Keratin YK93-5 as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention belongs to the field of biological pharmacy, and provides keratin YK93-5, a coding nucleic acid molecule thereof, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector or genome integrated with the nucleic acid molecule, and a preparation method and a pharmaceutical composition of the keratin YK93-5. The invention also provides application of the keratin YK93-5 and other products in preparation of drugs for treating lung cancer, lymphoma, breast cancer, melanoma, hysteromyoma, prostatic hyperplasia and the like.

Description

Keratin YK93-5, preparation method, pharmaceutical composition and use thereof Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals and relates to keratin YK93-5, a nucleic acid molecule encoding keratin YK93-5, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or having the nucleic acid molecule integrated into its genome, as well as a preparation method of keratin YK93-5, a pharmaceutical composition containing the keratin, and use of the keratin and the pharmaceutical composition in preparing drugs for preventing or treating prostate hyperplasia, uterine fibroids, lymphoma, breast cancer, lung cancer, lactation, and coagulation. Background Art

[0002] Keratin is a structural protein of ectodermal cells and is divided into two categories, soft keratin and hard keratin, depending on whether it is fibrotic or not. Hard keratin is the main component of hair, feathers, hooves, shells, claws, horns, scales, etc. It is a key structural protein in connective tissue and plays a protective role in the body.

[0003] Since hard keratin is not easily soluble in various solvents and is generally more resistant to enzymatic hydrolysis by proteases than other proteins, current research on keratin focuses more on the study of soft keratin in cells. However, there is less research on the hard keratin resources that account for the vast majority in nature, namely the hard keratin in animal body coverings (hooves, horns, nails, shells, etc.), and it has not been effectively utilized and is usually discarded as waste.

[0004] With the rapid development of modern biotechnologies such as genomics, proteomics, genetic engineering, and microbial engineering, keratin applications in materials, biology, and medicine are gaining increasing attention. Therefore, utilizing protein expression systems to produce hard keratin, study its pharmacological effects, and promote the development of innovative drugs using hard keratin as an active ingredient are both novel and innovative.

[0005] Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a keratin YK93-5, a nucleic acid molecule encoding keratin YK93-5, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or whose genome is integrated with the nucleic acid molecule, as well as a preparation method of keratin YK93-5, a pharmaceutical composition containing keratin YK93-5, and the use of the above keratin YK93-5, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the preparation of drugs for prostate hyperplasia, uterine fibroids, lymphoma, breast cancer, lung cancer, lactation, and coagulation.

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

[0008] The first aspect of the technical solution of the present invention is to provide a keratin YK93-5, characterized in that the amino acid sequence of the keratin YK93-5 is:

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

[0010] (2) An amino acid sequence substantially retaining the same biological function as that of the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing, formed by replacing, deleting or adding 1 to 35 amino acids.

[0011] Furthermore, conventional modifications can be performed on keratin YK93-5; or a tag for detection or purification can be connected to keratin YK93-5.

[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, and Profinity eXact.

[0013] The second aspect of the technical solution of the present invention is to provide a nucleic acid molecule encoding the keratin YK93-5 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-30a(+).

[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 cells include bacteria, yeast, Aspergillus, plant cells, or insect cells.

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

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

[0024] The fifth aspect of the technical solution of the present invention is to provide a method for preparing the keratin YK93-5 described in the first aspect, characterized in that it comprises the following steps:

[0025] A. synthesizing a nucleic acid molecule corresponding to the keratin YK93-5 described in the first aspect, linking the nucleic acid molecule into a corresponding expression vector, transforming the expression vector into a host cell, culturing the host cell carrying the expression vector in a fermentation device under certain conditions and inducing expression of keratin YK93-5 to obtain a crude protein solution containing keratin YK93-5;

[0026] B. Separate, purify and dry the crude protein solution expressed in step A to obtain keratin YK93-5.

[0027] Furthermore, in step A, the host cell is mainly selected from Escherichia coli, the keratin YK93-5 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shake flask or a fermentation tank.

[0028] Furthermore, in step A, after inducing the expression of keratin YK93-5, impurities can be cleaned with a detergent and dissolved with a solution to obtain a crude protein solution.

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

[0030] Furthermore, the inducer 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 disrupted, 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 YK93-5 crude protein solution.

[0032] The buffer is preferably buffer A, and its dosage is: fermentation liquid volume: buffer A volume = 1 to 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 1M urea solution (which may contain 1% Triton), and the dosage is: fermentation broth volume: 1M urea (which may contain 1% Triton) volume = 0.2-100:1, preferably 1-15:1;

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

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

[0036] Furthermore, 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 YK93-5 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 YK93-5.

[0040] Preferably, the purification is performed by microfiltration membrane twice, with the pore size of the first membrane being 1000-1500 nm and the pore size of the second membrane being 20-50 nm.

