Application of Ms-Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitors or therapeutic drugs

Abstract

The present invention discloses Ms Application of ‑Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitors or therapeutic drugs, characterized in that it provides Ms Application of ‑Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitors and Ms Application of ‑Piscidin antimicrobial peptide in the preparation of therapeutic drugs for largemouth bass rhabdovirus infection, Ms The amino acid sequence of the antimicrobial peptide ‑Piscidin is shown in SEQ ID NO.1: FLKHIKSFWRGAKAIFRGARQGWREHR, and its advantages are Ms ‑Piscidin antimicrobial peptide can inhibit the proliferation of MSRV and improve the survival rate of largemouth bass infected with MSRV, and can be used as a potential drug for the prevention and treatment of MSRV-infected diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture and specifically relates to a Ms -Application of Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitors or therapeutic drugs. Background Art

[0002] Piscidin antimicrobial peptide is an important natural immune molecule in fish, playing a key role in maintaining fish immune homeostasis and resisting pathogen infection. Piscidin antimicrobial peptide is composed of a signal peptide at the amino terminal, a mature peptide, and a pro-domain at the carboxyl terminal, of which the mature peptide is the structural region of Piscidin with antibiological activity.

[0003] As a class of broad-spectrum antimicrobial molecules, Piscidin antimicrobial peptides exhibit significant biological functions in the antiviral process. Generally, Piscidin antimicrobial peptides directly act on the viral envelope through their unique amphipathic structure, destroying the structural integrity of the virus, thereby preventing the virus from entering the host cell and inhibiting its replication. Piscidin antimicrobial peptides can not only directly interact with the phospholipids in the viral envelope, leading to the lysis and inactivation of the virus, but also can regulate the body's immune system and activate key components of the innate immune response of fish, such as macrophages and natural killer cells, further enhancing the body's antiviral defense mechanism. Studies have shown that Piscidin antimicrobial peptides can induce the production of reactive oxygen species in host cells, destroy the replication environment of the virus, and activate host immune signaling pathways, such as the NF-κB signal transduction pathway, thereby promoting local inflammatory responses and limiting the spread of the virus. In addition, Piscidin antimicrobial peptides can also directly bind to viral proteins or nucleic acids, interfering with their normal functions and further inhibiting the proliferation of the virus.

[0004] The broad-spectrum antiviral activity and low drug resistance of Piscidin antimicrobial peptides make them potential applications in aquaculture, especially in controlling fish virus outbreaks. However, although the antiviral mechanism of Piscidin antimicrobial peptides in vivo has been gradually revealed, its specific application in the prevention and treatment of fish viruses is still in the exploratory stage.

[0005] Largemouth bass rhabdovirus ( Micropterus salmoides rhabdovirusMSRV (abbreviated as MSRV) is one of the main pathogens that cause viral diseases of largemouth bass, often leading to serious deaths of important freshwater economic fish such as largemouth bass. The outbreak of this virus not only brings significant economic losses, but also poses a threat to the sustainable development of largemouth bass aquaculture industry. As an emerging high-quality freshwater aquaculture species in my country, largemouth bass has the characteristics of rapid spread and high mortality due to viral diseases, which has attracted widespread attention from relevant scientific research and aquaculture units. At present, although there has been some progress in virus isolation, tissue pathology research, genome analysis, etc., no effective prevention and control strategy has been found. In-depth research on the pathogenic characteristics of MSRV and the proposal of targeted prevention and control plans have become an urgent need for the largemouth bass aquaculture industry to respond to virus outbreaks and reduce economic losses. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a Ms The application of -Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitors or therapeutic drugs can be used to prevent and treat aquatic animal diseases caused by largemouth bass rhabdovirus.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: the present invention provides Ms -Application of Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitors.

[0008] Furthermore, the Ms The amino acid sequence of -Piscidin antimicrobial peptide is shown in SEQ ID NO.1: FLKHIKSFWRGAKAIFRGARQGWREHR.

[0009] The present invention also provides Ms -Application of Piscidin antimicrobial peptide in the preparation of therapeutic drugs for largemouth bass rhabdovirus infection.

[0010] Furthermore, the Ms -Piscidin antimicrobial peptide is used in a dosage of 0.1-10 mg per kg of largemouth bass.

