Application of largemouth bass hepcidin-1 and synthetic polypeptide thereof in preparation of anti-fish virus drugs

Abstract

This invention provides the application of Hepcidin-1 (Ms-Hepcidin-1) and its synthetic polypeptide in the preparation of antiviral drugs for largemouth bass. The invention reveals that the Ms-Hepcidin-1 synthetic polypeptide exhibits significant antiviral activity and is a novel antimicrobial peptide with the function of inhibiting fish viruses. Both the Ms-Hepcidin-1 synthetic polypeptide and its recombinant eukaryotic expression vector can significantly reduce the transcriptional levels of MCP, MMP, and DNMT genes and the MCP protein level of LMBV, inhibit LMBV replication, and reduce viral titers, demonstrating significant antiviral activity against fish viruses. Furthermore, the polypeptide synthesized from Ms-Hepcidin-1 is simple to prepare, low in cost, safe to use, and easy to store. As a novel antiviral functional gene product, it can be applied to the preparation of antiviral drugs for fish, which is of great significance for the prevention and control of iridovirus disease in fish.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of largemouth bass Hepcidin-1 (Ms-Hepcidin-1) and its synthetic polypeptide in the preparation of antiviral drugs for fish. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Largemouth bass are an important economically important freshwater aquaculture fish in my country, highly favored by the market due to their high nutritional value and delicious meat. However, with the continuous expansion of aquaculture scale, increased stocking density, deterioration of the aquaculture environment, and overuse of antibiotics, viral diseases of largemouth bass have frequently broken out and spread, seriously restricting the healthy and sustainable development of the aquaculture industry. Among them, largemouth bass virus (LMBV) is one of the most important viral pathogens. Currently, the control of fish diseases still mainly relies on antibiotics and chemical drugs, but these traditional methods are prone to causing a series of problems such as drug residues, increased pathogen resistance, and water pollution. Therefore, it is crucial to carry out basic research on fish immunity and disease resistance and to develop new disease-resistant genetically engineered products.

[0003] Because fish are poikilothermic animals, their adaptive immune systems are defective, making their innate immune systems play a more crucial role in defending against pathogens. Antimicrobial peptides (AMPs), as innate immune molecules, play a key role in the host's fight against pathogen infection. These gene-encoded polypeptides are widely distributed in the mucous membranes, skin, and immune cells of fish and can be induced to express upon exposure to environmental pathogens. AMPs can not only directly inhibit microbial growth but also enhance host immunity. To date, researchers have identified a large number of AMPs in fish, generally classified into several different families. Hepcidin is a cysteine-rich cationic antimicrobial peptide in fish, primarily secreted by the liver. Mature hepcidin typically consists of 20-25 amino acids and also has iron metabolism regulatory functions. Structurally diverse secreted antimicrobial peptides can serve as candidate molecules for novel drug development to treat infections caused by drug-resistant pathogens. Although studies have shown that the Hepcidin family has a certain inhibitory effect on the replication of some fish viruses, the specific inhibitory effects and mechanisms of action require further investigation. Currently, there is limited research on the functional effects of the antimicrobial peptide hepcidin in largemouth bass, especially regarding its antiviral properties in fish. To better control largemouth bass iridovirus, it is necessary to develop more antiviral drugs for fish and provide new strategies for the prevention and control of largemouth bass viral diseases. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of existing drugs for the prevention and control of largemouth bass iridovirus disease and the research on the antiviral effect of largemouth bass Hepcidin (Ms-Hepcidin-1). The present invention provides the application of Ms-Hepcidin-1 and its synthetic polypeptide in the preparation of antiviral drugs for fish.

[0005] The first objective of this invention is to provide a synthetic peptide of Ms-Hepcidin-1.

[0006] A second objective of this invention is to provide applications for the synthesis of Ms-Hepcidin-1 polypeptides.

[0007] A third objective of this invention is to provide applications of Ms-Hepcidin-1.

