Keratin BD-3, method of preparation thereof, pharmaceutical composition and uses

Abstract

The present invention provides keratin BD-3, a nucleic acid molecule encoding the same, an expression vector, a host cell, a method for producing said keratin, and a pharmaceutical composition containing said keratin. said keratin BD-3 can be used in the production of antipyretic analgesic, antitussive expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs.

Description

Technology Field

[0001] The present invention relates to keratin BD-3, a nucleic acid molecule encoding keratin BD-3, an expression vector containing said nucleic acid molecule, a host cell containing said nucleic acid molecule incorporated into said expression vector or genome, a method for producing keratin BD-3, a pharmaceutical composition containing said keratin, and the application of said keratin and said pharmaceutical composition in the manufacture of antipyretic analgesic, antitussive expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs. Background Technology

[0002] Keratin is a type of protein that is widely present in the epidermis of humans and animals, and is also the main component of hair, feathers, hooves, shells, claws, and horns. It is an extremely important structural protein of connective tissue that plays a role in protecting the organism.

[0003] Keratin is a renewable resource widely present in living organisms with high utility value, but it is not being utilized extensively and effectively. The main reasons for this are that keratin is insoluble in various solvents and generally resists enzymatic hydrolysis by proteases more than other proteins. Therefore, extracting and manufacturing natural keratin is very difficult.

[0004] With the rapid advancement of modern biological technologies such as genomics, proteomics, genetic engineering, and microbial engineering, an increasing number of genes are being discovered. The preparation and production of target proteins using protein expression systems is an important means of studying the biological functions of genes or proteins.

[0005] The preparation of target keratin using a protein expression system and the subsequent study of its structure and function has not been reported in other literature, thus possessing novelty and inventiveness.

[0006] The technical problem that the present invention aims to solve is to provide keratin BD-3, a nucleic acid molecule encoding keratin BD-3, an expression vector containing said nucleic acid molecule, a host cell containing said nucleic acid molecule integrated into said expression vector or genome, a method for producing keratin BD-3, a pharmaceutical composition containing keratin BD-3, and the application of said keratin BD-3, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the production of antipyretic analgesic, antitussive expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs.

[0007] To solve the technical problem of the present invention, the present invention provides the following technical solution.

[0008] A first embodiment of the technical solution means of the present invention is keratin BD-3, wherein

[0009] The amino acid sequence of the above keratin BD-3 is,

[0010] (1) Amino acid sequence indicated by SEQ ID NO.1 of the sequence list;

[0011] (2) Provides a keratin BD-3 characterized by having an amino acid sequence that basically maintains the same biological function as formed by substituting, deleting, or adding 1 to 35 amino acids to the amino acid sequence indicated by SEQ ID NO.1 of the sequence list.

[0012] Furthermore, conventional modifications can be applied to keratin BD-3; or a tag for detection or purification is additionally attached to keratin BD-3.

[0013] Furthermore, the above conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorophore modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamination, formation of disulfide bonds or cleavage of disulfide bonds; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, Profinity eXact.

[0014] A second embodiment of the technical solution of the present invention provides a nucleic acid molecule encoding keratin BD-3 according to the first embodiment.

[0015] Furthermore, the nucleotide sequence of the above nucleic acid molecule is,

[0016] (1) Nucleotide sequence indicated by SEQ ID NO.2 of the sequence list;

[0017] (2) Nucleotide sequence obtained by sequence optimization based on the nucleotide sequence indicated by SEQ ID NO.2;

[0018] (3) A nucleotide sequence that is complementary to the nucleotide sequence of (1) or (2) above.

[0019] A third aspect of the technical solution of the present invention provides an expression vector, wherein the expression vector is characterized by containing a nucleic acid molecule according to the second aspect.

[0020] Furthermore, the expression vector may be 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, pPIC3.5K, etc.; the preferred expression vector is the pET series vector; and the most preferred expression vector is pET-28a(+).

[0021] A fourth aspect of the technical solution of the present invention provides a host cell, wherein the host cell is characterized by containing an expression vector according to the third aspect or a nucleic acid molecule according to the second aspect integrated into the genome.

[0022] Furthermore, the host cell includes bacteria, yeast, Aspergillus, plant cells, or insect cells.

[0023] Furthermore, the bacteria mentioned above include E. coli or yeast.

[0024] Competent host cells may be BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; preferred expression competency cells are BL21 (DE3) and Transetta (DE3).

[0025] A fifth aspect of the technical solution of the present invention is a method for producing keratin BD-3 according to the first aspect, wherein

[0026] A. A step of synthesizing a nucleic acid molecule corresponding to keratin BD-3 according to the first embodiment, linking the nucleic acid molecule to a corresponding expression vector, transforming the expression vector into a host cell, culturing the host cell having the expression vector in a fermentation apparatus under predetermined conditions, and inducing the expression of keratin BD-3 to obtain a crude protein solution containing keratin BD-3; and

[0027] B. Provides a method characterized by including the step of isolating, purifying, and drying the crude protein solution expressed in step A to obtain keratin BD-3.

[0028] Furthermore, in step A, the host cell is mainly selected from E. coli, the keratin BD-3 is expressed in an E. coli inclusion body, and the fermentation device includes a shake flask or a fermentation tank.

[0029] Furthermore, in step A, after inducing the expression of keratin BD-3, impurities can be removed with a detergent and dissolved in a solution to obtain a crude protein solution.

[0030] Furthermore, in step A, the medium may be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Red Bengal medium, high salt Chashi medium, DOBA medium, rice koji medium, and improved formulations thereof; for shake flask fermentation, LB medium and TB medium are preferred, and TB medium is most preferred; and for the fermentation tank, LB medium and improved formulations thereof are preferred.

[0031] Furthermore, in step A, the inducer may be IPTG, lactose, arabinose, etc.; preferably, it is IPTG or lactose.

[0032] Furthermore, in step A, the obtained fermentation solution is centrifuged to remove the supernatant; the precipitate is suspended in a buffer solution, the bacteria are crushed, and then centrifuged to remove the supernatant; the precipitate is washed with a detergent and dissolved in a urea solution to obtain a BD-3 crude protein solution.

[0033] Here, the buffer is preferably buffer A, and the dosage is fermentation liquid volume: buffer A volume = 1 to 100:1, preferably 10:1;

[0034] The detergent may be a urea solution, guanidine hydrochloride solution, Triton, buffer A, etc., preferably a urea solution, most preferably a 2M urea solution (which may contain 1% Triton), and the dosage is fermentation liquid volume : 2M urea volume = 0.2 to 100:1, preferably 1 to 15:1;

[0035] The urea solution is preferably an 8M urea solution, and the dosage is fermentation liquid volume: 8M urea volume = 0.2 to 100:1, preferably 2 to 15:1.

[0036] Furthermore, in step B, the separation and purification methods include ultrafiltration and microfiltration membrane purification methods, column chromatography purification methods, salting out and dialysis methods.

[0037] Furthermore, in step B, the separation and purification methods are as follows.

[0038] (1) The above dialysis method is a method to obtain a target protein BD-3 solution by purifying the crude protein solution obtained in step A using dialysis.

[0039] The molecular weight cutoff of the dialysis bag may be 0.5 to 10 kD, the preferred molecular weight cutoff of the dialysis bag is 3.5 to 10 kD, and the most preferred molecular weight cutoff of the dialysis bag is 10 kD.

[0040] (2) The above ultrafiltration and microfiltration methods are methods to obtain a concentrated solution of the target protein BD-3 by purifying the crude protein solution obtained in step A using membrane technology such as an ultrafiltration membrane or a microfiltration membrane.

[0041] Preferably, microfiltration membrane purification is performed twice, with the membrane pore size being 1000 to 1500 nm in the first step and 20 to 50 nm in the second step.

[0042] (3) The above column chromatography method is a method of obtaining the target protein BD-3 by passing the crude protein solution obtained in step A through various exchange columns or exclusion column chromatography and separating and purifying it.

[0043] Preferred exclusion columns are dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns are anion exchange resin columns, ion exchange resin columns such as 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.