[0011] Compared with the prior art, the present invention has the advantages that: the present invention discloses for the first time Ms -Piscidin antimicrobial peptide is used as a new drug to prevent and treat largemouth bass rhabdovirus (abbreviated as MSRV) infection. The results of in vitro experiments show that Ms -Piscidin antimicrobial peptide can significantly inhibit the replication of MSRV and alleviate the degree of cytopathic effect; experimental animal survival rate model confirmed Ms -Piscidin antimicrobial peptide plays a protective role against MSRV infection. Largemouth bass infected with MSRV were injected Ms-Piscidin antimicrobial peptide significantly reduced the mortality rate caused by viral infection. Ms -Piscidin antimicrobial peptide can inhibit the proliferation of MSRV and improve the survival rate of largemouth bass infected with MSRV, and can be used as a potential drug for the prevention and treatment of MSRV-infected diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For Example 1 Ms -The effect of Piscidin antimicrobial peptide on the proliferation activity of MSRV in vitro;

[0013] Figure 2 For different concentrations in Example 1 Ms -Optical microscopy of MSRV-infected lesions treated with Piscidin antimicrobial peptide;

[0014] Figure 3 For Example 2 Ms -The results of the effect of Piscidin antimicrobial peptide on the survival rate of largemouth bass infected with MSRV. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below with reference to the accompanying drawings.

[0016] Embodiment 1: Ms -Effect of Piscidin antimicrobial peptide on the infection activity of MSRV.

[0017] Largemouth bass Piscidin Ms -Piscidin, with the accession number of the gene bank UAD82650.1, is 76 amino acid residues in total length, consisting of a signal peptide (1-22 amino acids), a mature peptide (23-49 amino acids) and a pro-domain (50-76 amino acids). The signal peptide and the pro-domain need to be removed to release the mature peptide and exert its biological activity. Therefore, the solid phase peptide synthesis method was used to synthesize Ms -Piscidin mature peptide, defined as Ms -Piscidin antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1: FLKHIKSFWRGAKAIFRGARQGWREHR.

[0018] Epithelioma papulosum cyprini (EPC) cells were seeded in 6-well plates and cultured to monolayer cells using DMEM medium containing 10 wt % fetal bovine serum.

[0019] Control group: 50 μL MSRV virus solution was mixed with equal volumes of PBS buffer to prepare 100 μL MSRV virus suspension. Figure 1 The middle one is marked as PBS;

[0020] Experimental group: 50 μL MSRV virus suspension, 10 μM Ms -Piscidin solution and PBS buffer were mixed to a total volume of 100 μL of virus-antimicrobial peptide mixture and treated at 22°C for 1 h. The experimental groups were set as 3 groups. Ms -Piscidin final concentrations were 0.01 μM, 0.1 μM and 1 μM, Figure 1 The corresponding markers are 10nM, 100nM and 1 μM.

[0021] 100 μL of the MSRV virus suspension of the control group and the virus-antimicrobial peptide mixture of the three experimental groups were inoculated into the monolayer EPC cells. After infection at 22°C for 2 hours, the MSRV virus suspension was removed and washed three times with PBS buffer, and then DMEM medium containing 10wt% fetal bovine serum was added. After further culture for 48 hours, the cells were collected after observation under an optical microscope, and the expression of the MSRV virus G gene was detected by qRT-PCR. RNA was extracted using the TriQuick kit (manufactured by Solebo), and then reverse transcribed using the HiScript® III All-in-one RT SuperMix Perfect for qPCR kit (manufactured by Norwexan), and random primers were used to synthesize cDNA. The reverse transcription reaction system was as follows: 5× All-in-one qRT SuperMix 4 μL, Enzyme Mix 1 μL, RNA 1 μg, and ddH2O was supplemented to 20 μL. The reaction conditions were: 50°C for 15 minutes, and 85°C for 20 seconds to inactivate the reverse transcriptase activity.

[0022] The cDNA obtained by reverse transcription was used as a template, and qRT-PCR detection was performed using Taq Pro Universal SYBR qPCR Master Mix (manufacturer: Novozyme). The reaction system was: 2× Rapid Taq Master Mix 10 μL, 0.4 μL each of forward and reverse primers, 5 μL of cDNA template, and ddH2O supplemented to 20 μL. The PCR reaction program was: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 10 seconds, annealing and extension at 60℃ for 30 seconds, for a total of 40 cycles. The nucleotide sequence of the qRT-PCR forward amplification primer is shown in SEQ ID NO.2: 5'-AAGAGCCCGAGAGAAAAAT-3'; The nucleotide sequence of the qRT-PCR reverse amplification primer is shown in SEQ ID NO.3: 5'-TGAATAGCGGTCCATCAAC-3', which specifically amplifies the MSRV virus G gene. The internal reference control is β-actin, the nucleotide sequence of the β-actin internal reference forward amplification primer is shown in SEQ ID NO.4: 5'-GCTATGTGGCTCTTGACTTCGA-3', and the nucleotide sequence of the β-actin internal reference reverse amplification primer is shown in SEQ ID NO.5: 5'-CCGTCAGGCAGCTCATAGCT-3'.

