Use of hnf4a or hnf4a-containing biomaterials in the preparation of products for inhibiting bacteria and / or viruses

Abstract

The application provides an application of Hnf4 alpha or a biological material containing Hnf4 alpha in preparation of a product for inhibiting bacteria and / or viruses, and belongs to the technical field of biological medicines. Through in-vivo and in-vitro experiments, it is found that overexpression of Hnf4 alpha can not only effectively prevent infection of Aeromonas salmonicida and grass carp hemorrhagic disease virus, but also can significantly inhibit co-infection of Aeromonas salmonicida and grass carp hemorrhagic disease virus. The bacterial or viral load after infection is significantly reduced, and the activity of cells and the survival rate of young fish are improved. The application provides a molecular target for prevention and control of bacterial infectious diseases, viral infectious diseases and mixed infectious diseases of Cyprinidae.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of products that inhibit bacteria and / or viruses. Background Technology

[0002] In recent years, with the rapid development of modern fisheries and the leapfrog development of aquaculture scale, the world's fishery output has grown rapidly, continuously providing high-quality protein for human tables. However, due to the large number of aquaculture species, the large scale, and the continuous deterioration of the aquaculture environment, various fish diseases have occurred frequently and are complex, causing significant economic losses to aquaculture and seriously affecting the development of the aquaculture industry.

[0003] Aeromonas salmonidida belongs to the phylum Proteobacteria, family Aeromonaceae, and genus Aeromonas. It is a Gram-negative short bacillus. Besides infecting salmonid fish, it can also infect carp, bass, mullet, and catfish. Grass carp and zebrafish are also susceptible to Aeromonas salmonidida infection. Infection with Aeromonas salmonidida causes scabies or ulcers in fish, is contagious, and causes significant economic losses to the aquaculture industry. Grass carp reovirus (GCRV) is a double-stranded RNA (dsRNA) virus belonging to the genus Aquareovirus. It is the most virulent aquatic reovirus, causing hemorrhagic disease in grass carp, leading to severe hemorrhagic symptoms and death, with a high mortality rate. GCRV exists in three genotypes: GCRV-I, GCRV-II, and GCRV-III, with representative strains being GCRV-873 (type I), GCRV-HZ08 (type II), and GCRV104 (type III), respectively. In aquaculture, due to the unique environment in which fish grow, mixed infections with multiple pathogens are common, seriously jeopardizing the profitability of aquaculture.

[0004] Hepatocyte nuclear factors (HNFs) are a class of transcription factors that regulate gene-specific expression in the liver. These transcription factors and their interactions with other genes' DNA or proteins form a complex regulatory network that precisely controls liver development and hepatocyte function. Hnf4 belongs to the nuclear receptor superfamily 2A subfamily (NR2A), including three members: Hnf4α, Hnf4β, and Hnf4γ. Hnf4α and Hnf4γ are widely distributed in mammals, while Hnf4β is only found in fish, amphibians, and birds. Hnf4α is closely associated with type 1 juvenile diabetes (MODY1) and is also involved in the development of liver fibrosis, cirrhosis, hepatocellular carcinoma, and fatty liver disease. Some studies have shown that HNF4α expression decreases during liver cancer development, and overexpression of HNF4α inhibits the invasiveness and reduces the proliferation rate of liver cancer cells, indicating a certain anti-tumor effect. However, reports on HNF4α in fish diseases are scarce, and other studies have not extensively discussed the link between HNF4α and apoptosis-related molecules. Furthermore, some research results indicate that certain genes, while enhancing protection against bacterial diseases, increase susceptibility to viral diseases, and vice versa. Therefore, screening for target molecules that can simultaneously resist bacterial and viral diseases is crucial for the prevention and control of infectious diseases in fish, especially bacterial-viral co-infections. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of products that inhibit bacteria and / or viruses.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of products that inhibit bacteria and / or viruses.

[0008] This invention provides the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of at least one of the following products (1) to (3).

[0009] (1) Prevention and treatment of bacterial infections;

[0010] (2) Prevention and treatment of viral infections;

[0011] (3) Prevention and treatment of diseases caused by co-infection of bacteria and viruses.

[0012] This invention provides an application of Hnf4α or Hnf4α-containing biomaterials in at least one of the following I to III.

[0013] I. Promote fish growth and reproduction;

[0014] II. Preparation of products that promote fish growth and reproduction;

[0015] III. Prepare products that enhance fish's disease resistance.

[0016] Preferably, the bacteria is Aeromonas salmonii; the virus is grass carp reovirus.

[0017] Preferably, the Hnf4α-containing biomaterial includes any one of the following a to c:

[0018] a. Expression vectors containing Hnf4α;

[0019] b. Host bacteria containing Hnf4α;

[0020] c. Host cells containing Hnf4α.

[0021] Preferably, in III, the product can improve fish resistance to diseases caused by at least one of Aeromonas salmonidae infection, grass carp reovirus infection, and Aeromonas salmonidae-grass carp reovirus co-infection.

[0022] Preferably, the fish is a carp.

[0023] Preferably, the fish includes grass carp and / or zebrafish.

[0024] This invention provides a method for breeding disease-resistant fish, comprising the following steps: increasing the expression level and / or activity of Hnf4α in recipient fish.

