A keratin YK93-2, its preparation method, pharmaceutical composition thereof, and its uses

Abstract

This invention belongs to the field of biopharmaceuticals and provides a keratin YK93-2, its encoding nucleic acid molecule, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or whose genome integrates the nucleic acid molecule, as well as a preparation method and a pharmaceutical composition thereof. It also provides the application of the above-mentioned keratin YK93-2 and other products in the preparation of drugs for treating lung cancer, lymphoma, breast cancer, melanoma, uterine fibroids, benign prostatic hyperplasia, etc.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals and relates to a keratin YK93-2, a nucleic acid molecule encoding keratin YK93-2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or whose genome integrates the nucleic acid molecule, a method for preparing keratin YK93-2, a pharmaceutical composition containing this keratin, and the use of this keratin and the pharmaceutical composition in the preparation of drugs for the prevention or treatment of benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, breast cancer, lactation, analgesia, uterine fibroids, and coagulation. Background Technology

[0002] Keratin is a structural protein of ectoderm cells, and is divided into two main categories based on whether it becomes fibrotic: soft keratin and hard keratin. Hard keratin is a major component of hair, feathers, hooves, shells, claws, horns, scales, etc., and is an important structural protein in connective tissue, playing a role in protecting the body.

[0003] Because hard keratin is not easily soluble in various solvents, and keratin is generally more resistant to enzymatic hydrolysis by proteases than other proteins, current research on keratin focuses more on intracellular soft keratin. However, research on hard keratin, which accounts for the majority of natural resources, namely hard keratin in animal body coverings (hooves, horns, shells, etc.), is limited and has not been effectively utilized, and is usually discarded as waste.

[0004] With the rapid development of modern biotechnology such as genomics, proteomics, genetic engineering, and microbial engineering, the application research of keratin in materials, biology, and medicine has gradually attracted attention. Therefore, the preparation and production of keratin using protein expression systems, followed by the study of its pharmacological effects and the development of new drugs using keratin as the active ingredient, is novel and innovative. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a keratin YK93-2, a nucleic acid molecule encoding keratin YK93-2, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or whose genome integrates the nucleic acid molecule, a method for preparing keratin YK93-2, a pharmaceutical composition containing keratin YK93-2, and the application of the above-mentioned keratin YK93-2, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the preparation of drugs for benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, breast cancer, lactation, analgesia, uterine fibroids, and coagulation.

[0006] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0007] The first aspect of the present invention is to provide a keratin YK93-2, characterized in that the amino acid sequence of the keratin YK93-2 is as follows:

[0008] (1) The amino acid sequence shown in SEQ ID NO.1 of the sequence listing;

[0009] (2) The amino acid sequence that retains the same biological function as the amino acid sequence shown in SEQ ID NO.1 in the sequence listing, formed by replacing, deleting or adding 1-35 amino acids.

[0010] Furthermore, conventional modifications can be performed on keratin YK93-2; or tags for detection or purification can be attached to keratin YK93-2.

[0011] 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, formation of disulfide bonds, or disulfide bond cleavage; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

[0012] The second aspect of the present invention is to provide a nucleic acid molecule encoding the keratin YK93-2 described in the first aspect.

[0013] Furthermore, the nucleotide sequence of the nucleic acid molecule is as follows:

[0014] (1) The nucleotide sequence shown in SEQ ID NO.2 in the sequence listing;

[0015] (2) The nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO.2;

[0016] (3) A nucleotide sequence complementary to the nucleotide sequence in (1) or (2) above.

[0017] A third aspect 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.

[0018] 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-30a(+).

[0019] The fourth aspect of the present invention is to provide 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 the genome.

[0020] Furthermore, the host cells include bacteria, yeast, Aspergillus, plant cells, or insect cells.

[0021] Furthermore, the bacteria mentioned include Escherichia coli or yeast.

[0022] The 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.; the preferred competent expression cells are BL21(DE3) and Transetta(DE3).

[0023] The fifth aspect of the present invention is to provide a method for preparing the keratin YK93-2 described in the first aspect, characterized by comprising the following steps:

[0024] A. Synthesize the nucleic acid molecule corresponding to keratin YK93-2 as described in the first aspect, link the nucleic acid molecule into the corresponding expression vector, transform the expression vector into host cells, culture the host cells with the expression vector in a fermentation device under certain conditions and induce the expression of keratin YK93-2 to obtain a crude protein solution containing keratin YK93-2;

[0025] B. The crude protein solution expressed in step A was separated, purified, and dried to obtain keratin YK93-2.

[0026] Furthermore, in step A, the host cell is mainly selected from Escherichia coli, the keratin YK93-2 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shake flask or a fermenter.

[0027] Furthermore, in step A, after inducing the expression of keratin YK93-2, impurities can be cleaned with a cleaning agent, and a crude protein solution can be obtained by dissolving the protein in a solution.

[0028] 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's medium, DOBA medium, koji medium and their modified formulations, etc.; LB medium is preferred for shake flask fermentation; LB medium and its modified formulations are preferred for fermentation tanks.

[0029] Furthermore, the inducing agent in step A can be IPTG, lactose, arabinose, etc.; preferably IPTG or lactose.

[0030] Further, in step A, the obtained fermentation broth is centrifuged and the supernatant is discarded; the precipitate is suspended in buffer solution, the cells are broken, centrifuged again, and the supernatant is discarded; the precipitate is washed with a cleaning agent and then dissolved in urea solution to obtain YK93-2 crude protein solution.

[0031] The buffer solution is preferably buffer A, and its volume is: fermentation broth volume: buffer A volume = 1~100:1, preferably 10:1;

[0032] 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 liquid volume : 1M urea (which may contain 1% Triton) volume = 0.2~100:1, preferably 1~15:1;

[0033] The preferred urea solution is an 8M urea solution, and its dosage is: fermentation liquid volume : 8M urea volume = 0.2~100:1, preferably 2~15:1.

