Antibacterial peptide for resisting spring viremia of carp virus and application of antibacterial peptide

Abstract

The invention belongs to the technical field of molecular biology and microbiology, and particularly relates to an antibacterial peptide for resisting spring viremia of carp virus and application thereof, and the antibacterial peptide is any one of the following: 1) an amino acid sequence as shown in SEQ ID NO.1; and 2) a nucleotide sequence for coding the amino acid sequence as shown in SEQ ID NO. 1. The invention reveals that the antibacterial peptide NKLj27 has remarkable anti-SVCV activity for the first time, can effectively inhibit infection of SVCV on EPC cells at the cellular level, and alleviates cytopathy and plaque formation caused by viruses. Meanwhile, the NKLj27 shows a good protection effect on SVCV infection at a carp living body level, the death rate caused by virus infection can be remarkably reduced, and the evidences show that the NKLj27 has remarkable anti-SVCV activity. The invention provides a new candidate drug for prevention and control of fish virus diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology and microbiology, and particularly relates to an antibacterial peptide against spring viremia of carp virus and application thereof. BACKGROUND

[0002] Aquaculture is threatened by various viral diseases worldwide, among which the harm of spring viremia of carp is particularly serious. Spring viremia of carp is an infectious fish disease caused by spring viremia of carp virus. Spring viremia of carp virus, abbreviated as SVCV, belongs to the family of rhabdoviridae and the genus of vesiculovirus, and is a pathogen with strong infectivity and high pathogenicity. The virus has broken out and spread in many places in Asia, Europe and America. The virus has a wide host range and can infect common farmed fish such as carp, crucian carp, grass carp, silver carp and bighead carp. It is extremely harmful to young fish, and the mortality rate after infection can be as high as 90% or more. China has listed it as a class II animal epidemic.

[0003] At present, there is no specific treatment drug for SVCV infection, and the prevention measures mainly rely on strengthening breeding management, quarantine isolation and water disinfection. Therefore, the development of new antiviral preparations with high efficiency and safety has become an urgent need for the prevention and control of aquatic epidemic diseases. SUMMARY

[0004] The purpose of the present application is to provide an antibacterial peptide against spring viremia of carp virus, which solves the problem of lack of specific and efficient treatment drugs for SVCV in the prior art.

[0005] The technical solution adopted by the present application is as follows: The present application provides an antibacterial peptide against spring viremia of carp virus, which is any one of the following: 1) the amino acid sequence shown in SEQ ID NO. 1; 2) the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1.

[0006] The present application also provides an application of the antibacterial peptide, which is used for at least one of the following: 1) preparing a drug for preventing and / or treating spring viremia of carp; 2) preparing a drug against spring viremia of carp virus.

[0007] Preferably, the fish to which the drug is applicable includes at least one of crucian carp, grass carp, silver carp and bighead carp.

[0008] Preferably, the preparation method of the drug is as follows: The antibacterial peptide is dissolved in PBS or DMEM medium to obtain a solution of 150 μM-170 μM, and the drug is obtained.

[0009] Preferably, the preparation method of the medicine is as follows: The antibacterial peptide is dissolved in PBS or DMEM medium to obtain a solution of 160 μM, and the medicine is obtained.

[0010] Preferably, the medicine further comprises a pharmaceutically acceptable excipient.

[0011] Preferably, the pharmaceutically acceptable excipient comprises at least one of a filler, a flavoring agent, a binder, a disintegrant, an antacid, and a nutritional fortifier.

[0012] Preferably, the acceptable dosage form of the medicine comprises one of a tablet, a capsule, a granule, an injection, a pill, and a powder.

[0013] Preferably, the filler comprises any one of microcrystalline cellulose, starch, lactose, and mannitol.

[0014] Preferably, the flavoring agent comprises any one of steviol glycoside, aspartame, and sucrose.

[0015] Preferably, the binder comprises any one of starch paste and hydroxypropyl methylcellulose.

[0016] Preferably, the disintegrant comprises any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0017] Preferably, the antacid comprises any one of calcium carbonate and aluminum hydroxide.

[0018] Preferably, the nutritional fortifier comprises any one of vitamin A, vitamin C, and vitamin D.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application provides an antibacterial peptide against spring viremia of carp virus, which is any one of the following: 1) an amino acid sequence shown in SEQ ID NO. 1; and 2) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1. The present application first discloses that the antibacterial peptide NKLj27 has significant anti-SVCV activity, can effectively inhibit the infection of EPC cells by SVCV at the cellular level, and reduce the cytopathic effect and plaque formation caused by the virus. At the same time, NKLj27 shows good protective effect on SVCV infection at the level of live carp, and can significantly reduce the mortality caused by viral infection. These evidences show that NKLj27 has significant anti-SVCV activity. The present application provides a new candidate drug for the prevention and control of fish viral diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1The results of bioinformatics analysis of NKLj27 are as follows: A: Sequence alignment of NKLj27 with NK-lysin-derived peptides from bony fishes, using different colors to characterize residue similarity levels: Dark blue: 100% similarity; Pink: ≥75% similarity; Light blue: ≥50% similarity; Yellow: ≥33% similarity. B: Predicted secondary structure of NKLj27; C: Extended α-helix configuration in the tertiary structure of NKLj27; D: Bent α-helix configuration in the tertiary structure of NKLj27.

