WO2021083193A1 - 一种角蛋白bd-4、制法和其药物组合物与用途 - Google Patents

一种角蛋白bd-4、制法和其药物组合物与用途 Download PDF

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WO2021083193A1
WO2021083193A1 PCT/CN2020/124329 CN2020124329W WO2021083193A1 WO 2021083193 A1 WO2021083193 A1 WO 2021083193A1 CN 2020124329 W CN2020124329 W CN 2020124329W WO 2021083193 A1 WO2021083193 A1 WO 2021083193A1
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Prior art keywords
keratin
protein
solution
nucleic acid
host cell
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English (en)
French (fr)
Inventor
庾石山
王晓良
屈晶
李密
苏国柱
王玲
蔡杰
徐少峰
符江
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Institute of Materia Medica of CAMS and PUMC
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Institute of Materia Medica of CAMS and PUMC
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Priority to BR112022008192A priority Critical patent/BR112022008192A2/pt
Priority to CN202080075287.8A priority patent/CN114599669B/zh
Priority to AU2020373859A priority patent/AU2020373859A1/en
Priority to US17/771,994 priority patent/US12098176B2/en
Priority to CA3159335A priority patent/CA3159335A1/en
Priority to KR1020227016958A priority patent/KR20220088737A/ko
Priority to EP20881931.8A priority patent/EP4053155A4/en
Priority to JP2022525473A priority patent/JP7402977B2/ja
Publication of WO2021083193A1 publication Critical patent/WO2021083193A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1748—Keratin; Cytokeratin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/10—Expectorants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/14—Antitussive agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the present invention relates to a keratin BD-4, a nucleic acid molecule encoding keratin BD-4, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or genome integrating the nucleic acid molecule, and keratin BD-4
  • Keratin is a kind of protein, which is widely found in the epidermis of humans and animals, and is the main component of hair, feathers, hoofs, shells, claws, horns, etc. It is a very important structural protein for 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 used. The main reason is that keratin is insoluble in various solvents, and keratin is generally more resistant to enzymatic hydrolysis by proteases 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 BD-4, a nucleic acid molecule encoding keratin BD-4, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or genome integrating the nucleic acid molecule, and
  • the preparation method of keratin BD-4, the pharmaceutical composition containing keratin BD-4, and the above-mentioned keratin BD-4, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition are used in the preparation of antipyretic, analgesic, and antitussive Application in expectorant, anticonvulsant, antiepileptic, blood pressure lowering, anti-inflammatory, and antiviral drugs.
  • the present invention provides the following technical solutions:
  • the first aspect of the technical solution of the present invention is to provide a keratin BD-4, characterized in that the amino acid sequence of the keratin BD-4 is:
  • amino acid sequence shown in SEQ ID NO. 1 in the sequence list is formed by substitution, deletion or addition of 1-35 amino acids to form an amino acid sequence that basically maintains the same biological function.
  • the conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, immobilization modification, Sulfation, oxidation, methylation, deamination, formation of disulfide bonds or disulfide bond breakage;
  • 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 provides a nucleic acid molecule encoding the keratin BD-4 of the first aspect.
  • nucleic acid molecule is:
  • the third aspect of the technical solution of the present invention provides an expression vector, which is 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 of the third aspect or the nucleic acid molecule of the second aspect is integrated into the genome.
  • the host cell includes bacteria, yeast, Aspergillus, plant cells, or insect cells.
  • bacteria include Escherichia coli or yeast.
  • 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.; preferably The expression competent cells are BL21 (DE3), Transetta (DE3).
  • the fifth aspect of the technical solution of the present invention provides a method for preparing the keratin BD-4 of the first aspect, which is characterized in that it comprises the following steps:
  • step B Separating, purifying and drying the crude protein solution expressed in step A to obtain keratin BD-4.
  • the host cells are mainly selected from Escherichia coli, the keratin BD-4 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shaker flask or a fermentor.
  • step A after the expression of keratin BD-4 is induced, the impurities can be washed with a cleaning agent and dissolved in a solution to obtain a crude protein solution.
  • the medium in step A may be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, red bengal medium, high salt Chashi medium , DOBA medium, rice koji medium and its modified formula, etc.; shake flask fermentation preferably LB medium, TB medium, most preferably TB medium; fermenter preferably LB medium and its modified formula.
  • the inducer in step A can be IPTG, lactose, arabinose, etc.; preferably, IPTG, lactose.
  • step A the obtained fermentation broth is centrifuged, and the supernatant is discarded; the precipitate is suspended in the buffer, the cells are broken, and then centrifuged to discard the supernatant; the precipitate is washed with a cleaning agent, and then used The urea solution is dissolved to obtain a crude protein solution of BD-4.
  • 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 that is, the crude protein solution obtained in step A is purified by dialysis to obtain the target protein BD-4 solution.
  • the molecular weight cut-off of the dialysis bag can be 0.5-10 kD, the preferred molecular weight cut-off of the dialysis bag is 3.5-10 kD, and the most preferred molecular weight cut-off of the dialysis bag is 10 kD.
  • the ultrafiltration and microfiltration method that is, the crude protein solution obtained in step A is purified by membrane technology such as ultrafiltration membrane or microfiltration membrane to obtain a concentrated solution of the target protein BD-4.
  • the microfiltration membrane purification is performed 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 pass the crude protein solution obtained in step A through column chromatography, such as various exchange columns or exclusion column chromatography, to separate and purify the target protein BD-4.
  • the preferred exclusion column is a dextran gel column, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.
  • the preferred exchange column is an ion exchange resin column: anion exchange resin column, 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 column, HiTrap SP FF, HiTrap Capto SP ImpRes, CaptoSP, Impopearl, HiT -650M, Toyopearl Super SP-650M.
  • the most preferred is an anion exchange resin column.
  • the eluents commonly used in the art can be used, such as water, salt solutions, and the salt solutions include sodium chloride solution, sodium dihydrogen phosphate solution, disodium 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 method to obtain the target protein BD-4 suspension.
  • the salting-out agent can be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, and the like.
  • 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%, more preferably 25%.
  • the number of salting out is 1 to 3 times, preferably 2 times.
  • the precipitate is washed with pure water, and the washing frequency is 2 to 5 times, preferably 3 times.
  • the target protein BD-4 solution purified 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.
  • the sixth aspect of the technical solution of the present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin BD-4 described in the first aspect or the nucleic acid molecule described in the second aspect or the first aspect
  • the expression vector of the third aspect or the host cell of the fourth aspect and a pharmaceutically acceptable carrier or excipient is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin BD-4 described in the first aspect or the nucleic acid molecule described in the second aspect or the first aspect.
  • the keratin obtained in the above steps of the present invention can be freeze-dried or vacuum-dried into a dry powder, or the concentrated liquid can be directly spray-dried into a dry powder, and then made into various dosage forms.
  • the present invention relates to a pharmaceutical composition, comprising any 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 0.1-100.0% of the total weight of the pharmaceutical composition.
  • the present invention also provides a pharmaceutical composition, which includes a pharmaceutical 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 that can be used as human or veterinary medicine. form.
  • the keratin of the present invention or the pharmaceutical composition containing it can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs, skin, vagina, For peritoneal, rectal, etc., oral administration is preferred.
  • the keratin protein of the present invention or the pharmaceutical composition containing it can be administered by injection.
  • Injections include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, and acupoint injection.
  • the dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil and double emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops Lotion, nasal drops, lotion and liniment, etc.