[0034] Furthermore, in step B, the separation and purification methods include ultrafiltration / microfiltration membrane purification, column chromatography purification, salting out, and dialysis.

[0035] Furthermore, in step B, the separation and purification methods are as follows:

[0036] (1) The dialysis method is to purify the crude protein solution obtained in step A by dialysis to obtain the target protein YK93-2 solution.

[0037] The molecular weight cutoff of the dialysis bag can be 0.5-10kD, with a preferred molecular weight cutoff of 3.5-10kD and an optimal molecular weight cutoff of 10kD.

[0038] (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 YK93-2.

[0039] Preferably, the purification is carried out by two microfiltration membranes, with the first membrane having a pore size of 1000-1500 nm and the second membrane having a pore size of 20-50 nm.

[0040] (3) The column chromatography method is to pass the crude protein solution obtained in step A through column chromatography, such as various exchange columns or size exclusion columns, to separate and purify the target protein YK93-2.

[0041] Preferred size exclusion columns are dextran gel columns, such as Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, and Superose 6 Increase; preferred exchange columns are ion exchange resin columns: anion exchange resin columns, such as HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, and Toyopearl SuperQ-650M; and cation exchange resin columns, such as HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, and Toyopearl Super SP-650M. The most preferred are anion exchange resin columns.

[0042] The eluent can be a commonly used eluent in the art, such as water or a salt solution, including sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, etc.

[0043] (4) The salting-out method is to purify the crude protein solution obtained in step A by salting out to obtain the target protein YK93-2 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. A saturated ammonium sulfate aqueous solution is added to achieve a final ammonium sulfate concentration of 10–50%, preferably 20–30%, and more preferably 25%.

[0045] The salting-out process is performed 1 to 3 times, preferably 2 times.

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

[0047] Furthermore, the target protein YK93-2 solution obtained from step B purification 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 present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition contains keratin YK93-2 as described in the first aspect, or a nucleic acid molecule as described in the second aspect, or an expression vector as described in the third aspect, or a host cell as described in the fourth aspect, as well as a pharmaceutically acceptable carrier or excipient.

[0049] The keratin obtained in the above steps of this 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] This invention relates to a pharmaceutical composition comprising any keratin obtained in the above steps and a pharmaceutically acceptable carrier.

[0051] This invention also relates to pharmaceutical compositions containing the keratin of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants. Typically, the keratin of the present invention comprises 0.1% to 100.0% of the total weight of the pharmaceutical composition.

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

[0053] The pharmaceutical compositions described in this invention can be prepared according to methods known in the art. For this purpose, if desired, the proteins of this invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to formulate suitable administration or dosage forms for use as human or veterinary medicine.

[0054] The keratin of the present invention or a pharmaceutical composition containing it can be administered in unit dose form, and the route of administration can be enteric or non-enteric, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, skin, vagina, peritoneum, rectum, etc., with oral administration being preferred.

[0055] The keratin or pharmaceutical composition containing it of the present invention can be administered via injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, and acupoint injection.

[0056] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil, and double 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, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays; semi-solid dosage forms can include ointments, gels, and pastes.

[0057] The keratin of this invention can be formulated into ordinary preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0058] To formulate unit-dose dosage forms into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate; the lubricant and flow aid can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0059] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0060] To formulate the drug delivery unit into a pellet, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol glycerol laurate, kaolin, talc, etc.; binders such as gum arabic, yellow gum, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

[0061] To formulate the drug delivery unit into a suppository, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.

[0062] To encapsulate the drug delivery unit, the active ingredient, 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 a soft capsule. Alternatively, the active ingredient, keratin of the present invention, can be formulated as a microcapsule, suspended in an aqueous medium to form a suspension, or filled into a hard capsule or formulated as an injectable preparation.

[0063] For example, the keratin of this invention can be formulated into injectable preparations, such as solutions, suspension solutions, emulsions, and lyophilized powder injections. These preparations can be aqueous or non-aqueous and may contain one or more pharmacodynamically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. Diluents may be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Furthermore, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. In addition, conventional solubilizers, buffers, pH adjusters, etc., can also be added. These excipients are commonly used in the art.

[0064] In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0065] To achieve the purpose of medication and enhance the therapeutic effect, the keratin or pharmaceutical composition of the present invention can be administered using any known method of administration.

[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 frequency of administration, and the therapeutic purpose. Therefore, the therapeutic dosage of the present invention can vary widely. Generally speaking, the dosage of the pharmaceutical components used in the present invention is well known to those skilled in the art. The actual amount of drug contained in the final formulation of the keratin composition according to the present invention can be appropriately adjusted to achieve the required therapeutic dose and accomplish the preventive or therapeutic purpose of the present invention. The suitable daily dosage range of the keratin of the present invention is: 0.01–1000 mg / kg body weight, preferably 5–1000 mg / kg body weight, more preferably 10–500 mg / kg body weight, and most preferably 20–300 mg / kg body weight. The above dosage can be administered in a single dose or divided into several doses, such as two, three, or four doses, depending on the clinical experience of the pharmacist and the administration regimen, including the use of other treatment methods. The total dose required for each treatment can be divided into multiple doses or administered as a single dose. The protein or pharmaceutical composition of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs, with dosage adjustments.

[0067] The seventh aspect of the present invention is to provide the use of the keratin YK93-2 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 drugs for benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, breast cancer, lactation, analgesia, uterine fibroids, and coagulation.

[0068] To achieve the objectives of this invention, the following technical solution is adopted. Specifically, the preparation of keratin YK93-2 of this invention includes the following steps:

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

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

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

[0072] The expression vectors 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 vectors are pET series vectors; the most preferred expression vector is pET-30a(+).

[0073] (3) Transfect the expression vector into the host cell;

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

[0075] 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).

