WO2024074102A1 - 一种角蛋白yk93-6、制法和其药物组合物与用途 - Google Patents

一种角蛋白yk93-6、制法和其药物组合物与用途 Download PDF

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Publication number
WO2024074102A1
WO2024074102A1 PCT/CN2023/121364 CN2023121364W WO2024074102A1 WO 2024074102 A1 WO2024074102 A1 WO 2024074102A1 CN 2023121364 W CN2023121364 W CN 2023121364W WO 2024074102 A1 WO2024074102 A1 WO 2024074102A1
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Prior art keywords
keratin
nucleic acid
acid molecule
expression vector
host cell
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PCT/CN2023/121364
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English (en)
French (fr)
Inventor
庾石山
陈晓光
金晶
李密
史国茹
刘云宝
王晓婧
姜慧敏
李勇
屈晶
郝梦瑶
王明晋
马双刚
苏国柱
李树颖
符江
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中国医学科学院药物研究所
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Publication of WO2024074102A1 publication Critical patent/WO2024074102A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention belongs to the field of biopharmaceuticals, and relates to keratin YK93-6, a nucleic acid molecule encoding keratin YK93-6, 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 YK93-6, a pharmaceutical composition containing the keratin, and use of the keratin and the pharmaceutical composition in preparing drugs for preventing or treating prostate hyperplasia, lymphoma, melanoma, analgesia, lactation, breast cancer, lung cancer, uterine fibroids, and coagulation.
  • Keratin is a type of protein that is widely found in the epidermis of humans and animals. It is the main component of hair, feathers, hooves, shells, claws, horns, etc. It is an extremely important structural protein of connective tissue and plays a role in protecting the body.
  • Keratin is widely present in organisms and is a renewable resource with great utilization value, but it has not been widely and effectively utilized. The main reason is that keratin is insoluble in various solvents and is generally more resistant to protease hydrolysis than other proteins. Therefore, it is very difficult to extract and prepare natural keratin.
  • the technical problem solved by the present invention is to provide a keratin YK93-6, a nucleic acid molecule encoding keratin YK93-6, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or the genome integrated with the nucleic acid molecule, as well as a preparation method of keratin YK93-6, a pharmaceutical composition containing keratin YK93-6, and the use of the above keratin YK93-6, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition in the preparation of prostate hyperplasia, lymphoma, melanoma, analgesia, lactation, breast cancer, lung cancer, uterine fibroids, and coagulation drugs.
  • the present invention provides the following technical solutions:
  • the first aspect of the technical solution of the present invention provides a keratin YK93-6, characterized in that the amino acid sequence of the keratin YK93-6 is:
  • the conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, immobilization modification, sulfation, oxidation, methylation, deamination, disulfide bond formation or disulfide bond cleavage;
  • the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, Profinity eXact.
  • the second aspect of the technical solution of the present invention is to provide a nucleic acid molecule encoding the keratin YK93-6 described in the first aspect.
  • nucleic acid molecule is:
  • 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.
  • the expression vector can be pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZ ⁇ /A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, pPIC3.5K, etc.; the preferred expression vector is the pET series vector; the most preferred expression vector is pET-28a(+).
  • 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.
  • the host cell includes bacteria, yeast, Aspergillus, plant cells, or insect cells.
  • bacteria include Escherichia coli or yeast.
  • the competent host cells can be BL21 series, Transetta series, Rosetta series, DH5 ⁇ series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; the preferred expression competent cells are BL21 (DE3) and Transetta (DE3).
  • the fifth aspect of the technical solution of the present invention provides a method for preparing the keratin YK93-6 described in the first aspect, characterized in that it comprises the following steps:
  • step B Separate, purify and dry the crude protein solution expressed in step A to obtain keratin YK93-6.
  • the host cell is mainly selected from Escherichia coli
  • the keratin YK93-6 is expressed in Escherichia coli inclusion bodies
  • the fermentation equipment includes a shake flask or a fermenter.
  • step A after inducing the expression of keratin YK93-6, impurities can be cleaned with a cleaning agent and dissolved with a solution to obtain a crude protein solution.
  • the culture medium in step A can be LB culture medium, TB culture medium, SB culture medium, SOB culture medium, SOC culture medium, PDA culture medium, YPD culture medium, Bengal rose culture medium, high salt Czapek culture medium, DOBA culture medium, rice koji culture medium and improved formula thereof; LB culture medium is preferred for shake flask fermentation; LB culture medium and improved formula thereof are preferred for fermentation tank.
  • the inducing agent in step A can be IPTG, lactose, arabinose, etc.; preferably IPTG and lactose.
  • step A the fermentation broth obtained is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacteria are broken, and the precipitate is centrifuged again, and the supernatant is discarded; the precipitate is washed with a detergent and then dissolved with a urea solution to obtain a YK93-6 crude protein solution.
  • the separation and purification method includes ultrafiltration microfiltration membrane technology purification method, column chromatography purification method, salting out method, and dialysis method.
  • step B the separation and purification method is as follows:
  • the dialysis method is to purify the crude protein solution obtained in step A by dialysis to obtain the target protein YK93-6 solution.
  • 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.
  • 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 YK93-6.
  • the purification is performed by microfiltration membrane twice, the pore size of the first membrane is 1000-1500 nm, and the pore size of the second membrane is 20-50 nm.
  • 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 YK93-6.
  • Preferred exclusion columns are dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns are ion exchange resin columns: anion exchange resin columns, HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, Toyopearl SuperQ-650M, etc.; cation exchange resin columns, HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M. The most preferred is an anion exchange resin column.
  • the eluent may be any eluent commonly used in the art, such as water, saline solution, wherein the saline solution includes sodium chloride solution, sodium dihydrogen phosphate solution, sodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.
  • 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 YK93-6.
  • the salting-out agent can be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc.
  • the preferred salting-out agent is ammonium sulfate and its aqueous solution. Saturated ammonium sulfate aqueous solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, more preferably 25%.
  • the number of salting-outs is 1 to 3 times, preferably 2 times.
  • the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.
  • the target protein YK93-6 solution obtained by purification in step B can be freeze-dried or vacuum-dried into dry powder, or the concentrated solution can be directly spray-dried into dry powder.
  • the sixth aspect of the technical solution of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin YK93-6 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.
  • 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.
  • the present invention relates to a pharmaceutical composition, comprising any one keratin obtained in the above steps and a pharmaceutically acceptable carrier.
  • 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.
  • the keratin of the present invention accounts for 0.1 to 100.0% of the total weight of the pharmaceutical composition.
  • the present invention also provides a pharmaceutical composition, which comprises a pharmaceutically effective dose of protein as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be prepared according to methods known in the art.
  • the protein of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare an appropriate administration form or dosage form that can be used as a human or veterinary drug.
  • the keratin or pharmaceutical composition containing the keratin of the present invention can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, skin, vagina, peritoneum, rectum, etc., preferably oral administration.
  • enteral or parenteral such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, skin, vagina, peritoneum, rectum, etc., preferably oral administration.
  • the administration route of the keratin or the pharmaceutical composition containing the keratin of the present invention can be injection, which includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, peritoneal injection and acupoint injection.
  • the dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.
  • Solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.
  • the keratin of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
  • diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the wetting agent can be water, ethanol, isopropanol, etc.
  • the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl Cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • disintegrants can be dry starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the wetting agent can be water, ethanol, isopropanol, etc.
  • the binder can be
  • 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.
  • carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, lauric acid macrogol glyceride, kaolin, talc, etc.; binders, such as gum arabic, yellow gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, lauric acid macrogol glyceride, kaolin, talc, etc.
  • binders such as gum arabic, yellow gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.
  • disintegrants such as
  • various carriers known in the art can be widely used.
  • carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.
  • the active ingredient keratin of the present invention is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule.
  • the active ingredient keratin of the present invention can also be prepared into a microcapsule, suspended in an aqueous medium to form a suspension, or loaded into a hard capsule or prepared as an injection for use.
  • the keratin of the present invention is made into an injection preparation, such as a solution, a suspension solution, an emulsion, or a freeze-dried powder injection.
  • This preparation can be aqueous or non-aqueous, and can contain one and/or more pharmacodynamically acceptable carriers, diluents, adhesives, lubricants, preservatives, surfactants, or dispersants.
  • the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, and the like.
  • an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation.
  • conventional cosolvents, buffers, pH regulators, and the like can also be added. These adjuvants are commonly used in the art.
  • colorants may be added to the pharmaceutical preparations.
  • preservatives perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.
  • the keratin or pharmaceutical composition of the present invention can be administered by any known administration method.
  • the dosage of the keratin pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the pharmaceutical ingredients in the present invention is well known to those skilled in the art. It can be appropriately adjusted according to the actual amount of the drug contained in the final preparation of the keratin composition of the present invention to achieve the requirements of its therapeutically effective amount and achieve the prevention or treatment purpose of the present invention.
