WO2021083191A1 - Keratin bd-2, preparation method therefor, pharmaceutical composition comprising same, and use thereof - Google Patents

Abstract

The present invention provides a keratin BD-2, an encoding nucleic acid molecule thereof, an expression vector thereof, a host cell thereof, and a pharmaceutical composition comprising the keratin. The keratin BD-2 can be used for preparing antipyretic and analgesic, antitussive and expectorant, and antiepileptic drugs.

Description

A kind of keratin BD-2, its preparation method and its pharmaceutical composition and use Technical field

The present invention relates to a keratin BD-2, a nucleic acid molecule encoding keratin BD-2, 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-2 The preparation method of the keratin-containing pharmaceutical composition, and the keratin and the pharmaceutical composition are used in the preparation of antipyretic and analgesic, antitussive and expectorant, anticonvulsant, antiepileptic, hypotensive, anti-inflammatory, and anti-inflammatory Application of viral drugs.

Background technique

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.

With the rapid development of modern biotechnology such as genomics, proteomics, genetic engineering, and microbial engineering, more and more genes have been discovered. The use of protein expression systems to prepare and produce target proteins is an important method for studying the biological functions of genes or proteins.

Using the protein expression system to prepare the target keratin, and then to study its structure and function, there is no other literature report, it is novel and creative.

Summary of the invention

The technical problem solved by the present invention is to provide a keratin BD-2, a nucleic acid molecule encoding keratin BD-2, 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-2, the pharmaceutical composition containing keratin BD-2, and the above-mentioned keratin BD-2, 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.

In order to solve the technical problems of the present invention, 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-2, characterized in that the amino acid sequence of the keratin BD-2 is:

(1) The amino acid sequence shown in SEQ ID NO. 1 in the sequence table;

(2) The 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.

Further, conventional modification can be carried out on keratin BD-2; or a label useful for detection or purification can be attached to keratin BD-2.

Furthermore, the conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, immobilization modification, Sulfation, oxidation, methylation, deamination, formation of disulfide bonds or disulfide bond 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 is to provide a nucleic acid molecule encoding the keratin BD-2 of the first aspect.

Further, the nucleotide sequence of the nucleic acid molecule is:

(1) The nucleotide sequence shown in SEQ ID NO. 2 in the sequence table;

(2) A nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO.2;

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

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.

Further, the expression vector can be pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZα/A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, pPIC3.5K, etc.; the preferred expression vector is the pET series vector; the most preferred expression vector is pET-28a(+).

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.

Further, the host cell includes bacteria, yeast, Aspergillus, plant cells, or insect cells.

Furthermore, the bacteria include Escherichia coli or yeast.

Competent host cells can be BL21 series, Transetta series, Rosetta series, DH5α series, JM series, Top series, Orgami series, Trans1-T1, TG1, TB1; Y11430, MG1003, GS115 (AOX1), KM71, SMD1168, etc.; 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-2 of the first aspect, which is characterized in that it comprises the following steps:

A. Synthesize the nucleic acid molecule corresponding to the keratin BD-2 described in the first aspect, connect the nucleic acid molecule to the corresponding expression vector, transform the expression vector into the host cell, and cultivate the expression vector in the fermentation equipment under certain conditions Host cells and induce expression of keratin BD-2 to obtain a crude protein solution containing keratin BD-2;

B. Separating, purifying and drying the crude protein solution expressed in step A to obtain keratin BD-2.

Further, in step A, the host cells are mainly selected from Escherichia coli, the keratin BD-2 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment includes a shaker flask or a fermentation tank.

Further, in step A, after the expression of keratin BD-2 is induced, the impurities can be washed with a cleaning agent and dissolved in a solution to obtain a crude protein solution.

Further, 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.

Further, the inducer in step A can be IPTG, lactose, arabinose, etc.; preferably, IPTG, lactose.

Further, in 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-2.

Among them, the buffer is preferably buffer A, and its dosage is: fermentation broth volume: buffer A volume = 1-100:1, preferably 10:1;

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

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

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

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

(1) The dialysis method, that is, the crude protein solution obtained in step A is purified by a dialysis method to obtain the target protein BD-2 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.

(2) 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-2.

Preferably, 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.

(3) The column chromatography method, that is, the crude protein solution obtained in step A is separated and purified by column chromatography, such as various exchange columns or exclusion column chromatography, to obtain the target protein BD-2.

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.

(4) 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-2 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.

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

Further, the target protein BD-2 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 provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin BD-2 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.

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. Generally, 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. When used for this purpose, if necessary, the protein of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare an appropriate administration form or dosage 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.

