CN108715599B - Synthetic polypeptide with uric acid reducing effect, preparation method and application thereof, and gene encoding polypeptide - Google Patents
Synthetic polypeptide with uric acid reducing effect, preparation method and application thereof, and gene encoding polypeptide Download PDFInfo
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- CN108715599B CN108715599B CN201810511559.9A CN201810511559A CN108715599B CN 108715599 B CN108715599 B CN 108715599B CN 201810511559 A CN201810511559 A CN 201810511559A CN 108715599 B CN108715599 B CN 108715599B
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- uric acid
- polypeptide
- synthetic polypeptide
- preparation
- reducing
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Abstract
The invention belongs to the field of biological pharmacy, and discloses a synthetic polypeptide with a uric acid reducing effect, a preparation method and application thereof, and a gene for encoding the polypeptide. The amino acid sequence of the synthetic polypeptide is shown as SEQ ID NO:1 is shown. The invention provides a solid-phase synthesis method for synthesizing novel uric acid-reducing tripeptide, and an in-vitro uric acid-reducing experiment proves that the tripeptide has the effect of obviously reducing uric acid. The action mechanism is analyzed by a computer molecular docking simulation technology, and the result shows that the hydrophobic interaction is the key for playing the role of reducing uric acid. The invention can be used for preparing the fields of medicines and health-care foods related to the hyperuricemia and gout resistant diseases, has wide application range and high safety, and has good social and economic benefits.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a synthetic method, application and an action mechanism of polypeptide with a uric acid reducing effect.
Background
Hyperuricemia is a purine metabolic disease caused by overhigh serum uric acid concentration. Uric acid in human bodies mainly exists in the form of urate, and under physiological conditions, the normal blood uric acid concentration of females is 178.4-297.4 mu mol/L (3-5mg/dL), and the normal blood uric acid concentration of males is 237.9-356.9 mu mol/L (4-6 mg/dL). The blood uric acid concentration of the male is obviously higher than that of the female, and the hyperuricemia is when the serum uric acid concentration of the female is higher than 360 mu mol/L (6mg/dL) and the serum uric acid concentration of the male is higher than 420 mu mol/L (7 mg/dL). When the concentration of uric acid in serum is too high, urate is deposited at joints, so that tophus is formed, gout is caused, and various metabolic syndromes such as diabetes, hypertension, cardiovascular and cerebrovascular diseases, coronary heart disease, kidney pathological changes and the like can be accompanied. Uric acid has antioxidant effect, and can directly scavenge oxygen free radicals, singlet oxygen and peroxide in vivo, and protect ascorbic acid in blood from oxidation. Therefore, human serum uric acid levels should be maintained within the normal range.
Uric acid is produced mainly by the liver in humans, and in the last step of purine metabolism, xanthine or hypoxanthine is catalyzed by Xanthine Oxidase (XOD) to finally produce uric acid. There are two ways for uric acid production, one is through purine food metabolism (exogenesis), accounting for 20% of total uric acid in human body; the second is produced by the catabolism of nucleic acids in the body (endogenous), accounting for 80% of the total amount. Uric acid synthesized by the body is 2/3 excreted through the kidney, and the rest 1/3 excreted from the intestinal tract. Therefore, hyperuricemia, gout, and other diseases can be alleviated by reducing the production of uric acid and promoting the catabolism of uric acid. The drugs for reducing uric acid production mainly comprise Allopurinol (Allopurinol), Febuxostat (Febuxostat), Topiroxostat (Topiroxostat) and the like, and reduce uric acid production mainly by inhibiting Xanthine Oxidase (XOD) activity. Examples of the drugs for promoting uric acid excretion include Probenecid (Probenecid), Benzbromarone (benzbrolone), RDEA594, and the like. Examples of the drug for promoting the decomposition of uric acid include labyrinase (Rasburicase), polyethylene glycol uricase (PEG-uricase), and pegolose (Pegloticase). The medicines can reduce the serum uric acid level, but have certain toxic and side effects on human organs, and are easy to cause serious allergic and inflammatory reactions. Therefore, the natural product uric acid reducing substance gradually receives attention of researchers due to small toxic and side effects.
