Keratin CF2, preparation method, pharmaceutical composition and application thereof

Abstract

Belongs to the technical field of medicines, discloses keratin CF2, a preparation method, a pharmaceutical composition and application thereof, and particularly relates to keratin CF2, a nucleic acid molecule for encoding the keratin CF2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or genome integrated with the nucleic acid molecule, and a preparation method of the keratin CF2. The invention relates to a keratin CF2-containing pharmaceutical composition and application of the keratin CF2, nucleic acid molecules, expression vectors, host cells or the pharmaceutical composition in preparation of antipyretic and analgesic, cough-relieving and phlegm-eliminating drugs, anticonvulsion, antiepilepsy, blood pressure lowering drugs, anti-inflammatory drugs and antiviral drugs, in particular to application of the keratin CF2, the nucleic acid molecules, the expression vectors, the host cells or the pharmaceutical composition in preparation of antipyretic and analgesic drugs.

Description

Keratin CF2, preparation method, pharmaceutical composition and use thereof Technical Field

[0001] The present invention belongs to the field of medical technology and relates to keratin CF2, a nucleic acid molecule encoding keratin CF2, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or having the nucleic acid molecule integrated into its genome, as well as a method for preparing keratin CF2, a pharmaceutical composition containing the keratin, and use of the keratin and the pharmaceutical composition in preparing antipyretic and analgesic, antitussive and expectorant, anticonvulsant, antiepileptic, blood pressure-lowering, anti-inflammatory, and antiviral drugs. Background Art

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

[0003] Keratin is widely present in living organisms and is a renewable resource with great potential, yet it has not been widely and effectively utilized. This is primarily due to its insolubility in various solvents and its greater resistance to protease degradation than other proteins. Consequently, extracting and preparing natural keratin is extremely challenging.

[0004] With the rapid development of modern biotechnology such as genomics, proteomics, genetic engineering, and microbial engineering, more and more genes have been discovered. The use of protein expression systems to produce target proteins is an important means to study the biological functions of genes or proteins.

[0005] The use of a protein expression system to prepare the target keratin and then study its structure and function has not been reported in other literature and is novel and creative.

[0006] Summary of the Invention

[0007] The technical problem solved by the present invention is to provide a keratin CF2, a nucleic acid molecule encoding keratin CF2, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or having the nucleic acid molecule integrated into its genome, as well as a method for preparing keratin CF2, a pharmaceutical composition containing keratin CF2, and the use of the above keratin CF2, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the preparation of antipyretic and analgesic, antitussive and expectorant, anticonvulsant, antiepileptic, blood pressure-lowering, anti-inflammatory, and antiviral drugs.

[0008] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0009] The first aspect of the technical solution of the present invention is to provide a keratin CF2, characterized in that the amino acid sequence of the keratin CF2 is:

[0010] (1) the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing;

[0011] (2) An amino acid sequence substantially retaining the same biological function as that of the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing, formed by replacing, deleting or adding 1 to 35 amino acids.

[0012] Furthermore, conventional modifications can be performed on keratin CF2; or a tag for detection or purification can be connected to keratin CF2.

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

[0014] The second aspect of the technical solution of the present invention is to provide a nucleic acid molecule encoding the keratin CF2 described in the first aspect.

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

[0016] (1) the nucleotide sequence shown in SEQ ID NO. 2 in the sequence listing;

[0017] (2) a nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO. 2;

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

[0019] The third aspect of the technical solution of the present invention is to provide an expression vector, characterized in that the expression vector contains the nucleic acid molecule described in the second aspect.

[0020] Furthermore, the expression vector can be 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; the most preferred expression vector is pET-28a(+).

[0021] The fourth aspect of the technical solution of the present invention provides a host cell, characterized in that the host cell contains the expression vector described in the third aspect or the nucleic acid molecule described in the second aspect is integrated into its genome.

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

[0023] Furthermore, the bacteria include Escherichia coli or yeast.

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

[0025] The fifth aspect of the technical solution of the present invention is to provide a method for preparing the keratin CF2 described in the first aspect, characterized in that it comprises the following steps:

[0026] A. synthesizing a nucleic acid molecule corresponding to the keratin CF2 described in the first aspect, linking the nucleic acid molecule into a corresponding expression vector, transforming the expression vector into a host cell, culturing the host cell carrying the expression vector in a fermentation device under certain conditions and inducing expression of keratin CF2 to obtain a crude protein solution containing keratin CF2;

[0027] B. Separate, purify and dry the crude protein solution expressed in step A to obtain keratin CF2.

[0028] Furthermore, in step A, the host cell is mainly selected from Escherichia coli, the keratin CF2 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shake flask or a fermentation tank.

[0029] Furthermore, in step A, after inducing the expression of keratin CF2, impurities can be cleaned with a detergent and then dissolved with a urea solution to obtain a crude protein solution.

[0030] Furthermore, the culture medium in step A can be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Bengal red medium, high salt Czapek medium, DOBA medium, rice koji medium and improved formulas thereof; LB medium and TB medium are preferred for shake flask fermentation, and TB medium is most preferred; LB medium and improved formulas thereof are preferred for fermentation tanks.

