Application of Micropterus salmoides Piscidin polypeptide in preparation of anti-fish virus drugs

Abstract

The invention discloses an application of Micropterus salmoides Piscidin polypeptide in preparation of an anti-fish virus drug. The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 2 or SEQ ID NO. 3. The research finds that the micropterus salmoides Piscidin is an antibacterial peptide with an antiviral function, the polypeptide synthesized by the artificially synthesized micropterus salmoides Piscidin has the characteristic of inhibiting fish DNA virus replication, has a remarkable inhibiting effect on fish iridovirus replication at low concentration, and can be applied to preparation, research and development of fish antiviral drugs. Research of the antibacterial peptide has important scientific value and application value for further research and development of disease-resistant functional gene products.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of Piscidin polypeptide in the preparation of antiviral drugs for fish. Background Technology

[0002] Largemouth bass (Micropterus salmoides) is an important freshwater aquaculture fish in my country, highly favored by the market for its high nutritional value and delicious meat. However, with the continuous degradation of fish quality, high-density farming, deteriorating farming environments, and the unregulated use of antibiotics and other drugs, viral diseases in largemouth bass frequently break out and spread, severely hindering the healthy and sustainable development of its aquaculture industry. Among these, largemouth bass virus (LMBV) is one of the most important viral pathogens. Due to its rapid onset and high mortality rate, there are currently no effective control measures. Therefore, it is urgent to conduct basic research on the immune and disease resistance mechanisms of largemouth bass and to develop novel disease-resistant genetically engineered products using screened disease-resistant molecular targets.

[0003] Antimicrobial peptides (AMPs), as innate immune molecules, are a class of small molecule polypeptides widely used by plants and animals to defend against external microbial invasion. They have advantages such as low toxicity and low likelihood of developing drug resistance. Recent studies have found that antimicrobial peptides not only make them the most powerful alternative to antibiotics due to their unique antibacterial mechanisms, but they can also inactivate or inhibit viral infection through multiple mechanisms: (1) disrupting the integrity of the viral envelope, thereby preventing viral infection of host cells; (2) blocking the interaction between the virus and cell surface receptors, inhibiting viral adsorption; (3) weakening gene transcription and viral assembly after viral entry into cells, inhibiting viral replication and proliferation; (4) interfering with or modifying the antiviral immune signaling pathways of host cells, etc. Researchers have identified several AMPs in fish, which are divided into different families. These polypeptides are widely distributed in the mucous membranes, skin, and immune cells of fish and can be induced to express upon contact with external pathogens. The Piscidin family, a type of AMP unique to and widely distributed in bony fish (especially perciformes), consists of small polypeptides of 20–50 amino acid residues with an α-helix structure, exhibiting broad-spectrum antibacterial, antitumor, and antiviral activities. Previous studies have shown that multiple subtypes of the Piscidin gene exist in fish, and some members have a certain inhibitory effect on the replication of fish viruses. However, there are currently no clear reports on the role of Piscidin in the control of iridoviruses in largemouth bass. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of Piscidin and its synthetic polypeptide in the preparation of antiviral drugs for fish.

[0005] The first objective of this invention is to provide a largemouth bass Piscidin polypeptide, the amino acid sequence of which is shown in SEQ ID NO.3.

[0006] Preferably, the nucleotide sequence of the gene encoding the polypeptide is shown in SEQ ID NO.4.

[0007] A second object of the present invention is to provide a recombinant plasmid comprising the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.4.

[0008] A third objective of this invention is to provide recombinant bacteria containing the aforementioned recombinant plasmids.

[0009] A fourth objective of this invention is to provide the application of the above-described recombinant plasmid or recombinant bacteria in the preparation of a drug against largemouth bass iridovirus (LMBV).

[0010] The fifth object of the present invention is to provide the use of the largemouth bass Piscidin polypeptide in the preparation of a drug against largemouth bass iridovirus (LMBV), the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0011] Preferably, the concentration of the largemouth bass Piscidin polypeptide used is 80-120 μg / mL, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.3.

[0012] Preferably, the anti-Largemouth bass iridovirus (LMBV) is used to inhibit the replication of the Largemouth bass iridovirus (LMBV).

[0013] Preferably, the anti-largemouth bass iridovirus LMBV is largemouth bass anti-largemouth bass iridovirus LMBV.

[0014] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0015] Preferably, the dosage form of the drug is any one of oral liquid, powder, injection, and capsule.

[0016] The present invention has the following advantages:

[0017] 1. The novel largemouth bass Piscidin synthetic polypeptide of the present invention has the ability to resist largemouth bass iridovirus LMBV at the cellular level.

