Polypeptide and application thereof

Abstract

The invention provides a polypeptide and application thereof. The polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 not only can remarkably remove ABTS free radicals and DPPH free radicals and play a role in resisting oxidation, but also can activate ADH enzyme and ALDH enzyme and play a role in promoting ethanol metabolism, so that the alcoholic liver disease is effectively resisted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceutical manufacturing. More specifically, it relates to a polypeptide and its application. BACKGROUND

[0002] Alcoholic liver disease (ALD) is a chronic liver injury caused by long-term excessive alcohol consumption, and is one of the important causes of liver disease and liver failure. There are many pathogenesis mechanisms of alcoholic liver disease, for example: (1) alcohol metabolite toxicity: alcohol (ethanol) is converted to acetaldehyde in the liver by alcohol dehydrogenase (ADH), and acetaldehyde, as the main toxic substance, will accumulate in the body if it is not converted into nontoxic acetic acid by acetaldehyde dehydrogenase (ALDH) in time, directly damaging the liver cell membrane, mitochondria and DNA, interfering with protein synthesis, and inducing inflammatory response; (2) oxidative stress and lipid peroxidation: alcohol metabolism consumes a large amount of antioxidant substances (such as glutathione), leading to accumulation of free radicals, triggering lipid peroxidation, and damaging the structure of liver cells.

[0003] Polypeptides are intermediate products of protein hydrolysis, and have been widely concerned in the field of biopharmaceutical manufacturing due to their various biological activities, rapid onset, and obvious advantages such as less adverse reactions and drug resistance. However, there are still few polypeptides that can resist alcoholic liver disease. SUMMARY

[0004] The present application aims to provide a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which can not only significantly scavenge ABTS free radicals and DPPH free radicals, and play an antioxidant role, but also activate ADH enzyme and ALDH enzyme, and play a role in promoting ethanol metabolism, thereby effectively resisting alcoholic liver disease.

[0005] The first object of the present application is to provide a polypeptide.

[0006] The second object of the present application is to provide the use of the above-mentioned polypeptide in the preparation of products for resisting alcoholic liver disease.

[0007] The third object of the present application is to provide related biological materials of the above-mentioned polypeptide.

[0008] The fourth object of the present application is to provide the use of the above-mentioned related biological materials in the preparation of products for resisting alcoholic liver disease.

[0009] The fifth object of the present application is to provide a product.

[0010] The above-mentioned objects of the present application are achieved by the following technical solutions: The present application provides a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which can not only significantly scavenge ABTS free radicals and DPPH free radicals, and play an antioxidant role, but also activate ADH enzyme and ALDH enzyme, and play a role in promoting ethanol metabolism, thereby effectively resisting alcoholic liver disease.

[0011] Based on this, the present application also provides an application of the polypeptide in the preparation of a product for resisting alcoholic liver disease, a related biological material of the polypeptide and an application of the related biological material in the preparation of a product for resisting alcoholic liver disease, and a product containing the polypeptide or the related biological material. The related biological material is a nucleic acid molecule capable of expressing the polypeptide, or a transgenic cell line, a genetically engineered bacterium, a recombinant vector or an expression construct containing the nucleic acid molecule.

[0012] Preferably, the alcoholic liver disease is promoting ethanol metabolism and / or antioxidant.

[0013] Further preferably, the promoting ethanol metabolism is activating ADH enzyme.

[0014] Further preferably, the promoting ethanol metabolism is activating ALDH enzyme.

[0015] Further preferably, the antioxidant is scavenging ABTS free radicals.

[0016] Further preferably, the antioxidant is scavenging DPPH free radicals.

[0017] Preferably, the product is a pharmaceutical product.

[0018] Preferably, the pharmaceutical product is one or more of oral liquid, spirit agent, tincture, aerosol, powder aerosol, injection, sterile powder for injection, suppository. The type of the above preparation can be understood according to the relevant definition in Pharmaceutical Chemistry (6th edition, People's Medical Publishing House, Cui Fude), and the preparation of the above preparation can be prepared according to the method of the relevant preparation in Pharmaceutical Chemistry (6th edition, People's Medical Publishing House, Cui Fude).

[0019] Further preferably, the pharmaceutical product further contains an excipient.

