Use of antibacterial peptide WK-13-3D in preparation of a medicine for inhibiting Japanese encephalitis virus infection

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to application of antibacterial peptide WK-13-3D in preparation of a medicine for inhibiting Japanese encephalitis virus infection. 50 The application surprisingly finds that the antibacterial 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 yield of infectious JEV, inhibit the adhesion of JEV to host cells, and the IC 50 value of the antiviral activity is 2.339 micromoles, 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 good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological gene engineering, and particularly relates to application of an antibacterial peptide WK-13-3D in preparation of a Japanese encephalitis virus infection inhibiting medicine. BACKGROUND

[0002] Japanese encephalitis virus (JEV) is also called B-type encephalitis virus, belongs to flavivirus of flavivirus family, and is a single-stranded RNA virus with envelope. JEV is a pathogen of Japanese encephalitis (JE), is mainly transmitted by mosquitoes, causes permanent neurological deficits in some surviving patients, including seizures, paralysis and low intelligence, and can also cause various pathological damages such as abortion of pregnant sows, death of piglets and orchitis of boars, which leads to the decline of production performance of pigs, and causes great threat to human health and healthy development of animal husbandry.

[0003] At present, an effective JE specific drug treatment method needs to be found, early intervention by antibodies or drugs is performed to reduce the sequelae of patients as much as possible.

[0004] Antibacterial peptides are small molecular polypeptides which are widely present in organisms, are coded by specific genes, have the effects of resisting invasion of external microorganisms and eliminating mutant cells in the body, and are important components of biological natural immune defense system. Researches show that WK-13-3D is a bacterial membrane inhibition related antibacterial peptide, can inhibit bacteria and their biofilms, especially methicillin-resistant Staphylococcus aureus, and the antibacterial peptide WK-13-3D can inhibit the proliferation of triple-negative breast cancer MDA-MB-231 cells, inhibit the growth of tumors in vivo, promote the apoptosis of MDA-MB-231 cells, and then play an anti-tumor effect.

[0005] The application accidentally finds that the antibacterial 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 can inhibit the infection of Japanese encephalitis virus. SUMMARY

[0006] In view of the above technical problems, the application accidentally finds that the antibacterial 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 can inhibit the infection of Japanese encephalitis virus. The specific contents include the following contents:

[0007] In a first aspect, the present application provides use of an antibacterial peptide WK-13-3D or a composition containing the antibacterial peptide WK-13-3D in the preparation of a medicament for inhibiting viral infection, wherein the sequence of the antibacterial peptide WK-13-3D is: WKRIVRRIKRWLR-NH 2.

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

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

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

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

[0012] Preferably, the antibacterial peptide WK-13-3D or the composition containing the antibacterial peptide WK-13-3D is added to a pharmaceutically acceptable adjuvant to form a pharmaceutically acceptable dosage form.

[0013] In a second aspect, the present application provides a pharmaceutical composition for inhibiting viral infection, wherein the active ingredient of the pharmaceutical composition comprises an antibacterial peptide WK-13-3D, and the sequence of the antibacterial peptide WK-13-3D is: WKRIVRRIKRWLR-NH 2.

[0014] Preferably, the active ingredient of the pharmaceutical composition further comprises one or more of a small molecule compound against Japanese encephalitis virus, a Japanese encephalitis virus vaccine, and a monoclonal antibody against Japanese encephalitis virus.

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

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

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

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

[0019] The beneficial effects of the present application are: the present application unexpectedly finds that the antibacterial 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 yield of infectious JEV, inhibit JEV adhesion to host cells, and the IC 50The value is 2.339 muM, can inhibit the infection of Japanese encephalitis virus, can be used as a drug for treating Japanese encephalitis virus infection, and provides a new drug and treatment scheme for clinical prevention and treatment of Japanese encephalitis virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Cytotoxicity of antibacterial peptide WK-13-3D on human glioma cell line T98G;

[0021] Figure 2 Effect of antibacterial peptide WK-13-3D on JEV RNA, protein and virus yield;

[0022] Figure 3 Effect of antibacterial peptide WK-13-3D on JEV infection of T98G cells;

[0023] Figure 4 Effect of antibacterial peptide WK-13-3D on JEV adhesion and internalization of T98G cells. DETAILED DESCRIPTION

[0024] To make the purpose, technical scheme and advantages of the present application clearer, the embodiments of the present application will be described in detail below with reference to the drawings. However, those skilled in the art can understand that in the embodiments of the present application, many technical details are proposed in order to make the reader better understand the present application. However, the technical scheme claimed by the present application can be realized even without these technical details and various changes and modifications based on the following embodiments.

[0025] In the following examples, the experimental methods are conventional methods unless otherwise specified; and in the following examples, the test materials used are obtained from conventional biochemical reagent companies unless otherwise specified.