[0021] Figure 2 The effect of NKLj27 on SVCV G gene mRNA expression.

[0022] Figure 3 The effect of NKLj27 on EPC lesions.

[0023] Figure 4 Crystal violet staining results showing the effect of NKLj27 on EPC plaques. A: EPC after 24 hours of culture; B: EPC after 48 hours of culture.

[0024] Figure 5 Safety evaluation of NKLj27.

[0025] Figure 6 The anti-SVCV effect of NKLj27 in carp. Detailed Implementation

[0026] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0027] The inventive concept of this invention is as follows: Antimicrobial peptides are an important component of the body's natural immune defense system. They have broad-spectrum antimicrobial activity, unique mechanisms of action, and are less likely to induce drug resistance, showing great potential in the development of antiviral drugs.

[0028] This invention systematically evaluates the antiviral effects of NK-lysin-derived peptide NKLj27 from sturgeon against SVCV at both cellular and in vivo levels, providing new candidate molecules and technical support for the prevention and control of viral diseases in fish.

[0029] This invention first predicted and synthesized a 27-amino acid polypeptide NKLj27 based on the amino acid sequence of the SapB domain of NK-lysin in sea bass. Bioinformatics analysis showed that NKLj27 has a molecular weight of 3073.77 Da, a theoretical isoelectric point of 9.79, and a net charge of +5. Its secondary and tertiary structures are predominantly α-helical, accounting for 88.89%. Sequence homology comparison results showed that NKLj27 has a sequence identity of 66.67%–79.17% with known NK-lysin polypeptides in bony fishes, including a high degree of similarity with the oval pomfret (…). Trachinotus ovatus ) and Georgian icefish ( Pseudochaenichthys georgianus It has the highest homology, demonstrating its high degree of conservation in the evolutionary process.

[0030] In this invention, pretreatment with 160 μM NKLj27 can significantly inhibit the infection of carp spring viremia virus on carp epithelioma cells and effectively reduce the formation of plaques caused by the virus. In in vivo experiments on carp, NKLj27 pretreatment can significantly reduce the mortality rate of carp after SVCV infection, indicating that it has significant anti-SVCV activity in vivo. This invention provides a new candidate drug for the prevention and control of viral diseases in fish.

[0031] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0032] The list of abbreviations for this invention is shown in Table 1.

[0033] Table 1. List of abbreviations for this invention Example 1 An antimicrobial peptide against spring viremia virus in carp and its application are detailed below: 1. Bioinformatics analysis of NKLj27.

[0034] Analysis of the physicochemical parameters of NKLj27 showed that its molecular weight was 3.07 kDa, its theoretical isoelectric point was 9.79, and its net charge was +5. DNAMAN was used to compare the amino acid sequences of NKLj27 with those of NK-lysin in other bony fishes. The results showed that NKLj27 is similar to that of the oval pomfret (…). Trachinotus ovatus Georgian icefish ( Pseudochaenichthys georgianus ), Redfin Pufferfish ( Takifugu rubripes African toothed carp ( Nothobranchius furzeri ), pikeperch ( Sander lucioperca ), Drillmouth Fish ( Chelmon rostratus ), Leopard Gill Spinach ( Plectropomus leopardus ), Burr's shoulder hole Antarctic fish (Trematomus bernacchii ), Pointed-headed naked dragonfish ( Gymnodraco acuticeps ), eel ( Monopterus albus ), Yellowfin pufferfish ( Takifugu flavidus ), naked scaly fish ( Anoplopoma fimbria ), sea bream ( Embiotoca jacksoni Yellow perch ( Perca flavescens ), spotted-tailed damselfish ( Stegastes partitus ) and perch ( Perca fluviatilis The sequence identity rates were 79.17%, 79.17%, 78.26%, 78.26%, 77.78%, 76.92%, 74.07%, 74.07%, 74.07%, 73.91%, 73.91%, 73.08%, 73.08%, 70.37%, 70.37%, and 66.67%, respectively.

[0035] The secondary and tertiary structures of NKLj27 were predicted using I-TASSER. Secondary structure prediction showed that NKLj27 exhibits a typical α-helix-dominated conformation, containing α-helix structures and random coils, with α-helices accounting for 11.11% and random coils accounting for 88.89%. Tertiary structure prediction showed that the polypeptide's spatial conformation is centered on α-helices, a result highly consistent with the secondary structure prediction. These results are shown in [link to results]. Figure 1 .

