阅读说明：本技术 多肽及其制备方法和用途 (Polypeptide and preparation method and application thereof ) 是由 王少平 张加余 代龙 杨爱琳 姜珊 董萍萍 于盈盈 于 2020-04-13 设计创作，主要内容包括：本发明公开了一种多肽及其制备方法和用途。该多肽具有SEQ ID NO:1所示的氨基酸序列。该多肽具有活血化瘀的功效。本发明还公开了上述多肽的制备方法,该方法包括：(1)酶解；(2)吸附层析；(3)分子筛层析等步骤。该方法得到的产物中多肽纯度高。(The invention discloses a polypeptide and a preparation method and application thereof. The polypeptide has an amino acid sequence shown as SEQ ID NO. 1. The polypeptide has effects of promoting blood circulation and removing blood stasis. The invention also discloses a preparation method of the polypeptide, which comprises the following steps: (1) carrying out enzymolysis; (2) adsorption chromatography; (3) molecular sieve chromatography and the like. The product obtained by the method has high purity of polypeptide.)

1. A polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence shown as SEQ ID NO. 1.

2. An isolated nucleic acid molecule encoding the polypeptide of claim 1.

3. A pharmaceutical composition comprising:

(1) the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; and

(2) pharmaceutically acceptable adjuvants.

4. The method for producing the polypeptide of claim 1, comprising the steps of:

(1) carrying out enzymolysis on the ground beetle slurry under the action of pepsin to obtain a first enzymolysis product; carrying out enzymolysis on the first enzymolysis product under the action of trypsin to obtain a second enzymolysis product;

(2) adsorbing and chromatographing the second enzymolysis product by adopting a DA201-C resin column, and eluting the DA201-C resin column after adsorption is finished to obtain a first eluent;

(3) performing molecular sieve chromatography on the first eluent by using a sephadex G25 column, and then eluting the sephadex G25 column to obtain a second eluent; and separating solid matters in the second eluent to obtain a product.

5. The method for producing the polypeptide of claim 1, comprising the steps of:

(1) carrying out enzymolysis on the ground beetle slurry under the action of pepsin to obtain a first enzymolysis product; carrying out enzymolysis on the first enzymolysis product under the action of trypsin to obtain a second enzymolysis product;

(2) adsorbing and chromatographing the second enzymolysis product by adopting a DA201-C resin column, and eluting the DA201-C resin column after adsorption is finished to obtain a first eluent;

(3) performing molecular sieve chromatography on the first eluent by using a sephadex G25 column, and then eluting the sephadex G25 column to obtain a second eluent;

(4) separating the second eluent by a reversed-phase high performance liquid chromatograph, and then eluting the chromatographic column to obtain a third eluent; the chromatographic column is a C18 chromatographic column, the mobile phase A is trifluoroacetic acid aqueous solution, and the mobile phase B is trifluoroacetic acid aqueous solution and acetonitrile; and separating solid matters in the third eluent to obtain a product.

6. The production method according to any one of claims 4 to 5, characterized in that:

in the step (2), the mass-to-volume ratio of the DA201-C resin to the second enzymolysis product is 5-20: 1g/ml, and ethanol is adopted to elute the DA201-C resin column;

in the step (3), the first eluent is added into a sephadex G25 column for multiple times, the volume ratio of the filling mass of sephadex G25 in the sephadex G25 column to the first eluent added into the sephadex G25 column is 0.2-12 kg/ml, and deionized water is adopted to elute the sephadex G25 column.

7. The preparation method according to claim 5, wherein the chromatographic column in the step (4) is a Waters ZOBRX-300SB C18 chromatographic column, the detector is an ultraviolet detector, and the detection wavelength is 200-240 nm and 260-300 nm.

8. The method of claim 5, wherein the polypeptide of claim 1 is present in an amount greater than 92 wt% of the product.

9. Use of the polypeptide or pharmaceutically acceptable salt according to claim 1 for the manufacture of a medicament for activating blood circulation to dissipate blood stasis.

10. Use of the polypeptide or pharmaceutically acceptable salt according to claim 1 in the manufacture of a medicament for the treatment or prevention of hyperlipidemia.

Technical Field

The invention relates to a polypeptide and a preparation method and application thereof, in particular to a polypeptide extracted from ground beetles and a preparation method and application thereof.

