CN109369783B - Polypeptide RDP1, and purification method and application thereof - Google Patents
Polypeptide RDP1, and purification method and application thereof Download PDFInfo
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- CN109369783B CN109369783B CN201811456981.5A CN201811456981A CN109369783B CN 109369783 B CN109369783 B CN 109369783B CN 201811456981 A CN201811456981 A CN 201811456981A CN 109369783 B CN109369783 B CN 109369783B
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- rdp1
- polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses an anti-hyperuricemia polypeptide RDP1, wherein the amino acid sequence of the polypeptide RDP1 is shown as SEQ No. 1. The invention also discloses a purification method and application of the polypeptide RDP 1. The anti-hyperuricemia peptide RDP1 can relieve hyperuricemia and relieve kidney injury, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to polypeptide RDP1, and a purification method and application thereof.
Background
According to the 'white paper of China gout status report' in 2017, Chinese gout patients already exceed 8000 thousands of people, and gout becomes the second largest metabolic disease second to diabetes in China. Gout develops due to accumulation and crystallization of uric acid in blood in joints or soft tissues, and its prophase symptom is hyperuricemia. Hyperuricemia is generally caused by overproduction or underproduction of uric acid, and xanthine, which is a key enzyme in uric acid production, is most commonly used as a drug target for anti-hyperuricemia. The anti-hyperuricemia is a main target of non-acute anti-gout, but the clinically existing anti-hyperuricemia drugs have certain defects, including low activity, high preparation cost, large side effect, difficult reversion and the like. Therefore, it is urgently needed to search or develop a novel anti-hyperuricemia drug with less adverse reactions, economy and practicality.
Substances reported to have anti-hyperuricemic activity are mainly small molecule compounds, but most are difficult to store and produce. Over the last decades, many studies have shown that: short peptides containing 5-10 amino acids are easier to absorb and often easier to detect specific activity. The researchers found that: short peptides generally have high activity, high stability and specificity, and are also more economical and easy to mass-produce; therefore, short peptides have a wide range of application values, which has attracted a high degree of attention from researchers all over the world. Some polypeptide drugs have been widely used clinically, such as exenatide, insulin, etc. Meanwhile, a large number of biological peptides with other activities, such as antibacterial peptides, analgesic peptides and the like, are found, but the research and discovery of polypeptides with anti-hyperuricemia activity are still in the initial stage.
Therefore, we have developed an anti-hyperuricemia active polypeptide which can effectively relieve hyperuricemia and has high stability and kidney protection activity.
Disclosure of Invention
The first purpose of the invention is to provide a polypeptide RDP1, wherein the polypeptide RDP1 comprises an amino acid sequence shown as SEQ No. 1.
The second purpose of the invention is to provide a method for purifying the polypeptide RDP1, which is characterized by comprising the following steps:
(1) soaking rice in deionized water overnight, vacuum freeze drying the filtered supernatant to obtain rice extract lyophilized powder, and storing at-80 deg.C;
(2) dissolving the rice extract freeze-dried powder in deionized water, and performing chromatographic separation by using Sephadex G50 column;
(3) performing reverse phase chromatography on the separation product obtained in the step (2) by using high performance liquid chromatography for the first time, and collecting active components;
(4) and carrying out reverse phase chromatography on the active component by using high performance liquid chromatography for the second time to obtain purified polypeptide RDP 1.
The third purpose of the invention is to provide an application of the polypeptide RDP1 in resisting hyperuricemia.
The fourth purpose of the invention is to provide a medicament containing the polypeptide RDP 1.
Drawings
FIG. 1 is a Sephadex G50 molecular sieve diagram of an anti-hyperuricemia active polypeptide RDP1 of the invention;
in the figure, the arrow indicates the absorption peak of RDP 1.
FIG. 2 is a HPLC reversed phase C18 column chromatography chart of the anti-hyperuricemia active polypeptide RDP1 of the invention.
In the figure, the arrow indicates the absorption peak of RDP 1.