[0041] (3) The column chromatography method is to separate and purify the crude protein solution obtained in step A through column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein YK93-5.

[0042] Preferred 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, such as 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. Cation exchange resin columns include HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, and Toyopearl Super SP-650M. Anion exchange resin columns are most preferred.

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

[0044] (4) The salting-out method is to purify the crude protein solution obtained in step A by salting-out to obtain a suspension of the target protein YK93-5.

[0045] The salting-out agent can be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc. The preferred salting-out agent is ammonium sulfate and its aqueous solution. A saturated aqueous ammonium sulfate solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, and more preferably 25%.

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

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

[0048] Furthermore, the target protein YK93-5 solution obtained by purification in step B can be freeze-dried or vacuum-dried into a dry powder, or the concentrated solution can be directly spray-dried into a dry powder.

[0049] The sixth aspect of the technical solution of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin YK93-5 described in the first aspect or the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect or the host cell described in the fourth aspect and a pharmaceutically acceptable carrier or excipient.

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

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

[0052] The present invention also relates to a pharmaceutical composition containing the keratin of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants. Typically, the keratin of the present invention accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0067] The dosage of the keratin pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, the number of doses, and the intended treatment. Therefore, the therapeutic dosage of the present invention can vary widely. Generally speaking, the dosages of the pharmaceutical ingredients of the present invention are well known to those skilled in the art. The dosage can be appropriately adjusted based on the actual amount of drug contained in the final formulation of the keratin composition of the present invention to achieve a therapeutically effective amount and achieve the preventive or therapeutic purpose of the present invention. A suitable daily dosage range for the keratin of the present invention is 0.01 to 1000 mg / kg body weight, preferably 5 to 1000 mg / kg body weight, more preferably 10 to 500 mg / kg body weight, and most preferably 20 to 300 mg / kg body weight. This dosage can be administered as a single dose or divided into several doses, such as two, three, or four doses, depending on the clinical experience of the administering physician and the dosing regimen, including the use of other therapeutic modalities. The total dosage required for each treatment can be administered in multiple doses or as a single dose. The protein or pharmaceutical composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage can be adjusted.

[0068] The seventh aspect of the technical solution of the present invention provides the use of the keratin YK93-5 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 prostate hyperplasia, uterine fibroids, lymphoma, breast cancer, lung cancer, lactation, and coagulation.

[0069] In order to achieve the purpose of the present invention, the present invention adopts the following technical solution. Specifically, the preparation of the keratin YK93-5 of the present invention includes the following steps:

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

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

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

[0073] The expression vector can be pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZα / A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, pPIC3.5K, etc.; the preferred expression vector is the pET series vector; the most preferred expression vector is pET-30a(+).

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

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

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

[0077] (4) fermenting and culturing the host cells under appropriate conditions to induce expression of the target protein YK93-5;

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

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

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

[0081] (5) Enrichment of target protein YK93-5 product;

[0082] The fermentation broth obtained in step (4) is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacteria are broken, and the precipitate is centrifuged again and the supernatant is discarded; the precipitate is washed with a detergent and then dissolved with a urea solution to obtain a YK93-5 crude protein solution.

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

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

[0085] The urea solution is preferably an 8M urea solution, and its dosage is: fermentation liquid volume: 8M urea volume = 0.2-100:1, preferably 2-15:1.

[0086] (6) Isolation and purification of target protein YK93-5:

[0087] The crude protein solution obtained in step (5) needs to be purified to obtain the target protein YK93-5. The purification can be carried out by dialysis, ultrafiltration, microfiltration, column chromatography, or salting out.

[0088] A. The dialysis step is to purify the crude protein solution obtained in step (5) by dialysis to obtain the target protein YK93-5 solution.

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

[0090] B. The ultrafiltration and microfiltration steps are to purify the crude protein solution obtained in step (5) using membrane technology such as ultrafiltration membrane or microfiltration membrane to obtain a concentrated solution of the target protein YK93-5.

[0091] Preferably, the purification is performed by microfiltration membrane twice, with the pore size of the first membrane being 1000-1500 nm and the pore size of the second membrane being 20-50 nm.

[0092] C. The column chromatography step is to separate and purify the crude protein solution obtained in step (5) through column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein YK93-5.

[0093] Preferred 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, such as 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. Cation exchange resin columns include HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, and Toyopearl Super SP-650M. Anion exchange resin columns are most preferred.

[0094] The eluent may be any eluent commonly used in the art, such as water, saline solution, including sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.

[0095] D. The salting-out step is to purify the crude protein solution obtained in step (5) by salting-out to obtain a suspension of the target protein YK93-5.