[0018] 2. The largemouth bass Piscidin synthetic polypeptide of the present invention is simple and convenient to prepare, the product is safe, non-toxic, non-polluting, and non-diffusion-free, can be produced on a large scale, and is easy to store.

[0019] 3. This invention has discovered that Piscidin, a novel antimicrobial peptide with antiviral function, is synthesized from largemouth bass. This synthetically produced Piscidin polypeptide exhibits the ability to inhibit the replication of fish DNA viruses, showing significant inhibitory effects on fish iridovirus replication even at low concentrations. It can be applied to the preparation and development of antiviral drugs for fish. The research on this antimicrobial peptide will have significant scientific and application value for further research and development of disease-resistant gene products. Attached Figure Description

[0020] Figure 1 The image shows the multiple sequence alignment results of Piscidin-like peptides from different fish species.

[0021] Figure 2 Figure A shows the distribution of Piscidin in various tissues of largemouth bass; Figure 2 In Figures B and C, the relative expression levels of the major LMBV capsid protein (MCP) (B) and Piscidin (C) in MSF cells changed over time after LMBV infection.

[0022] Figure 3 Piscidin exerts an antiviral effect in largemouth bass iridovirus infection.

[0023] Figure 4 Image A shows a quality control report for the synthetic largemouth bass Piscidin polypeptide. Figure 4 Image B is a mass spectrometry image of the synthetic peptide of Piscidin from largemouth bass.

[0024] Figure 5 The image shown in Figure A is a high-performance liquid chromatography (HPLC) chromatogram of the purified Piscidin polypeptide from largemouth bass. Figure 5 In Figure B, different concentrations of Piscidin-synthesized peptides from largemouth bass are shown to affect cell viability.

[0025] Figure 6 The figure in A shows that the Piscidin synthetic polypeptide of largemouth bass inhibits the replication of LMBV at the transcriptional level. Figure 6 In the middle B, the Piscidin-synthesized polypeptide of largemouth bass attenuated the LMBV-induced CPE changes, and the white arrows indicate LMBV-induced cell rounding and cell aggregation. Figure 6 In the middle, C represents the Piscidin synthesis peptide of largemouth bass, which inhibits the synthesis of the major capsid protein of LMBV. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. These embodiments are intended to illustrate the invention and are not intended to limit the invention in any way.

[0027] The conventional experimental methods and materials used in the embodiments of this invention are as follows:

[0028] 1. Cell lines and viruses: The cell lines were the largemouth bass fin (Micropterus salmoides fin, MSF) cell line (preserved by the Aquatic Animal Medicine Laboratory of South China Agricultural University, see Yang J, Xu W, Wang W, Pan Z, Qin Q, Huang X, Huang Y. Largemouth Bass Virus Infection Induced Non-Apoptotic Cell Deathin MsF Cells.Viruses. 2022 Jul 19;14(7):1568) and the fatheadmonnow (FHM) cell line (preserved by the Aquatic Animal Medicine Laboratory of South China Agricultural University, see Zhang X, Wang L, Liu J, et al. Generation and identification of novel DNA aptamers with antiviral activities against largemouth bass). LMBV virus [J]. Aquaculture, 2022, 547: 737478, was cultured at 28°C in Leibovitz L-15 medium (Sigma) containing 10% fetal bovine serum (FBS, Gibco). The virus used in the experiment was laboratory-preserved LMBV. MSF and FHM cells were infected with LMBV, and after three freeze-thaw cycles, the cells were collected and stored at -80°C. The half-maximal dose (TCID) method was used for infection. 50 The viral titer was determined to be 2.37 × 10⁻⁶. 8 TCID 50 / mL.

[0029] 2. Experimental fish: Largemouth bass were purchased from a largemouth bass farm in Dongcheng District, Sihui City, Guangzhou, China. The species was largemouth bass (Micropterus salmoides), which was cultured in the aquaculture system of the Guangzhou Nansha Huanong Fisheries Research Institute. The fish weighed 3-4g and had a body length of 7.0-8.0cm.

[0030] 3. Eukaryotic expression bacteria: Escherichia coli strain DH5α, a product of Beijing Qingke Biotechnology Co., Ltd., stored at -80℃.