[0008] A fourth object of the present invention is to provide the use of a formulation expressing Ms-Hepcidin-1 as an anti-largemouth bass iridovirus formulation.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention relates to a synthetic polypeptide of Hepcidin-1 from largemouth bass, the amino acid sequence of which is shown in SEQ ID NO.3.

[0011] This invention reveals that Ms-Hepcidin-1 is a novel antimicrobial peptide with the function of inhibiting the replication of fish viruses. The nucleotide sequence of Ms-Hepcidin-1 is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. By preparing a recombinant eukaryotic expression vector for Ms-Hepcidin-1 and transfecting it into cells, it was found that expression of Ms-Hepcidin-1 significantly reduced the transcriptional levels of MCP, MMP, and DNMT genes of largemouth bass iridovirus (LMBV) and the MCP protein synthesis level, and significantly reduced the viral titer in virus-infected cells. This indicates that Ms-Hepcidin-1 can significantly inhibit the infection and replication of LMBV in cells and has a significant antiviral effect. Simultaneously, this invention synthesizes a Ms-Hepcidin-1 polypeptide through the Hepcidin-1 gene of largemouth bass. The amino acid sequence of this polypeptide is shown in SEQ ID NO.3. Studies have shown that the Ms-Hepcidin-1 polypeptide has good biocompatibility and no significant toxicity to fish cells. Treatment of cells with low concentrations of the synthetic polypeptide can significantly inhibit LMBV infection and replication, significantly reduce the transcriptional expression levels of LMBV MCP, MMP, and DNMT genes and the viral MCP protein level, and reduce the number of rounded cells in LMBV-infected cells, demonstrating anti-LMBV virus infection activity. The synthetic polypeptide provided by this invention, as a novel antiviral functional gene product, can be applied to the preparation of antiviral drugs for fish. The preparation of this polypeptide is simple, low-cost, safe to use, and easy to store, and has important application value for the prevention and control of largemouth bass iridovirus disease. Therefore, this invention discloses for the first time the novel application of Ms-Hepcidin-1 and its synthetic polypeptide in the prevention and control of fish iridovirus, which can be used to prepare more anti-fish iridovirus drugs and is of great significance for the prevention and control of fish iridovirus disease.

[0012] This invention provides the application of the Ms-Hepcidin-1 synthetic peptide as an anti-largemouth bass iridovirus preparation.

[0013] This invention provides the application of the Ms-Hepcidin-1 synthetic peptide in the preparation of a drug for treating infection with largemouth bass iridovirus.

[0014] This invention provides the use of the Ms-Hepcidin-1 synthetic peptide in the preparation and / or treatment of largemouth bass iridovirus disease.

[0015] Preferably, the product is a feed additive or a medicine.

[0016] This invention provides the application of Ms-Hepcidin-1 synthetic peptide in the treatment of largemouth bass iridovirus for non-disease diagnosis and treatment purposes.

[0017] In particular, the Ms-Hepcidin-1 synthetic peptide can be used for laboratory in vitro anti-LMBV studies.

[0018] This invention provides the application of Ms-Hepcidin-1 in the preparation of drugs against largemouth bass iridovirus.

[0019] Preferably, the drug against largemouth bass iridovirus is prepared by in vitro expression of Ms-Hepcidin-1.

[0020] This invention provides the use of a formulation expressing Ms-Hepcidin-1 as an anti-largemouth bass iridovirus formulation.

[0021] This invention provides the application of a formulation expressing Ms-Hepcidin-1 in the preparation of a drug for treating infection with largemouth bass iridovirus.

[0022] This invention provides the application of a formulation expressing Ms-Hepcidin-1 in the preparation of a drug for treating infection with largemouth bass iridovirus.

[0023] This invention provides the use of formulations expressing Ms-Hepcidin-1 in the preparation of products for the prevention and / or treatment of iridovirus disease in largemouth bass.

[0024] Preferably, the preparation is a plasmid, a vector, or a recombinant bacterium.

[0025] More preferably, the formulation is a Ms-Hepcidin-1 recombinant eukaryotic expression plasmid (Hepcidin-1-C1) containing the nucleotide sequence shown in SEQ ID NO.1 or the amino acid sequence shown in SEQ ID NO.2.

