Keratin CF1, its manufacturing method, pharmaceutical composition containing it, and its use

Abstract

This application relates to the pharmaceutical technology, specifically to keratin CF1, nucleic acid molecules encoding keratin CF1, expression vectors containing nucleic acid molecules, host cells containing expression vectors or nucleic acid molecules incorporated into a genome, methods for producing keratin CF1, pharmaceutical compositions containing keratin CF1, and the use of the above-mentioned keratin CF1, nucleic acid molecules, expression vectors, host cells, or pharmaceutical compositions in the manufacture of antipyretics, analgesics, antitussives, expectorants, anticonvulsants, antiepileptics, antihypertensives, anti-inflammatorys, and antiviral agents.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and relates to keratin CF1, a nucleic acid molecule encoding keratin CF1, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or the nucleic acid molecule integrated into the genome, a method for producing keratin CF1, a pharmaceutical composition containing keratin, and the use of keratin and the pharmaceutical composition in the production of antipyretics, analgesics, antitussives, expectorants, antiepileptics, antiepileptics, antihypertensives, anti-inflammatory agents and antiviral agents.

Background Art

[0002] Keratin is a kind of protein widely found in the epidermis of humans and animals, and is a major component of hair, feathers, hooves, shells, claws, horns, etc. It is a very important structural protein for connective tissues and plays a role in protecting the body.

[0003] Keratin is a kind of renewable resource that widely exists in organisms and has excellent utilization value, but it has not been widely and effectively utilized. The main reason is that keratin does not dissolve in various solvents and generally has high resistance to hydrolysis by proteases compared to other proteins. Therefore, it is very difficult to extract and manufacture natural keratin.

[0004] With the rapid development of modern biotechnology such as genomics, proteomics, genetic engineering, and microbial engineering, the number of discovered genes is increasing. Producing target proteins using protein expression systems is an important means in the study of the biological functions of genes or proteins.

[0005] Producing target keratin using a protein expression system and further studying its structure and function is novel and creative and has not been reported in other documents.

Summary of the Invention

[0006] The technical problem that this invention solves is to provide keratin CF1, a nucleic acid molecule encoding keratin CF1, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or a nucleic acid molecule incorporated into the genome, a method for producing keratin CF1, a pharmaceutical composition containing keratin CF1, and the use of the above keratin CF1, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the production of antipyretics, analgesics, antitussives, expectorants, anticonvulsants, antiepileptics, antihypertensives, anti-inflammatorys, and antiviral agents.

[0007] To solve the technical problems of this invention, the present invention provides the following technical solutions: The first aspect of the technical solution of the present invention is keratin CF1, wherein the amino acid sequence of keratin CF1 is (1) The amino acid sequence shown in Sequence ID No. 1 of the sequence listing; or, (2) An amino acid sequence in which 1 to 35 amino acids are substituted, deleted, or added in the amino acid sequence shown in Sequence ID No. 1 of the sequence listing, and which maintains essentially the same biological function; The objective is to provide keratin CF1 characterized by the following:

[0008] Furthermore, conventional modifications can be applied to keratin CF1; or tags for detection or purification can be attached to keratin CF1.

[0009] Furthermore, conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, immobilization modification, sulfation, oxidation, methylation, deamination, disulfide bond formation or disulfide bond cleavage; tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

[0010] A second aspect of the technical solution of the present invention is to provide a nucleic acid molecule encoding keratin CF1 as described in the first aspect.

[0011] Furthermore, the nucleotide sequence of nucleic acid molecules is, (1) Nucleotide sequence shown in Sequence ID No. 2 of the sequence listing (2) Sequence-optimized nucleotide sequence based on the nucleotide sequence shown in Sequence ID No. 2 (3) A nucleotide sequence complementary to the nucleotide sequence in (1) or (2) above. That is the case.

[0012] A third aspect of the technical solution of the present invention is to provide an expression vector characterized by comprising the nucleic acid molecule described in the second aspect.

[0013] Furthermore, expression vectors such as the pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZα / A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, and pPIC3.5K can be used; the preferred expression vector is the pET series vector; the most preferred expression vector is pET-28a(+).

[0014] A fourth aspect of the technical solution of the present invention is to provide a host cell characterized by containing an expression vector of the third aspect or a nucleic acid molecule of the second aspect incorporated into the genome.

[0015] Furthermore, the host cells include bacteria, yeast, Aspergillus, plant cells, or insect cells.

[0016] Furthermore, the bacteria include E. coli or yeast.

[0017] Competent host cells that can be used include the BL21 lineage, Transetta lineage, Rosetta lineage, DH5α lineage, JM lineage, Top lineage, Orgami lineage, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115(AOX1), KM71, SMD1168, etc. Preferred competent cells for expression are BL21(DE3) and Transetta(DE3).

[0018] A fifth aspect of the technical solution of the present invention is the following steps: A. Synthesize the nucleic acid molecule corresponding to keratin CF1 as described in the first side, ligate the nucleic acid molecule to the corresponding expression vector, transform the host cell with the expression vector, culture the host cell containing the expression vector in a fermentation apparatus under specific conditions to induce keratin CF1 expression, and obtain a crude protein solution containing keratin CF1; B. The crude protein solution expressed in step A is separated, purified, and dried to obtain keratin CF1; The objective is to provide a method for producing keratin CF1 of the first side, characterized by including [a specific element].

[0019] Furthermore, in step A, the host cells are mainly selected from E. coli, keratin CF1 is expressed in E. coli inclusion bodies, and the fermentation apparatus includes a shaking flask or fermentation tank.