  • the daily suitable 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 dosage form or divided into several, such as two, three or four dosage forms, depending on the clinical experience of the administering doctor and the administration scheme including the use of other treatment methods.
  • the total dosage required for each treatment can be divided into multiple doses or administered in one 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.
  • the seventh aspect of the technical solution of the present invention provides the use of the keratin YK93-6 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, lymphoma, melanoma, analgesia, lactation, breast cancer, lung cancer, uterine fibroids, and coagulation.
  • the preparation of keratin YK93-6 of the present invention comprises the following steps:
  • the preferred nucleotide sequence is shown as SEQ ID No.2.
  • the expression vector can be pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZ ⁇ /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(+).
  • the host cell may be Escherichia coli or yeast; the preferred host cell is Escherichia coli;
  • the competent cells can be BL21 series, Transetta series, Rosetta series, DH5 ⁇ series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; the preferred expression competent cells are BL21 (DE3) and Transetta (DE3).
  • Fermentation equipment can be shake flasks or fermentation tanks
  • the culture medium can be LB culture medium, TB culture medium, SB culture medium, SOB culture medium, SOC culture medium, PDA culture medium, YPD culture medium, Bengal rose culture medium, high salt Czapek culture medium, DOBA culture medium, rice koji culture medium and improved formula thereof; LB culture medium is preferred for shake flask fermentation; LB culture medium and improved formula thereof are preferred for fermentation tank.
  • the inducer may be IPTG, lactose, arabinose, etc.; preferably IPTG and lactose.
  • the fermented bacterial liquid obtained in step (4) is centrifuged and the supernatant is discarded; the precipitate is suspended in a buffer solution, the bacterial cells are broken, and the mixture is centrifuged again, and the supernatant is discarded; the precipitate is washed with a detergent and then dissolved with a urea solution to obtain a YK93-6 crude protein solution.
  • the crude protein solution obtained in step (5) needs to be purified to obtain the target protein YK93-6.
  • the purification can be carried out by Dialysis, or ultrafiltration, microfiltration, or column chromatography, or salting out steps are performed.
  • the dialysis step is to purify the crude protein solution obtained in step (5) by dialysis to obtain the target protein YK93-6 solution.
  • 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.
  • 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 YK93-6.
  • the purification is performed by microfiltration membrane twice, the pore size of the first membrane is 1000-1500 nm, and the pore size of the second membrane is 20-50 nm.
  • the column chromatography step is to separate and purify the crude protein solution obtained in step (5) by column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein YK93-6.
  • Preferred exclusion columns are dextran gel columns, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.; preferred exchange columns are ion exchange resin columns: anion exchange resin columns, HiTrap Q FF, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE, Toyopearl Q-650M, Toyopearl SuperQ-650M, etc.; cation exchange resin columns, HiTrap SP FF, HiTrap Capto SP ImpRes, Capto SP ImpRes, HiTrap Capto SP, Toyopearl SP-650M, Toyopearl Super SP-650M. The most preferred is an anion exchange resin column.
  • the eluent may be any eluent commonly used in the art, such as water, saline solution, wherein the saline solution includes sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.
  • 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 YK93-6.
  • the salting-out agent can be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, etc.
  • the preferred salting-out agent is ammonium sulfate and its aqueous solution. Saturated ammonium sulfate aqueous solution is added to make the final concentration of ammonium sulfate reach 10-50%, preferably 20-30%, more preferably 25%.
  • the number of salting-outs is 1 to 3 times, preferably 2 times.
  • the precipitate is washed with pure water for 2 to 5 times, preferably 3 times.
  • the target protein YK93-6 solution obtained by purification in steps A to D can be freeze-dried or vacuum-dried into dry powder, or the concentrated solution can be directly spray-dried into dry powder.
  • 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.
  • the present invention studies the efficacy of protein YK93-6 on a mouse prostate hyperplasia model, proving that protein YK93-6 can significantly reduce the wet weight of the prostate;
  • the present invention studies the efficacy of protein YK93-6 on Lewis lung cancer, proving that protein YK93-6 has a significant effect of inhibiting the proliferation of lung cancer;
  • the present invention studies the effect of protein YK93-6 on lactation in pregnant mice and proves that protein YK93-6 can affect the content of prolactin in maternal serum. The amount had no significant effect;
  • the present invention studies the efficacy of protein YK93-6 on EL-4 lymphoma, proving that protein YK93-6 can significantly inhibit the proliferation of lymphoma.
  • the present invention studies the efficacy of protein YK93-6 on a mouse analgesic model, proving that protein YK93-6 can reduce the number of writhings to a certain extent.
  • the present invention studies the efficacy of protein YK93-6 on B16F10 melanoma, proving that protein YK93-6 has a significant effect of inhibiting melanoma proliferation.
  • the recombinant strain was streaked on an LBA plate containing kanamycin, and the plate was inverted and placed in a 37°C constant temperature incubator for overnight culture for 16 hours.
  • the crude solution A of protein YK93-6 was analyzed by reducing SDS-PAGE, the separation gel concentration was 12.5%, and the Coomassie Brilliant Blue R250 method was used for staining; an obvious blue band was shown near the molecular weight of 52KD.
  • 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 expression vector was added to LB medium and cultured in a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.
  • glycerol bacteria Take out a glycerol bacteria from the working cell bank, take 100 ⁇ l, add it to 40 ml LB medium, add kanamycin (final concentration 50 ⁇ g/ml), and culture it in an oscillator at 37°C and 220 rpm for 6 hours to obtain the primary seed solution.
  • the bacterial solution was centrifuged at 7000rpm for 5 minutes, and the supernatant was discarded after sterilization; the precipitate was suspended in about 200ml buffer A, filtered with an 80-100 mesh screen, and the filtrate was crushed with a high-pressure crusher at a pressure of 800-1000bar, twice, each time for 2 minutes. After crushing, the bacterial solution was centrifuged at 7000rpm for 30 minutes, and the supernatant was discarded.
  • the precipitate was washed twice with 1% Triton solution, 600 ml each time, centrifuged, and the supernatant was discarded.
  • the precipitate was dissolved once with 4M urea solution and twice with 8M urea solution, each with a volume of 600 ml. The three solutions were combined, centrifuged at 7000 rpm for 30 minutes, the precipitate was discarded, and the supernatant was the crude protein solution B.
  • the crude solution B of protein YK93-6 was analyzed by reducing SDS-PAGE, the separation gel concentration was 12.5%, and it was stained with Coomassie Brilliant Blue R250 method; an obvious blue band was shown near the molecular weight of 52KD.
  • Example 3 Protein YK93-6 was prepared from crude protein solution B by membrane technology
  • 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 YK93-6.
  • the protein YK93-6 solution was analyzed by reducing SDS-PAGE, the separation gel concentration was 12.5%, and the Coomassie brilliant blue R250 method was used for staining.
  • the molecular weight of the YK93-6 band was around 52KD.
  • Main materials acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Pepsin, Elastase;
  • Protein YK93-6 was pre-treated by dissolution replacement, reductive alkylation, and multiple proteolysis to obtain enzyme-cleaved peptides; the enzyme-cleaved peptide solution was analyzed by liquid chromatography-tandem mass spectrometry, and the mass spectrum raw file was analyzed using Byonic software to identify The result showed a coverage rate of 100%, confirming that it was consistent with the target sequence SEQ ID No. 1.
  • Example 4 Purification of crude protein solution A to obtain protein YK93-6
  • the crude protein solution A obtained in Example 1 was purified by the following three methods:
  • the first method dialysis
  • the crude protein solution A was filtered with a 0.45 ⁇ m filter membrane, and the filtrate was dialyzed with water for more than 72 hours.
  • the inner solution was freeze-dried to obtain the target protein YK93-6.
  • the second method salting out
  • the crude protein solution A is placed in a stirred container for two salting-outs: a saturated ammonium sulfate solution is slowly added along the wall to make the final concentration of ammonium sulfate 25% or 50%. During the salting-out process, the protein is precipitated. After the salting-out is complete, the solution is filtered to complete the first salting-out. 400 ml of pure water is added to the precipitate for suspension, and a saturated ammonium sulfate solution is slowly added along the wall again to make the final concentration of ammonium sulfate 25%. The solution is salted-out for the second time, filtered, and the precipitate is the crude protein extract. The crude protein extract is washed three times with water: 200 ml of pure water is added for suspension, stirred, allowed to stand, and filtered. After repeating this three times, the precipitate is freeze-dried to obtain the target protein YK93-6.
  • the third method column chromatography
  • the crude protein solution A was purified by anion exchange resin columns such as HiTrap Q FF 16/10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, and HiTrap DEAE.