[0025] Preferably, the disease-resistant fish is resistant to diseases caused by at least one of Aeromonas salmonidae infection, grass carp reovirus infection, and Aeromonas salmonidae-grass carp reovirus co-infection.

[0026] Preferably, the recipient fish includes grass carp and / or zebrafish.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of products that inhibit bacteria and / or viruses. Through in vivo and in vitro experiments, this invention found that overexpression of Hnf4α not only effectively prevents infection by Aeromonas salmonid and grass carp hemorrhagic septicemia virus, but also significantly inhibits co-infection by these two viruses. It significantly reduces the bacterial or viral load after infection, improves cell activity, and increases the survival rate of juvenile fish. This invention provides a molecular target for the prevention and control of bacterial, viral, and mixed infectious diseases in cyprinid fish. Attached Figure Description

[0029] Figure 1 Electrophoresis diagram of the protein from the gcHnf4α-FLAG recombinant expression vector;

[0030] Figure 2 To illustrate the expression and subcellular localization of gcHnf4α, A represents the constitutive expression of gcHnf4α in healthy grass carp; B represents the induced expression of gcHnf4α in grass carp after infection with Aeromonas salmonidus and / or grass carp hemorrhagic disease virus for 6 hours (6 hpi) and 24 hours (24 hpi); C represents the subcellular localization of grass carp infected with or without Aeromonas salmonidus after overexpression of FLAG and gcHnf4α-FLAG in CIK cells; and D represents the subcellular localization of grass carp hemorrhagic disease virus after overexpression of FLAG and gcHnf4α-FLAG in CIK cells.

[0031] Figure 3 To investigate the effects of gcHnf4α overexpression in CIK cells on Aeromonas salmonid proliferation and cell survival, A represents the effect of FLAG and gcHnf4α-FLAG overexpression on bacterial proliferation in CIK cells at 3 hours (3 hpi) and 6 hours (6 hpi) after Aeromonas salmonid infection; B represents the effect of FLAG and gcHnf4α-FLAG overexpression on cell death rate after Aeromonas salmonid infection in CIK cells; and C represents the effect of FLAG and gcHnf4α-FLAG overexpression on cell viability at 6, 24, and 48 h after infection with or without Aeromonas salmonid infection in CIK cells.

[0032] Figure 4 To illustrate the effect of gcHnf4α overexpression in CIK cells on GCRV replication and cell survival, A shows the crystal violet staining of CIK cells after GCRV-I infection with overexpressed FLAG and gcHnf4α-FLAG, and B shows the detection of GCRV-I titer after overexpression of FLAG and gcHnf4α-FLAG in CIK cells.

[0033] Figure 5To assess the effect of gcHnf4α overexpression in CIK cells on co-infection with Aeromonas salmonicida and GCRV-I, A shows crystal violet staining of FLAG and gcHnf4α-FLAG overexpression in CIK cells after Aeromonas salmonicida and / or GCRV-I infection; B shows the effect of FLAG and gcHnf4α-FLAG overexpression in CIK cells on cell viability under Aeromonas salmonicida and / or GCRV-I infection; C shows the effect of FLAG and gcHnf4α-FLAG overexpression in CIK cells on bacterial proliferation after single-pathogen Aeromonas salmonicida infection or co-infection with Aeromonas salmonicida and GCRV-I; D shows the effect of FLAG and gcHnf4α overexpression on bacterial proliferation. Overexpression of nf4α-FLAG in CIK cells was followed by GCRV-I single pathogen infection or co-infection with Aeromonas salmonicida and GCRV-I to verify gcHnf4α overexpression. E represents the overexpression of FLAG and gcHnf4α-FLAG in CIK cells. After GCRV-I single pathogen infection or co-infection with Aeromonas salmonicida and GCRV-I, the relative expression of the key molecule NS38 of GCRV-I was evaluated. F represents the relative expression of the key molecule NS80 of GCRV-I after GCRV-I single pathogen infection or co-infection with Aeromonas salmonicida and GCRV-I following the overexpression of FLAG and gcHnf4α-FLAG in CIK cells.

[0034] Figure 6 To assess the effect of gcHnf4α overexpression in CIK cells on co-infection with Aeromonas salmonicida and GCRV-I under caspase 3 inhibition, A shows crystal violet staining after infection with Aeromonas salmonicida and / or GCRV-I following caspase 3 inhibition of FLAG and gcHnf4α-FLAG overexpression in CIK cells. B shows the effect of FLAG and gcHnf4α-FLAG overexpression in CIK cells on cell viability after infection with Aeromonas salmonicida and / or GCRV-I following caspase 3 inhibition. C shows the effect of FLAG and gcHnf4α-FLAG overexpression in CIK cells on bacterial proliferation after single-pathogen infection with Aeromonas salmonicida or co-infection with Aeromonas salmonicida and GCRV-I in the presence of DEVD. D shows the effect of caspase 3 inhibition after FLAG and gcHnf4α-FLAG overexpression in CIK cells. 3. Perform single pathogen infection with GCRV-I or co-infection with Aeromonas salmonii and GCRV-I to verify the overexpression of gcHnf4α and assess the relative expression changes of the key molecules NS38 and NS80 of GCRV-I.

