Artificially synthesized polypeptide P2 and application thereof in resisting herpes virus infection

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an artificially synthesized polypeptide P2 and application thereof in resisting herpes virus infection. According to the invention, a polypeptide P2 of key amino acid residues (77N, 80I, 82N, 84N, 85N and 129F) combined with virus glycoprotein gD in a specific targeting host cell surface receptor Nectin-1 is designed and obtained through an RFdiffusion algorithm; the polypeptide P2 can significantly inhibit PRV infection, and has no significant cytotoxicity; the polypeptide P2 can be used for remarkably down-regulating PRV UL42 protein expression and gB mRNA (messenger Ribonucleic Acid) level; based on the conservative property of Nectin-1 in a herpes virus invasion mechanism, the polypeptide P2 can also be used for inhibiting infection of other herpes viruses (such as HSV-1 and HSV-2), and an important strategy is provided for developing novel anti-herpes virus drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an artificially synthesized polypeptide P2 and its application in anti-herpes virus infection. Background Art

[0002] Pseudorabies virus (PRV) belongs to the subfamily Alphaherpesvirinae and can infect various mammals such as pigs and cattle, causing neurological damage and significant economic losses. PRV mediates virus invasion by binding its envelope glycoprotein gD to the host cell surface receptor Nectin-1. Currently, the prevention and treatment of PRV mainly rely on vaccines and nucleic acid inhibitors, but there are problems such as immune escape, drug resistance, and hepatotoxicity and nephrotoxicity.

[0003] Herpes simplex virus (HSV-1 / HSV-2) also relies on Nectin-1 as the main host receptor, and its infection mechanism is highly conserved with that of PRV. Existing anti-HSV drugs such as acyclovir need to be activated by viral thymidine kinase, which is prone to cause drug-resistant mutants, and there is an urgent need to develop new inhibitors that do not rely on viral enzymes.

[0004] Studies have shown that the key residues (77N, 80I, 82N, 84N, 85N, 129F) in the gD binding domain of Nectin-1 are molecular "hotspots" for virus invasion. Based on this, the present invention designed a polypeptide P2, which precisely binds to the above sites through conformational optimization of the spatial conformation, blocking virus adsorption and internalization. Summary of the Invention

[0005] Based on the above technical problems, the present invention provides an artificially synthesized polypeptide P2, which efficiently blocks herpes virus (PRV and HSV) infection by competitively inhibiting the interaction between viral gD and host Nectin-1. The specific contents are as follows:

[0006] In the first aspect, the present invention provides an artificially synthesized polypeptide P2, and the amino acid sequence of the polypeptide P2 is: PSVPSTSPISELSKLLEELLEELLG.

[0007] In the second aspect, the present invention provides the application of the polypeptide P2 described in the first aspect above in the preparation of antiviral drugs.

[0008] Preferably, the virus is a herpes virus.

[0009] Preferably, the herpes virus is selected from pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

[0010] Preferably, the herpes virus is pseudorabies virus.

[0011] Preferably, the polypeptide P2 inhibits the adsorption of virus particles to host cells or blocks the replication of the viral genome.

[0012] Preferably, the polypeptide P2 blocks the interaction between the viral glycoprotein gD and Nectin-1 to inhibit viral infection.

[0013] Preferably, the polypeptide P2 specifically targets the key residues 77N, 80I, 82N, 84N, 85N, and 129F of the gD-binding domain of the host cell surface receptor Nectin-1.

[0014] In a third aspect, the present invention provides a pharmaceutical composition, and the active ingredient of the pharmaceutical composition comprises the polypeptide P2 described in the first aspect above.

[0015] In a fourth aspect, the present invention provides the use of the pharmaceutical composition described in the third aspect above in the preparation of an antiviral drug.

[0016] Preferably, the virus is a herpes virus.

[0017] Preferably, the herpes virus is selected from pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

[0018] Preferably, the herpes virus is pseudorabies virus.

[0019] In a fifth aspect, the present invention provides a pharmaceutical preparation, and the pharmaceutical preparation comprises the polypeptide P2 described in the first aspect above or the pharmaceutical composition described in the third aspect above and a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the dosage form of the pharmaceutical preparation includes injection, nasal spray, oral tablet, or sustained-release microsphere preparation.