  • the eluent was a NaCl solution gradient elution, and 20 mM NaH 2 PO 4 /Na 2 HPO 4 buffer (pH 8.0) was added.
  • Each elution fraction was combined by SDS-PAGE electrophoresis detection, and the combined eluate was centrifuged twice at 7000 rpm, each time for 1 hour; the supernatant was filtered with a 0.45 ⁇ m filter membrane, and the filtrate was combined.
  • the filtrate was dialyzed and concentrated with water, the molecular weight cutoff of the dialysis bag was 10 kD, and the inner solution was freeze-dried to obtain the target protein YK93-6.
  • the product protein YK93-6 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.
  • the crude protein solution B obtained in Example 2 is placed in a stirred container for two salting-outs: a saturated ammonium sulfate solution is slowly added along the wall to make the final concentration of ammonium sulfate 25%, and the protein is precipitated during the salting-out process. After the salting-out is complete, it is filtered to complete the first salting-out; 400 ml of pure water is added to the precipitate for suspension, and a saturated ammonium sulfate solution is slowly added along the wall again to make the final concentration of ammonium sulfate 25%, and the second salting-out is performed, filtered, and the precipitate is the crude protein extract.
  • the crude protein extract is washed three times with water: 200 ml of pure water is added for suspension, stirred, allowed to stand, and filtered; after repeating this three times, the precipitate is freeze-dried to obtain the target protein YK93-6.
  • the product protein YK93-6 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.
  • Testosterone propionate injection Neingbo Second Hormone Factory
  • Finasteride Merck
  • Model group androgen load model
  • Preparation of experimental animals The experimental animals were acclimatized in the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day.
  • Subcutaneous injection of testosterone propionate to replicate the model of prostatic hyperplasia in mice The mice were randomly divided into groups before modeling, with 10 mice in each group. Testosterone propionate (0.01 mg/kg) was injected subcutaneously in soybean oil once a day for 30 consecutive days. The control group was subcutaneously injected with an equal volume of sodium carboxymethyl cellulose. Finasteride 1 mg/kg, finasteride 3 mg/kg and YK93-6 0.5 g/kg were orally administered immediately after modeling. Body weight was weighed every other day. After 30 days of administration, the mice were weighed, the prostate was removed through a midline incision in the lower abdomen, the wet weight was weighed, and the prostate index was calculated as prostate wet weight (mg)/mouse body weight (g).
  • Cyclophosphamide (CTX) for injection YK93-6;
  • Preparation of experimental animals The experimental animals were acclimatized in the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day.
  • Lewis cell suspension was inoculated subcutaneously to replicate the mouse breast cancer model: Lewis tumor tissue was ground into a suspension using a glass homogenizer, and the tumor suspension was drawn with a syringe to count the number of live cells. The cell concentration was adjusted to 2.6*10 6 /ml with normal saline, and then the suspension was injected into the left armpit of the mouse at 0.2ml/mouse. The mice were randomly divided into a control group, a cyclophosphamide (100mg/kg) group, and a YK93-6 (0.5g/kg) group on the second day. Cyclophosphamide was injected intraperitoneally once, and the YK93-6 group was gavaged daily for 10 consecutive days.
  • the control group was orally administered with an equal volume of sodium carboxymethyl cellulose.
  • the body weight was measured on the day of treatment, 20 ⁇ L of blood was obtained by orbital blood sampling and blood routine analysis was performed, the tumor mass was peeled off, weighed and photographed, and the tumor inhibition rate was calculated.
  • 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%. TTEST was used to compare the data among groups. P ⁇ 0.05 was considered to be significantly different.
  • YK93-6 (0.5g/kg) can significantly inhibit the growth of lung cancer, with a tumor inhibition rate of 25.31%, and a significant reduction in tumor weight, compared with the control group, P ⁇ 0.05, with statistical significance. YK93-6 can also significantly inhibit the number of peripheral blood neutrophils, P ⁇ 0.05, with statistical significance.
  • Preparation of experimental animals The experimental animals were adapted 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 randomly divided into the control group and YK 93-6 group according to the birth date.
  • YK93-6 (2g/kg) was administered by gavage, and the weight and weight gain of the young mice were recorded every day. After 13 days of administration, the mother mice were killed by cervical dislocation, 400 ⁇ l of blood was collected from the eye sockets, left to stand at room temperature for 1-2h, and centrifuged at 1000g for 20min; serum was collected to measure the prolactin content. Data statistics:
  • 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, and the data of each group were compared among the groups using T.TEST. P ⁇ 0.05 was considered to be a significant difference.
  • Preparation of experimental animals The experimental animals were acclimatized in the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day.
  • EL-4 tumor tissue was ground into suspension using a glass homogenizer, and the tumor suspension was drawn with a syringe to count the number of live cells. The cell concentration was adjusted to 2.0*10 6 /ml with normal saline, 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 YK93-6 (2g/kg) group.
  • Cyclophosphamide was injected intraperitoneally once, and the YK93-6 (2g/kg) group was gavaged daily for 13 consecutive days.
  • the control group was orally administered with an equal volume of sodium carboxymethyl cellulose.
  • the body weight was measured on the day of treatment, 20 ⁇ L of blood was obtained by orbital blood sampling and blood routine analysis was performed, the tumor mass was peeled off, weighed and photographed, and the tumor inhibition rate was calculated.
  • 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%. TTEST was used to compare the data among groups. P ⁇ 0.05 was considered to be significantly different.
  • EL-4 cells were subcutaneously inoculated to establish a mouse T cell lymphoma model.
  • the positive drug cyclophosphamide group can significantly inhibit tumor growth, significantly reduce tumor weight, and the tumor inhibition rate is 95.67%, compared with the control group, P ⁇ 0.05, with statistical difference.
  • the cyclophosphamide group can also significantly reduce white blood cells, lymphocytes, monocytes, and neutrophils, P ⁇ 0.05, with statistical difference.
  • YK93-6 can significantly inhibit the growth of lymphoma, significantly reduce the tumor weight, and the tumor inhibition rate is 29.33%, P ⁇ 0.05, with statistical significance. YK93-6 has no significant effect on peripheral blood leukocytes.
  • Drugs Tramadol hydrochloride sustained-release tablets, glacial acetic acid, YK93-6;
  • Preparation of experimental animals The experimental animals were acclimatized in the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day.
  • the YK93-6 (2g/kg) group was given oral administration, and the model group was given an equal volume of CMC-Na, once a day for 7 consecutive days. 30 minutes after the last administration, 1% acetic acid was intraperitoneally injected, and the number of twisting times of mice within 15 minutes was recorded, and the inhibition rate of drug twisting reaction was calculated.
  • the observation criteria were abdominal concavity, extension of hind limbs, and elevation of hips.
  • 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 the data of each group were compared between groups using TTEST. P ⁇ 0.05 was considered to be significantly different.
  • YK93-6 had a certain effect on alleviating pain and the number of writhing events was reduced.
  • Cyclophosphamide (CTX) for injection YK93-6;
  • Instrument electronic balance, five-category counter
  • Preparation of experimental animals The experimental animals were acclimatized in the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day.
  • Subcutaneous inoculation of B16F10 cell suspension to replicate mouse melanoma model Tumor mass was removed under sterile conditions, ground into tumor fluid, and the concentration was adjusted to 2.58 ⁇ 10 6 /ml with normal saline, and 0.2ml was evenly inoculated subcutaneously on the back of the mouse armpit. The animals were randomly divided into groups on the second day and drug administration began (recorded as day 0). CTX (100 mg/kg) was administered once a week, YK-93-6 (2 g/kg) was administered daily, and the control group was orally administered with an equal volume of sodium carboxymethyl cellulose. Body weight was measured on the day of treatment, 20 ⁇ L of blood was obtained through orbital blood sampling and blood routine analysis was performed, tumor mass was peeled, weighed and photographed, and tumor inhibition rate was calculated.
  • 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%. TTEST was used to compare the data among groups. P ⁇ 0.05 was considered to be significantly different.
  • YK93-6 (2g/kg) can significantly inhibit the growth of lung cancer, significantly reduce tumor weight, and the tumor inhibition rate is 66.11%, compared with the control group, P ⁇ 0.05, with statistical difference.
  • YK93-6 significantly reduces the number of peripheral blood lymphocytes, P ⁇ 0.05, with statistical difference.