Background

The Eupolyphaga Seu Steleophaga is traditional Chinese medicine, has effects of removing blood stasis, promoting reunion of fractured tendons and bones, and can be used for treating traumatic injury, tendon injury fracture, blood stasis amenorrhea, puerperal blood stasis abdominal pain, and addiction abdominal lump. Modern researches have shown that Eupolyphaga Seu Steleophaga has effects of treating blood and cerebrovascular diseases, such as atherosclerosis, hyperlipemia, cerebral thrombosis, etc. The traditional Chinese medicine is generally taken directly after decocting raw medicinal materials in water or taken after extracting the raw medicinal materials in water, so that the utilization rate of the medicinal materials is low and the side effect is great.

CN104055800A discloses a ground beetle protease zymolyte, which is prepared by the following method: (1) cleaning Eupolyphaga Seu Steleophaga, and pulverizing into fine powder; (2) placing Eupolyphaga Seu Steleophaga fine powder in artificial gastric juice, adding pepsin for hydrolysis to obtain pepsin enzymatic hydrolysate, and centrifuging; (3) taking the supernatant, and drying to obtain ground beetle pepsin zymolyte; (4) transferring the residue into artificial intestinal juice, adding trypsin, and hydrolyzing to obtain Eupolyphaga Seu Steleophaga trypsin zymolyte. And (4) obtaining the pepsin zymolyte obtained in the step (3) and the trypsin zymolyte obtained in the step (4) as the ground beetle protease zymolyte. Although the utilization rate of the medicinal materials is improved to a certain extent, the purity of the effective components in the protein zymolyte is still not high.

CN101647822A discloses a bionic enzymatic hydrolysate of ground beetles, which is prepared by the following method: adding water into ground beetles, homogenizing, adjusting the pH value to 1.0-3.0, adding pepsin, performing enzymolysis for 0.5-4 hours at the temperature of 35-45 ℃, adjusting the pH value to 7.5-8.5, adding pancreatin or trypsin, performing enzymolysis for 2-8 hours at the temperature of 40-50 ℃, and obtaining the traditional Chinese medicine. The enzymolysis product obtained by the method has low purity of effective components.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a polypeptide, which has the effects of promoting blood circulation and removing blood stasis.

Another object of the present invention is to provide a nucleic acid molecule encoding the above polypeptide.

It is a further object of the present invention to provide a pharmaceutical composition comprising the above polypeptide.

It is still another object of the present invention to provide a method for producing the above polypeptide, which can produce a product having high purity.

It is still another object of the present invention to provide uses of the above polypeptides.

The invention provides a polypeptide or pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence shown as SEQ ID NO. 1.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a polypeptide as described above.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising:

(1) the above polypeptide or a pharmaceutically acceptable salt thereof; and

(2) pharmaceutically acceptable adjuvants.

In another aspect, the present invention provides a method for preparing the above polypeptide, comprising the steps of:

(1) carrying out enzymolysis on the ground beetle slurry under the action of pepsin to obtain a first enzymolysis product; carrying out enzymolysis on the first enzymolysis product under the action of trypsin to obtain a second enzymolysis product;

(2) adsorbing and chromatographing the second enzymolysis product by adopting a DA201-C resin column, and eluting the DA201-C resin column after adsorption is finished to obtain a first eluent;

(3) performing molecular sieve chromatography on the first eluent by using a sephadex G25 column, and then eluting the sephadex G25 column to obtain a second eluent; and separating solid matters in the second eluent to obtain a product.

The preparation method of the polypeptide of the invention can also comprise the following steps:

(1) carrying out enzymolysis on the ground beetle slurry under the action of pepsin to obtain a first enzymolysis product; carrying out enzymolysis on the first enzymolysis product under the action of trypsin to obtain a second enzymolysis product;

(2) adsorbing and chromatographing the second enzymolysis product by adopting a DA201-C resin column, and eluting the DA201-C resin column after adsorption is finished to obtain a first eluent;

(3) performing molecular sieve chromatography on the first eluent by using a sephadex G25 column, and then eluting the sephadex G25 column to obtain a second eluent;

(4) separating the second eluent by a reversed-phase high performance liquid chromatograph, and then eluting the chromatographic column to obtain a third eluent; the chromatographic column is a C18 chromatographic column, the mobile phase A is trifluoroacetic acid aqueous solution, and the mobile phase B is trifluoroacetic acid aqueous solution and acetonitrile; and separating solid matters in the third eluent to obtain a product.

According to the preparation method provided by the invention, preferably, in the step (2), the mass-volume ratio of the DA201-C resin to the second enzymolysis product is 5-20: 1g/ml, and ethanol is adopted to elute the DA201-C resin column; in the step (3), the first eluent is added into a sephadex G25 column for multiple times, the volume ratio of the filling mass of sephadex G25 in the sephadex G25 column to the first eluent added into the sephadex G25 column is 0.2-12 kg/ml, and deionized water is adopted to elute the sephadex G25 column.