[0035] Figure 7To assess the effect of gcHnf4α overexpression in CIK cells on co-infection with Aeromonas salmonicida and GCRV-I under caspase 9 inhibition, A shows crystal violet staining after LEHD treatment with overexpression of FLAG and gcHnf4α-FLAG in CIK cells followed by infection with Aeromonas salmonicida and / or GCRV-I. B shows the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells on cell viability after infection with Aeromonas salmonicida and / or GCRV-I following caspase 9 inhibition. C shows the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells on bacterial proliferation after single-pathogen infection with Aeromonas salmonicida or co-infection with Aeromonas salmonicida and GCRV-I in the presence of LEHD. D shows the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells after caspase 9 inhibition. 9. Perform single pathogen infection with GCRV-I or co-infection with Aeromonas salmonii and GCRV-I to verify the overexpression of gcHnf4α and assess the relative expression changes of the key molecules NS38 and NS80 of GCRV-I.

[0036] Figure 8 The effects of gcHnf4α overexpression in zebrafish on juvenile survival under bacterial and viral infections are as follows: A: Survival rate of wild-type zebrafish with overexpression of FLAG and gcHnf4α-FLAG in the uninfected state; B: Effect of FLAG and gcHnf4α-FLAG overexpression in wild-type zebrafish on juvenile survival rate after single pathogen infection with Aeromonas salmonicidae; C: Effect of FLAG and gcHnf4α-FLAG overexpression in wild-type zebrafish on juvenile survival rate after single pathogen infection with GCRV-II; D: Effect of FLAG and gcHnf4α-FLAG overexpression in wild-type zebrafish on juvenile survival rate after co-infection with Aeromonas salmonicidae and GCRV-II.

[0037] Figure 9To investigate the effects of inhibiting caspase 3 overexpression of gcHnf4α in zebrafish on juvenile survival after bacterial and viral infections, the following studies were conducted: A) Survival rate of uninfected juveniles in the presence of DEVD after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish; B) Survival rate of juveniles after single-pathogen infection with Aeromonas salmonicidae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish with caspase 3 inhibition; C) Survival rate of juveniles after single-pathogen infection with GCRV-II after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish with caspase 3 inhibition; D) Survival rate of juveniles after co-infection with Aeromonas salmonicidae and GCRV-II after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish with caspase 3 inhibition.

[0038] Figure 10 To investigate the effects of bacterial and viral post-infection on juvenile survival after suppressing caspase 9 overexpression of gcHnf4α in zebrafish, the following studies were conducted: A) Survival rate of uninfected juveniles in the presence of LEHD after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish; B) Survival rate of juveniles after single-pathogen infection with Aeromonas salmonicidae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish with caspase 9 suppression; C) Survival rate of juveniles after single-pathogen infection with GCRV-II after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish with caspase 9 suppression; D) Survival rate of juveniles after co-infection with Aeromonas salmonicidae and GCRV-II after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish with caspase 9 suppression. Detailed Implementation

[0039] This invention provides the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of products that inhibit bacteria and / or viruses.

[0040] This invention provides the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of at least one of the following products (1) to (3).

[0041] (1) Prevention and treatment of bacterial infections;

[0042] (2) Prevention and treatment of viral infections;

[0043] (3) Prevention and treatment of diseases caused by co-infection of bacteria and viruses.

[0044] This invention provides an application of Hnf4α or Hnf4α-containing biomaterials in at least one of the following I to III.

[0045] I. Promote fish growth and reproduction;

[0046] II. Preparation of products that promote fish growth and reproduction;

[0047] III. Prepare products that enhance fish's disease resistance.

[0048] In this invention, the amino acid sequence of Hnf4α is shown in SEQ ID No. 1, and the nucleotide sequence of Hnf4α is shown in SEQ ID No. 2.

[0049] In this invention, the bacteria is Aeromonas salmonidae, and the virus is grass carp reovirus. III. The product can improve fish resistance to diseases caused by at least one of Aeromonas salmonidae infection, grass carp reovirus infection, and Aeromonas salmonidae-grass carp reovirus co-infection.

[0050] In this invention, the Hnf4α-containing biomaterial includes any one of the following a to c:

[0051] a. Expression vectors containing Hnf4α;

[0052] b. Host bacteria containing Hnf4α;

[0053] c. Host cells containing Hnf4α.

[0054] In this invention, an expression vector containing Hnf4α can be constructed using existing mammalian cell expression vectors, and the empty vector of the mammalian cell expression vector is preferably p3×FLAG-CMV. TM -14; A host bacterium containing Hnf4α can be constructed using a prokaryotic system, preferably Escherichia coli, such as Escherichia coli Top 10; the host cell is preferably CIK cell.

[0055] In the aforementioned biomaterials, as a preferred embodiment, the preparation of the Hnf4α-containing expression vector of the present invention includes inserting Hnf4α into p3×FLAG-CMV. TM The step involves inserting Hnf4α into the -14 vector to obtain an expression vector containing Hnf4α. The Hnf4α is inserted into p3×FLAG-CMV. TMThe restriction endonuclease for the -14 vector is between Hind III and BamHI. The preparation of the host bacterium containing Hnf4α includes the step of transforming the Hnf4α-containing expression vector into *E. coli* Top 10 competent cells. The preparation of the host cells containing Hnf4α includes the step of transfecting the Hnf4α-containing expression vector into CIK cells. This invention utilizes the above-mentioned expression vector, host bacterium, or host cells to ensure both the overexpression and activity of Hnf4α.