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Abstract

本发明属于生物制药领域，提供了一种角蛋白YK93-6、其编码核酸分子、含有该核酸分子的表达载体、含有该表达载体或基因组整合该核酸分子的宿主细胞，以及其制备方法和药物组合物，还提供了上述角蛋白YK93-6等产品在制备治疗前列腺增生、淋巴癌、黑色素瘤、镇痛、泌乳、乳腺癌、肺癌、子宫肌瘤、凝血等药物中的应用。

Description

一种角蛋白YK93-6、制法和其药物组合物与用途 技术领域
本发明属于生物制药领域，涉及一种角蛋白YK93-6，编码角蛋白YK93-6的核酸分子，含有该核酸分子的表达载体，以及含有该表达载体或基因组整合该核酸分子的宿主细胞，以及角蛋白YK93-6的制备方法，含有这种角蛋白的药物组合物，以及这种角蛋白和所述药物组合物在制备预防或治疗前列腺增生、淋巴癌、黑色素瘤、镇痛、泌乳、乳腺癌、肺癌、子宫肌瘤、凝血药物中的应用。
背景技术
角蛋白是蛋白质的一种，广泛存在于人和动物的表皮，并且是毛发、羽毛、蹄、壳、爪、角等的主要成分，是结缔组织极重要的结构蛋白质，起着保护机体的作用。
角蛋白广泛存在于生物体中，是一种可再生资源，具有很大的利用价值，但并没有得到广泛有效的利用。主要原因是角蛋白不溶于各种溶剂，并且角蛋白一般都较其他蛋白质更耐蛋白酶的酶解。因此提取制备天然角蛋白的难度非常大。
随着基因组学、蛋白质组学、基因工程、微生物工程等现代生物技术的飞速发展，越来越多的基因被发现。而利用蛋白表达系统制备生产目的蛋白是研究基因或蛋白的生物功能的重要手段。
利用蛋白表达系统制备目的角蛋白，进而研究其结构与功能，未见其他文献报道，具有新颖性和创造性。
发明内容
本发明解决的技术问题是提供一种角蛋白YK93-6，编码角蛋白YK93-6的核酸分子，含有该核酸分子的表达载体，以及含有该表达载体或基因组整合该核酸分子的宿主细胞，以及角蛋白YK93-6的制备方法，含有角蛋白YK93-6的药物组合物，以及上述角蛋白YK93-6、核酸分子、表达载体、宿主细胞、或药物组合物在制备前列腺增生、淋巴癌、黑色素瘤、镇痛、泌乳、乳腺癌、肺癌、子宫肌瘤、凝血药物中的应用。
为解决本发明的技术问题，本发明提供如下技术方案：
本发明技术方案的第一方面是提供了一种角蛋白YK93-6，其特征在于，所述角蛋白YK93-6的氨基酸序列为：
(1)序列表中SEQ ID NO.1所示的氨基酸序列；
(2)序列表中SEQ ID NO.1所示的氨基酸序列经替换、缺失或添加1-35个氨基酸形成的基本上保持相同生物学功能的氨基酸序列。
进一步的，在角蛋白YK93-6上可进行常规修饰；或者在角蛋白YK93-6上还连接有用于检测或纯化的标签。
更进一步的，所述的常规修饰包括乙酰化、酰胺化、环化、糖基化、磷酸化、烷基化、生物素化、荧光基团修饰、聚乙二醇PEG修饰、固定化修饰、硫酸化、氧化、甲基化、脱氨化、形成二硫键或二硫键断裂；所述的标签包括His6、GST、EGFP、MBP、Nus、HA、 IgG、FLAG、c-Myc、Profinity eXact。
本发明技术方案的第二方面是提供了一种编码第一方面所述角蛋白YK93-6的核酸分子。
进一步的，所述的核酸分子的核苷酸序列为：
(1)序列表中SEQ ID NO.2所示的核苷酸序列；
(2)基于SEQ ID NO.2所示的核苷酸序列进行序列优化得到的核苷酸序列；
(3)与上述(1)或(2)中的核苷酸序列互补的核苷酸序列。
本发明技术方案的第三方面是提供了一种表达载体，其特征在于，所述的表达载体含有第二方面所述的核酸分子。
进一步的，表达载体可以为pET系列、pUC系列、pQE系列、pBV系列、pMAL系列，pPIC9、pPIC9K、pHIL-S1、pPICZα/A、pYAM75P，pHIL-D2、pA0815、pPIC3K、pPICZ、pHWO10、pGAPZ、pGAPZa、pPIC3.5K等；优选的表达载体为pET系列载体；最优选的表达载体为pET-28a(+)。
本发明技术方案的第四方面是提供了一种宿主细胞，其特征在于，所述的宿主细胞含有第三方面所述的表达载体或者基因组中整合有第二方面所述的核酸分子。
进一步的，所述的宿主细胞包括细菌、酵母、曲霉菌、植物细胞、或昆虫细胞。
更进一步的，所述的细菌包括大肠杆菌或酵母。
感受态宿主细胞可以为BL21系列，Transetta系列，Rosetta系列，DH5α系列，JM系列，Top系列，Orgami系列，Trans1-T1，TG1，TB1；Y11430，MG1003，GS115(AOX1)，KM71，SMD1168等；优选的表达感受态细胞为BL21(DE3)，Transetta(DE3)。
本发明技术方案的第五方面是提供了一种制备第一方面所述角蛋白YK93-6的方法，其特征在于，包括以下步骤：
A.合成第一方面所述的角蛋白YK93-6对应的核酸分子，将核酸分子连入相应的表达载体，将表达载体转化到宿主细胞，在一定条件下在发酵设备中培养带表达载体的宿主细胞并诱导表达角蛋白YK93-6，得到含有角蛋白YK93-6的粗蛋白溶液；
B.对步骤A中所表达的粗蛋白溶液进行分离纯化干燥得到角蛋白YK93-6。
进一步的，在步骤A中，所述的宿主细胞主要为选自大肠杆菌，所述的角蛋白YK93-6在大肠杆菌包涵体中表达，所述的发酵设备包括摇瓶或发酵罐。
进一步的，在步骤A中，诱导表达角蛋白YK93-6后，可用清洗剂清洗杂质，用溶液溶解得到粗蛋白溶液。
进一步的，步骤A中的培养基可以为LB培养基、TB培养基、SB培养基、SOB培养基、SOC培养基，PDA培养基、YPD培养基、孟加拉红培养基、高盐察氏培养基、DOBA培养基、米曲培养基及其改良配方等；摇瓶发酵优选LB培养基；发酵罐优选LB培养基及其改良配方。
进一步的，步骤A中的诱导剂可以为IPTG、乳糖、阿拉伯糖等；优选为IPTG、乳糖。
进一步的，步骤A中，获得的发酵菌液，离心，弃去上清液；沉淀悬浮于缓冲液中，破碎菌体，再离心，弃去上清液；沉淀用清洗剂清洗后，再用尿素溶液溶解，得到YK93-6粗蛋白溶液。
其中，缓冲液优选为buffer A，其用量为：发酵液体积:buffer A体积＝1～100:1，优选为10:1；
清洗剂可以为尿素溶液、盐酸胍溶液、Triton、buffer A等，最优选为1％Triton溶液，其用量为：发酵液体积:1％Triton体积＝0.2～100:1，优选为1～15:1；
尿素溶液优选为8M尿素溶液，其用量为：发酵液体积:8M尿素体积＝0.2～100:1，优选为2～15:1。
进一步的，在步骤B中，所述的分离纯化的方法包括超滤微滤膜技术纯化方法、柱色谱纯化方法、盐析方法、透析方法。
进一步的，步骤B中，分离纯化方法如下：
(1)所述透析方法，即将步骤A中获得的粗蛋白溶液用透析的方法纯化，得到目标蛋白YK93-6溶液。
透析袋截留分子量可以为0.5-10kD，优选的透析袋截留分子量为3.5-10kD，最优选的透析袋截留分子量为10kD。
(2)所述超滤微滤方法，即将步骤A中获得的粗蛋白溶液用超滤膜或微滤膜等膜技术纯化，得到目标蛋白YK93-6浓缩溶液。
优选的是两次微滤膜纯化，第一次膜孔径1000～1500nm，第二次膜孔径20～50nm。
(3)所述柱色谱方法，即将步骤A中获得的粗蛋白溶液通过柱色谱，例如各种交换柱或排阻柱色谱，分离纯化得到目标蛋白YK93-6。
优选的排阻柱是葡聚糖凝胶柱，Superdex 30 Increase，Superdex 75 Increase，Superdex 200 Increase，Superose 6 Increase等；优选的交换柱是离子交换树脂柱：阴离子交换树脂柱，HiTrap Q FF，HiTrap Capto Q ImpRes，Capto Q ImpRes，HiTrap Capto Q，HiTrap DEAE，Toyopearl Q-650M，Toyopearl SuperQ-650M等；阳离子交换树脂柱，HiTrap SP FF，HiTrap Capto SP ImpRes，Capto SP ImpRes，HiTrap Capto SP，Toyopearl SP-650M，Toyopearl Super SP-650M。最优选的是阴离子交换树脂柱。
洗脱剂可以使用本领域常用的洗脱剂，例如水、盐溶液，所述盐溶液包括氯化钠溶液、磷酸二氢钠溶液、磷酸氢二钠溶液、醋酸钠、醋酸等。
(4)所述盐析方法，即将步骤A中获得的粗蛋白溶液中用盐析的方法纯化，得到目标蛋白YK93-6混悬液。
盐析剂可以为硫酸铵、硫酸钠、氯化钠、氯化镁、硫酸铝，硝酸铵、氯化铵、硫酸镁等。优选的盐析剂为硫酸铵及其水溶液。加入饱和硫酸铵水溶液使硫酸铵终浓度达到10～50％，优选为20～30％，更优选为25％。