FIG. 3 is a 2 nd HPLC reversed phase C18 column chromatography chart of anti-hyperuricemia active polypeptide RDP1 of the invention.
In the figure, the arrow indicates the absorption peak of RDP 1.
FIG. 4 is a mass spectrum of the anti-hyperuricemia active polypeptide RDP 1.
FIG. 5 is a primary structural diagram of the anti-hyperuricemia active polypeptide RDP1 of the present invention.
FIG. 6 is a diagram showing the uric acid lowering results of the anti-hyperuricemia active polypeptide RDP1 of the present invention. In the figure, denotesP<0.001（tTest).
FIG. 7 is a graph showing the results of the creatinine reduction using the anti-hyperuricemia active polypeptide RDP1 of the present invention. In the figure, denotesP<0.05, representsP<0.01, representsP<0.001（tTest).
FIG. 8 is a graph showing the results of HE staining of the anti-hyperuricemia active polypeptide RDP1 of the present invention.
FIG. 9 shows the results of in vitro inhibition of xanthine oxidase activity by the anti-hyperuricemia active polypeptide RDP1 of the present invention. In the figure, denotesP<0.01, representsP<0.001（tTest).
FIG. 10 shows the anti-hyperuricemia effect of the present inventionResults of in vivo inhibition of xanthine oxidase activity in rat serum after administration of the active polypeptide RDP 1. In the figure, denotesP<0.05, representsP<0.01（tTest).
Detailed Description
The invention is further described with reference to the accompanying drawings, which are not intended to be limiting in any way, and any alterations and modifications based on the teachings of the invention are intended to be included within the scope of the invention.
The polypeptide RDP1 is derived from rice extract, and the amino acid sequence of the polypeptide RDP1 is shown as SEQ No. 1.
The invention also provides a purification method for purifying the polypeptide RDP1 from a rice extract, which comprises the following steps.
(1) Soaking rice in deionized water overnight, vacuum freeze drying the filtered supernatant to obtain rice extract lyophilized powder, and storing at-80 deg.C.
(2) Dissolving the rice extract freeze-dried powder in deionized water, and performing chromatographic separation by using a Sephadex G50 column, specifically dissolving the rice extract freeze-dried powder in deionized water, taking 1mL of Sephadex G50 column (the length is 40cm, the inner diameter width is 1.5 cm) which is balanced by 20mmol/L Tris-HCl buffer solution (the pH is =7.8 and contains 0.1mol/L NaCl) in advance, eluting by using the same buffer solution, wherein the flow rate is 3mL/10min, and collecting for 1 time every 10 min.
(3) The isolated product obtained in step (2) was previously eluted with acetonitrile (containing 0.1% trifluoroacetic acid) under a linear gradient (0-100%, 100 min) using Hypersil ODS 25 mm column equilibrated with ultrapure water (containing 0.1% trifluoroacetic acid) in a Waters 1525 high pressure liquid phase system at a flow rate of 1mL/min, and the active product was collected at a monitoring wavelength of 220 nm.
(4) And (4) dissolving the active product obtained in the step (3) in deionized water after vacuum freeze drying, and then repeating the process in the step (3) to obtain the purified RDP1 polypeptide.
Besides being obtained from rice extract, the polypeptide RDP1 of the invention can also be obtained by artificial synthesis. There are many methods for preparing bioactive peptides, including protected chemical synthesis, hydrolysis, recombinant DNA technology, etc., which are suitable for preparing the polypeptide RDP1 of the present invention.
The polypeptide RDP1 disclosed by the invention is simple in structure and has activity of resisting hyperuricemia, the purified active polypeptide RDP1 shows stronger activity of resisting hyperuricemia in an animal model experiment of hyperuricemia induced by potassium oxonate, and the application prospect of the polypeptide RDP1 disclosed by the invention in the aspect of resisting hyperuricemia is huge.
The present invention will be further described with reference to specific examples.
Example 1: the anti-hyperuricemia active polypeptide RDP1 is separated, purified and identified.