[0096] The salting-out agent can be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc. The preferred salting-out agent is ammonium sulfate and its aqueous solution. A saturated aqueous ammonium sulfate solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, and more preferably 25%.

[0097] The number of salting-outs is 1 to 3 times, preferably 2 times.

[0098] After salting out, the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.

[0099] The target protein YK93-5 solution obtained by purification in steps A to D can be freeze-dried or vacuum-dried to form a dry powder, or the concentrated solution can be directly spray-dried to form a dry powder.

[0100] Beneficial technical effects of the present invention:

[0101] 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.

[0102] 2. The present invention studies the pharmacodynamics of protein YK93-5 on a mouse prostate model, demonstrating that protein YK93-5 can significantly improve the pathological damage of mice with benign prostatic hyperplasia;

[0103] 3. The present invention studies the pharmacodynamics of protein YK93-5 on a mouse pain model, demonstrating that protein YK93-5 can reduce the number of writhings in mice, but there is no statistically significant difference;

[0104] 4. The present invention studies the effect of protein YK93-5 on lactation in pregnant mice, and proves that protein YK93-5 can increase the content of prolactin in the serum of maternal mice to a certain extent.

[0105] 5. The present invention studies the efficacy of protein YK93-5 on EL-4 lymphoma, demonstrating that protein YK93-5 can inhibit lymphoma proliferation to a certain extent.

[0106] 6. The present invention studies the efficacy of protein YK93-5 on B16F10 melanoma, demonstrating that protein YK93-5 has a significant effect in inhibiting melanoma proliferation.

[0107] 7. The present invention studies the efficacy of protein YK93-5 on Lewis lung cancer, proving that protein YK93-5 can inhibit the proliferation of lung cancer to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1. Analysis of the expressed protein YK93-5 by reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

[0109] (M: protein molecular weight standard; S: expressed protein YK93-5)

[0110] Figure 2. Effects of YK93-5 on mouse prostate histopathology

[0111] Figure 3. Effect of YK93-5 on histopathological scores of mouse prostate tissues

[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. Inhibitory effect of YK93-5 on mouse pain model

[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. Effect of YK93-5 on lactation in maternal 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. Effect of YK93-5 on serum prolactin in maternal 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 7. Effect of YK93-5 on blood routine of EL-4 lymphoma 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)

[0121] Figure 8. Effect of YK93-5 on blood routine of B16F10 melanoma mice

[0122] (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)

[0123] Figure 9. Effect of YK93-5 on blood routine in Lewis lung cancer mice

[0124] (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 DESCRIPTION

[0125] 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.

[0126] 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.

[0127] Example 1 Preparation of Protein YK93-5 Crude Solution A by Shake Flask Fermentation (LB Medium)

[0128] The nucleotide sequence shown in SEQ ID No. 2 was synthesized and transferred into the pET-30a(+) vector. Sequencing confirmed the expression vector containing the correct sequence. The expression vector was transfected into BL21(DE3) cells to generate expression-competent host cells containing the target nucleotide sequence. The recombinant strain was obtained by adding the recombinant strain to LB medium and incubating at 37°C and 220 rpm on a shaker for 1 hour.

[0129] The recombinant strain was streaked onto an LBA plate containing kanamycin, and the plate was inverted and placed in a 37°C constant temperature incubator for overnight culture for 16 hours.

[0130] Prepare 400 ml of LB medium and divide into two 200 ml bottles. Add kanamycin (final concentration 50 μg / ml) to each 200 ml bottle of LB medium. Take a single colony from the plate and add it to the LB medium. Incubate overnight on a shaker at 37°C and 220 rpm to obtain the seed solution.

[0131] Prepare 9.6 L of LB medium and divide it into 48 bottles, 200 ml per bottle. Add kanamycin (final concentration 50 μg / ml) to each bottle (200 ml) of LB medium, then add 2 ml of seed solution, and culture in a shaker at 37°C and 220 rpm for 2-3 hours. Monitor OD 600 , when OD 600When the concentration reaches about 1.0, add the inducer and induce protein expression in a shaker. The induction conditions are selected from the table below.

[0132] Combine the bacterial suspensions from each vial and centrifuge at 7000 rpm for 5 minutes. Sterilize and discard the supernatant. Resuspend the precipitate in approximately 1 L of buffer and filter through an 80-100 mesh screen. The filtrate is crushed using a high-pressure crusher at 800-1000 bar twice for 2 minutes each. Centrifuge the crushed suspension 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 detergent, centrifuge, and discard the supernatant. Dissolve the precipitate three times in urea solution, each volume reaching 600 ml. Combine the three solutions, centrifuge at 7000 rpm for 30 minutes, and discard the precipitate. The supernatant is crude protein solution A.