[0044] The eluent may be a solution commonly used in the industry, such as water or a salt solution, and the salt solution includes a sodium chloride solution, a sodium dihydrogen phosphate solution, a disodium hydrogen phosphate solution, sodium acetate, acetic acid, etc.

[0045] (4) The above salting-out method is a method of obtaining a target protein BD-3 suspension by purifying the crude protein solution obtained in step A using the salting-out method.

[0046] The salting-out agent may be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc. A preferred salting-out agent is ammonium sulfate and an aqueous solution thereof. A saturated aqueous solution of ammonium sulfate is added so that the final concentration of ammonium sulfate reaches 10 to 50%, preferably 20 to 30%, and more preferably 25%.

[0047] The number of salting cycles is 1 to 3 times, preferably 2 times.

[0048] After salting out, the precipitate is washed by adding pure water, with the number of washes ranging from 2 to 5 times, preferably 3 times.

[0049] Furthermore, the target protein BD-3 solution purified in step B can be prepared into a dry powder by freeze-drying or vacuum drying, or by directly spray-drying the concentrate into a dry powder.

[0050] A sixth aspect of the technical solution of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises keratin BD-3 according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect and a pharmaceutically acceptable vector or excipient.

[0051] The keratin obtained in the above steps of the present invention can be prepared into a dried powder by freeze-drying or vacuum drying, or into a dried powder by directly spray-drying the concentrate, and then prepared into various formulations.

[0052] The present invention relates to a pharmaceutical composition comprising any keratin obtained in the above step and a pharmaceutically acceptable vector.

[0053] The present invention also relates to a pharmaceutical composition containing the keratin of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants. Generally, the keratin of the present invention accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.

[0054] The present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of protein as an active ingredient and a pharmaceutically acceptable vector.

[0055] The pharmaceutical composition of the present invention may be prepared according to methods known in the art. When used for such purposes, the protein of the present invention may be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants as needed to be prepared in a suitable dosage form or dosage form that can be used in drugs for humans or animals.

[0056] The keratin of the present invention or a pharmaceutical composition containing it may be administered in the form of a unit dose, and the route of administration may be oral administration or parenteral administration such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, skin, vagina, peritoneum, rectum, etc., and preferably oral administration.

[0057] The route of administration of the keratin of the present invention or a pharmaceutical composition containing it may be injection. Injections include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, and acupoint injection, etc.

[0058] The formulation for administration may be a liquid formulation, a solid formulation, or a semi-solid formulation. Liquid formulations may include liquid preparations (including pure liquid and colloidal solutions), emulsions (including oil in water, water in oil and double emulsions), suspensions, injectables (including water injectables, powder injectables and infusions), eye drops, nasal drops, detergents, and topical agents. Solid formulations may include tablets (including regular tablets, enteric-coated tablets, oral tablets, dispersed tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, pellets, drop preparations, suppositories, films, patches, air (powder) sprays, sprays, etc.; semi-solid formulations may include ointments, gels, pastes, etc.

[0059] The keratin of the present invention can be manufactured into general formulations, sustained-release formulations, long-acting formulations, targeted formulations, and various microparticle administration systems.

[0060] To manufacture a unit dosage form into a tablet, a wide range of excipients known in the art, such as diluents, binders, wetting agents, disintegrants, lubricants, and lubricants, may be used. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropanol, etc.; and the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene dipropanol, etc.; The disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate; the lubricant and lubricant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0061] Tablets can also be further manufactured as coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0062] Various vectors known in the art may be used to make the dosage unit into a pill. Examples of vectors include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, macrogolglyceryl laurate, kaolin, talc, etc.; binders such as gum arabic, xanthan gum, gelatin, ethanol, honey, liquid sugar, rice paste or dough, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose, etc.

[0063] To manufacture a dosage unit as a suppository, a wide variety of vectors known in the art can be used. Examples of vectors include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.

[0064] In order to manufacture a dosage unit as a capsule, the keratin of the present invention, which is the active ingredient, is mixed with the various vectors above, and the mixture obtained therefrom is placed in a hard gelatin capsule or a soft capsule. The keratin of the present invention, which is the active ingredient, can be prepared as a microcapsule, suspended in an aqueous medium to form a suspension, and may be applied by placing it in a hard capsule or by manufacturing it as an injectable.

[0065] For example, the keratin of the present invention is prepared into injectable formulations such as liquids, suspensions, emulsions, and lyophilized powder injectables, and such formulations may be aqueous or non-aqueous and may contain one and / or more pharmaceutically acceptable vectors, diluents, binders, lubricants, preservatives, surfactants, or dispersants. For example, diluents are selected from water, ethanol, polyethylene glycol, l,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. Additionally, to prepare an isotonic injectable solution, an appropriate amount of sodium chloride, glucose, or glycerin may be added to the injectable formulation, and conventional solubilizers, buffers, pH adjusters, etc. may also be added. These adjuvants are adjuvants commonly used in the art.

[0066] In addition, if necessary, coloring agents, preservatives, flavorings, deodorizers, sweeteners, or other materials may be added to the drug preparation.

[0067] To achieve medicinal purposes and enhance therapeutic effects, the keratin or pharmaceutical composition of the present invention may be administered by any known method of administration.

[0068] The dosage of the keratin pharmaceutical composition of the present invention is determined by many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, temperament and individual response of the patient or animal, the route of administration, the frequency of administration, and the purpose of treatment. Accordingly, the therapeutic dosage of the present invention may vary significantly. In general, the dosage of the pharmaceutical component of the present invention is known to those skilled in the art. By appropriately adjusting according to the actual amount of the drug contained in the final formulation of the keratin composition of the present invention, the requirements for a therapeutically effective dosage can be reached and the preventive or therapeutic purpose of the present invention can be achieved. Appropriate daily dosage range of the keratin of the present invention: The dosage of the 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 may be administered as a single dose or divided into multiple doses, such as two, three, or four doses, which is determined by the administering physician's clinical experience and administration methods, including the use of other therapeutic means. The total dosage required for each treatment may be divided into multiple doses or single doses. The protein or pharmaceutical composition of the present invention may be taken alone or in combination with other therapeutic drugs or symptomatic drugs to adjust the dosage.

[0069] A seventh embodiment of the technical solution of the present invention provides the application of keratin BD-3 according to the first embodiment, a nucleic acid molecule according to the second embodiment, an expression vector according to the third embodiment, a host cell according to the fourth embodiment, or a pharmaceutical composition according to the sixth embodiment in the manufacture of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, and antiviral drugs.

[0070] To achieve the objective of the present invention, the present invention applies the following technical solutions. Specifically, the method for manufacturing keratin BD-3 of the present invention includes the following steps.

[0071] (1) A step of synthesizing a nucleotide sequence and measuring the accuracy of the sequence;

[0072] The desired nucleotide sequence was indicated as SEQ ID No. 2.

[0073] (2) A step of transferring a nucleotide sequence to an expression vector;

[0074] The expression vector may be 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, pPIC3.5K, etc.; the preferred expression vector is the pET series vector; and the most preferred expression vector is pET-28a(+).

[0075] (3) Step of transfecting a host cell with an expression vector;

[0076] The host cell may be E. coli or yeast; the preferred host cell is E. coli;

[0077] Competent cells may be BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; preferred expression competency cells are BL21 (DE3) and Transetta (DE3).

[0078] (4) A step of inducing the expression of the target protein BD-3 by fermenting and culturing host cells under appropriate conditions;

[0079] The fermentation equipment may utilize a shake flask or a fermentation tank;

[0080] The medium may be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Red Bengal medium, high-salt Chasi medium, DOBA medium, rice koji medium, and improved formulations thereof; for shake flask fermentation, LB medium and TB medium are preferred, and TB medium is most preferred; for fermentation tank, LB medium and improved formulations thereof are preferred.

[0081] The inducer may be IPTG, lactose, arabinose, etc.; preferably, it is IPTG or lactose.