[0031] Furthermore, the inducer in step A can be IPTG, lactose, arabinose, etc.; preferably IPTG and lactose.

[0032] Furthermore, in step A, the fermentation broth obtained is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacteria are broken, and the precipitate is centrifuged again, and the supernatant is discarded; the precipitate is washed with a detergent and then dissolved with a urea solution to obtain a CF2 crude protein solution.

[0033] The buffer is preferably buffer A, and its dosage is: fermentation broth volume: buffer A volume = 1-100:1, preferably 10:1;

[0034] The cleaning agent can be urea solution, guanidine hydrochloride solution, Triton, buffer A, etc., preferably urea solution, most preferably 2M urea solution (which may contain 1% Triton) and 4M urea solution, and the dosage is: fermentation broth volume: urea solution volume = 0.2-100:1, preferably 1-15:1;

[0035] The urea solution is preferably a 4M to 8M urea solution, most preferably an 8M urea solution, and its dosage is: fermentation broth volume: 8M urea volume = 0.2 to 100:1, preferably 2 to 15:1.

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

[0037] Furthermore, in step B, the separation and purification method is as follows:

[0038] (1) The dialysis method is to purify the crude protein solution obtained in step A by dialysis to obtain the target protein CF2 solution.

[0039] The molecular weight cut-off of the dialysis bag can be 0.5-10 kD, preferably 3.5-10 kD, and most preferably 10 kD.

[0040] (2) The ultrafiltration and microfiltration method is to purify the crude protein solution obtained in step A using membrane technology such as ultrafiltration membrane or microfiltration membrane to obtain a concentrated solution of the target protein CF2.

[0041] (3) The column chromatography method is to separate and purify the crude protein solution obtained in step A through column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein CF2.

[0042] Preferred exclusion columns are dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns are ion exchange resin columns: anion exchange resin columns, HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, Toyopearl SuperQ-650M, etc.; cation exchange resin columns, HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SPImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M.

[0043] The eluent may be any eluent commonly used in the art, such as water, saline solution, including sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.

[0044] (4) The salting-out method is to purify the crude protein solution obtained in step A by salting-out to obtain a target protein CF2 suspension.

[0045] The salting-out agent can 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 ammonium sulfate solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, and more preferably 25%.

[0046] The number of salting-outs is 1 to 3 times, preferably 2 times.

[0047] After salting out, the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.

[0048] Furthermore, the target protein CF2 solution obtained by purification in step B can be freeze-dried or vacuum-dried into a dry powder, or the concentrated solution can be directly spray-dried into a dry powder.

[0049] The sixth aspect of the technical solution of the present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin CF2 described in the first aspect or the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect or the host cell described in the fourth aspect and a pharmaceutically acceptable carrier or excipient.

[0050] The keratin obtained in the above steps of the present invention can be freeze-dried or vacuum-dried into dry powder, or the concentrated liquid can be directly spray-dried into dry powder and then made into various dosage forms.

[0051] The present invention relates to a pharmaceutical composition comprising any one keratin obtained in the above steps and a pharmaceutically acceptable carrier.

[0052] 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. Typically, the keratin of the present invention accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.

[0053] The present invention also provides a pharmaceutical composition comprising a pharmaceutically effective dose of protein as an active ingredient and a pharmaceutically acceptable carrier.

[0054] The pharmaceutical compositions of the present invention can be prepared according to methods known in the art. For this purpose, the protein of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants, if necessary, to prepare an appropriate administration form or dosage form for use as a human or veterinary drug.

[0055] The keratin or pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, skin, vagina, peritoneum, rectum, etc., preferably oral administration.

[0056] The keratin or the pharmaceutical composition containing the keratin of the present invention can be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection and acupoint injection.

[0057] Dosage forms can be liquid, solid, or semisolid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments. Solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays. Semisolid dosage forms can include ointments, gels, and pastes.

[0058] The keratin of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0059] In order to prepare the unit dosage form into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. 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, isopropyl alcohol, etc.; the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, 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 tectonic acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0060] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0061] To prepare the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, macrogol laurate, kaolin, talc, etc.; binders such as gum arabic, calcite, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.

[0062] In order to prepare the administration unit into a suppository, various carriers known in the art can be widely used, such as polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.

[0063] To prepare a dosing unit as a capsule, the active ingredient, the keratin of the present invention, is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or soft capsule. Alternatively, the active ingredient, the keratin of the present invention, can be formulated into microcapsules and suspended in an aqueous medium to form a suspension, which can then be encapsulated in a hard capsule or formulated as an injectable.

[0064] For example, the keratin of the present invention can be prepared into an injectable preparation, such as a solution, suspension solution, emulsion, or freeze-dried powder injection. This preparation can be aqueous or non-aqueous and can contain one or more pharmacologically acceptable carriers, diluents, adhesives, lubricants, preservatives, surfactants, or dispersants. For example, the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. In addition, to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injectable preparation. In addition, conventional cosolvents, buffers, pH regulators, and the like can also be added. These excipients are commonly used in the art.

