Keratin CF3 as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to keratin CF3, a preparation method, a pharmaceutical composition and application thereof. In particular to keratin CF3, a nucleic acid molecule for coding the keratin CF3, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or genome integrated with the nucleic acid molecule, a preparation method of the keratin CF3 and a pharmaceutical composition containing the keratin CF3. The invention also discloses application of the keratin CF3, the nucleic acid molecule, the expression vector, the host cell or the pharmaceutical composition in preparation of antipyretic and analgesic drugs, antitussive and expectorant drugs, anticonvulsion drugs, antiepileptic drugs, antihypertensive drugs, anti-inflammatory drugs and antiviral drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology. It relates to a keratin CF3, a nucleic acid molecule encoding keratin CF3, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or the genome of which the nucleic acid molecule is integrated, as well as a preparation method of keratin CF3, a pharmaceutical composition containing the keratin, and the use of the keratin and the pharmaceutical composition in the preparation of 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 found 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 organisms and is a renewable resource with great utilization value, but it has not been widely and effectively utilized. The main reason is that keratin is insoluble in various solvents and is generally more resistant to protease hydrolysis than other proteins. Therefore, it is very difficult to extract and prepare 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 discovered. The use of protein expression systems to prepare and 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. Summary of the invention

[0006] The technical problem solved by the present invention is to provide a keratin CF3, a nucleic acid molecule encoding keratin CF3, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or the genome integrated with the nucleic acid molecule, as well as a preparation method of keratin CF3, a pharmaceutical composition containing keratin CF3, and the use of the above keratin CF3, nucleic acid molecules, expression vectors, host cells, or pharmaceutical compositions in the preparation of antipyretic and analgesic, antitussive and expectorant, anticonvulsant, antiepileptic, blood pressure lowering, anti-inflammatory, and antiviral drugs.

[0007] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention provides a keratin CF3, characterized in that the amino acid sequence of the keratin CF3 is:

[0009] (1) the amino acid sequence shown in SEQ ID NO.1 in the sequence listing;

[0010] (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 35 amino acids.

[0011] Furthermore, conventional modifications can be performed on keratin CF3; or a tag for detection or purification can be connected to keratin CF3.

[0012] 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, Profinity eXact.

[0013] The second aspect of the technical solution of the present invention is to provide a nucleic acid molecule encoding the keratin CF3 described in the first aspect.

[0014] Furthermore, the nucleotide sequence of the nucleic acid molecule is:

[0015] (1) the nucleotide sequence shown in SEQ ID NO.2 in the sequence listing;

[0016] (2) a nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO.2;

[0017] (3) A nucleotide sequence complementary to the nucleotide sequence in (1) or (2) above.

[0018] 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.

[0019] 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(+).

[0020] 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.

[0021] Furthermore, the host cell includes bacteria, yeast, Aspergillus, plant cells, or insect cells.

[0022] Furthermore, the bacteria include Escherichia coli or yeast.

[0023] The competent host cells can be BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; the preferred expression competent cells are BL21 (DE3) and Transetta (DE3).

[0024] The fifth aspect of the technical solution of the present invention provides a method for preparing the keratin CF3 described in the first aspect, characterized in that it comprises the following steps:

[0025] A. synthesizing the nucleic acid molecule corresponding to the keratin CF3 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 the expression of keratin CF3, and obtaining a crude protein solution containing keratin CF3;

[0026] B. Separate, purify and dry the crude protein solution expressed in step A to obtain keratin CF3.

[0027] Furthermore, in step A, the host cell is mainly selected from Escherichia coli, the keratin CF3 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shake flask or a fermenter.

[0028] Furthermore, in step A, after inducing the expression of keratin CF3, impurities can be cleaned with a cleaning agent and then dissolved with a urea solution to obtain a crude protein solution.

[0029] Furthermore, the culture medium in step A can be LB culture medium, TB culture medium, SB culture medium, SOB culture medium, SOC culture medium, PDA culture medium, YPD culture medium, Bengal rose culture medium, high salt Czapek culture medium, DOBA culture medium, rice koji culture medium and improved formula thereof; LB culture medium and TB culture medium are preferred for shake flask fermentation, and TB culture medium is most preferred; LB culture medium and improved formula thereof are preferred for fermentation tank.

[0030] Furthermore, the inducing agent in step A can be IPTG, lactose, arabinose, etc.; preferably IPTG and lactose.

[0031] 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 CF3 crude protein solution.

[0032] The buffer is preferably buffer A, and its dosage is: fermentation liquid volume: buffer A volume = 1-100:1, preferably 10:1;

[0033] The cleaning agent can be urea solution, guanidine hydrochloride solution, Triton, buffer A, etc., preferably urea solution, most preferably 2M urea solution (may contain 1% Triton) and 4M urea solution, and the dosage is: fermentation liquid volume: urea solution volume = 0.2-100:1, preferably 1-15:1;

[0034] The urea solution is preferably a 6M to 8M urea solution, and most preferably an 8M urea solution, and its dosage is: fermentation liquid volume: 8M urea volume = 0.2 to 100:1, preferably 2 to 15:1.