[0020] Furthermore, in step A, after inducing keratin CF1 expression, impurities are washed away with a detergent, and then the protein is dissolved using a urea solution to obtain a crude protein solution.

[0021] Furthermore, the culture medium used in step A can be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, rose bengal medium, salt Czapek Dox medium, DOBA medium, rice koji medium, and their modifications; in shaking flask fermentation, LB medium and TB medium are preferred, with TB medium being the most preferred; in fermentation tanks, LB medium and its modifications are preferred.

[0022] Furthermore, the inducer in step A can be IPTG, lactose, arabinose, etc.; preferably, IPTG and lactose.

[0023] Furthermore, in step A, the obtained fermentation culture solution is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacterial cells are crushed and centrifuged, and the supernatant is discarded; the precipitate is washed with a washing agent and dissolved in urea solution to obtain a crude CF1 protein solution.

[0024] Among these, the buffer solution is preferably buffer solution A, and its usage amount is such that the volume ratio of the fermentation culture solution to buffer solution A is 1 to 100:1, preferably 10:1.

[0025] As the detergent, a urea solution, a guanidine hydrochloride solution, TRITON (registered trademark), buffer solution A, etc. can be used. Preferably, a urea solution is used, most preferably a 2M urea solution (containing 1% TRITON (registered trademark)) and a 4M urea solution. The usage amount is such that the volume ratio of the fermentation culture solution to the urea solution is 0.2 to 100:1, preferably 1 to 15:1.

[0026] The urea solution is preferably a 4M to 8M urea solution, most preferably an 8M urea solution, and its usage amount is such that the volume ratio of the fermentation culture solution to 8M urea is 0.2 to 100:1, preferably 2 to 15:1.

[0027] Furthermore, in step B, the methods of separation and purification include ultrafiltration or microfiltration membrane purification method, column chromatography purification method, salting-out method, and dialysis method.

[0028] Furthermore, the methods of separation and purification in step B are as follows: (1) The dialysis method is to purify the crude protein solution obtained in step A by dialysis to obtain the target protein CF1 solution.

[0029] The molecular weight cut-off of the dialysis bag can be 0.5 to 10 kD. The preferred molecular weight cut-off of the dialysis bag is 3.5 to 10 kD, and the most preferred molecular weight cut-off of the dialysis bag is 10 kD.

[0030] (2) The ultrafiltration method and the microfiltration method are to purify the crude protein solution obtained in step A by membrane technologies such as an ultrafiltration membrane or a microfiltration membrane to obtain a concentrated solution of the target protein CF1.

[0031] (3) The column chromatography method involves separating and purifying the crude protein solution obtained in step A using various exchange columns or exclusion column chromatography to obtain the target protein CF1.

[0032] Preferred exclusion columns include dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, and Superose 6 Increase; preferred exchange columns include ion exchange resin columns: anion exchange resin columns, HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, Toyopearl SuperQ-650M, etc.; and cation exchange resin columns: HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, and Toyopearl Super SP-650M.

[0033] The eluent can be any eluent commonly used in the relevant art, such as water or a salt solution. Salt solutions include sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, and acetic acid.

[0034] (4) The salting-out method involves purifying the crude protein solution obtained in step A by salting-out to obtain a suspension of the target protein CF1.

[0035] Suitable salting-out agents include ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, and magnesium sulfate. Preferred salting-out agents are ammonium sulfate and its aqueous solution. A saturated aqueous solution of ammonium sulfate is added to achieve a final concentration of ammonium sulfate of 10-50%, preferably 20-30%, and more preferably 25%.

[0036] The salting-out process is performed 1 to 3 times, preferably 2 times.

[0037] After salting out, the precipitate is washed by adding pure water 2 to 5 times, preferably 3 times.

[0038] Furthermore, the target protein CF1 solution purified in step B can be freeze-dried or vacuum-dried to obtain a dry powder, or the concentrated solution can be directly spray-dried to obtain a dry powder.

[0039] A sixth aspect of the technical solution of the present invention is to provide a pharmaceutical composition characterized by comprising keratin CF1 as described in the first aspect, a nucleic acid molecule as described in the second aspect, an expression vector as described in the third aspect or a host cell as described in the fourth aspect, and a pharmaceutically acceptable carrier or additive.

[0040] The keratin obtained in the above steps of the present invention can be freeze-dried or vacuum-dried to obtain a dry powder, or the concentrated liquid can be directly spray-dried to obtain a dry powder, which can then be made into various dosage forms.

[0041] The present invention relates to a pharmaceutical composition comprising keratin obtained in the above-mentioned process and a pharmaceutically acceptable carrier.

[0042] The present invention also relates to a pharmaceutical composition comprising the keratin of the present invention as an active ingredient and a conventional pharmaceutical additive or adjuvant. The keratin of the present invention typically accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.

[0043] The present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of protein as an active ingredient and a pharmaceutically acceptable carrier.

[0044] The pharmaceutical compositions of the present invention can be manufactured by methods known in the art. For this purpose, the proteins of the present invention can, if necessary, be combined with one or more solid or liquid pharmaceutical additives and / or adjuvants to produce suitable dosage forms or unit dosage forms that can be used as human or veterinary pharmaceuticals.

[0045] The keratin or pharmaceutical composition containing the same according to the present invention can be administered in unit dosage form. The route of administration can be enteral or parenteral, including oral, intramuscular, subcutaneous, intranasal, oral mucosa, intraocular, lung, transdermal, vaginal, abdominal cavity, and rectal, with oral administration being preferred.

[0046] The keratin protein of the present invention or a pharmaceutical composition containing the same can be administered by injection. Injections include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, and acupoint injection.

