Keratin CF2, its manufacturing method, pharmaceutical composition containing it, and its use

Abstract

The present invention relates to the pharmaceutical technology. Keratin CF2, methods for producing the same, pharmaceutical compositions containing the same and the use thereof are disclosed, and the present invention particularly relates to keratin CF2, nucleic acid molecules encoding the keratin CF2, expression vectors containing the nucleic acid molecule, host cells containing the expression vector or in which the nucleic acid molecule is incorporated into the genome, methods for producing the keratin CF2, pharmaceutical compositions containing the keratin CF2, and the use of the keratin CF2, nucleic acid molecules, expression vectors, host cells or pharmaceutical compositions in the manufacture of antipyretics, analgesics, antitussives, expectorants, anticonvulsants, antiepileptics, antihypertensives, anti-inflammatorys or antivirals.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and relates to keratin CF2, a nucleic acid molecule encoding the keratin CF2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or in which the nucleic acid molecule is integrated into the genome, a method for producing the keratin CF2, a pharmaceutical composition containing the keratin, and the use of the keratin and the pharmaceutical composition in the production of antipyretic agents, analgesics, antitussives, expectorants, antispasmodics, antiepileptic drugs, antihypertensive drugs, anti-inflammatory drugs and antiviral drugs.

Background Art

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

[0003] Keratin is a renewable resource that is widely present in organisms and has high utilization value, but it has not been widely and effectively utilized. The main reason is that keratin is insoluble in various solvents and generally has higher resistance to proteolytic degradation than 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, more and more genes have been identified, and the use of protein expression systems for manufacturing and producing target proteins is an important method for studying the biological functions of genes or proteins.

[0005] After manufacturing the target keratin, there is no literature reporting the use of a protein expression system for studying its structure and function, and it is obvious that it has novelty and progressiveness.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The technical problems to be solved by the present invention are to provide keratin CF2, a nucleic acid molecule encoding the keratin CF2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or in which the nucleic acid molecule is incorporated into the genome, a method for producing the keratin CF2, a pharmaceutical composition containing the keratin CF2, and the use of the above-mentioned keratin CF2, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the production of antipyretics, analgesics, antitussives, expectorants, anticonvulsants, antiepileptics, antihypertensives, anti-inflammatory drugs, or antiviral drugs. [Means for solving the problem]

[0007] To solve the technical problems of the present invention, the present invention provides the following technical solutions.

[0008] A first aspect of the technical solution of the present invention is to provide keratin CF2, wherein the amino acid sequence of the keratin CF2 is (1) The amino acid sequence shown in Sequence ID No. 1 in the sequence listing, (2) The amino acid sequence is characterized by having 1 to 35 amino acids substituted, deleted, or added to the amino acid sequence shown in Sequence ID No. 1 in the sequence listing, and substantially maintaining the same biological function as keratin CF2.

[0009] Furthermore, the keratin CF2 may have conventional modifications, or the keratin CF2 may be further linked to a tag for detection or purification.

[0010] Furthermore, the above conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorophore group modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamination, disulfide bond formation, or disulfide bond cleavage, and the above tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

[0011] A second aspect of the technical solution of the present invention provides a nucleic acid molecule encoding keratin CF2 according to the first aspect.

[0012] Furthermore, the nucleotide sequence of the above nucleic acid molecule is, (1) The nucleotide sequence shown in Sequence ID No. 2 in the sequence listing, (2) A nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in Sequence ID No. 2, (3) A nucleotide sequence that is complementary to the nucleotide sequence of (1) or (2) above.

[0013] A third aspect of the technical solution of the present invention provides an expression vector characterized by comprising the nucleic acid molecule described in the second aspect.

[0014] 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(+).

[0015] A fourth aspect of the technical solution of the present invention provides a host cell characterized by comprising an expression vector of the third aspect or by having a nucleic acid molecule of the second aspect incorporated into the genome.

[0016] Furthermore, the host cells mentioned above are selected from the group consisting of bacteria, yeast, Aspergillus, plant cells, and insect cells.

[0017] Furthermore, the bacteria mentioned above are either E. coli or yeast.

[0018] Competent host cells may include the BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Origami series, Trans1-T1, TG1, TB1, Y11430, MG1003, GS115(AOX1), KM71, SMD1168, etc., with preferred expression competent cells being BL21(DE3) or Transetta(DE3).

[0019] A fifth aspect of the technical solution of the present invention provides a method for producing keratin CF2 according to the first aspect, characterized by comprising the following steps.

[0020] A. A nucleic acid molecule corresponding to keratin CF2 as described in the first embodiment is synthesized, the nucleic acid molecule is linked to an appropriate expression vector, the expression vector is transformed into host cells, the host cells holding the expression vector are cultured in a fermentation apparatus under specific conditions to induce the expression of keratin CF2 and obtain a crude protein solution containing keratin CF2.

[0021] B. The crude protein solution obtained in step A is separated and purified, then dried to obtain the keratin CF2.

[0022] Furthermore, in step A, the host cells are mainly selected from E. coli, the keratin CF2 is expressed as an inclusion body in E. coli, and the fermentation apparatus includes a shaking flask or a fermenter.

[0023] Furthermore, in step A, after inducing the expression of keratin CF2, impurities may be washed away with a washing solution, and then dissolved in urea solution to obtain a crude protein solution. Furthermore, the culture medium in step A may be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Rose Bengal medium, high-salt Zapek medium, DOBA medium, rice koji medium, and modified formulations thereof. Shaking flask fermentation is preferably performed using LB medium or TB medium, with TB medium being the most preferred medium. Fermentation vessels are preferably LB medium and modified formulations thereof.

[0024] Furthermore, the inducer in Step A may be IPTG, lactose, arabinose, etc., with IPTG or lactose being preferred.