[0036] 2. In vitro experiments: The antimicrobial peptide NKLj27 inhibited the infection of EPC cells by SVCV and the formation of plaques.

[0037] This embodiment evaluates the antiviral activity of the antimicrobial peptide NKLj27 against carp spring viremia virus through in vitro experiments, including its safety evaluation against carp epithelioma cells and its inhibitory effect on the SVCV infection process. The amino acid sequence of the antimicrobial peptide NKLj27 used in this embodiment is shown in SEQ ID NO.1. Its synthesis and purification were carried out according to existing literature methods, and the purity was not less than 95% as determined by high performance liquid chromatography.

[0038] SEQ ID NO. 1: KLTSKLKSVCNEMGLLKSLCHKFVTKH.

[0039] 2.1 Experimental materials.

[0040] Antimicrobial peptide NKLj27: Dissolved in serum-free DMEM medium, prepared as a 160 μM stock solution, aliquoted and stored at -20 °C, then brought to room temperature before experimentation.

[0041] Target cells: EPC cells, preserved in our laboratory, were cultured in DMEM medium containing 10% v / v fetal bovine serum at 26℃, 5% CO2, and logarithmic growth phase cells were used for experiments.

[0042] Virus: SVCV virus, a preserved strain in our laboratory, was amplified by EPC cells, and the virus titer was 1×10 6 PFU / mL, preserved at -80℃.

[0043] 2.2, Anti-SVCV activity detection.

[0044] (1) Effect of NKLj27 on the expression of SVCV G gene mRNA.

[0045] EPC cells were inoculated in a 6-well plate at a density of 2×10 5 cells / well, and cultured in a 26℃, 5% CO2 incubator for 24h until the cells were completely adherent. The original culture medium was discarded, and 2mL of culture medium containing 160μM NKLj27 was added to each well, and pre-treated for 1h. 2mL of 10 -3 diluted SVCV virus liquid was added, and after 26℃ adsorption for 1h, the virus liquid was discarded, and fresh complete culture medium was replaced for continuous culture. At the same time, an equal volume of PBS was used to replace NKLj27 as a control group.

[0046] Cells were collected at 3h, 24h and 48h after virus liquid infection, total RNA was extracted by Trizol method, and reverse transcribed into cDNA. The cDNA was used as a template, and the mRNA expression level of SVCV G gene was detected by real-time quantitative PCR, and the internal reference gene β-actin was standardized. The primer sequences of SVCV G gene are shown in SEQ ID NO.2 and SEQ ID NO.3, and the primer sequences of β-actin gene are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0047] SEQ ID NO.2: Upstream primer, 5'-CGACCTGGATTAGACTTG-3.

[0048] SEQ ID NO.3: Downstream primer, 5'-AATGTTCCGTTTCTCACT-3'.

[0049] SEQ ID NO.4: Upstream primer, 5'-TGAAGATCCTGACCGAGCGT -3'.

[0050] SEQ ID NO.5: Downstream primer, 5'-GGAAGAAGAGGCAGCGGTTC-3'.

[0051] The2⁻ΔΔCt The relative expression of SVCV G gene was calculated, and the results are shown in Figure 2 Table 1. The mRNA expression levels of SVCV G gene in NKLj27+SVCV treatment group were significantly lower than those in control group at 3h, 24h and 48h after infection, p <0.01, and the inhibitory effect was more obvious with the extension of time, indicating that NKLj27 had time-dependent anti-SVCV activity.

[0052] (2) EPC plaque formation observation.

[0053] EPC cells were inoculated in 12-well plates at 1x10 5 cells / well and cultured for 24h to adhere. The original culture medium was discarded, and 1mL of NKLj27 culture medium with a concentration of 160μM was added for 1h of incubation. 1mL of 10 -3 diluted SVCV virus solution was added, and after 1h of adsorption, complete culture medium was replaced for continued culture for 24h and 48h. After completion of the culture, the cells were fixed with 4% paraformaldehyde solution, stained with 1% crystal violet, washed, and photographed to record the plaque formation. At the same time, the normally cultured cells were recorded as the Control group; the treatment group without the addition of NKLj27 culture medium but with the addition of 1mL of 10 -3 diluted SVCV virus solution was recorded as the SVCV group.

[0054] Further observation of the effect of the addition of NKLj27 on the plaque state of EPC cells infected with SVCV. The results of microscopic observation are shown in Figure 3 , showing that the cell morphology of the NKLj27+SVCV group was relatively complete, and the cell density was higher, while the cells of the SVCV group showed more pathological changes and death.

