Grass carp IFNf interferon and truncated protein, and application thereof in antiviral or antibacterial treatment

Abstract

The invention relates to the field of biological medicine and aquatic products, and discloses a grass carp IFNf interferon and truncated protein and application thereof in virus resistance or bacterium resistance. The applicant discovers the new subtype IFNf of the grass carp interferon for the first time, the protein coded by the new gene has an efficient antiviral effect, and the derived peptide AMP-G2 of the grass carp interferon has broad-spectrum antibacterial activity. The sensitivity of the grass carp interferon IFNf to grass carp reovirus (GCRV) infection is higher than that of other interferon, and the grass carp interferon IFNf shows obvious antiviral activity on the cellular level and can be used for preparing antiviral drugs. The interferon-derived peptide provided by the invention is a host natural peptide, is small in molecular weight and simple to synthesize, has a remarkable bactericidal effect on various bacteria and fungi, and can be used for preparing broad-spectrum antibacterial agents or antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and aquaculture, and specifically relates to grass carp IFNf interferon and truncated proteins thereof, and their applications in antiviral or antibacterial activities. Background Art

[0002] Interferons (IFNs) are a class of cytokines with multiple functions such as antiviral, antibacterial, and immunomodulatory activities. The vertebrate interferon family can be divided into four types, namely type I, type II, type III, and type IV interferons. However, fish lack type III interferon and only have three types of interferons, namely type I, type II, and type IV. Among them, type I interferon 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 teleost type I interferon family. Currently, it has only been reported in basal ray-finned fish (Acipenser sinensis, Lepisosteus oculatus) and three teleost species (Anguilla japonica, Salmo salar, Oncorhynchus mykiss). Fish IFNf is more sensitive to RNA virus infection than other interferons and plays a crucial role in the body's first line of defense against RNA viruses. In addition, interferon-derived polypeptides possess physicochemical properties similar to antimicrobial peptides (AMPs) and broad-spectrum antibacterial functions. Therefore, interferon IFNf and its natural-derived peptides have broad application prospects in disease prevention and control and drug research and development.

[0003] Cypriniformes is the most widely distributed and species-rich freshwater fish group in the teleost class, containing many species with significant scientific research value and economic value. Among them, grass carp (Ctenopharyngodon idella) is the freshwater fish with the highest aquaculture production, and zebrafish (Danio rerio) has become one of the most important model species due to its unique biological characteristics such as strong reproductive ability, rapid development, and transparent embryos. Current research shows that both grass carp and zebrafish have only 7 interferon genes, and type I interferon only includes 3 subtypes, namely IFNa (IFN1), IFNc (IFN2, IFN3), and IFNd (IFN4). There have been no reports on other subtypes, especially the earlier-originated IFNf.

[0004] The present invention first discovered a new interferon subtype - IFNf in the important economic species grass carp and the important model species zebrafish, and reported the antiviral function of this gene and the broad-spectrum antibacterial function of its derived peptides. This new gene and its natural-derived peptides will contribute to the development of more efficient and safer antiviral and antibacterial drugs, and are expected to solve problems such as drug resistance and side effects of current antiviral drugs and antibiotics, and promote the healthy and sustainable development of the fishery. Summary of the Invention

[0005] The object of the present invention is to provide grass carp IFNf interferon, and the interferon is shown as SEQ ID NO.2.

[0006] Another object of the present invention is to provide the application of grass carp IFNf interferon in antiviral.

[0007] Another object of the present invention is to provide truncated proteins AMP-G1 and AMP-G2 of grass carp IFNf interferon, where AMP-G1 is shown as SEQ ID NO.5 and AMP-G2 is shown as SEQ ID NO.6.

[0008] The last object of the present invention is to provide the application of the truncated protein of grass carp IFNf interferon in antibacterial. To achieve the above object, the present invention takes the following technical measures:

[0009] The protection scope of the present invention includes:

[0010] Grass carp IFNf interferon, and the interferon is shown as SEQ ID NO.2.

[0011] The application of grass carp IFNf interferon in the preparation of drugs for treating or preventing grass carp reovirus infection.

[0012] The protection scope of the present invention also includes:

[0013] The antibacterial peptide shown as SEQ ID NO.5.

[0014] The application of the antibacterial peptide shown as SEQ ID NO.5 in the preparation of drugs for treating or preventing bacterial infection, and the bacteria include Aeromonas hydrophila or Vibrio cholerae.