[0133] The crude solution A of protein YK93-5 was analyzed by reducing SDS-PAGE with a separation gel concentration of 12.5% ​​and stained with Coomassie Brilliant Blue R250; a clear blue band was shown near the molecular weight of 55 kD.

[0134] Example 2 Preparation of Protein YK93-5 Crude Solution B in a Fermentation Tank

[0135] In Example 1, an expression vector containing the sequence shown in SEQ ID No. 2 was synthesized and sequenced to obtain the expression vector. The expression vector was transfected into BL21(DE3) cells to obtain expression-competent host cells containing the target nucleotide sequence. The cells were added to LB medium and cultured on a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.

[0136] Add 100 μl of the recombinant strain to an LBA plate containing kanamycin and spread evenly with a spreader until dry. Incubate the plate upside down at 37°C in a constant-temperature incubator overnight. Streak three individual colonies onto the plate containing kanamycin and culture overnight. After three batches of shake flask fermentation and expression verification, preserve the strain in 15% glycerol and aliquot into 1 ml tubes to obtain a working cell bank. Store frozen at -80°C until needed.

[0137] Take out one glycerol strain from the working cell bank, take 100 μl, add it to 40 ml LB medium, add kanamycin (final concentration 50 μg / ml), and culture it in an oscillator at 37°C and 220 rpm for 6 hours to obtain the first-level seed liquid.

[0138] Take 1.2 ml of the first-level seed solution and add it to 120 ml of LB medium. Add kanamycin (final concentration 50 μg / ml) and culture it in an oscillator at 37°C and 220 rpm for 7 hours to obtain the second-level seed solution.

[0139] To a 5-L fermentor, add 3 L of modified LB broth, 120 mL of secondary seed solution, and 3 mL of kanamycin (final concentration 50 μg / mL). Cultivate at 37°C, 30% dissolved oxygen (tandem speed) for approximately 4 hours. Monitor the OD value around 20. Induce with lactose as the inducer at 20°C, feed at 30 mL / hour, and incubate at 20°C for 24 hours.

[0140] Centrifuge the bacterial suspension at 7000 rpm for 5 minutes, sterilize and discard the supernatant. Resuspend the pellet in approximately 200 ml of buffer A and filter through an 80-100 mesh sieve. Disintegrate the filtrate using a high-pressure crusher at 800-1000 bar twice for 2 minutes each. Centrifuge the resulting suspension at 7000 rpm for 30 minutes, and discard the supernatant.

[0141] Wash the pellet twice with 1M urea solution (containing 1% Triton), 600 ml each time, centrifuge, and discard the supernatant. Dissolve the pellet once with 4M urea solution and twice with 8M urea solution, each volume being 600 ml. Combine the three solutions and centrifuge at 7000 rpm for 30 minutes. Discard the pellet and the supernatant is crude protein solution B.

[0142] The crude solution B of protein YK93-5 was analyzed by reducing SDS-PAGE with a separation gel concentration of 12.5% ​​and stained with Coomassie Brilliant Blue R250; a clear blue band was shown near the molecular weight of 55 kD.

[0143] Example 3: Protein YK93-5 was prepared from crude protein solution B by membrane technology

[0144] The crude protein solution B obtained in Example 2 was purified by microfiltration membrane technology: solid-liquid separation was first performed using a 1500 nm or 1000 nm ceramic membrane core; the inner liquid was discarded, and the outer liquid was repeatedly microfiltered using a 20 nm or 50 nm ceramic membrane core to remove urea; the inner liquid after the second microfiltration was freeze-dried to obtain the target protein YK93-5.

[0145] Protein YK93-5 structure confirmation:

[0146] 1. Reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

[0147] Instrument: Protein electrophoresis apparatus (Bio-Rad).

[0148] Methods and Results: The YK93-5 protein solution was analyzed by reducing SDS-PAGE with a separation gel concentration of 12.5% ​​and stained with Coomassie Brilliant Blue R250. The molecular weight of the YK93-5 band was around 55 kD.

[0149] 2. Protein full sequence analysis based on LC-MS / MS

[0150] Main materials: acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin;

[0151] Main instruments: nanoflow liquid chromatograph (Thermo EastnLC1200), mass spectrometer (Thermo Orbitrap Fusion Lumos), constant temperature incubator (Zhongyi Guoke (Beijing) Technology Co., Ltd., DHP-9052).

[0152] Methods and Results:

[0153] Protein YK93-5 was pre-treated by dissolution displacement, reductive alkylation, and multiple proteolysis to obtain enzymatic peptides. The enzymatic peptide solution was analyzed by liquid chromatography-tandem mass spectrometry, and the mass spectrometry raw file was analyzed using Byonic software. The identification result had a coverage rate of 100%, confirming that it was consistent with the target sequence SEQ ID No. 1.