[0035] Furthermore, in step B, the separation and purification method includes ultrafiltration microfiltration membrane technology purification method, column chromatography purification method, salting out method, and dialysis method.

[0036] Further, in step B, the separation and purification method is as follows:

[0037] (1) The dialysis method is to purify the crude protein solution obtained in step A by dialysis to obtain the target protein CF3 solution.

[0038] 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.

[0039] (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 CF3.

[0040] (3) The column chromatography method is to separate and purify the crude protein solution obtained in step A by column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein CF3.

[0041] Preferred exclusion columns are dextran gel columns, Superdex 30Increase, Superdex 75Increase, Superdex 200Increase, Superose 6Increase, 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, HiTrapSP FF, HiTrap Capto SPImpRes, Capto SPImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M.

[0042] The eluent may be any eluent commonly used in the art, such as water, a saline solution, and the saline solution includes a sodium chloride solution, a sodium dihydrogen phosphate solution, a disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.

[0043] (4) The salting-out method is to purify the crude protein solution obtained in step A by salting-out to obtain a target protein CF3 suspension.

[0044] 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. Saturated ammonium sulfate aqueous solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, more preferably 25%.

[0045] The number of salting-outs is 1 to 3 times, preferably 2 times.

[0046] After salting out, the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.

[0047] Furthermore, the target protein CF3 solution obtained by purification in step B can be freeze-dried or vacuum-dried into dry powder, or the concentrated solution can be directly spray-dried into dry powder.

[0048] The sixth aspect of the technical solution of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin CF3 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.

[0049] 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.

[0050] The present invention relates to a pharmaceutical composition, comprising any one keratin obtained in the above steps and a pharmaceutically acceptable carrier.

[0051] 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. Usually, the keratin of the present invention accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.

[0052] The present invention also provides a pharmaceutical composition, which comprises a pharmaceutically effective dose of protein as an active ingredient and a pharmaceutically acceptable carrier.

[0053] The pharmaceutical composition of the present invention can be prepared according to methods known in the art. For this purpose, if necessary, the protein of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to prepare an appropriate administration form or dosage form that can be used as a human or veterinary drug.

[0054] The keratin or pharmaceutical composition containing the keratin of the present invention 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.

[0055] The administration route of the keratin or the pharmaceutical composition containing the keratin of the present invention can be injection, which includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, peritoneal injection and acupoint injection.

[0056] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc. Solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0057] The keratin of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0058] In order to prepare the unit dosage form into tablets, various excipients 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, isopropanol, 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, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and tectonic acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0059] 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.

[0060] In order to make 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, lauric acid macrogol glyceride, kaolin, talc, etc.; binders, such as gum arabic, yellow gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.

[0061] In order to prepare the administration unit as a suppository, various carriers known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.

[0062] In order to prepare the dosing unit into a capsule, the active ingredient keratin of the present invention is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient keratin of the present invention can also be prepared into a microcapsule, suspended in an aqueous medium to form a suspension, or loaded into a hard capsule or prepared as an injection for use.

[0063] For example, the keratin of the present invention is made into an injection preparation, such as a solution, a suspension solution, an emulsion, or a freeze-dried powder injection. This preparation can be aqueous or non-aqueous, and can contain one and / or more pharmacodynamically 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 sorbitol fatty acid esters, and the like. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH regulators, and the like can also be added. These adjuvants are commonly used in the art.

[0064] In addition, if necessary, colorants, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

[0065] 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.

[0066] 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 administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the pharmaceutical ingredients in the present invention is well known to those skilled in the art. It can be appropriately adjusted according to the actual amount of the drug contained in the final preparation of the keratin composition of the present invention to achieve the requirements of its therapeutic effective amount and complete the prevention or treatment purpose of the present invention. The daily suitable dosage range of the keratin of the present invention: the dosage of the keratin of the present invention is 0.01-500 mg / kg body weight, preferably 0.5-100 mg / kg body weight, more preferably 1-50 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms, depending on the clinical experience of the administering doctor and the dosage regimen including the use of other treatment methods. The total dosage required for each treatment can be divided into multiple doses or administered in 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.

[0067] The seventh aspect of the technical solution of the present invention provides the use of the keratin CF3 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.

[0068] In order to achieve the purpose of the present invention, the present invention adopts the following technical scheme. Specifically, the preparation of keratin CF3 of the present invention comprises the following steps:

[0069] (1) Synthesize nucleotide sequences and determine the accuracy of the sequences;

[0070] The preferred nucleotide sequence is shown as SEQ ID No.2.

[0071] (2) transferring the nucleotide sequence into an expression vector;

[0072] 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(+).