[0056] This invention provides the application of Hnf4α or Hnf4α-containing biomaterials in the preparation of products that enhance fish disease resistance. These products can improve fish resistance to diseases caused by at least one of Aeromonas salmonid infection, grass carp reovirus infection, and Aeromonas salmonid-grass carp reovirus co-infection.

[0057] In this invention, the product is a drug, feed, or feed additive.

[0058] In this invention, the fish is a carp, such as grass carp and / or zebrafish.

[0059] This invention provides a method for breeding disease-resistant fish, comprising the following steps: increasing the expression level and / or activity of Hnf4α in recipient fish.

[0060] In this invention, the expression level and / or activity of Hnf4α in the recipient fish is increased by introducing the aforementioned biological material into the recipient fish, such as by injecting the aforementioned Hnf4α-containing expression vector into the recipient fish. The biological materials have been described above and will not be repeated here.

[0061] In this invention, the disease-resistant fish is resistant to diseases caused by at least one of Aeromonas salmonid infection, grass carp reovirus infection, and Aeromonas salmonid-grass carp reovirus co-infection. The recipient fish is a cyprinid fish, such as grass carp and / or zebrafish.

[0062] In this invention, the grass carp reovirus is GCRV type I and / or GCRV-II, wherein GCRV type I is GCRV-873 and GCRV-II is GCRV-GD108.

[0063] This invention improves the resistance of recipient fish to diseases caused by at least one of Aeromonas salmonid infection, grass carp reovirus infection, and Aeromonas salmonid-grass carp reovirus co-infection by overexpressing Hnf4α, thereby promoting the survival rate and growth of recipient fish.

[0064] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0065] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] In the following embodiments, the Aeromonas salmonidae is ATCC 33658; the GCRV-I is GCRV-873; and the GCRV-II is GCRV-GD108.

[0067] Example 1

[0068] Construction of grass carp Hnf4α eukaryotic expression plasmid and verification of its protein expression

[0069] This embodiment uses the nucleotide sequence of grass carp Hnf4α downloaded from the NCBI database and combined with the eukaryotic expression plasmid p3×FLAG-CMV TM The multiple cloning site at -14 was used to design primers using SnapGene software. The ORF region of Hnf4α was amplified using grass carp cDNA as a template, and after double digestion with Hind III and BamHI enzymes, p3×FLAG-CMV was inserted. TM The -14 expression vector was transformed into *E. coli* Top 10 competent cells, and single clones were selected for verification. Based on the sequencing results, strains with correct sequencing were selected, and plasmids were extracted using the OMEGA kit. The concentration and purity were detected using a micro-spectrophotometer. The grass carp Hnf4α plasmid was named gcHnf4α-FLAG. gcHnf4α-FLAG was transfected into CIK cells. After 48 hours of transfection, cells were collected, and cellular proteins were extracted using RIPA lysis buffer and verified for expression by Western blotting.

[0070] Experimental results are as follows Figure 1 As shown, the protein size detected by Western blotting matches the predicted size of the gcHnf4α-FLAG protein, and the band is single, approximately 50 kDa. Sequencing results show that the amino acid sequence of grass carp Hnf4α (gcHnf4α) is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2. This indicates that the grass carp Hnf4α plasmid was successfully constructed.

[0071] The amino acid sequence of grass carp Hnf4α (gcHnf4α):

[0072] MEMADYSEALDPAYTTLEFENMQVLAMSTDSSPAESANMNAANHLGAGTLCAICGDRATGKHYGASSCDGCKGFFRRSVRKNHMYSCRFNRQCIVDKDKRNQCRYCRLKKCFRAGMKKEAVQNERDRISTRRSSYEDSSLPSINALIQADVLSRQISSPGPIMNGDIRTKKVATITDVCESMKQQLLVLVEWAKYIPAFCDLPLDDQVALLRAHAGEHLLLGAAKRSMLYKDILLLGNDHIVPRNCPELEVSRVAVRILDELVLPFQDLQIDDNEYACLKAIVFFDPDAKGLSDPSKIKRMRYQVQVSLEDYINDRQYDSRGRFGELLLLLPTLQSITWQMIEQIQFVKLFGMAKIDNLLQEMLLGGSANEAPHSHHSLHPHLVQEHLSNNVIVTTNMATPIHNGQMSTPETPIPSPPTASGSDHYKMAPGVIATVPKQPSSIPQPTITKQEAI(SEQ ID No.1).

[0073] Nucleotide sequence of grass carp Hnf4α (gcHnf4α):

[0074]

[0075] Example 2

[0076] Constitutive and inducible expression of Hnf4α in grass carp, and its subcellular localization before and after cell infection.

[0077] In this embodiment, healthy grass carp (average weight 10±1g) were raised in aerated water at a temperature of 25±2℃ and fed with 3% of their body weight of commercial pellet feed daily.

[0078] For constitutive expression analysis, three grass carp were randomly selected, and seven tissues, including liver, heart, intestine, spleen, brain, gills and kidney, were collected. All samples were stored at -80℃, and RNA was extracted and reverse transcribed for qRT-PCR detection.