1. And (5) separating and purifying.
Soaking rice in deionized water overnight, filtering, vacuum freeze drying the supernatant, and storing at-80 deg. The obtained rice extract lyophilized powder was dissolved in deionized water, and 1mL of Sephadex G50 (GE Healthcare, ultra-fine) column (length 40cm, inner diameter width 1.5 cm) which had been equilibrated in advance with 20mmol/L Tris-HCl buffer (pH 7.8, containing 0.1mol/L NaCl) for 24 hours was eluted with the same buffer at a flow rate of 3mL/10min and collected 1 time per 10 min. And detecting the absorbance of each tube, wherein the detection wavelength is 280 nm, and combining the samples under the absorption peak indicated by an arrow in the figure 1.
The obtained sample was loaded on a Hypersil ODS 25 mm column (Erilter product, size 4.6 mm × 300 mm) equilibrated in advance with ultrapure water (containing 0.1% trifluoroacetic acid), the experimental apparatus was a Waters 1525 high pressure liquid phase system, elution was performed with acetonitrile (containing 0.1% trifluoroacetic acid) under a linear gradient (0-100%, 100 min) at a flow rate of 1mL/min, the monitored wavelength was 220nm, and the obtained separation and purification profile was as shown in FIG. 2: the arrow indicates the peak RDP 1. The peaks indicated by the arrows in FIG. 2 were collected, lyophilized in vacuo and dissolved in deionized water, and the 1 st HPLC procedure was repeated, and the obtained separation and purification pattern is shown in FIG. 3, and the arrows indicate the peaks of purified RDP 1.
2. And (4) determining the amino acid sequence.
The molecular weight of the sample was determined by mass spectrometry and found to be 785.91 Da (FIG. 4). mu.L of the sample was mixed with 1. mu.L of α -cyano-4-hydroxyoctanoic acid (5 mg/mL in 50% ACN, 0.1% TFA) and spotted on a sample plate. The crystallized samples were analyzed on an AutoFlex Speed MALDI TOF/TOF mass spectrometer.
To determine the amino acid sequence, the samples were dissolved in 25 mmol/L NH4HCO3In (1), reducing with dithiothreitol at 37 ℃ for 1h, blocking with iodoacetamide for 30min, then mixing the sample with alpha-cyano-4-hydroxyoctanoic acid, and performing tandem mass spectrometry on the same equipment.
The results are shown in FIG. 5: the amino acid sequence of the active polypeptide RDP1 for resisting hyperuricemia is AAAAGAKAR, which is shown as SEQ No. 1.
Example 2: determination of the anti-hyperuricemic Activity of polypeptide RDP 1.
1. Serum uric acid and creatinine content determination.
The anti-hyperuricemia activity of the polypeptide RDP1 is detected by using a hyperuricemia animal model induced by potassium oxonate. SPF-grade SD (Spodoptera frugiperda) big white mice weighing 100-150 g are selected for experiment, the rats are randomly divided into 6 groups, each group comprises 5 rats, and the 6 groups are blank control, negative control, positive control and 3 RDP1 treatment groups (10, 100 and 1000 mug/kg). The blank control is administrated with equal amount of normal saline by intragastric administration every day, and after the other groups are administrated with potassium oxonate (450 mg/kg) and adenine (100 mg/kg) by intragastric administration every day for 1h, the administration by intragastric administration is respectively carried out: the negative control group was given with the same amount of physiological saline, the positive control group was given with 10 mg/kg allopurinol, and the 3 RDP1 treatment groups were given with 10, 100, 1000 mug/kg RDP1, respectively, for a total of 7 days. 1h after the end of the day 7 dosing (saline, allopurinol, RDP 1), rats were anesthetized with 1% sodium pentobarbital (0.3 mL/100 g) by intraperitoneal injection, and then whole blood was taken from the femoral artery of the rats. The whole blood was centrifuged at 6000 r/min at room temperature for 5 min to obtain serum. The kit is used for detecting the content of uric acid and creatinine in partial rat serum, and the results are shown in fig. 6 and 7: the serum uric acid and creatinine contents of the positive control group and the RDP 1-treated group are obviously lower than those of the negative control group. The remaining serum was used for in vivo assay of xanthine oxidase inhibitory activity.