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

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

[0028] The antibacterial peptide WK-13-3D was synthesized by a commissioned company, and the specific sequence information of the antibacterial peptide WK-13-3D is as follows:

[0029] WKRIVRRIKRWLR-NH2.

[0030] In order to verify the cytotoxicity of antibacterial peptide WK-13-3D on T98G cells, the T98G cells were treated with the antibacterial peptide synthesized by the company in a dose-increasing manner for 24 hours, and then the cell viability of each dose treatment group was determined by CCK-8 method, and the CC50 Values.

[0031] Results as shown in Figure 1 Figure 2A, the cytotoxicity of the antibacterial peptide WK-13-3D to T98G cells was low, and the CC 50 value was 18.94 μM.

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

[0033] The different doses of the antibacterial peptide WK-13-3D and MOI = 1.0 JEV were incubated at 37 °C for 1 h, and then the mixture of the antibacterial peptide and the virus was incubated in T98G cells for 90 min. After washing twice with PBS, the cells were cultured in DMEM medium containing 2% FBS for 48 h. The cell precipitate and the virus supernatant were collected, and the effects of the antibacterial peptide WK-13-3D on JEV RNA production were detected by RT-qPCR. The effects of the antibacterial peptide WK-13-3D on the expression level of the JEV coding non-structural protein NS3 were verified by immunoblotting. The collected virus supernatant was subjected to virus plaque assay to determine the virus titer, and the effects of the antibacterial peptide WK-13-3D on JEV production were analyzed.

[0034] The results of real-time quantitative PCR are shown in Figure 2 A, and the antibacterial peptide WK-13-3D can significantly reduce the production of JEV RNA in a dose-dependent manner.

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

[0036] The collected cell culture supernatant was subjected to virus plaque assay to determine the production of infectious virions, and the results are shown in Figure 2 C, and the antibacterial peptide WK-13-3D can significantly reduce the production of infectious JEV in a dose-dependent manner.

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

[0038] Example 3 Effects of the antibacterial peptide WK-13-3D on JEV infected T98G cells

[0039] The different doses of the antibacterial peptide WK-13-3D and the MOI = 1.0 JEV were incubated at 37°C for 1 h, respectively, and then the mixture of the antibacterial peptide and the virus was incubated in the T98G cells for 90 min. After being washed twice with PBS, the cells were continuously cultured in the DMEM medium containing 2% FBS for 36 h. The fluorescence intensity of the NS3 protein was observed by immunofluorescence using the rabbit-derived NS3 protein antibody as the primary antibody, so as to analyze the influence of the antibacterial peptide WK-13-3D on the JEV infection of the T98G cells.

[0040] The immunofluorescence results are shown in Figure 3 It can be seen that the antibacterial peptide WK-13-3D can significantly reduce the fluorescence intensity of the JEV NS3 protein in a dose-dependent manner, which indicates that the antibacterial peptide WK-13-3D can significantly inhibit the infection of the JEV.

[0041] Example 4: Influence of the antibacterial peptide WK-13-3D on the adhesion and internalization of JEV to the T98G cells

[0042] The 0 μM or 5 μM antibacterial peptide WK-13-3D was mixed with the MOI = 1.0 JEV, respectively, and then the JEV was incubated at 4°C for 30 min and washed or incubated at 4°C for 30 min and washed, and then incubated at 37°C for 1 h. The cell precipitate was collected, and the abundance of the adhered or internalized viral RNA was analyzed by the real-time quantitative PCR technique, so as to analyze the influence of the antibacterial peptide WK-13-3D on the adhesion and internalization of the JEV to the T98G cells.

[0043] The real-time quantitative PCR results are shown in Figure 4 The results show that the antibacterial peptide WK-13-3D can significantly inhibit the adhesion of the JEV to the T98G cells, but has no significant influence on the internalization process of the JEV to the T98G cells.

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

Claims

1. The use of antimicrobial peptide WK-13-3D or a composition containing antimicrobial peptide WK-13-3D in the preparation of a medicament for inhibiting viral infection, wherein the antimicrobial peptide WK-13-3D is: WKRIVRRIKRWLR-NH2; and the virus is Japanese encephalitis virus.

2. The application as described in claim 1, characterized in that, The antimicrobial peptide WK-13-3D reduces the RNA content of Japanese encephalitis virus.

3. The application as described in claim 1, characterized in that, The antimicrobial peptide WK-13-3D inhibits the expression of the non-structural protein NS3 of Japanese encephalitis virus.

4. The application as described in claim 1, characterized in that, The antimicrobial peptide WK-13-3D inhibits the adhesion of Japanese encephalitis virus to host cells.

5. The application as described in any one of claims 1-4, characterized in that, The antimicrobial peptide WK-13-3D is delivered via a drug delivery carrier.

6. The application as described in claim 5, characterized in that, The drug delivery carrier is a liposome nanoparticle.

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