[0065] In addition, if necessary, colorants, preservatives, perfumes, flavorings, sweeteners or other materials may be added to the pharmaceutical preparations.

[0066] In order to achieve the purpose of medication and enhance the therapeutic effect, the keratin or pharmaceutical composition of the present invention can be administered by any known administration method.

[0067] The dosage of the keratin pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, the number of doses, and the intended treatment. Therefore, the therapeutic dosage of the present invention can vary widely. Generally speaking, the dosages of the pharmaceutical ingredients of the present invention are well known to those skilled in the art. The dosage can be appropriately adjusted based on the actual amount of drug contained in the final formulation of the keratin composition of the present invention to achieve a therapeutically effective amount and achieve the preventive or therapeutic purpose of the present invention. A suitable daily dosage range for 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. This dosage can be administered as a single dose or in divided doses, such as two, three, or four doses, depending on the clinical experience of the administering physician and the dosing regimen, including the use of other therapeutic modalities. The total dosage required for each treatment can be administered in divided doses or as a single dose. The protein or pharmaceutical composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage can be adjusted.

[0068] The seventh aspect of the technical solution of the present invention provides the use of the keratin CF2 described in the first aspect or the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect or the host cell described in the fourth aspect or the pharmaceutical composition described in the sixth aspect in the preparation of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs.

[0069] In order to achieve the purpose of the present invention, the present invention adopts the following technical solution. Specifically, the preparation of keratin CF2 of the present invention includes the following steps:

[0070] (1) Synthesize nucleotide sequences and determine the accuracy of the sequences;

[0071] The preferred nucleotide sequence is shown in SEQ ID No.2.

[0072] (2) transferring the nucleotide sequence into an expression vector;

[0073] The expression vector can be 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; the most preferred expression vector is pET-28a(+).

[0074] (3) transfecting the expression vector into host cells;

[0075] The host cell can be Escherichia coli or yeast; the preferred host cell is Escherichia coli;

[0076] Competent cells can be BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Organami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; preferred expression competent cells are BL21 (DE3) and Transetta (DE3).

[0077] (4) fermenting and culturing the host cells under appropriate conditions to induce expression of the target protein CF2;

[0078] Fermentation equipment can be shake flasks or fermentation tanks;

[0079] The culture medium can be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Bengal rose medium, high salt Czapek medium, DOBA medium, rice koji medium and improved formulas thereof; LB medium and TB medium are preferred for shake flask fermentation, and TB medium is most preferred; LB medium and improved formulas thereof are preferred for fermentation tanks.

[0080] The inducer can be IPTG, lactose, arabinose, etc.; preferably IPTG and lactose.

[0081] (5) Enrichment of target protein CF2 product;

[0082] The fermentation broth obtained in step (4) is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacteria are broken, and the precipitate is centrifuged again and the supernatant is discarded; the precipitate is washed with a detergent and then dissolved with a urea solution to obtain a CF2 crude protein solution.

[0083] The buffer is preferably buffer A, and its dosage is: fermentation broth volume: buffer A volume = 1-100:1, preferably 5-10:1;

[0084] The cleaning agent can be urea solution, guanidine hydrochloride solution, Triton, buffer A, etc., preferably urea solution, most preferably 2M urea solution (which may contain 1% Triton), and its dosage is: fermentation broth volume: urea solution volume = 0.2-100:1, preferably 1-15:1; the number of cleaning times is 3-10 times, preferably 6 times;

[0085] The urea solution is preferably a 4M to 8M urea solution, most preferably an 8M urea solution, and its dosage is: fermentation broth volume: 8M urea solution volume = 0.2 to 100:1, preferably 2 to 15:1.

[0086] (6) Isolation and purification of target protein CF2:

[0087] The crude protein solution obtained in step (5) needs to be purified to obtain the target protein CF2. The purification can be carried out by dialysis, ultrafiltration, microfiltration, column chromatography, or salting out.

[0088] A. The dialysis step is to purify the crude protein solution obtained in step (5) by dialysis to obtain the target protein CF2 solution.

[0089] The molecular weight cut-off of the dialysis bag can be 0.5-10 kD, preferably 3.5-10 kD, and most preferably 10 kD.

[0090] B. The ultrafiltration and microfiltration steps are to purify the crude protein solution obtained in step (5) using membrane technology such as ultrafiltration membrane or microfiltration membrane to obtain a concentrated solution of the target protein CF2.

[0091] C. The column chromatography step is to separate and purify the crude protein solution obtained in step (5) through column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein CF2.

[0092] Preferred size exclusion columns are dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns are ion exchange resin columns, such as anion exchange resin columns, HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, Toyopearl SuperQ-650M, etc.; and cation exchange resin columns, such as HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SPImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M. Most preferred are anion exchange resin columns.

[0093] The eluent may be any eluent commonly used in the art, such as water, saline solution, including sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.