[0047] The dosage form may be a liquid, solid, or semi-solid form. Liquid dosage forms may include liquids (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil, and complex emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and topical applications. Solid dosage forms may include tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, drops, suppositories, films, patches, aerosols (dry powders), and sprays; semi-solid dosage forms may include ointments, gels, and pastes.

[0048] The keratin of the present invention can be formulated into conventional pharmaceutical formulations, and can also be used in sustained-release formulations, controlled-release formulations, targeted formulations, and various particle delivery systems.

[0049] Various additives known in the art, including diluents, binders, wetting agents, disintegrants, lubricants, and fluidizers, can be used to form a tablet for a unit-dose formulation. Diluents may include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, crystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; Wetting agents may include water, ethanol, isopropanol, etc.; Binders may include starch slurry, dextrin, syrup, honey, glucose solution, crystalline cellulose, gum arabic, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene dipropyl alcohol, etc.; Disintegrants may include dried starch, crystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium, carboxymethyl starch sodium, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium lauryl sulfate; Lubricants and fluidizers may include talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0050] The tablets may also be coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or two-layer tablets and multi-layer tablets.

[0051] Various carriers known in the art can be used to form the dosage unit into a pill. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol laurate, kaolin, and talc; binders such as gum arabic, xanthan gum, gelatin, ethanol, honey, liquid sugar, rice paste, or wheat flour paste; and disintegrants such as powdered agar, dried starch, alginate, sodium lauryl sulfate, methylcellulose, and ethylcellulose.

[0052] Various carriers known in the art can be used to provide suppositories as the dosage unit. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0053] To form the dosage unit into a capsule, the keratin of the present invention, which is the active ingredient, is mixed with the various carriers mentioned above, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The keratin of the present invention, which is the active ingredient, can be microencapsulated, suspended in an aqueous medium to form a suspension, filled into a hard capsule, or used as an injectable preparation.

[0054] For example, the keratin of the present invention can be manufactured into injectable formulations such as liquids, suspensions, emulsions, and lyophilized powder injections. Such formulations may be aqueous or non-aqueous and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. For example, diluents can be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, and the like. In addition, appropriate amounts of sodium chloride, glucose, or glycerin can be added to the injection to produce an isotonic injection. Furthermore, common solubilizers, buffers, pH adjusters, etc., can also be added. These additives are commonly used in the art.

[0055] In addition, colorants, preservatives, flavoring agents, taste enhancers, sweeteners, or other ingredients may be added to the formulation as needed.

[0056] To achieve the purpose of drug administration and enhance the therapeutic effect, the keratin or pharmaceutical composition of the present invention can be administered by known methods.

[0057] The dosage of the keratin pharmaceutical composition of the present invention depends on many factors, including the nature and severity of the disease to be prevented or treated, the sex, age, weight, temperament and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary considerably. Generally speaking, the dosage of the pharmaceutical component of the present invention is well known to those skilled in the art. To achieve the preventive or therapeutic objectives of the present invention, the actual amount of drug contained in the final formulation of the keratin composition of the present invention can be appropriately adjusted to the amount required for treatment. Appropriate daily dose of keratin of the present invention: The dosage of keratin of the present invention is 0.01 to 500 mg / kg body weight, preferably 0.5 to 100 mg / kg body weight, more preferably 1 to 50 mg / kg body weight, and most preferably 2 to 30 mg / kg body weight. The above dosage can be administered in one dose or in multiple doses, such as two, three, or four times, depending on the clinical experience of the administering physician and the drug regimen, including other treatments. The total dose required for each treatment can be administered in multiple doses or in a single dose. The protein or pharmaceutical composition of the present invention can be taken alone or used in combination with other therapeutic or symptomatic agents by adjusting the dosage.

[0058] A seventh aspect of the technical solution of the present invention is to provide the use of keratin CF1 (by the first aspect), nucleic acid molecules (by the second aspect), expression vectors (by the third aspect), host cells (by the fourth aspect), or pharmaceutical compositions (by the sixth aspect) in the manufacture of antipyretics, analgesics, antitussives, expectorants, anticonvulsants, antiepileptics, antihypertensives, anti-inflammatory agents, and antiviral agents.

[0059] To achieve the objectives of the present invention, the present invention employs the following technical solutions. Specifically, the production of keratin CF1 according to the present invention includes the following steps.

[0060] (1) Synthesize the nucleotide sequence and determine the accuracy of the sequence; A preferred nucleotide sequence is shown in Sequence ID No. 2.

[0061] (2) Transfer the nucleotide sequence into the expression vector; Expression vectors that can be used include the pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZα / A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, and pPIC3.5K. The preferred expression vector is the pET series vector; the most preferred expression vector is pET-28a(+).

[0062] (3) Transfect the host cell with the expression vector; The host cell can be E. coli or yeast; the preferred host cell is E. coli.

[0063] Competent cells that can be used include the BL21 lineage, Transetta lineage, Rosetta lineage, DH5α lineage, JM lineage, Top lineage, Orgami lineage, Trans1-T1, TG1, TB1, Y11430, MG1003, GS115(AOX1), KM71, and SMD1168. Preferred competent expression cells are BL21(DE3) and Transetta(DE3).

[0064] (4) Ferment the host cells under appropriate conditions to induce the expression of the target protein CF1; Fermentation equipment can be a shaking flask or a fermentation tank.

[0065] Suitable culture media include LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, rose bengal medium, salt Czapek Dox medium, DOBA medium, rice koji medium, and their modifications; for shaking flask fermentation, LB medium and TB medium are preferred, with TB medium being the most preferred; for fermentation tanks, LB medium and its modifications are preferred.