According to the preparation method of the invention, preferably, in the step (4), the chromatographic column is a Waters ZOBRX-300SB C18 chromatographic column, the detector is an ultraviolet detector, and the detection wavelength is 200-240 nm and 260-300 nm.

According to the preparation method of the present invention, preferably, the content of the polypeptide in the product is more than 92 wt%.

In another aspect, the invention provides the use of the polypeptide or the pharmaceutically acceptable salt thereof in preparing a medicament for promoting blood circulation to remove blood stasis.

The invention also provides the application of the polypeptide or the pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing hyperlipidemia.

The invention provides a polypeptide with the functions of promoting blood circulation and removing blood stasis, which is safe and effective and has small side effect. The invention also provides a preparation method of the polypeptide, and the purity of the active polypeptide in the product prepared by the method is high.

Drawings

FIG. 1 is a graph showing the results of chromatography at a detection wavelength of 220 nm.

FIG. 2 is a diagram showing the results of separation by semi-preparative reverse phase HPLC.

FIG. 3 is a liquid phase primary mass spectrum.

FIG. 4 is a matrix-assisted laser desorption ionization-liquid phase secondary mass spectrum.

Fig. 5 is a thrombocyte count chart.

Fig. 6 is a blood viscosity graph.

FIG. 7 is a graph of hemoglobin and hematocrit.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.

The inventor determines the polypeptide structure with the function of promoting blood circulation to remove blood stasis in the ground beetle through long-term research on the medicinal mechanism of the ground beetle, and separates the active substance from the raw material medicine of the ground beetle with high purity by a proper method.

< polypeptide or pharmaceutically acceptable salt thereof >

The polypeptide of the present invention has an amino acid sequence shown in SEQ ID NO. 1 or a variant thereof. The variant forms include but are not limited to deletion, insertion and/or substitution of 1-5 amino acids; preferably 1-3 amino acid deletions, insertions and/or substitutions; more preferably 1 to 2 amino acids are deleted, inserted and/or substituted. According to one embodiment of the invention, the polypeptide of the invention has the amino acid sequence shown in SEQ ID NO. 1.

The molecular mass of the polypeptide can be 1300-1600 Da; preferably 1400-1500 Da; more preferably 1431 Da.

The polypeptides of the invention can also be used in the form of pharmaceutically acceptable acid or base derived salts. These salts include, but are not limited to, salts formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulfonic, ethanesulfonic, benzenesulfonic or isethionic acid; salts with the following alkali or alkaline earth metals: sodium, potassium, calcium or magnesium.

< nucleic acid molecule and pharmaceutical composition >

The invention provides a nucleic acid molecule which can code an amino acid sequence shown as SEQ ID NO. 1.

The pharmaceutical composition of the invention comprises: (1) a polypeptide of the invention or a pharmaceutically acceptable salt thereof; and (2) pharmaceutically acceptable adjuvants. The dosage of the polypeptide is usually 0.001-10 g/dose; preferably 0.01 to 1 g/dose; more preferably 0.05 to 0.5 g/dose.

The pharmaceutically acceptable excipients do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such adjuvants are well known to those skilled in the art, for example solvents, pH adjusters, surfactants, suspending agents, preservatives, antioxidants, isotonicity adjusting agents, topical analgesics, disintegrants, binders, lubricants, diluents and the like. Examples of solvents include, but are not limited to, water, saline, glucose, glycerol, ethanol, or combinations thereof. Examples of pH adjusting agents include, but are not limited to, hydrochloric acid, sodium hydroxide, sodium bicarbonate, citric acid buffer, tartaric acid buffer, hydrochloric acid buffer, or combinations thereof. Examples of surfactants include, but are not limited to, tween-80, cremophor, poloxamer 188, lecithin, or combinations thereof. Examples of suspending agents include, but are not limited to, polyvinylpyrrolidone, gelatin, methylcellulose, sodium carboxymethylcellulose, or a combination thereof. Examples of preservatives include, but are not limited to, phenol, chlorobutanol, thimerosal, cresol, benzyl alcohol, or combinations thereof. Examples of antioxidants include, but are not limited to, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, or combinations thereof. Examples of isotonic adjusting agents include, but are not limited to, sodium chloride, glucose, or combinations thereof. Examples of topical analgesics include, but are not limited to, benzyl alcohol, chlorobutanol, procaine hydrochloride, lidocaine, or combinations thereof. Examples of disintegrants include, but are not limited to, croscarmellose sodium, crospovidone, starch, sodium carboxymethyl starch, hydroxypropyl starch, low substituted hydroxypropyl cellulose, or combinations thereof. Examples of binders include, but are not limited to, starch slurry, povidone, polyethylene glycol, sodium carboxymethylcellulose, or combinations thereof. Examples of lubricants include, but are not limited to, magnesium stearate, polyethylene glycol, magnesium lauryl sulfate, colloidal silica, talc, or combinations thereof. Examples of diluents include, but are not limited to, starch, compressible starch, dextrin, lactose, mannitol, microcrystalline cellulose, or combinations thereof.