[0015] The antibacterial peptide shown as SEQ ID NO.6.

[0016] The fusion protein obtained by fusing the antibacterial peptide shown as SEQ ID NO.6 with a protein tag.

[0017] The coding gene of the antibacterial peptide shown as SEQ ID NO.6 or the above fusion protein.

[0018] The expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell having the above coding gene.

[0019] The application of the antibacterial peptide shown as SEQ ID NO.6, the fusion protein, the coding gene of the antibacterial peptide shown as SEQ ID NO.6 or the fusion protein, and the expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell having the above coding gene in the preparation of drugs for treating or preventing bacterial infection.

[0020] In the above-mentioned application, preferably, the bacteria include: Escherichia coli, Aeromonas hydrophila, Pseudomonas putida, and / or Vibrio cholerae.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention discovers a new subtype of grass carp and zebrafish interferon, IFNf, for the first time. The protein encoded by this new gene has a highly efficient antiviral effect, and the grass carp interferon-derived peptide AMP-G2 has a broad-spectrum antibacterial activity. Grass carp interferon IFNf is more sensitive to grass carp reovirus (GCRV) infection than other interferons and shows obvious antiviral activity at the cellular level, and can be used to prepare antiviral drugs. The interferon-derived peptide provided by the present invention is a natural peptide of the host, with a small molecular weight and simple synthesis, and has a significant bactericidal effect on a variety of bacteria and fungi, and can be used to prepare broad-spectrum antibacterial agents or antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Cloning of grass carp and zebrafish interferon IFNf genes;

[0024] Among them: the left figure shows the size of the grass carp interferon CiIFNf coding gene; the right figure shows the size of the zebrafish interferon DrIFNf coding gene.

[0025] Figure 2 Expression levels of IFNf in different tissues at different time points before and after grass carp infection with GCRV.

[0026] Figure 3 Changes in the expression levels of 8 interferon genes in different tissues at different time points before and after grass carp infection with GCRV.

[0027] Figure 4 Schematic diagram of eukaryotic expression of grass carp and zebrafish IFNf proteins;

[0028] Among them: the left figure is grass carp interferon CiIFNf; the right figure is zebrafish interferon DrIFNf.

[0029] Figure 5 Detection of the anti-GCRV activity of grass carp interferon IFNf fusion protein.

[0030] Figure 6 Effect diagram of the activity test of grass carp interferon-derived peptide AMP-G2 inhibiting the growth of Pseudomonas putida.

[0031] Figure 7Effect test diagram of the activity of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Vibrio cholerae.

[0032] Figure 8 Effect test diagram of the activity of grass carp interferon-derived peptide AMP-G1 in inhibiting the growth of Aeromonas hydrophila.

[0033] Figure 9 Effect test diagram of the activity of grass carp interferon-derived peptide AMP-G1 in inhibiting the growth of Vibrio cholerae.

[0034] Figure 10 Effect test diagram of the activity of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Escherichia coli.

[0035] Figure 11 Effect test diagram of the activity of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Aeromonas hydrophila.

[0036] Figure 12 Effect test diagram of the activity of zebrafish interferon-derived peptide AMP-Z1 in inhibiting the growth of Pseudomonas putida.

[0037] Figure 13 Effect test diagram of the activity of zebrafish interferon-derived peptide AMP-Z1 in inhibiting the growth of Vibrio cholerae.

[0038] Figure 14 Effect test diagram of the activity of zebrafish interferon-derived peptide AMP-Z2 in inhibiting the growth of Vibrio cholerae.

[0039] Figure 15 Effect test diagram of the activity of zebrafish interferon-derived peptide AMP-Z2 in inhibiting the growth of Pichia pastoris. Detailed implementation mode

[0040] The technical solutions described in the present invention are the prior art in the field if not otherwise specified; the reagents or materials are all from commercial channels if not otherwise specified.

[0041] Example 1: Obtaining of grass carp and zebrafish IFNf sequences:

[0042] Total RNA was separately extracted from grass carp gills and zebrafish whole fish homogenates, and the RNA quality and integrity were detected by agarose gel electrophoresis and spectrophotometry. Using 1 μg of total RNA as a template, the first-strand cDNA synthesis kit Hifair III (YEASEN, Shanghai, China) was used to synthesize grass carp and zebrafish cDNA. Using this cDNA as a template, the coding regions of the IFNf genes of grass carp (primers were CiIFNf-F and CiIFNf-R) and zebrafish (primers were DrIFNf-F and DrIFNf-R) were amplified. PCR reaction program: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 40 s, 39 cycles; 72°C for 5 min.