In order to make a unit dosage form into a tablet, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluent 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, microcrystalline cellulose, low-substituted hydroxypropyl cellulose , Cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and rafter acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, dodecyl Sodium sulfonate; lubricant and glidant can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

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.

In order to make the administration unit into a pill, various carriers known in the art can be widely used. Examples of 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.

In order to make the administration unit into a suppository, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.

In order to make the administration unit into a capsule, 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.

For example, the keratin of the present invention is prepared into 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. For example, 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. In addition, in order to prepare an isotonic injection, 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.

In addition, if necessary, coloring agents, preservatives, flavors, flavors, sweeteners, or other materials can also be added to the pharmaceutical preparations.

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

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: the dosage 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-2 of the first aspect or the nucleic acid molecule of the second aspect or the expression vector of the third aspect or the host cell of the fourth aspect or Application of the pharmaceutical composition of the sixth aspect in the preparation of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs.

In order to accomplish the purpose of the present invention, the present invention adopts the following technical solutions. Specifically, the preparation of keratin BD-2 of the present invention includes the following steps:

(1) Synthesize the nucleotide sequence and determine the accuracy of the sequence;

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

(2) Transfer the nucleotide sequence into the expression vector;

The expression vector can be pET series, pUC series, pQE series, pBV series, pMAL series, pPIC9, pPIC9K, pHIL-S1, pPICZα/A, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, pPIC3 .5K, etc.; the preferred expression vector is the pET series vector; the most preferred expression vector is pET-28a(+).

(3) Transfect the expression vector into the host cell;

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, DH5α 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).

(4) The host cell will be fermented and cultured under appropriate conditions to induce the expression of the target protein BD-2;

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.

(5) Enrichment of target protein BD-2 product;

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-2 crude protein solution.

Among them, the buffer is preferably buffer A, and its dosage is: fermentation broth volume: buffer A volume = 1-100:1, preferably 10:1;

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

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

(6) Isolation and purification of target protein BD-2:

The crude protein solution obtained in step (5) needs to be purified to obtain the target protein BD-2. The purification can be performed by dialysis, or ultrafiltration and microfiltration, or column chromatography, or salting out steps.

A. 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-2 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.

B. 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 the target protein BD-2.

Preferably, 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.

C. 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-2.

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.

D. 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-2 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.

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

The target protein BD-2 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 beneficial technical effects of the present invention:

1. The protein of the present invention is keratin obtained for the first time, and the preparation method of the present invention has the characteristics of high yield and high sample purity.

2. According to the present invention, the pharmacodynamic test study of protein BD-2 on the fever model of SD rats induced by yeast and lipopolysaccharide proves that protein BD-2 can significantly inhibit the increase of body temperature at most time points after modeling. And has a strong effect;

3. The present invention uses protein BD-2 to test the effects of pilocarpine (PLO) and pentylenetetrazol (PTZ) on convulsions and epilepsy in mice, and proves that protein BD-2 can significantly prolong the level of epilepsy in mice. Onset latency

4. The present invention proves that protein BD-2 has a tendency to increase the excretion of phenol red and has the potential of expectorant effect through the experimental study of the effect of protein BD-2 on the expectorant of phenol red excretion method in mice;

5. According to the experimental study of the effect of protein BD-2 on the antitussive of mice induced by ammonia water, it is proved that protein BD-2 can effectively prolong the incubation period, significantly reduce the number of coughs, and has a significant antitussive effect;

6. According to the pharmacodynamic study of protein BD-2 on acetic acid writhing in ICR mice, the present invention proves that protein BD-2 can significantly reduce the number of writhing times in mice and has a significant analgesic effect.

Description of the drawings

Figure 1: Analysis of expressed protein BD-2 reduced SDS polyacrylamide gel electrophoresis (SDS-PAGE)

(M: protein molecular weight standard; S: expressed protein BD-2)

Figure 2. The effect of protein BD-2 on the yeast-induced fever model in rats

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

Figure 3. The effect of protein BD-2 on lipopolysaccharide (LPS) induced fever in rats

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

Detailed ways

The following examples and pharmacological activity test examples are used to further illustrate the present invention, but this does not mean any limitation to the present invention.

The experimental methods in the following examples and pharmacological activity test examples are conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, are purchased from conventional biochemical reagent companies.

Example 1 Preparation of Protein BD-2 Crude Solution A (TB Medium) by Shaking Flask Fermentation

Synthesize the nucleotide sequence shown in SEQ ID No.2 and transfer it into the pET-28a(+) vector; confirm the sequence to obtain an expression vector containing the correct sequence; transfect the expression vector into BL21(DE3) cells, Obtain expression competent host cells 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.

Dip the recombinant strain into an LBA plate containing Kanamycin, and place the plate upside down in a 37°C constant temperature incubator overnight for 16 hours.

