Zebra fish IFNf interferon truncated protein and antibacterial application thereof

Abstract

The invention relates to the field of biological medicine and aquatic products, and discloses zebra fish IFNf interferon truncated protein and application thereof in antibiosis. The zebra fish interferon derived peptide AMP-Z2 can specifically have an inhibition effect on fungi. 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 invention relates to the fields of biomedicine and aquatic products, and in particular to a zebrafish IFNf interferon truncated protein and an application thereof in antibacterial treatment. Background Art

[0002] Interferon (IFN) is a type of cytokine with multiple functions such as antiviral, antibacterial and immunomodulatory. The vertebrate interferon family can be divided into four types, namely type I, type II, type III and type IV. 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 is also the oldest member of the teleost type I interferon family. It has only been reported in basal ray-finned fish (Chinese sturgeon, gar) and three teleost fish (Japanese eel, Atlantic salmon, rainbow trout). Fish IFNf is more sensitive to RNA virus infection than other interferons, and plays a vital role in the body's first line of defense against RNA viruses. In addition, interferon-derived peptides have similar physical and chemical properties to antimicrobial peptides (AMPs) and broad-spectrum antimicrobial functions. Therefore, interferon IFNf and its naturally derived peptides have broad application prospects in disease prevention and control and drug development.

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

[0004] This paper first discovered a new subtype of interferon, IFNf, in the important economic species grass carp and the important model species zebrafish, and reported the antiviral function of the gene and the broad-spectrum antibacterial function of its derived peptides. The new gene and its naturally derived peptides will help develop more efficient and safer antiviral and antibacterial drugs, and are expected to solve the problems of drug resistance, toxic side effects, etc. of current antiviral drugs and antibiotics, and promote the healthy and sustainable development of fisheries. Summary of the invention

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

[0006] The last object of the present invention is to provide the application of the truncated protein of zebrafish IFNf interferon in antibacterial.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The protection scope of the present invention includes:

[0009] An artificially synthesized truncated protein of zebrafish IFNf interferon, the truncated protein is AMP-Z1 or / and AMP-Z2, the 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-mentioned application, preferably, when AMP-Z1 is used, the bacteria include Pseudomonas putida or Vibrio cholerae.

[0012] A 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 above fusion protein.

[0014] An expression cassette, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell having the above coding gene.

[0015] The truncated protein or fusion protein shown in SEQ ID NO.8, the coding gene of the truncated protein or fusion protein shown in SEQ ID NO.8, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the above coding gene in the preparation of a drug for treating or preventing bacterial infection.

[0016] In the above application, preferably, the bacteria include: Vibrio cholerae.

[0017] The truncated protein or fusion protein shown in SEQ ID NO.8, the coding gene of the truncated protein or fusion protein shown in SEQ ID NO.8, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the above coding gene in the preparation of a drug for treating or preventing fungal infection.

[0018] In the above application, preferably, the fungus includes: yeast (saccharomyces).

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

[0020] The present invention discovers for the first time a new subtype of interferon IFNf in grass carp and zebrafish. The protein encoded by the new gene has a highly effective antiviral effect, and the zebrafish interferon-derived peptide AMP-Z2 can specifically inhibit fungi. The interferon-derived peptide provided by the present invention is a natural peptide of the host, has a small molecular weight, is simple to synthesize, has a significant bactericidal effect on a variety of bacteria and fungi, and can be used to prepare a broad-spectrum antibacterial agent or antibacterial drug. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Among them: the left picture shows the size of the gene encoding grass carp interferon CiIFNf; the right picture shows the size of the gene encoding zebrafish interferon DrIFNf.

[0023] Figure 2 The expression levels of IFNf in different tissues of grass carp at different time points before and after infection with GCRV.

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

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

[0026] Among them: the left picture is: grass carp interferon CiIFNf; the right picture is: zebrafish interferon DrIFNf.

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

[0028] Figure 6 Activity test effect diagram of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Pseudomonas putida.

[0029] Figure 7 Activity test effect diagram of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Vibrio cholerae.

[0030] Figure 8 Activity test effect diagram of grass carp interferon-derived peptide AMP-G1 in inhibiting the growth of Aeromonas hydrophila.

[0031] Fig. 9 Activity test effect diagram of grass carp interferon-derived peptide AMP-G1 in inhibiting the growth of Vibrio cholerae.

[0032] Fig.10 Activity test effect diagram of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Escherichia coli.

[0033] Fig.11 Activity test effect diagram of grass carp interferon-derived peptide AMP-G2 in inhibiting the growth of Aeromonas hydrophila.

[0034] Fig.12 Activity test effect diagram of zebrafish interferon-derived peptide AMP-Z1 in inhibiting the growth of Pseudomonas putida.

[0035] Fig.13 Activity test effect diagram of zebrafish interferon-derived peptide AMP-Z1 in inhibiting the growth of Vibrio cholerae.