[0076] (4) Under appropriate conditions, host cells will be fermented and cultured to induce the expression of the target protein YK93-2;

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

[0078] The culture medium can be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, Bengal red medium, high-salt Czapek's medium, DOBA medium, koji medium and their modified formulations, etc.; LB medium is preferred for shake flask fermentation; LB medium and its modified formulations are preferred for fermentation tanks.

[0079] The inducer can be IPTG, lactose, arabinose, etc.; IPTG and lactose are preferred.

[0080] (5) Enrichment of the target protein YK93-2;

[0081] The fermentation broth obtained in step (4) is centrifuged and the supernatant is discarded; the precipitate is suspended in buffer solution, the cells are broken, centrifuged again, and the supernatant is discarded; the precipitate is washed with a cleaning agent and then dissolved in urea solution to obtain YK93-2 crude protein solution.

[0082] The buffer solution is preferably buffer A, and its volume is: fermentation broth volume: buffer A volume = 1~100:1, preferably 10:1;

[0083] 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 liquid volume : 1M urea (which may contain 1% Triton) volume = 0.2~100:1, preferably 1~15:1;

[0084] The preferred urea solution is an 8M urea solution, and its dosage is: fermentation liquid volume : 8M urea volume = 0.2~100:1, preferably 2~15:1.

[0085] (6) Isolation and purification of target protein YK93-2:

[0086] The crude protein solution obtained in step (5) needs to be purified to obtain the target protein YK93-2. The purification can be carried out by dialysis, ultrafiltration / microfiltration, column chromatography, or salting out.

[0087] A. The dialysis step involves purifying the crude protein solution obtained in step (5) by dialysis to obtain the target protein YK93-2 solution.

[0088] The molecular weight cutoff of the dialysis bag can be 0.5-10kD, with a preferred molecular weight cutoff of 3.5-10kD and an optimal molecular weight cutoff of 10kD.

[0089] B. The ultrafiltration and microfiltration steps involve purifying the crude protein solution obtained in step (5) using membrane technology such as ultrafiltration or microfiltration to obtain a concentrated solution of the target protein YK93-2.

[0090] Preferably, the purification is carried out by two microfiltration membranes, with the first membrane having a pore size of 1000-1500 nm and the second membrane having a pore size of 20-50 nm.

[0091] C. The column chromatography step involves passing the crude protein solution obtained in step (5) through column chromatography, such as various exchange columns or size exclusion columns, to separate and purify the target protein YK93-2.

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

[0093] The eluent can be a commonly used eluent in the art, such as water or a salt solution, including sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, etc.

[0094] D. The salting-out step involves purifying the crude protein solution obtained in step (5) by salting out to obtain a suspension of the target protein YK93-2.

[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 ammonium sulfate aqueous solution is added to achieve a final ammonium sulfate concentration of 10–50%, preferably 20–30%, and more preferably 25%.

[0096] The salting-out process is performed 1 to 3 times, preferably 2 times.

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

[0098] The target protein YK93-2 solution obtained from purification 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.

[0099] Beneficial technical effects of the present invention:

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

[0101] 2. This invention demonstrates, through pharmacological studies of protein YK93-2 on a mouse model of benign prostatic hyperplasia, that protein YK93-2 can significantly reduce prostate wet weight and prostate index.

[0102] 3. This invention demonstrates, through the pharmacodynamic study of protein YK93-2 on Lewis lung cancer, that protein YK93-2 has a significant inhibitory effect on lung cancer proliferation.

[0103] 4. This invention demonstrates, through research on the effect of protein YK93-2 on lactation in pregnant mice, that protein YK93-2 can increase the content of prolactin in the serum of female mice to a certain extent.

[0104] 5. This invention demonstrates, through the pharmacodynamic study of protein YK93-2 on EL-4 lymphoma, that protein YK93-2 has a significant inhibitory effect on lymphoma proliferation.

[0105] 6. This invention demonstrates, through pharmacodynamic studies of protein YK93-2 on a mouse analgesia model, that protein YK93-2 can reduce the number of writhing movements to a certain extent and has a certain analgesic effect.

[0106] 7. This invention demonstrates, through the efficacy study of protein YK93-2 against B16F10 melanoma, that protein YK93-2 has a significant inhibitory effect on melanoma proliferation. Attached Figure Description

[0107] Figure 1 Analysis of expressed protein YK93-2 by reduced SDS-PAGE

[0108] (M: Protein molecular weight standard; S: Expressed protein YK93-2)

[0109] Figure 2 Effects of YK93-2 on blood routine tests in Lewis lung cancer mice

[0110] (Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001)

[0111] Figure 3 Effects of YK93-2 on milk production in female mice

[0112] (Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001)

[0113] Figure 4 Effects of YK93-2 on serum prolactin in female rats

[0114] (Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001)

[0115] Figure 5 Effects of YK93-2 on blood routine tests in EL-4 lymphoma mice

[0116] (Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001)

[0117] Figure 6 Inhibitory effect of YK93-2 on acetic acid-induced writhing in mice

[0118] (Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001)

[0119] Figure 7Effects of YK93-2 on blood routine tests in B16F10 melanoma mice

[0120] (Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001) Detailed Implementation

[0121] The following examples and pharmacological activity test cases are used to further illustrate the present invention, but they do not imply any limitation on the present invention.

[0122] Unless otherwise specified, the experimental methods used in the following examples and pharmacological activity test cases are conventional methods; unless otherwise specified, the experimental materials used were purchased from conventional biochemical reagent companies.

[0123] Example 1: Preparation of crude protein solution A (LB medium) A by shake-flask fermentation

[0124] The nucleotide sequence shown in SEQ ID No. 2 was synthesized and transformed into the pET-30a(+) vector; sequencing confirmed that the expression vector contained the correct sequence was obtained; the expression vector was transfected into BL21(DE3) cells to obtain 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 the recombinant strain.