[0094] D. The salting-out step is to purify the crude protein solution obtained in step (5) by salting-out to obtain a target protein CF2 suspension.

[0095] The salting-out agent can 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 ammonium sulfate solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, and more preferably 25%.

[0096] The number of salting-outs is 1 to 3 times, preferably 2 times.

[0097] After salting out, the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.

[0098] The target protein CF2 solution obtained by purification in steps A to D can be freeze-dried or vacuum-dried to form a dry powder, or the concentrated solution can be directly spray-dried to form a dry powder.

[0099] Beneficial technical effects of the present invention:

[0100] 1. The protein of the present invention is keratin obtained for the first time, and the preparation method of the present invention has the characteristics of high yield and high sample purity.

[0101] 2. The present invention has demonstrated that protein CF2 has a significant effect of reducing the elevated body temperature of the model animals 8 hours after model establishment through efficacy studies on the yeast-induced SD rat fever model; and has demonstrated that protein CF2 has a significant effect of reducing the elevated body temperature of the model rats 2 hours after model establishment through efficacy studies on the lipopolysaccharide (LPS)-induced SD rat fever model.

[0102] 3. The present invention studies the efficacy of protein CF2 on epilepsy induced by pentylenetetrazol (PTZ) in mice, demonstrating that protein CF2 can significantly prolong the latency period of grade III epileptic seizures in mice; BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1. Effects of protein CF2 on lipopolysaccharide (LPS)-induced fever model in rats

[0104] (Compared with the normal control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01)

[0105] Figure 2. Effect of protein CF2 on yeast-induced fever model in rats

[0106] (Compared with the normal control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01) DETAILED DESCRIPTION

[0107] The following examples and pharmacological activity test examples are used to further illustrate the present invention, but they do not mean any limitation of the present invention.

[0108] The experimental methods in the following examples and pharmacological activity test examples are conventional methods unless otherwise specified; the experimental materials used are purchased from conventional biochemical reagent companies unless otherwise specified.

[0109] Example 1 Preparation of Protein CF2 Crude Solution A by Shake Flask Fermentation (TB Medium)

[0110] The nucleotide sequence shown in SEQ ID No. 2 was synthesized and transferred into the pET-28a(+) vector. Sequencing confirmed the expression vector containing the correct sequence. The expression vector was transfected into BL21(DE3) cells to generate expression-competent host cells containing the target nucleotide sequence. The recombinant strain was obtained by adding the recombinant strain to LB medium and incubating at 37°C and 220 rpm on a shaker for 1 hour.

[0111] The recombinant strain was streaked onto an LBA plate containing kanamycin, and the plate was inverted and placed in a 37°C constant temperature incubator for overnight culture for 16 hours.

[0112] Prepare 400 ml of TB medium and aliquot it into two 200 ml bottles. Add kanamycin (final concentration 50 μg / ml) to each 200 ml bottle. Add a single colony from the plate to the TB medium and culture overnight on a shaker at 37°C and 220 rpm to obtain the seed solution.

[0113] Prepare 28.8L of TB medium and distribute it into 144 bottles, 200ml per bottle. Add kanamycin (final concentration 50μg / ml) to each bottle (200ml) of TB medium, then add 2ml of seed solution, and culture in a shaker at 37℃ and 220rpm for 2-3 hours. Monitor OD 600 , when OD 600 When the concentration reaches about 1.0, an inducer is added to induce protein expression in a shaker. The induction conditions are selected from the table below.

[0114] Combine the bacterial suspensions from each bottle and centrifuge at 7000 rpm for 5 minutes. Sterilize and discard the supernatant. Resuspend the precipitate in approximately 3 L of buffer and filter through an 80-100 mesh screen. Disintegrate the filtrate using a high-pressure crusher at 800-1000 bar twice for 2 minutes each. Centrifuge the crushed bacterial suspension at 7000 rpm for 30 minutes, discard the supernatant, and obtain the precipitate (i.e., inclusion bodies). Wash the precipitate with detergent (3 times with 1 L of 2M urea-1% triton solution and twice with 1 L of 3M urea solution), centrifuge, and discard the supernatant. Dissolve the precipitate in 1 L of 8M urea solution to obtain crude protein solution A.

[0115] Example 2 Preparation of Protein CF2 Crude Solution B by Shake Flask Fermentation (Other Culture Medium)

[0116] In Example 1, an expression vector containing the sequence shown in SEQ ID No. 2 was synthesized and sequenced; the expression vector was transfected into BL21 (DE3) cells to obtain expression competent host cells containing the target nucleotide sequence.

[0117] Prepare 20 ml of LB medium, take 800 μl, add 50 μl of host cells containing the target coding sequence, and culture in a shaker at 37°C and 220 rpm for 1 hour.

[0118] The above bacterial solution was dipped and streaked on an LBA plate containing kanamycin, and the plate was inverted and placed in a 37°C constant temperature incubator for overnight culture for 16 hours.

[0119] Take 10 ml of LB medium, add kanamycin (final concentration 50 μg / ml), take a single colony from the plate and add it to the LB medium, and culture it overnight on a shaker at 37°C and 220 rpm for 15 hours to obtain seed liquid.