Configure 400ml TB medium, divided into 2 bottles, each bottle of 200ml. Kanamycin (final concentration 50μg/ml) was added to each bottle (200ml) of TB medium. A single colony on the plate was added to the TB medium. The culture was expanded overnight at 37°C and 220rpm in a shaker. Get the seed liquid.

Configure 28.8L TB medium, divided into 144 bottles, each bottle of 200ml. Kanamycin (final concentration 50μg/ml) was added to each bottle (200ml) of TB medium, and then 2ml of seed solution was added, and cultured in a shaker at 37°C and 220rpm for 2-3 hours. Monitor the OD ₆₀₀ , when the OD ₆₀₀ reaches about 1.0, add an inducer to induce protein expression in a shaker, and the induction conditions are selected from the following table.

Combine each bottle of bacterial liquid, centrifuge at 7000 rpm for 5 minutes, and discard the supernatant after sterilization; the precipitate is suspended in about 3L of buffer, filtered with an 80-100 mesh screen, and the filtrate is crushed with a high-pressure crusher at a pressure of 800-1000 bar. 2 times, 2 minutes each time. After crushing, the bacterial liquid was centrifuged at 7000 rpm for 30 minutes, and the supernatant was discarded to obtain a precipitate (ie, inclusion bodies). The pellet was washed twice with 1L detergent, centrifuged, and the supernatant was discarded. The precipitate was added to the urea solution and dissolved 4 times, respectively, 800ml, 600ml, 400ml, 400ml. The four solutions were combined, centrifuged at 7000 rpm for 30 minutes, the precipitate was discarded, and the supernatant was the crude protein solution A.

Protein BD-2 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 2 Preparation of Protein BD-2 Crude Solution B (Other Medium) by Shaking Flask Fermentation

In 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.

Prepare 20 ml of LB medium, take 800 μl, add 50 μl of host cells containing the target coding sequence, and incubate in a shaker at 37° C. and 220 rpm for 1 hour.

Dip the above bacterial liquid and streak it in an LBA plate containing Kanamycin, and place the plate upside down in a 37°C constant temperature incubator overnight for 16 hours.

Take 10ml of LB medium, add Kanamycin (final concentration 50μg/ml), take a single colony on the plate and add it to LB medium, in a shaker, at 37℃, 220rpm conditions overnight expansion and culture for 15 hours to obtain seeds liquid.

Configure 1L of the medium shown in the table below, and divide it into 10 bottles of 100ml each. Kanamycin (final concentration 50μg/ml) was added to each bottle (100ml) of culture medium, and then 1ml of seed solution was added, and cultured in a shaker at 37°C and 220rpm for 2-3 hours. Monitor the OD ₆₀₀ , when the OD ₆₀₀ reaches about 1.0, add the inducer IPTG (final concentration 0.5 mM), in a shaker, induce protein expression at 37° C. and 220 rpm.

Culture medium

LB medium, SOB medium, SOC medium

Combine each bottle of bacteria liquid, centrifuge at 10000 rpm for 10 minutes, and discard the supernatant after sterilization; the precipitate is suspended in about 100 mL of buffer, filtered with an 80-100 mesh screen, and the filtrate is crushed with a high-pressure crusher at a pressure of 800-1000 bar. 2 times, 2 minutes each time. The broken bacteria liquid was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was discarded.

Add 40mL detergent buffer A to the precipitate and wash it 3 times, centrifuge and discard the supernatant; add 40mL detergent 2M urea solution to the sediment for 2 times, centrifuge and discard the supernatant; add 40mL 4M urea solution to the precipitate and wash it twice , Centrifuge, discard the supernatant; add 8M urea solution (containing 50mM Tris/HCl buffer) to the pellet to dissolve 3 times, respectively, 40ml, 30ml, 30ml; combine the solution, centrifuge at 7000rpm for 30 minutes, discard the pellet, and discard the supernatant The solution is the crude protein solution B.

Protein BD-2 crude solution B, 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 3 Preparation of protein BD-2 crude solution C in a fermentor

In 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.

In the LBA plate containing Kanamycin, add 100 μl of the recombinant strain, spread the spreader until it becomes evenly dry, and place the plate upside down in a constant temperature incubator at 37°C for overnight culture. Take three single colonies and streak them on a Kanamycin-containing plate, culture the plate overnight, and confirm the correct expression by three batches of shake flask fermentation, save the strains with 15% glycerol, and divide them into 0.8ml each to obtain Working cell bank, frozen at -80 ℃ refrigerator for later use.