盐析次数为1～3次，优选为2次。
盐析后沉淀加入纯水清洗，清洗次数为2～5次，优选为3次。
进一步的，步骤B纯化得到的目标蛋白YK93-6溶液可经冷冻干燥或真空干燥成干粉，也可将浓缩液直接喷雾干燥成干粉。
本发明技术方案的第六方面是提供了一种药物组合物，其特征在于，所述的药物组合物含有第一方面所述的角蛋白YK93-6或第二方面所述的核酸分子或第三方面所述的表达载体或第四方面所述的宿主细胞以及药学上可接受的载体或赋形剂。
本发明上述步骤中得到的角蛋白可经冷冻干燥或真空干燥成干粉，也可把浓缩液体直接喷雾干燥成干粉，然后制成各种剂型。
本发明涉及一种药物组合物，包括上述步骤中得到的任意一种角蛋白及药学上可接受的载体。
本发明还涉及含有作为活性成份的本发明角蛋白以及常规药物赋形剂或辅剂的药物组合物。通常本发明角蛋白占药物组合物总重量的0.1～100.0％。
本发明还提供一种药物组合物，它包括药物有效剂量的作为活性成分的蛋白质及药学上可接受的载体。
本发明所述的药物组合物可根据本领域公知的方法制备。用于此目的时，如果需要，可将本发明蛋白质与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人药或兽药使用的适当的施用形式或剂量形式。
本发明角蛋白或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、肌肉、皮下、鼻腔、口腔粘膜、眼、肺、皮肤、阴道、腹膜、直肠等，优选口服给药。
本发明角蛋白或含有它的药物组合物的给药途径可为注射给药。注射包括静脉注射、肌肉注射、皮下注射、皮内注射、腹腔注射和穴位注射等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括水包油型、油包水型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等。固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明角蛋白可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基 纤维素、丙烯酸树脂、卡波姆、聚乙烯毗咯烷酮、聚乙二丙醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯毗咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与构椽酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、月桂酸聚乙二醇甘油酯、高岭土、滑石粉等；粘合剂，如阿拉伯胶、黄菩胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。
为了将给药单元制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。
为了将给药单元制成胶囊，将有效成分本发明角蛋白与上述的各种载体混合，并将由此得到的混合物置于硬的明胶胶囊或软胶囊中。也可将有效成分本发明角蛋白制成微囊剂，混悬于水性介质中形成混悬剂，亦可装入硬胶囊中或制成注射剂应用。
例如，将本发明角蛋白制成注射用制剂，如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂，这种制剂可以是含水或非水的，可含一种和/或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、l,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。这些辅料是本领域常用的。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
为达到用药目的，增强治疗效果，本发明的角蛋白或药物组合物可用任何公知的给药方法给药。
本发明角蛋白药物组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重、性格及个体反应，给药途径、给药次数、治疗目的，因此本发明的治疗剂量可以有大范围的变化。一般来讲，本发明中药学成分的使用剂量是本领域技术人员公知的。可以根据本发明角蛋白组合物中最后的制剂中所含有的实际药物数量，加以适当的调整，以达到其治疗有效量的要求，完成本发明的预防或治疗目的。本发明角蛋白的每天的合适剂量范围：本发明的角蛋白的用量为0.01～1000mg/kg体重，优选为5～1000mg/kg体重，更优选为10～500mg/kg体重，最优选为20～300mg/kg体重。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药，这取决于给药医生的临床经验以及包括运用其它治疗手段的给药方案。每一种治疗所需总剂量可分成多次或按一次剂量给药。本发明的蛋白质或药物组合物可单独服用，或与其他治疗药物或对症药物合并使用并调整剂量。
本发明技术方案的第七方面是提供了第一方面所述的角蛋白YK93-6或第二方面所述的核酸分子或第三方面所述的表达载体或第四方面所述的宿主细胞或第六方面所述的药物组合物在制备前列腺增生、淋巴癌、黑色素瘤、镇痛、泌乳、乳腺癌、肺癌、子宫肌瘤、凝血药物中的应用。
为了完成本发明之目的，本发明采取如下技术方案，具体地讲，制备本发明角蛋白YK93-6，包括如下步骤：
(1)合成核苷酸序列，并测定序列的准确性；
优选的核苷酸序列如SEQ ID No.2所示。
(2)将核苷酸序列转入表达载体中；
表达载体可以为pET系列、pUC系列、pQE系列、pBV系列、pMAL系列，pPIC9、pPIC9K、pHIL-S1、pPICZα/A、pYAM75P，pHIL-D2、pA0815、pPIC3K、pPICZ、pHWO10、pGAPZ、pGAPZa、pPIC3.5K等；优选的表达载体为pET系列载体；最优选的表达载体为pET-30a(+)。
(3)将表达载体转染入宿主细胞中；
宿主细胞可以为大肠杆菌或酵母；优选的宿主细胞为大肠杆菌；
感受态细胞可以为BL21系列，Transetta系列，Rosetta系列，DH5α系列，JM系列，Top系列，Orgami系列，Trans1-T1，TG1，TB1；Y11430，MG1003，GS115(AOX1)，KM71，SMD1168等；优选的表达感受态细胞为BL21(DE3)，Transetta(DE3)。
(4)将在适当的条件下，发酵培养宿主细胞，诱导表达目标蛋白YK93-6；
发酵设备可以采用摇瓶或发酵罐；
培养基可以为LB培养基、TB培养基、SB培养基、SOB培养基、SOC培养基，PDA培养基、YPD培养基、孟加拉红培养基、高盐察氏培养基、DOBA培养基、米曲培养基及其改良配方等；摇瓶发酵优选LB培养基；发酵罐优选LB培养基及其改良配方。
诱导剂可以为IPTG、乳糖、阿拉伯糖等；优选为IPTG、乳糖。
(5)目标蛋白YK93-6产物富集；
步骤(4)获得的发酵菌液，离心，弃去上清液；沉淀悬浮于缓冲液中，破碎菌体，再离心，弃去上清液；沉淀用清洗剂清洗后，再用尿素溶液溶解，得到YK93-6粗蛋白溶液。
其中，缓冲液优选为buffer A，其用量为：发酵液体积:buffer A体积＝1～100:1，优选为10:1；
清洗剂可以为尿素溶液、盐酸胍溶液、Triton、buffer A等，最优选为1％Triton溶液，其用量为：发酵液体积:1％Triton体积＝0.2～100:1，优选为1～15:1；
尿素溶液优选为8M尿素溶液，其用量为：发酵液体积:8M尿素体积＝0.2～100:1，优选为2～15:1。
(6)分离纯化目标蛋白YK93-6：
步骤(5)获得的粗蛋白溶液，需经过纯化获得目标蛋白YK93-6。所述纯化可以通过 透析、或超滤微滤、或柱色谱、或盐析步骤进行。
A.所述透析步骤，即将步骤(5)获得的粗蛋白溶液用透析的方法纯化，得到目标蛋白YK93-6溶液。
透析袋截留分子量可以为0.5-10kD，优选的透析袋截留分子量为3.5-10kD，最优选的透析袋截留分子量为10kD。
B.所述超滤微滤步骤，即将步骤(5)获得的粗蛋白溶液用超滤膜或微滤膜等膜技术纯化，得到目标蛋白YK93-6浓缩溶液。
优选的是两次微滤膜纯化，第一次膜孔径1000～1500nm，第二次膜孔径20～50nm。
C.所述柱色谱步骤，即将步骤(5)获得的粗蛋白溶液通过柱色谱，例如各种交换柱或排阻柱色谱，分离纯化得到目标蛋白YK93-6。