[0154] Example 4: Purification of crude protein solution A to obtain protein YK93-5

[0155] The crude protein solution A obtained in Example 1 was purified by the following three methods:

[0156] The first method: dialysis;

[0157] The crude protein solution A was filtered through a 0.45 μm filter membrane, and the filtrate was dialyzed against water for more than 72 hours. The inner solution was freeze-dried to obtain the target protein YK93-5.

[0158] The second method: salting out;

[0159] The crude protein solution A is placed in a stirred container and salted out twice: saturated ammonium sulfate solution is slowly added along the wall to a final concentration of 25% or 50%. During the salting-out process, the protein precipitates. After the salting-out is complete, the solution is filtered to complete the first salting-out. 400ml of pure water is added to the precipitate to suspend it. Saturated ammonium sulfate solution is then slowly added along the wall to a final concentration of 25%. A second salting-out is performed, and the precipitate is filtered to obtain the crude protein extract. The crude protein extract is then washed three times with water: 200ml of pure water is added to suspend it, stirred, allowed to stand, and filtered. After repeating this process three times, the precipitate is freeze-dried to obtain the target protein YK93-5.

[0160] The third method: column chromatography;

[0161] Crude protein solution A was purified using anion exchange resin columns, including HiTrap Q FF 16 / 10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, and HiTrap DEAE. The eluent was a NaCl gradient elution solution supplemented with 20 mM NaH2PO4 / Na2HPO4 buffer (pH 8.0). The fractions were combined and analyzed by SDS-PAGE electrophoresis. The combined eluate was centrifuged twice at 7000 rpm for 1 hour each. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrates were combined. The filtrate was concentrated by dialyzing with water using a 10 kD molecular weight cutoff. The resulting solution was freeze-dried to obtain the target protein YK93-5.

[0162] The product protein YK93-5 obtained by the three methods was confirmed to have the same amino acid sequence as the protein prepared in Example 3 by the same structure confirmation method as in Example 3.

[0163] Example 5 Protein YK93-5 was obtained by salting out the crude protein solution B

[0164] The crude protein solution B obtained in Example 2 was placed in a stirred container and subjected to two salting-outs: a saturated ammonium sulfate solution was slowly added along the wall to a final concentration of 25%. During the salting-out process, protein precipitated. After the salting-out was complete, the solution was filtered to complete the first salting-out. 400 ml of pure water was added to the precipitate to suspend it, and a saturated ammonium sulfate solution was slowly added along the wall again to a final concentration of 25%. A second salting-out was performed, and the solution was filtered to obtain the crude protein extract. The crude protein extract was washed three times with water: 200 ml of pure water was added to suspend it, stirred, allowed to stand, and filtered. After repeating this process three times, the precipitate was freeze-dried to obtain the target protein YK93-5.

[0165] The product protein YK93-5 was confirmed to have the same amino acid sequence as the protein prepared in Example 3 by the same structure confirmation method as in Example 3.

[0166] Pharmacological tests

[0167] Experimental Example 1: Efficacy of YK93-5 (Protein from Example 4) on a Mouse Prostatic Hyperplasia Model

[0168] Animals: Male Kunming mice, 18-20 grams

[0169] Drugs: Testosterone propionate injection (Ningbo Second Hormone Factory); Finasteride (Merck); YK93-5

[0170] Instruments: electronic balance, timer

[0171] Experimental groups:

[0172] Normal control group;

[0173] Model group: androgen load model;

[0174] Positive control group: finasteride (1 mg / kg) group;

[0175] Protein YK93-5 (0.5g / kg) group.

[0176] method:

[0177] Preparation of experimental animals: Experimental animals were acclimatized to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for 1 day.

[0178] Subcutaneous injection of testosterone propionate to establish a benign prostatic hyperplasia model in mice: mice were randomly divided into groups before modeling, with 10-12 mice in each group. Testosterone propionate (0.01 mg / kg) prepared in soybean oil was injected subcutaneously once a day for 30 consecutive days. The control group was given an equal volume of sodium carboxymethylcellulose subcutaneously. Finasteride 1 mg / kg and YK93-5 0.5 g / kg were orally administered immediately after modeling. Body weight was measured every other day. After 30 days of administration, the mice were weighed and sacrificed. The prostate was removed through a midline incision in the lower abdomen, and the wet weight was measured. The prostate index was calculated as the wet weight of the prostate (mg) / mouse body weight (g). The prostate was dehydrated and fixed with 4% paraformaldehyde, embedded in paraffin, and the largest surface was cut into thin sections. Tissue sections were stained with hematoxylin and eosin to examine whether there were any pathological changes.

[0179] Statistics:

[0180] The mean, standard deviation and standard error of the prostate index and pathological score of each group were calculated, and the data of each group were compared between groups using TTEST. P < 0.05 was considered to be a significant difference.