[0026] Preferably, the dosage form of the above-mentioned drug is any one of oral liquid, powder, and injection.

[0027] Preferably, the above-mentioned drug contains a pharmaceutically acceptable carrier or excipient.

[0028] The present invention has the following beneficial effects:

[0029] This invention is the first to discover that Ms-Hepcidin-1 and its synthetic polypeptide possess significant antiviral activity, representing a novel antimicrobial peptide with inhibitory function against fish viruses. Both the recombinant eukaryotic expression vector of Ms-Hepcidin-1 and the synthetic polypeptide significantly reduced the transcriptional levels of MCP, MMP, and DNMT genes and the MCP protein level in LMBV, inhibited LMBV replication in FHM cells, and reduced viral titers in virus-infected cells, demonstrating significant antiviral activity against fish viruses. Furthermore, the polypeptide synthesized from Ms-Hepcidin-1 is simple to prepare, low in cost, safe to use, and easy to store. It significantly inhibits LMBV infection and replication, reduces viral titers, and resists viral infection. As a novel antiviral functional gene product, it can be applied to the preparation of antiviral drugs for fish, which is of great significance for the prevention and control of iridovirus disease in fish. Attached Figure Description

[0030] Figure 1 The image shows the sequence alignment results of Ms-Hepcidin-1 and Hepcidin-1 from different fish species (the image shows the multiple sequence alignment results of Hepcidin-1 from different fish species).

[0031] Figure 2 The figure shows the effect of Ms-Hepcidin-1 recombinant plasmid expression on LMBV replication (A in the figure is the expression detection of the Hepcidin-1-C1 eukaryotic vector recombinant plasmid; B is the effect of Ms-Hepcidin-1 expression on LMBV gene transcription; C is the effect of Ms-Hepcidin-1 expression on LMBV MCP protein synthesis; D is the effect of Ms-Hepcidin-1 expression on progeny virus production).

[0032] Figure 3 Figure 1 shows the purification and identification results of the Ms-Hepcidin-1 synthetic peptide (A is the mass spectrometry detection of the Ms-Hepcidin-1 synthetic peptide; B is the HPLC detection of the Ms-Hepcidin-1 synthetic peptide).

[0033] Figure 4 The figure shows the effect of different concentrations of Ms-Hepcidin-1 synthetic peptides on cell viability.

[0034] Figure 5The figure shows the effect of Ms-Hepcidin-1 synthetic peptide on LMBV replication (A in the figure shows the effect of Ms-Hepcidin-1 synthetic peptide on LMBV gene transcription; B shows the effect of Ms-Hepcidin-1 synthetic peptide on virus-induced CPE, and the white arrows indicate LMBV-induced cell rounding and cell aggregation; C shows the effect of Ms-Hepcidin-1 synthetic peptide on LMBV MCP protein synthesis). Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Example 1: Structural characteristics of Ms-Hepcidin-1 and construction of recombinant plasmid

[0038] 1. Homology analysis of Hepcidin-1 (Ms-Hepcidin-1) in largemouth bass

[0039] Based on the EST sequence of the largemouth bass transcriptome, the Ms-Hepcidin-1 gene was obtained by PCR amplification. The nucleotide sequence of this gene is shown in SEQ ID NO.1: ATGAAGACACTCAGTGTTGCAGTTGCAGTGGCCGTCGTGCTCACCTTTATTTGCCTTCCGGAGAGCTCTGCTGTCCCAGTCACTGAGGTGCAGGAGCTGGAGGAGCCAATGAGCGATGACAATCTAGCTGCTGCACACGTAGACATGTCAGTGGAATCCTGGAAGATGCCATATAACAACAGACAGAAGCGCGGCATTAAATGTCGCTTTTGCTGTGGCTGCTGCATCCCCGGCGTCTGTGGATTGTGCTGCAGGTTCTGA. The encoded amino acid sequence is shown in SEQ ID NO.2: MKTLSVAVAVAVVLTFICLPESSAVPVTEVQELEEPMSDDNLAAAHVDMSVESWKMPYNNRQKRGIKCRFCCGCCIPGVCGLCCRF.

