A polypeptide for inhibiting influenza virus neuraminidase 2 activity

Abstract

The present application belongs to the technical field of microbiology, and particularly relates to a polypeptide for inhibiting the activity of influenza virus neuraminidase 2. The polypeptide of polyporus umbellatus is extracted, the polyporus umbellatus polypeptide is fractionated by using a Bio-Rad high-pressure chromatography system, and the polypeptide sequence with a higher inhibition rate and higher abundance is picked for full synthesis under the guidance of influenza virus neuraminidase 2 inhibition activity screening, so as to obtain a polypeptide monomer as shown in the sequence table SEQ ID NO. 1. The results of the examples show that the polypeptide monomer can effectively inhibit the activity of influenza virus neuraminidase 2, and further provides a new direction for an anti-influenza virus active substance.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, specifically relating to a polypeptide for inhibiting the activity of influenza virus neuraminidase 2. Background Technology

[0002] Influenza viruses cause numerous deaths every year, and the recent global H1N1 pandemic has resulted in significant losses of life and economic property. Therefore, effectively suppressing the spread of influenza viruses is of great importance to promoting and protecting human civilization. Currently available effective influenza medications include... Amantadine (Adamantane) Oseltamivir (Oseltamivir) Zanamivir Drugs such as zanamivir target the ion channel protein (M2) and surface protein (NA) of the influenza virus, respectively. However, these small molecule drugs also have some problems, such as the emergence of resistant viruses. Therefore, more methods are needed to inhibit the spread of the influenza virus.

[0003] Influenza virus polymerase is a heterotrimeric complex composed of three proteins (PB2, PB1, and PA), and is a crucial complex responsible for influenza virus replication and transcription. After infecting a cell, the virus utilizes the host cell's protein synthesis system to synthesize its own proteins, including newly synthesized PB2, PB1, and PA proteins. The newly synthesized proteins PB1 and PA assemble into dimers in the cytoplasm, and then assemble with PB2 in the nucleus to form a fully functional trimer to perform transcription and replication. The function of influenza virus polymerase in vivo also requires the participation of NP proteins, which are mainly involved in RNA binding and interaction with PB2 host factors.

[0004] With a deeper understanding of the structure and function of influenza virus polymerases, they have become a new and effective target for the development of influenza drugs. The assembly of PA and PB1 is mainly mediated by amino acids 1-25 at the C-terminus of PA and the N-terminus of PB1 (hereinafter referred to as PB11-25). Fragments of the influenza virus polymerase subunits are named similarly, and the crystal structure of this complex has been resolved. The assembly of PB1 and PB2 is mainly mediated by amino acids 676-757 of PB1 (hereinafter referred to as PB1c) and amino acids 1-40 of PB2 (hereinafter referred to as PB2n), and their crystal structures have also been resolved. The crystal structures of both complexes are excellent targets for peptide or small molecule drugs. Evolutionarily, the structure and function of influenza virus polymerases are highly conserved, and drugs targeting influenza virus polymerases have better broad-spectrum activity and resistance compared to drugs targeting surface proteins. Based on its surface spike protein hemagglutinin ( hemagglutinin HA) and neuraminidase ( neuraminidase, different from NA), influenza viruses are often classified into four types: A, B, C, and D. HA and NA are also the targets for influenza virus treatment. Therefore, exploring substances with anti-influenza virus efficacy is one of the effective ways to combat influenza.

[0005] Polyporus umbellatus ( Polyporus umbellatus (Pers.) Fr.) is a perennial fungus of the genus Polyporus. After the sclerotium germinates, it differentiates into white polyporus, and after two autumn and winter seasons, it develops into mature black polyporus, which is the medicinal part of Polyporus umbellatus. Polyporus umbellatus has activities such as diuresis, anti-tumor, and enhancing immunity. White polyporus is white, tender, with a mushroom aroma and is edible.

[0006] Currently, there is no report on Polyporus umbellatus containing substances with anti-influenza virus activity. Summary of the Invention

[0007] The purpose of the present invention is to provide a polypeptide for inhibiting the activity of influenza virus neuraminidase 2. The polypeptide provided by the present invention is derived from white polyporus of Polyporus umbellatus and can efficiently inhibit the activity of influenza virus neuraminidase 2.

[0008] To achieve the above purpose, the present invention provides the following technical solutions: The present invention provides a polypeptide for inhibiting the activity of influenza virus neuraminidase 2, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0009] Preferably, the polypeptide is taken from white polyporus of Polyporus umbellatus.

[0010] The present invention also provides a nucleotide molecule, which encodes the above polypeptide.

[0011] The present invention also provides an expression vector including the above nucleotide molecule.

[0012] The present invention also provides a host cell containing the above expression vector.