When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition may be used depending on the use situation. Preferably, the injection can be injection or powder injection.

According to one embodiment of the invention, the pharmaceutical composition is an injection comprising the polypeptide of the invention, a solvent and a preservative. The dosage of the polypeptide is 1.0-5.0 wt%; preferably 2.0 to 4.0 wt%; more preferably 3.0 wt%. The amount of the solvent is 40-99 wt%; preferably 50 to 99 wt%; more preferably 60 to 99 wt%. The dosage of the preservative is 0.01-1.20 wt%; preferably 0.40 to 1.0 wt%; more preferably 0.5 wt%. The solvent may be selected from water, saline, glucose, glycerol, ethanol, or combinations thereof; preferably water. The preservative may be selected from phenol, chlorobutanol, thimerosal, cresol, benzyl alcohol or combinations thereof; preferably chlorobutanol.

According to another embodiment of the present invention, the pharmaceutical composition is a powder injection, and the polypeptide of the present invention is lyophilized by a lyophilizer.

< preparation method >

The preparation method of the polypeptide comprises the following steps: (1) carrying out enzymolysis; (2) adsorption chromatography; (3) performing molecular sieve chromatography; and optionally (4) separating by reversed phase high performance liquid chromatography.

Enzymolysis

Carrying out enzymolysis on the ground beetle slurry under the action of pepsin to obtain a first enzymolysis product; and carrying out enzymolysis on the first enzymolysis product under the action of trypsin to obtain a second enzymolysis product.

According to one embodiment of the invention, the ground beetle slurry is prepared by the following steps of crushing the ground beetle, mixing the ground beetle with water to prepare a homogenate, and sterilizing the homogenate at 80-130 ℃ for 10-40 min.

The dosage of the pepsin is 0.5-3 wt% of the weight of the ground beetle, preferably 0.5-2.5 wt%, more preferably 0.5-2 wt%, the enzymolysis temperature of the pepsin can be 30-50 ℃, preferably 35-45 ℃, more preferably 38-42 ℃, the enzymolysis pH of the pepsin can be 1-3, preferably 1.5-2.5, more preferably 3, the enzymolysis time of the pepsin can be 40-90 min, preferably 50-80 min, more preferably 50-70 min, the pH value can be adjusted by adopting a hydrochloric acid solution, and the concentration of the hydrochloric acid solution can be 0.01-1 mol/L, preferably 0.05-0.5 mol/L.

The using amount of the trypsin is 0.5-3 wt% of the weight of the ground beetle; preferably 0.5 to 2.5 wt%; more preferably 0.5 to 2 wt%. The temperature of the trypsin enzymolysis can be 30-50 ℃; preferably 35-45 ℃; more preferably 38 to 42 ℃. The pH value of the trypsin enzymolysis can be 8-10; preferably 8.5 to 9.5; more preferably 9. The enzymolysis time of the trypsin can be 150-240 min; preferably 150-200 min; more preferably 160-190 min. The pH can be adjusted with sodium hydroxide solution. The concentration of the sodium hydroxide solution can be 1-5 wt%; preferably 1 to 3 wt%.

The product after enzymolysis under the action of trypsin can be concentrated, so that the second enzymolysis product has solid matters with proper concentration, and the solid matters contain polypeptides after enzymolysis by pepsin and trypsin. The concentration of solid matters in the second enzymolysis product can be 20-80 mg/ml; preferably 30-70 mg/ml; more preferably 40 to 60 mg/ml. This facilitates subsequent separation.

The pepsin and trypsin enzymolysis can be carried out in a constant temperature enzymolysis instrument.

Adsorption chromatography

And adsorbing and chromatographing the second enzymolysis product by adopting a DA201-C resin column, and eluting the DA201-C resin column after adsorption is finished to obtain a first eluent.

The mass-volume ratio of the DA201-C resin to the second enzymolysis product is 5-20: 1 g/ml; preferably 6-15: 1 g/ml; more preferably 8-12: 1 g/ml.