[0181] Experimental results:

[0182] The results are shown in Table 1, Figures 2 and 3.

[0183] Table 1. Effects of YK93-5 on body weight, prostate wet weight and prostate index in mice

[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) Androgen loading can successfully induce benign prostatic hyperplasia in mice. The prostate wet weight and prostate index of the model group mice were significantly increased after modeling. Papillary hyperplasia and calcification were observed in the prostate cavity, and fibrous tissue and smooth muscle tissue proliferation were observed around the gland. Compared with the normal group, P < 0.05, there was a statistically significant difference.

[0187] 2) The positive drug finasteride group effectively reduced the wet weight and index of the prostate in the model mice after modeling, with a statistically significant difference (P<0.01) compared with the model group. Papillary and interstitial tissue decreased, and hyperplasia showed an improvement trend compared with the model group, but no statistical difference was found.

[0188] 3) Compared with the model group, the YK 93-5 group showed weakened papillary hyperplasia and calcification in the prostate cavity, decreased proliferation of fibrous tissue and smooth muscle tissue around the gland, and significantly improved the pathological damage of prostatic hyperplasia (P<0.05).

[0189] Experimental Example 2: Efficacy of protein YK93-5 (protein from Example 4) on a mouse pain model

[0190] Animals: Male Kunming mice

[0191] Drugs: Tramadol hydrochloride sustained-release tablets, glacial acetic acid, YK93-5;

[0192] Instruments: electronic balance, timer

[0193] Experimental groups:

[0194] control group;

[0195] Positive control group: tramadol hydrochloride group;

[0196] YK93-5(2g / kg) group.

[0197] method:

[0198] Preparation of experimental animals: Experimental animals were acclimatized to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for 1 day.

[0199] The YK93-5 group (2g / kg) was gavage-administered, while the model group received an equal volume of CMC-Na, once daily for 7 consecutive days. Thirty minutes after the last dose, 1% acetic acid was intraperitoneally injected into the abdomen. The number of writhing movements in the mice was recorded over a 15-minute period, and the inhibition rate of the writhing reaction was calculated. Observation criteria included abdominal concavity, hind limb extension, and hip elevation.

[0200] Statistics:

[0201] The inhibition rate was calculated as (average number of writhing reactions in the control group - average number of writhing reactions in the drug-treated group) / average number of writhing reactions in the control group × 100%. The mean, standard deviation and standard error of the writhing times were recorded and compared between groups using TTEST. P < 0.05 was considered to be a significant difference.

[0202] Experimental results:

[0203] The results are shown in Table 2 and Figure 4.

[0204] Table 2. Inhibitory effect of YK93-5 on mouse pain model

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

[0206] Experimental conclusion:

[0207] 1) Intraperitoneal injection of 1% acetic acid successfully induced a pain model in mice. The average number of writhing times over 15 minutes was 28.60, indicating a stable model.

[0208] 2) The positive tool drug tramadol hydrochloride group can significantly relieve pain, the number of writhing events is significantly reduced, and the pain inhibition rate is 93.88%. Compared with the control group, P<0.01, there is a statistically significant difference.

[0209] 3) Compared with the control group, YK93-5 could effectively relieve pain and reduce the number of writhing events, with an inhibition rate of 34.44%, but there was no statistical difference.

[0210] Experimental Example 3: Effect of Protein YK93-5 (Protein from Example 4) on Lactation in Pregnant Rats

[0211] Animal: Female Kunming pregnant rat, 30-50g

[0212] Drug: YK93-5

[0213] Instrument: Electronic balance

[0214] Experimental groups:

[0215] control group;

[0216] YK93-5(2g / kg) group

[0217] method:

[0218] Preparation of experimental animals: Experimental animals were acclimated to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for 1 day. The birth date of each pregnant mouse was recorded and the mice were randomly divided into a control group and a YK93-5 group according to their birth date.

[0219] YK93-5 (2g / kg) was administered orally, and the weight and weight gain of the pups were recorded daily. After 13 days of administration, the female mice were killed by cervical dislocation, and 400μl of blood was collected from the orbits, allowed to stand at room temperature for 1-2 hours, and centrifuged at 1000g for 20 minutes. Serum was then collected for prolactin measurement.

[0220] Statistics:

[0221] The average daily weight gain of the pups in each group (the amount of milk produced by the mothers) and the serum prolactin level of the mothers were calculated. The data of each group were compared among the groups using T.TEST. P < 0.05 was considered to be a significant difference.

[0222] Experimental results:

[0223] The results are shown in Figures 5 and 6.

[0224] Experimental conclusion:

[0225] 1) YK93-5 had no significant effect on the weight gain of young mice.