[0020] Further, the adjuvant is one or more of solid lubricants (such as stearic acid and / or magnesium stearate, etc. for reducing friction to ensure the success of drug manufacturing), vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and / or cocoa butter, etc. used as solvents or carriers to help drug ingredients better dissolve or disperse), polyols (such as propylene glycol, glycerol, sorbitol, mannitol and / or polyethylene glycol, etc. for improving the stability of the drug, preventing the drug from losing water or deteriorating during storage, and also for improving the taste and solubility of the drug), alginic acid (maintaining the physical stability of the drug to prevent the drug particles from settling or aggregating), emulsifiers (such as Tween and / or polyoxyethylene castor oil, etc. for reducing interfacial tension to facilitate the emulsification of the dispersed phase and maintain the stability of the emulsion), wetting agents (such as sodium lauryl sulfate, etc. for helping drug ingredients better disperse and dissolve), colorants (for improving the appearance of the drug color, making it easier to identify and distinguish), flavorings (for improving the taste of the drug, improving the patient's medication compliance), tabletting agents (helping to press loose granular material into solid tablets, making it easier for patients to take and carry, also helping to improve the stability and bioavailability of the drug), stabilizers (increasing the physical stability of the drug to prevent the drug from deteriorating or degrading during storage or transportation), antioxidants (for preventing the drug from deteriorating due to oxidation during storage or use, prolonging the shelf life of the drug), isotonic salt solutions (for adjusting the osmotic pressure of the drug to meet the physiological requirements of the human body, thereby reducing the irritation and adverse reactions to the human body), phosphate buffer (for adjusting the pH value of the drug to meet the physiological requirements of the human body, thereby reducing the irritation and adverse reactions to the human body), sugars (such as lactose, glucose and / or sucrose, etc. for helping drug ingredients better disperse and dissolve, while providing necessary energy support), starches (such as corn starch and / or potato starch, etc. for helping the tablet to disintegrate rapidly after taking and release the drug ingredients, thereby improving the bioavailability of the drug), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and / or methyl cellulose, etc. for improving the compressibility and formability of the drug, also for controlling the release rate of the drug), binding agents (such as gelatin, etc. for helping drug ingredients better bind together), lubricants (such as talc, etc. for reducing the friction of the drug). The type of adjuvant can be selected according to the needs of improving the stability, activity and / or bioavailability of the drug, etc.

[0021] The present application has the following beneficial effects: The polypeptide with the amino acid sequence shown in SEQ ID NO: 1 can not only significantly scavenge ABTS free radicals and DPPH free radicals to play an antioxidant role, but also activate ADH enzymes and ALDH enzymes to play a role in promoting ethanol metabolism, thereby effectively resisting alcoholic liver disease. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Mass spectrometry identification result of the polypeptide.

[0023] Figure 2 Purity analysis result of the polypeptide.

[0024] Figure 3 ABTS free radical scavenging rate result graph of the polypeptide.

[0025] Figure 4 DPPH free radical scavenging rate result graph of the polypeptide.

[0026] Figure 5 ADH activation rate result graph of the polypeptide.

[0027] Figure 6 ALDH enzyme activity result graph after the polypeptide acts. DETAILED DESCRIPTION

[0028] The present application is further illustrated in conjunction with the accompanying drawings and specific examples of the specification, but the examples do not make any form of limitation to the present application. Unless specifically stated, the reagents, methods and equipment used in the present application are the conventional reagents, methods and equipment in the technical field.

[0029] Unless specifically stated, the reagents and materials used in the following examples are commercially available.

[0030] Example 1 Synthesis and identification of the polypeptide In this example, the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 is synthesized by solid phase synthesis (assisted by Nanjing Peptide Valley Biotechnology Co., Ltd.), and the synthesized polypeptide is identified by mass spectrometry and analyzed for purity. Among them, SEQ ID NO: 1: PVPDP.

[0031] I. Mass spectrometry identification of the polypeptide ESI-MS mass spectrometry is used to identify the polypeptide, and whether it is the target product is determined according to the molecular weight on the mass spectrum and the theoretical value. Mass spectrometry conditions: mobile phase A: water (containing 0.1% (v / v) formic acid); mobile phase B: acetonitrile (containing 0.1% (v / v) formic acid); flow rate: 0.2 mL / min; running time: 1 min; positive ion mode; scan range: 0-2000 Da. v / v v / v

[0032] The mass spectrometry identification result of the polypeptide is shown in Figure 1 As can be seen from Figure 1 , the quasi-molecular ion peak (M+H)+ of the polypeptide of the present application is 524.46, with one charge, which is consistent with the theoretical value of its molecular weight.