The bioactive peptide has the physiological functions of resisting tumor, lowering blood pressure, lowering blood sugar, resisting inflammation, resisting oxidation, resisting senility, etc. and is easy to be absorbed by human body compared with protein and amino acid and has no toxic side effect. However, relatively few reports on uric acid-lowering bioactive peptides have been reported, and these food-derived bioactive peptides have xanthine oxidase inhibitory activities in vitro and efficacy in lowering serum uric acid levels in hyperuricemic mice.
The bioactive peptide can be obtained by enzymolysis of marine fishes, the bioactive peptide can be obtained by using marine fish protein such as Liuyang and the like as raw materials, and the anti-gout peptide with good activity is prepared by optimizing an enzymolysis process by a response surface analysis method, and has obvious in-vitro XOD (x-ray diffraction order) inhibition activity. Further research on the action mechanism shows that the marine fish protein peptide can effectively inhibit the XOD activity of hyperuricemia rats, reduce the expression level of ADA and XOD mRNA in the livers of the rats, and has good kidney protection effect. In addition, after the enzymatic hydrolysate is purified and identified, the synthesized polypeptide sequence also has the in vitro xanthine oxidase inhibitory activity and the effect of reducing the serum uric acid level of the hyperuricemia mice. It was reported that 18 synthetic polypeptides each containing an aromatic amino acid such as Tyr, Trp, Phe, etc. were identified in an aqueous extract of shark cartilage. The activity of the synthesized polypeptide is evaluated by a hyperuricemia animal model, and the result shows that the blood uric acid content of a rat with hyperuricemia can be obviously reduced after the rat with the hyperuricemia is treated by a Tyr-Leu-Asp-Asn-Tyr (YLDNY) and Ser-Pro-Pro-Tyr-Trp-Pro-Tyr (SPPYWPY) in an intravenous injection mode, and the blood uric acid content of the rat with the hyperuricemia can also be reduced by the treatment of YLDNY in an oral mode. Further research shows that certain dipeptide or tripeptide derived from YLDNY has better XOD inhibitory activity of Asp-Asn fragment, which indicates that YLDNY can reduce rat serum uric acid by inhibiting XOD activity after being absorbed in vivo possibly in a dipeptide or tripeptide mode. Therefore, the polypeptide has wide market prospect in the development of health products and medicines for reducing uric acid.
Disclosure of Invention
In view of the above, the invention provides a preparation method and application of a synthetic polypeptide, and an action mechanism of the synthetic polypeptide for reducing uric acid is analyzed by a molecular docking technology.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme.
A synthetic polypeptide with uric acid lowering effect has an amino acid sequence shown in SEQ ID NO 1. The amino acid sequence is Trp-Met-Leu, and has the activity of reducing uric acid in vitro.
Wherein, Trp represents the corresponding residue of amino acid with English name of Tryptophan and Chinese name of Tryptophan;
met represents the corresponding residue of the amino acid with the English name methionine and the Chinese name methionine;
leu represents the corresponding residue of the amino acid called Leucine in England and Leucine in Chinese.
The gene for coding the synthetic polypeptide of claim 1, wherein the gene sequence tggatgctg is shown as the sequence table SEQ ID No. 2. The present invention provides DNA molecules encoding synthetic polypeptides. Due to the degeneracy of the codons, a variety of nucleotide sequences are possible. The DNA molecule code of the polypeptide of the invention determines and provides the DNA molecule of the polypeptide amino acid sequence by selecting the codon of the amino acid residue of the amino acid sequence, and the DNA molecule can be prepared and synthesized by the technicians in the field by using the existing method.
The method for preparing the synthetic polypeptide with the effect of reducing uric acid is implemented by a solid-phase synthesis method according to the sequence shown in SEQ ID NO:1, fluorenylmethyloxycarbonyl (Fmoc) is used as a protecting group of amino acid, the amino acid is synthesized from a C end to an N end in a mode of coupling the amino acid one by one, the Fmoc protecting group is removed, meanwhile, solid resin is coupled to form polypeptide resin, the outer layer resin is cracked under the action of a cracking agent and a side chain protecting group removing agent, and the Fmoc protecting group of a side chain is removed, so that the synthesized polypeptide can be obtained.
The solid-phase synthesis method of the polypeptide is characterized in that the polypeptide is synthesized by coupling amino acids one by one from the C end to the N end of the amino acid sequence of the synthesized polypeptide.
Further, the solid carrier resin is 4- (hydroxymethyl) phenoxymethyl polystyrene resin (Wang resin), is connected with the C end of the amino acid in a covalent bond mode, and is used as a carrier for one-by-one extension coupling peptide-connecting reaction in the polypeptide synthesis process.