[0082] (5) A step of concentrating the target protein BD-3 product;

[0083] (4) The fermented bacterial solution obtained in step (4) is centrifuged to remove the supernatant; the precipitate is suspended in a buffer solution, the bacteria are crushed, and then centrifuged to remove the supernatant; the precipitate is washed with detergent and dissolved in a urea solution to obtain a BD-3 crude protein solution.

[0084] Here, the buffer is preferably buffer A, and the dosage is fermentation liquid volume: buffer A volume = 1 to 100:1, preferably 10:1;

[0085] The detergent may be a urea solution, guanidine hydrochloride solution, Triton, buffer A, etc., preferably a urea solution, most preferably a 2M urea solution (which may contain 1% Triton), and the dosage is fermentation liquid volume : 2M urea volume = 0.2 to 100:1, preferably 1 to 15:1;

[0086] The urea solution is preferably an 8M urea solution, and the dosage is fermentation liquid volume: 8M urea volume = 0.2 to 100:1, preferably 2 to 15:1.

[0087] (6) Step to isolate and purify the target protein BD-3:

[0088] (5) The crude protein solution obtained in step must undergo purification to obtain the target protein BD-3. The purification can be performed through dialysis, ultrafiltration and microfiltration, column chromatography, or a salting-out step.

[0089] A. The above dialysis step is a step of obtaining the target protein BD-3 by purifying the crude protein solution obtained in step (5) by dialysis.

[0090] The molecular weight cutoff of the dialysis bag may be 0.5 to 10 kD, the preferred molecular weight cutoff of the dialysis bag is 3.5 to 10 kD, and the most preferred molecular weight cutoff of the dialysis bag is 10 kD.

[0091] B. The above ultrafiltration and microfiltration steps are steps to obtain a target protein BD-3 concentrated solution by purifying the crude protein solution obtained in step (5) using membrane technology such as an ultrafiltration membrane or a microfiltration membrane.

[0092] Preferably, microfiltration membrane purification is performed twice, with the membrane pore size being 1000 to 1500 nm in the first step and 20 to 50 nm in the second step.

[0093] C. The above column chromatography step is a step of obtaining the target protein BD-3 by passing the crude protein solution obtained in step (5) through column chromatography, such as various exchange column or exclusion column chromatography, and separating and purifying it.

[0094] Preferred exclusion columns are dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns are anion exchange resin columns, ion exchange resin columns such as 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.

[0095] The eluent may be a solution commonly used in the industry, such as water or a salt solution, and the salt solution includes a sodium chloride solution, a sodium dihydrogen phosphate solution, a disodium hydrogen phosphate solution, sodium acetate, acetic acid, etc.

[0096] D. The above salting-out step is a step of obtaining a target protein BD-3 suspension by purifying the crude protein solution obtained in step (5) using the salting-out method.

[0097] The salting-out agent may be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc. A preferred salting-out agent is ammonium sulfate and an aqueous solution thereof. A saturated aqueous solution of ammonium sulfate is added so that the final concentration of ammonium sulfate reaches 10 to 50%, preferably 20 to 30%, and more preferably 25%.

[0098] The number of salting cycles is 1 to 3 times, preferably 2 times.

[0099] After salting out, the precipitate is washed by adding pure water, with the number of washes ranging from 2 to 5 times, preferably 3 times.

[0100] The target protein BD-3 solution purified in steps A to D can be prepared into a dry powder by freeze-drying or vacuum drying, or by directly spray-drying the concentrate into a dry powder.

[0101] The beneficial technical effects of the present invention are as follows.

[0102] 1. The protein of the present invention is the first obtained keratin, and the manufacturing method of the present invention is characterized by a high yield and high sample purity.

[0103] 2. The present invention demonstrated, through pharmacodynamic testing of protein BD-3 on a lipopolysaccharide (LPS)-induced SD rat fever model, that protein BD-3 has a significant effect in reducing the rise in body temperature after 2 and 4 hours of modeling; and through pharmacodynamic testing of protein BD-3 on a yeast-induced SD rat fever model, that protein BD-3 can significantly suppress the rise in body temperature after both 2 and 4 hours of modeling and has a strong effect.

[0104] 3. The present invention demonstrated, through pharmacodynamic studies of protein BD-3 on mouse seizures and epilepsy induced by the convulsant pilocarpine (PLO), that protein BD-3 can significantly extend the seizure latency period of grade III and grade IV epilepsy in mice.

[0105] 4. The present invention demonstrated that protein BD-3 has a significant expectorant effect through pharmacodynamic testing of protein BD-3 on expectoration by phenol red excretion method in mice.

[0106] 5. The present invention demonstrated that protein BD-3 has a significant antitussive effect by significantly extending the incubation period and significantly reducing the frequency of coughing through ammonia water cough induction in mice, through a pharmacodynamic study of protein BD-3.

[0107] 6. The present invention demonstrated that protein BD-3 has a significant analgesic effect by significantly reducing the number of squirming movements in mice through pharmacodynamic testing of protein BD-3 on acetic acid squirming in ICR mice. Brief explanation of the drawing

[0108] Figure 1 is a diagram of the reduced form of the expressed protein BD-3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. (M: Protein molecular weight standard; S: Expression protein BD-3) Figure 2 is a diagram illustrating the effect of protein BD-3 on a lipopolysaccharide (LPS)-induced rat fever model. (Comparison with normal control group, *** P<0.001; Comparison with model group, ## P<0.01, ### P<0.001) Figure 3 is a diagram illustrating the effect of protein BD-3 on a yeast-induced rat fever model. (Comparison with normal control group, ** P<0.01, *** P<0.001; Comparison with model group, # P<0.05, ## P<0.01, ### P<0.001) Specific details for implementing the invention

[0109] The following examples and pharmacological activity test examples are intended to further explain the present invention and are not intended to limit the present invention.

[0110] The experimental methods for the following examples and pharmacological activity test examples are conventional methods unless otherwise specified, and the experimental materials used were purchased from conventional biochemical reagent companies unless specifically noted otherwise.

[0111] Example 1Preparation of Protein BD-3 Solution A (TB Medium) via Shake Flask Fermentation

[0112] A nucleotide sequence denoted by SEQ ID No. 2 was synthesized and delivered to a pET-28a(+) vector; sequencing was performed to determine an expression vector containing the correct sequence; and the expression vector was transfected into BL21(DE3) cells to obtain expression competent host cells containing the target nucleotide sequence. These were added to LB medium and cultured in a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.

[0113] After immersing the recombinant strain and streaking it onto an LBA plate containing kanamycin, the plate was inverted and incubated overnight for 16 hours in a 37°C incubator.

[0114] 400 ml of TB medium was prepared and divided into two bottles, each containing 200 ml. Kanamycin (final concentration 50 μg / ml) was added to the TB medium in each bottle (200 ml). A single colony was taken from a plate, added to the TB medium, and then incubated overnight in a shaker at 37°C and 220 rpm to obtain the seed solution.

[0115] 28.8 L of TB medium was prepared and divided into 144 bottles, with 200 ml per bottle. After adding kanamycin (final concentration 50 μg / ml) to the TB medium in each bottle (200 ml), 2 ml of seed solution was added, and the mixture was incubated in a shaker at 37°C and 220 rpm for 2 to 3 hours. OD 600 By monitoring OD 600 When this reached about 1.0, an inducer was added to induce protein expression in a shaker. The induction conditions were selected from the table below.

[0116]

[0117] The bacterial suspensions from each bottle were combined and centrifuged at 7,000 rpm for 5 minutes, and the supernatant was removed after sterilization; the precipitate was suspended in approximately 3 L of buffer solution and filtered through an 80–100 mesh sieve, and the filtrate was crushed twice using a high-pressure crusher at a pressure of 800–1,000 bar for 2 minutes per cycle. The crushed bacterial suspension was centrifuged at 7,000 rpm for 30 minutes, and the supernatant was removed to obtain the precipitate (i.e., inclusion bodies). 1 L of detergent was added to the precipitate and washed twice, and the supernatant was removed by centrifugation. Urea solution was added to the precipitate again and dissolved four times in amounts of 800 ml, 600 ml, 400 ml, and 400 ml, respectively; the solutions were combined and centrifuged at 7,000 rpm for 30 minutes to remove the precipitate, and the supernatant is protein solution A.