[0033] II. Purity analysis of the polypeptide ​​The purity of the polypeptide was analyzed by high performance liquid chromatography; analysis conditions: column: Kromasil C18 (4.6*150 mm, 5 μm); mobile phase A: acetonitrile (containing 0.1% (TFA) trifluoroacetic acid); mobile phase B: water (containing 0.1% (TFA) trifluoroacetic acid); elution gradient: 0→0.01 min, 5% (A), 0.01→25 min, 50% (A), 25→30 min, 90% (A); flow rate: 1 mL / min; detection wavelength: 214 nm; sample loading amount: 20 μL. v / v v / v v / v v / v v / v

[0034] The purity analysis results of the polypeptide are shown in Table 1. Figure 2 As shown in Table 1, in the polypeptide of the present application, the main peak accounts for the main proportion, only a few impurity peaks appear, and the purity is as high as 98.28%. Figure 2

[0035] In summary, the polypeptide synthesized in this embodiment is the polypeptide with the amino acid sequence shown in SEQ ID NO: 1, and the purity meets the requirements of subsequent experiments.

[0036] Example 2: ABTS free radical scavenging test of polypeptide I. Solution preparation Preparation of potassium persulfate solution: 0.017 g of potassium persulfate (K2S2O8) solid was dissolved in 25 mL of ultrapure water to obtain the potassium persulfate solution.

[0037] Preparation of ABTS stock solution: 0.096 g of ABTS (2,2-amino diphenyl-1-benzyl hydrazine) solid was dissolved in 25 mL of ultrapure water to obtain the ABTS stock solution.

[0038] Preparation of ABTS free radical working solution: the ABTS stock solution and the potassium persulfate solution were mixed in a volume ratio of 1:1, and the system was placed in the dark until it turned blue-green to obtain the ABTS free radical working solution.

[0039] Preparation of polypeptide solution: the polypeptide obtained in Example 1 was dissolved in ultrapure water to obtain polypeptide solutions with concentrations of 0.005, 0.01, 0.1, 0.5 and 1 mg / mL, respectively, which were stored at -20°C for standby use.

[0040] II. Determination method Blank group: in a 96-well plate, 150 μL of ABTS free radical working solution was mixed with 50 μL of ultrapure water, and then incubated in the dark for 40 min. The absorbance A0 was measured in an enzyme marker with a wavelength of 734 nm.

[0041] ​​​​​​Control group: in 96-well plates, 150 μL ultrapure water was mixed with 50 μL polypeptide solution, and the absorbance A1 was measured in an enzyme marker at a wavelength of 734 nm after incubation in the dark for 40 min.

[0042] Sample group: in 96-well plates, 150 μL ABTS free radical working solution was mixed with 50 μL polypeptide solution, and the absorbance A2 was measured in an enzyme marker at a wavelength of 734 nm after incubation in the dark for 40 min.

[0043] Four parallel samples were set in each group, and the ABTS free radical scavenging rate of the polypeptide solution was calculated according to the formula "ABTS free radical scavenging rate (%) = [1- (A2-A1) / A0] x 100%".

[0044] III. Determination results As shown in the results Figure 3 , the ABTS free radical scavenging rate of the polypeptide solution of 0.005-1 mg / mL reached (49.7%±2.9%)-(90.8%±6.6%), indicating that the polypeptide of the present application can significantly scavenge ABTS free radicals, play an antioxidant role, and effectively resist alcoholic liver disease, and is very suitable for preparing drugs for resisting alcoholic liver disease.

[0045] Example 3: DPPH free radical scavenging test of polypeptide I. Solution preparation Preparation of DPPH solution: 4 mg of DPPH (2,2-diphenyl-1-picrylhydrazyl) solid was dissolved in 100 mL of anhydrous ethanol to obtain a DPPH solution, which was stored at 4°C in the dark for standby use.

