Anti-gout active polypeptide RDP3, and preparation method and application thereof
阅读说明：本技术 一种抗痛风活性多肽rdp3及其制备方法与应用 (Anti-gout active polypeptide RDP3, and preparation method and application thereof ) 是由 杨新旺 王滢 孙俊 刘乃心 唐璟 于 2020-05-21 设计创作，主要内容包括：本发明公开了一种抗痛风活性多肽RDP3及其制备方法与应用。所述的抗痛风活性多肽RDP3的氨基酸序列为AAAAMAGPK-NH2。本发明纯化得到的抗痛风活性多肽RDP3在氧嗪酸钾诱发的高尿酸血症动物模型实验中显示了较强的降尿酸活性,在单钠尿酸盐晶体诱发的痛风发作动物模型中表现出了较好的缓解能力,揭示本发明抗痛风活性多肽RDP3有潜在的应用前景。本发明所述的抗痛风活性多肽RDP3具有结构简单、抗痛风活性强的有益特点。(The invention discloses an anti-gout active polypeptide RDP3, and a preparation method and application thereof. The amino acid sequence of the anti-gout active polypeptide RDP3 is AAAAMAGPK-NH 2. The purified anti-gout active polypeptide RDP3 shows stronger uric acid reduction activity in hyperuricemia animal model experiments induced by potassium oxonate, shows better relieving capability in an animal model of gout attack induced by mono-sodiuurate crystals, and reveals that the anti-gout active polypeptide RDP3 has potential application prospect. The anti-gout active polypeptide RDP3 has the beneficial characteristics of simple structure and strong anti-gout activity.)
1. An anti-gout active polypeptide RDP3, which is characterized in that the amino acid sequence of the anti-gout active polypeptide RDP3 is AAAAMAGPK-NH 2.
2. A method for preparing the anti-gout active polypeptide RDP3 of claim 1, which is characterized by comprising the following steps:
A. soaking rice in deionized water overnight, and vacuum freeze-drying the filtered supernatant to obtain a material a, and storing at-80 deg.C;
B. dissolving the material a in water to obtain a material b, eluting the material b with a Tris-HCl buffer solution, and collecting an eluent to obtain a material c;
C. performing high performance liquid chromatography reverse phase chromatography on the material c for the first time to obtain a material d;
D. and (5) performing reverse phase chromatography on the material d by using high performance liquid chromatography for the second time to obtain the target anti-gout active polypeptide RDP 3.
3. The method according to claim 2, wherein the water in step B is deionized water.
4. The method according to claim 2, wherein the elution in step B is performed by eluting the material B on a Sephadex G50 column previously equilibrated with 20 mmol/L Tris-HCl buffer solution for 24 hours, followed by elution with Tris-HCl buffer solution.
5. The method according to claim 4, wherein the flow rate of elution is 3 ml/10 min.
6. The method according to claim 2 or 4, wherein the Tris-HCl buffer has a pH of 7.8 and contains 0.1 mol/L NaCl.
7. The method according to claim 2, wherein the first reversed-phase HPLC chromatography is performed by loading the material c on a Hypersil ODS25mm column equilibrated in advance with ultrapure water containing 0.1% trifluoroacetic acid, eluting with acetonitrile containing 0.1% trifluoroacetic acid under a linear gradient at a flow rate of 0.5 to 1.5ml/min, and detecting the wavelength at 220 nm.
8. The method according to claim 2, wherein the second reversed-phase HPLC chromatography is performed by vacuum freeze-drying the material d, dissolving in deionized water, and repeating the first reversed-phase HPLC chromatography.
9. The application of the anti-gout active polypeptide RDP3 of claim 1, wherein the anti-gout active polypeptide RDP3 is used for preparing medicines for preventing/treating gout.
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-gout active polypeptide RDP3, and a preparation method and application thereof.
Background
With the economic development and the change of the dietary structure of Chinese people, gout has gradually evolved from western frequently-occurring gout to global frequently-occurring gout. The Chinese gout report in 2017 shows that the Chinese gout patients are more than 8000 thousands of people, and the gout is the second most metabolic disease next 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. Gout not only can induce severe pain in the acute attack stage of gout, which affects the quality of life of people and causes the reduction of joint function; and in its occult, i.e. non-onset, stage, the sustained high uric acid also increases the risk of cardiovascular disease and even diabetes.
Uric acid is the end product of purine metabolism in humans, while hyperuricemia is generally caused by either overproduction or underexcretion of uric acid. Clinically, drugs for controlling uric acid mainly start from two aspects, namely, reducing uric acid production by inhibiting xanthine oxidase, a key enzyme in uric acid production, and increasing uric acid excretion by inhibiting uric acid reabsorption protein, namely, urate transporter (URAT 1). However, the existing anti-gout drugs in clinic are basically developed from one side, or the uric acid level is controlled in the hiding period, or the inflammation is inhibited in the attack period, and the development of the overall anti-gout drugs with double functions is still blank. Therefore, it is urgently needed to search or develop a novel anti-gout drug with less adverse reaction and economical and practical use.
Therefore, an anti-gout active polypeptide is developed, which can effectively relieve gout attack and has higher activity of reducing uric acid.