[0055] Crystal violet staining is shown in Figure 4 , and the observation shows that the pathological area of the NKLj27 treatment group is relatively small, and with the extension of the infection time, the difference in the degree of pathological changes is more obvious; especially at 48h, the number of plaques in the NKLj27 treatment group is significantly less than that in the SVCV infection group only. This result directly reflects that NKLj27 has a significant inhibitory effect on SVCV infection, and this inhibitory effect can be reflected in the early and late stages of infection.

[0056] (3) Safety evaluation of NKLj27 on EPC cells.

[0057] In order to evaluate the safety of NKLj27 on EPC cells, different concentrations of NKLj27 were incubated with EPC cells for 12h, and then CCK-8 reagent was added, and the absorbance at 450nm was measured at 1h, 1.5h, 2h and 4h of incubation.

[0058] The results are shown inFigure 5 With the change of NKLj27 concentration and the extension of CCK-8 incubation time, A 450 showed a certain regularity, but there was no significant difference compared with the Control group, indicating that NKLj27 had no significant cytotoxicity in the experimental concentration range. The Control group was the normal culture group of cells

[0059] 3. In vivo experiment: prevention and control of SVCV infection in carp.

[0060] This example verifies the prevention and control effect of antibacterial peptide NKLj27 on carp spring viremia virus infection at the in vivo level through carp live infection experiment, and provides experimental basis for its practical application.

[0061] 3.1. Experimental materials and conditions.

[0062] Experimental animals: 150 healthy carps, body weight 12±0.8g, body length 12±1cm, purchased from a certain aquaculture base in Qingdao, Shandong Province. The experimental fish was temporarily raised in an indoor recirculating water culture system for 7 days, the water temperature was maintained at 20±1℃, commercial extruded feed was fed twice a day, natural light cycle, and no abnormality and death phenomenon during the temporary raising period.

[0063] NKLj27 preparation: the purified NKLj27 was dissolved in sterile PBS buffer, and an injection solution with a final concentration of 160μM was prepared, and it was prepared on demand.

[0064] Virus solution: prepared after SVCV virus was amplified by EPC cells, the titer was 1×10 6 PFU / mL, stored at -80℃.

[0065] 3.2. Experimental grouping and treatment.

[0066] Select 100 healthy carps, randomly divide them into 2 groups, 50 in each group, and the specific treatment is as follows: NKLj27+SVCV group: each fish was injected with 100μL of 160μM NKLj27 injection solution through abdominal cavity, 1h later, 100μL of SVCV virus solution was injected at the same site.

[0067] PBS+SVCV group: each fish was injected with 100μL of sterile PBS through abdominal cavity, 1h later, 100μL of SVCV virus solution was injected.

[0068] After injection, the death of carps was observed and recorded in detail for 15d in succession to analyze the anti-SVCV effect of NKLj27 in carps.

[0069] 3.3. Observation and statistics.

[0070] Observe the fish continuously for 15 days after injection, record the number of dead fish in each group daily, and remove dead fish in a timely manner. After the experiment, calculate the cumulative mortality rate and plot the survival curve.

[0071] 3.4 Results.

[0072] like Figure 6 As shown, within 15 days post-infection, the cumulative mortality rate in the PBS+SVCV group was significantly higher, while the mortality rate in the NKLj27+SVCV group was significantly lower. These results indicate that intraperitoneal injection of NKLj27 can effectively improve the resistance of carp to SVCV infection and significantly reduce mortality caused by the virus, further confirming that NKLj27 has significant antiviral protective effects at the in vivo level and possesses the potential to be developed into a drug for treating SVCV infection in fish.

[0073] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0074] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide against spring viremia virus of carp, characterized in that, The antimicrobial peptide is any one of the following: 1) The amino acid sequence shown in SEQ ID NO.1; 2) The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

1.

2. The application of the antimicrobial peptide as described in claim 1, characterized in that, The antimicrobial peptide is used in at least one of the following: 1) Prepare drugs for the prevention and / or treatment of spring viremia in carp; 2) Preparation of drugs against carp spring viremia virus.

3. The application as described in claim 2, characterized in that, The fish species for which the drug is applicable include at least one of crucian carp, grass carp, silver carp, and bighead carp.

4. The application as described in claim 2, characterized in that, The preparation method of the drug is as follows: The antimicrobial peptide is dissolved in PBS or DMEM medium to obtain a 150 μM to 170 μM solution, which is the drug.

5. The application as described in claim 4, characterized in that, The preparation method of the drug is as follows: The antimicrobial peptide was dissolved in PBS or DMEM medium to obtain a 160 μM solution, which is the drug.

6. The application as described in claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application as described in claim 6, characterized in that, Pharmaceutically acceptable excipients include at least one of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

8. The application as described in claim 2, characterized in that, The acceptable dosage forms of the drug include one of the following: tablets, capsules, granules, injections, pills, and powders.

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