优选的排阻柱是葡聚糖凝胶柱，Superdex 30 Increase，Superdex 75 Increase，Superdex 200 Increase，Superose 6 Increase等；优选的交换柱是离子交换树脂柱：阴离子交换树脂柱，HiTrap Q FF，HiTrap Capto Q ImpRes，Capto Q ImpRes，HiTrap Capto Q，HiTrap DEAE，Toyopearl Q-650M，Toyopearl SuperQ-650M等；阳离子交换树脂柱，HiTrap SP FF，HiTrap Capto SP ImpRes，Capto SP ImpRes，HiTrap Capto SP，Toyopearl SP-650M，Toyopearl Super SP-650M。最优选的是阴离子交换树脂柱。
洗脱剂可以使用本领域常用的洗脱剂，例如水、盐溶液，所述盐溶液包括氯化钠溶液、磷酸二氢钠溶液、磷酸氢二钠溶液、醋酸钠、醋酸等。
D.所述盐析步骤，即将步骤(5)获得的粗蛋白溶液中用盐析的方法纯化，得到目标蛋白YK93-6混悬液。
盐析剂可以为硫酸铵、硫酸钠、氯化钠、氯化镁、硫酸铝，硝酸铵、氯化铵、硫酸镁等。优选的盐析剂为硫酸铵及其水溶液。加入饱和硫酸铵水溶液使硫酸铵终浓度达到10～50％，优选为20～30％，更优选为25％。
盐析次数为1～3次，优选为2次。
盐析后沉淀加入纯水清洗，清洗次数为2～5次，优选为3次。
步骤A～D纯化得到的目标蛋白YK93-6溶液可经冷冻干燥或真空干燥成干粉，也可将浓缩液直接喷雾干燥成干粉。
本发明的有益技术效果：
1、本发明蛋白为首次获得的角蛋白，本发明的制备方法具备收率高、样品纯度高的特点。
2、本发明通过蛋白YK93-6对小鼠前列腺增生模型的药效研究，证明蛋白YK93-6可显著降低前列腺湿重；
3、本发明通过蛋白YK93-6对Lewis肺癌药效研究，证明蛋白YK93-6具有明显的抑制肺癌增殖作用；
4、本发明通过蛋白YK93-6对孕鼠泌乳研究，证明蛋白YK93-6对母鼠血清泌乳素的含 量无明显影响；
5、本发明通过蛋白YK93-6对EL-4淋巴瘤药效研究，证明蛋白YK93-6能显著抑制淋巴瘤的增殖。
6、本发明通过蛋白YK93-6对小鼠镇痛模型的药效研究，证明蛋白YK93-6可一定程度减少扭体次数。
7、本发明通过蛋白YK93-6对B16F10黑色素瘤药效研究，证明蛋白YK93-6具有明显的抑制黑色素瘤增殖作用。
附图说明
图1.表达蛋白YK93-6还原型SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)分析
(M：蛋白分子量标准；S：表达蛋白YK93-6)
图2.YK93-6对Lewis肺癌小鼠血常规的影响
(与正常对照组比较，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
图3.YK93-6对母鼠泌乳量的影响
(与正常对照组比较，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
图4.YK93-6对母鼠血清泌乳素的影响
(与正常对照组比较，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
图5.YK93-6对EL-4淋巴瘤小鼠血常规的影响
(与正常对照组比较，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
图6.YK93-6对小鼠醋酸扭体的抑制作用
(与正常对照组比较，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
图7.YK93-6对B16F10黑色素瘤小鼠血常规的影响
(与正常对照组比较，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
具体实施方式
下面的实施例及药理活性试验例用来进一步说明本发明，但这并不意味着对本发明的任何限制。
下述实施例及药理活性试验例中的实验方法，如无特殊说明，均为常规方法；所用的实验材料，如无特殊说明，均购自常规生化试剂公司。
实施例1摇瓶发酵制备蛋白YK93-6粗溶液A(LB培养基)
合成如SEQ ID No.2所示的核苷酸序列，将其转入pET-30a(+)载体中；测序确定得到含有正确序列的表达载体；将表达载体转染入BL21(DE3)细胞，得到含有目标核苷酸序列的表达感受态宿主细胞。加入LB培养基中，于摇床中，在37℃、220rpm条件下培养1小时，得到重组菌株。
蘸取重组菌株在含有Kana霉素的LBA平板中划线，平板倒置于37℃恒温培养箱过夜培养16小时。
配置400ml LB培养基，分装2瓶，每瓶200ml。在每瓶(200ml)LB培养基中加入Kana霉素(终浓度50μg/ml)，取平板上单一菌落加入LB培养基中，于摇床中，在37℃、220rpm条件下过夜扩增培养，得到种子液。
配置9.6L LB培养基，分装于48瓶，每瓶200ml。在每瓶(200ml)LB培养基中加入Kana霉素(终浓度50μg/ml)，再加入2ml种子液，于摇床中，在37℃、220rpm条件下培养2-3小时。监测OD600，当OD600达到1.0左右时，加入诱导剂，于摇床中诱导表达蛋白，诱导条件选自下表。
合并各瓶菌液，7000rpm离心5分钟，上清液灭菌后弃去；沉淀悬浮于约1L缓冲液中，用80-100目筛网过滤，滤液用高压破碎仪破碎，压力800-1000bar，2次，每次2分钟。破碎后菌液7000rpm离心30分钟，弃去上清液，得到沉淀(即包涵体)。沉淀加入600ml清洗剂清洗2次，离心，弃去上清液。沉淀再加入尿素溶液溶解3次，体积均为600ml。合并3次溶液，7000rpm离心30分钟，沉淀弃去，上清液即为蛋白粗溶液A。
蛋白YK93-6粗溶液A，用还原型SDS-PAGE分析，分离胶浓度为12.5％，考马斯亮蓝R250法染色；在分子量52KD附近显示明显蓝色条带。
实施例2发酵罐制备蛋白YK93-6粗溶液B
实施例1中合成并测序确定得到含有如SEQ ID No.2所示的序列的表达载体；将表达载体转染入BL21(DE3)细胞，得到含有目标核苷酸序列的表达感受态宿主细胞。加入LB培养基中，于摇床中，在37℃、220rpm条件下培养1小时，得到重组菌株。
在含有Kana霉素的LBA平板中，加入重组菌株100μl，涂布器涂至均匀变干，平板 倒置于37℃恒温培养箱过夜培养。分别取三个单菌落在含有Kana霉素的平板中划线，平板过夜培养，经三批摇瓶发酵表达验证确认无误后，用15％甘油保存菌株，分装成每支1ml，即得工作细胞库，冻存于-80℃冰箱备用。
从工作细胞库中取出1支甘油菌，取100μl，加入40ml LB培养基中，加入Kana霉素(终浓度50μg/ml)，于振荡器中，以37℃、220rpm条件培养6小时，得一级种子液。
取一级种子液1.2ml，加入120ml LB培养基中，加入Kana霉素(终浓度50μg/ml)，于振荡器中，以37℃、220rpm条件培养7小时，得二级种子液。
5L发酵罐中加入3L改良的LB培养液，再加入120ml二级种子液，3ml Kana霉素(终浓度50μg/ml)，以37℃、溶氧30％(串联转速)条件培养约4小时。监测OD值在20左右，3g乳糖作为诱导剂，于20℃进行诱导，以30ml/小时的速率进行补料，20℃培养24小时。
菌液7000rpm离心5分钟，上清液灭菌后弃去；沉淀悬浮于约200ml buffer A中，用80-100目筛网过滤，滤液用高压破碎仪破碎，压力800-1000bar，2次，每次2分钟。破碎后菌液7000rpm离心30分钟，弃去上清液。
沉淀加入1％Triton溶液清洗2次，每次600ml，离心，弃去上清液。沉淀依次加入4M尿素溶液溶解1次，8M尿素溶液溶解2次，体积均为600ml。合并三次溶液，7000rpm离心30分钟，沉淀弃去，上清液即为蛋白粗溶液B。
蛋白YK93-6粗溶液B，用还原型SDS-PAGE分析，分离胶浓度为12.5％，考马斯亮蓝R250法染色；在分子量52KD附近显示明显蓝色条带。
实施例3蛋白粗溶液B通过膜技术制备得蛋白YK93-6
实施例2获得的蛋白粗溶液B，通过微滤膜技术进行纯化：先用1500nm或1000nm陶瓷膜芯进行固液分离；内液弃去，外液再用20nm或50nm陶瓷膜芯反复微滤除去尿素；二次微滤的内液冷冻干燥，即得目标蛋白YK93-6。
蛋白YK93-6结构确证：
1、还原型SDS-聚丙烯酰胺凝胶电泳法(SDS-PAGE)分析
仪器：蛋白质电泳仪(Bio-Rad)。
方法和结果：蛋白YK93-6溶液，用还原型SDS-PAGE分析，分离胶浓度为12.5％，考马斯亮蓝R250法染色。YK93-6条带分子量在52KD附近。