After blood is taken, the rat kidney is taken and fixed in 4% formaldehyde for 24-48 h, tissues are dehydrated through gradient ethanol, and dimethylbenzene is transparent. And (5) slicing by using a paraffin embedding method to 5 mu m, and carrying out HE staining. The results were observed by means of an upright microscope at a magnification of 200. The nephroprotective activity of polypeptide RDP1 is shown in figure 8: the kidney of the rat in the negative control group (model group) can see disappearance of brush border and renal tubular atrophy compared with the kidney of the blank group; significant reduction in renal pathology was observed in the positive control and RDP1 compared to the negative control.
2. And measuring the xanthine oxidase inhibition rate.
The test is divided into a negative control group, a positive control group and 3 RDP1 treatment groups, and the in vivo and in vitro xanthine oxidase activity inhibition assay is respectively carried out; at the same time, 50 mmol/L Tris-HCl pH 8 buffer solution is prepared for standby.
(1) In vitro experiments.
Negative control: 128 mU L2 mmol/L xanthine oxidase solution +16 mU L0.52 mU/mL xanthine oxidase solution +928 mU L buffer solution +32 mU L buffer solution.
Positive control: 128 μ L of 2 mmol/L xanthine solution +16 μ L of 0.52 mU/mL xanthine oxidase solution +928 μ L buffer solution +32 μ L of 1 mg/mL allopurinol solution.
3 RDP1 treatment groups: 128 mU L2 mmol/L xanthine oxidase solution +16 mU L0.52 mU/mL xanthine oxidase solution +928 mU L buffer solution +32 mU L1/10/1000 mU g/kg RDP1 solution.
(2) In vivo experiments.
Negative control: negative control group rat serum of 128 muL 2 mmol/L xanthine solution +16 muL 0.52 mU/mL xanthine oxidase solution +928 muL buffer solution +32 muL.
Positive control: serum of a positive control group rat of 128 muL 2 mmol/L xanthine solution +16 muL 0.52 mU/mL xanthine oxidase solution +928 muL buffer solution +32 muL 1 mg/mL.
3 RDP1 treatment groups: 928 μ L buffer +32 μ L1/10/1000 μ g/kg RDP1 treated group rat sera.
Each test group was incubated at 37 ℃ for 15 min, the reaction was stopped by adding 48 μ L of 1mol/L HCl, and the absorbance was measured at 292 nm. The inhibitory activity was calculated as follows:
xanthine oxidase inhibition (%) = (negative control OD) 292nmValue ‒ Positive control or treatment OD 292nmValue)/negative control OD 292nmValue X100%.
The polypeptide RDP1 can inhibit the xanthine oxidase activity in vitro as shown in FIG. 9: the positive control shows the highest xanthine oxidase inhibition rate, and the subsequent treatment group with RDP1 shows higher xanthine oxidase inhibition rate than the negative control.
In vivo inhibition of xanthine oxidase activity is shown in figure 10: the inhibition rate of xanthine oxidase in a 1000 mug/kg RDP1 group in serum is the highest, and then the inhibition rate is 100 mug/kg RDP1 group, a positive control group and a 10 mug/kg RDP1 group.
In conclusion, the anti-hyperuricemia active polypeptide RDP1 disclosed by the invention has the beneficial characteristics of simple structure, high activity, simple administration mode and the like.