[0226] 2) Compared with the control group, YK93-5 could increase the serum prolactin level of maternal rats to a certain extent, but P>0.05, indicating no statistical difference.

[0227] Experimental Example 4: Efficacy test of YK93-5 (protein from Example 3) in EL-4 lymphoma model

[0228] Animals: Male C57BL / 6J mice

[0229] Drug: Cyclophosphamide (CTX) for injection, YK93-5

[0230] Instruments: electronic balance, five-category counter

[0231] Experimental groups:

[0232] control group;

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

[0234] YK93-5(2g / kg) group.

[0235] method:

[0236] Preparation of experimental animals: Experimental animals were acclimatized to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for 1 day.

[0237] Subcutaneous inoculation of EL-4 cell suspension to establish a mouse lymphoma model: EL-4 tumor tissue was ground into a suspension using a glass homogenizer. The tumor suspension was aspirated with a syringe, the number of viable cells was counted, and the cell concentration was adjusted to 2.0*10 with physiological saline. 6 / ml, and then the suspension was injected into the left armpit of the mouse at 0.2ml / mouse. The next day, the mice were randomly divided into a control group, a cyclophosphamide (100mg / kg) group, and a YK93-5 (2g / kg) group. Cyclophosphamide was injected intraperitoneally once, and the YK93-5 (2g / kg) group was gavaged daily for 13 consecutive days. The control group was orally administered with an equal volume of sodium carboxymethylcellulose. Body weight was measured on the day of treatment, 20μL of blood was obtained by orbital bleeding and routine blood analysis was performed, the tumor mass was peeled off, 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 by (average weight of control group - average weight of experimental group) / average weight of control group × 100%. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be significantly different.

[0239] Experimental results:

[0240] The results are shown in Table 3 and Figure 7.

[0241] Table 3. Inhibitory effect of YK93-5 on mouse EL-4 lymphoma

[0242] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0243] Experimental conclusion:

[0244] 1) EL-4 cells were subcutaneously inoculated to establish a mouse T cell lymphoma model.

[0245] 2) The positive drug cyclophosphamide group significantly inhibited tumor growth and reduced tumor weight, with a tumor inhibition rate of 95.67% (P < 0.05), which was statistically significant compared with the control group. The cyclophosphamide group also significantly reduced the number of white blood cells, lymphocytes, monocytes, and neutrophils (P < 0.05), which was statistically significant.

[0246] 3) Compared with the control group, YK93-5 showed a trend of inhibiting the growth of lymphoma, with tumor weight reduced and a tumor inhibition rate of 19.95%, but compared with the control group, P>0.05, there was no statistical difference. YK93-5 had no significant effect on peripheral blood leukocytes.

[0247] Experimental Example 5: Efficacy of YK93-5 (Protein from Example 3) on B16F10 Melanin Model

[0248] Animals: Female C57BL / 6J mice

[0249] Drugs: Cyclophosphamide (CTX) for injection, YK93-5;

[0250] Instruments: electronic balance, five-category counter

[0251] Experimental groups:

[0252] control group;

[0253] Positive control group: cyclophosphamide (100 mg / kg)

[0254] YK93-5(2g / kg) group.

[0255] method:

[0256] Preparation of experimental animals: Experimental animals were acclimatized to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for 1 day.

[0257] Subcutaneous inoculation of B16F10 cell suspension to establish a mouse melanoma model: the tumor mass was removed under sterile conditions, ground into tumor fluid, and the concentration was adjusted to 2.58×10 6 / ml, 0.2ml was evenly inoculated subcutaneously on the back of the mouse's armpit. The next day, the animals were randomly divided into groups and drug administration began (recorded as day 0). CTX (100mg / kg) was administered once a week, YK93-5 (2g / kg) was administered daily, and the control group was orally administered with an equal volume of sodium carboxymethylcellulose. On the day of treatment, body weight was measured, 20μL of blood was obtained by orbital bleeding and routine blood analysis was performed, the tumor mass was peeled, weighed and photographed, and the tumor inhibition rate was calculated.

[0258] Statistics:

[0259] The mean, standard deviation and standard error of tumor inhibition rate and tumor weight were calculated by (average weight of control group - average weight of experimental group) / average weight of control group × 100%. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be significantly different.

[0260] Experimental results:

[0261] The results are shown in Table 4 and Figure 8.

[0262] Table 4. Inhibitory effect of YK93-5 on mouse B16F10 melanoma

[0263] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0264] Experimental conclusion:

[0265] 1) Subcutaneous inoculation of B16F10 cells can successfully establish a mouse melanoma model.

[0266] 2) The positive drug cyclophosphamide group could significantly inhibit tumor growth and reduce tumor weight significantly, with a tumor inhibition rate of 85.82%. Compared with the control group, P<0.05, there was a statistically significant difference.