[0046] Preparation of polypeptide solution: the polypeptide obtained in Example 1 was dissolved in ultrapure water to obtain polypeptide solutions of 0.005, 0.01, 0.1, 0.5 and 1 mg / mL, respectively, which were stored at -20°C for standby use.

[0047] II. Determination method Blank group: in 96-well plates, 150 μL DPPH solution was mixed with 50 μL ultrapure water, and the absorbance A0 was measured in an enzyme marker at a wavelength of 517 nm after incubation in the dark for 40 min.

[0048] Control group: in 96-well plates, 150 μL anhydrous ethanol was mixed with 50 μL polypeptide solution, and the absorbance A1 was measured in an enzyme marker at a wavelength of 517 nm after incubation in the dark for 40 min.

[0049] Sample group: in 96-well plates, 150 μL DPPH solution was mixed with 50 μL polypeptide solution, and the absorbance A2 was measured in an enzyme marker at a wavelength of 517 nm after incubation in the dark for 40 min.

[0050] Each group is set four parallel samples, and the DPPH free radical scavenging rate of the polypeptide solution is calculated according to the formula "DPPH free radical scavenging rate (%) = [1- (A2-A1) / A0] x 100%".

[0051] III. Determination results The results are shown in Figure 4 As shown, the DPPH free radical scavenging rate of the polypeptide solution of 0.005-1 mg / mL reaches (10.0%±1.6%)-(50.9%±3.5%), indicating that the polypeptide of the present application can significantly scavenge DPPH free radicals, play an antioxidant role, and effectively resist alcoholic liver disease, and is very suitable for preparing drugs for resisting alcoholic liver disease.

[0052] Example 4: ADH enzyme activation test of polypeptide I. Solution preparation ADH enzyme solution was prepared: ADH enzyme (360 U / mg) was dissolved in ultrapure water to obtain an ADH enzyme solution of 0.2 U / mL, which was stored at -20 ℃ for standby.

[0053] ADH enzyme working solution was prepared: it was prepared according to the instructions of the ethanol dehydrogenase (ADH) kit.

[0054] Polypeptide solution was prepared: the polypeptide obtained in Example 1 was dissolved in ultrapure water to obtain polypeptide solutions of 0.01, 0.1, 1, 2 and 4 mg / mL, respectively, which were stored at -20 ℃ for standby.

[0055] II. Determination method Control group: in a 96-well plate, 150 μL of ADH enzyme working solution was mixed with 50 μL of ultrapure water, incubated at 37 ℃ for 5 min, then 50 μL of ADH enzyme solution was added for reaction, and the absorbance was immediately determined in a VIRIOSKAN LUX enzyme marker at a wavelength of 340 nm, scanned once every 15 seconds, for a duration of 10 min, a total of 41 times. The first derivative of the curve at 0 min was obtained by fitting the reaction kinetics curve, and the initial reaction rate V0 was obtained.

[0056] Sample group: in a 96-well plate, 150 μL of ADH enzyme working solution was mixed with 50 μL of polypeptide solution, incubated at 37 ℃ for 5 min, then 50 μL of ADH enzyme solution was added for reaction, and the absorbance was immediately determined in a VIRIOSKAN LUX enzyme marker at a wavelength of 340 nm, scanned once every 15 seconds, for a duration of 10 min, a total of 41 times. The first derivative of the curve at 0 min was obtained by fitting the reaction kinetics curve, and the initial reaction rate V1 was obtained.

[0057] The ADH activation rate of the polypeptide solution was calculated according to the formula "ADH activation rate (%) = [(V1-V0) / V0]x100%".

[0058] III. Determination results The results, as shown in Table 1, show that the ADH activation rate of the polypeptide solution at 0.01-4 mg / mL reaches (8.9±0.3)%-(41.7±1.3)%, indicating that the polypeptide of the present application can effectively activate ADH enzyme, play a role in promoting the metabolism of ethanol into acetaldehyde, and further effectively resist alcoholic liver disease, and is very suitable for preparing a drug for resisting alcoholic liver disease. Figure 5 Example 5: ALDH enzyme activation test of the polypeptide

[0059] I. Solution preparation Preparation of ALDH enzyme solution: ALDH enzyme (15 U / mg) was dissolved in ultrapure water to obtain an ALDH enzyme solution of 0.2 U / mL, which was stored at -20°C for standby. Preparation of ALDH enzyme inactivation solution: ALDH enzyme (15 U / mg) was dissolved in ultrapure water to obtain an ALDH enzyme solution of 0.2 U / mL, which was inactivated at 100°C for 5 min and then stored at -20°C for standby.