Take 1 strain of glycerol bacteria from the working cell bank, take 100μl, add 40ml LB medium, add Kanamycin (final concentration 50μg/ml), incubate in a shaker at 37℃, 220rpm for 6 hours, get one Grade seed liquid.

Take 1.2ml of the first-level seed solution, add 120ml LB medium, add Kanamycin (final concentration 50μg/ml), incubate in a shaker at 37°C and 220rpm for 7 hours to obtain the second-level seed solution.

Add 3L of modified LB broth to a 5L fermentor, then add 120ml of secondary seed solution, 3ml of Kanamycin (final concentration 50μg/ml), and incubate for about 8 hours at 37°C and 30% dissolved oxygen (series speed). Monitor the OD value at about 20, 3g lactose as an inducer, induce at 20°C, feed at a rate of 30ml/hour, and incubate at 20°C for 24 hours.

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.

Add 2M urea solution (including 1% Triton) to the precipitate and wash it twice, 1L each time; add 1L 2M urea solution to wash once, centrifuge, and discard the supernatant. The precipitate was dissolved by adding 8M urea solution 4 times, respectively, 400ml, 300ml, 200ml, 100ml. The four solutions were combined, centrifuged at 7000 rpm for 30 minutes, the precipitate was discarded, and the supernatant was the crude protein solution C.

Protein BD-2 crude solution C 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 4 Protein crude solution A was prepared by dialysis to obtain protein BD-2

The crude protein solution A obtained in Example 1 was filtered with a 0.45 μm filter membrane, and the filtrate was combined. The filtrate was dialyzed with water, the dialysis bag had a molecular weight cutoff of 10kD, dialyzed for 72 hours, and the inner liquid was freeze-dried to obtain the target protein BD-2; the purity measured by electrophoresis was 97.1%.

Confirmation of protein BD-2 structure:

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

Apparatus: Protein electrophoresis instrument (Bio-Rad).

Methods and results: The protein BD-2 solution was analyzed by reduced SDS-PAGE, the separation gel concentration was 12.5%, and it was stained with Coomassie Brilliant Blue R250. The molecular weight of BD-2 band is around 45kD.

2. Complete protein sequence analysis based on LC-MS/MS

Main materials: Acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trypsin, chymotrypsin, Glu-C, Asp-N;

Main instruments: Capillary High Performance Liquid Chromatograph (Thermo Ultimate 3000), Electrospray-Combined Ion Trap Orbitrap Mass Spectrometer (Thermo Q Exative Hybrid Quadrupole-Orbitrap Mass Spectrometer).

Methods and results:

Protein BD-2 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. The original mass spectrometry file uses Maxquant (1.6.2.10 ) Search the protein database to analyze the data, and the identification result confirms that it is consistent with the target sequence SEQ ID No. 1.

Example 5 The crude protein solution A was purified by other methods to prepare protein BD-2

The crude protein solution A obtained in Example 1 was purified by the following two methods:

The first 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-2.

The second 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 μm 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-2.

The product protein BD-2 obtained by the two 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 6 Protein BD-2 was prepared by purification of crude protein solution B

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 0.45μm 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-2.

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 μm 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-2.

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-2.

The product protein BD-2 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 Protein BD-2 was prepared by purification of crude protein solution C

The crude protein solution C obtained in Example 3 was purified by the following two methods:

The first method: microfiltration membrane technology;

The crude protein solution C is purified by microfiltration membrane technology: first use 1500nm or 1000nm ceramic membrane cores for solid-liquid separation; discard the inner liquid, and then use 20nm or 50nm ceramic membrane cores for repeated microfiltration to remove urea; secondary microfiltration The filtered inner liquid is freeze-dried to obtain the target protein BD-2.

The second method: salting out;

The crude protein solution C is 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 will precipitate. After the salting-out is complete, filter it. 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 the precipitate is the crude protein extraction Things. 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-2.

The product protein BD-2 obtained by the two 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.

Pharmacological test

Experimental Example 1 The efficacy test of protein BD-2 (Example 4 protein) on yeast-induced fever in SD rats

Animals: 230-260 grams of male SD rats;

Medicines: Yeast (OXOID LP0021), Aspirin (SIGMA A2093), Protein BD-2;

Instruments: electronic balance (SARTORIUS BP121S type), electronic clinical thermometer (CITIZEN CT-513W type).

Experiment grouping:

Normal control group;

Model group: yeast fever model;

Positive control group: Aspirin 300mg/kg group;

Protein BD-2, 10mg/kg group, 50mg/kg group.

method:

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 ℃, and randomly divide them into groups, each with 8 rats. After oral administration of aspirin and different doses of protein BD-2, immediately subcutaneously injected 20% yeast suspension (10ml/kg), and the normal control group was injected subcutaneously with an equal volume of normal saline. After 2 hours, the rats' body temperature was monitored at intervals of 2 hours. The body temperature was monitored once for a total of 8 hours. Statistics:

According to the body temperature measured at each time point on the day of the experiment, the mean, standard deviation and standard error of the body temperature of each group of rats were calculated, and the data of each group was compared with each other using TTEST. P<0.05 was considered as a significant difference.