[0031] 5. Experimental reagents: Leibovitz's L-15 medium Products of Sigma Corporation; trypsin (AmRESCO, 0458); FBS Purchased from GIBCO; LMBV-MCP antibody Prepared in the laboratory (refer to patent: Monoclonal antibody against largemouth bass iridovirus LMBV and its application, patent number: ZL202210174632.4), and used at a concentration ratio of 1:3000; Cell culture medium Leibovitz's L-15 medium containing 10% FBS (by volume) should be stored at 4°C for later use. LB medium 10g tryptone, 5g yeast extract, 10g sodium chloride (add 20g agar powder when preparing plates), dissolved in ddH2O, pH adjusted to 7.4 with 1mol / L NaOH, brought to a final volume of 1000mL, autoclaved at 120℃ for 20min, and stored at 4℃ for later use. 5×SDS-PAGE electrophoresis Buffer solution: Weigh 94.0g of Gly, 15.1g of Tris, and 5g of SDS, dissolve them in 800mL of ddH2O, stir thoroughly, then add water to make up to 1000mL, and store at room temperature until needed. Membrane transfer buffer Add 2.9g of Gly, 5.8g of Tris, and 0.37g of SDS to 800mL of ddH2O, stir thoroughly, and add 200mL of methanol before use. TBST 50 mL TrisHCl (1 M, pH 7.5), 8 g NaCl, 0.2 g KCl, 0.5 mL Tween, add distilled water to a final volume of 1 L; 5% skim milk Add 5g of skim milk to 100mL of TBST, dissolve completely, and store at 4℃.

[0032] 6. Cell transfection: MSF or 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 hours. Observation under a microscope was performed; once the cells had grown to a confluent monolayer, transfection could begin. Lipofectamine was used in this experiment. TM Use Invitrogen 2000 reagent (11668019) to transfect plasmids, following the instructions in the manual.

[0033] 7. Total RNA extraction from cells: Cell RNA was extracted using the SVTotal RNAIsolation Kit (Promega, Z3100), following the instructions in the manufacturer's manual.

[0034] 8. The quantitative real-time PCR (qRT-PCR) experiment was performed using a 2×SYBRGreen Real-time PCR Mix kit (TOYOBO, Japan, TYB-QPK-201), following the instructions.

[0035] 9. SDS-PAGE analysis and Western blotting electrophoresis gels were prepared using the kit (KGC4711-1) from Jiangsu Kaiji Biotechnology Co., Ltd., and the experimental procedures were performed according to the SDS-PAGE and Western blotting instructions. Finally, photographs were taken using a Tanon 5200 (Shanghai, China) electrophoresis system.

[0036] Example 1: Study on the in vitro antiviral activity of Piscidin eukaryotic expression protein

[0037] 1) Construction of eukaryotic recombinant plasmids

[0038] Construction of plasmid Piscidin-C1: Based on the EST sequence of the largemouth bass transcriptome, the largemouth bass Piscidin gene (its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2) was obtained by PCR amplification (primer sequences are shown in Table 1, namely Piscidin-F and Piscidin-R). Simultaneously, multiple sequence alignment analysis was performed to determine its structural characteristics. Figure 1 ).

[0039] The PCR amplification system is as follows:

[0040] Template (Msf cell cDNA): 2 μL

[0041] Primers: 1 μL each of Piscidin-F and Piscidin-R

[0042] 2×GS Taq PCRMix (Beijing Jinsha Biotechnology Co., Ltd., ST111): 12.5μL

[0043] Add 8.5 μL of sterile water to a total volume of 25 μL, mix well and set aside.

[0044] The PCR amplification program is: 95℃, 5 min; 95℃, 30s; 60℃, 30s; 72℃, 30s [35 cycles in total]; 72℃, 10 min; 16℃, 30 min.

[0045] The fragment was then inserted into plasmid pEGFP-C1 via homologous recombination (upstream restriction site XhoI, downstream restriction site KpnI) using primers Piscidin-C1-F and Piscidin-C1-R, sequences of which are shown in Table 1. The ligation product was transformed into E. coli DH5α and cultured on LB solid medium containing a final concentration of 50 μg / mL kanamycin. Successfully transformed strains were screened by picking single colonies and sequencing, and these strains contained the recombinant plasmid Piscidin-C1.

[0046] 2) Analysis of Piscidin Expression Pattern in Largemouth Bass

[0047] Real-time PCR analysis:

[0048] Total RNA was extracted from various tissues of healthy largemouth bass and from MSF cells infected with LMBV (see "Cell Transfection" above) at different time points (0, 6, 12, 24, 36, 48 h) according to the instructions using the Cell Total RNA Isolation Kit (Foregene). Subsequently, using... cDNA templates were obtained by reverse transcription using the qPCR RT Kit (TOYOBO) according to the manufacturer's instructions. 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, Japan, TYB-QPK-201) according to the manufacturer's instructions. The reaction conditions were as follows: 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.

