Application of antibacterial peptide WK-13-3D in preparation of drugs for inhibiting Japanese encephalitis virus infection

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to application of an antibacterial peptide WK-13-3D in preparation of a medicine for inhibiting Japanese encephalitis virus infection. The invention accidentally finds that the antibacterial peptide WK-13-3D can obviously reduce the production of JEV RNA in a dose-dependent manner, inhibit the expression of JEV non-structural protein NS3, obviously reduce the yield of infectious JEV and inhibit the JEV from adhering to host cells, the IC50 value of the antiviral activity is 2.339 mu M, and the antibacterial peptide WK-13-3D can inhibit the infection of Japanese encephalitis virus, can be used as a medicine for treating Japanese encephalitis virus infection, and has a wide application prospect. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to the application of an antimicrobial peptide WK-13-3D in the preparation of a drug for inhibiting Japanese encephalitis virus infection. Background Art

[0002] Japanese encephalitis virus (JEV), also known as Japanese encephalitis virus, belongs to the Flaviviridae family and is an enveloped single-stranded positive-sense RNA virus. JEV is the pathogen of Japanese encephalitis (JE), which is mainly transmitted by mosquitoes. Some surviving patients suffer from permanent neurological deficits, including epileptic seizures, paralysis and mental retardation. It can also cause abortion in pregnant sows, death of piglets and orchitis in boars, and other pathological damages that lead to reduced pig production performance, posing a huge threat to human health and the healthy development of animal husbandry.

[0003] Currently, there is an urgent need to find an effective JE-specific drug treatment method to minimize the sequelae of patients through early intervention with antibodies or drugs.

[0004] Antimicrobial peptides are a class of small molecule peptides widely present in organisms and encoded by specific genes. They have the function of resisting external microbial invasion and removing mutant cells in the body. They are an important part of the biological natural immune defense system. Studies have shown that WK-13-3D is an antimicrobial peptide related to bacterial membrane inhibition, inhibiting bacteria and their biofilms, especially methicillin-resistant Staphylococcus aureus, and the antimicrobial peptide WK-13-3D can inhibit the proliferation of triple-negative breast cancer MDA-MB-231 cells, inhibit the growth of tumors in the body, and promote the apoptosis of MDA-MB-231 cells, thereby exerting an anti-tumor effect.

[0005] The present invention unexpectedly discovered that the antimicrobial peptide WK-13-3D can significantly reduce the production of JEV RNA, inhibit the expression of JEV non-structural protein NS3, reduce the yield of infectious JEV, and inhibit the infection of Japanese encephalitis virus. Summary of the invention

[0006] In response to the above technical problems, the present invention unexpectedly discovered that the antimicrobial peptide WK-13-3D can significantly reduce the production of JEV RNA, inhibit the expression of JEV non-structural protein NS3, reduce the production of infectious JEV, and inhibit the infection of Japanese encephalitis virus. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides an antimicrobial peptide WK-13-3D or a composition containing the antimicrobial peptide WK-13-3D for use in preparing a drug for inhibiting viral infection. The sequence of the antimicrobial peptide WK-13-3D is: WKRIVRRIKRWLR-NH 2.

[0008] Preferably, the virus is Japanese encephalitis virus.

[0009] Preferably, the antimicrobial peptide WK-13-3D reduces the RNA content of Japanese encephalitis virus.

[0010] Preferably, the antimicrobial peptide WK-13-3D inhibits the expression of Japanese encephalitis virus non-structural protein NS3.

[0011] Preferably, the antimicrobial peptide WK-13-3D inhibits Japanese encephalitis virus from adhering to host cells.

[0012] Preferably, the antimicrobial peptide WK-13-3D or a composition containing the antimicrobial peptide WK-13-3D is added with pharmaceutically acceptable excipients to prepare any pharmaceutically acceptable dosage form.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for inhibiting viral infection, wherein the active ingredient of the pharmaceutical composition comprises an antimicrobial peptide WK-13-3D, and the sequence of the antimicrobial peptide WK-13-3D is: WKRIVRRIKRWLR-NH2.

[0014] Preferably, the active ingredients of the pharmaceutical composition further include one or more of small molecule compounds against Japanese encephalitis virus, Japanese encephalitis virus vaccines, and monoclonal antibodies against Japanese encephalitis virus.