[0043] After PCR product sequencing identification ( Figure 1 ), the full-length coding region of the grass carp IFNf gene (named CiIFNf) was 492 bp, and the specific nucleotide sequence was as shown in SEQ ID NO.1, and the amino acid sequence it encoded was as shown in SEQ ID NO.2; the full-length coding region of the zebrafish IFNf gene (named DrIFNf) was 498 bp, and the specific nucleotide sequence was as shown in SEQ ID NO.3, and the amino acid sequence it encoded was as shown in SEQ ID NO.4.

[0044] Table 1: Primer sequences used in this application

[0045]

[0046]

[0047] Example 2: GCRV infection activates the expression of the grass carp interferon gene IFNf

[0048] Three-month-old grass carp (about 10 cm) were challenged with GCRV by gavage. Grass carp individuals at 0 days (i.e., not infected) and 1, 4, 7, 14, and 27 days after infection were collected. Total RNA of the spleen, head kidney, gills, foregut, midgut, and hindgut was separately extracted and reverse transcribed into cDNA. Primers for 8 grass carp interferon genes (IFNf, IFN1, IFN2, IFN3, IFN4, IFNγ, IFNγrel, IFNυ) were designed (specific primers are shown in Table 1). Using β-actin as an internal reference gene, the above-synthesized cDNA was used as a template to perform real-time fluorescence quantitative PCR detection on each tissue sample (6 biological replicates were set in each group). Reaction program: 95°C for 5 min; 95°C for 10 s, 55°C for 30 s, a total of 35 cycles. The relative expression level of the interferon gene was calculated using 2 -ΔΔCt for calculation. One-way ANOVA and paired-sample t-tests were used for the differential analysis of gene expression levels between the control group and the infected group, and a heat map of gene expression differences was plotted using the average -ΔΔCt of each group of samples.

[0049] The results showed that the expression level of IFNf gene in healthy grass carp was low in all tissues, but after GCRV infection, the expression level of IFNf was significantly up-regulated and reached the peak on the 7th day (the peak of infection). As the infection entered the regression period (the 14th day), the expression of IFNf in most tissues except the midgut decreased, and finally returned to the baseline level after the 21st day, indicating that the infection had been cleared or a persistent infection had been established ( Figure 2 ). This result indicated that grass carp interferon IFNf could be activated by virus infection. In addition, the expression levels of other interferon genes in grass carp also showed significant up-regulation in specific tissues or at specific time points after GCRV infection, but the up-regulation amplitude of IFNf expression was the most significant compared with the other seven interferons ( Figure 3 ), indicating that IFNf might play a more important role in grass carp against GCRV infection than other interferons.

[0050] Example 3:

[0051] Expression of IFNf proteins in grass carp and zebrafish

[0052] Construct eukaryotic expression vectors of IFNf in grass carp and zebrafish. Primers with BamHⅠ and KpnⅠ double digestion sites were designed according to the coding region sequences of grass carp CiIFNf and zebrafish DrIFNf genes (Table 1), and eukaryotic expression plasmids p3xFLAG-CMV-14-CiIFNf and p3xFLAG-CMV-14-DrIFNf were constructed.

[0053] Specifically: CiIFNf and DrIFNf genes were amplified using grass carp gill and zebrafish whole fish homogenate cDNA as templates respectively. The PCR reaction program was: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, for 35 cycles; 72℃ for 5 min. After purification, the PCR products were subjected to homologous recombination 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 taken and spread on LB solid medium containing ampicillin, and then cultured for another 12 h. Monoclonal colonies were randomly selected for PCR and sequencing identification.

[0054] With a density of 5×10 5HEK-293T cells at a density of

[0055] were seeded into 6-well cell culture plates and cultured in complete DMEM medium at 28 °C under 5% CO₂. When the cell confluence reached approximately 80%, p3xFLAG-CMV-14-CiIFNf, p3xFLAG-CMV-14-DrIFNf, and empty vector were transfected into the cells respectively. The transfection system was: 2 μg plasmid, 4 μL transfection reagent (Lipofectamine 2000), and 200 μL culture medium (Opti-MEM).