2、基于LC-MS/MS的蛋白质全序列分析
主要材料：乙腈、甲酸、碳酸氢铵、二硫苏糖醇(DTT)、碘乙酰胺(IAA)、胰蛋白酶、糜蛋白酶、Glu-C、Pepsin、Elastase；
主要仪器：纳流液相色谱仪(Thermo EastnLC1200)，质谱仪(Thermo Orbitrap Fusion Lumos)，恒温培养箱(中仪国科(北京)科技有限公司，DHP-9052)。方法和结果：
蛋白YK93-6通过溶解置换、还原烷基化、多种蛋白酶解等前处理，得到酶切肽段；酶切肽段溶液，经液相色谱串联质谱分析，质谱原始文件使用Byonic软件分析数据，鉴定 结果覆盖率100％，确定其与目标序列SEQ ID No.1一致。
实施例4蛋白粗溶液A纯化制备得蛋白YK93-6
实施例1获得的蛋白粗溶液A通过下述三种方法进行纯化：
第一种方法：透析；
蛋白粗溶液A用0.45μm滤膜过滤，滤液用水透析，透析72小时以上，内液冷冻干燥，即得目标蛋白YK93-6。
第二种方法：盐析；
蛋白粗溶液A置于带搅拌的容器中进行两次盐析：沿壁缓慢加入硫酸铵饱和溶液，使硫酸铵终浓度为25％或50％，盐析过程中蛋白析出，待盐析完全后，过滤，完成第一次盐析；沉淀中加入400ml纯水混悬，再次沿壁缓慢加入硫酸铵饱和溶液，使硫酸铵终浓度为25％，进行第二次盐析，过滤，沉淀即为蛋白粗提物。蛋白粗提物用水进行三次清洗：加200ml纯水混悬，搅拌，静置，过滤；如此重复三次后，沉淀冷冻干燥即得目标蛋白YK93-6。
第三种方法：柱色谱；
蛋白粗溶液A分别经过HiTrap Q FF 16/10，HiTrap Capto Q ImpRes，Capto Q ImpRes，HiTrap Capto Q，HiTrap DEAE等阴离子交换树脂柱纯化。洗脱液为NaCl溶液梯度洗脱，加20mM NaH2PO4/Na2HPO4缓冲液(pH 8.0)。各洗脱流份用SDS-PAGE电泳检测合并，合并的洗脱液7000rpm离心2次，每次1小时；上清液用0.45μm滤膜过滤，合并滤液。滤液用水透析浓缩，透析袋截留分子量10kD，内液冷冻干燥，即得目标蛋白YK93-6。
三种方法得到的产物蛋白YK93-6，经与实施例3相同的结构确证方法，确认其与实施例3制备得到的蛋白具有相同的氨基酸序列。
实施例5蛋白粗溶液B通过盐析方法得蛋白YK93-6
实施例2获得的蛋白粗溶液B置于带搅拌的容器中进行两次盐析：沿壁缓慢加入硫酸铵饱和溶液，使硫酸铵终浓度为25％，盐析过程中蛋白析出，待盐析完全后，过滤，完成第一次盐析；沉淀中加入400ml纯水混悬，再次沿壁缓慢加入硫酸铵饱和溶液，使硫酸铵终浓度为25％，进行第二次盐析，过滤，沉淀即为蛋白粗提物。蛋白粗提物用水进行三次清洗：加200ml纯水混悬，搅拌，静置，过滤；如此重复三次后，沉淀冷冻干燥即得目标蛋白YK93-6。
产物蛋白YK93-6，经与实施例3相同的结构确证方法，确认其与实施例3制备得到的蛋白具有相同的氨基酸序列。
药理试验
实验例1 YK93-6(实施例3蛋白)对小鼠前列腺增生模型的药效试验
动物：雄性昆明种小鼠18-20克
药品：丙酸睾酮注射液(宁波第二激素厂)；非那雄胺(默沙东)：
仪器：电子天平
实验分组：
正常对照组；
模型组：雄激素负荷模型；
阳性对照组：非那雄胺(1mg/kg)组；非那雄胺(3mg/kg)组；
YK93-6(0.5g/kg)组。
方法：
实验动物的准备：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天。
皮下注射丙酸睾酮复制小鼠前列腺增生模型：造模前随机分组，每组10只。皮下注射丙酸睾酮(0.01mg/kg)，大豆油配制，每日一次，连续30日。对照组皮下注射等体积羧甲基纤维素钠。造模后立即口服给予非那雄胺1mg/kg、非那雄胺3mg/kg及YK93-6 0.5g/kg。隔日称量体重。给药30天后称重，处死小鼠，通过下腹正中切口切除前列腺，称取湿重，以前列腺湿重(mg)/小鼠体重(g)计算前列腺指数。
数据统计：
计算各组前列腺湿重、前列腺指数的均值、标准差及标准误，应用T.TEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果：
结果见表1。
表1.YK93-6对小鼠体重、前列腺湿重和前列腺指数的影响
(与正常对照组比较，*P<0.05，**P<0.01，***P<0.001；与模型组比较，#P<0.05，##P<0.01，###P<0.001)
实验结论：
1)雄激素负荷法可成功诱导小鼠前列腺增生，模型组小鼠造模后前列腺湿重与前列腺指数明显升高，与正常组比较，P<0.05，有统计学差异。
2)阳性药非那雄胺组低剂量和高剂量在造模后均可以有效降低模型小鼠前列腺湿重与指数，且高剂量效果略好，与模型组比较，P<0.05，有统计学差异。
3)与模型组比较，YK 93-6明显降低小鼠的前列腺湿重(P<0.05)。对前列腺指数无明显影响，P>0.05，无统计学差异。
实验例2YK93-6(实施例3蛋白)对Lewis肺癌模型的药效试验
动物：雄性C57BL/6J小鼠
药品：注射用环磷酰胺(CTX)，YK93-6；
仪器：电子天平、五分类计数仪
实验分组：
对照组；
阳性对照组：环磷酰胺(100mg/kg)
YK93-6(0.5g/kg)组。
方法：
实验动物的准备：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天。
皮下接种Lewis细胞悬液复制小鼠乳腺癌模型：将Lewis瘤组织用玻璃匀浆器研磨制成悬液，用注射器吸取瘤悬液，计算活细胞数，用生理盐水调整细胞浓度为2.6*106/ml，再将悬液按照0.2ml/只注射到小鼠左侧腋下。第二天随机分成对照组、环磷酰胺(100mg/kg)组和YK93-6(0.5g/kg)组。环磷酰胺单次腹腔注射，YK93-6组每日灌胃，连续10日，对照组口服等量体积羧甲基纤维素钠。处理当天测量体重，通过眼眶取血得到20μL血液并进行血常规分析，剥取瘤块，称重拍照，计算抑瘤率。
数据统计：
以(对照组平均质量-实验组平均质量)/对照组平均质量×100％计算抑瘤率及瘤重的均值、标准差及标准误，应用TTEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果：
结果见表2、图2。
表2.YK93-6对小鼠Lewis肺癌的抑制作用
(与对照组比较，*P<0.05，**P<0.01，***P<0.001)
实验结论：
1)皮下接种Lewis细胞可成功建立小鼠肺癌模型。
2)阳性药环磷酰胺组可以显著抑制肿瘤生长，瘤重明显降低，抑瘤率达95.35％，与对照组比较，P<0.05，有统计学差异。
3)与对照组比较，YK93-6(0.5g/kg)可显著抑制肺癌的增长，抑瘤率达25.31％，瘤重明显降低，与对照组比较，P<0.05，有统计学差异。YK93-6还可显著抑制外周血中性粒细胞的数量，P<0.05，有统计学差异。
实验例3蛋白YK93-6(实施例3蛋白)对孕鼠泌乳的药效试验
动物：雌性昆明种孕鼠30-50克
药品：YK93-6
仪器：电子天平
实验分组：
对照组；
YK93-6(2g/kg)组
方法：
实验动物的准备：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天。记录每只孕鼠生产日期，按照出生日期进行随机分为对照组、YK 93-6组。
YK93-6(2g/kg)灌胃给药，每天记录幼鼠体重及体重增加量。给药13天后颈椎脱臼法处死母鼠，眼眶取血400μl，室温静置1-2h，1000g离心20min；取血清测泌乳素的含量。数据统计：
计算各组幼鼠每天平均体重增加量(母鼠泌乳量)与母鼠血清泌乳素含量，应用T.TEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果：
结果见图3、图4。
实验结论：
1)YK93-6对幼鼠的体重增长无明显影响。
2)与对照组比较，YK93-6对母鼠血清泌乳素无明显影响。
实验例4 YK93-6(实施例3蛋白)EL-4淋巴瘤模型的药效试验
动物：雄性C57BL/6J小鼠
药品：注射用环磷酰胺(CTX)，YK93-6
仪器：电子天平、五分类计数仪
实验分组：
对照组；
阳性对照组：环磷酰胺(100mg/kg)
YK93-6(2g/kg)组。
方法：
实验动物的准备：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天。
