Zebrafish ifn f interferon truncated protein and its application in antibiosis

Abstract

The application relates to the fields of biological medicine and aquaculture, and discloses a zebrafish IFNf interferon truncated protein and application thereof in antibiosis. The zebrafish interferon derivative peptide AMP-Z2 can specifically inhibit fungi. The interferon derivative peptide provided by the application is a host natural peptide, has small molecular weight, is simple to synthesize, has significant bactericidal effects on various bacteria and fungi, and can be used for preparing broad-spectrum antibacterial agents or antibacterial drugs.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and aquaculture, specifically to zebrafish IFNf interferon truncated protein and its application in antibacterial activity. Background Technology

[0002] Interferons (IFNs) are a class of cytokines with multiple functions, including antiviral, antibacterial, and immunomodulatory effects. The vertebrate interferon family can be divided into four types: type I, type II, type III, and type IV. However, fish lack type III interferons, possessing only type I, type II, and type IV. Type I interferons can be further divided into group 1 (IFNa, IFNd, IFNe, IFNh), group 2 (IFNb, IFNc, and IFNi), and group 3 (IFNf). The IFNf subtype is the only member of group 3 and the oldest member of the type I interferon family in bony fish, currently reported only in basal ray-finned fish (Chinese sturgeon and gar) and three species of teleost fish (Japanese eel, Atlantic salmon, and rainbow trout). Fish IFNf exhibits higher susceptibility to RNA virus infection than other interferons, playing a crucial role in the body's first line of defense against RNA viruses. In addition, interferon-derived peptides possess similar physicochemical properties and broad-spectrum antibacterial functions to antimicrobial peptides (AMPs). Therefore, interferon IFNf and its naturally derived peptides have broad application prospects in disease control and drug development.

[0003] Cypriniformes is the most widely distributed and diverse freshwater fish group within the class Osteichthyes, encompassing many species of significant scientific and economic value. Among them, the grass carp (Ctenopharyngodon idella) is the most farmed freshwater fish, while the zebrafish (Danio rerio) is one of the most important model species due to its unique biological characteristics, such as high reproductive rate, rapid development, and transparent embryos. Current research shows that both grass carp and zebrafish possess only seven interferon genes, with type I interferons comprising only three subtypes: IFNa (IFN1), IFNc (IFN2, IFN3), and IFNd (IFN4). No reports have been found on other subtypes, especially the earlier-originating IFNf.

[0004] This invention marks the first discovery of a novel interferon subtype, IFNf, in the important economic species grass carp and the important model species zebrafish. The antiviral function of this gene and the broad-spectrum antibacterial function of its derived peptides are also reported. This new gene and its naturally derived peptides will contribute to the development of more efficient and safer antiviral and antibacterial drugs, potentially addressing issues such as drug resistance and toxicity associated with current antiviral drugs and antibiotics, and promoting the healthy and sustainable development of fisheries. Summary of the Invention

[0005] The purpose of this invention is to provide truncated proteins AMP-Z1 and AMP-Z2 of zebrafish IFNf interferon, wherein AMP-Z1 is shown in SEQ ID NO.7 and AMP-Z2 is shown in SEQ ID NO.8.

[0006] The final objective of this invention is to provide the application of a truncated protein of zebrafish IFNf interferon in antibacterial activity.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] The scope of protection of this invention includes:

[0009] A synthetically produced truncated protein of zebrafish IFNf interferon, wherein the truncated protein is AMP-Z1 or / and AMP-Z2, wherein AMP-Z1 is shown in SEQ ID NO.7 and AMP-Z2 is shown in SEQ ID NO.8.

[0010] The above-mentioned stage proteins are used in the preparation of drugs for treating or preventing bacterial infections.

[0011] In the above-described applications, preferably, when using AMP-Z1, the bacteria include Pseudomonas putida or Vibrio cholerae.

[0012] The fusion protein obtained by fusing the truncated protein shown in SEQ ID NO.8 with a protein tag.

[0013] The gene encoding the truncated protein shown in SEQ ID NO.8 or the aforementioned fusion protein.

[0014] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0015] The use of the truncated protein, fusion protein, encoding gene of the truncated protein or fusion protein shown in SEQ ID NO.8, expression cassette, recombinant vector, recombinant microorganism or ex vivo recombinant cell having the above encoding gene in the preparation of drugs for treating or preventing bacterial infections.