  • the solid dosage form can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules Preparations, powders, pellets, dripping pills, suppositories, films, patches, air (powder) sprays, sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc.
  • the keratin of the present invention can be made into ordinary preparations, slow-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.
  • diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene dipropanol, etc.
  • disintegrant can be dry starch, micro
  • the tablets can also 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 In order to make the administration unit into a pill, various carriers known in the art can be widely used.
  • carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol laurate, kaolin, talc, etc.; binders, such as Gum arabic, xanthan gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dried starch, alginate, sodium lauryl sulfonate, methyl cellulose, Ethyl cellulose and so on.
  • diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol laurate, kaolin, talc, etc.
  • binders such as Gum arabic, xanthan gum, ge
  • various carriers known in the art can be widely used.
  • carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.
  • the active ingredient of the keratin of the present invention is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule.
  • the active ingredient keratin of the present invention can also be made into microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules or made into injections for application.
  • the keratin of the present invention is prepared into injection preparations, such as solutions, suspension solutions, emulsions, and lyophilized powder injections.
  • injection preparations such as solutions, suspension solutions, emulsions, and lyophilized powder injections.
  • Such preparations may be aqueous or non-aqueous, and may contain one and/or more A pharmacologically acceptable carrier, diluent, binder, lubricant, preservative, surfactant or dispersant.
  • the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters and the like.
  • an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation, and in addition, conventional solubilizers, buffers, pH adjusters, etc. can also be added. These auxiliary materials are commonly used in this field.
  • coloring agents can also 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 gender, age, weight, personality and individual response of the patient or animal, the route of administration, and the number of administrations. , The purpose of treatment, so the therapeutic dose of the present invention can have a wide range of changes. Generally speaking, the dosage of the pharmaceutical ingredients of the present invention is well known to those skilled in the art. Appropriate adjustments can be made according to the actual amount of the drug contained in the final preparation of the keratin composition of the present invention to achieve the requirement of the therapeutically effective amount, and to accomplish the prevention or treatment purpose of the present invention.
  • the appropriate daily dosage range of keratin of the present invention is 0.01-500 mg/kg body weight, preferably 0.5-100 mg/kg body weight, more preferably 1-50 mg/kg body weight, and most preferably 2- 30mg/kg body weight.
  • the above-mentioned dosage can be administered in a single dosage form or divided into several, for example, two, three or four dosage forms, depending on the clinical experience of the administering doctor and the dosage regimen including the use of other treatment means.
  • the total dose required for each treatment can be divided into multiple or single doses.
  • the protein or pharmaceutical composition of the present invention can be taken alone, or combined with other therapeutic drugs or symptomatic drugs, and the dosage is adjusted.
  • the seventh aspect of the technical solution of the present invention provides the keratin BD-4 according to the first aspect or the nucleic acid molecule according to the second aspect or the expression vector according to the third aspect or the host cell according to the fourth aspect or Application of the pharmaceutical composition of the sixth aspect in the preparation of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, and antiviral drugs.
  • the preparation of keratin BD-4 of the present invention includes the following steps:
  • the preferred nucleotide sequence is shown in 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-28a(+).
  • the host cell can be E. coli or yeast; the preferred host cell is E. coli;
  • 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.; preferred
  • the expression competent cells are BL21 (DE3), Transetta (DE3).
  • the host cell will be fermented and cultured under appropriate conditions to induce the expression of the target protein BD-4;
  • Fermentation equipment can use shaker flasks or fermentation tanks;
  • the medium can be LB medium, TB medium, SB medium, SOB medium, SOC medium, PDA medium, YPD medium, red bengal medium, high salt Chashi medium, DOBA medium, rice koji Medium and its modified formula, etc.; LB medium and TB medium are preferred for shake flask fermentation, and TB medium is most preferred; LB medium and its modified formula are preferred for fermentation tanks.
  • the inducer can be IPTG, lactose, arabinose, etc.; preferably, IPTG, lactose.
  • Step (4) The obtained fermentation bacteria liquid is centrifuged, and the supernatant is discarded; the precipitate is suspended in the buffer, the cells are broken, and then centrifuged, and the supernatant is discarded; the precipitate is washed with a cleaning agent, and then dissolved with a urea solution , Get BD-4 crude protein solution.
  • the crude protein solution obtained in step (5) needs to be purified to obtain the target protein BD-4.
  • the purification can be performed by dialysis, or ultrafiltration and microfiltration, or column chromatography, or salting out steps.
  • the dialysis step that is, the crude protein solution obtained in step (5) is purified by a dialysis method to obtain the target protein BD-4 solution.
  • the molecular weight cut-off of the dialysis bag can be 0.5-10 kD, the preferred molecular weight cut-off of the dialysis bag is 3.5-10 kD, and the most preferred molecular weight cut-off of the dialysis bag is 10 kD.
  • the ultrafiltration and microfiltration step that is, the crude protein solution obtained in step (5) is purified by membrane technology such as ultrafiltration membrane or microfiltration membrane to obtain a concentrated solution of target protein BD-4.
  • the microfiltration membrane purification is performed 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 that is, the crude protein solution obtained in step (5) is passed through column chromatography, such as various exchange columns or exclusion column chromatography, to separate and purify the target protein BD-4.
  • the preferred exclusion column is a dextran gel column, Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, Superose 6 Increase, etc.
  • the preferred exchange column is an ion exchange resin column: anion exchange resin column, 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 column, HiTrap SP FF, HiTrap Capto SP ImpRes, CaptoSP, Impopearl, HiT -650M, Toyopearl Super SP-650M.
  • the most preferred is an anion exchange resin column.
  • the eluents commonly used in the art can be used, such as water, salt solutions, and the salt solutions include sodium chloride solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium acetate, acetic acid, and the like.
  • the salting-out step that is, the crude protein solution obtained in step (5) is purified by salting-out method to obtain the target protein BD-4 suspension.
  • the salting-out agent can be ammonium sulfate, sodium sulfate, sodium chloride, magnesium chloride, aluminum sulfate, ammonium nitrate, ammonium chloride, magnesium sulfate, and the like.
  • 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%, more preferably 25%.
  • the number of salting out is 1 to 3 times, preferably 2 times.
  • the precipitate is washed with pure water, and the washing frequency is 2 to 5 times, preferably 3 times.
  • the target protein BD-4 solution purified 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 BD-4 on the fever model of SD rats induced by yeast and lipopolysaccharide, which proves that protein BD-4 can significantly inhibit the increase in body temperature 4 hours and 8 hours after the yeast is modeled. Has a significant antipyretic effect;
  • the present invention respectively tested the effect of protein BD-4 on pilocarpine (PLO) and pentylenetetrazole (PTZ) induced convulsions and epilepsy in mice, and proved that protein BD-4 can significantly prolong the level of epilepsy in mice. And the incubation period of grade III onset;
  • the present invention proves that the protein BD-4 has obvious expectorant effects through the experimental study of the effect of protein BD-4 on the expectorant of phenol red excretion method in mice;
  • protein BD-4 can significantly reduce the number of coughs and prolong the incubation period of coughs, and has a significant antitussive effect
  • the present invention proves that the protein BD-4 can significantly reduce the number of writhing in mice and has a significant analgesic effect through the pharmacodynamic test study of the protein BD-4 on the acetic acid writhing of ICR mice.
  • Protein BD-4 crude solution A analyzed by reduced SDS-PAGE, the separation gel concentration was 12.5%, stained with Coomassie brilliant blue R250 method; a clear blue band was shown near the molecular weight of 45kD.