Experimental results:

After oral administration of aspirin (300mg/kg) and protein BD-2 (10mg/kg, 50mg/kg), immediately subcutaneously inject 20% yeast to make the model, and monitor it at 2 hours, 4 hours, 6 hours, and 8 hours after the model is made. Animal body temperature. The results are shown in Table 1 and Figure 2.

Table 1. The effects of the tested drugs on the yeast-induced fever model in rats

(Compared with the normal control group, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001) Experimental conclusion:

After oral administration of aspirin (300mg/kg) and protein BD-2 (10mg/kg, 50mg/kg), immediately subcutaneously inject 20% yeast to make the model, and monitor it at 2 hours, 4 hours, 6 hours, and 8 hours after the model is made. Animal body temperature. The results show that:

1) The body temperature of rats in the model group increased significantly at 2 hours, 4 hours, 6 hours, and 8 hours after modeling. Compared with the normal group, P<0.05, which was statistically different. The model was successfully established and was stable and reliable.

2) 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 aspirin Stable performance.

3) Different doses of protein BD-2 can inhibit the increase in body temperature of model rats to varying degrees after modeling, and the 10mg/kg dose group has a stronger effect. It can inhibit the model at 2 hours, 6 hours, and 8 hours after modeling. The body temperature of the mice increased, and compared with the model group, P<0.05, there was a statistical difference.

Experimental Example 2 The pharmacodynamic test of protein BD-2 (Example 4 protein) on lipopolysaccharide (LPS) induced fever in SD rats

Animals: 230-260 grams of male SD rats;

Drugs: lipopolysaccharide (LPS, SIGMA L-2880), aspirin (SIGMA A2093), protein BD-2;

Instruments: electronic balance (SARTORIUS BP121S type), electronic clinical thermometer (CITIZEN CT-513W type).

Experiment grouping:

Normal control group;

Model group: lipopolysaccharide fever model;

Positive control group: Aspirin 300mg/kg group;

Protein BD-2, 10mg/kg group, 50mg/kg group.

Method: Lipopolysaccharide was injected into the abdominal cavity to replicate the rat fever model. Method:

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 ℃, and randomly group them into groups of 8 rats. After oral administration of aspirin and different doses of protein BD-2, lipopolysaccharide (20μg/kg, 2ml/kg) was injected intraperitoneally immediately, and 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.

Statistics:

According to the body temperature measured at each time point on the day of the experiment, the mean, standard deviation and standard error of the body temperature of each group of rats were calculated, and the data of each group was compared with each other using TTEST. P<0.05 was considered as a significant difference.

Experimental results:

Oral administration of aspirin (300mg/kg), protein BD-2 (10mg/kg, 50mg/kg) immediately followed by intraperitoneal injection of 20μg/kg lipopolysaccharide to make the model, respectively 2 hours, 4 hours, 6 hours, 8 hours after the model was made Monitor the animal's body temperature. The results are shown in Table 3 and Figure 3.

Table 2. Effects of test drugs on lipopolysaccharide (LPS) induced fever in rats

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

Experimental results:

After oral administration of aspirin (300mg/kg) and protein BD-2 (10mg/kg, 50mg/kg), 20μg/kg lipopolysaccharide was injected into the intraperitoneal cavity immediately to make the model, and 2 hours, 4 hours, 6 hours, 8 hours after the model was made Hourly monitor the animal's body temperature, the results show:

1) Intraperitoneal injection of 20μg/kg lipopolysaccharide can successfully induce the body temperature of rats. The body temperature of rats in the model group increased significantly after 2 hours, 4 hours, 6 hours, and 8 hours after modeling. Compared with the normal group, P<0.05, There are statistical differences and the model is stable.

2) 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.

3) The BD-2 10mg/kg dose group had a tendency to lower the body temperature of the model rats at 2 and 8 hours after modeling, but there was no statistical significance.

Experimental example 3 The pharmacodynamic test of protein BD-2 (Example 4 protein) on convulsive epilepsy in mice caused by the convulsant Pilocarpine (PLO)

Animals: male ICR mice;

Drugs: Pilocarpine HCl (PLO, pilocarpine, pilocarpine hydrochloride), Diazepam (diazepam tablets), protein BD-2.