[0049] Piscidin transcripts were detected in all tissues of healthy largemouth bass using qPCR technology. Figure 2 As shown in Figure A, piscidin is mainly expressed in the gills, intestine, heart, and skin. To reveal the expression pattern of piscidin during viral infection, the transcriptional level of LMBV-infected MSF cells was detected at different time points. Figure 2 B, 2C). The results showed that the expression level of the LMBV MCP gene gradually increased with the duration of infection, indicating efficient viral replication. Figure 2B); It was also found that, compared with the control group (0h after infection), the expression level of Piscidin began to rise after LMBV virus infection, reaching a peak at 12h, which was 3.5 times that of the control group. Figure 2 C).

[0050] 3) Study on the in vitro antiviral function of Piscidin

[0051] To determine the role of Piscidin in the process of fish virus infection, the effects of Piscidin overexpression on viral gene transcription and protein expression were detected by qPCR and WB, respectively.

[0052] FHM cells were digested with 0.25% trypsin and transferred to 24-well cell culture plates. The plates were incubated at 28°C for approximately 18 hours. Cells were observed under a microscope until they formed a confluent monolayer, at which point transfection could begin. Lipofectamine was used in this experiment. TM 2000 reagent (Invitrogen) for Piscidin-C1 ( Figure 3 Transfection with Piscidin-C1 was performed according to the instructions. Empty vector pEGFP-C1 was used as a control.

[0053] Real-time PCR is performed as described in step 2 above.

[0054] Western blot analysis:

[0055] The collected infected cells were lysed in RIPA buffer (Thermo Fisher Scientific), and 10 μL of 5× Loading Buffer was added and thoroughly vortexed. The denatured cell samples were then boiled. The samples were subsequently 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.

[0056] In Piscidin-transfected cells (Piscidin-C1), the transcriptional level of Piscidin was significantly upregulated compared with that of the empty vector (pEGFP-C1), indicating that Piscidin was successfully expressed in the transfected cells. Figure 3 A). Piscidin expression significantly reduced the transcriptional levels of MCP, MMP, and DNMT in LMBV. Figure 3 B) and the protein expression level of MCP ( Figure 3 C), while Piscidin expression significantly reduced the viral yield of LMBV ( Figure 3 D).

[0057] In conclusion, Piscidin exerts an antiviral effect in largemouth bass red color virus infection.

[0058] Table 1 Primers for amplification of internal control and viral genes

[0059]

[0060] Example 2: Study on the in vitro antiviral function of Piscidin synthetic peptides from largemouth bass

[0061] 1) Artificial synthesis of Piscidin polypeptide from largemouth bass

[0062] Piscidin polypeptide from largemouth bass was obtained by chemical synthesis at Anhui Guoping Pharmaceutical Co., Ltd. Its sequence is: EGFLGTLLHGAVHVSKILHGIMGGDHGVQEQEEQLDKRSTDYNSGRPGFS (SEQ ID No. 3, nucleotide sequence as shown in SEQ ID NO. 4). The quality of the synthesized polypeptide was determined using a mass spectrometer (Shimadzu LCMS-2020) according to the literature (Wang C, Wang S, Li D, Chen P, Han S, Zhao G, Chen Y, Zhao J, Xiong J, Qiu J, Wei DQ, Zhao J, Wang J. ACS Infect Dis. 2021 Jun 11;7(6):1545-1554. doi:10.1021 / acsinfecdis.1c00096). Figure 4 Purified using HPLC (Shimadzu HPLC-20AD / AT) Figure 5 A). After the largemouth bass Piscidin synthetic peptide was correctly tested, it was vacuum dried into powder, dispensed into 1 mg vials, and protected with nitrogen to prevent oxidative denaturation of the synthetic peptide. It was then stored in an ultra-low temperature freezer at -80°C.