[0125] The recombinant strain was streaked onto an LBA plate containing Kanamycin, and the plate was inverted and incubated overnight at 37°C for 16 hours.

[0126] Prepare 400ml LB medium and dispense it into two 200ml bottles. Add Kanamycin (final concentration 50μg / ml) to each 200ml LB medium bottle. Take a single colony from the plate and add it to the LB medium. Incubate overnight on a shaker at 37℃ and 220rpm to obtain the seed culture.

[0127] Prepare 9.6 L LB medium, aliquoting it into 48 200 ml bottles. Add Kanamycin (final concentration 50 μg / ml) to each 200 ml LB medium bottle, followed by 2 ml of seed culture. Incubate on a shaker at 37°C and 220 rpm for 2-3 hours. Monitor OD. 600 When OD 600 When the protein expression reaches approximately 1.0, an inducer is added, and the protein is induced to express in a shaker. The induction conditions are selected from the table below.

[0128]

[0129] Combine the bacterial cultures from each bottle, centrifuge at 7000 rpm for 5 minutes, and discard the supernatant after sterilization. Resuspend the precipitate in approximately 1 L of buffer solution, filter through an 80-100 mesh sieve, and disrupt the filtrate using a high-pressure homogenizer at 800-1000 bar twice, 2 minutes each time. Centrifuge the disrupted bacterial culture at 7000 rpm for 30 minutes, discard the supernatant, and obtain the precipitate (i.e., inclusion bodies). Wash the precipitate twice with 600 ml of washing solution, centrifuge, and discard the supernatant. Dissolve the precipitate three times with urea solution, each time in 600 ml volume. Combine the three solutions, centrifuge at 7000 rpm for 30 minutes, discard the precipitate, and the supernatant is the crude protein solution A.

[0130]

[0131] The crude solution A of protein YK93-2 was analyzed by reducing SDS-PAGE with a separating gel concentration of 12.5% ​​and stained with Coomassie Brilliant Blue R250. A distinct blue band was observed near the molecular weight of 55 kDa.

[0132] Example 2: Preparation of crude protein YK93-2 solution B in a fermenter

[0133] 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 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 the recombinant strain.

[0134] Add 100 μl of the recombinant strain to an LBA plate containing Kanamycin, spread it evenly until dry, and incubate the plate inverted at 37°C overnight. Streak three single colonies onto a Kanamycin-containing plate, incubate overnight, and verify the expression through three batches of shake-flask fermentation. After confirmation, preserve the strain in 15% glycerol, aliquot it into 1 ml vials to obtain a working cell bank, and freeze at -80°C for later use.

[0135] Take one glycerol bacterium from the working cell bank, take 100 μl, add it to 40 ml of LB medium, add Kanamycin (final concentration 50 μg / ml), and incubate in a shaker at 37℃ and 220 rpm for 6 hours to obtain the first-stage seed culture.

[0136] Take 1.2 ml of the primary seed culture and add it to 120 ml of LB medium. Add Kanamycin (final concentration 50 μg / ml) and incubate in a shaker at 37 °C and 220 rpm for 7 hours to obtain the secondary seed culture.

[0137] Add 3L of modified LB medium to a 5L fermenter, then add 120ml of secondary seed culture and 3ml of Kanamycin (final concentration 50μg / ml). Incubate at 37℃ and 30% dissolved oxygen (tandem rotation speed) for about 4 hours. Monitor the OD value to around 20, then use 15g of lactose as an inducer and induce at 20℃. Feed the mixture at a rate of 30ml / hour and incubate at 20℃ for 24 hours.

[0138] Centrifuge the bacterial culture at 7000 rpm for 5 minutes, sterilize the supernatant and discard it; suspend the precipitate in approximately 200 ml of buffer A, filter through an 80-100 mesh sieve, and crush the filtrate using a high-pressure grinder at 800-1000 bar twice, 2 minutes each time. After crushing, centrifuge the bacterial culture at 7000 rpm for 30 minutes and discard the supernatant.

[0139] Wash the precipitate three times with 1M urea solution (containing 1% Triton), 600ml each time, centrifuge, and discard the supernatant. Dissolve the precipitate once with 4M urea solution and twice with 8M urea solution, 600ml each time. Combine the three solutions, centrifuge at 7000rpm for 30 minutes, discard the precipitate, and the supernatant is the crude protein solution B.

[0140] The crude solution B of protein YK93-2 was analyzed by reducing SDS-PAGE with a separating gel concentration of 12.5% ​​and stained with Coomassie Brilliant Blue R250. A distinct blue band was observed near the molecular weight of 55 kDa.

[0141] Example 3: Protein YK93-2 was prepared from crude protein solution B using membrane technology.

[0142] The crude protein solution B obtained in Example 2 was purified by microfiltration membrane technology: first, solid-liquid separation was performed using a 1500nm or 1000nm ceramic membrane core; the inner liquid was discarded, and the outer liquid was repeatedly microfiltered using a 20nm or 50nm ceramic membrane core to remove urea; the inner liquid after the second microfiltration was freeze-dried to obtain the target protein YK93-2.

[0143] Structure confirmation of protein YK93-2:

[0144] 1. Analysis using reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

[0145] Instrument: Bio-Rad protein electrophoresis apparatus.

[0146] Methods and Results: Protein YK93-2 solution was analyzed by reducing SDS-PAGE with a separating gel concentration of 12.5% ​​and stained with Coomassie Brilliant Blue R250. The molecular weight of the YK93-2 band was around 55 kDa.

[0147] 2. Protein full-sequence analysis based on LC-MS / MS

[0148] Main materials: acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Pepsin, Elastase;

[0149] Main instruments: Nanoflow liquid chromatograph (Thermo EastnLC1200), mass spectrometer (Thermo OrbitrapFusion Lumos), constant temperature incubator (Zhongyi Guoke (Beijing) Technology Co., Ltd., DHP-9052).