[0120] Prepare 1L of the culture medium shown in the table below and divide it into 10 bottles, 100ml per bottle. Add kanamycin (final concentration 50μg / ml) to each bottle (100ml) of culture medium, then add 1ml of seed solution, and culture in a shaker at 37℃ and 220rpm for 2-3 hours. Monitor OD 600 , when OD 600 When the C value reached about 1.0, the inducer IPTG (final concentration 0.5 mM) was added and the protein was induced to express in a shaker at 37°C and 220 rpm.

[0121] Combine the bacterial suspensions from each vial and centrifuge at 10,000 rpm for 10 minutes. Sterilize and discard the supernatant. Resuspend the pellet in approximately 100 mL of buffer and filter through an 80-100 mesh sieve. Disintegrate the filtrate using a high-pressure crusher at 800-1000 bar for two cycles of 2 minutes each. Centrifuge the resulting suspension at 10,000 rpm for 30 minutes, and discard the supernatant.

[0122] The precipitate was first washed with 40 mL of detergent buffer A, centrifuged, and the supernatant was discarded; the precipitate was washed twice with 40 mL of detergent 2M urea solution (containing 1% triton), centrifuged, and the supernatant was discarded; the precipitate was then washed twice with 40 mL of 3M urea solution, centrifuged, and the supernatant was discarded; the precipitate was then added with 40 mL of 8M urea solution to obtain crude protein solution B.

[0123] Example 3 Preparation of protein CF2 crude solution C in a fermenter

[0124] In Example 1, an expression vector containing the sequence shown in SEQ ID No. 2 was synthesized and sequenced to obtain the expression vector. The expression vector was transfected into BL21(DE3) cells to obtain expression-competent host cells containing the target nucleotide sequence. The cells were added to LB medium and cultured on a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.

[0125] Add 100 μl of the recombinant strain to an LBA plate containing kanamycin and spread evenly with a spreader until dry. Incubate the plate upside down at 37°C in a constant-temperature incubator overnight. Streak three individual colonies onto the plate containing kanamycin and culture overnight. After three batches of shake flask fermentation and expression verification, preserve the strain in 15% glycerol and aliquot into 0.8 ml vials to obtain a working cell bank. Store frozen at -80°C until needed.

[0126] Take out one glycerol strain from the working cell bank, take 100 μl, add it to 40 ml LB medium, add kanamycin (final concentration 50 μg / ml), and culture it in an oscillator at 37°C and 220 rpm for 6 hours to obtain the first-level seed liquid.

[0127] Take 1.2 ml of the first-level seed solution and add it to 120 ml of LB medium. Add kanamycin (final concentration 50 μg / ml) and culture it in an oscillator at 37°C and 220 rpm for 7 hours to obtain the second-level seed solution.

[0128] To a 5-L fermentor, add 3 L of modified LB broth, 120 ml of secondary seed solution, and 3 ml of kanamycin (final concentration 50 μg / ml). Cultivate at 37°C and 30% dissolved oxygen (tandem speed) for approximately 8 hours. Monitor the OD value around 20. Induce with 3 g of lactose as an inducer at 20°C, feed at 30 ml / hour, and incubate at 20°C for 24 hours.

[0129] Centrifuge the bacterial suspension at 7000 rpm for 5 minutes, sterilize and discard the supernatant. Resuspend the pellet in approximately 600 ml of buffer A and filter through an 80-100 mesh sieve. Disintegrate the filtrate using a high-pressure crusher at 800-1000 bar twice for 2 minutes each. Centrifuge the resulting suspension at 7000 rpm for 30 minutes, and discard the supernatant.

[0130] The pellet was washed three times with 1 L of 2M urea solution (containing 1% Triton); then washed twice with 1 L of 3M urea solution, centrifuged, and the supernatant discarded; then washed once with 1 L of 8M urea solution, centrifuged, and the supernatant discarded. The pellet was then added with 2 L of 8M urea solution to obtain crude protein solution C.

[0131] Example 4: Preparation of Protein CF2 by Dialysis of Crude Protein Solution A

[0132] The crude protein solution A obtained in Example 1 was filtered through a 0.45 μm filter membrane, and the filtrates were combined and dialyzed against water using a dialysis bag with a molecular weight cutoff of 10 kD for 72 hours. The resulting solution was freeze-dried to obtain the target protein CF2.

[0133] Protein CF2 Structure Confirmation--Protein Sequence Analysis Based on LC-MS / MS

[0134] Main materials: acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Asp-N;

[0135] Main instruments: capillary high performance liquid chromatography (Thermo Ultimate 3000), electrospray ion trap-Orbitrap mass spectrometer (Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer).

[0136] Methods and Results:

[0137] Protein CF2 was pre-treated by dissolution displacement, reductive alkylation, and multiple proteolysis to obtain enzymatic peptides. The enzymatic peptide solution was analyzed by liquid chromatography-tandem mass spectrometry, and the mass spectrum raw file was retrieved using Maxquant (1.6.2.10) to retrieve the protein database for data analysis. The identification results confirmed that it was consistent with the target sequence SEQ ID No. 1.