[0015] Preferably, the pharmaceutical composition comprises a synthetic antimicrobial peptide, or a recombinant vector containing an antimicrobial peptide.

[0016] Preferably, the synthetic antimicrobial peptide or the recombinant vector carrying the antimicrobial peptide is delivered into the body via a drug delivery vector to inhibit viral infection.

[0017] Preferably, the drug delivery vehicle is a liposome nanoparticle.

[0018] In a third aspect, the present invention provides use of the pharmaceutical composition described in the second aspect in the preparation of a drug for treating Japanese encephalitis virus infection.

[0019] The beneficial effects of the present invention are as follows: the present invention unexpectedly found that the antimicrobial peptide WK-13-3D can significantly reduce the production of JEV RNA in a dose-dependent manner, inhibit the expression of JEV non-structural protein NS3, significantly reduce the production of infectious JEV, inhibit JEV adhesion to host cells, and have an antiviral activity of IC 50The value is 2.339 μM, which can inhibit the infection of Japanese encephalitis virus and can be used as a drug for the treatment of Japanese encephalitis virus infection, providing new drugs and treatment options for the clinical prevention and treatment of Japanese encephalitis virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Cytotoxicity of the antimicrobial peptide WK-13-3D against the human glioma cell line T98G;

[0021] Figure 2 Effects of the antimicrobial peptide WK-13-3D on JEV RNA, protein, and virus yields;

[0022] Figure 3 Effects of antimicrobial peptide WK-13-3D on JEV-infected T98G cells;

[0023] Figure 4 Effects of the antimicrobial peptide WK-13-3D on JEV adhesion and internalization into T98G cells. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are provided to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0026] The Japanese encephalitis virus JEV described in the following examples is the JEV-GS strain, which is derived from mosquitoes collected in Pingliang, Gansu Province.

[0027] Example 1 Cytotoxicity of the antimicrobial peptide WK-13-3D on human glioma cell line T98G

[0028] The company was commissioned to synthesize the antimicrobial peptide WK-13-3D. The specific sequence information of the antimicrobial peptide WK-13-3D is as follows:

[0029] WKRIVRRIKRWLR-NH2.

[0030] In order to verify the cytotoxicity of the antimicrobial peptide WK-13-3D on T98G cells, the antimicrobial peptide synthesized by the company was used to treat T98G cells in an increasing dose manner for 24 hours, and the cell viability of the cells in each dose treatment group was determined by the CCK-8 method. CC was calculated.50 value.

[0031] The results are as follows Figure 1 As shown, the antimicrobial peptide WK-13-3D described in the present application has low cytotoxicity to T98G cells, CC 50 The value is 18.94 μM.

[0032] Example 2 Effect of antimicrobial peptide WK-13-3D on JEV RNA, protein and virus production

[0033] At 37°C and 5% CO2, different doses of antimicrobial peptide WK-13-3D and MOI=1.0 JEV were incubated at 37°C for 1 hour, and then the antimicrobial peptide and virus mixture was incubated in T98G cells for 90 minutes. After washing with PBS twice, the cells were cultured in DMEM culture medium containing 2% FBS for another 48 hours, and the cell pellets and virus supernatants were collected. The effect of antimicrobial peptide WK-13-3D on the production of JEV RNA was detected by RT-qPCR; the effect of antimicrobial peptide WK-13-3D on the expression level of JEV-encoded non-structural protein NS3 was verified by immunoblotting; the collected virus supernatant was used to determine the virus titer by viral plaque technique to analyze the effect of antimicrobial peptide WK-13-3D on JEV production.

[0034] Real-time quantitative PCR results Figure 2 As shown in middle A, the antimicrobial peptide WK-13-3D can significantly reduce the production of JEV RNA in a dose-dependent manner.

[0035] The results of immunoblotting were Figure 2 As shown in Figure B, the antimicrobial peptide WK-13-3D can significantly inhibit the expression of JEV non-structural protein NS3 in a dose-dependent manner.

[0036] The collected cell culture supernatant was used to determine the yield of infectious virus particles by virus plaque assay. Figure 2 As shown in C, the antimicrobial peptide WK-13-3D can significantly reduce the yield of infectious JEV in a dose-dependent manner.