[0056] At 24 h after transfection, the cells and cell culture supernatants were collected, and the expression levels of interferon proteins in the cells and secreted into the medium were detected respectively. First, the medium samples were aspirated for standby, and then 200 μL of RIPA cell lysate was added to the cells and incubated for 30 min. The lysed cell samples were taken for standby. The medium samples and cell lysate samples were centrifuged at 12000 rpm for 10 min, and the supernatants were taken and added with SDS-PAGE protein loading buffer, and heated at 96 °C for 10 min. The protein samples treated as above were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Mouse anti-FLAG antibody was used as the primary antibody and HRP-goat anti-mouse IgG was used as the secondary antibody. Immunoblotting was used to detect the expression of the fusion proteins CiIFNf and DrIFNf. The fusion protein CiIFNf contains the amino acid sequence shown in SEQ ID NO.2, and DrIFNf contains the amino acid sequence shown in SEQ ID NO.4.

[0056] As shown in Figure 4 the figure, the cell lysate samples and medium samples (lanes 3 and 4) transfected with the recombinant protein expression vectors of grass carp or zebrafish IFNf showed target bands near 25 kDa, while no protein bands were detected in the cell and medium samples transfected with the empty vector (lanes 1 and 2), indicating that the grass carp CiIFNf and zebrafish DrIFNf fusion proteins were successfully synthesized in the cells and further secreted into the cell culture medium.

[0057] Example 4:

[0058] Detection of the anti-GCRV virus activity of grass carp IFNf fusion protein

[0059] GCO cells at a density of 5×10 5 cells / mL were seeded into 12-well cell culture plates and cultured in complete M199 medium at 28 °C under 5% CO₂. When the cell confluence reached approximately 80%, p3xFLAG-CMV-14-CiIFNf plasmid and empty vector were transfected into the cells respectively. The transfection system was: 1 μg plasmid, 3 μL transfection reagent (FuGENE), and 42 μL culture medium (Opti-MEM). At 24 h after transfection, the cells were infected with serially diluted GCRV virus (10-3 , 10 -4 , 10 -5 ) Infect the cells and observe and record the cell status under a microscope. The CPE phenomenon of the cells was clearly visible 48 h after infection. Aspirate the culture medium and wash the cells with PBS. Finally, stain the cells with 0.1% crystal violet solution for 8 min.

[0060] The results are as Figure 5 shown. GCRV infection led to the detachment of GCO cells and the CPE phenomenon in different groups. The higher the virus concentration, the more obvious the CPE phenomenon. The cells transfected with the eukaryotic expression vector of grass carp IFNf had less detachment and fewer CPE plaques than the control group transfected with the empty vector, indicating that the grass carp IFNf fusion protein could effectively protect GCO cells from GCRV virus infection, and grass carp IFNf had antiviral activity.

[0061] Example 5:

[0062] Obtaining of grass carp IFNf-derived peptides (AMP-G1, AMP-G2) and zebrafish IFNf-derived peptides (AMP-Z1, AMP-Z2)

[0063] The applicant effectively truncated the IFNf of grass carp and zebrafish, and obtained grass carp interferon IFNf-derived peptides AMP-G1 (shown in SEQ ID NO.5) and AMP-G2 (shown in SEQ ID NO.6); zebrafish interferon IFNf-derived peptides AMP-Z1 (shown in SEQ ID NO.7) and AMP-Z2 (shown in SEQ ID NO.8).

[0064] The full sequences of the above 4 interferon IFNf-derived peptides were synthesized by the standard amino acid polypeptide solid-phase resin chemical synthesis method and customized by Shanghai Gil Biochemical Co., Ltd. The synthesized polypeptide products were purified using a high-performance liquid chromatography system (HPLC), and then eluted with an acetonitrile gradient at 1 mL / min. The purity of the AMP-G1, AMP-G2, AMP-Z1, and AMP-Z2 polypeptide powders determined by HPLC-MS / MS was above 95%. The molecular weights of the synthesized linear peptides were 2337.75, 2388.72, 2347.73, and 2515.91 daltons (Da), respectively. The polypeptide powders were fully dissolved in phosphate buffer (PBS) and serially diluted to multiple concentrations (2, 0.5, 0.125, 0.0625 mg / mL) for standby.