[0040] The obtained Ms-Hepcidin-1 gene was compared with the amino acid sequences of Hepcidin-1 from different fish species using ClustalX 1.83 and GENEDOC for multiple sequence alignment analysis to determine structural characteristics. The results showed that the full-length Ms-Hepcidin-1 sequence obtained by PCR amplification contained 261 bases, encoding an 86-amino acid polypeptide. The amino acid sequence alignment results are shown below. Figure 1 As shown, Ms-Hepcidin-1 is associated with the golden-headed seabream ( Sparus aurata The homology of () was 66.28%.

[0041] 2. Construction of Ms-Hepcidin-1 eukaryotic expression recombinant plasmid

[0042] Subsequently, the Ms-Hepcidin-1 gene sequence was inserted into the plasmid pEGFP-C1 via homologous recombination. The resulting recombinant plasmid was transformed into Escherichia coli DH5α and cultured on LB solid medium containing kanamycin. Successfully transformed strains were then screened by bacterial PCR and sequencing to obtain the Ms-Hepcidin-1 eukaryotic expression recombinant plasmid Hepcidin-1-C1.

[0043] Example 2: Antiviral function of Ms-Hepcidin-1 in vitro cells

[0044] 1. Ms-Hepcidin-1 gene transcription and protein expression

[0045] (1) Cell transfection

[0046] Fathead Monnow (FHM) cells were digested with 0.25% trypsin and transferred into 24-well cell culture plates. The plates were incubated at 28 °C for approximately 18 h. Microscopic observation was performed until the cells reached confluent concentration and formed a monolayer, at which point transfection could begin. The cells were transfected using Lipofectamine according to the reagent instructions. TM The Hepcidin-1-C1 recombinant plasmid and the pEGFP-C1 empty vector were transfected into FHM cells using the 2000 reagent (Invitrogen).

[0047] 24 h after infection, cells were collected and the transcription and protein expression of the Ms-Hepcidin-1 gene in transfected cells were detected by quantitative real-time PCR and Western blotting, respectively.

[0048] (2) Real-time PCR analysis

[0049] Total RNA was extracted from the cells of each treatment group using the Cell Total RNA Isolation Kit (Foregene) according to the manufacturer's instructions. Subsequently, ReverTra Ace was used to analyze the RNA. ® cDNA templates were obtained by reverse transcription using the qPCR RT Kit (TOYOBO). qPCR reactions were performed on an Applied Biosystems QuantStudio 5 real-time PCR instrument (Thermo, USA) using a 2×SYBR Green Real-time PCR Mix kit (TOYOBO) under the following conditions: 95℃, 1 min; 95℃, 5 s; 60℃, 15 s; 72℃, 45 s; 40 cycles. β-actin was used as an internal control. The internal control and primers used for viral gene amplification are shown in Table 1. The final detection data represent the mean ± standard deviation of three replicate experiments.

[0050] Table 1 Primers for amplification of internal control and viral genes

[0051]

[0052] (3) Western blot analysis

[0053] The collected infected cells were lysed in RIPA buffer (Thermo Fisher Scientific), and after adding 10 μL of 5× Loading buffer, the mixture was thoroughly vortexed and boiled to remove the denatured cells. The samples were then separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). The membrane containing the samples was blocked by incubating with 5% skim milk at room temperature for 2 h, followed by overnight incubation at 4°C with the corresponding primary antibodies, including anti-β-tubulin antibody (1:3000, Abcam) and anti-LMBV-MCP antibody (1:2000, prepared in our laboratory, derived from existing patent technology: CN114230660A). After washing three times with PBST, the membrane was incubated with the corresponding secondary antibodies at room temperature for 1 h, including horseradish peroxidase (HRP)-labeled goat anti-rabbit or goat anti-mouse IgG antibody (1:5000, Proteintech). Finally, the color was developed using ECL colorimetric solution (Bioscience) on a Tanon 5200 automated chemiluminescence imaging system.