[0073] (3) transfecting the expression vector into host cells;

[0074] The host cell may be Escherichia coli or yeast; the preferred host cell is Escherichia coli;

[0075] The competent cells can be BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; the preferred expression competent cells are BL21 (DE3) and Transetta (DE3).

[0076] (4) fermenting and culturing the host cells under appropriate conditions to induce the expression of the target protein CF3;

[0077] Fermentation equipment can be shake flasks or fermentation tanks;

[0078] The culture medium can be LB culture medium, TB culture medium, SB culture medium, SOB culture medium, SOC culture medium, PDA culture medium, YPD culture medium, Bengal rose culture medium, high salt Czapek culture medium, DOBA culture medium, rice koji culture medium and improved formula thereof; LB culture medium and TB culture medium are preferred for shake flask fermentation, and TB culture medium is most preferred; LB culture medium and improved formula thereof are preferred for fermentation tank.

[0079] The inducer may be IPTG, lactose, arabinose, etc.; preferably IPTG and lactose.

[0080] (5) Enrichment of target protein CF3 product;

[0081] The fermented bacterial liquid obtained in step (4) is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacterial cells are broken, and the mixture 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 CF3 crude protein solution.

[0082] The buffer is preferably buffer A, and its dosage is: fermentation liquid volume: buffer A volume = 1-100:1, preferably 5-10:1;

[0083] The cleaning agent can be urea solution, guanidine hydrochloride solution, Triton, buffer A, etc., preferably urea solution, most preferably 3M urea solution (may contain 1% Triton), and the dosage is: fermentation liquid volume: urea solution volume = 0.2-100:1, preferably 1-15:1; the number of cleaning times is 3-10 times, preferably 6 times;

[0084] The urea solution is preferably a 6M to 8M urea solution, and most preferably an 8M urea solution. The dosage thereof is: fermentation broth volume: 8M urea solution volume = 0.2 to 100:1, and preferably 2 to 15:1.

[0085] (6) Isolation and purification of target protein CF3:

[0086] The crude protein solution obtained in step (5) needs to be purified to obtain the target protein CF3. The purification can be performed by dialysis, ultrafiltration, microfiltration, column chromatography, or salting out.

[0087] A. The dialysis step is to purify the crude protein solution obtained in step (5) by dialysis to obtain the target protein CF3 solution.

[0088] 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.

[0089] B. The ultrafiltration and microfiltration step is 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 CF3.

[0090] C. The column chromatography step is to separate and purify the crude protein solution obtained in step (5) by column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein CF3.

[0091] Preferred exclusion columns are dextran gel columns, Superdex 30Increase, Superdex 75Increase, Superdex 200Increase, Superose 6Increase, 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, HiTrapSP FF, HiTrap Capto SPImpRes, Capto SPImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M. Most preferred is an anion exchange resin column.

[0092] The eluent may be any eluent commonly used in the art, such as water, a saline solution, and the saline solution includes a sodium chloride solution, a sodium dihydrogen phosphate solution, a disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.

[0093] D. The salting-out step is to purify the crude protein solution obtained in step (5) by salting-out to obtain a target protein CF3 suspension.

[0094] 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. Saturated ammonium sulfate aqueous solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, more preferably 25%.

[0095] The number of salting-outs is 1 to 3 times, preferably 2 times.

[0096] After salting out, the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.

[0097] The target protein CF3 solution obtained by purification in steps A to D can be freeze-dried or vacuum-dried into dry powder, or the concentrated solution can be directly spray-dried into dry powder.

[0098] Beneficial technical effects of the present invention:

[0099] 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.

[0100] 2. The present invention studies the efficacy of protein CF3 on the yeast-induced SD rat fever model, proving that protein CF3 (10 mg / kg) has a significant effect of reducing the elevated body temperature of the model animals 4 hours after modeling;

[0101] 3. The present invention uses a pharmacodynamic study of protein CF3 on convulsions and epilepsy caused by the convulsant pilocarpine (PLO) in mice, proving that protein CF3 (200 mg / kg) can significantly prolong the latency of epilepsy grade IV in mice; and uses a pharmacodynamic study of protein CF3 on epilepsy caused by pentylenetetrazol (PTZ) in mice, proving that protein CF3 (200 mg) can significantly prolong the latency of epilepsy grade III in mice;

[0102] 4. The present invention uses a drug efficacy test of protein CF3 on ammonia-induced cough in mice to prove that protein CF3 (20 mg / kg) can reduce the number of coughs and has an antitussive effect;

[0103] 5. The present invention studies the efficacy of protein CF3 on expectoration by phenol red excretion method in mice, and proves that protein CF3 (20 mg / kg, 50 mg / kg) has obvious expectorant effect at two doses; BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 . Effect of protein CF3 on yeast-induced fever model in rats (compared with the normal control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01) DETAILED DESCRIPTION

[0105] 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.