[0065] Example 6:

[0066] Antibacterial activity detection of grass carp IFNf-derived peptides AMP-G1, AMP-G2, zebrafish IFNf-derived peptides AMP-Z1, AMP-Z2

[0067] Test bacteria: Escherichia coli, Aeromonas hydrophila, Pseudomonas putida, Vibrio cholerae, Staphylococcus aureus; test fungus: Pichia pastoris. All the above strains are stored in a -80°C ultra-low temperature refrigerator in our laboratory.

[0068] The specific steps are as follows:

[0069] (1) Take the above-mentioned preserved bacterial liquid, dissolve it at 4°C and then take it out to equilibrate to room temperature. Inoculate the bacterial liquid into fresh medium for activation, and culture it in a constant temperature shaker at 30°C for 18 hours until the logarithmic growth phase. Inoculate the bacterial liquid by streaking onto agar solid medium and culture it at 30°C for 18 hours.

[0070] (2) Pick single bacterial colonies and inoculate them into MHB liquid medium (for bacteria) or YPD liquid medium (for fungi) at a ratio of 1:50 respectively, and culture until the logarithmic growth phase.

[0071] (3) Centrifuge the bacterial liquid, take the precipitate and dilute it with PBS to 10 7 CFU / mL. Take 10 μL of the bacterial suspension and mix it with 10 μL of polypeptide dilutions at different concentrations. Do 3 parallels for each group and incubate for 30 minutes, using PBS buffer as the blank control.

[0072] (4) Transfer the 20 μL mixture after co-incubation to a 96-well plate containing 180 μL of liquid medium in each well, place it in a multi-functional microplate reader (Biotek, Winooski, VT, USA), set it to culture statically at a constant temperature of 30°C, shake for 10 seconds every 30 minutes and detect OD600 once. Use Prism (v.9.0) to statistically analyze the results, and use paired-samples t-test to compare the significance of differences between groups.

[0073] The results are shown in Table 2 and Figures 6 - 15As shown, the four interferon-derived peptides have obvious inhibitory effects on different types of bacteria or fungi. Among them, AMP-G2 shows the broadest antibacterial effect and can simultaneously inhibit Escherichia coli, Aeromonas hydrophila, Pseudomonas putida, and Vibrio cholerae. AMP-G1 has a significant inhibitory effect on Aeromonas hydrophila and Vibrio cholerae. AMP-Z1 and AMP-Z2 have significant inhibitory effects on Pseudomonas putida and Pichia pastoris at the final concentrations of 3.125 μg / ml and 6.25 μg / ml, respectively, indicating that they can play an antibacterial role at lower concentrations. All four interferon-derived peptides can 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 the present invention, especially AMP-G2, all show broad-spectrum antibacterial activity, and at the same time, AMP-Z2 has antifungal activity.

[0074] Table 2 Antibacterial activities of interferon-derived peptides from grass carp and zebrafish

[0075]

[0076] Note * represents having antibacterial activity, and N represents having no activity.

Claims

1. Grass carp IFNf interferon, the interferon protein is shown in SEQ ID NO.

2.

2. Use of the grass carp IFNf interferon according to claim 1 in the preparation of a medicament for treating or preventing grass carp reovirus infection.

3. The antimicrobial peptide shown in SEQ ID NO.

5.

4. Use of the antimicrobial peptide shown in SEQ ID NO.5 in the preparation of a drug for treating or preventing bacterial infection, wherein the bacteria include Aeromonas hydrophila or Vibrio cholerae.

5. The antimicrobial peptide shown in SEQ ID NO.

6.

6. A fusion protein obtained by fusing the antimicrobial peptide shown in SEQ ID NO.6 with a protein tag.

7. A gene encoding the antimicrobial peptide according to claim 5 or the fusion protein according to claim 6.

8. An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 7.

9. Use of the antimicrobial peptide according to claim 5, the fusion protein according to claim 6, the encoding gene according to claim 7, or the expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell according to claim 8 in the preparation of a drug for treating or preventing bacterial infection.

10. The use according to claim 9, wherein the bacteria is: Escherichia coli ( Escherichia coli )、Aeromonas hydrophila( Aeromonas hydrophila ), Pseudomonas putida ( Pseudomonas putida ), and / or Vibrio cholerae ( Vibrio cholerae ).

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