[0118]

[0119] As a result of analyzing protein BD-3 solution A by reduced SDS-PAGE, the separation gel concentration was 12.5%, and when stained with Coomassie Brilliant Blue R250, a distinct blue band was observed around the molecular weight of 43 kD.

[0120] Example 2 Preparation of Protein BD-3 Solution B (Other Media) via Shake Flask Fermentation

[0121] In Example 1, an expression vector containing the sequence denoted by SEQ ID No. 2 was determined by synthesis and sequencing; the expression vector was transfected into BL21(DE3) cells to obtain an expression competent host cell containing the target nucleotide sequence.

[0122] 20 ml of LB medium was prepared, 800 μl was taken and added to 50 μl of host cells containing the target coding sequence, and cultured in a shaker at 37°C and 220 rpm for 1 hour.

[0123] After immersing the above bacterial solution and streaking it onto an LBA plate containing kanamycin, the plate was inverted and incubated overnight for 16 hours in a 37°C constant temperature incubator.

[0124] 10 ml of LB medium was taken and kanamycin (final concentration 50 μg / ml) was added. A single colony was taken from the plate and added to the LB medium, and then amplified overnight in a shaker at 37°C and 220 rpm for 15 hours to obtain a seed solution.

[0125] 1 L of medium was prepared as shown in the table below and divided into 10 bottles, with 100 ml per bottle. After adding kanamycin (final concentration 50 μg / ml) to the medium in each bottle (100 ml), 1 ml of seed solution was added, and the samples were incubated in a shaker at 37°C and 220 rpm for 2 to 3 hours. OD 600 By monitoring OD 600 When this level was reached about 1.0, the inducer IPTG (final concentration 0.5 mM) was added, and protein expression was induced in a shaker at 37°C and 220 rpm.

[0126]

[0127] The bacterial suspensions from each bottle were combined and centrifuged at 10,000 rpm for 10 minutes, and the supernatant was removed after sterilization; the precipitate was suspended in approximately 100 mL of buffer solution and filtered through an 80 to 100 mesh sieve, and the filtrate was crushed twice using a high-pressure crusher at a pressure of 800 to 1,000 bar for 2 minutes each time. The crushed bacterial suspension was centrifuged at 10,000 rpm for 30 minutes to remove the supernatant.

[0128] First, 40 mL of detergent buffer A was added to the precipitate and washed three times, and the supernatant was removed by centrifugation; 40 mL of detergent 2M urea solution was added to the precipitate and washed twice, and the supernatant was removed by centrifugation; 40 mL of 4M urea solution was added to the precipitate again and washed twice, and the supernatant was removed by centrifugation; 8M urea solution (including 50 mM Tris / HCl buffer) was added to the precipitate and dissolved three times in amounts of 40 mL, 30 mL, and 30 mL, respectively; the solutions were combined and centrifuged at 7000 rpm for 30 minutes to remove the precipitate, and the supernatant was the protein solution B.

[0129] As a result of reducing SDS-PAGE analysis of protein BD-3 solution B, the separation gel concentration was 12.5%, and when stained with Coomassie Brilliant Blue R250, a distinct blue band was observed around the molecular weight of 43 kD.

[0130] Example 3 Preparation of Protein BD-3 Solution C in a Fermentation Tank

[0131] In Example 1, an expression vector containing the sequence denoted by SEQ ID No. 2 was determined by synthesis and sequencing; the expression vector was transfected into BL21(DE3) cells to obtain expression competent host cells containing the target nucleotide sequence. After adding this to LB medium, the cells were cultured in a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.

[0132] 100 μl of the recombinant strain was added to an LBA plate containing kanamycin and spread with a spreader until uniformly dried. The plate was then inverted and incubated overnight in a 37°C incubator. Three single colonies were each taken and streaked onto a plate containing kanamycin. The plate was then incubated overnight. After confirming that shake flask fermentation and expression were correct over three batches, the strain was preserved in 15% glycerin and divided into 0.8 ml portions to obtain a working cell bank. This was then frozen and stored in a refrigerator at -80°C.

[0133] One glycerol bacterium was taken from a working cell bank, 100 μl was taken and added to 40 ml of LB medium, kanamycin (final concentration 50 μg / ml) was added, and the mixture was cultured in a shaker at 37°C and 220 rpm for 6 hours to obtain a Grade 1 seed solution.

[0134] 1.2 ml of Grade 1 seed solution was taken and 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 Grade 2 seed solution.

[0135] After adding the improved 3 L LB culture medium to a 5 L fermentation tank, 120 ml of Grade 2 seed solution and 3 ml of kanamycin (final concentration 50 μg / ml) were added, and the mixture was cultured for approximately 8 hours under conditions of 37°C and 30% dissolved oxygen (series rate). The OD value was monitored to approximately 20, and 3 g of lactose was used as an inducer to induce the culture at 20°C. Feeding was performed at a rate of 30 ml / hour, and the mixture was cultured at 20°C for 24 hours.

[0136] The bacterial suspension was centrifuged at 7,000 rpm for 5 minutes, and the supernatant was removed after sterilization; the precipitate was suspended in approximately 200 ml of buffer A and filtered through an 80-100 mesh sieve, and the filtrate was crushed twice using a high-pressure crusher at a pressure of 800-1,000 bar for 2 minutes each time. The crushed bacterial suspension was centrifuged at 7,000 rpm for 30 minutes to remove the supernatant.

[0137] The precipitate was washed twice by adding 1 L of 2M urea solution (containing 1% Triton) per wash; then, 1 L of 2M urea solution was added for one wash, and the supernatant was removed by centrifugation. 8M urea solution was added to the precipitate and dissolved four times in amounts of 400 ml, 300 ml, 200 ml, and 100 ml, respectively. The four solutions were combined and centrifuged at 7000 rpm for 30 minutes to remove the precipitate, and the supernatant was the protein solution C.

[0138] As a result of reducing SDS-PAGE analysis of protein BD-3 solution C, the separation gel concentration was 12.5%, and when stained with Coomassie Brilliant Blue R250, a distinct blue band was observed around the molecular weight of 43 kD.

[0139] Example 4 Protein BD-3 is prepared by dialyzing protein solution A.

[0140] Protein solution A obtained in Example 1 was filtered through a 0.45 μml filtration membrane, and the filtrate was combined. The filtrate was dialyzed with water, with a molecular weight cutoff of 10 kD in the dialysis bag, for 72 hours, and the liquid was freeze-dried to obtain the target protein BD-3; the purity measured by electrophoresis was 96.5%.

[0141] Structural confirmation of protein BD-3:

[0142] 1. Analysis by reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

[0143] Device: Protein electrophoresis (Bio-Rad).

[0144] Methods and Results: Analysis of the protein BD-3 solution by reduced SDS-PAGE showed a separation gel concentration of 12.5%, and staining with Coomassie Brilliant Blue R250 revealed that the molecular weight of the BD-3 band was approximately 43 kD.

[0145] 2. Whole protein sequence analysis based on LC-MS / MS

[0146] Main ingredients: Acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Asp-N;

[0147] Key Instruments: Capillary High Performance Liquid Chromatograph (Thermo Ultimate 3000 Type), Electron Spray Coupled Ion Trap Orbitrap Mass Spectrometer (Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer).

[0148] Method and Results:

[0149] Protein BD-3 was pretreated to obtain enzyme-cleaved peptides through lysis substitution, reductive alkylation, and various protein degradations; the enzyme-cleaved peptide solution was analyzed by liquid chromatography tandem mass spectrometry, and the original mass spectrometry file was analyzed by searching the protein database using Maxquant (1.6.2.10), and the identification results confirmed that it matched the target sequence SEQ ID No. 1.

[0150] Example 5 Protein BD-3 is prepared by purifying protein solution A by other methods.

[0151] Protein solution A obtained in Example 1 was purified by the following two methods.