[0021] In a sixth aspect, the present invention provides the use of the pharmaceutical preparation described in the fifth aspect above in the preparation of an antiviral drug.

[0022] Preferably, the virus is a herpes virus.

[0023] Preferably, the herpes virus is selected from pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

[0024] Preferably, the herpes virus is pseudorabies virus.

[0025] The beneficial effects of the present invention are:

[0026] (1) The polypeptide P2 of the present invention was designed and obtained by the RFdiffusion algorithm, which specifically targets the key amino acid residues (77N, 80I, 82N, 84N, 85N, 129F) in the host cell surface receptor Nectin-1 that bind to the viral glycoprotein gD; the polypeptide P2 forms a high-affinity complex with the gD binding domain of Nectin-1 (predicted binding free energy ΔG = -8.6 kcal / mol);

[0027] (2) The polypeptide P2 of the present invention significantly inhibits the infection of Hela and A549 cells by PRV at a concentration of 400 μM, and the inhibition rates are 66% and 67% respectively, and there is no cytotoxicity (determined by CCK8);

[0028] (3) The polypeptide P2 can reduce the expression of PRV UL42 protein and the level of gB mRNA, indicating that it inhibits both the early adsorption and late replication stages of the virus.

[0029] (4) Based on structural similarity, the polypeptide P2 of the present invention can also inhibit the replication of HSV-1 and HSV-2, and can be used to prepare drugs for anti-herpes virus infection. Description of the Drawings

[0030] Figure 1 Schematic diagram of the polypeptide P2 targeting the gD binding domain of Nectin-1;

[0031] Figure 2 Results of detecting the cytotoxicity of the polypeptide P2 by the CCK8 method (concentration 0 - 400 μM);

[0032] Figure 3 Results of observing PRV-infected cells by fluorescence microscopy (the virus is labeled with green fluorescence);

[0033] Figure 4 Results of quantitatively detecting the inhibition rate of PRV infection by flow cytometry;

[0034] Figure 5 Results of detecting the expression of PRV UL42 protein by Western blot;

[0035] Figure 6 Results of detecting the level of PRV gB mRNA by qPCR. Detailed Embodiments

[0036] The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples are all obtained from regular biochemical reagent companies unless otherwise specified.

[0037] Example 1 Synthesis and Cytotoxicity Determination of Polypeptide P2

[0038] 1. Synthesis of polypeptide P2

[0039] In this example, polypeptide P2 (PSVPSTSPISELSKLLEELLEELLG) that specifically targets the key amino acid residues (77N, 80I, 82N, 84N, 85N, 129F) in the host cell surface receptor Nectin-1 that binds to the viral glycoprotein gD was designed and obtained through the RFdiffusion algorithm; the schematic diagram of the gD binding domain of polypeptide P2 targeting Nectin-1 is as shown in Figure 1 shown.

[0040] GenScript Biotech Corporation was commissioned for the artificial synthesis of polypeptide P2. After HPLC purification, the purity of polypeptide P2 was > 97%, and the molecular weight identified by mass spectrometry was consistent with the theoretical value (2681.08 Da).

[0041] 2. Cytotoxicity

[0042] Hela cells were seeded in 96-well plates (1×10 4 / well), and 0 - 400 μM of polypeptide P2 (containing 0.24% DMSO) was added respectively. After incubation for 24 h, CCK8 reagent was added, and the absorbance was measured at 450 nm.

[0043] The results are as shown in Figure 2 shown. Polypeptide P2 had no significant effect on the activity of Hela cells and had good safety.

[0044] Example 2 In vitro experiment on the inhibition of PRV infection by polypeptide P2

[0045] Hela and A549 cells were seeded in 48-well plates at a density of 5×104 / well. After culturing for 24 h, they were pre-incubated with 400 μM of polypeptide P2 for 2 h respectively, and then PRV-GFP (MOI = 0.1) was inoculated. PRV-GFP was a recombinant virus constructed using homologous recombination technology based on the PRV strain isolated in the laboratory. After 1 h of inoculation, the medium was replaced with fresh medium. After 24 h, the PRV infection situation was observed and the cells were collected.