[0138] Example 5: Protein CF2 was prepared by purifying the crude protein solution A by salting out.

[0139] The crude protein solution A is placed in a stirred container and salted out twice: saturated ammonium sulfate solution is slowly added along the wall to a final concentration of 25% or 50%. During the salting-out process, the protein precipitates. After the salting-out is complete, the solution is filtered to complete the first salting-out. 400ml of pure water is added to the precipitate to suspend it. Saturated ammonium sulfate solution is then slowly added along the wall to a final concentration of 25%. A second salting-out is performed, and the precipitate is filtered to obtain the crude protein extract. The crude protein extract is then washed three times with water: 200ml of pure water is added to suspend it, stirred, allowed to stand, and filtered. After repeating this process three times, the precipitate is freeze-dried to obtain the target protein CF2.

[0140] The same structure confirmation method as in Example 4 confirmed that the protein had the same amino acid sequence as the protein prepared in Example 4.

[0141] Example 6: Purification of crude protein solution B to obtain protein CF2

[0142] The crude protein solution B obtained in Example 2 was purified by the following two methods:

[0143] The first method: dialysis;

[0144] The crude protein solution B was filtered through a 0.45 μm membrane, and the filtrate was dialyzed against water for more than 72 hours. The inner solution was freeze-dried to obtain the target protein CF2.

[0145] The second method: salting out;

[0146] The crude protein solution B is placed in a stirred container and salted out twice: saturated ammonium sulfate solution is slowly added along the wall to a final concentration of 25% or 50%. During the salting-out process, the protein precipitates. After the salting-out is complete, the solution is filtered to complete the first salting-out. 400ml of pure water is added to the precipitate to suspend it. Saturated ammonium sulfate solution is again slowly added along the wall to a final concentration of 25%. Salting-out is performed a second time, and the precipitate is filtered to obtain the crude protein extract. The crude protein extract is washed three times with water: 200ml of pure water is added to suspend it, stirred, allowed to stand, and filtered. After repeating this process three times, the precipitate is freeze-dried to obtain the target protein CF2.

[0147] The product protein CF2 obtained by the two methods was confirmed to have the same amino acid sequence as the protein prepared in Example 4 by the same structure confirmation method as in Example 4.

[0148] Example 7: Purification of crude protein solution C to obtain protein CF2

[0149] The crude protein solution C is purified by microfiltration membrane technology: urea is removed by repeated microfiltration using a 20nm or 50nm ceramic membrane core; the internal solution is freeze-dried to obtain the target protein CF2.

[0150] The same structure confirmation method as in Example 4 confirmed that the protein had the same amino acid sequence as the protein prepared in Example 4.

[0151] Pharmacological tests

[0152] Experimental Example 1: Efficacy of Protein CF2 (Protein from Example 7) on Lipopolysaccharide (LPS)-Induced Fever Model in SD Rat

[0153] Animals: Male Sprague-Dawley rats, 230-260 g;

[0154] Drugs: Lipopolysaccharide (LPS, SIGMA L-2880), aspirin (SIGMACF2093), protein CF2;

[0155] Instruments: electronic balance (SARTORIUSBP121S), electronic thermometer (CITIZEN CT-513W).

[0156] Experimental groups:

[0157] Normal control group;

[0158] Model group: lipopolysaccharide fever model;

[0159] Positive control group: Aspirin 300 mg / kg group;

[0160] Protein CF2, 10mg / kg group, 50mg / kg group.

[0161] Methods: Intraperitoneal injection of lipopolysaccharide to induce fever model in rats.

[0162] Animal Preparation: After acclimating to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for one day, animals were pre-acclimated to rectal temperature measurement at 8:00 AM and 3:00 PM. Animals were fasted but not watered for 12 hours prior to the experiment, and their feces were emptied before rectal temperature measurement. Before each temperature measurement, the electronic thermometer probe was coated with petroleum jelly and inserted 2 cm into the rat's rectum (a mark at the 2 cm mark was provided to ensure consistent insertion depth). Temperature was recorded after the reading stabilized.

[0163] Intraperitoneal injection of lipopolysaccharide to induce a fever model in rats: Before modeling, rat body temperature was measured, and rats with a body temperature between 36.2 and 37.3°C were screened and randomly divided into groups of 8. Immediately after oral administration of aspirin and varying doses of protein CF2, lipopolysaccharide (20 μg / kg, 2 ml / kg) was injected intraperitoneally. A normal control group received an equal volume of saline. Body temperature was monitored starting 2 hours later for a total of 8 hours.

[0164] Statistics:

[0165] According to the body temperature values ​​measured at each time point on the day of the experiment, the mean, standard deviation and standard error of body temperature of rats in each group were calculated. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be significantly different.