[0150] Methods and Results:

[0151] Protein YK93-2 was pretreated by dissolution and displacement, reductive alkylation, and various proteolytic digestion to obtain enzymatically digested peptides. The enzymatically digested peptide solutions were analyzed by liquid chromatography-tandem mass spectrometry. The raw mass spectrometry data were analyzed using Byonic software, and the identification results showed 100% coverage, confirming that it was consistent with the target sequence SEQ ID No.1.

[0152] Example 4: Protein YK93-2 was prepared by purifying crude protein solution A.

[0153] The crude protein solution A obtained in Example 1 was purified using the following three methods:

[0154] The first method: dialysis;

[0155] The crude protein solution A was filtered through a 0.45 μm filter membrane, and the filtrate was dialyzed with water for more than 72 hours. The inner liquid was then freeze-dried to obtain the target protein YK93-2.

[0156]

[0157] The second method: salting out;

[0158] The crude protein solution A was placed in a stirred container for two salting-out processes: A saturated ammonium sulfate solution was slowly added along the wall to achieve a final ammonium sulfate concentration of 25% or 50%. During salting-out, the protein precipitated out. After complete salting-out, the solution was filtered, completing the first salting-out process. 400 ml of pure water was added to the precipitate for suspension, and another saturated ammonium sulfate solution was slowly added along the wall to achieve a final ammonium sulfate concentration of 25%, resulting in a second salting-out process. After filtration, the precipitate was the crude protein extract. The crude protein extract was washed three times with water: 200 ml of pure water was added for suspension, the mixture was stirred, allowed to stand, and then filtered. This process was repeated three times. The precipitate was then freeze-dried to obtain the target protein YK93-2.

[0159] The third method: column chromatography;

[0160] The crude protein solution A was purified using HiTrap Q FF 16 / 10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, and HiTrap DEAE anion exchange resin columns. The eluent was a gradient elution with NaCl solution, followed by 20 mM NaH2PO4 / Na2HPO4 buffer (pH 8.0). Each eluent fraction was analyzed by SDS-PAGE electrophoresis and combined. The combined eluent was centrifuged twice at 7000 rpm for 1 hour each time. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrates were combined. The filtrate was concentrated by dialyzing with water, with a molecular weight cutoff of 10 kDa in the dialysis bag. The inner liquid was freeze-dried to obtain the target protein YK93-2.

[0161] The protein YK93-2 obtained by the three methods was confirmed to have the same amino acid sequence as the protein prepared in Example 3 by the same structural confirmation method as in Example 3.

[0162] Example 5: Protein YK93-2 was obtained from crude protein solution B by salting out.

[0163] The crude protein solution B obtained in Example 2 was placed in a stirred container for two salting-out processes: A saturated ammonium sulfate solution was slowly added along the wall to bring the final ammonium sulfate concentration to 25%. During salting-out, the protein precipitated. After complete salting-out, the solution was filtered, completing the first salting-out process. 400 ml of pure water was added to the precipitate for suspension, and another saturated ammonium sulfate solution was slowly added along the wall to bring the final ammonium sulfate concentration to 25%, resulting in a second salting-out process. After filtration, the precipitate was the crude protein extract. The crude protein extract was washed three times with water: 200 ml of pure water was added for suspension, the mixture was stirred, allowed to stand, and then filtered. This process was repeated three times. The precipitate was then freeze-dried to obtain the target protein YK93-2.

[0164] The product protein YK93-2 was confirmed to have the same amino acid sequence as the protein prepared in Example 3 by the same structural confirmation method as in Example 3.

[0165] Pharmacological test

[0166] Experiment 1: Efficacy test of YK93-2 (protein of Example 3) in a mouse model of benign prostatic hyperplasia

[0167] Animals: Male Kunming mice, 18-20 grams

[0168] Drugs: Testosterone propionate injection (Ningbo No. 2 Hormone Factory); Finasteride (Merck):

[0169] Instrument: Electronic balance

[0170] Experimental Groups:

[0171] Normal control group;

[0172] Model group: androgen load model;

[0173] Positive control groups: finasteride (1 mg / kg) group; finasteride (3 mg / kg) group;

[0174] YK93-2 (0.5g / kg) group.

[0175] method:

[0176] Preparation of laboratory animals: Laboratory animals were allowed to acclimatize to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day.

[0177] A mouse model of benign prostatic hyperplasia (BPH) was established by subcutaneous injection of testosterone propionate. Mice were randomly divided into groups of 10 mice before modeling. Testosterone propionate (0.01 mg / kg), prepared with soybean oil, was injected subcutaneously once daily for 30 days. The control group received an equal volume of sodium carboxymethyl cellulose subcutaneously. Immediately after modeling, mice were orally administered finasteride 1 mg / kg, finasteride 3 mg / kg, and YK93-2 0.5 g / kg. Body weight was measured every other day. After 30 days of treatment, mice were weighed, sacrificed, and the prostate was removed via a midline incision in the lower abdomen. Wet weight was recorded, and the prostate index was calculated as prostate wet weight (mg) / mouse body weight (g).

[0178] Data statistics:

[0179] The mean, standard deviation, and standard error of prostate wet weight and prostate index were calculated for each group. The T.TEST was used to compare the data between groups, and P<0.05 was considered to be statistically significant.

[0180] Experimental results:

[0181] The results are shown in Table 1.

[0182] Table 1. Effects of YK93-2 on mouse body weight, prostate wet weight, and prostate index

[0183]

[0184] (Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001)

[0185] Experimental conclusion:

[0186] 1) The androgen loading method successfully induced prostatic hyperplasia in mice. After modeling, the wet weight of the prostate and the prostate index of the mice in the model group were significantly increased, which was statistically different from that in the normal group (P<0.05).