[0066] Inducers that can be used include IPTG, lactose, and arabinose; IPTG and lactose are preferred.

[0067] (5) Enrich the target protein CF1 product; The fermentation culture obtained in step (4) is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacterial cells are crushed and centrifuged, and the supernatant is discarded; the precipitate is washed with a washing agent and dissolved in urea solution to obtain a crude CF1 protein solution.

[0068] Here, the buffer solution is preferably buffer solution A, and the amount used is such that the volume ratio of fermentation culture solution to buffer solution A is 1 to 100:1, preferably 5 to 10:1.

[0069] The cleaning agent can be urea solution, guanidine hydrochloride solution, TRITON®, or buffer A, preferably urea solution, most preferably 2M urea solution (which may contain 1% TRITON®). Amount used: The volume ratio of fermentation culture solution to urea solution is 0.2 to 100:1, preferably 1 to 15:1; the number of washes is 3 to 10, preferably 6.

[0070] The urea solution is preferably a 4M to 8M urea solution, and most preferably an 8M urea solution. The amount used is such that the volume ratio of fermentation culture solution to 8M urea solution is 0.2 to 100:1, preferably 2 to 15:1.

[0071] (6) Isolation and purification of the target protein CF1 The crude protein solution obtained in step (5) needs to be purified to obtain the target protein CF1. Purification can be carried out by dialysis, ultrafiltration and microfiltration, column chromatography, or salting out.

[0072] A. The dialysis process involves purifying the crude protein solution obtained in step (5) by dialysis to obtain the target protein CF1 solution.

[0073] The molecular weight cutoff for the dialysis bag can be 0.5 to 10 kD, the preferred molecular weight cutoff for the dialysis bag is 3.5 to 10 kD, and the most preferred molecular weight cutoff for the dialysis bag is 10 kD.

[0074] B. The ultrafiltration and microfiltration steps involve purifying the crude protein solution obtained in step (5) using membrane technology such as an ultrafiltration membrane or a microfiltration membrane to obtain a concentrated solution of the target protein CF1.

[0075] C. The column chromatography step involves separating and purifying the crude protein solution obtained in step (5) using various exchange columns or exclusion column chromatography to obtain the target protein CF1.

[0076] Preferred exclusion columns include dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns include ion exchange resin columns: anion exchange resin columns, HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, Toyopearl SuperQ-650M, etc.; cation exchange resin columns: HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M. Most preferably, anion exchange resin columns are used.

[0077] The eluent can be any eluent commonly used in the art, such as water or a salt solution. Salt solutions include sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, and acetic acid.

[0078] D. The salting-out step involves purifying the crude protein solution obtained in step (5) by salting-out to obtain a suspension of the target protein CF1.

[0079] Suitable salting-out agents include ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, and magnesium sulfate. Preferred salting-out agents are ammonium sulfate and its aqueous solution. A saturated aqueous solution of ammonium sulfate is added to achieve a final concentration of ammonium sulfate of 10-50%, preferably 20-30%, and more preferably 25%.

[0080] The salting-out process is performed 1 to 3 times, preferably 2 times.

[0081] After salting out, the precipitate is washed by adding pure water 2 to 5 times, preferably 3 times.

[0082] The target protein CF1 solution purified in steps A to D can be freeze-dried or vacuum-dried to obtain a dry powder, or the concentrated solution can be directly spray-dried to obtain a dry powder.

[0083] Beneficial technical effects of the present invention: 1. The protein of the present invention is a keratin obtained for the first time, and the production method of the present invention is characterized by high yield and high sample purity. 2. The present invention demonstrates that the protein CF1 significantly reduces the rise in body temperature of model animals 8 hours after modeling through efficacy testing of the protein CF1 in a yeast-induced fever model of SD rats; and that the protein CF1 significantly reduces the body temperature of model rats 2 hours after modeling through efficacy testing of the protein CF1 in a lipopolysaccharide (LPS)-induced fever model of SD rats. 3. The efficacy test of protein CF1 against ammonia-water induced cough in mice in this invention demonstrates that protein CF1 can reduce the number of coughs and has an antitussive effect. [Brief explanation of the drawing]

[0084] [Figure 1]Figure 1: Effect of protein CF1 on a lipopolysaccharide (LPS)-induced fever model in rats. (Compared to normal control group, **P<0.01; compared to model group, #P<0.05, ##P<0.01) [Figure 2] Figure 2: Effect of protein CF1 on a rat yeast-induced fever model. (Compared to normal control group, **P<0.01; compared to model group, #P<0.05, ##P<0.01) [Examples]

[0085] The following examples and pharmacological test examples further illustrate the present invention and do not limit it.

[0086] The experimental methods in the following examples and pharmacological test examples are conventional methods unless otherwise specified; the experimental materials used are purchased from conventional biochemical reagent companies unless otherwise specified.

[0087] Example 1: Preparation of crude protein CF1 solution A by shaking flask fermentation (TB medium) The nucleotide sequence shown in Sequence ID No. 2 was synthesized and transferred into a pET-28a(+) vector. The correct sequence was confirmed to be present in the expression vector by sequencing. The expression vector was transfected into BL21(DE3) cells to obtain competent host cells for expression containing the target nucleotide sequence. The competent host cells for expression were added to LB medium and cultured at 37°C and 220 rpm for 1 hour using a shaker to obtain recombinant cells.

[0088] Recombinant strains were collected, streaked onto LBA plates containing kanamycin, and then inverted into a 37°C constant temperature incubator and incubated overnight for 16 hours.