[0152] First method: Salting out;

[0153] Protein crude solution A was placed in a stirred vessel and salted out twice as follows. Saturated ammonium sulfate solution was slowly added along the wall until the final ammonium sulfate concentration reached 25% or 50%, and the protein was precipitated during the salting-out process. After the salting-out was completed, 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 along the wall again until the final ammonium sulfate concentration reached 25% to perform the second salting-out. After filtration, the precipitate was the protein crude extract. The protein crude extract was washed three times with water as follows. After repeating the process of adding 200 ml of pure water, suspending, stirring, standing, and filtration three times, the precipitate was freeze-dried to obtain the target protein BD-3.

[0154] Second method: Column chromatography;

[0155] Protein solution A was purified using anion exchange resin columns such as HiTrap Q FF 16 / 10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, and HiTrap DEAE. The eluent was prepared using an NaCl gradient eluent, to which 20 mM NaH2PO4 / Na2HPO4 buffer (pH 8.0) was added. Each eluent fraction was detected by SDS-PAGE electrophoresis and combined. The combined eluent was centrifuged twice at 7000 rpm for 1 hour per cycle; the supernatant was filtered through a 0.45 μm membrane, and the filtrates were combined. The filtrate was concentrated by dialysis with water, with a molecular weight cutoff of 10 kD in the dialysis bag, and the liquid was freeze-dried to obtain the target protein BD-3.

[0156] The product protein BD-3 obtained by two methods was confirmed to have the same amino acid sequence as the protein prepared in Example 4 through the same structural confirmation method as in Example 4.

[0157] Example 6Protein BD-3 is prepared by purifying protein solution B.

[0158] Protein solution B obtained in Example 2 was purified by the following three methods.

[0159] First method: Dialysis;

[0160] Protein solution B was dialyzed with water through a 0.45 μm membrane for more than 72 hours, and the liquid was freeze-dried to obtain the target protein BD-3.

[0161]

[0162] Second method: Column chromatography;

[0163] Protein solution B was purified using anion exchange resin columns such as HiTrap Q FF 16 / 10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, and HiTrap DEAE. The eluent was prepared using an NaCl gradient eluent, to which 20 mM NaH2PO4 / Na2HPO4 buffer (pH 8.0) was added. Each eluent fraction was detected by SDS-PAGE electrophoresis and combined; the combined eluent was centrifuged at 7,000 rpm for 1 hour per cycle; the supernatant was filtered through a 0.45 μm membrane, and the filtrates were combined. The filtrate was concentrated by dialyzing with water, with a molecular weight cutoff of 10 kD in the dialysis bag, and the liquid was freeze-dried to obtain the target protein BD-3.

[0164] Third method: Salting out;

[0165] The protein crude solution was placed in a stirred vessel B and salted out twice as follows. Saturated ammonium sulfate solution was slowly added along the wall until the final ammonium sulfate concentration reached 25% or 50%, and the protein was precipitated during the salting-out process. After the salting-out was complete, 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 until the final ammonium sulfate concentration reached 25% to perform the second salting-out. After filtration, the precipitate was the crude protein extract. The crude protein extract was washed three times with water as follows. After repeating the process of adding 200 ml of pure water, suspending, stirring, standing, and filtration three times, the precipitate was freeze-dried to obtain the target protein BD-3.

[0166] The product protein BD-3 obtained by the three methods was confirmed to have the same amino acid sequence as the protein prepared in Example 4 through the same structural confirmation method as in Example 4.

[0167] Example 7 Protein BD-3 is prepared by purifying protein solution C.

[0168] The protein solution C obtained in Example 3 was purified by the following two methods.

[0169] First method: Microfiltration membrane technology;

[0170] Protein solution C was purified using microfiltration membrane technology as follows. First, solid-liquid separation was performed using a 1500 nm or 1000 nm ceramic membrane core; after removing the inner liquid, urea was removed from the outer liquid through repeated microfiltration using a 20 nm or 50 nm ceramic membrane core; and the inner liquid, microfiltered twice, was freeze-dried to obtain the target protein BD-3.

[0171] Second method: Salting out;

[0172] Protein crude solution C was placed in a stirred vessel and salted out twice as follows. Saturated ammonium sulfate solution was slowly added along the wall until the final ammonium sulfate concentration reached 25%, and the protein was precipitated during the salting-out process. After the salting-out was completed, 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 along the wall again until the final ammonium sulfate concentration reached 25% to perform the second salting-out. After filtration, the precipitate was the crude protein extract. The crude protein extract was washed three times with water as follows. After repeating the process of adding 200 ml of pure water, suspending, stirring, standing, and filtration three times, the precipitate was freeze-dried to obtain the target protein BD-3.

[0173] The product protein BD-3 obtained by two methods was confirmed to have the same amino acid sequence as the protein prepared in Example 4 through the same structural confirmation method as in Example 4.

[0174] Pharmacological test

[0175] Experimental Example 1 Pharmacodynamic test of protein BD-3 in a lipopolysaccharide (LPS)-induced SD rat fever model

[0176] Animal: Male SD rat weighing 230–260g;

[0177] Drugs: Lipopolysaccharide (LPS, SIGMA L-2880), Aspirin (SIGMA A2093), Protein BD-3;

[0178] Devices: Electronic scale (SARTORIUS BP121S type), electronic thermometer (CITIZEN CT-513W type).

[0179] Experimental Grouping:

[0180] Normal control group;

[0181] Model group: Lipopolysaccharide fever model;

[0182] Positive control group: Aspirin 300 mg / kg group;

[0183] Protein BD-3, 10 mg / kg group, 50 mg / kg group.

[0184] Method: Method of intraperitoneal injection of lipopolysaccharide to replicate rat fever model:

[0185] Preparation of experimental animals: After acclimatizing the experimental animals to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for one day, pre-acclimatization was performed for measuring rectal temperature at 8:00 AM and 15:00, respectively. The animals were fasted for 12 hours prior to the experiment, but water was not prohibited, and they were allowed to empty their feces before measuring rectal temperature. Before each temperature measurement, Vaseline was applied to the electronic thermometer probe and inserted 2 cm into the rat's rectum (a mark could be made at the 2 cm position to ensure consistent insertion depth each time), and the body temperature was recorded after the readings stabilized.

[0186] Intraperitoneal injection of lipopolysaccharide to replicate a rat fever model: Before modeling, rat body temperatures were measured to select eligible rats with temperatures ranging from 36.2 to 37.3°C, and they were randomly grouped into groups of 8. Immediately after oral administration of aspirin and different doses of protein BD-3, lipopolysaccharide (20 μg / kg, 2 ml / kg) was injected intraperitoneally, while the normal control group was injected intraperitoneally with an equal amount of physiological saline. Rat body temperatures were monitored starting 2 hours later and continued for a total of 8 hours.

[0187] Data Statistics:

[0188] Based on the body temperature values ​​measured at each time point on the day of the experiment, the mean body temperature, standard deviation, and standard error of each group of rats were calculated, and the data of each group were compared by group using TTEST, with P<0.05 considered a significant difference.

[0189] Experimental Results:

[0190] After orally administering aspirin (300 mg / kg) and protein BD-3 (10 mg / kg, 50 mg / kg), 20 μg / kg lipopolysaccharide was immediately injected intraperitoneally to model, and the body temperature of the animals was monitored at 2, 4, 6, and 8 hours after modeling, respectively. The results are shown in Table 1 and Figure 2.

[0191] By group N Basal body temperature (°C) Body temperature (°C) 2 hours after modeling Body temperature (°C) 4 hours after modeling Body temperature (°C) 6 hours after modeling Body temperature (°C) 8 hours after modeling Normal control group 8 36.9±0.1 36.7±0.1 36.8±0.05 36.6±0.05 36.7±0.03 Model group 8 36.8±0.1 37.6±0.1*** 37.8±0.1*** 37.8±0.2*** 37.8±0.2*** positive control group 8 36.9±0.1 36.8±0.1### 36.9±0.2### 36.8±0.2## 36.9±0.1### BD-3-10mg / kg 8 36.9±0.1 37.6±0.1 37.9±0.2 37.9±0.2 37.6±0.2 BD-3-50mg / kg 8 36.9±0.1 37.2±0.2# 37.3±0.2# 37.7±0.3 37.5±0.2

[0192] Effects of the test drug on a lipopolysaccharide (LPS)-induced rat fever model

[0193] (Comparison with normal control group, *** P<0.001; Comparison with model group, # P<0.05, ## P<0.01, ### P<0.001)

[0194] Experimental Conclusion:

[0195] The following can be learned from the results of modeling by immediately injecting 20 μg / kg of lipopolysaccharide into the peritoneal cavity after orally administering aspirin (300 mg / kg) and protein BD-3 (10 mg / kg, 50 mg / kg), respectively, and monitoring the body temperature of the animals 2, 4, 6, and 8 hours after modeling.