[0187] 2) Both low and high doses of the positive control drug finasteride effectively reduced the wet weight and index of the prostate in model mice after modeling, with the high dose showing slightly better results. Compared with the model group, P<0.05, indicating a statistically significant difference.

[0188] 3) Compared with the model group, YK 93-2 significantly reduced the wet weight of the prostate and the prostate index in mice (P<0.05).

[0189] Experiment 2: Efficacy test of YK93-2 (protein from Example 3) in a Lewis lung cancer model

[0190] Animals: Male C57BL / 6J mice

[0191] Drug: Cyclophosphamide for injection (CTX), YK93-2;

[0192] Instruments: Electronic balance, five-part separator

[0193] Experimental Groups:

[0194] Control group;

[0195] Positive control group: Cyclophosphamide (100 mg / kg)

[0196] YK93-2 (0.5g / kg) group.

[0197] method:

[0198] Preparation of laboratory animals: Laboratory animals were allowed to acclimatize to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day.

[0199] Subcutaneous inoculation of Lewis cell suspension to establish a mouse breast cancer model: Lewis tumor tissue was homogenized using a glass homogenizer to prepare a suspension. The tumor suspension was aspirated with a syringe, the number of viable cells was counted, and the cell concentration was adjusted to 2.6*10⁻⁶ with physiological saline. 6 The suspension was then injected at a rate of 0.2 ml / mouse into the left axilla. The mice were randomly divided into a control group, a cyclophosphamide (100 mg / kg) group, and a YK93-2 (0.5 g / kg) group the next day. Cyclophosphamide was administered via a single intraperitoneal injection, the YK93-2 group was administered via gavage daily for 10 consecutive days, and the control group received an equal volume of sodium carboxymethyl cellulose orally. On the day of treatment, body weight was measured, and 20 μL of blood was collected via orbital sampling for complete blood count analysis. Tumor masses were removed, weighed, photographed, and the tumor inhibition rate was calculated. Data statistics:

[0200] The mean, standard deviation, and standard error of tumor inhibition rate and tumor weight were calculated as (mean mass of control group - mean mass of experimental group) / mean mass of control group × 100%. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be statistically significant.

[0201] Experimental results:

[0202] The results are shown in Table 2. Figure 2 .

[0203] Table 2. Inhibitory effect of YK93-2 on Lewis lung cancer in mice

[0204]

[0205] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0206] Experimental conclusion:

[0207] 1) Subcutaneous inoculation of Lewis cells can successfully establish a mouse lung cancer model.

[0208] 2) The positive control group of cyclophosphamide can significantly inhibit tumor growth, significantly reduce tumor weight, and achieve a tumor inhibition rate of 95.35%. Compared with the control group, P<0.05, which shows a statistical difference.

[0209] 3) Compared with the control group, YK93-2 (0.5g / kg) significantly inhibited lung cancer growth, significantly reduced tumor weight, and achieved a tumor inhibition rate of 27.38%. The difference was statistically significant (P<0.05). However, YK93-2 did not reduce the number of leukocytes in the peripheral blood of animals.

[0210] Efficacy test of protein YK93-2 (protein of Example 3) on lactation in pregnant mice.

[0211] Animals: 30-50 grams of pregnant female Kunming rats

[0212] Medicine: YK93-2

[0213] Instrument: Electronic balance

[0214] Experimental Groups:

[0215] Control group;

[0216] YK93-2(2g / kg) group

[0217] method:

[0218] Preparation of experimental animals: The experimental animals were acclimatized to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day. The delivery date of each pregnant mouse was recorded, and they were randomly divided into a control group and a YK 93-2 group according to their birth date.

[0219] YK93-2 (2g / kg) was administered by gavage, and the weight and weight gain of the pups were recorded daily. Thirteen days after administration, the mother mice were euthanized by cervical dislocation, and 400μl of blood was collected from the orbital cavity. The blood was allowed to stand at room temperature for 1-2 hours and then centrifuged at 1000g for 20 minutes. The serum was then used to measure the prolactin content.

[0220] Data statistics:

[0221] The average daily weight gain of the pups (milk production of the mother mice) and the serum prolactin content of the mother mice were calculated for each group. The data of each group were compared between groups using T.TEST. P<0.05 was considered to be statistically significant.

[0222] Experimental results:

[0223] See results Figure 3 , Figure 4 .

[0224] Experimental conclusion:

[0225] 1) YK93-2 had no significant effect on the weight gain of young mice.

[0226] 2) Compared with the control group, YK93-2 can increase the content of prolactin in the serum of female mice to a certain extent.

[0227] Experiment 4: Efficacy test of YK93-2 (protein from Example 3) EL-4 lymphoma model

[0228] Animals: Male C57BL / 6J mice

[0229] Drug: Cyclophosphamide for Injection (CTX), YK93-2

[0230] Instruments: Electronic balance, five-part separator

[0231] Experimental Groups:

[0232] Control group;

[0233] Positive control group: Cyclophosphamide (100 mg / kg)

[0234] YK93-2(2g / kg) group.

[0235] method:

[0236] Preparation of laboratory animals: Laboratory animals were allowed to acclimatize to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day.

[0237] A mouse lymphoma model was established by subcutaneous inoculation of EL-4 cell suspension: EL-4 tumor tissue was homogenized using a glass homogenizer to prepare a suspension. The suspension was aspirated using a syringe, the number of viable cells was counted, and the cell concentration was adjusted to 2.0 x 10⁻⁶ cells / mL with physiological saline. 6The suspension was then injected at a rate of 0.2 ml / mouse into the left axilla. The mice were randomly assigned to a control group, a cyclophosphamide (100 mg / kg) group, or a YK93-2 (2 g / kg) group the following day. Cyclophosphamide was administered via a single intraperitoneal injection, the YK93-2 (2 g / kg) group was administered via gavage daily for 13 consecutive days, and the control group received an equal volume of sodium carboxymethyl cellulose orally. On the day of treatment, body weight was measured, and 20 μL of blood was collected via orbital sampling for complete blood count analysis. Tumor masses were removed, weighed, photographed, and the tumor inhibition rate was calculated. Data statistics:

[0238] The mean, standard deviation, and standard error of tumor inhibition rate and tumor weight were calculated as (mean mass of control group - mean mass of experimental group) / mean mass of control group × 100%. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be statistically significant.