[0089] 400 ml of TB medium was prepared and divided into two 200 ml bottles. Kanamycin (final concentration 50 μg / ml) was added to each bottle (200 ml) of TB medium. A single colony was taken from a plate, added to the TB medium, and cultured overnight at 37°C and 220 rpm using a shaker to obtain a seed solution.

[0090] 28.8 L of TB medium was prepared and divided into 144 200 ml bottles. Kanamycin (final concentration 50 μg / ml) was added to each bottle (200 ml) of TB medium, followed by 2 ml of seed solution. The cultures were then incubated at 37°C and 220 rpm for 2-3 hours using a shaker. OD 600 Monitor and OD 600 When the expression level reached approximately 1.0, an inducer was added to induce protein expression in the shaker. The induction conditions were selected from the table below.

[0091] [Table A]

[0092] The bacterial solutions from each bottle were combined and centrifuged at 7000 rpm for 5 minutes. The supernatant was sterilized and discarded. The precipitate was suspended in approximately 3 L of buffer solution and filtered through an 80-100 mesh sieve. The filtrate was ground twice at a pressure of 800-1000 bar for 2 minutes each time using a high-pressure grinder. The ground bacterial solution was centrifuged at 7000 rpm for 30 minutes, the supernatant was discarded, and the precipitate (i.e., inclusion bodies) was obtained. The precipitate was washed with a washing agent (three times with 1 L of 2 M urea-1% TRITON® solution and twice with 1 L of 3 M urea solution), centrifuged, and the supernatant was discarded. The precipitate was dissolved in 1 L of 8 M urea solution to obtain crude protein solution A.

[0093] Example 2: Preparation of crude protein CF1 solution B by shaking flask fermentation (other medium) In Example 1, an expression vector was synthesized and sequenced to confirm that an expression vector containing the sequence shown in SEQ ID NO: 2 was obtained. The expression vector was transfected into BL21(DE3) cells to obtain competent host cells for expression containing the target nucleotide sequence.

[0094] 20 ml of LB medium was prepared, and 50 μl of host cells containing the target nucleotide sequence were added to 800 μl of LB medium. The cells were then cultured at 37°C and 220 rpm for 1 hour using a shaker.

[0095] The bacterial solution described above was collected, streaked onto an LBA plate containing kanamycin, and the plate was inverted and placed in a 37°C constant temperature incubator for 16 hours, overnight.

[0096] 10 ml of LB medium was prepared and kanamycin (final concentration 50 μg / ml) was added. A single colony was taken from the plate, added to the LB medium, and cultured overnight for 15 hours at 37°C and 220 rpm using a shaker to obtain a seed solution.

[0097] Prepare 1 liter of the culture medium shown in the table below and divide it into 10 bottles of 100 ml each. Add kanamycin (final concentration 50 μg / ml) to the medium in each bottle (100 ml), add 1 ml of seed solution, and incubate using a shaker at 37°C and 220 rpm for 2-3 hours. OD 600 Monitor and OD 600 When the ratio reached approximately 1.0, the inducing agent IPTG (final concentration 0.5 mM) was added, and protein expression was induced using a shaker at 37°C and 220 rpm.

[0098] [Table B]

[0099] The bacterial solutions from each bottle were combined and centrifuged at 10,000 rpm for 10 minutes. The supernatant was sterilized and discarded. The precipitate was suspended in approximately 100 mL of buffer solution, filtered through an 80-100 mesh sieve, and the filtrate was ground twice using a high-pressure grinder at a pressure of 800-1000 bar for 2 minutes each time. The ground bacterial solution was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was discarded.

[0100] First, the precipitate was washed with 40 mL of buffer A as a washing agent, centrifuged, and the supernatant was discarded. Next, the precipitate was washed twice with 40 mL of 2 M urea solution (containing 1% TRITON®) as a washing agent, centrifuged, and the supernatant was discarded. Subsequently, the precipitate was washed twice with 40 mL of 3 M urea solution, centrifuged, and the supernatant was discarded. Finally, 40 mL of 8 M urea solution was added to the precipitate to obtain crude protein solution B.

[0101] Example 3: Production of crude protein CF1 solution C using a fermentation tank In Example 1, an expression vector was synthesized and sequenced to confirm that an expression vector containing the sequence shown in SEQ ID NO: 2 was obtained. The expression vector was transfected into BL21(DE3) cells to obtain competent host cells for expression containing the target nucleotide sequence. The competent host cells for expression were added to LB medium and cultured at 37°C and 220 rpm for 1 hour using a shaker to obtain recombinant cells.

[0102] 100 μl of recombinant strain was added to an LBA plate containing kanamycin, spread evenly using a spreader until dry, and then the plate was inverted and placed in a 37°C incubator for overnight incubation. Three single colonies were separately collected and streaked onto plates containing kanamycin, and the plates were incubated overnight. After verifying correct expression by three shaking flask fermentations, the strain was stored in 15% glycerin, divided into 0.8 ml portions to obtain a working cell bank, and stored in a -80°C freezer for later use.

[0103] A 100 μl glycerin-containing bacterial stock was taken from the working cell bank, 40 ml of LB medium was added, kanamycin (final concentration 50 μg / ml) was added, and the mixture was incubated in a shaker at 37°C and 220 rpm for 6 hours to obtain the first-stage seed solution.

[0104] 1.2 ml of the first-stage seed solution was added to 120 ml of LB medium, kanamycin (final concentration 50 μg / ml) was added, and the mixture was incubated in a shaker at 37°C and 220 rpm for 7 hours to obtain the second-stage seed solution.