  • Example 1 an expression vector containing the sequence shown in SEQ ID No. 2 was obtained by synthesis and sequencing; the expression vector was transfected into BL21 (DE3) cells to obtain an expression competent host cell containing the target nucleotide sequence.
  • Kanamycin final concentration 50 ⁇ g/ml
  • LB medium Take 10ml of LB medium, add Kanamycin (final concentration 50 ⁇ g/ml), take a single colony on the plate and add it to LB medium, in a shaker, at 37°C, 220rpm conditions overnight expansion and culture for 15 hours to obtain seeds liquid.
  • Protein BD-4 crude solution B was analyzed by reduced SDS-PAGE, and the separation gel concentration was 12.5%, stained with Coomassie Brilliant Blue R250 method; a clear blue band was shown near the molecular weight of 45kD.
  • Example 1 an expression vector containing the sequence shown in SEQ ID No. 2 was obtained by synthesis and sequencing; the expression vector was transfected into BL21 (DE3) cells to obtain an expression competent host cell containing the target nucleotide sequence. Add the LB medium and incubate in a shaker at 37°C and 220 rpm for 1 hour to obtain a recombinant strain.
  • the bacterial solution was centrifuged at 7000 rpm for 5 minutes, and the supernatant was sterilized and discarded; the precipitate was suspended in about 200 ml buffer A, filtered with an 80-100 mesh screen, and the filtrate was crushed with a high-pressure crusher at a pressure of 800-1000 bar, twice, each 2 minutes each time. After crushing, the bacterial liquid was centrifuged at 7000 rpm for 30 minutes, and the supernatant was discarded.
  • Protein BD-4 crude solution C analyzed by reduced SDS-PAGE, the separation gel concentration was 12.5%, stained with Coomassie Brilliant Blue R250; a clear blue band was shown near the molecular weight of 45kD.
  • Example 4 Protein crude solution C was prepared by membrane technology to obtain protein BD-4
  • the crude protein solution C obtained in Example 3 was purified by microfiltration membrane technology: firstly use a 1500nm or 1000nm ceramic membrane core for solid-liquid separation; discard the inner liquid, and then use a 20nm or 50nm ceramic membrane core for repeated microfiltration to remove the outer liquid Urea; the inner liquid of the second microfiltration is freeze-dried to obtain the target protein BD-4; the purity determined by electrophoresis is 95.6%.
  • the protein BD-4 solution was analyzed by reduced SDS-PAGE, the separation gel concentration was 12.5%, and it was stained with Coomassie brilliant blue R250 method.
  • the molecular weight of BD-4 band is around 45kD.
  • Main materials Acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Asp-N;
  • Protein BD-4 undergoes pretreatments such as dissolution replacement, reductive alkylation, and multiple proteolysis to obtain digested peptides; the digested peptide solution is analyzed by liquid chromatography tandem mass spectrometry, and the original mass spectrometry file uses Maxquant (1.6.2.10 ) Search the protein database to analyze the data, and the coverage of the identification results is 100%, and it is determined that it is consistent with the target sequence SEQ ID No.1.
  • Example 5 Protein BD-4 was prepared by purification of crude protein solution A
  • 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 l filter membrane, and the filtrate was combined.
  • the filtrate was dialyzed with water, the molecular weight cutoff of the dialysis bag was 10kD, dialyzed for 72 hours, and the inner liquid was freeze-dried to obtain the target protein BD-4; the purity measured by electrophoresis was 90.1%.
  • the second method salting out
  • the crude protein solution A is placed in a container with stirring for two salting out: slowly add ammonium sulfate saturated solution along the wall to make the final concentration of ammonium sulfate 25% or 50%.
  • the protein will precipitate during the salting out process, and wait until the salting out is complete.
  • Filter to complete the first salting out add 400ml of pure water to the precipitate to suspend, again slowly add a saturated solution of ammonium sulfate along the wall to make the final concentration of ammonium sulfate 25%, perform the second salting out, filter, the precipitation is Crude protein extract. Wash the crude protein extract three times with water: add 200ml of pure water to suspend, stir, stand, and filter; after repeating this three times, the precipitate is freeze-dried to obtain the target protein BD-4.
  • the third method column chromatography
  • the crude protein solution A was purified by HiTrap Q FF 16/10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE and other anion exchange resin columns.
  • the eluent is a gradient elution of NaCl solution, plus 20mM NaH 2 PO 4 /Na 2 HPO 4 buffer (pH 8.0).
  • the eluate fractions were detected and combined by SDS-PAGE electrophoresis, and the combined eluate was centrifuged twice at 7000 rpm for 1 hour each; the supernatant was filtered with a 0.45 ⁇ m filter membrane, and the filtrates were combined.
  • the filtrate is concentrated by dialysis with water, the molecular weight cut-off of the dialysis bag is 10kD, and the inner liquid is freeze-dried to obtain the target protein BD-4.
  • the product protein BD-4 obtained by the three methods was confirmed to have the same amino acid sequence as the protein prepared in Example 4 through the same structural confirmation method as in Example 4.
  • the crude protein solution B obtained in Example 2 was purified by the following three methods:
  • the first method dialysis
  • the crude protein solution B is filtered with a 0.45 ⁇ m membrane, the filtrate is dialyzed with water, dialyzed for more than 72 hours, and the inner solution is freeze-dried to obtain the target protein BD-4.
  • Dialysis bag Molecular weight cut-off 0.5kD, 3.5kD, 5kD, 10kD
  • the second method column chromatography
  • the crude protein solution B was purified by HiTrap Q FF 16/10, HiTrap Capto Q ImpRes, Capto Q ImpRes, HiTrap Capto Q, HiTrap DEAE and other anion exchange resin columns.
  • the eluent is a gradient elution of NaCl solution, plus 20mM NaH 2 PO 4 /Na 2 HPO 4 buffer (pH 8.0).
  • the eluate fractions were detected and combined by SDS-PAGE electrophoresis, and the combined eluate was centrifuged twice at 7000 rpm for 1 hour each; the supernatant was filtered with a 0.45 ⁇ m filter membrane, and the filtrates were combined.
  • the filtrate is concentrated by dialysis with water, the molecular weight cut-off of the dialysis bag is 10kD, and the inner liquid is freeze-dried to obtain the target protein BD-4.
  • the third method salting out
  • the crude protein solution B is placed in a container with stirring for two salting out: slowly add ammonium sulfate saturated solution along the wall to make the final concentration of ammonium sulfate 25% or 50%, the protein will precipitate during the salting out process, wait until the salting out is complete , Filter to complete the first salting out; add 400ml of pure water to the precipitate to suspend, again slowly add a saturated solution of ammonium sulfate along the wall to make the final concentration of ammonium sulfate 25%, perform the second salting out, filter, the precipitation is Crude protein extract. Wash the crude protein extract three times with water: add 200ml of pure water to suspend, stir, stand, and filter; after repeating this three times, the precipitate is freeze-dried to obtain the target protein BD-4.
  • the product protein BD-4 obtained by the three methods was confirmed to have the same amino acid sequence as the protein prepared in Example 4 through the same structural confirmation method as in Example 4.