[0036] Fig.14 Activity test effect diagram of zebrafish interferon-derived peptide AMP-Z2 in inhibiting the growth of Vibrio cholerae.

[0037] Fig.15 Activity test effect diagram of zebrafish interferon-derived peptide AMP-Z2 inhibiting the growth of Pichia pastoris. DETAILED DESCRIPTION

[0038] The technical solutions described in the present invention, unless otherwise specified, are prior arts in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0039] Example 1: Acquisition of grass carp and zebrafish IFNf sequences:

[0040] Total RNA was extracted from grass carp gills and zebrafish whole fish homogenate, and the RNA quality and integrity were detected by agarose gel electrophoresis and spectrophotometry. With 1 μg of total RNA as template, grass carp and zebrafish cDNA were synthesized using the Hifair III First Strand cDNA Synthesis Kit (YEASEN, Shanghai, China). The cDNA was used as a template to amplify the coding region of the IFNf gene of grass carp (primers CiIFNf-F and CiIFNf-R) and zebrafish (primers DrIFNf-F and DrIFNf-R). The PCR reaction program was: 95℃3min; 95℃30s, 55℃30s, 72℃40s, 39 cycles; 72℃5min.

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

[0045] Three-month-old grass carp (about 10 cm) were challenged with GCRV by oral gavage. Uninfected (i.e., day 0) and infected grass carp individuals on days 1, 4, 7, 14, and 27 were collected, and total RNA from spleen, head kidney, gill, foregut, midgut, and hindgut was extracted and reverse transcribed to synthesize cDNA. Primers for eight interferon genes (IFNf, IFN1, IFN2, IFN3, IFN4, IFNγ, IFNγrel, and IFNυ) of grass carp were designed (see Table 1 for specific primers). β-actin was used as the internal reference gene, and the above-synthesized cDNA was used as a template to perform real-time fluorescence quantitative PCR detection on each tissue sample (six biological replicates in each group). Reaction procedure: 95°C 5min; 95°C 10s, 55°C 30s, for a total of 35 cycles. The relative expression of interferon genes was measured using 2 -ΔΔCt The difference in gene expression between the control group and the infected group was calculated using one-way analysis of variance and paired sample t-test, and the heat map of gene expression difference was drawn using the average -ΔΔCt of each group of samples.

[0046] The results showed that the expression of IFNf gene in healthy grass carp was low in all tissues, but after GCRV infection, the expression of IFNf was significantly upregulated and reached its peak on the 7th day (peak of infection). As the infection entered the resolution phase (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 ). The results showed that grass carp interferon IFNf can be activated by viral infection. In addition, the expression levels of other interferon genes in grass carp 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 with the other seven interferons ( Figure 3), indicating that IFNf may play a more important role than other interferons in grass carp's resistance to GCRV infection.

[0047] Embodiment 3:

[0048] Expression of IFNf protein in grass carp and zebrafish

[0049] Construction of grass carp and zebrafish IFNf eukaryotic expression vectors. Primers with BamHⅠ and KpnⅠ double restriction 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.

[0050] Specifically, the cDNA of grass carp gill and zebrafish whole fish homogenate was used as template to amplify CiIFNf and DrIFNf genes. The PCR reaction program was: 95℃ 3min; 95℃ 30s, 55℃ 30s, 72℃ 40s, 35 cycles; 72℃ 5min. After purification, the PCR product was homologously recombined with the double-enzyme-digested empty p3xFLAG-CMV-14 and transformed into competent cells (DH5α). After culturing at 37℃ for 1h, 100μL of the transformation solution was spread on LB solid medium containing ampicillin. After continuing to culture for 12h, single clones were randomly picked for PCR and sequencing identification.

[0051] The density is 5×10 5 HEK-293T cells were inoculated into 6-well cell culture plates with 100 μg / mL of plasmid and cultured in DMEM complete medium at 28°C and 5% CO2. When the cell confluence was about 80%, p3xFLAG-CMV-14-CiIFNf, p3xFLAG-CMV-14-DrIFNf and empty vector were transfected into the cells. The transfection system was: 2 μg plasmid, 4 μL transfection reagent (Lipofectamine 2000) and 200 μL culture medium (Opti-MEM).

[0052] 24h after transfection, cells and cell culture fluid were collected to detect the expression of interferon proteins in cells and secreted into the culture medium. First, the culture medium sample was taken for use, and then 200μL RIPA cell lysis solution was added to the cells for treatment for 30min, and the cell samples after lysis were taken for use. The culture medium sample and the cell lysis sample were centrifuged at 12000rpm for 10min, the supernatant was taken, SDS-PAGE protein loading buffer was added, and heated at 96℃ for 10min. Each group of protein samples after the above treatment was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The mouse anti-FLAG antibody was used as the primary antibody and HRP-goat anti-mouse IgG was used as the secondary antibody. The expression of fusion proteins CiIFNf and DrIFNf was detected by immunoblotting. The fusion protein CiIFNf contains the amino acid sequence shown in SEQID NO.2, and DrIFNf contains the amino acid sequence shown in SEQ ID NO.4.