[0079] For the inducible expression analysis experiment, grass carp were randomly divided into 4 groups and injected intraperitoneally with PBS at a dose of 15 μL / g (as the control group), 1×10 5 Aeromonas salmonidida CFU / mL, 1.38 × 10⁻⁶ 9 Infection was performed using GCRV-II virus solution at concentrations of copies / µL, and a mixture of A. salmonicida bacterial solution and GCRV-II virus solution (at the same concentrations as in the first two experiments). Liver samples from three fish were collected at 6 and 24 hours post-infection, RNA was extracted, and used for qRT-PCR detection.

[0080] For subcellular localization experiments, CIK cells were seeded in 24-well plates. The following day, each well was transfected with 800 ng of the empty FLAG plasmid and the gcHnf4α-FLAG plasmid prepared in Example 1. After 36 h of transfection, cells were either left uninfected or infected with *A. salmonicida* (MOI = 0.5) or GCRV-I (MOI = 0.5), respectively. Once cytopathic effects appeared, cells were washed with PBS, and each well was fixed with 4% paraformaldehyde at room temperature for 1 h. The fixative was removed, and cells were washed three times with PBS, followed by permeabilization with 0.1% Triton X-100 for 10 min, and then washed three times with PBS. Next, each well was blocked with PBS containing 5% BSA at room temperature for 1 h, and then washed three times with PBS. Anti-FLAG antibody (1:1000) was added and incubated overnight (for samples infected with GCRV-I, GCRV-I NS38 antibody was added separately), and the cells were washed three times with PBST. Dilute the fluorescent 488-conjugated anti-mouse IgG (1:500) secondary antibody and the fluorescent 594-conjugated anti-rabbit IgG (1:500) secondary antibody with 2% BSA in PBST solution, incubate in the dark for 2 hours, and rinse three times with PBST at room temperature for 5 minutes each time. Then remove the slides, mount each slide with 10 μL of mounting medium containing DAPI, taking care to avoid air bubbles, incubate overnight at 4°C, and then photograph and observe using a fluorescence inverted microscope.

[0081] The results are as follows Figure 2 As shown, in grass carp, Hnf4α expression was highest in the liver, followed by the kidneys and intestines, and lowest in the gills (see...). Figure 2 (A) In grass carp, single pathogen infection with Aeromonas salmonicidae, single pathogen infection with GCRV-II, and co-infection with Aeromonas salmonicidae and GCRV-II were performed. At 6 h and 24 h post-infection with each pathogen, the expression level of Hnf4α in grass carp was significantly upregulated compared to the PBS-injected control group (see A). Figure 2 (See section B). Meanwhile, subcellular localization of grass carp HNF4α was examined in CIK cells, revealing that grass carp HNF4α was primarily localized in the nucleus in uninfected individuals, and remained localized in the nucleus after infection with Aeromonas salmonidone or GCRV-I, without any change in subcellular localization (see section B). Figure 2 (C and D in the text).

[0082] Example 3

[0083] Effects of gcHnf4α overexpression in CIK cells on Aeromonas salmonicida proliferation and cell survival

[0084] In this embodiment, CIK cells were seeded into 24-well plates, and the following day, each well was transfected with 800 ng of the empty FLAG plasmid and the gcHnf4α-FLAG plasmid prepared in Example 1. After 36 h of transfection, bacterial infection was performed by adding 250 μL of *A. salmonicida* with an MOI of 1 diluted in MEM medium to each well. The cells were cultured at 25°C for 2 h, and then an equal volume of MEM medium containing 4% FBS was added for continuous infection. At 3 and 6 hpi, the mixture of cells and culture supernatant was collected, thoroughly vortexed, and serially diluted with PBS. 100 μL of the mixture was seeded onto BHI agar plates containing ampicillin. The culture plates were inverted and placed in a 28°C bacterial incubator for 18 h, and colony counting was performed. The experiment was repeated three times.

[0085] Figure 3 The results showed that gcHnf4α had antibacterial activity in CIK cells, and overexpression of gcHnf4α inhibited bacterial proliferation at 3 h and 6 h post-A. salmonicida infection. At 3 h post-infection, the mean A. salmonicida load in the gcHnf4α overexpression group was 4.45 × 10⁻⁶. 4 The average bacterial load in the FLAG group was 1.82 × 10⁻⁶ CFU / mL. 5 CFU / mL. At 6 h of infection, the average A. salmonicida load in the gcHnf4α overexpression group was 2.95 × 10⁻⁶. 6 The average bacterial load in the FLAG group was 4.58 × 10⁻⁶ CFU / mL. 6 CFU / mL, overexpression of gcHnf4α can significantly inhibit the proliferation of A. salmonicida in cells.

[0086] In the cell death assay, CIK cells were transfected with the empty FLAG plasmid and the gcHnf4α-FLAG plasmid prepared in Example 1, respectively. After 36 h of transfection, *A. salmonicida* was infected. Cell supernatant was collected at 6 hpi, centrifuged at 600 × g for 5 min, and 50 μL of the clear supernatant was transferred to a 96-well plate (each sample was repeated 3 times). An equal volume of CytoTox was then added to each well. Reagents. Incubate at room temperature in the dark for 30 min, then add 50 μL of reaction stop solution. After 1 h, detect the absorbance signal at 490 nm using a microplate reader. Calculate the percentage of cell death according to the formula in the instructions: Percentage of cell death = (Sample A490 nm - Uninfected cell A490 nm) / (Lysate-treated cell A490 nm - Uninfected cell A490 nm) × 100.