[0025] Furthermore, in Step A, after centrifuging the obtained fermentation broth, the supernatant is discarded, the precipitate is suspended in a buffer, the cells are disrupted, centrifuged again, the supernatant is discarded, and after washing the precipitate with a washing solution, it is dissolved in a urea solution to obtain a crude protein solution of CF2.

[0026] Among them, the above buffer is preferably Buffer A, and its dosage is fermentation broth volume:Buffer A volume = 1 - 100:1, preferably 10:1.

[0027] The above washing solution may be a urea solution, guanidine hydrochloride solution, Triton, and Buffer A, etc., preferably a urea solution, most preferably a 2M urea solution (which may contain 1% Triton) and a 4M urea solution. The dosage is fermentation broth volume:urea solution volume = 0.2 - 100:1, preferably 1 - 15:1.

[0028] The above urea solution is preferably a 4M - 8M urea solution, most preferably an 8M urea solution, and its dosage is fermentation broth volume:8M urea volume = 0.2 - 100:1, preferably 2 - 15:1.

[0029] Furthermore, in Step B, examples of the above separation and purification methods include purification methods using ultrafiltration and microfiltration membrane technology, column chromatography purification methods, salting - out methods, and dialysis methods.

[0030] Furthermore, in Step B, the above separation and purification method is as follows.

[0031] (1) The above dialysis method means purifying the crude protein solution obtained in Step A by dialysis to obtain a target protein CF2 solution.

[0032] The molecular weight cutoff for the dialysis bag may 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.

[0033] (2) In the ultrafiltration / microfiltration method described above, the crude protein solution obtained in step A is purified by membrane technology such as an ultrafiltration membrane or a microfiltration membrane to obtain a concentrated solution of the target protein CF2.

[0034] (3) The above column chromatography method involves passing the crude protein solution obtained in step A through various exchange columns or exclusion column chromatography to separate and purify the target protein CF2.

[0035] Preferred exclusion columns include dextran gel columns, SUPERDEX 30 INCREASE, SUPERDEX 75 INCREASE, SUPERDEX 200 INCREASE, and SUPEROSE 6 INCREASE. Preferred exchange columns are ion exchange resin columns, including anion exchange resin columns such as HITRAP Q FF, HITRAP CAPTO Q ImpRes, CAPTO Q ImpRes, HITRAP CAPTO Q, HITRAP DEAE, TOYOPEARL Q-650M, and TOYOPEARL SUPER Q-650M, and cation exchange resin columns such as HITRAP SP FF, HITRAP CAPTO SP ImpRes, Capto SP ImpRes, HITRAP CAPTO SP, TOYOPEARL SP-650M, and TOYOPEARL SUPER SP-650M. The most preferred is anion exchange resin column.

[0036] As the eluent, water or a salt solution, or any other eluent commonly used in this field, may be used. Examples of the salt solution include sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, and acetic acid.

[0037] (4) The above salting-out method involves purifying the crude protein solution obtained in step A by salting-out to obtain a suspension of the target protein CF2.

[0038] The salting-out agent may be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc. The preferred salting-out agent is ammonium sulfate and its aqueous solution. A saturated aqueous solution of ammonium sulfate is added so that the final concentration of ammonium sulfate is 10-50%, preferably 20-30%, and more preferably 25%.

[0039] The number of salting-out steps is 1 to 3, preferably 2.

[0040] After salting out, the precipitate is washed with pure water, preferably 2 to 5 times, and more preferably 3 times.

[0041] Furthermore, the target protein CF2 solution purified in step B may be freeze-dried or vacuum-dried to obtain a dry powder, or the concentrated solution may be directly spray-dried to obtain a dry powder.

[0042] A sixth aspect of the technical solution of the present invention provides a pharmaceutical composition characterized by comprising keratin CF2 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 excipient.

[0043] The keratin obtained in the above steps of the present invention may be freeze-dried or vacuum-dried to obtain a dry powder, or the concentrated liquid may be directly spray-dried to obtain a dry powder and then made into various dosage forms.

[0044] The present invention relates to a pharmaceutical composition comprising any keratin obtained in the above process and a pharmaceutically acceptable carrier.

[0045] The present invention also relates to a pharmaceutical composition comprising the keratin of the present invention as an active ingredient and a conventional pharmaceutical excipient or adjuvant. Generally, the keratin of the present invention accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.

[0046] The present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of protein as an active ingredient and a pharmaceutically acceptable carrier.

[0047] The pharmaceutical compositions of the present invention can be manufactured according to methods recognized in the art. When used for this purpose, the proteins of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants as needed to produce suitable dosage forms or formulations that can be used as human or veterinary pharmaceutical forms.

[0048] The keratin or pharmaceutical composition containing the same of the present invention may be administered in unit dosage form. The route of administration may be enteral or parenteral, for example, oral administration, intramuscular administration, subcutaneous administration, nasal administration, oral mucosal administration, ocular administration, pulmonary administration, cutaneous administration, vaginal administration, peritoneal administration, and rectal administration, with oral administration being preferred.

[0049] The keratin protein of the present invention or a pharmaceutical composition containing the same may be administered by injection. Examples of such injections include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, and acupuncture injection.

[0050] The dosage form for administration may be a liquid, solid, or semi-solid form. The liquid dosage form may be a solution (such as a true solution and a colloidal solution), an emulsion (such as an oil-in-water, water-in-oil, and double emulsion), a suspension, an injection (such as a water injection, a powder injection, and an infusion), eye drops, nasal drops, lotions, and liniments. The solid dosage form may be a tablet (such as a regular tablet, an enteric-coated tablet, an oral tablet, a dispersible tablet, a chewable tablet, an effervescent tablet, orally disintegrating tablet), a capsule (such as a hard capsule, a soft capsule, or enteric-coated capsule), a granule, a powder, a pellet, a dropper pill, a suppository, a film, a patch, an aerosol (powder), or a spray. The semi-solid dosage form may be an ointment, a gel, a paste, or the like.