  • Example 7 Crude protein solution C obtained protein BD-4 by salting-out method
  • the crude protein solution C obtained in Example 3 was placed in a stirred container for two salting out: Slowly add saturated ammonium sulfate solution along the wall to make the final concentration of ammonium sulfate 25%. During the salting-out process, the protein would precipitate out. After completion, filter to complete the first salting out; add 400ml of pure water to the precipitate to suspend, and then slowly add ammonium sulfate saturated solution along the wall to make the final concentration of ammonium sulfate 25%, perform the second salting out, filter, and precipitate It is the crude protein extract. Wash the crude protein extract three times with water: add 200ml of pure water to suspend, stir, stand, and filter; after repeating this three times, the precipitate is freeze-dried to obtain the target protein BD-4.
  • the product protein BD-4 was confirmed to have the same amino acid sequence as the protein prepared in Example 4 through the same structural confirmation method as in Example 4.
  • yeast OXOID LP0021
  • aspirin SIGMA A2093
  • protein BD-4 protein BD-4
  • Model group yeast fever model
  • Positive control group Aspirin 300mg/kg group
  • Protein BD-4 10mg/kg group, 50mg/kg group.
  • Preparation of experimental animals After the experimental animals have been adapted to the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day, they are pre-adapted to measure rectal temperature at 8:00 and 15:00, respectively. The animals were fasted for the first 12 hours without water, and the animals were allowed to empty their feces before measuring the rectal temperature. Before each temperature measurement, apply petroleum jelly to the probe of the electronic thermometer and insert it into the rat rectum 2cm (can be marked at 2cm to ensure the same depth of insertion each time), and record the body temperature after the reading is stable.
  • Subcutaneous injection of dry yeast to replicate the rat fever model measure the rat's body temperature before modeling, select qualified rats with a body temperature of 36.2-37.3 °C, and randomly divide them into groups, each with 8 rats.
  • the rats' body temperature was monitored at intervals of 2 hours. The body temperature was monitored once for a total of 8 hours.
  • the positive tool drug aspirin group can effectively inhibit the increase in body temperature of model rats at 4 hours, 6 hours and 8 hours after modeling. Compared with the model group, P ⁇ 0.05, there is a statistical difference, and the positive tool drug aspirin has stable performance.
  • Protein BD-4 10mg/kg dose group can significantly inhibit the increase in body temperature of model rats 4 hours after modeling, compared with model group, P ⁇ 0.05, there is a statistical difference; 50mg/kg dose group after modeling 8 hours can significantly inhibit the increase in body temperature of model rats, and compared with the model group, P ⁇ 0.05, there is a statistical difference.
  • LPS lipopolysaccharide
  • SIGMA L-2880 aspirin
  • SIGMA A2093 protein BD-4;
  • Model group lipopolysaccharide fever model
  • Positive control group Aspirin 300mg/kg group
  • Protein BD-4 10mg/kg group, 50mg/kg group.
  • Lipopolysaccharide was injected into the abdominal cavity to replicate the rat fever model.
  • Preparation of experimental animals After the experimental animals have been adapted to the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day, they shall be pre-adapted to the operation of measuring rectal temperature at 8:00 and 15:00 respectively, before the experiment Fasting for 12 hours without water, let the animals empty their feces before measuring the rectal temperature. Before each temperature measurement, apply petroleum jelly to the probe of the electronic thermometer and insert it into the rat rectum 2cm (can be marked at 2cm to ensure the same depth of insertion each time), and record the body temperature after the reading is stable.
  • Intraperitoneal injection of lipopolysaccharide to replicate the rat fever model measure the rat's body temperature before modeling, and select qualified rats with a body temperature of 36.2-37.3 °C, and randomly group them into groups of 8 rats.
  • lipopolysaccharide (20 ⁇ g/kg, 2ml/kg) was injected intraperitoneally immediately.
  • the normal control group was injected intraperitoneally with an equal volume of normal saline. After 2 hours, the rats' body temperature was monitored for a total of 8 hours.
  • the positive tool drug aspirin group can effectively inhibit the increase in body temperature of model rats at 2 hours, 4 hours, 6 hours and 8 hours after modeling. Compared with the model group, P ⁇ 0.05, there is a statistical difference, the positive tool drug performance More stable.
  • Protein BD-4 has a tendency to lower the body temperature of model rats after modeling, but it has no statistical significance.
  • Pilocarpine HCl PLO, pilocarpine, pilocarpine hydrochloride
  • Diazepam diazepam tablets
  • protein BD-4 Protein BD-4.
  • Diazepam (Diazepam) 2mg/kg group;
  • Protein BD-4 50mg/kg group, 200mg/kg group.
  • PLO-225mg/kg (molding agent) was intraperitoneally injected 1 hour after gavage of the test drug on the day of modeling, and the positive drug was administered once 20 minutes before modeling. Observe for 30 minutes after PLO injection.
  • Level of seizure Refer to Racine grading standard: Level 0: No reaction; Level I: Twitching of facial muscles or the corners of the mouth; Level II: Nodding; Level III: Twitching of one limb; Level IV: Rigidity or twitching of the whole body ; Grade V: generalized epilepsy (generalized tonic convulsive seizures).
  • Positive drugs can completely inhibit the seizure rate of grade IV epilepsy, and significantly prolong the incubation period of grade II, grade III and grade IV epileptic seizures in mice.
  • PTZ Pentylenetetrazol
  • Retigabine Protein BD-4.
  • Protein BD-4 50mg/kg group, 200mg/kg group.
  • the drug was administered once in the afternoon the day before modeling, and PTZ-65 mg/kg (molding agent) was intraperitoneally injected 1 hour after gavage of the test drug on the day of modeling, and the positive drug was administered once half an hour before modeling. Observe for 15 minutes after PTZ injection.
  • Level of seizure Refer to Racine grading standard: Level 0: No reaction; Level I: Twitching of facial muscles or the corners of the mouth; Level II: Nodding; Level III: Twitching of one limb; Level IV: Rigidity or twitching of the whole body ; Grade V: full-blown epilepsy (generalized tonic convulsive seizures).
  • Positive drugs can significantly reduce the seizure rate of grade IV epilepsy, and significantly prolong the incubation period of grade III and IV seizures in mice.
  • Drugs and reagents Mucosultan (ambroxol hydrochloride tablets), phenol red, sodium bicarbonate, protein BD-4;
  • centrifuge Sigma-3K15 type
  • balance XS105DU type
  • enzyme label tester BIO-TEK type
  • Protein BD-4 20mg/kg group, 50mg/kg group.
  • mice were sacrificed by neck removal, and they were taken from subthyroid cartilage.
  • To a segment of the trachea before the trachea branch put the trachea into 3ml of 5% NaHCO 3 solution and let stand for 3 hours, take 1ml of the supernatant, centrifuge at 3000rpm for 5 minutes, measure and record the absorbance at 546nm. Calculate the excretion of phenol red according to the standard curve of phenol red. The results are shown in Table 7.
  • the BD-4 20mg/kg dose group showed a significant increase in phenol red excretion, P ⁇ 0.05, which was statistically significant.
  • Drugs and reagents dextromethorphan hydrobromide, ammonia, 0.2% CMC-Na, protein BD-4;
  • Apparatus Compressed atomizer (403T type), balance (XS105DU type).
  • Protein BD-4 20mg/kg group, 50mg/kg group.
  • Dextromethorphan and different doses of protein BD-4 were given orally in groups, and the solvent control group was given the same volume of distilled water. After 1 hour, it was placed in a sealed box and atomized with 10% ammonia water for 10 seconds. Zhong, then observe and record the incubation period of cough in mice and the number of coughs within 2 minutes.