Experiment grouping:

Model group

Diazepam (Diazepam) 2mg/kg group;

Protein BD-2, 50mg/kg group, 200mg/kg group.

method:

Model preparation and administration:

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.

Observation indicators: ①Seizure situation: the time of seizure of grade Ⅱ～Ⅳ; ②time of death.

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).

data processing:

The number of cases of class IV seizures and deaths in each group of mice was counted; the incubation period of class II, class III and class IV, and the incubation period of mice that did not seize to class IV was recorded as the maximum value of 1800 seconds. Chi-square test was used for statistics of the number of cases. The average value and standard error of the incubation period were calculated, and TTEST was used to compare the model group with other groups. P<0.05 was considered as a significant difference.

Experimental results: see Table 3 and Table 4.

Table 3. Experiments of tested drugs on PLO-induced epilepsy in mice-statistics of cases

Group	Number of experimental examples	Number of Grade IV cases	Grade IV seizure rate	Number of deaths	mortality rate
Model group	10	9	90%	0	0
Diazepam 2mg/kg	10	1**	10%**	0	0
BD-2-50mg/kg	10	7	70%	1	10%
BD-2-200mg/kg	10	8	80%	1	10%

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

Table 4. Experiments of tested drugs on PLO-induced epilepsy in mice-level II, level III and level IV seizure latency (mean±SEM)

Group	Level II onset incubation period (s)	Level III onset latency (s)	Level IV onset latency (s)
Model group	86±3	145±7	672±131
Diazepam 2mg/kg	94±7	176±13*	1691±109**
BD-2-50mg/kg	96±6	176±7**	976±189
BD-2-200mg/kg	90±3	161±6	935±180

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

Experimental results:

1) The experimental results show that the grade IV attack rate in the model group is 90%. Two of the 40 mice died.

2) Positive drugs can completely suppress the rate of class IV epileptic seizures, and significantly prolong the incubation period of class III and IV epileptic seizures in mice.

3) In the comparison of epilepsy grade III incubation period, the BD-2 50mg/kg group is statistically different from the model group.

Experimental example 4 The efficacy test of protein BD-2 (Example 4 protein) on pentylenetetrazole (PTZ)-induced epilepsy in mice

Animals: male ICR mice;

Drugs: Pentylenetetrazol (PTZ), Retigabine, Protein BD-2.

Experiment grouping:

Model group

Retigabine 60mg/kg group;

Protein BD-2, 50mg/kg group, 200mg/kg group.

method:

Model preparation and administration:

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. Observation indicators: ①Epilepsy status: seizure time of grade Ⅲ～Ⅵ; ②death situation

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).

data processing:

The number of seizures and deaths in each group of mice in the experiment was counted; the incubation period of grade III and IV, and the incubation period of mice that did not attack to grade IV was recorded as the maximum of 900 seconds. Chi-square test was used for statistics of the number of cases. The average value and standard error of the incubation period were calculated, and TTEST was used to compare the model group with other groups. P<0.05 was considered as a significant difference.

Experimental results: see Table 5 and Table 6.

Table 5. Experiments of tested drugs on PTZ-induced epilepsy in mice-statistics of cases

Group	Number of experimental examples	Number of Grade IV cases	Grade IV seizure rate	Number of deaths	mortality rate
Model group	10	9	90%	1	10%
Retigabine 60mg/kg	10	3	30%	0	0
BD-2-50mg/kg	10	8	80%	0	0
BD-2-200mg/kg	10	8	80%	0	0

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

Table 6. Experiment of the test drug on PTZ-induced epilepsy in mice-the incubation period of grade III and IV seizures (mean±SEM)

Group	Level III incubation period (seconds)	Level IV onset latency (seconds)
Model group	66±4	205±81
Retigabine 60mg/kg	106±16*	722±94**
BD-2-50mg/kg	94±8**	307±102
BD-2-200mg/kg	78±8	277±105

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

Experimental results:

1) Experimental results show that the grade IV attack rate in the model group is 90%. 1 out of 40 mice died.

2) Positive drugs can reduce the seizure rate of grade IV epilepsy, and significantly prolong the incubation period of grade III and IV seizures in mice.

3) In the comparison of the incubation period of grade III epilepsy, the BD-2 50mg/kg group and the model group are statistically different.