[0051] The keratin of the present invention can be used in conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various particle delivery systems.

[0052] To form the unit dosage form into tablets, a wide variety of excipients known in the art, such as diluents, binders, humectants, disintegrants, lubricants, and flow enhancers, can be widely used. The above diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc. The above humectant may be water, ethanol, isopropanol, etc. The above binder may be corn syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic, gelatin syrup, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc. The above disintegrant may be dried starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc. The above lubricant and flow promoter may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0053] The above tablets can also be made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, bilayer tablets, or multilayer tablets.

[0054] Various carriers known in this field can be widely used to form the dosage units into pills. Examples of the above carriers include, for example, as diluents and absorbents, glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol laurate, kaolin, talc, etc.; as binders, acacia gum, xanthan gum, gelatin, ethanol, honey, liquid sugar, rice paste, or wheat paste, etc.; and as disintegrants, agar powder, dried starch, alginate, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.

[0055] To prepare the dosage unit as a suppository, a wide variety of carriers known in this field can be widely used. Examples of such carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides.

[0056] To prepare the dosage unit as a capsule, the active ingredient keratin of the present invention is mixed with the various carriers described above, and the resulting mixture is then filled into a hard gelatin capsule or a soft capsule. Alternatively, the active ingredient keratin may be formulated as a microcapsule and suspended in an aqueous medium to form a suspension, or filled into a hard capsule, or prepared as an injectable preparation for administration.

[0057] For example, the keratin of the present invention may be prepared as an injectable formulation, such as a solution, suspension, emulsion, or lyophilized powder injection. Such formulations may be aqueous or non-aqueous and may contain one and / or more pharmacodynamically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. For example, the diluent may 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 may be added to the injectable formulation to produce an isotonic injection. Conventional solubilizers, buffers, pH adjusters, etc., may also be added. These auxiliary materials are commonly used in the art.

[0058] Furthermore, colorants, preservatives, fragrances, flavorings, sweeteners, or other ingredients may be added to the pharmaceutical preparation as needed.

[0059] To achieve the drug administration objective and enhance the therapeutic effect, the keratin or pharmaceutical composition of the present invention can be administered by known administration methods.

[0060] The dosage of the keratin pharmaceutical composition of the present invention can vary widely because it depends on many factors, including the nature and severity of the disease being 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 therapeutic objective. Generally speaking, the dosage of the pharmaceutical component of the present invention is well known to those skilled in the art. Depending on the actual amount of drug contained in the final formulation of the keratin composition of the present invention, it can be appropriately adjusted to meet the requirements for a therapeutically effective dose and achieve the preventive or therapeutic objective of the present invention. Appropriate daily dose range for keratin of the present invention: The dose 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 dose may be administered in a single dose or divided into multiple doses, for example, two, three or four doses, depending on the clinical experience of the administering physician and the administration plan, including the use of other treatments. The total dose required for each treatment may be divided into multiple doses or administered as a single dose. The protein or pharmaceutical composition of the present invention may be taken alone or in combination with other therapeutic or symptomatic agents to adjust the dosage.

[0061] A seventh aspect of the technical solution of the present invention provides the use of keratin CF2 according to the first aspect, nucleic acid molecules according to the second aspect, expression vectors according to the third aspect, host cells according to the fourth aspect, or pharmaceutical compositions according to the sixth aspect in the manufacture of antipyretics, analgesics, antitussives, expectorants, anticonvulsants, antiepileptics, antihypertensives, anti-inflammatorys, or antiviral drugs.

[0062] To achieve the objectives of the present invention, the present invention provides the following technical solutions. Specifically, the production of keratin CF2 according to the present invention includes the following steps.

[0063] (1) Synthesize the nucleotide sequence and determine the accuracy of the above sequence. A preferred nucleotide sequence is shown in Sequence ID No. 2.

[0064] (2) The above nucleotide sequence is transferred into the expression vector. 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(+).

[0065] (3) Transfect the host cells with the expression vector described above. The host cell may be E. coli or yeast, with E. coli being the preferred host cell. Competent cells may include the BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Origami series, Trans1-T1, TG1, TB1, Y11430, MG1003, GS115(AOX1), KM71, SMD1168, etc. Preferred competent expression cells are BL21(DE3) or Transetta(DE3).

[0066] (4) The host cells described above are cultured under appropriate conditions to induce the expression of the target protein CF2. The fermentation apparatus can be a shaking flask or a fermenter. The culture medium may be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Rose Bengal medium, high-salt Zapek medium, DOBA medium, rice koji medium, and modified formulations thereof. Shaking flask fermentation is preferably performed using LB medium or TB medium, with TB medium being the most preferred medium. Fermentation vessels are preferably LB medium and modified formulations thereof.

[0067] The inducing substance may be IPTG, lactose, arabinose, etc., and is preferably IPTG or lactose.

[0068] (5) Enrich the target protein CF2 product. The fermentation liquid obtained in step (4) is centrifuged, the supernatant is discarded, the precipitate is suspended in buffer solution, the microbial cells are then crushed, then centrifuged again, the supernatant is discarded, the precipitate is washed with washing solution, and then dissolved in urea solution to obtain a crude CF2 protein solution.

[0069] In this context, the buffer solution is preferably Buffer A, and its dosage is such that the ratio of the volume of the fermentation liquid to the volume of Buffer A is 1 to 100:1, preferably 5 to 10:1.