  • the incubation period of cough refers to the number of seconds from the start of the atomization of ammonia to the occurrence of cough.
  • the performance of coughing in mice is based on contraction of the abdominal muscles (breast reduction) and opening of the mouth at the same time. Calculate the mean and standard error of each group of data, use TTEST to compare the model group with other groups, and P ⁇ 0.05 is considered as a significant difference.
  • Protein BD-4 50mg/kg group, 200mg/kg group.
  • the experimental animals were given aspirin 300mg/kg, protein BD-4 50mg/kg, 200mg/kg, and the administration volume was 10ml/kg one hour in advance; then 0.6% acetic acid solution was injected intraperitoneally and observed within 15 minutes The incubation period (seconds) and frequency of animal writhing.
  • the 0.6% acetic acid solution was injected into the abdominal cavity of mice, which caused deep and long-term painful stimulation, which caused the mice to have a writhing response (the abdomen contracted into an "S" shape, the trunk and hind legs were stretched, the buttocks were raised and the Row).
  • the incubation time and the number of times of writhing in the mouse were used as the pain response index to determine whether the test sample has analgesic effect.
  • Aspirin 300mg/kg can significantly delay the latency of writhing and reduce the number of writhing, and has a certain analgesic effect. Compared with the model group, P ⁇ 0.05, which is statistically significant.
  • the BD-4 50mg/kg dose group can significantly reduce the number of writhing times in mice. Compared with the model group, P ⁇ 0.05, which is statistically significant.

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Abstract

提供了角蛋白BD-4、其编码核酸分子、表达载体、宿主细胞以及含有该角蛋白的药物组合物。角蛋白BD-4可用于制备解热镇痛、镇咳祛痰、抗癫痫药物。

Description

一种角蛋白BD-4、制法和其药物组合物与用途 技术领域
本发明涉及一种角蛋白BD-4，编码角蛋白BD-4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BD-4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解热镇痛、镇咳祛痰、抗惊厥、抗癫痫、降血压、抗炎、抗病毒药物中的应用。
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BD-4，编码角蛋白BD-4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BD-4的制备方法，含有角蛋白BD-4的药物组合物，以及上述角蛋白BD-4、核酸分子、表达载体、宿主细胞、或药物组合物在制备解热镇痛、镇咳祛痰、抗惊厥、抗癫痫、降血压、抗炎、抗病毒药物中的应用。
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BD-4，其特征在于，所述角蛋白BD-4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BD-4上可进行常规修饰；或者在角蛋白BD-4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BD-4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BD-4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BD-4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BD-4，得到含有角蛋白BD-4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BD-4。
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BD-4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BD-4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、TB培养基，最优选为TB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BD-4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，优选为尿素溶液，最优选为2M尿素溶液(可含有1％Triton)，其用量为：发酵液体积∶2M尿素体积＝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BD-4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BD-4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BD-4。
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BD-4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BD-4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BD-4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0.01～500mg/kg体重，优选为0.5～100mg/kg体重，更优选为1～50mg/kg体重，最优选为2～30mg/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BD-4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解热、镇痛、镇咳、祛痰、抗惊厥、抗癫痫、降血压、抗炎、抗病毒药物中的应用。
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BD-4，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-28a(+)。
(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BD-4；
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、TB培养基，最优选为TB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BD-4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BD-4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，优选为尿素溶液，最优选为2M尿素溶液(可含有1％Triton)，其用量为：发酵液体积∶2M尿素体积＝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BD-4：
步骤(5)获得的粗蛋白溶液，需经过纯化获得目标蛋白BD-4。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BD-4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BD-4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BD-4。