[0054] (4) Results Analysis

[0055] The results are as follows Figure 2As shown in Figure A, qPCR analysis revealed that the transcriptional level of Hepcidin-1 was significantly upregulated in Hepcidin-1-C1 transfected cells compared to cells transfected with the empty vector. Simultaneously, Western blot analysis indicated a significant increase in the protein level of Hepcidin-1 in Hepcidin-1-C1 transfected cells, demonstrating successful expression of Hepcidin-1 in FHM cells.

[0056] 2. Effects of Ms-Hepcidin-1 expression on viral gene transcription and expression

[0057] Based on the above experimental methods, largemouth bass virus (LMBV) with an infection index (MOI) of 2 was inoculated into transfected cells. Twenty-four hours after infection, virus-infected cells were harvested by trypsin digestion, and the virus was analyzed by quantitative real-time PCR (qPCR), Western blotting (WB), and total tissue infectious dose (TCID). 50 The transcription, protein expression, and viral titer of transfected cells were detected. The transcription levels of MCP, MMP, and DNMT genes related to LMBV were determined by qPCR, and the expression of LMBV MCP protein was determined by WB, using the same detection methods as above.

[0058] qPCR and Western blotting results of Ms-Hepcidin-1 expression on viral gene transcription and protein expression showed that, compared with cells transfected with empty vector, the transcription levels of MCP, MMP, and DNMT of LMBV were significantly reduced in cells expressing Ms-Hepcidin-1 (e.g., ...). Figure 2 (As shown in B in the figure). Western blotting analysis also showed that the MCP protein level of LMBV was significantly reduced in cells expressing Ms-Hepcidin-1 (e.g., as shown in B in the figure). Figure 2 (as shown in C).

[0059] 3. Effect of Ms-Hepcidin-1 expression on LMBV viral titer

[0060] The collected infected cells were subjected to three freeze-thaw cycles at -20°C, and the results were analyzed using the median tissue infectious dose (TCID). 50 The viral titer was determined using the following method: FHM cells were transferred to 96-well cell culture plates and cultured overnight at 28°C. The collected infected cell samples were serially diluted 10-fold. 100 µL of the diluted virus-infected cell sample was seeded into each well, and then incubated at 28°C. On day 5 post-infection, the number of wells exhibiting cytopathic effect (CPE) was observed and recorded, and the TCID in the infected samples was calculated using the Reed-Muench method. 50 .

[0061] The results are as follows Figure 2 As shown by D, the viral titer in cells transfected with the empty vector 24 h after infection is 10. 6.49 TCID 50 / mL, the viral titer decreased to 10 in Hepcidin-1-C1 transfected infected cells. 5.75 TCID 50 / mL.

[0062] In conclusion, Hepcidin-1 expression significantly inhibits LMBV replication in FHM cells, indicating that it has antiviral function.

[0063] Example 3 Synthesis and purification of Ms-Hepcidin-1 peptide

[0064] After homology comparison of the amino acid sequence of Ms-Hepcidin-1 (SEQ ID NO.2) with the amino acid sequences of Hepcidin-1 from other species, its mature peptide region was determined. The mature peptide of Ms-Hepcidin-1 was synthesized, purified and identified by chemical synthesis method of Shanghai Sangon Biotech Co., Ltd., and its sequence is shown in SEQ ID NO.3: MSDDNLAAAHVDMSVESWKMPYNNRQKRGIKCRFCCGCCIPGVCGLCCRF.

[0065] The mass spectrometry results of the synthesized Ms-Hepcidin-1 peptide are as follows: Figure 3 As shown, Figure 3 In this context, A represents a synthetically produced polypeptide whose mass is determined by mass spectrometry. Figure 3 B in the table represents the result of peptide determination after HPLC purification; the chromatographic detection results are shown in Table 2. After being identified and confirmed, the Ms-Hepcidin-1 synthetic peptide was vacuum dried into powder, dispensed into 1 mg tubes, and protected with nitrogen to prevent oxidative denaturation of the synthetic peptide. The powder was then stored in an ultra-low temperature freezer at -80°C for later use.