Experimental Example 5 The efficacy test of protein BD-2 (Example 4 protein) on the expectorant of phenol red excretion method in mice

Animals: male ICR mice;

Drugs and reagents: Mucosultan (ambroxol hydrochloride tablets), phenol red, sodium bicarbonate, protein BD-2;

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

Experiment grouping:

Solvent control group;

Mucosultan 30mg/kg group;

Protein BD-2, 20mg/kg group, 50mg/kg group.

method:

Model preparation and administration:

The animals were fasted and watered 16 hours before the experiment. Amucosultan and different doses of protein BD-2 (administration volume 10ml/kg) were given orally according to groups. The solvent control group was given the same volume of distilled water, and 2.5% phenol red solution was intraperitoneally injected 1 hour later. The mice were killed by neck removal 30 minutes later. , Taken from the thyroid cartilage to a section of trachea before the tracheal branch, put the trachea into 3ml of 5% NaHCO ₃ solution and let it stand for 3 hours, take 1ml of 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.

data processing:

Record the time point of oral administration, the time point of intraperitoneal injection of 2.5% phenol red solution, and the time point of trachea; the absorbance of samples of each group was measured at 546nm by the microplate reader, and the excretion of phenol red was calculated according to the standard curve of phenol red. Calculate the mean and standard error of the data of each group, use TTEST to compare the solvent control group with other groups, and P<0.05 is considered as a significant difference.

Experimental results:

Give Mucosultan (30mg/kg) and different doses of protein BD-2 (20mg/kg, 50mg/kg), and intraperitoneal injection of 2.5% phenol red solution after 1 hour. After 30 minutes, the mice were sacrificed by neck removal, which were taken from the 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.

Table 7. The effect of the test drug on the expectorant effect of the phenol red excretion method in mice (X±SEM)

Group	N	Phenol red excretion (μg/ml)	P
Solvent control group	10	0.715±0.087	---
Mucosultan 30mg/kg	10	1.169±0.109**	0.004
BD-2-20mg/kg	10	1.065±0.162	0.073
BD-2-50mg/kg	10	0.931±0.091	0.102

(Compared with solvent control group, *P<0.05, **P<0.01)

Experimental results:

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

2) Compared with the solvent control group, the BD-2 20mg/kg dose group showed an upward trend in the excretion of phenol red, but it was not statistically significant.

Experimental Example 6 The effect of protein BD-2 (Example 4 protein) on the antitussive effect of the method of inducing cough with ammonia water in mice

Animals: male ICR mice;

Drugs and reagents: dextromethorphan hydrobromide, ammonia, 0.2% CMC-Na, protein BD-2;

Apparatus: Compressed atomizer (403T type), balance (XS105DU type).

Experiment grouping:

Solvent control group;

Dextromethorphan 15mg/kg group;

Protein BD-2, 20mg/kg group, 50mg/kg group.

method:

Model preparation and administration:

Dextromethorphan and different doses of protein BD-2 (administration volume 10ml/kg) were given orally in groups. 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.

data processing:

Record the time point of oral administration, the time point of the nebulization experiment, the incubation period of the mouse cough 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.

Experimental results:

Give dextromethorphan (15mg/kg) and different doses of protein BD-2 (20mg/kg, 50mg/kg) in advance, put them in a sealed box after 1 hour, spray 10% ammonia water for 10 seconds, and then Observe and record the incubation period of cough in mice and the number of coughs within 2 minutes. The results are shown in Table 8.

Table 8. Antitussive effect experiment of tested drugs on mice cough induced by ammonia water (X±SEM)

Group	N	Latency (seconds)	P	Number of coughs	P
Solvent control group	9	25.3±1.9	---	66.8±3.9	---
Dextromethorphan 15mg/kg	9	38.0±2.57**	0.001	33.4±3.8**	0.001
BD-2-20mg/kg	9	32.4±2.3*	0.028	48.1±5.4*	0.013
BD-2-50mg/kg	9	31.0±2.3	0.074	53.2±3.0*	0.014

(Compared with solvent control group, *P<0.05, **P<0.01)

Experimental results:

1) The experimental results show that the dextromethorphan group and the solvent control group have a significant improvement in the incubation period and the number of coughs, P<0.05, which is statistically significant.

2) Compared with the solvent control group, different doses of BD-2 have a significant improvement in the incubation period, P<0.05, which is statistically significant; different doses of BD-2 have a significant improvement in the number of coughs compared with the solvent control group The effect, P<0.05, is statistically significant.

Experimental Example 7 The efficacy test of protein BD-2 (Example 4 protein) on acetic acid writhing in ICR mice

Animals: male ICR mice;

Medicines and reagents: aspirin, physiological saline, glacial acetic acid, protein BD-2.

Experiment grouping:

Model group

Aspirin 300mg/kg group;

Protein BD-2, 50mg/kg group, 200mg/kg group.

method:

After one day of adaptation to the environment, the experimental animals were orally given aspirin 300mg/kg, protein BD-2 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.

data processing:

Calculate the mean value and standard error of each group of data, using TTEST, compared with the model group, P<0.05 considered statistically different.