[0087] Figure 3The results of study B showed that gcHnf4α could inhibit cell death after infection with A. salmonicida. Cell death occurred after A. salmonicida infection, and the mortality rates of the FLAG overexpression group and the gcHnf4α overexpression group were 51.45% and 35.72%, respectively. Overexpression of gcHnf4α can reduce cell death caused by A. salmonicida infection, and the mortality rate decreased by 15.73% compared with the control group, which is beneficial to the survival of CIK cells.

[0088] In the cell viability assay, well-grown CIK cells were cultured at 1.5 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of 48 wells. The following day, each well was transfected with 400 ng of empty FLAG plasmid and gcHnf4α-FLAG plasmid prepared in Example 1. 36 h post-transfection, the culture medium was discarded, and 200 μL of *A. salmonicida* with an MOI of 0.5 diluted in MEM medium was added to each well as the infection group, or serum-free MEM medium was added as the uninfected group. The cells were cultured at 25°C for 2 h, and then 200 μL of MEM medium containing 4% FBS was added for further infection. At 6, 24, and 48 h post-infection, the culture medium was discarded, and the cells were washed twice with MEM medium. Then, 200 μL of CCK-8 solution diluted in MEM medium (medium medium: CCK-8 solution = 100:1) was added to each well. The cells were incubated at 37°C for 4 h, and the absorbance signal was measured at 450 nm using a microplate reader for cell viability analysis.

[0089] The results are as follows Figure 3 As shown in Figure C, overexpression of gcHnf4α can enhance the activity of both uninfected and infected cells. In the uninfected state, overexpression of gcHnf4α can enhance the activity of CIK cells. At 6 h, 24 h, and 48 h, compared with the corresponding control groups, the cell activity of the gcHnf4α group was increased by 17.53%, 25.88%, and 20.15%, respectively. At 24 h and 48 h after A. salmonicida infection, the cell activity of cells overexpressing gcHnf4α was increased by 32.24% and 25.82% compared with the FLAG control group, respectively.

[0090] In summary, gcHnf4α can inhibit bacterial proliferation, reduce cell death rate, and increase cell activity after bacterial infection.

[0091] Example 4

[0092] Effects of gcHnf4α overexpression on GCRV replication and cell survival in CIK cells

[0093] In the cell virus infection experiment, CIK cells in good growth condition were first divided into groups of 3 × 10⁻⁶ cells. 5 Cells were evenly seeded at a density of cells / well in 24-well plates. After 18 hours, 800 ng of FLAG or the gcHnf4α-FLAG plasmid prepared in Example 1 was transfected. After 36 hours of transfection, the cell culture medium was discarded, and 500 μL of serum-free MEM medium was added to each well to dilute GCRV-I virus to different gradients (multiples of infection of 0, 0.02, 0.2, and 2). The plates were incubated at 25°C. After 1 hour, the virus dilution was removed, and the medium was replaced with MEM medium containing 2% FBS for further culture. After the cells showed cytopathic effects, the supernatant was collected for titer determination, and the cells were fixed with 4% paraformaldehyde. After 1 hour, the fixative was removed, and the cells were stained overnight at room temperature with 1% crystal violet solution. After removing the crystal violet solution, the cells were air-dried and photographed.

[0094] The results are as follows Figure 4 As shown in the crystal violet staining results after viral infection, it can be seen that when cells overexpressing gcHnf4α were infected with GCRV-I at different multiplicity of infection, gcHnf4α could resist GCRV-I infection, and the number of remaining cells in the wells was greater than that in the control group (see...). Figure 4 (A) Collect the supernatant of the culture medium from the infected cells with MOI=0.2 and use TCID50. 50 The viral titer was detected using the method described above. It was found that overexpression of gcHnf4α significantly inhibited GCRV-I replication, reducing viral load by 18.1 times compared to the control group (see [link to data]). Figure 4 (B in the text) This indicates that gcHnf4α has antiviral activity in CIK cells.

[0095] Example 5

[0096] Evaluation of the effect of gcHnf4α overexpression in CIK cells on co-infection with Aeromonas salmonicidae and GCRV-I

[0097] In the single pathogen infection experiment of Aeromonas salmonicidae, CIK cells were prepared at a ratio of 3 × 10⁶ cells per well. 5 Cells were seeded in 24-well plates and cultured overnight, then transfected with 800 ng FLAG or gcHnf4α-FLAG. 36 h after transfection, 250 μL of *A. salmonicida* diluted with MEM medium (multiple of infection (MOF) of 0.5) was added to each well for infection. At 24 hpi, cells and supernatant were collected for crystal violet staining, colony counting, CCK8 cell viability assay, and qRT-PCR.

[0098] In the GCRV-I single pathogen infection experiment, CIK cells were prepared at a ratio of 3 × 10⁶ cells per well. 5Cells were seeded in 24-well plates and cultured overnight, then transfected with 800 ng FLAG or gcHnf4α-FLAG. 36 h after transfection, 250 μL of GCRV-I diluted with MEM medium at a multiplicity of infection (MOI) of 0.5 was added to each well for infection. One h later, the viral dilution was removed, and the medium was replaced with MEM medium containing 2% FBS for further culture. At 24 hpi, cells and supernatant were collected for crystal violet staining, colony counting, CCK8 cell viability assay, and qRT-PCR.