[0106] 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.

[0107] Example 1 Preparation of protein CF3 crude solution A by shake flask fermentation (TB medium)

[0108] The nucleotide sequence shown in SEQ ID No. 2 was synthesized and transferred into the pET-28a(+) vector; the expression vector containing the correct sequence was obtained by sequencing; 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 in a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.

[0109] The recombinant strain was 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.

[0110] Prepare 400 ml TB medium and divide it into 2 bottles, each containing 200 ml. Add kanamycin (final concentration 50 μg / ml) to each bottle (200 ml) of TB medium, take a single colony from the plate and add it to the TB medium, and culture it overnight in a shaker at 37°C and 220 rpm to obtain seed solution.

[0111] Prepare 28.8L TB medium and divide 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 at 37℃ and 220rpm in a shaker for 2-3 hours. Monitor OD 600 , when OD 600 When it reaches about 1.0, add the inducer and induce protein expression in a shaker. The induction conditions are selected from the table below.

[0112]

[0113] Combine the bacterial solutions from each bottle, centrifuge at 7000rpm for 5 minutes, and discard the supernatant after sterilization; suspend the precipitate in about 3L of buffer, filter with an 80-100 mesh screen, and crush the filtrate with a high-pressure crusher at a pressure of 800-1000 bar, twice, and 2 minutes each time. After crushing, centrifuge the bacterial solution at 7000rpm for 30 minutes, discard the supernatant, and obtain the precipitate (i.e., inclusion body). Add detergent to the precipitate for washing (1L 3M urea-1% triton solution 3 times, 1L 3M urea solution 2 times), centrifuge, and discard the supernatant. Add 1L 8M urea solution to dissolve the precipitate, which is the crude protein solution A.

[0114] Example 2 Preparation of protein CF3 crude solution B by shake flask fermentation (other culture medium)

[0115] In Example 1, an expression vector containing the sequence shown in SEQ ID No. 2 was synthesized and sequenced to obtain; the expression vector was transfected into BL21 (DE3) cells to obtain expression competent host cells containing the target nucleotide sequence.

[0116] Prepare 20 ml 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.

[0117] 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.

[0118] Take 10 ml of LB medium, add kanamycin (final concentration 50 μg / ml), take a single colony on the plate and add it to the LB medium, and culture it overnight in a shaker at 37°C and 220 rpm for 15 hours to obtain seed solution.

[0119] 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 at 37℃ and 220rpm in a shaker for 2-3 hours. Monitor OD 600 , when OD 600 When the concentration 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.

[0120] Culture medium LB medium, SOB medium, SOC medium

[0121] Combine the bacterial solutions from each bottle, centrifuge at 10,000 rpm for 10 minutes, and discard the supernatant after sterilization; suspend the precipitate in about 100 mL of buffer, filter with an 80-100 mesh screen, and crush the filtrate with a high-pressure crusher at a pressure of 800-1000 bar, twice, 2 minutes each time. After crushing, centrifuge the bacterial solution 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 3 M urea solution (containing 1% triton), centrifuged, and the supernatant was discarded; the precipitate was washed twice with 40 mL of 3 M urea solution, centrifuged, and the supernatant was discarded; the precipitate was then washed twice with 40 mL of 8 M urea solution, which was the crude protein solution B.

[0123] Example 3 Preparation of protein CF3 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 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 in 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 the LBA plate containing kanamycin, spread it with a spreader until it is evenly dry, and invert the plate to culture overnight in a 37°C constant temperature incubator. Take three single colonies and streak them on the plate containing kanamycin, culture them overnight on the plate, and after three batches of shake flask fermentation expression verification, preserve the strain with 15% glycerol and divide it into 0.8 ml each to obtain the working cell bank, which is frozen in a -80°C refrigerator for use.

[0126] Take out a glycerol bacteria 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 primary seed solution.

[0127] Take 1.2 ml of the primary seed solution, 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 secondary seed solution.

[0128] Add 3L of modified LB culture medium to a 5L fermenter, then add 120ml of secondary seed liquid, 3ml of Kanamycin (final concentration 50μg / ml), and culture at 37°C, 30% dissolved oxygen (tandem speed) for about 8 hours. Monitor the OD value at around 20, use 3g of lactose as an inducer, induce at 20°C, feed at a rate of 30ml / hour, and culture at 20°C for 24 hours.

[0129] The bacterial solution was centrifuged at 7000 rpm for 5 minutes, and the supernatant was discarded after sterilization; the precipitate was suspended in about 600 ml of buffer A, filtered with a 80-100 mesh screen, and the filtrate was crushed with a high-pressure crusher at a pressure of 800-1000 bar, twice, each time for 2 minutes. After crushing, the bacterial solution was centrifuged at 7000 rpm for 30 minutes, and the supernatant was discarded.