Further, the coupling agent adopted for the one-by-one extension coupling is a HOBT/HBTU mixed coupling agent.
Further, the cracking agent is piperidine N, N-dimethylformamide DMF solution with the mass concentration of 20%.
Further, the side chain protecting group remover is a mixed solution of trifluoroacetic acid, ethylenediamine tartrate, distilled water and TIS.
The synthetic polypeptide provided by the invention can be applied to preparation of a medicine for resisting hyperuricemia.
In the application, the in vitro uric acid reducing activity is determined by detecting the relative inhibition rate of the xanthine oxidase catalyzing the generation of uric acid. The inhibition mechanism of the synthetic polypeptide on xanthine oxidase is evaluated by adopting a molecular docking technology.
In a specific embodiment of the invention, the amino acid sequence according to SEQ ID NO:1, adopting Fmoc protecting group to protect amino group of amino acid, coupling the amino acid raw materials of Fmoc-Trp-OH, Fmoc-Met (trt) -OH, Fmoc-Leu (boc) -OH and Wang resin, specifically coupling the protected amino acid raw materials and the resin one by one according to the sequence of the amino acid from C end to N end, then removing the resin and the side chain protecting group from lysate to obtain crude polypeptide, and separating and purifying to obtain the target polypeptide.
The coupling agent for coupling one by one adopts a HOBT/HBTU mixed coupling agent, the coupling effect is better than that of a single coupling agent, and the dosage of the coupling agent is preferably excessive.
In the coupling mode in the polypeptide preparation method, the Fmoc removal treatment is carried out on the amino acid protected by Fmoc before coupling. In a specific embodiment, the Fmoc removing reagent is 20% piperidine N, N-Dimethylformamide (DMF) solution, and the Fmoc protecting group can be removed by stirring for 30 min.
Preferably, the cleavage remover for cleaving Wang resin in the present invention is a mixed solution of trifluoroacetic acid, ethylenediamine tartrate, distilled water and TIS, specifically trifluoroacetic acid: ethylenediamine tartrate: distilled water: mixed system of TIS 94.5:2.5:2:1, reaction time is 2 h.
In addition, the invention adopts an in vitro xanthine oxidase catalytic reaction method to detect the uric acid reducing activity of the synthesized polypeptide, and the result shows that the synthesized polypeptide can be combined with the xanthine oxidase through hydrophobic interaction and hydrogen bond interaction, so that the catalytic activity of the xanthine oxidase is inhibited, and the synthetic polypeptide has the obvious uric acid reducing effect.
Based on the excellent experimental results, the invention provides the application of the synthetic polypeptide in preparing the anti-hyperuricemia drug.
In the application, the invention provides a medicament, the synthetic polypeptide is mixed with various common auxiliary materials acceptable in medicine, such as a filling agent, a disintegrating agent, a lubricating agent, an adhesive and the like, and the filling agent is specifically starch, lactose, glucose, mannitol and silicic acid; disintegrating agents such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate, low-substituted hydroxypropylcellulose; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate; binding agents such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia.
The polypeptide uric acid-reducing medicine can be prepared into medicines of different dosage forms. Preferably, the pharmaceutical form of the present invention can be made into tablets, capsules, oral liquids, granules, pills, powders, and the like. The dosage form of the drug is not limited thereto, and those skilled in the art will recognize that a feasible dosage form is within the scope of the present invention.
According to the technical scheme, the invention provides a synthesis method and application of polypeptide with the effect of reducing uric acid. The amino acid sequence of the synthesized polypeptide is shown as SEQ ID NO. 1, and the polypeptide is prepared by adopting Fmoc solid phase synthesis.
Compared with the prior art, the invention has the following advantages and beneficial effects: the invention provides a solid-phase synthesis method for synthesizing novel uric acid-reducing tripeptide, and an in-vitro uric acid-reducing experiment proves that the tripeptide has the effect of obviously reducing uric acid. The action mechanism is analyzed by a computer molecular docking simulation technology, and the result shows that the hydrophobic interaction is the key for playing the role of reducing uric acid. The invention can be used for preparing the fields of medicines and health-care foods related to the hyperuricemia and gout resistant diseases, has wide application range and high safety, and has good social and economic benefits. The polypeptide has good xanthine oxidase inhibitory activity, can be used for preparing medicines for resisting hyperuricemia, gout and the like, has high safety and wide application range, and has good social and economic benefits.