[0105] 3 L of modified LB medium was added to a 5 L fermentation tank, followed by 120 ml of the second-stage seed solution and 3 ml of kanamycin (final concentration 50 μg / ml). The culture was incubated at 37°C with 30% dissolved oxygen (culture rate) for approximately 8 hours. The OD value was monitored to approximately 20, and 3 g of lactose was used as an inducer. Induction was induced by supplying lactose at 30 ml / hour at 20°C, and the culture was incubated at 20°C for 24 hours.

[0106] The bacterial suspension was centrifuged at 7000 rpm for 5 minutes, and the supernatant was sterilized and discarded. The precipitate was suspended in approximately 600 mL of buffer A, filtered through an 80-100 mesh sieve, and the filtrate was ground twice at a pressure of 800-1000 bar for 2 minutes each time using a high-pressure grinder. The ground bacterial suspension was centrifuged at 7000 rpm for 30 minutes, and the supernatant was discarded.

[0107] The precipitate was washed three times with 2M urea solution (containing 1% TRITON®) (1L each time); then, 1L of 3M urea solution was added to the precipitate and washed twice, followed by centrifugation and discarding the supernatant. Subsequently, 1L of 8M urea solution was added to the precipitate and washed once, followed by centrifugation and discarding the supernatant. Finally, 2L of 8M urea solution was added to the precipitate to obtain crude protein solution C.

[0108] Example 4: Production of protein CF1 by dialysis of crude protein solution A The crude protein solution A obtained in Example 1 was filtered through a 0.45 μm filter membrane, and the filtrates were combined. The filtrates were dialyzed with water using a dialysis bag with a molecular weight cutoff of 10 kD. After 72 hours of dialyzing, the filtrate was freeze-dried to obtain the target protein CF1.

[0109] Structural confirmation of protein CF1 - Whole protein sequence analysis by LC-MS / MS

[0110] Main ingredients: Acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Asp-N.

[0111] Main equipment: Capillary high-performance liquid chromatography (Thermo Ultimate 3000), electrospray-ion trap orbitrap mass spectrometer (Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer).

[0112] Methods and Results Protein CF1 was pretreated with various methods including dissolution and substitution, reductive alkylation, and various proteolytic processes to obtain enzymatically cleaved peptides. The enzymatically cleaved peptide solutions were analyzed using a liquid chromatography-tandem mass spectrometer. The raw mass spectrometry data were analyzed using the Maxquant (1.6.2.10) protein database. Identification confirmed that the peptides matched the target sequence shown in Sequence ID No. 1.

[0113] Example 5: Production of protein CF1 by purification of crude protein solution A by salting-out method Crude protein solution A was placed in a stirring vessel and salted out twice. Saturated ammonium sulfate solution was slowly added along the wall to achieve a final ammonium sulfate concentration of 25% or 50%. Protein precipitated by salting out. After the completion of salting out, the solution was filtered to complete the first salting out. Next, 400 ml of pure water was added to the precipitate to suspend it, and saturated ammonium sulfate solution was again slowly added along the wall to achieve a final ammonium sulfate concentration of 25%. A second salting out and filtration were performed to obtain a precipitate of crude protein extract. The crude protein extract was washed three times with water; 200 ml of pure water was added to suspend it, stirred, allowed to stand, and filtered. This was repeated three times, and the precipitate was freeze-dried to obtain the target protein CF1.

[0114] Using the same structural verification method as in Example 4, it was confirmed that it had the same amino acid sequence as the protein produced in Example 4.

[0115] Example 6: Production of protein CF1 by purification of crude protein solution B The crude protein solution B obtained in Example 2 was purified by the following two methods:

[0116] Method 1 Dialysis Crude protein solution B was filtered through a 0.45 μm membrane, the filtrate was dialyzed with water for at least 72 hours, and the internal solution was freeze-dried to obtain the target protein CF1.

[0117] [Table C]

[0118] Method 2 Salting out Crude protein solution B was placed in a stirring vessel and salted out twice. Saturated ammonium sulfate solution was slowly added along the wall to achieve a final ammonium sulfate concentration of 25% or 50%. Protein precipitated by salting out. After the completion of salting out, the solution was filtered to complete the first salting out. 400 ml of pure water was added to the precipitate to suspend it, and then saturated ammonium sulfate solution was slowly added again along the wall to achieve a final ammonium sulfate concentration of 25%. A second salting out and filtration were performed to obtain a precipitate of crude protein extract. The crude protein extract was washed three times with water. It was suspended in 200 ml of pure water, stirred, allowed to stand, and filtered. This was repeated three times, and the precipitate was freeze-dried to obtain the target protein CF1.

[0119] Using the same structural verification method as in Example 4, it was confirmed that the protein CF1 obtained by the two methods had the same amino acid sequence as the protein produced in Example 4.

[0120] Example 7: Production of protein CF1 by purification of crude protein solution C Crude protein solution C was purified using a microfiltration membrane; urea was removed by repeated microfiltration using a 20 nm or 50 nm ceramic membrane core; and the resulting solution was freeze-dried to obtain the target protein CF1.

[0121] Using the same structural verification method as in Example 4, it was confirmed that it had the same amino acid sequence as the protein produced in Example 4.