[0166] Experimental results:

[0167] After oral administration of aspirin (300 mg / kg) and protein CF2 (10 mg / kg and 50 mg / kg), 20 μg / kg of lipopolysaccharide (LPS) was immediately injected intraperitoneally to establish the model. The body temperature of the animals was monitored 2, 4, 6, and 8 hours after model establishment. The results are shown in Table 1 and Figure 1.

[0168] Table 1. Effects of the test drugs on the lipopolysaccharide (LPS)-induced fever model in rats

[0169] (Compared with the normal control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01)

[0170] Experimental conclusion:

[0171] After oral administration of aspirin (300 mg / kg) and protein CF2 (10 mg / kg, 50 mg / kg), 20 μg / kg lipopolysaccharide was immediately injected intraperitoneally to establish the model. The body temperature of the animals was monitored 2 hours, 4 hours, 6 hours, and 8 hours after model establishment. The results showed that:

[0172] 1) Intraperitoneal injection of 20 μg / kg lipopolysaccharide successfully induced an increase in body temperature in rats. The body temperature of the rats in the model group increased significantly 2 hours, 4 hours, 6 hours, and 8 hours after modeling. Compared with the normal group, P < 0.05, there was a statistically significant difference, and the model was stable.

[0173] 2) The positive tool drug aspirin group can effectively inhibit the increase in body temperature of model rats at 2 hours, 4 hours, 6 hours, and 8 hours after modeling. Compared with the model group, P<0.05, there is a statistically significant difference, and the positive tool drug performance is relatively stable.

[0174] 3) The protein CF2 10mg / kg dose group could significantly reduce the body temperature of the model rats 2 hours after modeling, and compared with the model group, P<0.05, there was a statistically significant difference.

[0175] Experimental Example 2: Efficacy of Protein CF2 (Protein from Example 7) on Yeast-Induced SD Rat Fever Model

[0176] Animals: Male Sprague-Dawley rats, 230-260 g;

[0177] Drugs: Yeast (OXOID LP0021), aspirin (SIGMACF2093), protein CF2;

[0178] Instruments: electronic balance (SARTORIUSBP121S), electronic thermometer (CITIZEN CT-513W).

[0179] Experimental groups:

[0180] Normal control group;

[0181] Model group: yeast fever model;

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

[0183] Protein CF2, 10mg / kg group, 50mg / kg group.

[0184] method:

[0185] Animal Preparation: After acclimating to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for one day, animals were pre-acclimated and rectal temperature was measured at 8:00 AM and 3:00 PM. Animals were fasted but not watered for 12 hours prior to the experiment, and their feces were emptied before rectal temperature measurement. Before each temperature measurement, the electronic thermometer probe was coated with petroleum jelly and inserted 2 cm into the rat's rectum (a mark at the 2 cm mark was provided to ensure consistent insertion depth). Temperature was recorded after the reading stabilized.

[0186] Subcutaneous injection of dry yeast to induce a fever model in rats: Before modeling, rat body temperature was measured. Rats with a body temperature between 36.2 and 37.3°C were selected and randomly divided into groups of 8. Following oral administration of aspirin and varying doses of protein CF2, a 20% yeast suspension (10 ml / kg) was immediately subcutaneously injected. A normal control group received an equal volume of saline. Body temperature was monitored beginning 2 hours later and every 2 hours for a total of 8 hours.

[0187] Statistics:

[0188] According to the body temperature values ​​measured at each time point on the day of the experiment, the mean, standard deviation and standard error of body temperature of rats in each group were calculated. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be significantly different.

[0189] Experimental results:

[0190] After oral administration of aspirin (300 mg / kg) and protein CF2 (10 mg / kg, 50 mg / kg), 20% yeast was injected subcutaneously to establish the model. The body temperature of the animals was monitored 2, 4, 6, and 8 hours after model establishment. The results are shown in Table 2 and Figure 2.

[0191] Table 2. Effects of the test drugs on yeast-induced fever model in rats

[0192] (Compared with the normal control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01)

[0193] Experimental conclusion:

[0194] After oral administration of aspirin (300 mg / kg) and protein CF2 (10 mg / kg, 50 mg / kg), 20% yeast was subcutaneously injected immediately to establish the model. The body temperature of the animals was monitored 2 hours, 4 hours, 6 hours, and 8 hours after model establishment.

[0195] The results show:

[0196] 1) The body temperature of the rats in the model group increased significantly 2 hours, 4 hours, 6 hours, and 8 hours after modeling, compared with the normal group, P < 0.05, with statistically significant differences. The model was successfully established and was stable and reliable.

[0197] 2) The positive tool drug aspirin group can effectively inhibit the increase in body temperature of model rats at 2 hours, 4 hours, 6 hours, and 8 hours after modeling. Compared with the model group, P<0.05, there is a statistically significant difference, and the positive tool drug aspirin performs stably.

[0198] 3) Protein CF2 showed a long-lasting effect in suppressing body temperature increases. The 10 mg / kg dose group significantly suppressed body temperature increases in model rats 2, 4, and 6 hours after modeling, with statistically significant differences (P < 0.05) compared to the model group. The 50 mg / kg dose group significantly suppressed body temperature increases in model rats 2, 4, and 8 hours after modeling, with statistically significant differences (P < 0.01) compared to the model group.