Drawings
FIG. 1 is a High Performance Liquid Chromatography (HPLC) chromatogram of a synthetic polypeptide.
FIG. 2 is a high resolution mass spectrometry (ESI-MS) profile of the synthesized polypeptide.
FIG. 3 in vitro Xanthine Oxidase (XOD) inhibitory activity of the polypeptide.
Detailed Description
The invention discloses a preparation method, application and an action mechanism of polypeptide with uric acid reducing effect. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are only some, and not all, embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. The in-vitro uric acid reduction experiment evaluation method and the molecular docking analysis technology adopted in the experiment can be widely used for evaluating the uric acid reduction activity of potential drugs and health-care foods, and provides a research method for seeking more effective and safe drugs for preventing and treating the hyperuricemia.
The synthetic polypeptide provided by the present invention, its synthetic method and application are further described below.
Example 1: solid phase synthesis of synthetic polypeptides:
adopting an Fmoc solid-phase synthesis strategy, taking Fmoc-protected amino acid as a raw material, selecting Wang resin as a carrier according to the sequence characteristics of an amino acid sequence Trp-Met-Leu, and using a HOBT/HBTU/DIEA mixed coupling agent to extend a peptide chain from a C end to an N end one by one, wherein the specific operation scheme is as follows:
(1) in a polypeptide synthesis tube, the dosage of each amino acid is 0.1mol, 0.3mol of Fmoc protected amino acid and 0.0125mmol of Wang resin are added, and the mixture is stirred for 30min to ensure that the resin is fully swelled.
(2) Adding 2mmol of Fmoc protected amino acid Fmoc-Trp-OH into a solid phase synthesis tube, adding a DMF solution containing 12mmol of DIEA, and washing with 20mL of DMF solution for three times to obtain Fmoc-Trp-CTC resin.
(3) 20% piperidine DMF solution (15ml/g) was added to the solid phase synthesis tube and stirred for 30min to remove the Fmoc protecting group from the polypeptide resin. And adding 0.259g of coupling agent HOBT, 0.728g of HBTU and 10mL of DMF, stirring and activating for 20min, and reacting for 4h to obtain the coupled polypeptide resin.
(5) Adding a mixed solution of a cracking removal agent trifluoroacetic acid (94.5%), ethylenediamine tartrate (2.5%), distilled water (2%) and TIS (1%) (volume fraction) and the obtained polypeptide chain into a solid-phase synthesis tube, uniformly mixing, reacting for 2h, eluting with DMF (dimethyl formamide) for three times, each time for 1min, and performing suction filtration and drying to obtain a crude polypeptide product from which resin and side chain protecting groups can be removed.
The above operation processes are all completed in a SYMPHONY type 12-channel polypeptide synthesizer, and crude polypeptide synthesized by the reaction is purified by an RP-HPLC reversed phase high performance liquid chromatograph and is shown in figure 1. The synthesized polypeptide is finally identified by high resolution mass spectrometry, and the result shows that the purity of the synthesized polypeptide reaches more than 99 percent, and fig. 2 is an ESI-MS mass spectrometry identification map.
Example 2: the in vitro uric acid reducing activity of the synthesized polypeptide is determined:
xanthine Oxidase (XOD) is a key enzyme for synthesizing uric acid, inhibits the activity of xanthine oxidase, and reduces the production of uric acid, so that the in vitro xanthine oxidase inhibition activity can be used as an effective method for evaluating the uric acid reducing activity of a sample. In the purine metabolism process, xanthine oxidase can oxidize xanthine and hypoxanthine into uric acid, and the reaction principle is as follows:
adopting a 96 enzyme-linked immunosorbent assay detection plate as a carrier for detecting an enzyme-substrate reaction system, respectively adding 50 mu L of a sample to be detected, 50 mu L of 0.05U/mL xanthine oxidase into the 96 enzyme-linked immunosorbent assay detection plate, incubating at 37 ℃ for 10min, adding 150 mu L of 0.4mmol/L xanthine solution, starting an enzymatic reaction, simultaneously recording the dynamic change of the light absorption value of the reaction system at 290nm within 2min, taking a pH 7.5PBS buffer solution as a reference, and recording the initial reaction rate of the enzymatic reaction system as V0The initial rate of the enzymatic reaction system in the presence of the sample is denoted VSThe inhibition rate of the sample on xanthine oxidase activity was calculated according to the following formula:
XOD inhibitory activity (%) - (V)0-VS)/V0
FIG. 3 shows the in vitro Xanthine Oxidase (XOD) inhibitory activity of the polypeptides. In an in-vitro uric acid reduction experiment for synthesizing the polypeptide, polypeptide solutions with different concentrations are added into a xanthine oxidase-xanthine reaction system, and the relative inhibition rate of the rate of catalyzing and generating uric acid by the xanthine oxidase is detected. One-way ANOVA was used to evaluate the effect of different concentrations of synthetic polypeptide on xanthine oxidase activity, all data are expressed as mean ± variance, where x represents P < 0.01. The synthetic polypeptide WML shows obvious dose-dependent effect on the inhibitory activity of xanthine oxidase, and the fact that the WML can interact and combine with the xanthine oxidase is proved to inhibit the xanthine to generate the uric acid.