[0070] The washing solution may be urea solution, guanidine hydrochloride solution, Triton, Buffer A, etc., preferably urea solution, most preferably 2M urea solution (which may contain 1% Triton). The dosage is fermentation broth volume : urea solution volume = 0.2 to 100:1, preferably 1 to 15:1. The washing procedure is performed 3 to 10 times, preferably 6 times.

[0071] The urea solution is preferably a 4M to 8M urea solution, most preferably an 8M urea solution. The dosage is such that the volume of the fermentation liquid : the volume of the 8M urea solution = 0.2 to 100:1, preferably 2 to 15:1.

[0072] (6) The target protein CF2 is isolated and purified. The crude protein solution obtained in step (5) needs to be purified to obtain the target protein CF2. Purification may be carried out by dialysis, ultrafiltration / microfiltration, column chromatography, or salting out.

[0073] A. In the dialysis process, the crude protein solution obtained in step (5) is purified by dialysis to obtain the target protein CF2 solution.

[0074] The molecular weight cutoff for the dialysis bag may 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.

[0075] B. In the ultrafiltration / microfiltration step, the crude protein solution obtained in step (5) is purified using membrane technology such as an ultrafiltration membrane or a microfiltration membrane to obtain a concentrated solution of the target protein CF2.

[0076] Preferably, microfiltration membrane purification is performed twice, with a membrane pore size of 1000-1500 nm for the first filtration and a membrane pore size of 20-50 nm for the second filtration.

[0077] C. In the column chromatography step, the crude protein solution obtained in step (5) is passed through various exchange columns or exclusion column chromatography to separate and purify the target protein CF2.

[0078] Preferred exclusion columns include dextran gel columns, SUPERDEX 30 INCREASE, SUPERDEX 75 INCREASE, SUPERDEX 200 INCREASE, and SUPEROSE 6 INCREASE. Preferred exchange columns are ion exchange resin columns, including anion exchange resin columns such as HITRAP Q FF, HITRAP CAPTO Q ImpRes, CAPTO Q ImpRes, HITRAP CAPTO Q, HITRAP DEAE, TOYOPEARL Q-650M, and TOYOPEARL SUPER Q-650M, and cation exchange resin columns such as HITRAP SP FF, HITRAP CAPTO SP ImpRes, CAPTO SP ImpRes, HITRAP CAPTO SP, TOYOPEARL SP-650M, and TOYOPEARL SUPER SP-650M. The most preferred is anion exchange resin column.

[0079] As the eluent, water or a salt solution, or any other eluent commonly used in this field, may be used. Examples of salt solutions include sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, and acetic acid.

[0080] D. In the salting-out step, the crude protein solution obtained in step (5) is purified by salting-out to obtain a suspension of the target protein CF2.

[0081] The salting-out agent may be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc. The preferred salting-out agent is ammonium sulfate and its aqueous solution. A saturated aqueous solution of ammonium sulfate is added so that the final concentration of ammonium sulfate is 10-50%, preferably 20-30%, and more preferably 25%.

[0082] The number of salting-out steps is 1 to 3, preferably 2.

[0083] After salting out, the precipitate is washed with pure water, preferably 2 to 5 times, and more preferably 3 times.

[0084] The target protein CF2 solution purified in steps A to D may be freeze-dried or vacuum-dried to obtain a dry powder, or the concentrated solution may be directly spray-dried to obtain a dry powder.

[0085] Beneficial technical effects of the present invention: 1. The protein of the present invention is the first keratin obtained, and the production method of the present invention is characterized by high yield and high purity.

[0086] 2. The present invention demonstrated, through pharmacodynamic testing of protein CF2 in a yeast-induced fever model of SD rats, that protein CF2 significantly reduces the rise in body temperature of the model animals 8 hours after modeling. Furthermore, the present invention demonstrated, through pharmacodynamic testing of protein CF2 in a lipopolysaccharide (LPS)-induced fever model of SD rats, that protein CF2 significantly reduces the body temperature of the model rats 2 hours after modeling. 3. The present invention demonstrates, through pharmacodynamic studies of protein CF2 in a mouse pentylenetetrazole (PTZ)-induced epilepsy model, that protein CF2 can significantly prolong the latency of grade III epileptic seizures in mice. [Brief explanation of the drawing]

[0087] [Figure 1] Effect of protein CF2 on a rat lipopolysaccharide (LPS)-induced fever model. (**P<0.01 compared to the normal control group; #P<0.05, ##P<0.01 compared to the model group). [Figure 2] Effect of protein CF2 on a rat yeast-induced fever model. (**P<0.01 compared to the normal control group; #P<0.05, ##P<0.01 compared to the model group). [Modes for carrying out the invention]

[0088] The following examples and pharmacological activity test examples are used to further illustrate the present invention, but do not imply any limitation to the present invention.

[0089] The experimental methods in the following examples and pharmacological activity test examples are conventional unless otherwise specified, and the experimental materials used are purchased from conventional biochemical reagent companies unless otherwise specified.

[0090] Example 1: Preparation of crude protein CF2 solution A (TB medium) by shaking flask fermentation The nucleotide sequence shown in Sequence ID No. 2 was synthesized, transferred into a pET-28a(+) vector, and the sequence was verified to obtain an expression vector containing the correct sequence. This expression vector was transfected into BL21(DE3) cells to obtain competent host cells expressing the target nucleotide sequence. LB medium was added, and the cells were incubated in a shaker at 37°C and 220 rpm for 1 hour to obtain recombinant cells.

[0091] Recombinant strains were streaked onto LBA plates containing kanamycin, and the plates were inverted and incubated overnight for 16 hours in a 37°C incubator.

[0092] 400 mL of TB medium was prepared and dispensed into two bottles, 200 mL each. Kanamycin (final concentration 50 μg / mL) was added to the TB medium (200 mL) in each bottle. A single colony was taken from a plate and added to the TB medium. The cells were cultured overnight at 37°C and 220 rpm to obtain a seed solution in a shaker.