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BD-4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BD-4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BD-4对酵母和脂多糖诱导的SD大鼠发热模型的药效试验研究，证明蛋白BD-4在酵母造模后4小时、8小时，可显著抑制体温升高，具有显著的解热作用；
3、本发明分别通过蛋白BD-4对匹罗卡品(PLO)和戊四唑(PTZ)致小鼠惊厥和癫痫的药效试验研究，证明蛋白BD-4能够显著延长小鼠癫痫II级和III级发作潜伏期；
4、本发明通过蛋白BD-4对小鼠酚红排泄法祛痰的药效试验研究，证明蛋白BD-4具有明显的祛痰作用；
5、本发明通过蛋白BD-4对小鼠氨水引咳法镇咳的药效试验研究，证明蛋白BD-4能够明显减少咳嗽次数和延长咳嗽潜伏期，具有显著的镇咳作用；
6、本发明通过蛋白BD-4对ICR小鼠醋酸扭体的药效试验研究，证明蛋白BD-4能明显减少小鼠扭体次数，具有显著的镇痛作用。
附图说明
图1：表达蛋白BD-4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BD-4)
图2.蛋白BD-4对酵母诱导大鼠发热模型的影响
(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.蛋白BD-4对脂多糖(LPS)诱导大鼠发热模型的影响
(与正常对照组比较，***P<0.001；与模型组比较，##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BD-4粗溶液A(TB培养基)
合成如SEQ ID No.2所示的核苷酸序列，将其转入pET-28a(+)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 TB培养基，分装2瓶，每瓶200ml。在每瓶(200ml)TB培养基中加入Kana霉素(终浓度50μg/ml)，取平板上单一菌落加入TB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28.8L TB培养基，分装于144瓶，每瓶200ml。在每瓶(200ml)TB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OD 600，当OD 600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。
Figure PCTCN2020124329-appb-000001
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3L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1L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4次，分别为800ml、600ml、400ml、400ml。合并4次溶液，7000rpm离心30分钟，沉淀弃去，上清液即为蛋白粗溶液A。
Figure PCTCN2020124329-appb-000002
蛋白BD-4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45kD附近显示明显蓝色条带。
实施例2 摇瓶发酵制备蛋白BD-4粗溶液B(其它培养基)
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。
配制20ml LB培养基，取800μl，加入含有目标编码序列的宿主细胞50μl，于摇床中，在37℃、220rpm条件下培养1小时。
蘸取上述菌液在含有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。
取10ml LB培养基，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15小时，得到种子液。
配置1L下表所示培养基，分装于10瓶，每瓶100ml。在每瓶(100ml)培养基中加入Kana霉素(终浓度50μg/ml)，再加入1ml种子液，于摇床中，在37℃、220rpm条件下培养2-3小时。监测OD 600，当OD 600达到1.0左右时，加入诱导剂IPTG(终浓度0.5mM)，于摇床中，在37℃、220rpm条件下诱导表达蛋白。
培养基 LB培养基、SOB培养基、SOC培养基
合并各瓶菌液，10000rpm离心10分钟，上清液灭菌后弃去；沉淀悬浮于约100mL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10000rpm离心30分钟，弃去上清液。
沉淀先加入40mL清洗剂buffer A清洗3次，离心，弃去上清液；沉淀加入40mL清洗剂2M尿素溶液清洗2次，离心，弃去上清液；沉淀再加入40mL 4M尿素溶液清洗2次，离心，弃去上清液；沉淀再加入8M尿素溶液(含50mM Tris/HCl缓冲液)溶解3次，分别为40ml、30ml、30ml；合并溶液，7000rpm离心30分钟，沉淀弃去，上清液即为蛋白粗溶液B。
蛋白BD-4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45kD附近显示明显蓝色条带。
实施例3 发酵罐制备蛋白BD-4粗溶液C
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0.8ml，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8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2M尿素溶液(含1％Triton)清洗2次，每次1L；再加入1L 2M尿素溶液清洗1次，离心，弃去上清液。沉淀加入8M尿素溶液溶解4次，分别为400ml、300ml、200ml、100ml。合并四次溶液，7000rpm离心30分钟，沉淀弃去，上清液即为蛋白粗溶液C。
蛋白BD-4粗溶液C，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45kD附近显示明显蓝色条带。
实施例4 蛋白粗溶液C通过膜技术制备得蛋白BD-4
实施例3获得的蛋白粗溶液C，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BD-4；电泳测定纯度95.6％。
蛋白BD-4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BD-4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。BD-4条带分子量在45kD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、Asp-N；
主要仪器：毛细管高效液相色谱仪(Thermo Ultimate 3000型)，电喷雾-组合型离子阱Orbitrap质谱仪(Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer)。
方法和结果：
蛋白BD-4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Maxquant(1.6.2.10)检索蛋白数据库分析数据，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5 蛋白粗溶液A纯化制备得蛋白BD-4
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 l滤膜过滤，合并滤液。滤液用水透析，透析袋截留分子量10kD，透析72小时，内液冷冻干燥，即得目标蛋白BD-4；电泳测得纯度90.1％。
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BD-4。
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 NaH 2PO 4/Na 2HPO 4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BD-4。
三种方法得到的产物蛋白BD-4，经与实施例4相同的结构确证方法，确认其与实施例4制备得到的蛋白具有相同的氨基酸序列。
实施例6 蛋白粗溶液B纯化制备得蛋白BD-4
实施例2获得的蛋白粗溶液B通过下述三种方法进行纯化：
第一种方法：透析；
蛋白粗溶液B，用0.45μm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BD-4。
透析袋 截留分子量：0.5kD、3.5kD、5kD、10kD
第二种方法：柱色谱；
蛋白粗溶液B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 NaH 2PO 4/Na 2HPO 4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BD-4。
第三种方法：盐析；
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％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BD-4。
三种方法得到的产物蛋白BD-4，经与实施例4相同的结构确证方法，确认其与实施例4制备得到的蛋白具有相同的氨基酸序列。
实施例7 蛋白粗溶液C通过盐析方法得蛋白BD-4
实施例3获得的蛋白粗溶液C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BD-4。
产物蛋白BD-4，经与实施例4相同的结构确证方法，确认其与实施例4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 蛋白BD-4(实施例4蛋白)对酵母诱导SD大鼠发热模型的药效试验
动物：雄性SD大鼠230-260克；
药品：酵母菌(OXOID LP0021)，阿司匹林(SIGMA A2093)，蛋白BD-4；
仪器：电子天平(SARTORIUS BP121S型)，电子体温计(CITIZEN CT-513W型)。
实验分组：
正常对照组；
模型组：酵母菌发热模型；