[0053] The results are as follows Figure 4 As shown, the cell lysate samples and culture medium samples (lanes 3 and 4) transfected with grass carp or zebrafish IFNf recombinant protein expression vectors showed the target band near 25 kDa, while no protein band was detected in the empty transfected cells and culture medium samples (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.

[0054] Embodiment 4:

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

[0056] The density is 5×10 5 GCO cells with a concentration of 1 μg / mL were inoculated into 12-well cell culture plates and cultured with M199 complete medium at 28°C and 5% CO2. When the cell confluence was about 80%, the 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). After transfection for 24 hours, the GCRV virus (10 -3 , 10 -4 , 10 -5 ) infected cells, observed and recorded the cell status under a microscope. 48h after infection, the CPE phenomenon of cells was clearly visible, the culture medium was removed and the cells were washed with PBS, and finally stained with 0.1% crystal violet stain for 8min.

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

[0058] Embodiment 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 grass carp and zebrafish IFNf to obtain 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), respectively.

[0061] The above four interferon IFNf derived peptides were synthesized using a standard amino acid peptide solid phase resin chemical synthesis method and were customized by Shanghai Jier Biochemical Co., Ltd. The synthesized peptide product was purified using a high performance liquid chromatography system (HPLC) and then eluted with a 1 mL / min acetonitrile gradient. The purity of the AMP-G1, AMP-G2, AMP-Z1 and AMP-Z2 polypeptide powders determined by HPLC-MS / MS was 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 polypeptide powders were fully dissolved in phosphate buffered saline (PBS) and gradiently diluted to multiple concentrations (2, 0.5, 0.125, 0.0625 mg / mL) for use.

[0062] Embodiment 6:

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

[0064] Test bacteria: Escherichia coli, Aeromonas hydrophila, Pseudomonas putida, Vibrio cholerae, Staphylococcus aureus; test fungi: Pichia pastoris. The above strains were stored in a -80℃ ultra-low temperature freezer in our laboratory.

[0065] The specific steps are as follows:

[0066] (1) Take the above seed culture solution and dissolve it at 4°C, then take it out and equilibrate it to room temperature. Inoculate the culture solution into fresh culture medium for activation, and culture it in a constant temperature shaker at 30°C for 18 hours until the logarithmic growth phase. Take the culture solution and streak it into agar solid culture medium, and culture it at 30°C for 18 hours.

[0067] (2) Pick 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 until the logarithmic growth phase.

[0068] (3) After centrifugation of the bacterial solution, the precipitate was taken and diluted to 10% with PBS. 7 CFU / mL. Take 10 μL of bacterial suspension and mix it with 10 μL of peptide dilutions of different concentrations. Make 3 parallels in each group and incubate for 30 minutes. Use PBS buffer as blank control.

[0069] (4) After co-incubation, 20 μL of the mixture was transferred to a 96-well plate containing 180 μL of liquid culture medium in each well and placed in a multifunctional microplate reader (Biotek, Winooski, VT, USA). The plate was incubated at a constant temperature of 30°C, with shaking for 10 seconds and OD600 measured every 30 minutes. The results were statistically analyzed using Prism (v.9.0), and the paired sample t-test was used to compare the significance of differences among the groups.

[0070] The results are shown in Table 2 and Figure 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, and AMP-Z1 and AMP-Z2 have significant inhibitory effects on Pseudomonas putida and Pichia pastoris at final concentrations of 3.125μg / ml and 6.25μg / ml, respectively, indicating that they can play an antibacterial role at lower concentrations. The four interferon-derived peptides can significantly inhibit the growth of Vibrio cholerae, but have no inhibitory effect on the Gram-positive bacteria 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 AMP-Z2 has an antifungal effect.

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

[0072]

[0073] Note: * indicates antibacterial activity, and N indicates no activity.

Claims

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

8.

2. Use of the truncated protein according to claim 1 in the preparation of a drug for treating or preventing bacterial infection.

3. The use according to claim 2, wherein when AMP-Z1 is used, the bacteria include Pseudomonas putida ( Pseudomonas putida ) and / or Vibrio cholerae ( Vibrio cholerae ).

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

5. A gene encoding the truncated protein shown in SEQ ID NO.8 or the fusion protein according to claim 4.

6. An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 5.

7. Use of the truncated protein shown in SEQ ID NO.8, the fusion protein according to claim 4, the coding gene according to claim 5, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 5 in the preparation of a drug for treating or preventing bacterial infection.

8. The use according to claim 7, wherein the bacteria comprises: Vibrio cholerae.

9. Use of the truncated protein shown in SEQ ID NO.8, the fusion protein according to claim 4, the coding gene according to claim 5, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the coding gene according to claim 5 in the preparation of a drug for treating or preventing fungal infection.

10. The use according to claim 9, wherein the fungus comprises: Yeast (saccharomyces).

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