Disclosure of Invention
The invention has the first aim of providing an anti-gout
The first object of the invention is realized by that the amino acid sequence of the anti-gout active polypeptide RDP3 is AAAAMAGPK-NH 2.
The second object of the present invention is achieved by comprising the steps of:
A. soaking rice in deionized water overnight, and vacuum freeze-drying the filtered supernatant to obtain a material a, and storing at-80 deg.C;
B. dissolving the material a in water to obtain a material b, eluting the material b with a Tris-HCl buffer solution, and collecting an eluent to obtain a material c;
C. performing high performance liquid chromatography reverse phase chromatography on the material c for the first time to obtain a material d;
D. and (5) performing reverse phase chromatography on the material d by using high performance liquid chromatography for the second time to obtain the target anti-gout
The third purpose of the invention is realized by the application of the anti-gout active polypeptide RDP3 in preparing medicines for preventing/treating gout.
The preparation method of the anti-gout active polypeptide RDP3 provided by the invention can be obtained from a rice extract by a biochemical separation and purification method. The anti-gout active polypeptide RDP3 prepared by the method can be identified by separation and purification and anti-gout activity.
The preparation of the anti-gout active polypeptide RDP3 can also be obtained by a conventional method in the field by adopting a chemical synthesis or gene expression mode, and the amino acid sequence is the same as the SEQ ID NO. 1.
The purified anti-gout active polypeptide RDP3 shows stronger uric acid reduction activity in hyperuricemia animal model experiments induced by potassium oxonate, and shows that the anti-gout active polypeptide RDP3 has potential application prospect except better relieving capacity in a gout attack animal model induced by mono-natriuretic urate crystals. The anti-gout active polypeptide RDP3 has the beneficial characteristics of simple structure and strong anti-gout activity.
Drawings
FIG. 1 is a Sephadex G50 molecular sieve diagram of anti-gout polypeptide RDP3 of the invention; in the figure, the arrow indicates the absorption peak of
FIG. 2 is a HPLC reversed phase C18 column chromatography chart of the anti-gout active polypeptide RDP3 of the invention; in the figure, the arrow indicates the absorption peak of
FIG. 3 is a 2 nd HPLC reversed phase C18 column chromatography chart of the anti-gout active polypeptide RDP3 of the invention; in the figure, the arrow indicates the absorption peak of
FIG. 4 is a diagram showing the uric acid lowering result of the anti-gout active polypeptide RDP3 of the invention; in the figure, # # #/. representsP<0.001（tChecking);
FIG. 5 is a xanthine oxidase activity diagram of mouse serum after administration of the anti-gout active polypeptide RDP3 of the invention; in the figure, # # #/. representsP<0.001（tChecking);
FIG. 6 is a xanthine oxidase activity map of mouse liver after administration of anti-gout active polypeptide RDP3 of the invention; in the figure, denotesP<0.01, # # #/# representsP<0.001（tChecking);
FIG. 7 is a diagram of the activity of the urate transporter (URAT 1) in the kidney of a mouse after administration of the anti-gout active polypeptide RDP3 of the invention; in the figure, #/. representsP<0.05, representsP<0.01（tChecking);
FIG. 8 is a graph showing the results of the anti-gout active polypeptide RDP3 of the invention against gout attacks. In the figure, # # #/. representsP<0.001（tChecking);
FIG. 9 is a graph showing the HE staining results of the anti-gout active polypeptide RDP3 of the present invention.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The amino acid sequence of the anti-gout active polypeptide RDP3 is AAAAMAGPK-NH 2.
The preparation method of the anti-gout active polypeptide RDP3 comprises the following steps:
A. soaking rice in deionized water overnight, and vacuum freeze-drying the filtered supernatant to obtain a material a, and storing at-80 deg.C;
B. dissolving the material a in water to obtain a material b, eluting the material b with a Tris-HCl buffer solution, and collecting an eluent to obtain a material c;
C. performing high performance liquid chromatography reverse phase chromatography on the material c for the first time to obtain a material d;
D. and (5) performing reverse phase chromatography on the material d by using high performance liquid chromatography for the second time to obtain the target anti-gout
And the water in the step B is deionized water.
And the elution in the step B is to take the material B on a Sephadex G50 column which is balanced by 20 mmol/L Tris-HCl buffer solution for 24 h in advance, and then to elute by using Tris-HCl buffer solution.
The flow rate of elution is 3 ml/10 min.
The pH value of the Tris-HCl buffer solution is 7.8, and the Tris-HCl buffer solution contains 0.1 mol/L NaCl.
The first high performance liquid chromatography reverse phase chromatography is to load the material c into a Hypersil ODS25mm column which is pre-balanced by ultrapure water containing 0.1% trifluoroacetic acid, and elute the material c under the condition of linear gradient by acetonitrile containing 0.1% trifluoroacetic acid at the flow rate of 0.5-1.5 ml/min, wherein the detection wavelength is 220 nm.
And the second high performance liquid chromatography reverse phase chromatography is to dissolve the material d in deionized water after vacuum freeze drying, and then repeat the first high performance liquid chromatography reverse phase chromatography process.
The application of the anti-gout active polypeptide RDP3 is the application of the anti-gout active polypeptide RDP3 in preparing medicines for preventing/treating gout. The present invention will be further described with reference to the following specific embodiments.