cacylate), CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolylactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.
7. The pharmaceutical composition of claim 5, wherein the carrier comprises an antimicrobial agent, an isotonic agent, an antioxidant, a local anesthetic, a suspending agent, a dispersing agent, an emulsifying agent, a chelating agent, a thickening agent, or a solubilizing agent.
8. The pharmaceutical composition of claim 5, wherein the excipient comprises a carbohydrate, a polymer, a lipid, or a mineral.
9. Use of a pharmaceutical composition according to any one of claims 4 to 8 in the manufacture of a medicament useful for the prevention or treatment of type 2 immune disorders.
10. The use according to claim 9, wherein said type 2 immune disorders comprise allergic diseases, mite infestations.
11. The use according to claim 10, wherein said type 2 immune disease is an allergic disease.
12. The use according to claim 11, wherein the allergic disease comprises chronic rhinosinusitis, asthma, allergic rhinitis, allergic dermatitis, food allergy.
13. The use according to claim 12, wherein the allergic disease is chronic rhinosinusitis.
14. The use of the polypeptide of claim 1, wherein said use comprises any one of the following:
use in the detection of CLC crystals for non-diagnostic purposes;
use in the preparation of a polypeptide derivative according to claim 2 or 3;
use in the manufacture of a pharmaceutical composition according to any one of claims 4 to 8;
the use in the manufacture of a medicament for the prevention or treatment of type 2 immune disorders;
use in the manufacture of a product useful in the diagnosis of type 2 immune disorders.
15. The use according to claim 14, wherein said type 2 immune disorders comprise allergic disorders, mite infestations.
16. The use according to claim 15, wherein said type 2 immune disease is an allergic disease.
17. The use of claim 16, wherein the allergic disease comprises chronic rhinosinusitis, asthma, allergic rhinitis, allergic dermatitis, food allergy.
18. The use according to claim 17, wherein the allergic disease is chronic rhinosinusitis.
19. Use of a polypeptide derivative according to claim 2 or 3, wherein said use comprises any of the following:
1) use in the detection of CLC crystals for non-diagnostic purposes;
2) use in the manufacture of a pharmaceutical composition according to any one of claims 4 to 8;
3) the use in the manufacture of a medicament for the prevention or treatment of type 2 immune disorders;
4) use in the manufacture of a product useful in the diagnosis of type 2 immune disorders.
20. The use according to claim 19, wherein said type 2 immune disorders comprise allergic diseases, acarid infections.
21. The use according to claim 20, wherein said type 2 immune disease is an allergic disease.
22. The use of claim 21, wherein said allergic disease comprises chronic rhinosinusitis, asthma, allergic rhinitis, allergic dermatitis, food allergy.
23. The use according to claim 22, wherein the allergic disease is chronic rhinosinusitis.
24. A method for the detection of CLC crystals for non-diagnostic purposes, the method comprising:
(1) contacting a sample with the polypeptide of claim 1, or the polypeptide derivative of claim 2 or 3;
(2) detecting the formation of a complex comprising the polypeptide of claim 1, or the polypeptide derivative of claim 2 or 3.
25. The method of claim 24, wherein the method of detecting the formation of a complex comprising the polypeptide of claim 1, or the polypeptide derivative of claim 2 or 3 comprises gel electrophoresis, chromatographic techniques, immunoblot analysis, immunohistochemistry, mass spectrometry, and/or high pressure liquid chromatography.
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| CN114681438B (en) * | 2022-05-17 | 2024-03-19 | 中国医学科学院基础医学研究所 | Use of metformin and other guanidine-containing compounds for reversing the crystallization tendency of Gal-10 and for alleviating the associated diseases |
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| CN1290749A (en) * | 1999-06-29 | 2001-04-11 | 复旦大学 | Charcot-leyden crystal IB and its coding sequence and producing method and use |
| CN112166126A (en) * | 2018-04-13 | 2021-01-01 | 阿根思公司 | Galectin-10 antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1290749A (en) * | 1999-06-29 | 2001-04-11 | 复旦大学 | Charcot-leyden crystal IB and its coding sequence and producing method and use |
| CN112166126A (en) * | 2018-04-13 | 2021-01-01 | 阿根思公司 | Galectin-10 antibody |
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| Title |
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| A Brief History of Charcot-Leyden Crystal Protein/Galectin-10 Research;Jiyong Su;《Molecules.》;20181109;第23卷(第11期);全文 * |
| 半乳糖凝集素-10 晶体结构及其与配体相互;高瑾;《中国优秀博硕士学位论文全文数据库(硕士)》;20190115;全文 * |
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