[0130] The precipitate was washed 3 times with 1L of 3M urea solution (containing 1% Triton); then washed twice with 1L of 3M urea solution, centrifuged, and the supernatant was discarded; then washed once with 1L of 4M urea solution, centrifuged, and the supernatant was discarded. The precipitate was added with 2L of 8M urea solution to obtain the crude protein solution C.

[0131] Example 4 Preparation of protein CF3 by dialysis of crude protein solution A

[0132] The crude protein solution A obtained in Example 1 was filtered with a 0.45 μm filter membrane, and the filtrates were combined. The filtrates were dialyzed with water, the molecular weight cutoff of the dialysis bag was 10 kD, and the dialysis was performed for 72 hours. The inner solution was freeze-dried to obtain the target protein CF3.

[0133] Protein CF3 structure confirmation--protein full 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 chromatograph (Thermo Ultimate 3000), electrospray ion trap-Orbitrap mass spectrometer (Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer).

[0136] Methods and Results:

[0137] Protein CF3 was pre-treated by dissolution replacement, reductive alkylation, and multiple proteolysis to obtain enzyme-cleaved peptides; the enzyme-cleaved 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, and the identification result confirmed that it was consistent with the target sequence SEQ ID No.1.

[0138] Example 5 Protein crude solution A was purified by salting out to obtain protein CF3

[0139] The crude protein solution A is placed in a stirred container for two salting-outs: a saturated ammonium sulfate solution is slowly added along the wall to make the final concentration of ammonium sulfate 25% or 50%. During the salting-out process, the protein is precipitated. After the salting-out is complete, it is filtered to complete the first salting-out; 400 ml of pure water is added to the precipitate for suspension, and a saturated ammonium sulfate solution is slowly added along the wall again to make the final concentration of ammonium sulfate 25%. The second salting-out is performed, filtered, and the precipitate is the crude protein extract. The crude protein extract is washed three times with water: 200 ml of pure water is added for suspension, stirred, allowed to stand, and filtered; after repeating this three times, the precipitate is freeze-dried to obtain the target protein CF3.

[0140] The same structure confirmation method as in Example 4 confirmed that it 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 CF3

[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 with a 0.45 μm membrane, and the filtrate was dialyzed with water for more than 72 hours. The inner solution was freeze-dried to obtain the target protein CF3.

[0145] Dialysis Bags Molecular weight cut-off: 0.5kD, 3.5kD, 5kD, 10kD

[0146] The second method: salting out;

[0147] The crude protein solution B is placed in a stirred container for two salting-outs: saturated ammonium sulfate solution is slowly added along the wall to make the final concentration of ammonium sulfate 25% or 50%. During the salting-out process, the protein is precipitated. After the salting-out is complete, it is filtered to complete the first salting-out; 400 ml of pure water is added to the precipitate for suspension, and saturated ammonium sulfate solution is slowly added along the wall again to make the final concentration of ammonium sulfate 25%. The second salting-out is performed, filtered, and the precipitate is the crude protein extract. The crude protein extract is washed three times with water: 200 ml of pure water is added for suspension, stirred, allowed to stand, and filtered; after repeating this three times, the precipitate is freeze-dried to obtain the target protein CF3.

[0148] The product protein CF3 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.

[0149] Example 7: Purification of crude protein solution C to obtain protein CF3

[0150] 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 inner solution is freeze-dried to obtain the target protein CF3.

[0151] The same structure confirmation method as in Example 4 confirmed that it had the same amino acid sequence as the protein prepared in Example 4.

[0152] Pharmacological tests

[0153] Experimental Example 1: Efficacy test of protein CF3 (protein of Example 7) on yeast-induced SD rat fever model

[0154] Animals: Male SD rats 230-260 g;

[0155] Drugs: yeast (OXOID LP0021), aspirin (SIGMA A2093), protein CF3;

[0156] Instruments: electronic balance (SARTORIUSBP121S), electronic thermometer (CITIZEN CT-513W).

[0157] Experimental groups:

[0158] Normal control group;

[0159] Model group: yeast fever model;

[0160] Positive control group: Aspirin 300 mg / kg group;

[0161] Protein CF3, 10mg / kg group, 50mg / kg group.

[0162] method:

[0163] Preparation of experimental animals: After the experimental animals were adapted to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day, they were pre-adapted to measure rectal temperature at 8:00 and 15:00 respectively. They were fasted but not watered for 12 hours before the experiment, and the animals were allowed to empty their feces before measuring rectal temperature. Before each temperature measurement, the electronic thermometer probe was coated with vaseline and inserted into the rat rectum 2cm (a mark can be made at 2cm to ensure the same insertion depth each time), and the body temperature value was recorded after the reading stabilized.