[0199] Experimental Example 4: Pharmacological Effect of Protein CF2 (Protein from Example 7) on Pentylenetetrazol (PTZ)-Induced Epilepsy in Mice

[0200] Animals: Male ICR mice;

[0201] Drugs: Pentylenetetrazol (PTZ), Retigabine, Protein CF2.

[0202] Experimental groups:

[0203] Model group;

[0204] Retigabine 60 mg / kg group;

[0205] Protein CF2, 50mg / kg group, 200mg / kg group.

[0206] method:

[0207] Model preparation and drug administration:

[0208] The drug was administered once in the afternoon of the day before modeling. On the day of modeling, PTZ-65 mg / kg (modeling agent) was injected intraperitoneally 1 hour after gavage with the test drug. The positive drug could be administered once half an hour before modeling. Observation was continued for 15 minutes after PTZ injection. Observation criteria included: ① Seizure severity (grades III to VI): duration of seizures; ② mortality. Seizure grade: Racine grading scale: Grade 0: no reaction; Grade I: twitching of facial muscles or corners of the mouth; Grade II: nodding; Grade III: convulsions of one limb; Grade IV: rigidity or generalized limb twitching; Grade V: generalized grand mal seizure (generalized tonic convulsion).

[0209] Data processing:

[0210] The number of mice experiencing seizures and deaths in each group was counted. The incubation periods for stage III and IV seizures were calculated, with the maximum latency for mice that did not develop a stage IV seizure being 900 seconds. The chi-square test was used to calculate the mean and standard error of the incubation period. TTEST was used to compare the model group with the other groups. A P < 0.05 was considered significant.

[0211] Experimental results: see Table 5 and Table 6.

[0212] Table 5. Statistics of the number of cases of the test drugs in the PTZ-induced epilepsy experiment in mice

[0213] (Compared with the model group: *P<0.05, **P<0.01)

[0214] Table 6. Effects of the test drugs on the PTZ-induced epilepsy test in mice - latency of grade III and grade IV epilepsy seizures (mean ± SEM)

[0215] (Compared with the model group, *P<0.05, **P<0.01)

[0216] Experimental conclusion:

[0217] 1) The experimental results showed that the grade IV attack rate in the model group was 80% and the mortality rate was 10%. The model was successfully established.

[0218] 2) Positive drugs can significantly prolong the latency period of grade III and grade IV epileptic seizures in mice.

[0219] 3) In the comparison of epilepsy grade III latency, there were significant differences between the CF2-50mg / kg and CF2-200mg / kg groups and the model group.

Claims

1. A keratin CF2, characterized in that: The amino acid sequence of the keratin CF2 is: (1) the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; (2) An amino acid sequence substantially maintaining the same biological function as that of SEQ ID NO. 1 in the sequence listing formed by replacing, deleting or adding 1 to 45 amino acids.

2. Keratin CF2 according to claim 1, characterized in that Conventional modifications may be performed on keratin CF2; or a tag for detection or purification may be connected to keratin CF2; or a homologous protein of keratin CF2 may be connected to keratin CF2.

3. Keratin CF2 according to claim 2, characterized in that The conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamination, disulfide bond formation or disulfide bond cleavage; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

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

5. A nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule is: (1) the nucleotide sequence shown in SEQ ID NO.2 in the sequence listing; (2) a nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence complementary to the nucleotide sequence in (1) or (2) above.

6. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to any one of claims 4-5.

7. A host cell, characterized in that The host cell contains the expression vector according to claim 6 or the nucleic acid molecule according to any one of claims 4-5 is integrated into its genome.

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

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

10. A method for preparing the keratin CF2 according to any one of claims 1 to 3, characterized in that: The following steps are involved: A. synthesizing the nucleic acid molecule corresponding to the keratin CF2 according to any one of claims 1 to 3, linking the nucleic acid molecule into a corresponding expression vector, transforming the expression vector into a host cell, culturing the host cell with the expression vector in a fermentation device under certain conditions and inducing the expression of keratin CF2, and obtaining a crude protein solution containing keratin CF2; B. Separate, purify and dry the crude protein solution expressed in step A to obtain keratin CF2.

11. The method according to claim 10, characterized in that In step A, the host cell is mainly selected from Escherichia coli, the keratin CF2 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shake flask or a fermenter.

12. The method according to claim 10, characterized in that In step A, after inducing the expression of keratin CF2, impurities can be washed with a cleaning agent and dissolved with a solution to obtain a crude protein solution.

13. The method according to claim 10, characterized in that In step B, the separation and purification methods include ultrafiltration and microfiltration membrane purification methods, column chromatography purification methods, salting out methods, and dialysis methods.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the keratin CF2 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

15. Use of the keratin CF2 described in any one of claims 1 to 3, or the nucleic acid molecule described in any one of claims 4-5, or the expression vector described in claim 6, or the host cell described in claims 7-9, or the pharmaceutical composition described in claim 14 in the preparation of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs.

Images