[0023] After the reaction, the expression of MSRV virus G gene was quantitatively analyzed, and the expression of β-actin was used as a reference. The ΔΔCt method was used to quantitatively analyze the expression of MSRV virus G gene. Figure 1 As shown, Ms -Piscidin pretreatment can significantly inhibit the infection activity of MSRV, and the inhibitory effect is Ms -Piscidin concentration-dependent, in Ms In the treatment group with -Piscidin concentration of 1 μM, the relative expression level of MSRV virus G gene was only 19.33% of that in the PBS control group.

[0024] like Figure 2 As shown in Figure 2, optical microscopy revealed that the degree of cytopathic effect caused by MSRV infection was Ms -Piscidin treatment significantly reduced the Ms -Piscidin concentration-dependent, compared with the control group, Ms In the group treated with -Piscidin at a concentration of 1 μM, only a small number of cell plaques appeared.

[0025] Embodiment 2: Ms -Effect of Piscidin antimicrobial peptide on mortality of largemouth bass infected with MSRV.

[0026] Healthy largemouth bass (about 2.5 g / fish) were cultured in a recirculating aquaculture system at a water temperature of 25°C. A positive control group, a negative control group and three experimental groups were set up, with 30 fish in each group.

[0027] In the positive control group, largemouth bass were intraperitoneally injected with 100 μL / tail of a mixture of MSRV virus suspension and PBS buffer at a volume ratio of 1:1. Figure 3 Marked as MSRV.

[0028] In the negative control group, largemouth bass were intraperitoneally injected with PBS buffer (100 μL / tail). Figure 3 The medium is marked as PBS.

[0029] In the experimental group, the largemouth bass were intraperitoneally injected with 100 μL / tail of the virus-antimicrobial peptide mixture prepared in Example 1. The experimental group was set to 3 groups. Ms -Piscidin final concentrations were 1 μM, 0.1 μM and 0.01 μM, Figure 3 The corresponding mark in Ms -Piscidin (10 mg / kg), Ms -Piscidin (1 mg / kg) and Ms -Piscidin (0.1 mg / kg).

[0030] After intraperitoneal injection, the fish were observed for 15 consecutive days, and the mortality of largemouth bass was recorded. Finally, the survival rate of largemouth bass was analyzed using the Mantel-Cox method. Figure 3 As shown in the figure, the largemouth bass in the positive control group died on the third day of infection, and the survival rate dropped to 0 after the 10th day of infection; in the experimental group, the largemouth bass also died on the third day of infection, but after the 5th, 6th, and 8th days of infection, the infected largemouth bass no longer died. Ms -The survival rate of the Piscidin (1 mg / kg) group was 63.33%, Ms -The survival rate of the Piscidin (10 mg / kg) group was 70%, and Ms -The survival rate of the Piscidin (0.1 mg / kg) group was 30%.

[0031] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A Ms -Application of Piscidin antimicrobial peptide in the preparation of largemouth bass rhabdovirus inhibitor, characterized in that: The Ms The amino acid sequence of -Piscidin antimicrobial peptide is shown in SEQ ID NO.1: FLKHIKSFWRGAKAIFRGARQGWREHR.

2. A Ms -Application of Piscidin antimicrobial peptide in the preparation of a drug for treating rhabdovirus infection of largemouth bass, characterized in that: The Ms The amino acid sequence of -Piscidin antimicrobial peptide is shown in SEQ ID NO.1: FLKHIKSFWRGAKAIFRGARQGWREHR.

3. According to claim 2 Ms -Application of Piscidin antimicrobial peptide in the preparation of a drug for treating rhabdovirus infection of largemouth bass, characterized in that: The Ms -Piscidin antimicrobial peptide is used in a dosage of 0.1-10 mg per kg of largemouth bass.

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