[0016] In the above-described applications, preferably, the bacteria include Vibrio cholerae.

[0017] The use of the truncated protein shown in SEQ ID NO.8, the fusion protein, the gene encoding the truncated protein or fusion protein shown in SEQ ID NO.8, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell in the preparation of a drug for treating or preventing fungal infections.

[0018] In the above-described applications, preferably, the fungus includes: saccharomyces.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention marks the first discovery of a novel subtype of interferon, IFNf, in grass carp and zebrafish. The protein encoded by this new gene exhibits highly effective antiviral activity, and the zebrafish interferon-derived peptide AMP-Z2 specifically inhibits fungal activity. The interferon-derived peptides provided by this invention are naturally occurring host peptides with small molecular weights and simple synthesis. They demonstrate significant bactericidal effects against various bacteria and fungi and can be used to prepare broad-spectrum antibacterial agents or antimicrobial drugs. Attached Figure Description

[0021] Figure 1 Cloning of interferon IFNf genes in grass carp and zebrafish;

[0022] The left image shows the size of the gene encoding interferon CiIFNf from grass carp; the right image shows the size of the gene encoding interferon DrIFNf from zebrafish.

[0023] Figure 2 Expression levels of IFNf in different tissues of grass carp before and after GCRV infection.

[0024] Figure 3 Changes in the expression levels of eight interferon genes in different tissues of grass carp before and after GCRV infection

[0025] Figure 4 Schematic diagram of eukaryotic expression of IFNf protein in grass carp and zebrafish;

[0026] The left image shows grass carp interferon CiIFNf, and the right image shows zebrafish interferon DrIFNf.

[0027] Figure 5 Detection of anti-GCRV activity of grass carp interferon IFNf fusion protein.

[0028] Figure 6 The effect of grass carp interferon-derived peptide AMP-G2 on inhibiting the growth of Pseudomonas putidae.

[0029] Figure 7 The effect of grass carp interferon-derived peptide AMP-G2 on inhibiting the growth of Vibrio cholerae.

[0030] Figure 8 The effect of grass carp interferon-derived peptide AMP-G1 on inhibiting the growth of Aeromonas hydrophila.

[0031] Figure 9 The effect of grass carp interferon-derived peptide AMP-G1 on inhibiting the growth of Vibrio cholerae.

[0032] Figure 10 The effect of grass carp interferon-derived peptide AMP-G2 on inhibiting the growth of Escherichia coli is shown in the figure.

[0033] Figure 11 The effect of grass carp interferon-derived peptide AMP-G2 on inhibiting the growth of Aeromonas hydrophila.

[0034] Figure 12 The effect of zebrafish interferon-derived peptide AMP-Z1 on inhibiting the growth of Pseudomonas putidae.

[0035] Figure 13 A graph showing the effect of zebrafish interferon-derived peptide AMP-Z1 on inhibiting the growth of Vibrio cholerae.

[0036] Figure 14 A graph showing the effect of zebrafish interferon-derived peptide AMP-Z2 on inhibiting the growth of Vibrio cholerae.

[0037] Figure 15 The effect of zebrafish interferon-derived peptide AMP-Z2 on inhibiting the growth of Pichia pastoris. Detailed Implementation

[0038] Unless otherwise specified, the technical solutions described in this invention are existing technologies in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0039] Example 1: Obtaining IFNf sequences from grass carp and zebrafish:

[0040] Total RNA was extracted from the gills of grass carp and the whole homogenate of zebrafish, and the quality and integrity of the RNA were detected by agarose gel electrophoresis and spectrophotometry. Using 1 μg of total RNA as a template, grass carp and zebrafish cDNA were synthesized using the Hifair III first-strand cDNA synthesis kit (YEASEN, Shanghai, China). Using this cDNA as a template, the coding regions of the IFNf gene in grass carp (primers CiIFNf-F and CiIFNf-R) and zebrafish (primers DrIFNf-F and DrIFNf-R) were amplified. PCR reaction program: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, 39 cycles; 72℃ for 5 min.