皮下接种EL-4细胞悬液复制小鼠淋巴瘤模型：将EL-4瘤组织用玻璃匀浆器研磨制成悬液，用注射器吸取瘤悬液，计算活细胞数，用生理盐水调整细胞浓度为2.0*106/ml，再将悬液按照0.2ml/只注射到小鼠左侧腋下。第二天随机分成对照组、环磷酰胺(100mg/kg)组、YK93-6(2g/kg)组。环磷酰胺单次腹腔注射，YK93-6(2g/kg)组每日灌胃，连续13日，对照组口服等量体积羧甲基纤维素钠。处理当天测量体重，通过眼眶取血得到20μL血液并进行血常规分析，剥取瘤块，称重拍照，计算抑瘤率。
数据统计：
以(对照组平均质量-实验组平均质量)/对照组平均质量×100％计算抑瘤率及瘤重的均值、标准差及标准误，应用TTEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果：
结果见表3、图5。
表3.YK93-6对小鼠EL-4淋巴瘤的抑制作用
(与对照组比较，*P<0.05，**P<0.01，***P<0.001)
实验结论：
1)皮下接种EL-4细胞建立小鼠T细胞淋巴瘤模型。
2)阳性药环磷酰胺组可以显著抑制肿瘤生长，瘤重明显降低，抑瘤率达95.67％，与对照组比较，P<0.05，有统计学差异。环磷酰胺组还可明显降低白细胞、淋巴细胞、单核细胞、中性粒细胞，P<0.05，有统计学差异。
3)与对照组比较，YK93-6能明显抑制淋巴瘤的生长，瘤重显著降低，抑瘤率达29.33％，P<0.05，有统计学差异。YK93-6对外周血白细胞无明显影响。
实验例5蛋白YK93-6(实施例3蛋白)对小鼠疼痛模型的药效试验
动物：雄性昆明小鼠
药品：盐酸曲马多缓释片，冰醋酸、YK93-6；
仪器：电子天平、计时器
实验分组：
对照组；
阳性对照组：盐酸曲马多组；
YK93-6(2g/kg)组。
方法：
实验动物的准备：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天。
YK93-6(2g/kg)组灌胃给药,模型组给予等体积的CMC-Na，每日1次,连续给药7日。末次给药30分钟后,均腹腔注射给予1％的醋酸,记录小鼠15min内扭体次数,并计算药物扭体反应抑制率。观察标准为腹腔内凹,伸展后肢,臀部抬高。
数据统计：
以(对照组扭体反应平均数-给药组扭体反应平均数)/对照组扭体反应平均数×100％计算抑制率。记录扭体次数的均值、标准差及标准误，应用TTEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果：
结果见表4、图6。
表4.YK93-6对小鼠疼痛模型的抑制作用
(与对照组比较，*P<0.05，**P<0.01，***P<0.001)
实验结论：
1)腹腔注射1％醋酸可成功诱导小鼠疼痛模型。15分钟平均扭体次数为19次。
2)阳性药盐酸曲马多组可以显著缓解疼痛，扭体发生次数明显减少，与对照组比较，P<0.05，有统计学差异。
3)与对照组比较，YK93-6对疼痛具有一定缓解作用，扭体发生次数减少。
实验例6 YK93-6(实施例3蛋白)对B16F10黑色素模型的药效试验
动物：雌性C57BL/6J小鼠
药品：注射用环磷酰胺(CTX)，YK93-6；
仪器：电子天平、五分类计数仪
实验分组：
对照组；
阳性对照组：环磷酰胺(100mg/kg)
YK93-6(2g/kg)组。
方法：
实验动物的准备：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天。
皮下接种B16F10细胞悬液复制小鼠黑色素瘤模型：无菌条件下取出瘤块，研磨成瘤液，生理盐水调整浓度至2.58×106/ml，取0.2ml均匀接种于小鼠腋背部皮下。第二天将动物随机分组，开始给药(记为第0天)。CTX(100mg/kg)每周给药一次，YK-93-6(2g/kg)每天给药，对照组口服等量体积羧甲基纤维素钠。处理当天测量体重，通过眼眶取血得到20μL血液并进行血常规分析，剥取瘤块，称重拍照，计算抑瘤率。
数据统计：
以(对照组平均质量-实验组平均质量)/对照组平均质量×100％计算抑瘤率及瘤重的均值、标准差及标准误，应用TTEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果：
结果见表5、图7。
表5.YK93-6对小鼠B16F10黑色素瘤的抑制作用
(与对照组比较，*P<0.05，**P<0.01，***P<0.001)
实验结论：
1)皮下接种B16F10细胞可成功建立小鼠黑色素瘤模型。
2)阳性药环磷酰胺组可以显著抑制肿瘤生长，瘤重明显降低，抑瘤率达85.82％，与对照组比较，P<0.05，有统计学差异。
3)与对照组比较，YK93-6(2g/kg)可显著抑制肺癌的生长，瘤重明显降低，抑瘤率达66.11％，与对照组比较，P<0.05，有统计学差异。YK93-6明显降低外周血淋巴细胞的数量，P<0.05，有统计学差异。

Claims (15)

  1. 一种角蛋白YK93-6，其特征在于，所述角蛋白YK93-6的氨基酸序列为：
    (1)序列表中SEQ ID NO.1所示的氨基酸序列；
    (2)序列表中SEQ ID NO.1所示的氨基酸序列经替换、缺失或添加1-35个氨基酸形成的基本上保持相同生物学功能的氨基酸序列。
  2. 根据权利要求1的角蛋白YK93-6，其特征在于，在角蛋白YK93-6上可进行常规修饰；或者在角蛋白YK93-6上还连接有用于检测或纯化的标签。
  3. 根据权利要求2的角蛋白YK93-6，其特征在于，所述的常规修饰包括乙酰化、酰胺化、环化、糖基化、磷酸化、烷基化、生物素化、荧光基团修饰、聚乙二醇PEG修饰、固定化修饰、硫酸化、氧化、甲基化、脱氨化、形成二硫键或二硫键断裂；所述的标签包括His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc、Profinity eXact。
  4. 一种编码权利要求1-3任一项所述角蛋白YK93-6的核酸分子。
  5. 根据权利要求4的核酸分子，其特征在于，所述的核酸分子的核苷酸序列为：
    (1)序列表中SEQ ID NO.2所示的核苷酸序列；
    (2)基于SEQ ID NO.2所示的核苷酸序列进行序列优化得到的核苷酸序列；
    (3)与上述(1)或(2)中的核苷酸序列互补的核苷酸序列。
  6. 一种表达载体，其特征在于，所述的表达载体含有权利要求4-5任一项所述的核酸分子。
  7. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求6所述的表达载体或者基因组中整合有权利要求4-5任一项所述的核酸分子。
  8. 根据权利要求7的宿主细胞，其特征在于，所述的宿主细胞包括细菌、酵母、曲霉菌、植物细胞、或昆虫细胞。
  9. 根据权利要求8的宿主细胞，其特征在于，所述的细菌包括大肠杆菌。
  10. 一种制备生产权利要求1～3任一项所述角蛋白YK93-6的方法，其特征在于，包括以下步骤：
    A.合成权利要求1～3任一项所述的角蛋白YK93-6对应的核酸分子，将核酸分子连入相应的表达载体，将表达载体转化到宿主细胞，在一定条件下在发酵设备中培养带表达载体的宿主细胞并诱导表达角蛋白YK93-6，得到含有角蛋白YK93-6的粗蛋白溶液；
    B.对步骤A中所表达的粗蛋白溶液进行分离纯化干燥得到角蛋白YK93-6。
  11. 根据权利要求10的方法，其特征在于，在步骤A中，所述的宿主细胞主要为选自大肠杆菌，所述的角蛋白YK93-6在大肠杆菌包涵体中表达，所述的发酵设备包括摇瓶或发酵罐。
  12. 根据权利要求10的方法，其特征在于，在步骤A中，诱导表达角蛋白YK93-6后，可用清洗剂清洗杂质，用溶液溶解得到粗蛋白溶液。
  13. 根据权利要求10的方法，其特征在于，在步骤B中，所述的分离纯化的方法包括超滤微滤膜技术纯化方法、柱色谱纯化方法、盐析方法、透析方法。
  14. 一种药物组合物，其特征在于，所述的药物组合物含有权利要求1～3任一项所述的角蛋白YK93-6及以及药学上可接受的载体或赋形剂。
  15. 权利要求1～3任一项所述的角蛋白YK93-6或权利要求4-5任一项所述的核酸分子或权利要求6所述的表达载体或权利要求7-9所述的宿主细胞或权利要求14所述的药物组合物在制备预防或治疗前列腺增生、淋巴癌、黑色素瘤、镇痛、泌乳、乳腺癌、肺癌、子宫肌瘤、凝血药物中的应用。
PCT/CN2023/121364 2022-10-04 2023-09-26 一种角蛋白yk93-6、制法和其药物组合物与用途 WO2024074102A1 (zh)

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