[0239] Experimental results:

[0240] The results are shown in Table 3. Figure 5 .

[0241] Table 3. Inhibitory effect of YK93-2 on EL-4 lymphoma in mice

[0242]

[0243] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0244] Experimental conclusion:

[0245] 1) Establish a mouse T-cell lymphoma model by subcutaneous inoculation of EL-4 cells.

[0246] 2) The positive control group (cyclophosphamide) significantly inhibited tumor growth and reduced tumor weight, with a tumor inhibition rate of 95.67%. Compared with the control group, the difference was statistically significant (P<0.05). The cyclophosphamide group also significantly reduced white blood cells, lymphocytes, monocytes, and neutrophils (P<0.05).

[0247] 3) Compared with the control group, YK93-2 inhibited lymphoma growth, significantly reduced tumor weight, and achieved a tumor inhibition rate of 25.59%. Compared with the control group, P<0.05, indicating a statistically significant difference. YK93-2 had no significant effect on peripheral blood leukocytes.

[0248] Experiment 5: Pharmacological test of protein YK93-2 (protein of Example 3) in a mouse pain model.

[0249] Animal: Male Kunming mouse

[0250] Medications: Tramadol hydrochloride sustained-release tablets, glacial acetic acid, YK93-2;

[0251] Instruments: Electronic balance, timer

[0252] Experimental Groups:

[0253] Control group;

[0254] Positive control group: Tramadol hydrochloride group;

[0255] YK93-2(2g / kg) group.

[0256] method:

[0257] Preparation of laboratory animals: Laboratory animals were allowed to acclimatize to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day.

[0258] The YK93-2 (2g / kg) group was administered the drug via gavage, while the model group received an equal volume of CMC-Na, once daily for 7 consecutive days. Thirty minutes after the last administration, all mice were given an intraperitoneal injection of 1% acetic acid. The number of writhing movements within 15 minutes was recorded, and the drug-induced inhibition rate of the writhing response was calculated. The criteria for observation were abdominal retraction, hind limb extension, and hip elevation.

[0259] Data statistics:

[0260] The inhibition rate was calculated as (mean number of writhing responses in the control group - mean number of writhing responses in the treatment group) / mean number of writhing responses in the control group × 100%. The mean, standard deviation, and standard error of the number of writhing responses were recorded. The data were compared between groups using TTEST, and a p-value < 0.05 was considered statistically significant.

[0261] Experimental results:

[0262] The results are shown in Table 4. Figure 6 .

[0263] Table 4. Inhibitory effect of YK93-2 on mouse pain model

[0264]

[0265] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0266] Experimental conclusion:

[0267] 1) Intraperitoneal injection of 1% acetic acid successfully induced a pain model in mice. The average number of writhing movements over 15 minutes was 19.

[0268] 2) The positive control group treated with tramadol hydrochloride significantly relieved pain and reduced the number of writhing episodes. Compared with the control group, the difference was statistically significant (P<0.05).

[0269] 3) Compared with the control group, YK93-2 can effectively relieve pain to a certain extent and reduce the number of twisting episodes.

[0270] Experiment 6: Efficacy test of YK93-2 (protein from Example 3) against a B16F10 melanoma model.

[0271] Animals: Female C57BL / 6J mice

[0272] Drug: Cyclophosphamide for injection (CTX), YK93-2;

[0273] Instruments: Electronic balance, five-part separator

[0274] Experimental Groups:

[0275] Control group;

[0276] Positive control group: Cyclophosphamide (100 mg / kg)

[0277] YK93-2(2g / kg) group.

[0278] method:

[0279] Preparation of laboratory animals: Laboratory animals were allowed to acclimatize to the experimental environment (temperature 22℃±2℃, relative humidity 50%±2%) for 1 day.

[0280] A mouse melanoma model was established by subcutaneous inoculation of B16F10 cell suspension: Tumor masses were removed under aseptic conditions, ground into a fluid, and the concentration was adjusted to 2.58 × 10⁻⁶ cells / mL with physiological saline. 6 / ml, 0.2ml was evenly injected subcutaneously into the axillary back of mice. The animals were randomly divided into groups the next day and administration began (day 0). CTX (100mg / kg) was administered weekly, YK-93-2 (2g / kg) was administered daily, and the control group received an equal volume of carboxymethyl cellulose sodium orally. On the day of treatment, body weight was measured, and 20μL of blood was collected via the orbital rim for complete blood count analysis. Tumor masses were removed, weighed, photographed, and the tumor inhibition rate was calculated.

[0281] Data statistics:

[0282] The mean, standard deviation, and standard error of tumor inhibition rate and tumor weight were calculated as (mean mass of control group - mean mass of experimental group) / mean mass of control group × 100%. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be statistically significant.

[0283] Experimental results:

[0284] The results are shown in Table 5. Figure 7 .

[0285] Table 5. Inhibitory effect of YK93-2 on B16F10 melanoma in mice

[0286]

[0287] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0288] Experimental conclusion:

[0289] 1) Subcutaneous inoculation of B16F10 cells can successfully establish a mouse melanoma model.

[0290] 2) The positive control group of cyclophosphamide can significantly inhibit tumor growth, significantly reduce tumor weight, and achieve a tumor inhibition rate of 85.82%. Compared with the control group, P<0.05, which shows a statistical difference.