[0041] Identification by PCR product sequencing Figure 1 The grass carp IFNf gene (named CiIFNf) has a coding region of 492 bp, the specific nucleotide sequence of which is shown in SEQ ID NO.1, and the encoded amino acid sequence of which is shown in SEQ ID NO.2; the zebrafish IFNf gene (named DrIFNf) has a coding region of 498 bp, the specific nucleotide sequence of which is shown in SEQ ID NO.3, and the encoded amino acid sequence of which is shown in SEQ ID NO.4.

[0042] Table 1: Primer sequences used in this application

[0043]

[0044] Example 2: GCRV infection activates the expression of grass carp interferon gene IFNf.

[0045] Three-month-old grass carp (approximately 10 cm) were challenged with GCRV via gavage. Grass carp individuals were collected from uninfected (day 0) and on days 1, 4, 7, 14, and 27 post-infection. Total RNA was extracted from the spleen, head kidney, gills, foregut, midgut, and hindgut, and cDNA was synthesized via reverse transcription. Primers for eight interferon genes (IFNf, IFN1, IFN2, IFN3, IFN4, IFNγ, IFNγrel, and IFNυ) were designed (see Table 1 for specific primers). β-actin was used as an internal control gene. Real-time quantitative PCR was performed on each tissue sample (six biological replicates per group) using the synthesized cDNA as a template. The reaction program was: 95℃ for 5 min; 95℃ for 10 s, 55℃ for 30 s, for a total of 35 cycles. The relative expression levels of interferon genes were measured using a 22 -ΔΔCt Calculations were performed, and the differences in gene expression levels between the control group and the infected group were analyzed using one-way ANOVA and paired-samples t-test. A heatmap of gene expression level differences was plotted using the mean -ΔΔCt of each group.

[0046] The results showed that the expression level of the IFNf gene in healthy grass carp was low in all tissues, but after GCRV infection, the expression level of IFNf was significantly upregulated, reaching its peak on day 7 (the peak of infection). As the infection entered the regression phase (day 14), the expression of IFNf in most tissues except the midgut decreased, and finally returned to baseline levels after day 21, indicating that the infection had been cleared or persistent infection had been established. Figure 2 This result indicates that grass carp interferon IFNf can be activated by viral infection. Furthermore, the expression levels of other grass carp interferon genes were also significantly upregulated in specific tissues or at specific time points after GCRV infection, but the upregulation of IFNf expression was the most significant compared to the other seven interferons. Figure 3This suggests that IFNf may play a more important role than other interferons in grass carp's resistance to GCRV infection.

[0047] Example 3:

[0048] IFNf protein expression in grass carp and zebrafish

[0049] Eukaryotic expression vectors for IFNf from grass carp and zebrafish were constructed. Primers with double restriction sites of BamHI and KpnHI were designed based on the coding region sequences of the grass carp CiIFNf and zebrafish DrIFNf genes (Table 1) to construct eukaryotic expression plasmids p3xFLAG-CMV-14-CiIFNf and p3xFLAG-CMV-14-DrIFNf.

[0050] Specifically, the CiIFNf and DrIFNf genes were amplified using cDNA homogenates from grass carp gills and zebrafish whole fish, respectively. The PCR reaction program was: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, 35 cycles; 72℃ for 5 min. After purification, the PCR products were homologously recombinated with the double-digested empty vector p3xFLAG-CMV-14 and transformed into competent cells (DH5α). After culturing at 37℃ for 1 h, 100 μL of the transformation solution was plated on LB solid medium containing ampicillin and cultured for another 12 h. Single clones were then randomly selected for PCR and sequencing identification.

[0051] With a density of 5×10 5 HEK-293T cells / mL were seeded into 6-well cell culture plates and cultured completely in DMEM at 28°C and 5% CO2. When the cells reached approximately 80% confluence, p3xFLAG-CMV-14-CiIFNf, p3xFLAG-CMV-14-DrIFNf, and the empty vector were transfected into the cells. The transfection system consisted of 2 μg plasmid, 4 μL transfection reagent (Lipofectamine 2000), and 200 μL culture medium (Opti-MEM).