Experimental results:

One hour after oral administration of aspirin 300 mg/kg and different doses of protein BD-2 (50 mg/kg, 200 mg/kg), 0.6% acetic acid solution was intraperitoneally injected to observe the writhing latency and frequency of ICR mice. The results are shown in Table 9.

Table 9. The effect of the tested drug on the acetic acid writhing test of ICR mice

Group	N	Weight (g)	Writhing latency (seconds)	Number of twists (times)
Model group 0.6% acetic acid	18	23.3±0.2	185.4±6.3	32.7±2.6
Aspirin 300mg/kg	18	24.3±0.2	281.0±26.5**	14.2±2.1***
BD-2-50mg/kg	12	23.5±0.4	233.1±37.6	21.7±4.3*
BD-2-200mg/kg	12	24.1±0.2	177.4±8.3	24.0±2.3*

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

Experimental results:

The 0.6% acetic acid solution was injected into the abdominal cavity of mice, which caused deep and long-lasting painful stimulation, which caused the mice to have a writhing response (the abdomen was contracted into an "S" shape, the trunk and hind legs were stretched, the buttocks were raised and wriggled. Row). The incubation time and the number of times the mice began to writhe were used as the pain response index to determine whether the test sample has analgesic effect. The results of this experiment show:

1) Aspirin 300mg/kg can significantly delay the latency of writhing and reduce the frequency of writhing, and has a significant analgesic effect. Compared with the model group, P<0.05, which is statistically significant.

2) BD-2 50mg/kg and 200mg/kg dose groups can significantly reduce the number of writhing times in mice. Compared with the model group, P<0.05, which is statistically significant.

Claims (15)

1. A keratin BD-2, characterized in that the amino acid sequence of the keratin BD-2 is:

   (1) The amino acid sequence shown in SEQ ID NO. 1 in the sequence table;

   (2) The 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.
2. The keratin BD-2 according to claim 1, characterized in that the keratin BD-2 can be conventionally modified; or a label for detection or purification is also attached to the keratin BD-2.
3. The keratin BD-2 according to claim 2, wherein the conventional modification includes 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.
4. A nucleic acid molecule encoding the keratin BD-2 of any one of claims 1-3.
5. The nucleic acid molecule according to claim 4, wherein the nucleotide sequence of the nucleic acid molecule is:

   (1) The nucleotide sequence shown in SEQ ID NO. 2 in the sequence table;

   (2) A nucleotide sequence obtained by sequence optimization based on the nucleotide sequence shown in SEQ ID NO.2;

   (3) A nucleotide sequence complementary to the nucleotide sequence in (1) or (2) above.
6. An expression vector, characterized in that the expression vector contains the nucleic acid molecule of any one of claims 4-5.
7. A host cell, characterized in that the host cell contains the expression vector of claim 6 or the nucleic acid molecule of any one of claims 4-5 is integrated into the genome.
8. The host cell according to claim 7, wherein said host cell comprises bacteria, yeast, Aspergillus, plant cells, or insect cells.
9. The host cell according to claim 8, wherein said bacteria comprise Escherichia coli.
10. A method for preparing and producing the keratin BD-2 of any one of claims 1 to 3, characterized in that it comprises the following steps:

    A. Synthesize the nucleic acid molecule corresponding to keratin BD-2 according to any one of claims 1 to 3, link the nucleic acid molecule into the corresponding expression vector, transform the expression vector into the host cell, and in a fermentation equipment under certain conditions Culturing host cells with expression vectors and inducing expression of keratin BD-2 to obtain a crude protein solution containing keratin BD-2;

    B. Separating, purifying and drying the crude protein solution expressed in step A to obtain keratin BD-2.
11. The method according to claim 10, characterized in that, in step A, the host cell is mainly selected from Escherichia coli, the keratin BD-2 is expressed in Escherichia coli inclusion bodies, and the fermentation equipment comprises Shake bottle or fermentation tank.
12. The method according to claim 10, characterized in that, in step A, after the expression of keratin BD-2 is induced, impurities can be washed with a cleaning agent and dissolved in a solution to obtain a crude protein solution.
13. The method according to claim 10, characterized in that, in step B, the separation and purification method includes ultrafiltration microfiltration membrane technology purification method, column chromatography purification method, salting out method, and dialysis method.
14. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the keratin BD-2 of any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.
15. The keratin BD-2 of any one of claims 1 to 3 or the nucleic acid molecule of any one of claims 4-5 or the expression vector of claim 6 or the host cell of claims 7-9 Or the use of the pharmaceutical composition according to claim 14 in the preparation of antipyretic, analgesic, antitussive, expectorant, anticonvulsant, antiepileptic, antihypertensive, anti-inflammatory, and antiviral drugs.

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