[0196] 1) Injecting 20 μg / kg lipopolysaccharide into the peritoneal cavity successfully induces an increase in body temperature in rats. The model group rats showed a significant increase in body temperature 2, 4, 6, and 8 hours after modeling, and compared to the normal group, there was a statistically significant difference with P<0.05, and the model is stable.

[0197] 2) The positive tool drug group, aspirin, can effectively suppress the rise in body temperature of model rats at 2, 4, 6, and 8 hours after modeling, and compared to the model group, there is a statistical difference with P<0.05, and the positive tool drug demonstrates stability.

[0198] 3) The group administered 50 mg / kg of protein BD-3 can reduce the body temperature of model rats 2 and 4 hours after modeling, and there is a statistical difference compared to the model group with P<0.05.

[0199] Experimental Example 2 Pharmacodynamic test of protein BD-3 (protein of Example 4) in a yeast-induced SD rat fever model

[0200] Animal: Male SD rat weighing 230–260g;

[0201] Drugs: Yeast (OXOID LP0021), Aspirin (SIGMA A2093), Protein BD-3;

[0202] Devices: Electronic scale (SARTORIUS BP121S type), electronic thermometer (CITIZEN CT-513W type).

[0203] Experimental Grouping:

[0204] Normal control group;

[0205] Model group: Yeast fever model;

[0206] Positive control group: Aspirin 300 mg / kg group;

[0207] Protein BD-3, 10 mg / kg group, 50 mg / kg group.

[0208] method:

[0209] Preparation of experimental animals: After acclimatizing the experimental animals to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for one day, pre-acclimatization was performed for measuring rectal temperature at 8:00 AM and 15:00, respectively. The animals were fasted for 12 hours prior to the experiment, but water was not prohibited, and they were allowed to empty their feces before measuring rectal temperature. Before each temperature measurement, Vaseline was applied to the electronic thermometer probe and inserted 2 cm into the rat's rectum (a mark could be made at the 2 cm position to ensure consistent insertion depth each time), and the body temperature was recorded after the readings stabilized.

[0210] Subcutaneous injection of dry yeast to replicate a rat fever model: Before modeling, rat body temperatures were measured, and eligible rats with a body temperature of 36.2–37.3°C were selected and randomly grouped into groups of 8. After oral administration of aspirin and different doses of protein BD-3, a 20% yeast suspension (10 ml / kg) was immediately injected subcutaneously, while the normal control group received an equal amount of physiological saline. After 2 hours, the rats' body temperatures were monitored once every 2 hours, and the rats were incubated for a total of 8 hours.

[0211] Data Statistics:

[0212] Based on the body temperature values ​​measured at each time point on the day of the experiment, the mean body temperature, standard deviation, and standard error of each group of rats were calculated, and the data of each group were compared by group using TTEST, with P<0.05 considered a significant difference.

[0213] Experimental Results:

[0214] After orally administering aspirin (300 mg / kg) and protein BD-3 (10 mg / kg, 50 mg / kg), 20% yeast was immediately injected subcutaneously to model, and the body temperature of the animals was monitored 2, 4, 6, and 8 hours after modeling, respectively. The results are shown in Table 2 and Figure 3.

[0215] By group N Basal body temperature (°C) Body temperature (°C) 2 hours after modeling Body temperature (°C) 4 hours after modeling Body temperature (°C) 6 hours after modeling Body temperature (°C) 8 hours after modeling Normal control group 8 36.6±0.1 36.6±0.1 36.7±0.1 36.6±0.1 36.5±0.04 Model group 8 36.7±0.1 37.5±0.1*** 37.7±0.1*** 37.7±0.1*** 37.6±0.1*** positive control group 8 36.7±0.1 37.0±0.1## 37.0±0.1### 37.0±0.1### 37.0±0.1### BD-3-10mg / kg 8 36.7±0.1 37.0±0.2## 37.3±0.1# 37.4±0.1 37.4±0.1 BD-3-50mg / kg 8 36.7±0.1 36.9±0.1## 37.2±0.1## 37.4±0.1 37.3±0.1

[0216] Effect of the test drug on a yeast-induced rat fever model

[0217] (Comparison with normal control group, ** P<0.01, *** P<0.001; Comparison with model group, # P<0.05, ## P<0.01, ### P<0.001)

[0218] Experimental Conclusion:

[0219] Modeling was performed by immediately injecting 20% ​​yeast subcutaneously after oral administration of aspirin (300 mg / kg) and protein BD-3 (10 mg / kg, 50 mg / kg), and the body temperature of the animals was monitored 2, 4, 6, and 8 hours after modeling, respectively. From the results, the following can be observed.

[0220] 1) In the model group rats, body temperature increased significantly 2, 4, 6, and 8 hours after modeling, and compared to the normal group, there was a statistical difference with P<0.05, and the model was successfully established and is also stable and reliable.

[0221] 2) The positive tool drug aspirin group can effectively suppress the rise in body temperature of model rats at 4, 6, and 8 hours after modeling, and compared to the model group, there is a statistical difference with P<0.05, and the positive tool drug aspirin demonstrates stability.

[0222] 3) Different doses of protein BD-3 can significantly suppress the rise in body temperature of model rats at both 2 and 4 hours after modeling, and there is a statistical difference with P<0.05 compared to the model group.

[0223] Experimental Example 3 Pharmacodynamic test of protein BD-3 (protein of Example 4) on mouse seizures and epilepsy induced by the convulsant pilocarpine (PLO)

[0224] Animal: Male ICR mouse;

[0225] Drugs: Pilocarpine HCl (PLO, pilocarpine, pilocarpine hydrochloride), Diazepam (diazepam tablets), protein BD-3.

[0226] Experimental Grouping:

[0227] Model group;

[0228] Diazepam 2 mg / kg group;

[0229] Protein BD-3, 50 mg / kg group, 200 mg / kg group.

[0230] method:

[0231] Model Preparation and Administration:

[0232] A single dose was administered on the afternoon of the day before modeling, and on the day of modeling, the test drug was administered gastrically, followed by an intraperitoneal injection of PLO-225 mg / kg (modeling agent) one hour later; the positive drug should be administered once 20 minutes prior to modeling. The patient was continuously observed for 30 minutes after the PLO injection.

[0233] Observation indicators: ① Epileptic seizure situation: Grade II to IV seizure duration; ② Time of death.

[0234] Seizure Grade: Refer to Racine Classification Standard: Grade 0: Unresponsive; Grade I: Presents as spasms of facial muscles or corners of the mouth; Grade II: Can nod head; Grade III: Spasms of one limb; Grade IV: Tonic or spasms of the entire limb; Grade V: Generalized epileptic grand mal seizure (generalized tonic seizure).

[0235] Data Processing:

[0236] In the experiment, the number of Grade IV seizures and deaths in mice of each group was statistically analyzed; the latency periods for Grade II, Grade III, and Grade IV mice, as well as the latency period for Grade IV mice without seizures, were recorded with a maximum value of 1800 seconds. The number of cases was statistically analyzed using the chi-square test. The mean and standard error of the latency periods were calculated, and the model group was compared to each other group using the TTEST, with P<0.05 considered a significant difference.

[0237] Experimental results: Shown in Tables 3 and 4.