[0122] Pharmacological tests Experimental Example 1: Efficacy study of protein CF1 (protein from Example 7) in a lipopolysaccharide (LPS)-induced fever model of SD rats. Animal: Male SD rat, weight 230-260g Drugs: Lipopolysaccharide (LPS, SIGMA L-2880), Aspirin (SIGMA A2093), Protein CF1 Equipment: Electronic balance (SARTORIUS BP121S), electronic thermometer (CITIZEN CT-513W) experimental group Normal control group Model group: Lipopolysaccharide thermogenic model Positive control group: Aspirin 300 mg / kg group Protein CF1, 10 mg / kg group, 50 mg / kg group

[0123] Method: Replicate a rat fever model by injecting lipopolysaccharide into the peritoneal cavity. Preparation of experimental animals: Experimental animals were allowed to acclimate for one day in the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%). For preliminary acclimatization, rectal temperature was measured at 8:00 AM and 3:00 PM. The animals were fasted for 12 hours prior to the experiment, but were allowed to drink water freely and were allowed to defecate before the rectal temperature measurement. Before each measurement, petroleum jelly was applied to the probe of the electronic thermometer and inserted 2 cm into the rat's rectum (a mark may be made at the 2 cm position to ensure a consistent insertion depth). After the reading stabilized, the body temperature was recorded.

[0124] A rat fever model was replicated by intraperitoneal injection of lipopolysaccharide; rat body temperature was measured before constructing the model. Acceptable rats with body temperature between 36.2 and 37.3°C were selected and randomly divided into groups of 8 rats each. After oral administration of aspirin and different doses of protein CF1, lipopolysaccharide (20 μg / kg, 2 ml / kg) was immediately injected intraperitoneally. The normal control group received the same volume of saline intraperitoneally. Body temperature was observed for 8 hours starting 2 hours later.

[0125] Data Statistics Based on the body temperature measured on the experimental day, the mean, standard deviation, and standard error of body temperature for each group were calculated. A t-test was applied to compare the data between groups, and a P<0.05 value was considered statistically significant.

[0126] Experimental results Immediately after oral administration of aspirin (300 mg / kg) and protein CF1 (10 mg / kg, 50 mg / kg), 20 μg / kg of lipopolysaccharide was injected intraperitoneally for modeling purposes. Body temperature was observed 2, 4, 6, and 8 hours after the modeling stage. The results are shown in Table 1 and Figure 1.

[0127] [Table 1]

[0128] Conclusion of the experiment Following oral administration of aspirin (300 mg / kg) and protein CF1 (10 mg / kg, 50 mg / kg), 20 μg / kg of lipopolysaccharide was immediately administered intraperitoneally for modeling purposes. Body temperature was monitored at 2, 4, 6, and 8 hours after the modeling stage. The results are as follows: 1) Intraperitoneal injection of 20 μg / kg of lipopolysaccharide successfully induced an increase in body temperature in rats. The increase in body temperature in the model group was significant at 2, 4, 6, and 8 hours after modeling. The model showed a statistically significant difference (P<0.05) compared to the normal group, indicating stability. 2) The positive control drug aspirin effectively suppressed the rise in body temperature in model rats at 2, 4, 6, and 8 hours of the model. The effect of the positive control drug was relatively stable, showing a statistical difference of P<0.05 compared to the model group. 3) The group administered 10 mg / kg of protein CF1 significantly reduced the body temperature of the model rats 2 hours after modeling. This showed a statistically significant difference (P<0.05) compared to the model group.

[0129] Experimental Example 2: Efficacy study of protein CF1 (protein from Example 7) in a yeast-induced fever model of SD rats. Animal: Male SD rat, weight 230-260g Drugs: Yeast (OXOID LP0021), Aspirin (SIGMA A2093), Protein CF1 Equipment: Electronic balance (SARTORIUS BP121S), electronic thermometer (CITIZEN CT-513W) experimental group Normal control group Model group: Yeast-induced fever model Positive control group: Aspirin 300 mg / kg group Protein CF1, 10 mg / kg group, 50 mg / kg group

[0130] method Preparation of experimental animals: Experimental animals were allowed to acclimate for one day in the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%). For preliminary acclimatization, rectal temperature was measured at 8:00 AM and 3:00 PM. The animals were fasted for 12 hours prior to the experiment, but were allowed to drink water freely and were allowed to defecate before the rectal temperature measurement. Before each measurement, petroleum jelly was applied to the probe of the electronic thermometer and inserted 2 cm into the rat's rectum (a mark may be made at the 2 cm position to ensure a consistent insertion depth). After the reading stabilized, the body temperature was recorded.

[0131] A rat fever model was replicated by subcutaneous injection of dried yeast; rat body temperature was measured before constructing the model. Passive rats with body temperatures of 36.2–37.3°C were selected and randomly divided into groups of 8 rats each. After oral administration of aspirin and different doses of protein CF1, a 20% yeast suspension (10 ml / kg) was immediately injected subcutaneously. The normal control group received the same volume of saline subcutaneously. Body temperature was observed every 2 hours starting 2 hours later, for a total of 8 hours.

[0132] Data Statistics Based on the body temperature measured on the experimental day, the mean, standard deviation, and standard error of body temperature for each group were calculated. A t-test was applied to compare the data between groups, and a P<0.05 value was considered statistically significant.

[0133] Experimental results Immediately after oral administration of aspirin (300 mg / kg) and protein CF1 (10 mg / kg, 50 mg / kg), a 20% yeast suspension was subcutaneously injected for modeling purposes. Body temperature was observed 2, 4, 6, and 8 hours after the modeling stage. The results are shown in Table 2 and Figure 2.

[0134] [Table 2]

[0135] Conclusion of the experiment Following oral administration of aspirin (300 mg / kg) and protein CF1 (10 mg / kg, 50 mg / kg), a 20% yeast suspension was immediately subcutaneously injected for modeling purposes. Body temperature was observed 2, 4, 6, and 8 hours after the modeling stage. The results are as follows: 1) The increase in body temperature in the model group was significant at 2, 4, 6, and 8 hours after modeling. It showed a statistically significant difference of P<0.05 compared to the normal group, and the model was successfully constructed, stable, and reliable. 2) The positive control drug aspirin effectively suppressed the rise in body temperature in model rats at 2, 4, 6, and 8 hours of the model. The effect of the positive control drug aspirin was relatively stable, showing a statistical difference of P<0.05 compared to the model group. 3) The group administered 10 mg / kg of protein CF1 significantly suppressed the rise in body temperature in model rats 2 and 8 hours after the modeling stage. This showed a statistically significant difference (P<0.05) compared to the model group.