Example 3: mechanism of inhibition of synthetic polypeptide on xanthine oxidase
The molecular docking technology is a computer simulation method for mutual recognition between ligand and receptor molecules through space matching and energy matching to form a molecular complex, and can integrally evaluate the capacity and the binding effect required by the interaction between the ligand and the receptor. Molecular docking simulation first requires a database of proteins (P)Protein data bank), removing water molecules and irrelevant ligands in a protein structure before a molecular docking experiment is carried out, and obtaining a complete structure of the xanthine oxidase by MGL Tools through hydrogenation and charging. Loading the 3D structure of the ligand synthetic polypeptide in a magnol website, performing visual analysis on the docking result between the xanthine oxidase and the ligand by using PyMOL software, finally performing computer simulation on the synthetic polypeptide and the xanthine oxidase through AutoDock to simulate the interaction of the synthetic polypeptide and the xanthine oxidase, and analyzing the interaction capacity and type result of the synthetic polypeptide and the xanthine oxidase. Xanthine oxidase contains two FAD, 2 molybdenum atoms and 8 iron atoms. Wherein the molybdenum atom in the enzyme is in the form of a molybdenum pterin cofactor, which is the active site of the enzyme, and the central amino acid residue of molybdenum pterin forms a hydrophobic cavity. The molybdopterin central hydrophobic cavity is mainly composed of amino acid residues: phe649, Phe914, Phe1009, Asn768, Val1011, Glu802, Ser876, Lys771, Leu873, Leu1014, Arg880, Thr1010, Glu1261 and the like. XOD inhibitors inhibit XOD activity by binding to these residues into the hydrophobic cavity. In order to determine the inhibition mechanism of polypeptide WML on XOD activity, the binding of synthetic polypeptide residue and molybdenum pterin residue is studied by utilizing molecular docking and whether the polypeptide molecule enters an active hydrophobic region and plays a role is identified. The hydrogen bond formed between the Leu residue of the synthesized polypeptide WML and the xanthine oxidase Asn768 residue is
And the binding energy is-7.34 kcal/mol, which is basically close to that of allopurinol serving as a clinical xanthine oxidase inhibitor. The synthetic polypeptide WML contains three hydrophobic amino acids: trp, Met and Leu show good affinity though only form a hydrogen bond with xanthine oxidase directly, which indicates that the acting force of WML entering the active hydrophobic region of xanthine oxidase is removed from the interaction force of hydrogen bond, wherein the acting force is mainly hydrophobic interaction force.
TABLE 1
Table 1 shows the molecular structural formula and LogP value of the synthesized polypeptide, and it can be seen from Table 1 that the LogP (>0 is hydrophilic; <0 is hydrophobic) value is negative, and WML is hydrophobic polypeptide, indicating that hydrophobic interaction and hydrogen bond interaction exist between WML and xanthine oxidase. The combination energy of the WML and the xanthine oxidase is-7.34 kcal/moL, the required combination energy is lower than the energy required by the combination of allopurinol, which is a clinically common xanthine oxidase inhibitor, and the enzyme, so that the WML has strong xanthine oxidase inhibition activity, but only forms a hydrogen bond, and the hydrophobic interaction becomes the main driving force of the polypeptide molecules entering a hydrophobic cavity.