[0099] In the cell co-infection experiment, CIK cells were arranged at a density of 3 × 10⁶ cells per well. 5 Cells were seeded in 24-well plates and cultured overnight, then transfected with 800 ng FLAG or gcHnf4α-FLAG. 36 h after transfection, 250 μL of A. salmonicida diluted with MEM medium (MOI = 0.5) was added to each well for infection. After 1.5 h of incubation at 25°C, an equal volume of MEM medium containing 4% fetal bovine serum was added for continued infection. Six hours later, the medium was carefully removed to retain A. salmonicida adsorbed onto the cells. 500 μL of MEM medium containing GCRV-I (MOI = 0.5) was added, and the cells were cultured for 1 h. The medium was discarded, and the cells were cultured in MEM medium containing 2% FBS at 25°C for 18 h. Cells and supernatant were then collected for crystal violet staining, colony counting, CCK8 cell viability assay, and qRT-PCR.

[0100] The results are as follows Figure 5 As shown, crystal violet staining and cell viability assays after overexpression of gcHnf4α, GCRV-I single pathogen infection, A. salmonicida single pathogen infection, and co-infection with GCRV-I and A. salmonicida showed that cell viability was higher than that of the control group in all infection modalities, with increases of 13.30%, 24.36%, and 13.17%, respectively (see Figure 1). Figure 5 (A and B in the text), and in cases of single infection with *A. salmonicida* and co-infection with GCRV-I, the bacterial load of gcHnf4α was 1.4 × 10⁻⁶. 3 and 1.7×10 3 It was much lower than the control group (see Figure 5 (C) In GCRV-I single pathogen infection and bacterial-viral co-infection, the overexpression of gcHnf4α was verified by real-time PCR, and the relative expression levels of GCRV-I non-structural proteins NS38 and NS80 were detected (the expression levels of viral proteins NS38 and NS80 were positively correlated with the GCRV-I load). It can be seen that gcHnf4α significantly reduced the expression levels of NS38 and NS80 (see C). Figure 5The D, E, and F values ​​indicate that gcHnf4α plays a role in resisting bacterial and / or viral infections, either alone or in co-infection.

[0101] Example 6

[0102] Effect of caspase 3 inhibition on the disease resistance of gcHnf4α overexpression in CIK cells

[0103] Based on the single infection and co-infection experimental methods in Example 5, cells were treated with 50 μM of the caspase 3 inhibitor Z-DEVD-FMK 1 hour before infection. Cell infection was then performed, and samples were collected after lesions appeared. The role of gcHnf4α in this process was assessed by crystal violet staining, colony counting, CCK8 cell viability assay, and qRT-PCR.

[0104] The results are as follows Figure 6 As shown, after casapse 3 was inhibited by the inhibitor, the role of gcHnf4α in resisting single bacterial or viral pathogen infections and co-infections, as originally demonstrated in Example 5, was suppressed. The results of the crystal violet staining experiment showed that gcHnf4α did not show significant differences in different infection modalities compared to the control group (see...). Figure 6 In the presence of the caspase 3 inhibitor Z-DEVD-FMK, the cell activity of gcHnf4α in different infection modalities was not significantly different from that in the control group (see A). Figure 6 In the case of B), while caspase 3 was inhibited, there was no difference in colony count after single bacterial pathogen infection with gcHnf4α, but in bacterial-viral co-infection, the bacterial load of the gcHnf4α-FLAG group was 2.54 × 10⁻⁶. 4 The CFU / mL concentration even exceeded that of the FLAG control group (1.96 × 10⁻⁶). 4 CFU / mL)(see Figure 6 (C) Regarding GCRV-I NS38, there was no difference in expression levels in single-pathogen viral infections. However, in bacterial-viral co-infections, the relative expression level of NS38 was higher than that of the control group, upregulated by 2.67-fold. Meanwhile, the relative expression level of NS80 was higher than that of the corresponding control groups in both single-pathogen viral infections and co-infections, upregulated by 4.07-fold and 3.03-fold, respectively (see C). Figure 6 The D in the figure indicates that when caspase 3 is inhibited, gcHnf4α cannot exert its original function of resisting single bacterial or viral pathogen infection or bacterial-viral co-infection.

[0105] Example 7

[0106] Effect of caspase 9 inhibition on the disease resistance of gcHnf4α overexpression in CIK cells

[0107] Based on the single infection and co-infection experimental methods in Example 5, cells were treated with the caspase 9 inhibitor Z-LEHD-FMK at 50 μM 1 hour before infection. Cell infection was then performed, and samples were collected after lesions appeared. The role of gcHnf4α in this process was assessed by crystal violet staining, CCK8 cell viability assay, colony counting, and qRT-PCR.