[0093] 28.8 L of TB medium was prepared and dispensed into 144 bottles, each containing 200 mL. Kanamycin (final concentration 50 μg / mL) was added to each bottle of TB medium (200 mL), followed by the addition of 2 mL of seed solution. The mixture was incubated in a shaker at 37°C and 220 rpm for 2-3 hours. OD 600 Monitor and OD 600 When the value reached approximately 1.0, an inducing substance was added to induce protein expression in a shaker, and the induction conditions were selected from the table below.

[0094] [Table 1]

[0095] The bacterial suspensions from each bottle were combined and centrifuged at 7000 rpm for 5 minutes. After sterilization, the supernatant was discarded, and the precipitate was suspended in approximately 3 L of buffer solution. The precipitate was filtered through an 80-100 mesh sieve, and the filtrate was crushed twice in a high-pressure crusher at a pressure of 800-1000 bar for 2 minutes each time. The crushed bacterial suspension was centrifuged at 7000 rpm for 30 minutes, and the supernatant was discarded to obtain the precipitate (i.e., inclusion bodies). The precipitate was washed with washing solution (three times with 1 L of 2 M urea-1% Triton solution, followed by two times with 1 L of 3 M urea solution), centrifuged, and the supernatant was discarded. The precipitate was then dissolved in 1 L of 8 M urea solution to obtain crude protein solution A.

[0096] Example 2 Preparation of crude protein CF2 solution B (other medium) by shaking flask fermentation In Example 1, synthesis and sequencing were performed to confirm that an expression vector containing the sequence shown in SEQ ID NO: 2 was obtained. The expression vector was transfected into Transetta(DE3) cells to obtain competent host cells expressing the target nucleotide sequence.

[0097] 20 mL of LB medium was prepared, 800 μL was taken, 50 μL of host cells containing the target coding sequence were added, and the mixture was incubated in a shaker at 37°C and 220 rpm for 1 hour.

[0098] The above bacterial suspension was taken, streaked onto an LBA plate containing kanamycin, and the plate was inverted and incubated overnight for 16 hours in a 37°C incubator.

[0099] 10 mL of LB medium was taken, kanamycin (final concentration 50 μg / mL) was added, a single colony was taken from the plate and added to the LB medium. The cells were cultured overnight at 37°C and 220 rpm for 15 hours, and a seed solution was obtained in a shaker.

[0100] Prepare 1 L of the culture medium shown in the table below and dispense 100 mL into 10 bottles. Add kanamycin (final concentration 50 μg / mL) to the culture medium (100 mL) in each bottle, then add 1 mL of seed solution. Incubate in a shaker at 37°C and 220 rpm for 2-3 hours. OD 600 Monitor and OD 600 When the pH reached approximately 1.0, the inducing agent IPTG (final concentration 0.5 mM) was added. Protein expression was induced in a shaker at 37°C and 220 rpm.

[0101] [Table 2]

[0102] The bacterial suspensions from each bottle were combined and centrifuged at 10,000 rpm for 10 minutes. After sterilization, the supernatant was discarded, the precipitate was suspended in approximately 100 mL of buffer solution, filtered through an 80-100 mesh sieve, and the filtrate was crushed twice in a high-pressure crusher at a pressure of 800-1000 bar for 2 minutes each time. The crushed bacterial suspension was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was discarded.

[0103] The precipitate was first washed with 40 mL of washing solution Buffer A, followed by centrifugation, and the supernatant was discarded. Next, 40 mL of washing solution 2 M urea solution (containing 1% Triton) was added to the precipitate and washed twice, centrifugation being performed each time, and the supernatant discarded. Then, 40 mL of washing solution 3 M urea solution was added to the precipitate and washed twice, centrifugation being performed each time, and the supernatant discarded. Finally, 40 mL of washing solution 8 M urea solution was added to the precipitate to obtain crude protein solution B.

[0104] Example 3 Preparation of crude protein CF2 solution C in a fermenter In Example 1, synthesis and sequencing were performed 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 containing the target nucleotide sequence. LB medium was added, and the cells were incubated in a shaker at 37°C and 220 rpm for 1 hour to obtain recombinant cells.

[0105] 100 μL of recombinant strain was added to an LBA plate containing kanamycin, spread with a spreader until uniformly dry, and the plate was inverted and incubated overnight in a 37°C incubator. Three single colonies were taken and streaked onto a plate containing kanamycin, and the plate was incubated overnight. After verifying the correctness of expression by three batches of shaking flask fermentation, the strain was stored in 15% glycerol, aliquoted into 0.8 mL portions to obtain a working cell bank, which was stored in a -80°C refrigerator for later use.

[0106] One glycerol stock was taken from the working cell bank, 100 μL was taken, and added to 40 mL of LB medium. Kanamycin (final concentration 50 μg / mL) was added, and this was incubated in a shaker at 37°C and 220 rpm for 6 hours to obtain a primary seed solution.

[0107] 1.2 mL of primary seed solution was taken and added to 120 mL of LB medium, kanamycin (final concentration 50 μg / mL) was added, and then the mixture was incubated in a shaker at 37°C and 220 rpm for 7 hours to obtain secondary seed solution.

[0108] 3 L of improved LB medium was added to a 5 L fermenter, followed by 120 mL of secondary seed solution and 3 mL of kanamycin (final concentration 50 μg / mL), and incubated at 37°C with 30% dissolved oxygen (series rotation speed) for approximately 8 hours. After monitoring the OD value to be approximately 20, induction was carried out at 20°C using 3 g of lactose as the inducer, with replenishment at a rate of 30 mL / hour, and incubated at 20°C for 24 hours.