[0052] 24 h post-transfection, cells and cell culture medium were collected, and the expression levels of interferon proteins intracellularly and secreted into the culture medium were detected. Culture medium samples were first collected and set aside. Then, 200 μL of RIPA cell lysis buffer was added to the cells and treated for 30 min. Lysed cell samples were collected. The culture medium samples and cell lysis samples were centrifuged at 12000 rpm for 10 min, and the supernatant was collected. SDS-PAGE protein loading buffer was added, and the mixture was heated at 96 °C for 10 min. The protein samples from each group were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Using mouse anti-FLAG antibody as the primary antibody and HRP-goat anti-mouse IgG as the secondary antibody, the expression of the fusion proteins CiIFNf and DrIFNf was detected by Western blotting. The fusion proteins CiIFNf contain the amino acid sequence shown in SEQ ID NO. 2, and DrIFNf contain the amino acid sequence shown in SEQ ID NO. 4.

[0053] The results are as follows Figure 4 As shown, cell lysate samples and culture medium samples (lanes 3 and 4) transfected with grass carp or zebrafish IFNf recombinant protein expression vectors both showed the target band near 25 kDa, while no protein band was detected in cells and culture medium samples (lanes 1 and 2) after empty vector transfection. This indicates that the grass carp CiIFNf and zebrafish DrIFNf fusion protein was successfully synthesized in cells and further secreted into the cell culture medium.

[0054] Example 4:

[0055] Detection of anti-GCRV virus activity of grass carp IFNf fusion protein

[0056] With a density of 5×10 5 GCO cells were seeded at 12-well plates and cultured in M199 at 28°C and 5% CO2. When the cells reached approximately 80% confluence, the p3xFLAG-CMV-14-CiIFNf plasmid and the empty vector were transfected into the cells. The transfection system consisted of 1 μg plasmid, 3 μL transfection reagent (FuGENE), and 42 μL culture medium (Opti-MEM). 24 h after transfection, the cells were further transfected with serially diluted GCRV virus (10... -3 10 -4 10 -5 Infected cells were observed and their state recorded under a microscope. Cell morphology (CPE) was clearly visible 48 hours post-infection. The culture medium was removed, and the cells were washed with PBS. Finally, the cells were stained with 0.1% crystal violet for 8 minutes.

[0057] The results are as follows Figure 5As shown, GCRV infection caused GCO cells to detach and exhibit CPE in different groups. The higher the viral concentration, the more pronounced the CPE phenomenon. Cells transfected with the grass carp IFNf eukaryotic expression vector had less detachment and fewer CPE plaques than the control group transfected with the empty vector. This indicates that the grass carp IFNf fusion protein can effectively protect GCO cells from GCRV virus infection, and that grass carp IFNf has antiviral activity.

[0058] Example 5:

[0059] Obtaining grass carp IFNf-derived peptides (AMP-G1, AMP-G2) and zebrafish IFNf-derived peptides (AMP-Z1, AMP-Z2).

[0060] The applicant effectively truncated the IFNf of grass carp and zebrafish to obtain grass carp interferon IFNf-derived peptides AMP-G1 (SEQ ID NO.5) and AMP-G2 (SEQ ID NO.6); and zebrafish interferon IFNf-derived peptides AMP-Z1 (SEQ ID NO.7) and AMP-Z2 (SEQ ID NO.8).

[0061] The four interferon IFNf-derived peptides were synthesized using a standard amino acid-peptide solid-phase resin chemical synthesis method, custom-made by Shanghai Jier Biochemical Co., Ltd. The synthesized peptide products were purified using high-performance liquid chromatography (HPLC) followed by elution with an acetonitrile gradient of 1 mL / min. The purity of the AMP-G1, AMP-G2, AMP-Z1, and AMP-Z2 peptide powders, as determined by HPLC-MS / MS, was all above 95%, and the molecular weights of the synthesized linear peptides were 2337.75, 2388.72, 2347.73, and 2515.91 Daltons (Da), respectively. The peptide powders were thoroughly dissolved in phosphate-buffered saline (PBS) and serially diluted to multiple concentrations (2, 0.5, 0.125, and 0.0625 mg / mL) for later use.

[0062] Example 6:

[0063] Antibacterial activity assay of grass carp IFNf-derived peptides AMP-G1 and AMP-G2, and zebrafish IFNf-derived peptides AMP-Z1 and AMP-Z2

[0064] The tested bacteria were: *Escherichia coli*, *Aeromonas hydrophila*, *Pseudomonas putida*, *Vibrio cholerae*, and *Staphylococcus aureus*; the tested fungus was *Pichia pastoris*. All the above bacterial strains were preserved in our laboratory at -80°C.