The DA201-C resin column can be eluted by using an ethanol solution in the invention. The concentration of the ethanol solution can be 30-80 vol%; preferably 30 to 70 vol%; more preferably 40 to 60 vol%.

Molecular sieve chromatography

And (3) carrying out chromatography on the first eluent by using a sephadex G25 column molecular sieve, and then eluting the sephadex G25 column to obtain a second eluent. In certain embodiments, the solid material in the second eluate is separated to produce the product. In other embodiments, it may be desirable to subject the second eluate to reverse phase high performance liquid chromatography.

In the invention, the first eluent can be added into the sephadex G25 column for multiple times, and the volume ratio of the filling mass of sephadex G25 in the sephadex G25 column to the first eluent added into the sephadex G25 column is 0.2-12 kg/ml; preferably 1-10 kg/ml; more preferably 1 to 4 kg/ml. The sephadex G25 column can be eluted with deionized water in the present invention.

The sephadex G25 column can be prepared by the following method: and washing the sephadex G25 with absolute ethyl alcohol and deionized water in sequence, and then filling the sephadex G25 into a column to obtain the sephadex G25 column.

Reversed phase high performance liquid chromatograph separation

And separating the second eluent by a reversed-phase high performance liquid chromatograph (RP-HP L C), eluting the chromatographic column to obtain a third eluent, and separating solid substances in the third eluent to obtain a product.

The chromatographic column is C18 chromatographic column. The length of the chromatographic column can be 120-200 mm, and the inner diameter can be 3-5.5 mm. Preferably, the length of the chromatographic column is 130-170 mm, and the inner diameter is 4-5 mm. According to one embodiment of the invention, the column is a Waters ZOBRX-300SB C18 column.

The mobile phase A can be aqueous trifluoroacetic acid, and the mobile phase B can be aqueous trifluoroacetic acid and acetonitrile. Preferably, the trifluoroacetic acid in the aqueous trifluoroacetic acid solution is present in an amount of 0.1 vol%.

The detector is an ultraviolet detector, and the detection wavelength is 200-240 nm and 260-300 nm. Preferably, the detection wavelengths are 210-230 nm and 270-290 nm. . More preferably, the detection wavelengths are 220nm and 280 nm.

In the product obtained by the preparation method, the content of the polypeptide with the amino acid sequence shown in SEQ ID NO. 1 is more than 92 wt%; preferably greater than 93 wt%; more preferably greater than 95 wt%.

< use >

The polypeptide of the invention has the efficacy of promoting blood circulation by removing blood stasis, so the polypeptide of the invention can be used for preparing the medicine with the efficacy of promoting blood circulation by removing blood stasis. The polypeptide of the invention can also be used for preparing medicines for preventing or treating hyperlipidemia.

Experimental example 1

(1) Taking 1kg of fresh Chinese wing beetle, crushing by using a high-pressure shearing machine, adding 10L deionized water, fully mixing to obtain homogenate, sterilizing the homogenate at 100 ℃ for 15min to obtain ground beetle slurry, cooling the ground beetle slurry to 40 ℃, adjusting the pH to 2.0 by using 0.1 mol/L HCl, then adding 10g of pepsin into the ground beetle slurry, carrying out enzymolysis for 60min on a constant-temperature enzymolysis instrument to obtain a first enzymolysis product, adding a2 wt% sodium hydroxide solution into the first enzymolysis product to adjust the pH of the first enzymolysis product to 9.0, adjusting the temperature of the first enzymolysis product to 40 ℃, then adding 10g of trypsin into the first enzymolysis product, carrying out enzymolysis for 180min on the constant-temperature enzymolysis instrument, then filtering, collecting filtrate, and concentrating the filtrate to a solid matter concentration of 50mg/ml to obtain a second enzymolysis product.

(2) And adding the second enzymolysis product into a DA201-C resin column, wherein the weight ratio of the DA201-C resin to the volume ratio of the second enzymolysis product is 10:1g/ml, and after adsorption, eluting the DA201-C resin column by adopting 50 vol% ethanol to obtain a first eluent.

(3) Performing molecular sieve chromatography on the first eluent by using a sephadex G25 column, wherein the filling amount of sephadex G25 in the sephadex G25 column is 1000G, the first eluent is added into the sephadex G25 column for multiple times, the adding amount is 0.5ml each time, and after adsorption, the sephadex G25 column is eluted by using deionized water to obtain a second eluent; and separating solid matters in the second eluent to obtain a product. The content of the polypeptide having the amino acid sequence shown in SEQ ID NO. 1 in the product was 69.23 wt%.