[0136] Experimental Example 3: Efficacy study of the antitussive effect of protein CF1 (protein from Example 7) on ammonia-water induced cough in mice. Animal: Male ICR mouse Drugs and reagents: Dextromethorphan hydrobromide, ammonia, 0.2% CMC-Na, protein CF1 Equipment: Compression nebulizer (403T), balance (XS105DU) experimental group solvent control group Dextromethorphan 15 mg / kg group Protein CF1, 20 mg / kg group, 50 mg / kg group

[0137] method Model building and medication Mice were orally administered dextromethorphan and different doses of protein CF1 (administered volume: 10 ml / kg). The solvent control group was administered the same volume of distilled water. After 1 hour, the mice were placed in a sealed chamber and sprayed with 10% ammonia water for 10 seconds. The cough latency and the number of coughs within 2 minutes were then observed and recorded.

[0138] Data processing The timing of oral administration, spraying, cough latency, and the number of coughs within 2 minutes were recorded. Cough latency refers to the number of seconds from the start of ammonia spraying to the onset of coughing. Coughing in mice was defined as abdominal muscle contraction (thoracic recession) and a wide-open mouth. The mean and standard error of the data for each group were calculated. A t-test was applied to compare the model group with other groups, and a P<0.05 was considered statistically significant.

[0139] Experimental results Dextromethorphan (15 mg / kg) and different doses of protein CF1 (20 mg / kg, 50 mg / kg) were administered beforehand. One hour later, the mice were placed in a sealed chamber and sprayed with 10% ammonia water for 10 seconds. Then, cough latency and the number of coughs within 2 minutes were observed and recorded. The results are shown in Table 3.

[0140] [Table 3]

[0141] Conclusion of the experiment 1) The experimental results demonstrate that the dextromethorphan group shows a statistically significant improvement in latency and cough frequency (P<0.05) compared to the solvent control group. 2) Compared to the solvent control group, the CF1 (20 mg / kg) administration group showed a statistically significant difference in the number of coughs, but this effect was weak.

Claims

1. Keratin CF1, wherein the amino acid sequence of the keratin CF1 is (1) The amino acid sequence shown in Sequence ID No. 1 of the sequence listing; or, (2) An amino acid sequence in which 1 to 45 amino acids are substituted, deleted, or added in the amino acid sequence shown in Sequence ID No. 1 of the sequence listing, and which maintains essentially the same biological function; Keratin CF1, characterized by being such.

2. The keratin CF1 according to claim 1, characterized in that the keratin CF1 may include conventional modifications; it may be bound to a tag for detection or purification; or it may be homologous to keratin CF1.

3. The conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, immobilization modification, sulfation, oxidation, methylation, deamination, formation of a disulfide bond or cleavage of a disulfide bond; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact; the keratin CF1 according to claim 2.

4. A nucleic acid molecule encoding keratin CF1 according to any one of claims 1 to 3.

5. The nucleotide sequence of the nucleic acid molecule is (1) The nucleotide sequence shown in Sequence ID No. 2 of the sequence listing; (2) A sequence optimized based on the nucleotide sequence shown in Sequence ID No. 2; or, (3) A nucleotide sequence complementary to the nucleotide sequence of (1) or (2) above; The nucleic acid molecule according to claim 4, characterized in that it is the same as described above.

6. An expression vector, characterized in that the expression vector contains the nucleic acid molecule described in claim 4 or 5.

7. A host cell, wherein the host cell contains the expression vector described in claim 6 or the nucleic acid molecule described in claim 4 or 5 incorporated into the genome.

8. The host cell according to claim 7, characterized in that the host cell includes bacteria, yeast, Aspergillus, plant cells, or insect cells.

9. The host cell according to claim 8, characterized in that the bacteria include Escherichia coli.

10. The following steps: A. Synthesize a nucleic acid molecule corresponding to keratin CF1 according to any one of claims 1 to 3, link the nucleic acid molecule to a corresponding expression vector, transform the expression vector into host cells, culture the host cells containing the expression vector in a fermentation apparatus under certain conditions to induce keratin CF1 expression and obtain a crude protein solution containing keratin CF1; B. The crude protein solution expressed in step A is separated, purified, and dried to obtain keratin CF1; A method for producing keratin CF1 according to any one of claims 1 to 3, characterized by including the following:

11. The method according to claim 10, characterized in that, in step A, the host cells are mainly selected from Escherichia coli, the keratin CF1 is expressed in inclusion bodies of Escherichia coli, and the fermentation apparatus includes a shaking flask or a fermentation tank.

12. The method according to claim 10, characterized in that in step A, after inducing the expression of keratin CF1, impurities are removed using a washing agent, and keratin CF1 is dissolved in a solution to obtain a crude protein solution.

13. The method according to claim 10, wherein step B includes ultrafiltration or microfiltration membrane purification, column chromatography purification, salting out, and dialysis.

14. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises keratin CF1 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or additive.

15. The use of keratin CF1 according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4 or 5, an expression vector according to claim 6, a host cell according to any one of claims 7 to 9, or a pharmaceutical composition according to claim 14 in the manufacture of an antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral agent.

Images