[0013] The present invention also provides the application of the above polypeptide in the preparation of drugs for detecting influenza virus.

[0014] Preferably, the drug can inhibit the activity of influenza virus neuraminidase 2.

[0015] Advantages of the present invention: The present invention first obtains a polypeptide from white polyporus of Polyporus umbellatus that can inhibit the activity of influenza virus neuraminidase 2, and the inhibition rate of the polypeptide monomer on the activity of influenza virus neuraminidase 2 is as high as 68%. Brief Description of the Drawings

[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 tandem mass spectrometry (MS / MS) fragmentation spectrum of peptides, i.e., a secondary mass spectrum. Detailed Implementation

[0018] This invention provides a polypeptide for inhibiting the activity of influenza virus neuraminidase 2. The polypeptide is derived from *Polyporus umbellatus* planted in Dundunshi Village, Liuba County, Shaanxi Province, and its specific amino acid sequence is: VDIWEHAFYL (SEQ ID NO.1).

[0019] The present invention uses a neuraminidase inhibitor screening kit to test the inhibitory activity of the above-mentioned peptide on the enzyme. The results of the examples show that the peptide has an inhibition rate of 68.0% against influenza virus neuraminidase 2.

[0020] In some embodiments, the present invention also provides a nucleotide molecule encoding the polypeptide.

[0021] Furthermore, expression vectors or host cells containing the aforementioned nucleotide molecules are also provided.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Example 1 1.1 Types of raw materials Poria cocos and Poria cocos var. cocos were harvested from the Poria cocos planting base in Dundunshi Village, Liuba County, Shaanxi Province.

[0026] 1.2 Peptide Preparation Methods and Process Flow (1) Clean the Poria cocos and Poria cocos, dry the surface moisture, freeze quickly with liquid nitrogen, and grind into fine powder; (2) Based on the ratio of 1 g of fine powder to 500 μL of total protein extract, add total protein extract (0.1 mol / L Tris-Cl (pH 7.5), 0.2 mol / L NaCl, 0.01 mol / L β-mercaptoethanol, 0.01 mol / L EDTA), suspend, and extract at 200 rpm with shaking in an ice bath for 30 min. Centrifuge at 20000 g and 4℃ for 30 min, collect the supernatant to obtain the total protein solution of Poria cocos and Poria cocos, and store at -80℃.

[0027] (3) Take an appropriate amount of total protein solution of *Polyporus umbellatus* and *Polyporus umbellatus*, with a total protein content of 100 mg. Adjust the pH of the solution to 7.5 with 0.01 mol / L sodium hydroxide solution, and add papain solution with an enzyme content of 2 mg. React at 55℃ in a water bath for 4 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 *Polyporus umbellatus* and *Polyporus umbellatus* polypeptide solution.

[0028] (4) The polypeptides of *Polyporus umbellatus* 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 0.5 ml / min, and detection wavelength 280 nm. Each 1 mL tube was collected as one fraction (F). The chromatographic peaks with the same retention time were combined. The collected and combined fractions were F13-16, F17-18, F19-20, F21, F22, F23-24, F25-26, and F27-28.

[0029] (5) The inhibitory activity of each component of *Polyporus umbellatus* 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 either *Polyporus umbellatus* polypeptide component F13-16, F17-18, F19-20, F21, F22, F23-24, F25-26, or F27-28 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.

[0030] 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 5.0 μL, respectively. 0–5.0 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 0–5.0 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%.

[0031] Calculations showed that the neuraminidase 2 inhibition rates of the papain-hydrolyzed polypeptides F13-16, F17-18, F19-20, F21, F22, F23-24, F25-26, and F27-28 from *Polyporus umbellatus* and *Polyporus umbellatus* were 38.7%, 32.5%, 33.0%, 29.4%, 42.9%, 55.2%, 34.8%, and 25.2%, respectively.

[0032] (6) High-resolution mass spectrometry was used to identify the peptides in F22 and F23-24. High-abundance VDIWEHAFYL was selected for total synthesis, and the monomer with a purity of 98% was obtained. The mass spectrometry sequence is shown below. Figure 1 As shown in Table 1.

[0033] Table 1

[0034] (7) The inhibitory activity of VDIWEHAFYL on neuraminidase 2 was determined by the above method, and the inhibition rate was 68.0%.

[0035] 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 for inhibiting 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 obtained from Poria cocos and Poria cocos.

3. A nucleotide molecule encoding the polypeptide of claim 1.

4. An expression vector comprising the nucleotide molecule according to claim 3.

5. A host cell containing the expression vector according to claim 4.

6. The use of the polypeptide of claim 1 in the preparation of a drug for detecting influenza virus.

7. The application according to claim 6, characterized in that, The drug can inhibit the activity of influenza virus neuraminidase 2.

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