[0109] The bacterial suspension was centrifuged at 7000 rpm for 5 minutes, 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 crushed twice in a high-pressure crusher at a pressure of 800-1000 bar for 2 minutes each time. The crushed bacterial suspension was centrifuged at 7000 rpm for 30 minutes, and the supernatant was discarded.

[0110] The precipitate was washed three times with 2M urea solution (containing 1% Triton), using 1 L each time. Next, it was washed twice with 1 L of 3M urea solution, centrifuged each time, and the supernatant was discarded. Then, it was washed once with 1 L of 8M urea solution, centrifuged, and the supernatant was discarded. Finally, 2 L of 8M urea solution was added to the precipitate to obtain crude protein solution C.

[0111] Example 4 Protein CF2 produced from crude protein solution A by dialysis 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, and the molecular weight cutoff of the dialysis bag was 10 kD. After 72 hours of dialysis, the filtrate was freeze-dried to obtain the target protein CF2.

[0112] Structural confirmation of protein CF2 by LC-MS / MS-based complete sequence analysis of the protein. Main ingredients: Acetonitrile, Formic acid, Ammonium bicarbonate, Dithiothreitol (DTT), Iodoacetamide (IAA), Trypsin, Chymotrypsin, Glu-C, Asp-N Main equipment: Capillary high-performance liquid chromatograph (Thermo Ultimate 3000), electrospray hybrid ion trap orbitrap mass spectrometer (Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer)

[0113] Methods and results: Protein CF2 was pretreated with various methods including dissolution substitution, reductive alkylation, and proteolysis to obtain enzyme-cleaved peptide segments. The solution of the enzyme-cleaved peptide segments was analyzed by liquid chromatography-tandem mass spectrometry, and the raw mass spectrometry files were searched for in a protein database using Maxquant (1.6.2.10) for data analysis. Identification results confirmed a match with the target sequence of Sequence ID No. 1.

[0114] Example 5 Protein CF2 prepared by purifying crude protein solution A by salting out. Crude protein solution A was placed in a stirring vessel and subjected to two salting-out processes. During salting-out, saturated ammonium sulfate solution was slowly added along the wall of the vessel until a final concentration of 25% or 50% was reached, allowing the protein to precipitate. The mixture was then filtered to complete the first salting-out. The protein obtained from the first salting-out was suspended in 400 mL of pure water. Saturated ammonium sulfate solution was again slowly added along the wall until a final concentration of 25%, and a second salting-out was performed to obtain a precipitate. After filtration, a crude protein extract was obtained. The crude protein extract was washed three times with water, then suspended in 200 mL of pure water, stirred, allowed to stand, and filtered. This procedure was repeated three times. The resulting precipitated protein was then freeze-dried to obtain the target protein CF2.

[0115] The product protein CF2 obtained by the salting-out method was confirmed to have the same amino acid sequence as the protein produced in Example 4, using the same structural verification method as in Example 4.

[0116] Example 6: Production of protein CF2 by purification of crude protein solution B The crude protein solution B obtained in Example 2 was purified by the following two methods.

[0117] Method 1: Dialysis Crude protein solution B was filtered through a 0.45 μm membrane, and the filtrate was dialyzed with water for more than 72 hours. The filtrate was then freeze-dried to obtain the target protein CF2.

[0118] [Table 3]

[0119] Method 2: Salting out Crude protein solution B was placed in a stirring vessel and subjected to two salting-out processes. During salting-out, saturated ammonium sulfate solution was slowly added along the wall of the vessel until a final concentration of 25% or 50% was reached, allowing the protein to precipitate. The mixture was then filtered to complete the first salting-out. The protein obtained from the first salting-out was suspended in 400 mL of pure water. Saturated ammonium sulfate solution was again slowly added along the wall until a final concentration of 25%, and a second salting-out was performed to obtain a precipitate. After filtration, a crude protein extract was obtained. The crude protein extract was washed three times with water, then suspended in 200 mL of pure water, stirred, allowed to stand, and filtered. This procedure was repeated three times. The resulting precipitated protein was then freeze-dried to obtain the target protein CF2.

[0120] The product protein CF2 obtained by the two methods was confirmed to have the same amino acid sequence as the protein produced in Example 4, using the same structural verification method as in Example 4.

[0121] Example 7: Production of protein CF2 by purification of crude protein solution C Crude protein solution C was purified using microfiltration membrane technology, and urea was removed by repeated microfiltration using a 20 nm or 50 nm ceramic membrane core. The resulting solution was freeze-dried to obtain the target protein CF2.

[0122] The product protein CF2 obtained by the microfiltration membrane technology was confirmed to have the same amino acid sequence as the protein produced in Example 4, using the same structural verification method as in Example 4.

[0123] Pharmacology Test Experimental Example 1: Pharmacodynamic study of protein CF2 (protein from Example 7) in a lipopolysaccharide (LPS)-induced fever model in SD rats. Animal: Male SD rat weighing 230-260 grams Drugs: Lipopolysaccharide (LPS, SIGMA L-2880), Aspirin (SIGMA CF2093), Protein CF2 Equipment: Electronic balance (SARTORIUS BP121S), electronic thermometer (CITIZEN CT-513W)

[0124] Experimental grouping: Normal control group Model group: Lipopolysaccharide thermogenic model Positive control group: Aspirin 300 mg / kg group Protein CF2 10 mg / kg group, 50 mg / kg group

[0125] Method: Method for intraperitoneal injection of lipopolysaccharide to create a rat fever model.