[0164] Subcutaneous injection of dry yeast to replicate the fever model in rats: The body temperature of rats was measured before modeling, and qualified rats with a body temperature of 36.2-37.3℃ were screened and randomly divided into groups of 8 rats in each group. After oral administration of aspirin and different doses of protein CF3, 20% yeast suspension (10ml / kg) was immediately subcutaneously injected, and the normal control group was subcutaneously injected with an equal volume of normal saline. The body temperature of rats was monitored 2 hours later, and the body temperature was monitored once every 2 hours for a total of 8 hours.

[0165] Statistics:

[0166] 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 among the groups using TTEST. P<0.05 was considered to be significantly different.

[0167] Experimental results:

[0168] After oral administration of aspirin (300 mg / kg) and protein CF3 (10 mg / kg, 50 mg / kg), 20% yeast was subcutaneously injected immediately to establish the model. The body temperature of the animals was monitored at 2 hours, 4 hours, 6 hours, and 8 hours after the establishment of the model. The results are shown in Table 1 and Figure 1 .

[0169] Table 1. Effects of the tested drugs on yeast-induced fever model in rats

[0170]

[0171] (Compared with the normal control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01)

[0172] Experimental conclusion:

[0173] After oral administration of aspirin (300 mg / kg) and protein CF3 (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.

[0174] The results show:

[0175] 1) The body temperature of the rats in the model group increased significantly at 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.

[0176] 2) The positive tool drug aspirin group could effectively inhibit the increase in body temperature of the model rats at 2 hours, 4 hours, 6 hours, and 8 hours after modeling. Compared with the model group, P<0.05, there was a statistically significant difference, and the positive tool drug aspirin performed stably.

[0177] 3) The 50 mg / kg dose group of protein CF3 at 2 hours after modeling and the 10 mg / kg dose group at 4 hours after modeling could significantly inhibit the increase in body temperature of the model rats, and compared with the model group, P<0.05, there was a statistically significant difference.

[0178] Experimental Example 2: Efficacy of protein CF3 (protein of Example 7) on convulsion epilepsy in mice induced by pilocarpine (PLO)

[0179] Animals: Male ICR mice;

[0180] Drugs: Pilocarpine HCl (PLO, pilocarpine, pilocarpine hydrochloride), Diazepam (diazepam tablets), protein CF3.

[0181] Experimental groups:

[0182] Model group;

[0183] Diazepam 2 mg / kg group;

[0184] Protein CF3, 50mg / kg group, 200mg / kg group.

[0185] method:

[0186] Model preparation and drug administration:

[0187] The drug was administered once in the afternoon of the day before modeling. On the day of modeling, PLO-225 mg / kg (modeling agent) was injected intraperitoneally 1 hour after oral administration of the test drug. The positive drug was administered once 20 minutes before modeling. After the injection of PLO, the animals were observed for 30 minutes.

[0188] Observation indicators: ① epileptic seizure condition: time of grade II to grade IV seizures; ② time of death.

[0189] Seizure level: Refer to the Racine grading standard: Level 0: no reaction; Level I: twitching of facial muscles or corners of mouth; Level II: can nod; Level III: convulsion of one limb; Level IV: rigidity or whole body limb convulsion; Level V: generalized epileptic seizure (generalized tonic convulsion).

[0190] Data processing:

[0191] The number of grade IV attacks and deaths in each group of mice in the experiment was counted; the incubation period of grades II, III and IV, and the incubation period of mice that did not attack grade IV was recorded as the maximum value of 1800 seconds. The number of cases was statistically analyzed using the chi-square test. The mean and standard error of the incubation period were calculated, and TTEST was used to compare the model group with other groups. P<0.05 was considered to be significantly different.

[0192] Experimental results: see Table 2 and Table 3.

[0193] Table 2. The effect of the tested drugs on PLO-induced epilepsy in mice - case statistics

[0194] Group Number of experimental cases Number of cases with grade IV Grade IV seizure rate Number of deaths mortality rate Model Group 8 7 88％ 0 0 Diazepam 2 mg / kg 8 0** 0 0 0 CF3 50mg / kg 8 7 88％ 1 13％ CF3 200mg / kg 8 3 38％ 1 13％

[0195] (Compared with the model group, **P<0.01)

[0196] Table 3. Effects of the tested drugs on PLO-induced epilepsy in mice - latency of grade II, grade III and grade IV epilepsy seizures (mean ± SEM)

[0197]

[0198] (Compared with the model group, *P<0.05, **P<0.01)

[0199] Experimental conclusion:

[0200] 1) The experimental results showed that the IV attack rate in the model group was 88%, and the mortality rate was 0.

[0201] 2) Positive drugs can significantly inhibit the incidence of grade IV epilepsy, and significantly prolong the latency period of grade III and grade IV epilepsy attacks in mice and reduce the grade of attacks.

[0202] 3) In the comparison of epilepsy grade IV latency, there was a statistically significant difference between the CF3 200 mg / kg dose group and the model group.

[0203] Experimental Example 3: Pharmacological Effect of Protein CF3 (Protein of Example 7) on Epilepsy in Mice Induced by Pentylenetetrazol (PTZ)

[0204] Animals: Male ICR mice;

[0205] Drugs: pentylenetetrazol (PTZ), diazepam, protein CF3.