阳性对照组：阿司匹林(Aspirin)300mg/kg组；
蛋白BD-4，10mg/kg组，50mg/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天后，分别于早8:00及15:00进行预适应测量肛温操作，实验前12小时禁食不禁水，测定肛温前让动物排空粪便。每次测温前电子体温计探头涂抹凡士林，插入大鼠直肠2cm(可在2cm处标记，确保每次插入深度一致)，待读数稳定以后记录体温值。
皮下注射干酵母复制大鼠发热模型：造模前测大鼠体温，筛选出体温在36.2-37.3℃的合格大鼠，随机分组，每组8只。口服给予阿司匹林、不同剂量蛋白BD-4后立即皮下注射20％酵母菌混悬液(10ml/kg)，正常对照组皮下注射等体积生理盐水，2小时后开始监测大鼠体温，每间隔2小时监测体温1次，共监测8小时。
数据统计：
根据实验当天测量的各时间点体温值，计算各组大鼠体温均值、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：
口服给予阿司匹林(300mg/kg)、蛋白BD-4(10mg/kg、50mg/kg)后立即皮下注射20％酵母菌造模，分别于造模后2小时、4小时、6小时、8小时监测动物体温。结果见表1和图2。
表1.受试药对酵母菌诱导大鼠发热模型的影响
Figure PCTCN2020124329-appb-000003
(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)
实验结论：
口服给予阿司匹林(300mg/kg)、蛋白BD-4(10mg/kg、50mg/kg)后立即皮下注射20％酵母菌造模，分别于造模后2小时、4小时、6小时、8小时监测动物体温。
结果显示：
1)模型组大鼠在造模4小时、6小时、8小时体温明显升高，与正常组比较，P<0.05，有统计学差异，模型建立成功且稳定可靠。
2)阳性工具药阿司匹林组在造模4小时、6小时、8小时均可以有效抑制模型大鼠体温升高，与模型组比较，P<0.05，有统计学差异，阳性工具药阿司匹林表现稳定。
3)蛋白BD-4 10mg/kg剂量组在造模后4小时可明显抑制模型大鼠体温升高，与模型组比较，P<0.05，有统计学差异；50mg/kg剂量组在造模后8小时可显著抑制模型大鼠体温升高，且与模型组比较，P<0.05，有统计学差异。
实验例2 蛋白BD-4(实施例4蛋白)对脂多糖(LPS)诱导SD大鼠发热模型的药效试验
动物：雄性SD大鼠230-260克；
药品：脂多糖(LPS，SIGMA L-2880)，阿司匹林(SIGMA A2093)，蛋白BD-4；
仪器：电子天平(SARTORIUS BP121S型)，电子体温计(CITIZEN CT-513W型)。
实验分组：
正常对照组；
模型组：脂多糖发热模型；
阳性对照组：阿司匹林(Aspirin)300mg/kg组；
蛋白BD-4，10mg/kg组，50mg/kg组。
方法：腹腔注射脂多糖复制大鼠发热模型方法：
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天后，分别于早8:00及15:00预适应测量肛温操作，实验前12小时禁食不禁水，测定肛温前让动物排空粪便。每次测温前电子体温计探头涂抹凡士林，插入大鼠直肠2cm(可在2cm处标记，确保每次插入深度一致)，待读数稳定以后记录体温值。
腹腔注射脂多糖复制大鼠发热模型：造模前测大鼠体温，筛选出体温在36.2-37.3℃的合格大鼠，随机分组，每组8只。口服给予阿司匹林及不同剂量蛋白BD-4后立即腹腔注射脂多糖(20μg/kg，2ml/kg)，正常对照组腹腔注射等体积生理盐水，2小时后开始监测大鼠体温，共监测8小时。
数据统计：
根据实验当天测量的各时间点体温值，计算各组大鼠体温均值、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：
口服给予阿司匹林(300mg/kg)、蛋白BD-4(10mg/kg、50mg/kg)后立即腹腔注射20μg/kg脂多糖造模，分别于造模后2小时、4小时、6小时、8小时监测动物体温。结果见表2和图3。
表2.受试药对脂多糖(LPS)诱导大鼠发热模型的影响
Figure PCTCN2020124329-appb-000004
(与正常对照组比较，***P<0.001；与模型组比较，##P<0.01，###P<0.001)
实验结论：
分别口服给予阿司匹林(300mg/kg)、蛋白BD-4(10mg/kg、50mg/kg)后立即腹腔注射20μg/kg脂多糖造模，并于造模后2小时、4小时、6小时、8小时监测动物体温，结果显示：
1)腹腔注射20μg/kg脂多糖可成功诱导大鼠体温升高，模型组大鼠在造模2小时、4小时、6小时、8小时体温明显升高，与正常组比较，P<0.05，有统计学差异，模型稳定。
2)阳性工具药阿司匹林组在造模2小时、4小时、6小时、8小时均可以有效抑制模型大鼠体温升高，与模型组比较，P<0.05，有统计学差异，阳性工具药表现较稳定。
3)蛋白BD-4在造模后有降低模型大鼠体温的趋势，但无统计学意义。
实验例3 蛋白BD-4(实施例4蛋白)对致惊剂匹罗卡品(Pilocarpine，PLO)致小鼠惊厥癫痫的药效试验
动物：雄性ICR小鼠；
药品：Pilocarpine HCl(PLO，匹罗卡品，盐酸毛果芸香碱)，Diazepam(地西泮片)，蛋白BD-4。
实验分组：
模型组；
地西泮(Diazepam)2mg/kg组；
蛋白BD-4，50mg/kg组，200mg/kg组。
方法：
模型制备及给药：
造模前一天下午给药一次，造模当天灌胃受试药后1小时腹腔注射PLO-225mg/kg(造模剂)，阳性药在造模前20分钟给药一次即可。注射PLO后持续观察30分钟。
观察指标：①癫痫发作情况：Ⅱ级～Ⅳ级发作时间；②死亡时间。
发作级别：参照Racine分级标准：0级：没有任何反应；I级：表现为面部肌肉或是嘴角的抽动；II级：可以点头；III级：一侧肢体抽搐；IV级：强直或全身肢体抽搐；V级：癫痫全面大发作(全身强直性惊厥发作)。
数据处理：
统计实验中各组小鼠IV级发作及死亡例数；II级、III级及IV级潜伏期，未发作至IV级的小鼠潜伏期记作最大值1800秒。例数统计用卡方检验统计。潜伏期计算均值及标准误，应用TTEST，将模型组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果：见表3和表4。
表3.受试药对PLO致小鼠癫痫实验－例数统计
组别 实验例数 IV级例数 IV级发作率 死亡例数 死亡率
模型组 10 7 70％ 1 10％
地西泮2mg/kg 10 0** 0** 0 0
BD-4-50mg/kg 10 7 70％ 0 0
BD-4-200mg/kg 10 9 90％ 1 10％
(与模型组比较，*P<0.05，**P<0.01)
表4.受试药对PLO致小鼠癫痫实验－II级，III级及IV级癫痫发作潜伏期(mean±SEM)
组别 II级发作潜伏期(s) III级发作潜伏期(s) IV级发作潜伏期(s)
模型组 82±4 124±5 958±205
地西泮2mg/kg 118±9** 172±13** 1800±0**
BD-4-50mg/kg 95±3* 162±5** 974±192
BD-4-200mg/kg 95±6 184±17** 765±135
(与模型组比较，*P<0.05，**P<0.01)
实验结论：
1)实验结果显示，模型组IV级发作率为70％。40只小鼠有2只死亡。
2)阳性药可以完全抑制癫痫IV级发作率，并显著延长小鼠II级、III级及IV级癫痫发作潜伏期。
3)在癫痫II级潜伏期比较中，BD-4 50mg/kg剂量组与模型组比较，有统计学差异；在III级潜伏期比较中，BD-4 50mg/kg、200mg/kg剂量组与模型组比较，均有统计学差异。
实验例4 蛋白BD-4(实施例4蛋白)对戊四唑(PTZ)致小鼠癫痫的药效试验
动物：雄性ICR小鼠；
药品：戊四唑(PTZ)，瑞替加滨，蛋白BD-4。
实验分组：
模型组；
瑞替加滨60mg/kg组；
蛋白BD-4，50mg/kg组，200mg/kg组。
方法：
模型制备及给药：
造模前一天下午给药一次，造模当天灌胃受试药后1小时腹腔注射PTZ-65mg/kg(造模剂)，阳性药在造模前半小时给药一次即可。注射PTZ后持续观察15分钟。
观察指标：①癫痫发作情况：Ⅲ级～Ⅵ级发作时间；②死亡情况
发作级别：参照Racine分级标准：0级：没有任何反应；I级：表现为面部肌肉或是嘴角的抽动；Ⅱ级：可以点头；Ⅲ级：一侧肢体抽搐；Ⅳ级：强直或全身肢体抽搐；V级：癫痫全面大发作(全身强直性惊厥发作)。
数据处理：
统计实验中各组小鼠发作及死亡例数；Ⅲ级及Ⅳ级潜伏期，未发作至Ⅳ级的小鼠潜伏期记作最大值900秒。例数统计用卡方检验统计。潜伏期计算均值及标准误，应用TTEST，将模型组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果：见表5和表6。
表5.受试药对PTZ致小鼠癫痫实验－例数统计
组别 实验例数 IV级例数 IV级发作率 死亡例数 死亡率
模型组 10 9 90％ 2 20％
瑞替加滨60mg/kg 10 1** 10％** 0 0
BD-4-50mg/kg 10 8 80％ 1 10％
BD-4-200mg/kg 10 7 70％ 0 0
(与模型组比较，#P<0.05，##P<0.01)
表6.受试药对PTZ致小鼠癫痫实验－III级及IV级癫痫发作潜伏期(mean±SEM)
组别 III级发作潜伏期(秒) IV级发作潜伏期(秒)
模型组 63±3 211±81
瑞替加滨60mg/kg 106±12** 819±81**
BD-4-50mg/kg 91±12* 308±103
BD-4-200mg/kg 89±8** 493±113
(与模型组比较，#P<0.05，##P<0.01)
实验结论：
1)实验结果显示，模型组IV级发作率为90％。40只小鼠有3只死亡。
2)阳性药可以显著降低癫痫IV级发作率，并显著延长小鼠III级及IV级癫痫发作潜伏期。
3)在癫痫III级潜伏期比较中，BD-4 50mg/kg、200mg/kg剂量组与模型组比较均有统计学差异。
实验例5 蛋白BD-4(实施例4蛋白)对小鼠酚红排泄法祛痰的药效试验
动物：雄性ICR小鼠；
药品与试剂：沐舒坦(盐酸氨溴索片)，苯酚红，碳酸氢钠，蛋白BD-4；
