Polypeptide from sclerotium of poria cocos inhibiting neuraminidase 2 activity and preparation method thereof
By extracting and screening the polypeptide KINRPRYRL from Poria cocos, the deficiency of Poria cocos polypeptide in anti-influenza virus neuraminidase 2 activity was solved, and a highly efficient inhibitory effect was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
- Filing Date
- 2026-04-28
- Publication Date
- 2026-05-29
AI Technical Summary
In the existing technology, there are few active peptides of Poria cocos in terms of anti-influenza virus activity, especially the inhibitory activity on neuraminidase 2 has not been fully explored.
Peptides were extracted from Poria cocos, cleaved by alkaline protease, and fractionated using a high-pressure chromatography system in Bio-Rad. The peptide KINRPRYRL (SEQ ID NO.1) with high inhibitory activity was screened out, and its sequence was identified by high-resolution mass spectrometry.
The selected peptide KINRPRYRL showed an inhibition rate of 66.9% against neuraminidase 2, providing an effective anti-influenza virus approach.
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Figure CN122103262A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of immunology technology, specifically relating to a polypeptide derived from the sclerotium of Poria cocos that inhibits the activity of neuraminidase 2 and its preparation method. Background Technology
[0002] Poria cocos is a fungus belonging to the Polyporaceae family. Poria cocos (Schw.) Wolf The dried sclerotia of Poria cocos are traditionally considered both a food and a medicinal herb (i.e., a dual-use substance). In terms of food, they are widely added to food products; in terms of medicinal use, they possess hepatoprotective, anti-cancer, anti-inflammatory, cardiovascular disease treatment, lipid metabolism regulation, and immune, intestinal, and nervous system regulation effects. Exploring the antiviral activity of Poria cocos can further enrich its application scope.
[0003] According to reports, the active ingredients of Poria cocos are mainly terpenes, polysaccharides, and sterols, while the number of Poria cocos proteins exceeds 2,000. The exploration of its functional proteins or peptides is still in its early stages, and there are very few active peptides that fight influenza viruses.
[0004] Based on its surface spike protein hemagglutinin ( hemagglutinin HA) and neuraminidase ( neuraminidase Influenza viruses are often classified into four types—A, B, C, and D—based on their differences in HA and NA (hypoallergenic and non-hypoallergenic receptors). HA and NA are also targets for influenza virus treatment. Therefore, screening for active ingredients that can effectively inhibit these targets is one of the effective methods to combat influenza. Summary of the Invention
[0005] The purpose of this invention is to provide a Poria cocos polypeptide that inhibits the activity of influenza virus neuraminidase 2. The Poria cocos polypeptide provided by this invention can effectively inhibit the activity of influenza virus neuraminidase 2.
[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a polypeptide that inhibits the activity of influenza virus neuraminidase 2, the amino acid sequence of which is shown in SEQ ID NO.1.
[0007] Preferably, the polypeptide is derived from Poria cocos.
[0008] The present invention also provides a method for preparing the above-mentioned polypeptide, comprising adding a total protein extract to Poria cocos ground into fine powder, reacting the extracted Poria cocos total protein in a water bath with a protease to obtain a Poria cocos enzyme-digested polypeptide solution, and obtaining the above-mentioned polypeptide by fractionation using a Bio-Rad high-pressure chromatography system and identification by high-resolution mass spectrometry.
[0009] Preferably, the protease is an alkaline protease;
[0010] This invention also provides the application of the above-mentioned polypeptide in the preparation of drugs for the prevention and treatment of influenza viruses.
[0011] Preferably, the drug achieves the purpose of preventing and treating influenza virus by inhibiting the activity of influenza virus neuraminidase 2.
[0012] The present invention also provides a nucleic acid molecule that encodes the aforementioned polypeptide.
[0013] The present invention also provides an expression vector comprising the above-mentioned nucleic acid molecules.
[0014] The beneficial effects of this invention are: This invention is the first to screen out a polypeptide monomer from Poria cocos that can inhibit influenza virus neuraminidase 2, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.
[0015] The test showed that KINRPRYRL (SEQ ID NO.1) inhibited neuraminidase 2 by 66.9%. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 This is a mass spectrometry identification result of the polypeptide monomer (KINRPRYRL). Detailed Implementation
[0018] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0019] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.
[0020] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.
[0021] Example 1 1.1 Types of raw materials Poria cocos was harvested from Tongjiang County, Sichuan Province.
[0022] 1.2 Peptide Preparation Methods and Process Flow 1.2.1 Alkaline protease digestion process and activity screening (1) Poria cocos, clean it, dry the surface moisture, cut it into small pieces, freeze it with liquid nitrogen, and grind it into fine powder.