[0066] Table 2 Chromatographic Peak Table

[0067]

[0068] Note: Detector A, channel 1, 220nm.

[0069] Example 4: Effect of Ms-Hepcidin-1 peptide on cell viability

[0070] The effect of different concentrations of Ms-Hepcidin-1 synthetic peptide treatment on cell viability was detected using the Cell Counting Kit-8 (CCK-8) assay. FHM cells were seeded in 96-well plates and cultured overnight at 28°C. Each well was then replaced with 100 μL of medium containing different concentrations (0, 25, 50, 100, 125, 250, 500, or 750 μg / mL) of Ms-Hepcidin-1 synthetic peptide, with four replicates per concentration. After 48 h of incubation, the peptide-containing medium was discarded, and 100 μL of pre-prepared 10% CCK-8 solution was added and incubated in the dark for 2 h. Finally, the absorbance (OD value) of each well was read at 450 nm using a multi-sensor microplate reader (ThermoFisher Science, USA). The effect of different concentrations of synthetic peptide treatment on cell viability was calculated using the formula (Cell viability = OD of crude polysaccharide group / Average OD of control group).

[0071] The results are as follows Figure 4 As shown, the Ms-Hepcidin-1 synthetic peptide has no significant toxicity to cells at concentrations of 25–750 μg / mL. Therefore, the concentration used in subsequent experiments was 100 μg / mL.

[0072] Example 5: Effect of Ms-Hepcidin-1 peptide on the replication of largemouth bass iridovirus

[0073] FHM cells were passaged into 24-well plates, with approximately 1 × 10⁶ cells seeded per well. 5 Cells were collected. After 16-24 hours, the cells formed a confluent monolayer. The original culture medium was discarded, and the cells were pretreated for 1 hour with serum-free medium containing 100 μg / mL Ms-Hepcidin-1 synthetic peptide. Cells were then infected with LMBV virus (MOI=2), with an equal volume of sterile water used as a control group. Infected cells were harvested 24 hours after infection by trypsin digestion and analyzed by quantitative real-time PCR, Western blotting, and TCID50. 50 To detect viral gene transcription, protein expression, and viral titer in transfected cells, qPCR, Western blotting, and TCID were performed. 50 The detection method and procedure are the same as in Example 2. Simultaneously, at 24 hours after infection, the lesions of infected cells in each group were observed using a phase-contrast microscope.

[0074] The results are as follows Figure 5 As shown, compared with the control group, the transcriptional expression levels of MCP, MMP, and DNMT genes of LMBV and the protein level of viral MCP were significantly reduced in the Ms-Hepcidin-1 synthetic peptide treatment group (e.g., Figure 5 A and Figure 5(As shown in C). Furthermore, compared to the control group infected cells, the Ms-Hepcidin-1 synthetic peptide-treated infected group had fewer rounded cells and only a small number of cells shed (e.g., ...). Figure 5 (As shown in B in the diagram).

[0075] In summary, the studies show that Ms-Hepcidin-1 and its synthetic peptides have significant antiviral effects, can significantly inhibit the replication of LMBV virus, and have good anti-LMBV infection and replication activity. They can be applied to the preparation of antiviral drugs for fish. As a new antiviral functional gene product, Ms-Hepcidin-1 and its synthetic peptides have important application value in the prevention and control of largemouth bass iridovirus disease.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A synthetic polypeptide of Hepcidin-1 from largemouth bass, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

2. The polypeptide according to claim 1, characterized in that, The polypeptide was synthesized from largemouth bass Hepcidin-1; the nucleotide sequence of the largemouth bass Hepcidin-1 is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

3. The use of the polypeptide of claim 1 in the preparation of an anti-largemouth bass iridovirus formulation.

4. The use of the polypeptide of claim 1 in the preparation of a drug for treating infection with largemouth bass iridovirus.

5. The use of the polypeptide of claim 1 in the preparation of products for the prevention and / or treatment of iridovirus disease in largemouth bass.

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