仪器：离心机(Sigma-3K15型)，天平(XS105DU型)，酶标测试仪(BIO-TEK型)。
实验分组：
溶剂对照组；
沐舒坦30mg/kg组；
蛋白BD-4，20mg/kg组，50mg/kg组。
方法：
模型制备及给药：
动物于实验前16小时禁食不禁水。按分组口服给予沐舒坦及不同剂量蛋白BD-4(给药体积10ml/kg)，溶剂对照组给予等量体积蒸馏水，1小时后腹腔注射2.5％酚红溶液，30分钟后脱颈处死小鼠，取自甲状软骨下至气管分支前一段气管，将气管放入3ml 5％NaHCO 3溶液静置3小时，取上清1ml，3000rpm离心5分钟后，546nm处测量并记录吸光度。根据酚红的标准曲线计算出酚红的排泄量。
数据处理：
分别记录口服给药时间点，2.5％酚红溶液腹腔注射时间点，取气管时间点；酶标仪546nm处测量得到各组样本吸光度，根据酚红的标准曲线计算出酚红的排泄量。各组数据计算均值及标准误，应用TTEST，将溶剂对照组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果：
给予沐舒坦(30mg/kg)及不同剂量蛋白BD-4(20mg/kg、50mg/kg)，1小时后腹腔注射2.5％酚红溶液，30分钟后脱颈处死小鼠，取自甲状软骨下至气管分支前一段气管，将气管放入3ml 5％NaHCO 3溶液静置3小时，取上清1ml，3000rpm离心5分钟后，546nm处测量并记录吸光度。根据酚红的标准曲线计算出酚红的排泄量。结果见表7。
表7.受试药对小鼠酚红排泄法祛痰药效实验(X±SEM)
组别 N 酚红排泄量(μg/ml) P
溶剂对照组 10 0.638±0.088 ---
沐舒坦30mg/kg 10 1.138±0.079** 0.001
BD-4-20mg/kg 10 0.898±0.076* 0.038
BD-4-50mg/kg 10 0.775±0.101 0.320
(与溶剂对照组比较，*P<0.05，**P<0.01)
实验结论：
1)实验结果显示，沐舒坦30mg/kg组与溶剂对照组比较，酚红排泄量明显升高，P<0.05，具有统计学意义。
2)BD-4 20mg/kg剂量组与溶剂对照组比较，酚红排泄量明显升高，P<0.05，具有统计学意义。
实验例6 蛋白BD-4(实施例4蛋白)对小鼠氨水引咳法镇咳的药效试验
动物：雄性ICR小鼠；
药品与试剂：氢溴酸右美沙芬，氨水，0.2％CMC-Na，蛋白BD-4；
仪器：压缩雾化器(403T型)，天平(XS105DU型)。
实验分组：
溶剂对照组；
右美沙芬15mg/kg组；
蛋白BD-4，20mg/kg组，50mg/kg组。
方法：
模型制备及给药：
按分组口服给予右美沙芬及不同剂量蛋白BD-4(给药体积10ml/kg)，溶剂对照组给予等量体积蒸馏水，1小时后放入密封盒内，通雾化10％浓度氨水10秒钟，然后观察和记录小鼠的咳嗽潜伏期和2分钟内咳嗽次数。
数据处理：
分别记录口服给药时间点，雾化实验时间点，小鼠的咳嗽潜伏期和2分钟内咳嗽次数。咳嗽潜伏期是指雾化氨水开始时间至发生咳嗽所需的秒数。小鼠咳嗽表现以其腹肌收缩(缩胸)，同时张大嘴为准。各组数据计算均值及标准误，应用TTEST，将模型组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果：
提前给予右美沙芬(15mg/kg)及不同剂量蛋白BD-4(20mg/kg、50mg/kg)，1小时后放入密封盒内，通雾化10％浓度氨水10秒钟，然后其对观察和记录小鼠的咳嗽潜伏期和2分钟内咳嗽次数。结果见表8。
表8.受试药对小鼠氨水引咳法的镇咳药效实验(X±SEM)
组别 N 潜伏期(秒) P 咳嗽次数 P
溶剂对照组 9 25.6±2.4 --- 74.4±4.1 ---
右美沙芬15mg/kg 9 39.2±2.8** 0.002 40.2±3.5** 0.001
BD-4-20mg/kg 9 34.0±2.4* 0.025 55.9±3.5** 0.003
BD-4-50mg/kg 9 33.2±3.9 0.116 62.1±3.7* 0.039
(与溶剂对照组比较，*P<0.05，**P<0.01)
实验结论：
1)实验结果显示，右美沙芬组与溶剂对照组在潜伏期和咳嗽次数上比较，有明显的改善作用，P<0.05，具有统计学意义。
2)BD-4 20mg/kg、50mg/kg剂量组，在咳嗽次数上与溶剂对照组比较，有明显改善作用，P<0.05，具有统计学意义；BD-4 20mg/kg剂量组，在潜伏期上与溶剂对照组比较，有明显改善作用，P<0.05，具有统计学意义。
实验例7 蛋白BD-4(实施例4蛋白)对ICR小鼠醋酸扭体的药效试验
动物：雄性ICR小鼠；
药品与试剂：阿司匹林、生理盐水、冰醋酸、蛋白BD-4。
实验分组：
模型组；
阿司匹林(Aspirin)300mg/kg组；
蛋白BD-4，50mg/kg组，200mg/kg组。
方法：
实验动物适应环境一天后，提前一小时分别口服给予阿司匹林300mg/kg，蛋白BD-4 50mg/kg、200mg/kg，给药体积为10ml/kg；而后腹腔注射0.6％醋酸溶液，观察15分钟内动物出现扭体潜伏期(秒)及次数。
数据处理：
各组数据计算均值及标准误，应用TTEST，与模型组比较，P<0.05认为有统计学差异。
实验结果：
口服给予阿司匹林300mg/kg及不同剂量蛋白BD-4(50mg/kg、200mg/kg)一小时后，腹腔注射0.6％醋酸溶液，观察ICR小鼠扭体潜伏期和次数。结果见表9。
表9.受试药对ICR小鼠醋酸扭体实验的影响
组别 N 体重(克) 扭体潜伏期(秒) 扭体次数(次)
模型组0.6％醋酸 22 23.8±0.3 234.6±26.0 29.3±3.6
阿司匹林300mg/kg 13 24.3±0.4 301.0±30.4 16.7±2.6*
BD-4-50mg/kg 15 24.6±0.2 315.2±66.3 16.9±3.7*
BD-4-200mg/kg 13 24.4±0.4 220.1±16.6 25.1±3.3
(与模型组比较，**P<0.01)
实验结论：
应用0.6％醋酸溶液注入小鼠腹腔内，引起深部的大面积而较久的疼痛刺激，致使小鼠产生扭体反应(腹部收缩成“S”形，躯干与后腿伸张、臀部高起及蠕行)。以小鼠开始出现扭体的潜伏时间及次数作为痛反应指标，判断受试样品是否具有镇痛作用。本次实验结果显示：
1)阿司匹林300mg/kg可明显后延扭体潜伏期和减少扭体次数，具有一定的镇痛作用，与模型组比较，P<0.05，有统计学意义。
2)BD-4 50mg/kg剂量组，可明显减少小鼠扭体次数，与模型组比较，P<0.05，有统计学意义。

Claims (15)

  1. 一种角蛋白BD-4，其特征在于，所述角蛋白BD-4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BD-4，其特征在于，在角蛋白BD-4上可进行常规修饰；或者在角蛋白BD-4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BD-4，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BD-4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BD-4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BD-4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BD-4，得到含有角蛋白BD-4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BD-4。
  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BD-4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BD-4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BD-4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BD-4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解热、镇痛、镇咳、祛痰、抗惊厥、抗癫痫、降血压、抗炎、抗病毒药物中的应用。
PCT/CN2020/124329 2019-10-28 2020-10-28 一种角蛋白bd-4、制法和其药物组合物与用途 Ceased WO2021083193A1 (zh)

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AU2020373859A AU2020373859A1 (en) 2019-10-28 2020-10-28 Keratin BD-4, preparation method, and pharmaceutical composition and use thereof
US17/771,994 US12098176B2 (en) 2019-10-28 2020-10-28 Keratin BD-4, preparation method, and pharmaceutical composition and use thereof
CA3159335A CA3159335A1 (en) 2019-10-28 2020-10-28 Keratin bd-4, its preparation and pharmaceutical compositions and use
KR1020227016958A KR20220088737A (ko) 2019-10-28 2020-10-28 케라틴 bd-4, 그의 제조 방법 및 약학적 조성물 및 용도
EP20881931.8A EP4053155A4 (en) 2019-10-28 2020-10-28 KERATIN BD-4, PREPARATION METHOD AND PHARMACEUTICAL COMPOSITION AND USE THEREOF
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