[0065] The specific steps are as follows:

[0066] (1) Take the above-mentioned bacterial culture solution, dissolve it at 4℃, take it out and bring it to room temperature. Inoculate the bacterial culture solution into fresh culture medium for activation, and incubate it in a shaker at 30℃ for 18 hours until the logarithmic growth phase. Take the bacterial culture solution and streak it onto agar solid medium, and incubate it at 30℃ for 18 hours.

[0067] (2) Select a single bacterial clone and inoculate it into MHB liquid medium (bacteria) or YPD liquid medium (fungus) at a ratio of 1:50 and culture it to the logarithmic growth phase.

[0068] (3) After centrifuging the bacterial culture, collect the precipitate and dilute it to 10 μL with PBS. 7 CFU / mL. Mix 10 μL of bacterial suspension with 10 μL of different concentrations of peptide dilution solution, perform 3 replicates for each group, and incubate for 30 minutes. Use PBS buffer as a blank control.

[0069] (4) Transfer the 20 μL mixture after co-incubation to a 96-well plate containing 180 μL of liquid culture medium in each well, place it in a multi-functional microplate reader (Biotek, Winooski, VT, USA), set it to 30℃ for static incubation, shake for 10 seconds every 30 minutes and measure OD600 once. Statistical analysis of the results was performed using Prism (v.9.0), and the significance of differences between groups was compared using a paired-samples t-test.

[0070] The results are shown in Table 2 and Figure 6-15As shown, the four interferon-derived peptides exhibit significant inhibitory effects against different types of bacteria or fungi. Among them, AMP-G2 shows the broadest-spectrum antibacterial effect, simultaneously inhibiting *Escherichia coli*, *Aeromonas hydrophila*, *Pseudomonas putida*, and *Vibrio cholerae*. AMP-G1 shows significant inhibitory effects against *Aeromonas hydrophila* and *Vibrio cholerae*. AMP-Z1 and AMP-Z2 show significant inhibitory effects against *Pseudomonas putida* and *Pichia pastoris* at final concentrations of 3.125 μg / ml and 6.25 μg / ml, respectively, indicating that they can exert antibacterial effects at low concentrations. All four interferon-derived peptides significantly inhibit the growth of *Vibrio cholerae*, but have no inhibitory effect on the Gram-positive bacterium *Staphylococcus aureus*. In summary, the four interferon IFNf-derived peptides provided by this invention, especially AMP-G2, all exhibit broad-spectrum antibacterial activity, while AMP-Z2 also has antifungal activity.

[0071] Table 2. Antibacterial activity of interferon-derived peptides from grass carp and zebrafish.

[0072]

[0073] * indicates antibacterial activity, N indicates no activity.

Claims

1. A synthetically produced truncated protein of zebrafish IFNf interferon, wherein the truncated protein is AMP-Z1 or / and AMP-Z2, wherein AMP-Z1 is shown in SEQ ID NO.7 and AMP-Z2 is shown in SEQ ID NO.

8.

2. The use of the truncated protein AMP-Z1 according to claim 1 in the preparation of a medicament for treating or preventing bacterial infections, wherein the bacteria is *Pseudomonas putida* (…). Pseudomonas putida ) and / or Vibrio cholerae ( Vibrio cholerae ).

3. The fusion protein obtained by fusing the truncated protein shown in SEQ ID NO.8 with a protein tag.

4. The gene encoding the truncated protein shown in SEQ ID NO.8 or the fusion protein of claim 3.

5. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the gene encoding as described in claim 4.

6. The truncated protein shown in SEQ ID NO.8, the fusion protein of claim 3, the encoding gene of claim 4, and the use of an expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the encoding gene of claim 4 in the preparation of a drug for treating or preventing bacterial infection, wherein the bacteria is Vibrio cholerae.

7. The truncated protein shown in SEQ ID NO. 8, the fusion protein of claim 3, the encoding gene of claim 4, and the use of an expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the encoding gene of claim 4 in the preparation of a medicament for treating or preventing fungal infections, wherein the fungus is Pichia pastoris (…). Pichia pastoris ).

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