[0206] Experimental groups:

[0207] Model group;

[0208] Diazepam 2 mg / kg group;

[0209] Protein CF3, 50mg / kg group, 200mg / kg group.

[0210] method:

[0211] Model preparation and drug administration:

[0212] The drug was given once in the afternoon before modeling. On the day of modeling, PTZ-65mg / kg (modeling agent) was injected intraperitoneally 1 hour after gavage of the test drug. The positive drug was given once half an hour before modeling. Observation continued for 15 minutes after injection of PTZ. Observation indicators: ① Epileptic seizure: Grade III to Grade VI seizure time; ② Death Seizure level: Refer to Racine grading standard: Grade 0: No reaction; Grade I: Twitching of facial muscles or corners of mouth; Grade II: Can nod; Grade III: Convulsion of one limb; Grade IV: Tonic or whole body limb convulsion; Grade V: Generalized epileptic seizure (generalized tonic convulsion).

[0213] Data processing:

[0214] The number of attacks and deaths of mice in each group in the experiment was counted; the incubation period of level III and level IV, the incubation period of mice that did not attack to level IV was recorded as the maximum value of 900 seconds. The number of cases was statistically analyzed using the chi-square test. The mean and standard error of the incubation period were calculated, and TTEST was used to compare the model group with other groups. P<0.05 was considered to be significantly different.

[0215] Experimental results: see Table 4 and Table 5.

[0216] Table 4. Number of cases of the tested drugs in the PTZ-induced epilepsy experiment in mice

[0217]

[0218]

[0219] (Compared with the model group: *P<0.05, **P<0.01)

[0220] Table 5. Effects of the tested drugs on PTZ-induced epilepsy in mice - latency of grade III and grade IV epilepsy seizures (mean ± SEM)

[0221] Group Level III seizure latency (seconds) Level IV seizure latency (seconds) Seizure level Model Group 63±3 302±118 3.8±0.1 Diazepam 2 mg / kg 187±26** 900±0** 3.0±0.0** CF3 50mg / kg 67±4 319±115 3.8±0.1 CF3 200mg / kg 70±2* 406±126 3.8±0.2

[0222] (Compared with the model group, *P<0.05, **P<0.01)

[0223] Experimental conclusion:

[0224] 1) The experimental results showed that the grade IV attack rate in the model group was 78% and the mortality rate was 0. The model was successfully established.

[0225] 2) Positive drugs can significantly prolong the latency period of grade III and IV epileptic seizures in mice.

[0226] 3) In the comparison of the latency period of epilepsy grade III, the latency period of the CF3 200mg / kg group was significantly prolonged, which was significantly different from the model group.

[0227] Experimental Example 4: Efficacy of protein CF3 (protein from Example 7) on antitussive effect of ammonia-induced cough in mice

[0228] Animals: Male ICR mice;

[0229] Drugs and reagents: dextromethorphan hydrobromide, ammonia water, 0.2% CMC-Na, protein CF3;

[0230] Instruments: Compressor nebulizer (403T model), balance (XS105DU model).

[0231] Experimental groups:

[0232] solvent control group;

[0233] Dextromethorphan 15 mg / kg group;

[0234] Protein CF3, 20mg / kg group, 50mg / kg group.

[0235] method:

[0236] Model preparation and drug administration:

[0237] Dextromethorphan and different doses of protein CF3 (dosage volume 10 ml / kg) were orally administered according to the grouping. The solvent control group was given an equal volume of distilled water. After 1 hour, the mice were placed in a sealed box and nebulized with 10% ammonia water for 10 seconds. Then the cough latency and the number of coughs within 2 minutes of the mice were observed and recorded.

[0238] Data processing:

[0239] The time points of oral administration, aerosol experiment, the cough latency of mice and the number of coughs within 2 minutes were recorded respectively. The cough latency refers to the number of seconds required from the start of aerosol ammonia to the occurrence of cough. The coughing of mice is characterized by the contraction of their abdominal muscles (chest shrinkage) and the opening of their mouths. The mean and standard error of each group of data were calculated, and TTEST was used to compare the model group with other groups. P<0.05 was considered to be significantly different.

[0240] Experimental results:

[0241] Dextromethorphan (15 mg / kg) and different doses of protein CF3 (20 mg / kg, 50 mg / kg) were given in advance, and 1 hour later, the mice were placed in a sealed box and atomized with 10% ammonia for 10 seconds, and then the cough latency and the number of coughs within 2 minutes of the mice were observed and recorded. The results are shown in Table 6.