Sequence listing
<110> university of southern China's science
<120> synthetic polypeptide with uric acid reducing effect, preparation method and application thereof, and gene encoding polypeptide
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tggatgctg 9
Claims (7)
1. The application of the synthetic polypeptide with the effect of reducing uric acid in preparing the anti-hyperuricemia drug is characterized in that the amino acid sequence of the synthetic polypeptide is Trp-Met-Leu, is shown as the sequence table SEQ ID No. 1, and has the in-vitro uric acid reducing activity.
2. The use of the synthetic polypeptide with uric acid lowering effect according to claim 1 in the preparation of an anti-hyperuricemia drug, wherein the preparation of the synthetic polypeptide comprises: by solid phase synthesis method, according to SEQ ID NO:1, fluorenylmethyloxycarbonyl Fmoc is used as a protecting group of amino acid, the Fmoc is synthesized from a C end to an N end in a mode of coupling the amino acid one by one, the Fmoc protecting group is removed and the solid resin is coupled to form polypeptide resin, the outer layer resin is cracked under the action of a cracking agent, and the Fmoc protecting group is removed by adopting a side chain protecting group removing agent, so that the synthesized polypeptide can be obtained.
3. The use of the synthetic polypeptide with uric acid lowering effect in the preparation of anti-hyperuricemia drugs according to claim 2, wherein the solid resin is 4- (hydroxymethyl) phenoxymethyl polystyrene resin which is covalently linked to the C-terminus of the amino acid, and can be used as a carrier for the one-by-one extension coupling peptide reaction during the polypeptide synthesis process.
4. The use of the synthetic polypeptide with uric acid lowering effect in the preparation of anti-hyperuricemia drugs according to claim 3, wherein the coupling agent used for the one-by-one extension coupling is a HOBT/HBTU mixed coupling agent.
5. The use of the synthetic polypeptide with uric acid lowering effect in the preparation of anti-hyperuricemia drugs according to claim 3, wherein the cleavage agent is piperidine N, N-dimethylformamide DMF solution with a mass concentration of 20%.
6. The use of the synthetic polypeptide with uric acid lowering effect in the preparation of anti-hyperuricemia drugs according to claim 3, wherein the side chain protecting group remover is a mixed solution of trifluoroacetic acid, ethylenediamine tartrate, distilled water and TIS.
7. The use of the synthetic polypeptide with uric acid lowering effect in the preparation of anti-hyperuricemia drugs according to claim 3, wherein the in vitro uric acid lowering activity of the synthetic polypeptide is determined by detecting the relative inhibition rate of the rate of production of uric acid catalyzed by xanthine oxidase.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341630A (en) * | 2000-09-05 | 2002-03-27 | 上海博德基因开发有限公司 | A novel polypeptide-human pancreatic island cell antigen p69 (ICAp69)28-9.13 and polynucleotide for coding said polypeptide |
| JP2007197434A (en) * | 2005-12-28 | 2007-08-09 | Univ Nagoya | Peptide having reactivity as substrate of transglutaminase and utilization of the same |
| CN106317178A (en) * | 2016-08-28 | 2017-01-11 | 华南理工大学 | Polypeptide with function of reducing uric acid and application of polypeptide |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341630A (en) * | 2000-09-05 | 2002-03-27 | 上海博德基因开发有限公司 | A novel polypeptide-human pancreatic island cell antigen p69 (ICAp69)28-9.13 and polynucleotide for coding said polypeptide |
| JP2007197434A (en) * | 2005-12-28 | 2007-08-09 | Univ Nagoya | Peptide having reactivity as substrate of transglutaminase and utilization of the same |
| CN106317178A (en) * | 2016-08-28 | 2017-01-11 | 华南理工大学 | Polypeptide with function of reducing uric acid and application of polypeptide |
Non-Patent Citations (3)
| Title |
|---|
| Anti-hyperuricemic peptides derived from bonito hydrolysates based on invivo hyperuricemic model and in vitro xanthine oxidase inhibitory activity;Yujuan Li等;《Peptides》;20180802;第107卷;第45-53页 * |
| 氨基酸分类与蛋白质二级结构相关性;王守源等;《内蒙古大学学报（自然科学版）》;20020731;第33卷(第4期);第2.1小节，第425页表4 * |
| 王守源等.氨基酸分类与蛋白质二级结构相关性.《内蒙古大学学报（自然科学版）》.2002,第33卷(第4期), * |
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