[0037] IC with antiviral activity 50 The value result is Figure 2 As shown in D, the statistical results show that the IC of the antiviral activity of the antimicrobial peptide WK-13-3D 50 The value is 2.339 μM.

[0038] Example 3 Effect of antimicrobial peptide WK-13-3D on JEV-infected T98G cells

[0039] At 37°C and 5% CO2, different doses of antimicrobial peptide WK-13-3D and MOI=1.0 JEV were incubated at 37°C for 1 hour, and then the antimicrobial peptide and virus mixture was incubated in T98G cells for 90 minutes. After washing twice with PBS, the cells were cultured in DMEM culture medium containing 2% FBS for another 36 hours. Rabbit NS3 protein antibody was used as the primary antibody, and the fluorescence intensity of NS3 protein was observed by immunofluorescence to analyze the effect of antimicrobial peptide WK-13-3D on JEV-infected T98G cells.

[0040] Immunofluorescence results Figure 3 As shown, the antimicrobial peptide WK-13-3D can significantly reduce the fluorescence intensity of JEV NS3 protein in a dose-dependent manner, indicating that the antimicrobial peptide WK-13-3D can significantly inhibit the infection of JEV.

[0041] Example 4 Effect of antimicrobial peptide WK-13-3D on JEV adhesion and internalization to T98G cells

[0042] 0 μM or 5 μM of the antimicrobial peptide WK-13-3D was mixed with MOI = 1.0 JEV, and JEV was incubated at 4°C for 30 min and then washed, or JEV was incubated at 4°C for 30 min and then washed. Then, the cells were incubated at 37°C for 1 hour, and the cell pellets were collected. The abundance of adhered or internalized viral RNA was analyzed by real-time quantitative PCR technology to analyze the effect of the antimicrobial peptide WK-13-3D on JEV adhesion and internalization to T98G cells.

[0043] Real-time quantitative PCR results Figure 4 As shown, A is the adhesion result and B is the internalization result. The results showed that the antimicrobial peptide WK-13-3D can significantly inhibit JEV adhesion to T98G cells, but has no significant effect on the virus internalization process of JEV infected T98G cells.

[0044] The above results indicate that the antimicrobial peptide WK-13-3D described in the present application has significant anti-JEV activity by reducing the production of JEV RNA, inhibiting the expression of JEV non-structural protein NS3, reducing the production of infectious JEV, and inhibiting JEV adhesion to host cells. It can be used as a drug for the treatment of Japanese encephalitis virus infection and has good application prospects.

Claims

1. Use of an antimicrobial peptide WK-13-3D or a composition containing the antimicrobial peptide WK-13-3D in the preparation of a drug for inhibiting viral infection, wherein the sequence of the antimicrobial peptide WK-13-3D is: WKRIVRRIKRWLR-NH2.

2. The use according to claim 1, characterized in that The virus is Japanese encephalitis virus.

3. The use according to claim 2, characterized in that The antimicrobial peptide WK-13-3D reduces the RNA content of Japanese encephalitis virus.

4. The use according to claim 2, characterized in that The antimicrobial peptide WK-13-3D inhibits the expression of the non-structural protein NS3 of Japanese encephalitis virus.

5. The use according to claim 2, characterized in that The antimicrobial peptide WK-13-3D inhibits Japanese encephalitis virus from adhering to host cells.

6. The use according to any one of claims 1 to 5, characterized in that: The antimicrobial peptide WK-13-3D is delivered via a drug delivery carrier.

7. The use according to claim 6, characterized in that The drug delivery carrier is liposome nanoparticles.

8. A pharmaceutical composition for inhibiting viral infection, characterized in that: The active ingredient of the pharmaceutical composition includes the antimicrobial peptide WK-13-3D, and the sequence of the antimicrobial peptide WK-13-3D is: WKRIVRRIKRWLR-NH2.

9. The pharmaceutical composition according to claim 8, characterized in that The active ingredients of the pharmaceutical composition also include one or more of small molecule compounds against Japanese encephalitis virus, Japanese encephalitis virus vaccines, and monoclonal antibodies against Japanese encephalitis virus.

10. Use of the pharmaceutical composition according to claim 8 or 9 in the preparation of a drug for treating Japanese encephalitis virus infection.

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