[0242] Table 6. Antitussive efficacy of the tested drugs on ammonia-induced cough in mice (X±SEM)

[0243] Group N Latency (seconds) P Number of coughs P Solvent control group 9 28.1±2.2 71.4±3.2 Dextromethorphan 15 mg / kg 9 36.5±2.0* 0.015 52.0±2.7** 0.001 CF3 20mg / kg 9 28.4±2.2 0.938 57.6±1.3** 0.001 CF3 50mg / kg 9 29.1±2.3 0.758 62.9±3.1 0.077

[0244] (Compared with the solvent control group, *P<0.05, **P<0.01)

[0245] Experimental conclusion:

[0246] 1) The experimental results showed that the dextromethorphan group had a significant improvement in the latency period and the number of coughs compared with the solvent control group (P<0.05), which was statistically significant.

[0247] 2) The number of coughs in the CF3 20 mg / kg group was statistically significant compared with the solvent control group.

[0248] Experimental Example 5: Efficacy of protein CF3 (protein of Example 7) on expectoration by phenol red excretion method in mice

[0249] Animals: Male ICR mice;

[0250] Drugs and reagents: Mucosolvan (Ambroxol Hydrochloride Tablets), phenol red, sodium bicarbonate, protein CF3;

[0251] Instruments: centrifuge (Sigma-3K15), balance (XS105DU), enzyme label tester (BIO-TEK).

[0252] Experimental groups:

[0253] solvent control group;

[0254] Mucosolvan 30 mg / kg group;

[0255] Protein CF3, 20mg / kg group, 50mg / kg group.

[0256] method:

[0257] Model preparation and drug administration:

[0258] Animals were fasted but not watered for 16 hours before the experiment. Mucosolvan and different doses of protein CF3 (administered in a volume of 10 ml / kg) were orally administered according to the grouping. The solvent control group was given an equal volume of distilled water. One hour later, 2.5% phenol red solution was injected intraperitoneally. The mice were killed by dislocation of the neck 30 minutes later. The trachea from the thyroid cartilage to the front of the tracheal branch was taken, and the trachea was placed in 3 ml of 5% NaHCO3 solution and allowed to stand for 3 hours. 1 ml of the supernatant was taken, and after centrifugation at 3000 rpm for 5 minutes, the absorbance was measured and recorded at 546 nm. The excretion of phenol red was calculated according to the standard curve of phenol red.

[0259] Data processing:

[0260] The time points of oral administration, intraperitoneal injection of 2.5% phenol red solution, and tracheal sampling were recorded respectively; the absorbance of each group of samples was measured at 546nm by an enzyme marker, and the excretion of phenol red was calculated according to the standard curve of phenol red. The mean and standard error of each group of data were calculated, and TTEST was used to compare the solvent control group with other groups. P<0.05 was considered to be significantly different.

[0261] Experimental results:

[0262] Mucosolvan (30 mg / kg) and different doses of protein CF3 (20 mg / kg, 50 mg / kg) were given, and 2.5% phenol red solution was injected intraperitoneally 1 hour later. The mice were killed by dislocation of the neck 30 minutes later. The trachea from the thyroid cartilage to the front of the tracheal branch was taken, and the trachea was placed in 3ml 5% NaHCO3 solution and left to stand for 3 hours. 1ml of the supernatant was taken, and after centrifugation at 3000rpm for 5 minutes, the absorbance was measured and recorded at 546nm. The excretion of phenol red was calculated according to the standard curve of phenol red. The results are shown in Table 7.

[0263] Table 7. Expectorant efficacy of the tested drugs in mice by phenol red excretion method (X±SEM)

[0264] Group N Phenol red excretion (μg / ml) P Solvent control group 8 0.499±0.054 Mucosolvan 30mg / kg 8 0.866±0.052** 0.001 CF3 20mg / kg 8 0.777±0.100* 0.028 CF3 50mg / kg 8 0.769±0.048** 0.002

[0265] (Compared with the solvent control group, *P<0.05, **P<0.01)

[0266] Experimental conclusion:

[0267] 1) The experimental results showed that the excretion of phenol red in the Mucosolvan 30 mg / kg group was significantly increased compared with the solvent control group, P<0.05, which was statistically significant.

[0268] 2) Compared with the solvent control group, the 20 mg / kg and 50 mg / kg CF3 groups showed significantly increased phenol red excretion, which was statistically significant.

Claims

1. A keratin CF3, characterized in that The amino acid sequence of the keratin CF3 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 CF3 according to claim 1, characterized in that Conventional modifications may be performed on keratin CF3; or a tag for detection or purification may be connected to keratin CF3; or a homologous protein of keratin CF3 may be connected to keratin CF3.

3. Keratin CF3 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 CF3 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 CF3 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 CF3 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 CF3, and obtaining a crude protein solution containing keratin CF3; B. Separate, purify and dry the crude protein solution expressed in step A to obtain keratin CF3.

11. The method according to claim 10, characterized in that In step A, the host cell is mainly selected from Escherichia coli, the keratin CF3 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 CF3, 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 CF3 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

15. Use of the keratin CF3 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.

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