[0060] Preparation of ALDH enzyme working solution: prepared according to the instructions of the acetaldehyde dehydrogenase (ALDH) kit.

[0061] Preparation of polypeptide solution: the polypeptide obtained in Example 1 was dissolved in ultrapure water to obtain polypeptide solutions of 0, 0.01, 2 and 4 mg / mL, respectively, which were stored at -20°C for standby.

[0062] II. Preparation of standard curve

[0063] The acetaldehyde dehydrogenase kit contains reagents: standard (powder, 1 bottle), reagent 2 (2.5 mL, 1 bottle), reagent 3 (70 mL, 1 bottle). Add 1.41 mL distilled water to the standard to dissolve to obtain a standard solution with a concentration of 1 nmol / μL, and then dilute it with distilled water into 6 concentration gradients of standard solution (0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol / μL). Take 20 μL of each gradient concentration, and then mix 6.5 μL of reagent 2 and 174 μL of reagent 3, incubate at 37°C for 5 min, and then read the absorbance at a wavelength of 450 nm. The standard concentration is taken as the abscissa x, and the absorbance is taken as the ordinate y, to prepare the standard curve y=ax+b. III. Determination method

[0064] ​Control group: In a 96-well plate, 180 μL of ALDH enzyme working solution was mixed with 10 μL of peptide solution, and then 10 μL of ALDH enzyme inactivation solution was added and mixed. After 30 s, the absorbance A1 was measured in a VIRIOSKAN LUX microplate reader at a wavelength of 450 nm. After 30 min, the absorbance A2 was measured again. The result was ΔA0 = A2 - A1.

[0065] Sample group: In a 96-well plate, 180 μL of ALDH enzyme working solution was mixed with 10 μL of peptide solution, and then 10 μL of ALDH enzyme solution was added and mixed. After 30 s, the absorbance A3 was measured in a VIRIOSKAN LUX microplate reader at a wavelength of 450 nm. After 30 min, the absorbance A4 was measured again. The result was ΔA1 = A4 - A3.

[0066] The ALDH enzyme activity after the polypeptide solution was reacted was calculated according to the formula “ALDH enzyme activity (nmol / min / mL) = (△Ab) / avt”, where △A = △A1 - △A0, v is the system volume (200 μL) and t is the reaction time (30 min).

[0067] IV. Measurement Results The results are as follows Figure 6 As shown, the ALDH enzyme activity reached (5.3±0.1) to (8.4±0.9) nmol / min / mL after treatment with a polypeptide solution of 0.01–4 mg / mL. This indicates that the polypeptide of the present invention can effectively activate ALDH enzyme, promote the conversion of acetaldehyde, a metabolite of ethanol, into acetic acid, and thus effectively combat alcoholic liver disease, making it highly suitable for the preparation of drugs for treating alcoholic liver disease.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The use of the polypeptide of claim 1 in the preparation of products for treating alcoholic liver disease.

3. The biomaterials related to the polypeptide of claim 1, characterized in that, The relevant biological material is a nucleic acid molecule capable of expressing the polypeptide, or a transgenic cell line, genetically engineered bacteria, recombinant vector, or expression construct containing the nucleic acid molecule.

4. The use of the biomaterial described in claim 3 in the preparation of products for treating alcoholic liver disease.

5. The application according to claim 2 or 4, characterized in that, The aforementioned anti-alcoholic liver disease refers to the promotion of ethanol metabolism and / or antioxidant effects.

6. The application according to claim 5, characterized in that, The process of promoting ethanol metabolism involves activating the ADH enzyme.

7. The application according to claim 5, characterized in that, The process of promoting ethanol metabolism involves activating the ALDH enzyme.

8. The application according to claim 5, characterized in that, The antioxidant is used to scavenge ABTS free radicals.

9. The application according to claim 5, characterized in that, The antioxidant is the scavenging of DPPH free radicals.

10. A product characterized in that, It comprises the polypeptide of claim 1 or the related biomaterial of claim 3.

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