[0291] 3) Compared with the control group, YK93-2 (2g / kg) significantly inhibited the growth of lung cancer, significantly reduced tumor weight, and achieved a tumor inhibition rate of 62.43%. Compared with the control group, P<0.05, indicating a statistically significant difference. YK93-2 significantly reduced the number of neutrophils in the peripheral blood of animals, P<0.05, indicating a statistically significant difference.

Claims

1. Application of keratin YK93-2 in the preparation of drugs for treating benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, and breast cancer. in, The amino acid sequence of the keratin YK93-2 is as shown in SEQ ID NO.1 in the sequence listing.

2. Application of keratin YK93-2 in the preparation of drugs for promoting lactation. in, The amino acid sequence of the keratin YK93-2 is as shown in SEQ ID NO.1 in the sequence listing.

3. Application of keratin YK93-2 in the preparation of drugs for analgesia. in, The amino acid sequence of the keratin YK93-2 is as shown in SEQ ID NO.1 in the sequence listing.

4. The application according to any one of claims 1-3, wherein, The keratin YK93-2 is routinely modified; or a tag for detection or purification is attached to the keratin YK93-2. The conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, immobilization modification, sulfation, oxidation, methylation, and deamination; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

5. The use of a nucleic acid molecule encoding keratin YK93-2 as defined in any one of claims 1-4 in the preparation of a medicament for treating benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, and breast cancer.

6. The use of a nucleic acid molecule encoding keratin YK93-2 as defined in any one of claims 1-4 in the preparation of a medicament for promoting lactation.

7. The use of a nucleic acid molecule encoding keratin YK93-2 as defined in any one of claims 1-4 in the preparation of a medicament for analgesia.

8. The application according to any one of claims 5-7, wherein, The nucleotide sequence of the nucleic acid molecule is as follows: (1) The nucleotide sequence shown in SEQ ID NO.2 of the sequence listing; (2) A nucleotide sequence complementary to the nucleotide sequence in (1) above.

9. The application of an expression vector in the preparation of drugs for treating benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, and breast cancer, wherein, The expression vector contains a nucleic acid molecule as defined in any one of claims 5-8.

10. The use of an expression vector in the preparation of a drug for promoting lactation, wherein, The expression vector contains a nucleic acid molecule as defined in any one of claims 5-8.

11. The use of an expression vector in the preparation of a drug for analgesia, wherein, The expression vector contains a nucleic acid molecule as defined in any one of claims 5-8.

12. The application according to any one of claims 9-11, wherein, The expression vectors are 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, or pPIC3.5K.

13. The application according to claim 12, wherein, The expression vector is pET-30a(+).

14. The application of a host cell in the preparation of drugs for treating benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, and breast cancer, wherein, The host cell contains an expression vector as defined in any one of claims 9-13 or has a nucleic acid molecule as defined in any one of claims 5-8 integrated into its genome.

15. The use of a host cell in the preparation of a drug for promoting lactation, wherein, The host cell contains an expression vector as defined in any one of claims 9-13 or has a nucleic acid molecule as defined in any one of claims 5-8 integrated into its genome.

16. The use of a host cell in the preparation of a drug for analgesia, wherein, The host cell contains an expression vector as defined in any one of claims 9-13 or has a nucleic acid molecule as defined in any one of claims 5-8 integrated into its genome.

17. The application according to any one of claims 14-16, wherein, The host cells include bacteria, yeast, Aspergillus, plant cells, or insect cells.

18. The application according to claim 17, wherein, The bacteria include Escherichia coli.

19. The application according to any one of claims 14-16, wherein, The host cell is a competent host cell, which is a BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Organami series, Trans1-T1, TG1, TB1, Y11430, MG1003, KM71 or SMD1168.

20. The application according to claim 19, wherein, The competent host cells are BL21 (DE3) or Transetta (DE3).

21. The use of a pharmaceutical composition in the preparation of a medicament for treating benign prostatic hyperplasia, lung cancer, lymphoma, melanoma, and breast cancer, wherein, The pharmaceutical composition contains keratin YK93-2 as defined in any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient.

22. The use of a pharmaceutical composition in the preparation of a medicament for promoting lactation, wherein, The pharmaceutical composition contains keratin YK93-2 as defined in any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient.

23. The use of a pharmaceutical composition in the preparation of a medicament for analgesia, wherein, The pharmaceutical composition contains keratin YK93-2 as defined in any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient.

24. The application according to any one of claims 21-23, wherein, The drug composition is administered via the intestinal route.

25. The application according to any one of claims 21-23, wherein, The drug composition is administered via a non-enteric route.

26. The application according to any one of claims 21-23, wherein, The drug composition can be administered orally, intramuscularly, subcutaneously, nasally, through the oral mucosa, via the eye, lung, skin, vagina, peritoneum, or rectum.

27. The application according to any one of claims 21-23, wherein, The drug composition is administered by injection.

28. The application according to claim 27, wherein, The injection administration includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, or acupoint injection.

29. The application according to any one of claims 21-23, wherein, The dosage form of the pharmaceutical composition is a liquid dosage form, a solid dosage form, or a semi-solid dosage form.

30. The application according to claim 29, wherein, The liquid dosage form is a solution, emulsion, suspension, injection, eye drops, nasal drops, lotion, or liniment.

31. The application according to claim 30, wherein, The solution is a true solution or a colloidal solution, and / or The emulsion is an oil-in-water emulsion, an oil-in-water emulsion, or a complex emulsion, and / or The injection is a water-based injection, a powder injection, or an infusion solution.

32. The application according to claim 29, wherein, The solid dosage form is a tablet, capsule, granule, powder, microcapsule, drop, suppository, film, patch, aerosol or spray.

33. The application according to claim 32, wherein, The tablets are ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, or orally disintegrating tablets, and / or The capsules are hard capsules, soft capsules, or enteric-coated capsules.

34. The application according to claim 29, wherein, The semi-solid dosage form is an ointment, gel, or paste.

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