[0046] 1. Observation of PRV infection by fluorescence microscopy

[0047] The cell culture plates inoculated with PRV-GFP for 24 h were placed on the stage of a fluorescence microscope, and the cell morphology and PRV-GFP infection situation were observed under ordinary light and fluorescence respectively.

[0048] The results are as shown in Figure 3 shown. Polypeptide P2 could significantly reduce the number of PRV-infected cells (viruses labeled with green fluorescence) and significantly reduce PRV infection.

[0049] 2. Flow cytometry for detecting the infection rate

[0050] Uninfected cells were used as a control (MOCK group), and the fluorescence intensity of GFP was detected using the B525-FITC channel of a Beckman flow cytometer. The infection rate of cells in each group was recorded and analyzed (reflected by the proportion of GFP-positive cells).

[0051] The results are as Figure 4 shown. The polypeptide P2 could significantly inhibit PRV-infected Hela and A549 cells, with inhibition rates of 66% and 67% respectively.

[0052] 3. Detection of UL42 protein by Western blot

[0053] Cells were lysed using a cell lysis buffer (1×SDS buffer). After quantifying the whole cell extract, SDS-PAGE analysis was carried out, and the results were transferred to a nitrocellulose membrane. Blotting was performed using primary antibodies against UL42 and GAPDH respectively, and chemiluminescence detection was carried out using an HRP-labeled secondary antibody.

[0054] The results are as Figure 5 shown. The polypeptide P2 could significantly reduce the expression level of PRV's UL42 protein and inhibit the early adsorption of PRV.

[0055] 4. Detection of gB mRNA by qPCR

[0056] Total cellular RNA was extracted using TRIzol reagent, and cDNA was synthesized using HiscriptⅡqPT SuperMixⅡ. Real-time fluorescence quantitative PCR analysis was carried out using QuantStudio 5 and a SYBR RT-PCR kit. According to the 2 -ΔΔCt calculation method, the relative RNA expression level was normalized to GAPDH.

[0057] The results are as Figure 6 shown. The polypeptide P2 could significantly reduce the level of PRV's gB mRNA. The inhibition rates of PRV-infected Hela and A549 cells were 70% and 88% respectively, significantly inhibiting the late replication of PRV.

[0058] The above experimental results indicate that the polypeptide P2 described in this application efficiently blocks PRV infection and significantly inhibits PRV replication by competitively inhibiting the interaction between viral gD and host Nectin-1, and can be used to prepare drugs against PRV infection.

[0059] Since Herpes simplex virus (HSV-1 / HSV-2) also relies on Nectin-1 as the main host receptor and its infection mechanism is highly conserved with PRV, the polypeptide P2 described in the present invention can also inhibit HSV replication and can be used to prepare drugs against HSV infection. Therefore, the polypeptide P2 described in the present invention is applicable to the treatment of livestock farming and human herpes virus infection and has broad market prospects.

Claims

1. An artificially synthesized polypeptide P2, characterized in that, The amino acid sequence of the polypeptide P2 is: PSVPSTSPISELSKLLEELLEELLG.

2. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition comprises the polypeptide P2 described in claim 1.

3. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the polypeptide P2 described in claim 1 or the pharmaceutical composition described in claim 2 and a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical preparation according to claim 3, characterized in that, The dosage forms of the pharmaceutical preparation include injections, nasal sprays, oral tablets or sustained-release microsphere preparations.

5. Use of the polypeptide P2 described in claim 1 or the pharmaceutical composition described in claim 2 or the pharmaceutical preparation described in claim 3 in the preparation of antiviral drugs.

6. The application according to claim 5, characterized in that, The virus is a herpes virus.

7. The application according to claim 6, characterized in that The herpes virus is selected from pseudorabies virus, herpes simplex virus type 1, and herpes simplex virus type 2.

8. The application according to claim 7, wherein The herpes virus is pseudorabies virus.

9. The application according to claim 6, characterized in that The polypeptide P2 inhibits the adsorption of virus particles to host cells or blocks the replication of the viral genome.

10. The application according to claim 6, characterized in that, The polypeptide P2 blocks the interaction between the viral glycoprotein gD and Nectin-1 to inhibit virus infection.

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