[0063] 2) Application of Piscidin synthetic peptide in largemouth bass against LMBV

[0064] The cytotoxicity of Piscidin synthetic peptides in largemouth bass was determined by the method described in the reference (Rajanbabu V, Chen JY. Fish Shellfish Immunol. 2011; 30(1):39-44. doi:10.1016 / j.fsi.2010.09.005.). The results showed that it had no significant cytotoxicity at concentrations of 25–750 μg / mL. Figure 5 (B) Therefore, this embodiment uses a 100 μg / mL concentration of Piscidin synthesized from largemouth bass to investigate the effect of Piscidin on LMBV virus replication. The specific experimental method is as follows: FHM cells are passaged into 24-well plates, and approximately 1 × 10⁶ cells are seeded in each well. 5 Cells were collected. After 18 hours, the cells formed a confluent monolayer. Before viral infection, cells were pretreated for 1 hour with a largemouth bass Piscidin synthetic peptide (100 μg / mL) in serum-free Leibovitz's L-15 cell culture medium at 28°C. Cells were then infected with LMBV (2 MOI). Simultaneously, 500 μL of Leibovitz's L-15 medium containing 10% FBS was added to each well of a 24-well plate to a final volume of 600 μL. In the control group, the Piscidin synthetic peptide was replaced with the same volume of sterile water; other procedures were the same as the experimental group. 24 hours post-infection, cell cultures were collected, with three replicates from each experimental and control group. Total RNA was extracted, and viral gene transcription was detected by quantitative real-time PCR, while viral protein expression levels were analyzed by Western blot (specific steps are as described in Example 1).

[0065] The effect of Piscidin synthetic peptides on LMBV virus replication in largemouth bass is as follows: Figure 6 As shown in the figure, compared with the control group, the relative expression (fold induction) of LMBV-MCP, LMBV-MMP, and LMBV-DNMT genes in the Piscidin-treated group was significantly reduced. Figure 6 A); meanwhile, the number of virus-induced rounded and floating cells in the Piscidin-treated group was significantly reduced compared to the control group. Figure 6 B); In addition, compared with the control group, the synthesis of the synthetic polypeptide Piscidi n treatment group significantly reduced the synthesis of the major viral capsid protein (B); Figure 6 C). It is evident that the synthetic peptide of Piscidin from largemouth bass can significantly inhibit the replication of LMBV virus, exhibiting good anti-LMBV activity.

[0066] SEQ ID NO.1 (nucleotide sequence of Piscidin)

[0067] ATGAAGTGTATTGTGATCTTTCTCGTGTTGT CCATGGTCGTGCTGGTCGTCCTCATGGCTGAACCTGGAGAAGGCTTTCTTGGAACGCTTCTCCATGGAGCTGTTCACGTCAGCAAGATACTCCATGGGATTATGGGTGGGGACCACGGTGTCCAAGAGCAGGAAGAGCAGCTGGACAAACGTTCAACCGATTACAACTCAG GGCGGCCTGGTTTTTCCT AG SEQ ID NO.2 (Amino acid sequence of Piscidin)

[0068] MKCIVIFLVLSMVVLVVLMAEPGEGFLGTLLHGAVHVSKILHGIMGGDHGVQEQEEQLDKRSTDYNSGRPGFS

[0069] SEQ ID NO.3 (Synthetic polypeptide sequence of Piscidin)

[0070] EGFLGTLLHGAVHVSKILHGIMGGDHGVQEQEEQLDKRSTDYNSGRPGFS

[0071] SEQ ID NO.4 (Nucleotide sequence of synthetic polypeptide of Piscidin)

[0072] GAAGGCTTTCTTGGAACGCTTCTCCATGGAGCTGTTCACGTCAGCAAGATACTCCATGGGATTATGGGTGGGGACCACGGTGTCCAAGAGCAGGAAGAGCAGCTGGACAAACGTTCAACCGATTACAACTCAGGGCGGCCTGGTTTTTCC

Claims

1. A Piscidin polypeptide for largemouth bass, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

2. The Piscidin polypeptide for largemouth bass according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the polypeptide is shown in SEQ ID NO.

4.

3. A recombinant plasmid, characterized in that, It contains the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.

4.

4. Recombinant bacteria containing the recombinant plasmid as described in claim 3.

5. The use of the recombinant plasmid of claim 3 or the recombinant bacteria of claim 4 in the preparation of a drug against largemouth bass iridovirus (LMBV).

6. The application of Piscidin polypeptide in the preparation of drugs against largemouth bass iridovirus (LMBV), characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.2 or SEQ ID NO.

3.

7. The application according to claim 6, characterized in that, The Piscidin polypeptide used in the largemouth bass is at a concentration of 80-120 μg / mL, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

8. The application according to claim 6, characterized in that, The anti-Largemouth Black Bass Rhinovirus (LMBV) is designed to inhibit the replication of LMBV.

9. The application according to claim 6, characterized in that, The LMBV anti-largemouth bass iridovirus is a largemouth bass anti-largemouth bass iridovirus LMBV.

10. The application according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients, and the dosage form of the drug is any one of oral liquid, powder, injection, and capsule.

Images