[0023] (2) Add total protein extract (Beibo Biotechnology) according to the ratio of 1 g fine powder to 500 μL total protein extract, suspend, and extract at 160 rpm with ice bath shaking for 30 min. Centrifuge at 14000 rpm and 4℃ for 25 min, take the supernatant to obtain Poria cocos total protein solution, and store at -80℃.
[0024] (3) Take an appropriate amount of Poria cocos total protein solution, with a total protein content of 100 mg. Adjust the pH of the solution to 10 with 0.01 mol / L sodium hydroxide solution, and add alkaline protease solution with an enzyme content of 1 mg. React at 55℃ in a water bath for 3 h, and adjust the pH to neutral with 0.01 mol / L hydrochloric acid. Filter through a 10 kDa ultrafiltration tube, collect the filtrate, and obtain the Poria cocos alkaline enzyme-digested polypeptide solution.
[0025] (4) The alkaline enzyme-digested peptides of Poria cocos were fractionated using a Bio-Rad high-pressure chromatography system (NGC Scout 10 Plus). The chromatographic conditions or parameters were as follows: Superdex 30 Increase 10 / 300 GL column, PBS buffer (pH 7.0) as the mobile phase, flow rate of 0.5 mL / min, and detection wavelength of 280 nm. Each 1 mL tube was collected as one fraction (F). Fractions with the same chromatographic peak were combined. The collected and combined fractions were F13-15, F16-17, F18-19, F20-21, F22-23, F25-26, and F28-29.
[0026] (5) The inhibitory activity of each component of the Poria cocos alkaline enzyme-digested polypeptide against the enzyme was tested using a neuraminidase inhibitor screening kit. 35 μL of neuraminidase 2 detection buffer was added to each well of a 96-well fluorescent microplate, followed by 5 μL of neuraminidase solution. After mixing, 5 μL of the test solution for each of the Poria cocos alkaline enzyme-digested polypeptide components F13-15, F16-17, F18-19, F20-21, F22-23, F25-26, or F28-29 was added, and the mixture was shaken for approximately 1 minute. The plate was incubated at 37°C for 10 minutes to allow sufficient interaction between the inhibitor and neuraminidase 2. 5 μL of neuraminidase 2 fluorescent substrate was added to each well, and the mixture was shaken for approximately 1 minute. The mixture was incubated at 37°C for 30 minutes. The fluorescence intensity was detected under the conditions of excitation wavelength of 322 nm and emission wavelength of 450 nm. Each test component was repeated in triplicate.
[0027] The same method was used to detect the standard curve. The volumes of neuraminidase 2 added to each well were 0, 0.5, 1.0, 2.5, 3.75, and 5 μL, respectively. 0–5 μL of ultrapure water was used instead of the sample solution. Fluorescence intensity values were measured, and the standard curve y = 4.042x + 8.933 (R²) was plotted. 2 =0.995), where x is the enzyme survival volume (V1), with a linear range of 0–5 μL, and y is the absorbance value. The enzyme survival volume V1 is calculated using this standard curve, and then the enzyme activity inhibition rate (%) is calculated according to the formula (1-V1 / V)*100%.
[0028] Calculations showed that the neuraminidase 2 inhibition rates of the alkaline enzyme-digested polypeptides F13-15, F16-17, F18-19, F20-21, F22-23, F25-26, and F28-29 were 23.3%, 11.7%, 20.8%, 32.2%, 49.6%, 40.8%, and 56.6%, respectively.
[0029] (6) High-resolution mass spectrometry was used to identify the polypeptides in F28-29. High-abundance KINRPRYRL (SEQ ID NO.1) was selected for total synthesis, and the monomer was purified to 98%. The mass spectrometry sequence was as follows: Figure 1 As shown.
[0030] (7) The inhibitory activity of KINRPRYRL (SEQ ID NO.1) on neuraminidase 2 was determined by the above method, and the inhibition rate was 66.9%.
[0031] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A polypeptide that inhibits the activity of influenza virus neuraminidase 2, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.
1.
2. The polypeptide according to claim 1, characterized in that, The polypeptide was derived from Poria cocos.
3. The method for preparing the polypeptide according to claim 1, characterized in that, The method involves adding a total protein extract to Poria cocos ground into a fine powder, reacting the extracted Poria cocos total protein in a water bath with a protease to obtain a Poria cocos enzyme-digested polypeptide solution, and then fractionating the polypeptide according to claim 1 using a Bio-Rad high-pressure chromatography system and identifying it by high-resolution mass spectrometry.
4. The preparation method according to claim 3, characterized in that, The protease is an alkaline protease.
5. The use of the polypeptide of claim 1 in the preparation of drugs for the prevention and treatment of influenza viruses.
6. The application according to claim 5, characterized in that, The drug achieves its purpose of preventing and treating influenza virus by inhibiting the activity of neuraminidase 2 in influenza virus.
7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1.
8. An expression vector comprising the nucleic acid molecule of claim 7.