[0126] Preparation of experimental animals: After acclimatizing the animals for one day in the experimental environment (temperature: 22°C ± 2°C, relative humidity: 50% ± 2%), the animals were pre-acclimatized to rectal temperature measurement procedures at 8:00 AM and 3:00 PM. The animals were fasted for 12 hours with free access to water before the experiment. The animals were allowed to defecate before temperature measurement. Before each measurement, the probe of the electronic thermometer was lightly coated with petroleum jelly and inserted 2 cm into the rat's rectum (a mark was made at the 2 cm position to ensure a consistent insertion depth). After stabilization, the temperature reading was recorded.

[0127] Establishment of a rat fever model using intraperitoneal lipopolysaccharide injection: Prior to establishing the model, rat body temperature was measured, and eligible rats with body temperatures between 36.2 and 37.3°C were selected and randomly divided into groups (8 rats per group). Immediately after oral administration of aspirin and various doses of protein CF2, lipopolysaccharide (20 μg / kg, 2 mL / kg) was administered intraperitoneally. The normal control group received an equal volume of saline via intraperitoneal injection. Monitoring of rat body temperature began 2 hours after injection and continued for 8 hours.

[0128] statistics: Based on body temperature values ​​measured at each point in time on the experimental day, the mean, standard deviation, and standard error of the body temperature of rats in each group were calculated. A t-test was used to compare the data between groups, and a P<0.05 value was considered statistically significant.

[0129] Experimental results: A model was established by oral administration of aspirin (300 mg / kg) and protein CF2 (10 mg / kg, 50 mg / kg), followed by intraperitoneal injection of 20 μg / kg of lipopolysaccharide. Body temperature was monitored 2, 4, 6, and 8 hours after modeling. The results are shown in Table 1 and Figure 1.

[0130] [Table 4]

[0131] Experimental results: Animals were orally administered aspirin (300 mg / kg) or protein CF2 (10 mg / kg, 50 mg / kg), respectively, followed immediately by intraperitoneal injection of LPS (20 μg / kg) to establish a model. Body temperature was monitored 2, 4, 6, and 8 hours after model establishment. The following results were obtained.

[0132] 1) Intraperitoneal injection of LPS (20 μg / kg) successfully induced an increase in body temperature in rats. The model group showed a significant increase in body temperature at 2, 4, 6, and 8 hours after modeling compared to the normal group (P<0.05), demonstrating a statistically significant difference and a stable model.

[0133] 2) The aspirin-positive control group effectively suppressed LPS-induced fever elevation in model rats at 2, 4, 6, and 8 hours after modeling. A statistically significant difference was observed compared to the model group (P<0.05), demonstrating consistent efficacy of the positive control drug.

[0134] 3) The protein CF2 (10 mg / kg) group significantly reduced the body temperature of the model rats 2 hours after modeling compared to the model group (P<0.05), demonstrating a statistically significant effect.

[0135] Experimental Example 2: Pharmacodynamic study of protein CF2 (protein from Example 7) in a yeast-induced SD rat fever model. Animal: Male SD rat weighing 230-260 grams Drugs: Yeast (OXOID LP0021), Aspirin (SIGMA CF2093), Protein CF2 Equipment: Electronic balance (SARTORIUS BP121S), electronic thermometer (CITIZEN CT-513W)

[0136] Experimental grouping: Normal control group Model group: Yeast fever model Positive control group: Aspirin 300 mg / kg group Protein CF2 10 mg / kg group, 50 mg / kg group

[0137] method: Preparation of experimental animals: After acclimatizing the animals for one day in the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%), the animals were pre-acclimatized to rectal temperature measurement procedures at 8:00 AM and 3:00 PM. The animals were fasted for 12 hours with free access to water before the experiment. The animals were allowed to defecate before temperature measurement. Before each measurement, the probe of the electronic thermometer was lightly coated with petroleum jelly and inserted 2 cm into the rat's rectum (a mark was made at the 2 cm position to ensure a consistent insertion depth). After stabilization, the temperature reading was recorded.

[0138] Subcutaneous injection of dried yeast to establish a rat fever model: Before modeling, body temperature was measured, and rats with a body temperature of 36.2–37.3°C were selected as eligible subjects and randomly divided into groups (8 rats per group). A 20% yeast suspension (10 mL / kg) was subcutaneously injected immediately after oral administration of aspirin and various doses of protein CF2. The normal control group received an equal volume of physiological saline subcutaneously. Body temperature monitoring began 2 hours after modeling and continued for 8 hours at 2-hour intervals.

[0139] statistics: Based on the body temperature measured at each point in time on the experimental day, the mean, standard deviation, and standard error of the body temperature of rats in each group were calculated. A t-test was used to compare the data between groups, and a P<0.05 value was considered statistically significant.

[0140] Experimental results: A model was established by subcutaneous injection of 20% yeast immediately after oral administration of aspirin (300 mg / kg) and protein CF2 (10 mg / kg, 50 mg / kg). Animal body temperature was monitored 2, 4, 6, and 8 hours after model establishment. The results are shown in Table 2 and Figure 2.

[0141] [Table 5]

[0142] Experimental results: A fever model was established by subcutaneous injection of a 20% yeast suspension immediately after oral administration of aspirin (300 mg / kg) or protein CF2 (10 mg / kg, 50 mg / kg). Body temperature was monitored 2, 4, 6, and 8 hours after modeling. The following results were shown.

[0143] 1) The model group showed a significant increase in body temperature compared to the normal group at 2, 4, 6, and 8 hours after modeling (P<0.05), demonstrating a statistically significant difference. This confirmed the successful establishment of a stable and reliable model.

[0144] 2) The positive control aspirin group effectively suppressed yeast-induced body temperature elevation in model rats at all monitored time points (2, 4, 6, and 8 hours). A statistically significant difference was observed compared to the model group (P<0.05), demonstrating the consistent efficacy of aspirin as a positive control drug.