SEQUENCE LISTING
<110> university of Kunming medical science
<120> polypeptide RDP1, and purification method and application thereof
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> Rice
<400> 1
Ala Ala Ala Ala Gly Ala Lys Ala Arg
1 5
Claims (4)
1. A polypeptide RDP1, wherein the amino acid sequence of the polypeptide RDP1 is shown as SEQ No. 1.
2. A method for purifying the polypeptide RDP1 of claim 1, comprising the steps of:
(1) soaking rice in deionized water overnight, vacuum freeze drying the filtered supernatant to obtain rice extract lyophilized powder, and storing at-80 deg.C;
(2) dissolving the rice extract freeze-dried powder in deionized water, and performing chromatographic separation by using Sephadex G50 column; dissolving the rice extract freeze-dried powder in deionized water, taking 1mL Sephadex G50 column which is balanced by 20mmol/L Tris-HCl buffer solution for 24h in advance, eluting by the same buffer solution, wherein the flow rate is 3mL/10min, and collecting 1 time every 10 min; the Tris-HCl buffer solution has pH of 7.8 and contains 0.1mol/L NaCl; the Sephadex G50 column has a length of 40cm and an inner diameter of 1.5 cm;
(3) performing reverse phase chromatography on the separation product obtained in the step (2) by using high performance liquid chromatography for the first time, and collecting active components; specifically, the separation product obtained in the step (2) is loaded on a Hypersil ODS 25 mm column which is pre-balanced by ultrapure water containing 0.1% trifluoroacetic acid, an experimental apparatus is a Waters 1525 high-pressure liquid phase system, under the condition of the flow rate of 1mL/min, acetonitrile containing 0.1% trifluoroacetic acid is used for elution under the condition of linear gradient, active components are collected, and the monitoring wavelength is 220 nm; the linear gradient is 0-100% for 100 min;
(4) and (3) performing reverse phase chromatography on the active component by using high performance liquid chromatography for the second time, specifically, dissolving the active component obtained in the step (3) in deionized water after vacuum freeze drying, and then repeating the step (3) to obtain the purified polypeptide RDP 1.
3. The use of the polypeptide RDP1 of claim 1 in the preparation of a medicament for treating hyperuricemia.
4. An anti-hyperuricemia drug comprising the polypeptide RDP1 of claim 1.
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| CN201811456981.5A CN109369783B (en) | 2018-11-30 | 2018-11-30 | Polypeptide RDP1, and purification method and application thereof |
| PCT/CN2019/122077 WO2020108629A1 (en) | 2018-11-30 | 2019-11-29 | Polypeptide rdp1 and purification method and use therefor |
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| CN109369783B (en) * | 2018-11-30 | 2021-09-10 | 昆明医科大学 | Polypeptide RDP1, and purification method and application thereof |
| CN111606973B (en) * | 2020-05-21 | 2022-03-18 | 昆明医科大学 | Anti-gout active polypeptide RDP3, and preparation method and application thereof |
| CN111533783B (en) * | 2020-05-21 | 2022-03-18 | 昆明医科大学 | Anti-gout active polypeptide RDP2, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103058944A (en) * | 2007-11-27 | 2013-04-24 | 亚德生化公司 | Novel compound and composition |
| WO2020108629A1 (en) * | 2018-11-30 | 2020-06-04 | 西安棣加生物科技有限公司 | Polypeptide rdp1 and purification method and use therefor |
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| US7214786B2 (en) * | 2000-12-14 | 2007-05-08 | Kovalic David K | Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103058944A (en) * | 2007-11-27 | 2013-04-24 | 亚德生化公司 | Novel compound and composition |
| WO2020108629A1 (en) * | 2018-11-30 | 2020-06-04 | 西安棣加生物科技有限公司 | Polypeptide rdp1 and purification method and use therefor |
Non-Patent Citations (3)
| Title |
|---|
| A new rice-derived short peptide potently alleviated hyperuricemia induced by potassium oxonate in rats;Liu Naixin等;《Journal of Agricultural and Food Chemistry》;20181218;全文 * |
| CLIP-associating protein 2 isoform X13 [Pelodiscus sinensis],NCBI Reference Sequence: XP_006121801.1;无;《NCBI》;20180604;全文 * |
| 降尿酸肽对高尿酸血症的作用及研究进展;骆贤亮 等;《食品科学》;20200417;全文 * |
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| CN109369783A (en) | 2019-02-22 |
| WO2020108629A8 (en) | 2020-07-30 |
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