[0238] By group Number of experimental cases Number of Class IV cases Grade IV seizure rate Number of death cases mortality rate Model group 10 8 80% 0 0 Diazepam 2 mg / kg 10 0** 0** 0 0 BD-3-50mg / kg 10 3 30% 0 0 BD-3-200mg / kg 10 6 60% 0 0

[0239] Experiment with test drugs for PLO-induced mouse epilepsy - Case count statistics

[0240] (Comparison with model group, * P<0.05, ** P<0.01)

[0241] By group Grade II seizure latency(s) Grade III seizure latency(s) Grade IV seizure latency(s) Model group 86±5 142±6 894±164 Diazepam 2 mg / kg 109±8* 182±14* 1800±0** BD-3-50mg / kg 91±6 155±12 1573±138** BD-3-200mg / kg 99±5 183±12** 1181±203

[0242] Experiment with test drugs on PLO-induced mouse epilepsy - Grade II, Grade III, and Grade IV epileptic seizure latency (mean±SEM)

[0243] (Comparison with model group, * P<0.05, ** P<0.01)

[0244] Experimental Conclusion:

[0245] 1) From the experimental results, it can be seen that the grade IV seizure rate of the model group was 80%, and that none of the 40 mice died.

[0246] 2) Positive drugs can completely suppress the rate of grade IV epileptic seizures and significantly extend the latency period of grade II, grade III, and grade IV epileptic seizures in mice.

[0247] 3) In the comparison of Grade III epilepsy latency periods, the BD-3 200 mg / kg administration group showed a statistical difference compared to the model group; in the comparison of Grade IV epilepsy latency periods, the BD-3 50 mg / kg administration group showed a statistical difference compared to the model group.

[0248] Experimental Example 4 Pharmacodynamic test of protein BD-3 (protein of Example 4) on pentylenetetrazole (PTZ)-induced mouse epilepsy

[0249] Animal: Male ICR mouse;

[0250] Drugs: Pentylenetetrazole (PTZ), Retigabin, Protein BD-3.

[0251] Experimental Grouping:

[0252] Model group;

[0253] Retigabine 60 mg / kg group;

[0254] Protein BD-3, 50 mg / kg group, 200 mg / kg group.

[0255] method:

[0256] Model Preparation and Administration:

[0257] A single dose was administered on the afternoon of the day before modeling, and on the day of modeling, the test drug was administered gastrically, followed by an intraperitoneal injection of PTZ-65 mg / kg (modeling agent) one hour later; the positive drug should be administered once half an hour before modeling. The patient was observed for 15 minutes after the PTZ injection.

[0258] Observation Indicators: ① Epileptic seizure situation: Grade III ~ VI; seizure duration; ② Death situation

[0259] Seizure Grade: Refer to Racine Classification Standard: Grade 0: Unresponsive; Grade I: Presents as spasms of facial muscles or corners of the mouth; Grade II: Can nod; Grade III: Spasms of one limb; Grade IV: Tonic or total limb spasms; Grade V: Generalized epileptic grand mal seizure (generalized tonic seizure).

[0260] Data Processing:

[0261] In the experiment, the number of seizure and death cases in mice of each group was statistically analyzed; the Grade III and Grade IV latency periods, as well as the latency period of Grade IV mice without seizures, were recorded with a maximum value of 900 seconds. The number of cases was statistically analyzed using the chi-square test. The mean and standard error of the latency periods were calculated, and the model group was compared with other groups using the TTEST, with P<0.05 considered a significant difference.

[0262] Experimental results are shown in Tables 5 and 6.

[0263] By group Number of experimental cases Number of IV surge cases Grade IV seizure rate Number of death cases mortality rate Model group 10 9 90% 1 10% Retigabine 60 mg / kg 10 2** 20%** 0 0 BD-3-50mg / kg 10 10 100% 1 10% BD-3-200mg / kg 10 7 70% 0 0

[0264] Experiment with Test Drugs on PTZ-Induced Mouse Epilepsy – Case Count Statistics

[0265] (Comparison with model group, * P<0.05, ** P<0.01)

[0266] By group Grade III seizure latency (seconds) Class IV seizure latency period (seconds) Model group 63±6 185±84 Retigabine 60 mg / kg 83±7* 745±103** BD-3-50mg / kg 75±6 162±58 BD-3-200mg / kg 62±5 327±125

[0267] Experiment with test drugs on PTZ-induced mouse epilepsy - Grade III and Grade IV epileptic seizure latency (mean±SEM)

[0268] (Comparison with model group, * P<0.05, ** P<0.01)

[0269] Experimental Conclusion:

[0270] 1) From the experimental results, it can be seen that the Grade IV seizure rate in the model group was 90%, with 2 out of 40 mice dying.

[0271] 2) Positive drugs can significantly suppress the rate of grade IV epilepsy seizures and significantly extend the latency period of grade III and grade IV epileptic seizures in mice.

[0272] 3) In the comparison of epilepsy grade IV latency periods, the BD-3 200 mg / kg administration group tended to prolong the latency period, but there was no statistical difference compared to the model group.

[0273] Experimental Example 5 Pharmacodynamic test of protein BD-3 (protein of Example 4) for expectoration by phenol red excretion method in mice

[0274] Animal: Male ICR mouse;

[0275] Drugs and Reagents: Mocosolban (Ambroxol Hydrochloride Tablets), Phenol Red, Sodium Bicarbonate, Protein BD-3;

[0276] Equipment: Centrifuge (Sigma-3K15 type), Balance (XS105DU type), Microplate Tester (BIO-TEK type).

[0277] Experimental Grouping:

[0278] Solvent control group;

[0279] Mocosolban 30 mg / kg group;

[0280] Protein BD-3, 20 mg / kg group, 50 mg / kg group.

[0281] method:

[0282] Model Preparation and Administration:

[0283] Animals were fasted for 16 hours prior to the experiment, but water was not restricted. Mocosolban and different dosages of protein BD-3 (administration volume 10 ml / kg) were orally administered to the groups, while the solvent control group was administered an equal amount of distilled water. One hour later, 2.5% phenol red solution was injected intraperitoneally, and 30 minutes later, the mice were sacrificed due to neck dislocation. The trachea was taken from below the thyroid cartilage through the anterior section of the tracheal branch, placed in 3 ml of 5% NaHCO3 solution, and left for 3 hours. 1 ml of the supernatant was taken, centrifuged at 3000 rpm for 5 minutes, and the absorbance was measured and recorded at 546 nm. The excretion amount of phenol red was calculated according to the standard curve of phenol red.

[0284] Data Processing:

[0285] The time of oral administration, intraperitoneal injection of 2.5% phenol red solution, and tracheal extraction were recorded, respectively; the absorbance of samples from each group was measured at 546 nm using a microplate tester, and the excretion amount of phenol red was calculated according to the standard curve of phenol red. The mean values ​​and standard errors of the data for each group were calculated, and the solvent control group was compared with each other using TTEST, with P<0.05 considered a significant difference.

[0286] Experimental Results:

[0287] Mocosolban (30 mg / kg) and different doses of protein BD-3 (20 mg / kg, 50 mg / kg) were administered. One hour later, a 2.5% phenol red solution was injected intraperitoneally, and the mice were sacrificed by neck dislocation 30 minutes later. The trachea was taken from below the thyroid cartilage through the anterior section of the tracheal branch, placed in 3 ml of 5% NaHCO3 solution, and left for 3 hours. 1 ml of the supernatant was taken, centrifuged at 3000 rpm for 5 minutes, and the absorbance was measured and recorded at 546 nm. The excretion amount of phenol red was calculated according to the standard curve of phenol red. The results are shown in Table 7.

[0288] By group N Phenol red excretion amount (μg / ml) P Solvent control group 10 0.506±0.040 --- Mocosolban 30 mg / kg 10 1.061±0.117** 0.001 BD-3-20mg / kg 10 0.750±0.085* 0.018 BD-3-50mg / kg 10 0.706±0.114 0.114

[0289] Pharmacodynamic experiment (X±SEM) of test drugs for expectoration by phenol red excretion method in mice

[0290] (Comparison with solvent control, * P<0.05, ** P<0.01)

[0291] Experimental Conclusion:

[0292] 1) From the experimental results, the 30 mg / kg mocosolban group showed a significantly increased phenol red excretion compared to the solvent control group, and with P<0.05, it has statistical significance.