[0145] 3) Protein CF2 showed a long-term inhibitory effect on the rise in body temperature. The 10 mg / kg dose group significantly suppressed the rise in body temperature in model rats at 2, 4, and 6 hours after modeling, showing a statistically significant difference compared to the model group (P<0.05). The 50 mg / kg dose group significantly suppressed the rise in body temperature at 2, 4, and 8 hours after modeling, showing a very statistically significant difference compared to the model group (P<0.01).

[0146] Experimental Example 4: Pharmacodynamic study of protein CF2 (protein from Example 7) against pentylenetetrazole (PTZ)-induced epilepsy in mice. Animal: Male ICR mouse Drugs: Pentylenetetrazole (PTZ), retigavine, protein CF2

[0147] Experimental grouping: Model group Retigavin 60 mg / kg group Protein CF2 50 mg / kg group, 200 mg / kg group method: Model creation and administration: Administration of the test drug as a single dose was initiated in the afternoon prior to model establishment. On the day of model establishment, the test drug was administered orally via gastric tube, followed by an intraperitoneal injection of PTZ (65 mg / kg, model inducer) one hour later. The positive control drug was administered once 30 minutes before model establishment. Animals were continuously observed for 15 minutes after PTZ injection.

[0148] Observation indicators: (1) Epileptic seizure status: Grade III to Grade VI seizure duration, (2) Mortality status Seizure severity (based on the Racine scale): Grade 0: No observable response, Grade I: Present as spasms of the facial muscles or corner of the mouth, Grade II: Head nodding, Grade III: Spasms of one limb, Grade IV: Rigidity or generalized seizure, Grade V: Generalized epilepsy (generalized tonic-clonic seizures)

[0149] Data processing: During the statistical experiment, the number of seizures and deaths observed in each group of mice, as well as the latencies for grade III and IV seizures, were recorded. For mice that did not progress to grade IV seizures, the latency was recorded with a maximum value of 900 seconds. Statistical analysis was performed using the chi-squared test to determine the sample size. Latency is expressed as mean ± standard error (SEM). Between-group comparisons were performed between the model group and other groups using t-tests. A P<0.05 was considered statistically significant.

[0150] Experimental results: Please refer to Tables 3 and 4.

[0151] [Table 6]

[0152] [Table 7]

[0153] Experimental results: 1) The experimental results showed that the grade IV seizure rate was 80% and the mortality rate was 10% in the model group, demonstrating the success of the model establishment.

[0154] 2) The positive drug significantly prolonged the latency of both grade III and grade IV seizures in mice.

[0155] 3) In a comparison of the latency of Grade III seizures, a significant difference was observed between the CF2-50 mg / kg group and the CF2-200 mg / kg group and the model group.

Claims

1. Keratin CF2, wherein the amino acid sequence of the above keratin CF2 is (1) The amino acid sequence shown in Sequence ID No. 1 in the sequence listing, or (2) Keratin CF2, which is an amino acid sequence that substantially maintains the same biological function, obtained by substituting, deleting, or adding 1 to 45 amino acids to the amino acid sequence shown in Sequence ID No. 1 in the sequence listing.

2. The keratin CF2 according to claim 1, wherein the keratin CF2 may have conventional modifications, or the keratin CF2 is further linked to a tag for detection or purification, or the keratin CF2 is a homologous protein thereof.

3. The above conventional modification includes acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorophore group modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamination, disulfide bond formation or disulfide bond cleavage, and the above tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc and Proficiency eXact, according to claim 2.

4. A nucleic acid molecule encoding keratin CF2 according to any one of claims 1 to 3.

5. The nucleotide sequence of the above nucleic acid molecule is (1) The nucleotide sequence shown in Sequence ID No. 2 in the sequence listing, (2) A nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in Sequence ID No. 2, or (3) The nucleic acid molecule according to claim 4, wherein the nucleotide sequence is complementary to the nucleotide sequence of (1) or (2) above.

6. An expression vector comprising a nucleic acid molecule according to any one of claims 4 to 5.

7. A host cell comprising the expression vector described in claim 6, or in which a nucleic acid molecule described in any one of claims 4 to 5 is incorporated into the genome.

8. The host cell according to claim 7, wherein the host cell is selected from the group consisting of bacteria, yeast, Aspergillus, plant cells, and insect cells.

9. The host cell according to claim 8, wherein the above-mentioned bacterium is Escherichia coli.

10. A method for producing keratin CF2 according to any one of claims 1 to 3, A. A step of synthesizing a nucleic acid molecule corresponding to keratin CF2 according to any one of claims 1 to 3, linking the nucleic acid molecule to an appropriate expression vector, transforming a host cell with the expression vector, culturing the host cell holding the expression vector under specific conditions in a fermentation apparatus, inducing the expression of keratin CF2, and obtaining a crude protein solution containing keratin CF2. B. A method comprising the steps of separating and purifying the crude protein solution obtained in step A, drying it, and obtaining the keratin CF2.

11. The method according to claim 10, wherein in step A, the host cells are mainly selected from Escherichia coli, the keratin CF2 is expressed as an inclusion body of Escherichia coli, and the fermentation apparatus includes a shaking flask or a fermenter.

12. The method according to claim 10, wherein in step A, after inducing the expression of the above-mentioned keratin CF2, impurities are washed away with a washing solution, and then the crude protein solution is obtained by dissolving it in a solution.

13. The method according to claim 10, wherein in step B, the separation and purification method is selected from the group consisting of purification by ultrafiltration / microfiltration membrane technology, column chromatography purification, salting-out method, and dialysis method.

14. A pharmaceutical composition comprising keratin CF2 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

15. The use of keratin CF2 according to any one of claims 1 to 3, or a nucleic acid molecule according to any one of claims 4 to 5, or an expression vector according to claim 6, or 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, or antiviral drug.

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