[0267] 3) Compared with the control group, YK93-5 (2g / kg) significantly inhibited lung cancer growth, significantly reducing tumor weight, with a tumor inhibition rate of 58.20%, P < 0.05. YK93-5 also increased the number of peripheral blood mononuclear cells, P < 0.05, also showing a statistically significant difference.

[0268] Experimental Example 6: Efficacy of YK93-5 (Protein from Example 3) on Lewis Lung Cancer Model

[0269] Animals: Male C57BL / 6J mice

[0270] Drugs: Cyclophosphamide (CTX) for injection, YK93-5;

[0271] Instruments: electronic balance, five-category counter

[0272] Experimental groups:

[0273] control group;

[0274] Positive control group: cyclophosphamide (100 mg / kg)

[0275] YK93-5(2g / kg) group.

[0276] method:

[0277] Preparation of experimental animals: Experimental animals were acclimatized to the experimental environment (temperature 22°C ± 2°C, relative humidity 50% ± 2%) for 1 day.

[0278] Lewis cell suspension was subcutaneously inoculated to replicate a mouse breast cancer model: Lewis tumor tissue was ground into a suspension using a glass homogenizer. 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 with normal saline. 6 / ml, and then the suspension was injected into the left armpit of the mouse at 0.2ml / mouse. The next day, the mice were randomly divided into a control group, a cyclophosphamide (100mg / kg) group, and a YK93-5 (2g / kg) group. Cyclophosphamide was injected intraperitoneally once, the YK93-5 group was gavaged daily for 17 consecutive days, and the control group was orally administered with an equal volume of sodium carboxymethylcellulose. Body weight was measured on the day of treatment, 20μL of blood was obtained by orbital sampling and routine blood analysis was performed, the tumor mass was peeled off, weighed, photographed, and the tumor inhibition rate was calculated. Data statistics:

[0279] The mean, standard deviation and standard error of tumor inhibition rate and tumor weight were calculated by (average weight of control group - average weight of experimental group) / average weight of control group × 100%. The data of each group were compared between groups using TTEST. P < 0.05 was considered to be significantly different.

[0280] Experimental results:

[0281] The results are shown in Table 5 and Figure 9.

[0282] Table 5. Inhibitory effect of YK93-5 on Lewis lung cancer in mice

[0283] (Compared with the control group, *P<0.05, **P<0.01, ***P<0.001)

[0284] Experimental conclusion:

[0285] 1) Subcutaneous inoculation of Lewis cells can successfully establish a mouse lung cancer model.

[0286] 2) The positive drug cyclophosphamide group could significantly inhibit tumor growth and reduce tumor weight significantly, with a tumor inhibition rate of 91.22%. Compared with the control group, P<0.05, there was a statistically significant difference.

[0287] 3) Compared with the control group, YK93-5 (2g / kg) inhibited lung cancer growth to a certain extent, reducing tumor weight and achieving a tumor inhibition rate of 28.67%. However, there was no statistical difference (P>0.05) compared with the control group. YK93-5 significantly reduced the number of peripheral blood leukocytes and neutrophils in the animals (P<0.05), which was statistically significant.

Claims

1. A keratin YK93-5, characterized in that The amino acid sequence of the keratin YK93-5 is: (1) the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing; (2) An amino acid sequence substantially retaining 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.

2. Keratin YK93-5 according to claim 1, characterized in that Conventional modifications can be performed on keratin YK93-5; or a tag for detection or purification can be linked to keratin YK93-5.

3. Keratin YK93-5 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. A nucleic acid molecule encoding the keratin YK93-5 according to any one of claims 1 to 3.

5. The 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 producing the keratin YK93-5 according to any one of claims 1 to 3, characterized in that: The following steps are involved: A. synthesizing a nucleic acid molecule corresponding to the keratin YK93-5 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 carrying the expression vector in a fermentation device under certain conditions and inducing expression of keratin YK93-5 to obtain a crude protein solution containing keratin YK93-5; B. Separate, purify and dry the crude protein solution expressed in step A to obtain keratin YK93-5.

11. The method according to claim 10, characterized in that In step A, the host cell is mainly selected from Escherichia coli, the keratin YK93-5 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shake flask or a fermentation tank.

12. The method according to claim 10, characterized in that In step A, after inducing the expression of keratin YK93-5, impurities can be washed with a detergent 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 YK93-5 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

15. Use of the keratin YK93-5 according to any one of claims 1 to 3, or the nucleic acid molecule according to any one of claims 4 to 5, or the expression vector according to claim 6, or the host cell according to claims 7 to 9, or the pharmaceutical composition according to claim 14 in the preparation of drugs for preventing or treating prostate hyperplasia, uterine fibroids, lymphoma, breast cancer, lung cancer, lactation, and coagulation.

Images