[0293] 2) The BD-3 20 mg / kg administration group showed a significantly increased phenol red excretion compared to the solvent control group, and with P<0.05, it is statistically significant.

[0294] Experimental Example 6 Pharmacodynamic test of protein BD-3 (protein of Example 4) for antitussiveness induced by ammonia water cough induction in mice

[0295] Animal: Male ICR mouse;

[0296] Drugs and Reagents: Dextromethorphan hydrobromide, ammonia water, 0.2% CMC-Na, Protein BD-3;

[0297] Equipment: Compressor sprayer (Type 403T), Scale (Type XS105DU).

[0298] Experimental Grouping:

[0299] Solvent control group;

[0300] Dextromethorphan 15 mg / kg group;

[0301] Protein BD-3, 20 mg / kg group, 50 mg / kg group.

[0302] method:

[0303] Model Preparation and Administration:

[0304] Dextromethorphan and different dosages of protein BD-3 (administration volume 10 ml / kg) were orally administered to the groups, and the solvent control group was administered an equal amount of distilled water. After 1 hour, the mice were placed in a sealed box, sprayed, and then immersed in a 10% concentration ammonia solution for 10 seconds. The cough incubation period and the number of coughs within 2 minutes were then observed and recorded.

[0305] Data Processing:

[0306] The time of oral administration, the time of the nebulization experiment, the cough latency, and the number of coughs within 2 minutes in mice were recorded, respectively. The cough latency refers to the number of seconds required from the start of nebulization until a cough occurs. A mouse cough is defined as opening the mouth wide simultaneously with abdominal muscle contraction (chest contraction). The mean values ​​and standard errors of the data for each group were calculated, and the model group was compared with other groups using TTEST; a P<0.05 value was considered a significant difference.

[0307] Experimental Results:

[0308] Dextromethorphan (15 mg / kg) and different doses of protein BD-3 (20 mg / kg, 50 mg / kg) were administered beforehand. After 1 hour, the mice were placed in a sealed box, sprayed, and then immersed in 10% ammonia solution for 10 seconds. Subsequently, the cough incubation period and the number of coughs within 2 minutes were observed and recorded. The results are shown in Table 8.

[0309] By group N Incubation period (seconds) P Cough frequency P Solvent control group 9 26.9±2.3 --- 67.1±5.7 --- Dextromethorphan 15 mg / kg 9 38.4±4.5* 0.037 34.0±2.9** 0.001 BD-3-20mg / kg 9 31.6±3.3 0.258 46.7±4.1* 0.010 BD-3-50mg / kg 9 34.2±2.1* 0.032 53.6±3.7 0.063

[0310] Pharmacodynamic experiment (X±SEM) of test drugs for antitussiveness induced by ammonia water cough induction in mice

[0311] (Comparison with solvent control, * P<0.05, ** P<0.01)

[0312] Experimental Conclusion:

[0313] 1) From the experimental results, the dextromethorphan group showed a significant improvement in terms of incubation period and cough frequency compared to the solvent control group, and with P<0.05, it has statistical significance.

[0314] 2) The BD-3 50 mg / kg administration group showed a significant improvement in terms of latency period compared to the solvent control group, with P<0.05, which is statistically significant; the 20 mg / kg administration group showed a significant improvement in terms of cough frequency compared to the solvent control group, with P<0.05, which is statistically significant.

[0315] Experimental Example 7 Pharmacodynamic test of protein BD-3 (protein of Example 4) on ICR mouse acetic acid struggle

[0316] Animal: Male ICR mouse;

[0317] Drugs and Reagents: Aspirin, Physiological Saline, Glacial Acetate, Protein BD-3.

[0318] Experimental Grouping:

[0319] Model group;

[0320] Aspirin 300 mg / kg group;

[0321] Protein BD-3, 50 mg / kg group, 200 mg / kg group.

[0322] method:

[0323] After acclimating the experimental animals to the environment for one day, 300 mg / kg of aspirin and 50 mg / kg and 200 mg / kg of protein BD-3 were orally administered one hour prior, with a dosage of 10 ml / kg; subsequently, a 0.6% acetic acid solution was injected intraperitoneally, and the incubation period (seconds) and frequency of writhing in the animals were observed within 15 minutes.

[0324] Data Processing:

[0325] The mean and standard error of the data for each group were calculated and compared with the model group using TTEST, and with P<0.05, it was considered that there was a statistically significant difference.

[0326] Experimental Results:

[0327] One hour after orally administering 300 mg / kg of aspirin and different doses of protein BD-3 (50 mg / kg, 200 mg / kg), a 0.6% acetic acid solution was injected intraperitoneally, and the latency period and frequency of thrashing in ICR mice were observed. The results are shown in Table 9.

[0328] By group N Body weight (g) Struggle Incubation Period (Seconds) Number of struggles (times) Model group 0.6% acetic acid 18 22.8±0.2 204.2±19.6 33.3±2.5 Aspirin 300 mg / kg 13 23.2±0.2 290.3±37.0* 15.2±2.9*** BD-3-50mg / kg 13 23.2±0.3 221.5±28.4 24.7±3.0* BD-3-200mg / kg 13 23.5±0.3 189.8±10.4 29.5±4.7

[0329] Effect of the test drug on the ICR mouse acetic acid struggle test

[0330] (Comparison with model group, * P<0.05, ** P<0.01, *** P<0.001)

[0331] Experimental Conclusion:

[0332] By injecting a 0.6% acetic acid solution into the abdominal cavity of mice to induce deep, wide, and prolonged pain and stimulation, a struggle response (the abdomen contracts in an "S" shape, the trunk and hind legs are extended, and the hips are lifted and hunched) was elicited in the mice. The incubation period and frequency of the mice's struggle were used as pain response indicators to determine whether the test sample had an analgesic effect. The following can be observed from the results of this experiment.

[0333] 1) Aspirin 300 mg / kg can significantly delay the latency period of thrashing and reduce the frequency of thrashing, thus having a consistent analgesic effect, and is statistically significant with P<0.05 compared to the model group.

[0334] 2) The BD-3 50 mg / kg administration group can significantly reduce the number of mouse struggles, and compared to the model group, it has statistical significance with P<0.05.

Claims

Claim 1 A pharmaceutical composition for use in antipyretic, analgesic, antitussive, expectorant, anticonvulsant, or antiepileptic therapy, comprising an effective amount of keratin BD-3 and a pharmaceutically acceptable carrier or excipient, wherein the amino acid sequence of keratin BD-3 is represented by SEQ ID NO:

1. Claim 2 A pharmaceutical composition for use in antipyretic, analgesic, antitussive, expectorant, anticonvulsant, or antiepileptic therapy, comprising an effective amount of a nucleic acid molecule encoding keratin BD-3 and a pharmaceutically acceptable carrier or excipient, wherein the amino acid sequence of keratin BD-3 is represented by SEQ ID NO:

1. Claim 3 A pharmaceutical composition according to paragraph 2, wherein the nucleotide sequence of the nucleic acid molecule is represented by SEQ ID NO:

2. Claim 4 A pharmaceutical composition for use in antipyretic, analgesic, antitussive, expectorant, anticonvulsant, or antiepileptic therapy, comprising an expression vector containing an effective amount of a nucleic acid molecule encoding keratin BD-3 and a pharmaceutically acceptable carrier or excipient, wherein the amino acid sequence of keratin BD-3 is represented by SEQ ID NO:

1. Claim 5 A pharmaceutical composition for use in antipyretic, analgesic, antitussive, expectorant, anticonvulsant, or antiepileptic therapy, comprising an effective amount of host cells expressing keratin BD-3 and a pharmaceutically acceptable carrier or excipient, wherein the amino acid sequence of keratin BD-3 is represented by SEQ ID NO:

1. Claim 6 A pharmaceutical composition according to claim 5, wherein the host cell is selected from the group consisting of bacteria, yeast, Aspergillus, plant cells, and insect cells. Claim 7 A pharmaceutical composition in which, in paragraph 6, the bacteria is E. coli. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete

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