[0108] The results are as follows Figure 7 As shown, after casapse 9 was inhibited by the inhibitor, the effect of gcHnf4α in resisting single bacterial or viral pathogen infections and mixed infections, as originally demonstrated in Example 5, was suppressed. The results of the crystal violet staining experiment showed that gcHnf4α did not show significant differences in different infection modalities compared to the control group (see...). Figure 7 In the presence of the caspase 9 inhibitor Z-LEHD-FMK, the cell viability of gcHnf4α gradually decreased in different modes of bacterial single-pathogen infection, viral single-pathogen infection, and bacterial-viral co-infection. In GCRV-I infection, gcHnf4α failed to increase cell viability (75.71%), a decrease of 9.41% compared to the control group (85.12%). After co-infection with GCRV-I and A. salmonicida, the cell viability of the gcHnf4α overexpression group and the FLAG group were 57.63% and 68.67%, respectively, with the gcHnf4α group showing an 11.04% decrease in cell viability (see A). Figure 7 (B in the text); while in the case of caspase 9 inhibition, bacterial proliferation increased after co-infection, but there was no difference in colony count between the gcHnf4α overexpression group and the FLAG control group (see B in the text); Figure 7 (C) Overexpression of gcHnf4α inhibits caspase 9. For GCRV-I NS38 and NS80, the expression levels of these two molecules were not different from the control group in both single viral infection and bacterial-viral co-infection (see C). Figure 7 (D in the text). These results also indicate that when caspase 9 is inhibited, gcHnf4α cannot exert its original function of resisting single bacterial or viral pathogen infections or bacterial-viral co-infections.

[0109] Example 8

[0110] The effect of gcHnf4α overexpression in zebrafish on the survival of juvenile fish under bacterial and viral infection conditions.

[0111] The empty FLAG plasmid or gcHnf4α-FLAG plasmid was diluted to 200 ng / μL and microinjected into zebrafish fertilized eggs at the single-cell or two-cell stage at a dose of 2 nL. The injected embryos were then reared at 28°C for 4 days post-flop (dpf). The juveniles were then divided into four groups: uninfected group, *A. salmonicida* infected group (bacterial infection group), GCRV-II infected group (viral infection group), and *A. salmonicida* and GCRV-II co-infected group (bacterial-viral co-infection group). The FLAG empty plasmid or gcHnf4α-FLAG plasmid was diluted to 200 ng / μL and microinjected into zebrafish fertilized eggs at the single-cell or two-cell stage at a final concentration of 1 × 10⁻⁶. 7 CFU / mL of A.salmonicida, 2.76×10 10 GCRV-II copies / mL or containing 1×10 7 A. salmonicida CFU / mL and 2.76×10 10 Zebrafish were infected with a bacterial and viral mixture containing copies / mL of GCRV-II, and 20mL of culture water was added 6 hours later. Mortality of the zebrafish juveniles was recorded daily, and the survival differences between groups were compared using a log-rank test for survival curve analysis.

[0112] according to Figure 8 The survival curve statistics show that, in the uninfected group, apart from the mortality of juvenile fish within the normal range during the growth stage, the survival rates of the Hnf4α-FLAG overexpression group and the FLAG empty plasmid overexpression group were 84.4% and 81.1%, respectively, with no difference between the two (see [link to data]). Figure 8 (A in the text). After infection with *A. salmonicida*, the survival rate of zebrafish juveniles overexpressing the empty plasmid FLAG was 42.2%, while the survival rate of zebrafish juveniles overexpressing Hnf4α-FLAG was 60.0%, which was 17.8% higher than the control group (p<0.01). Figure 8 (B) In the study, the survival rates of juvenile fish overexpressing gcHnf4α-FLAG or FLAG empty plasmid were 64.4% and 45.6% after GCRV-II infection, respectively. Overexpression of gcHnf4α significantly increased the survival rate by 18.8% (see section B). Figure 8 (C) In the case of co-infection with A. salmonicida and GCRV-II, the survival rate of juvenile fish overexpressing Hnf4α-FLAG was 44.4%, which was 23.3% higher than the survival rate of the control (21.1%), p<0.001 (see C). Figure 8 (D in the text). This demonstrates that overexpression of Hnf4α in wild-type zebrafish provides resistance to single pathogen infection by A. salmonicida and GCRV-II, as well as bacterial-viral co-infection, further validating the results of the in vitro experiments in Example 5.

[0113] Example 9

[0114] The effect of inhibiting caspase 3 on the overexpression of gcHnf4α in zebrafish on the survival of juvenile fish under bacterial and viral infections.

[0115] Following the method described in Example 8, the empty FLAG plasmid or the gcHnf4α-FLAG plasmid was overexpressed in zebrafish embryos. After hatching, the juveniles were treated with a 75 μM caspase 3 inhibitor, Z-DEVD-FMK, for 18 h. Subsequently, the juveniles were subjected to single-pathogen and mixed-pathogen infections with bacteria and / or viruses. Daily mortality rates of the zebrafish juveniles were recorded, survival curves were plotted, and log-rank tests were used to compare survival differences between groups.

[0116] according to Figure 9 The survival curve statistics show that after Z-DEVD-FMK treatment, in the uninfected group, the survival rate of the Hnf4α-FLAG overexpression group (66.6%) was 17.8% lower than that of the FLAG empty plasmid overexpression group (84.4%), p<0.01 (see [link to data]). Figure 9 (A in the text). Following infection with *A. salmonicida* with caspase 3 inhibition, the survival rate of the group overexpressing the empty plasmid FLAG was 73.3%, while the survival rate of juvenile zebrafish overexpressing Hnf4α-FLAG was 43.3%, a decrease of 30.0% compared to the control group (p < 0.001). Figure 9 (B) Survival rates of juvenile fish overexpressing gcHnf4α-FLAG or FLAG empty plasmid after GCRV-II infection were 12.2% and 34.4%, respectively. Inhibition of caspase 3 significantly reduced the survival rate of juvenile fish overexpressing gcHnf4α, decreasing the survival rate by 22.3% (p < 0.001). Figure 9 (C in the text). However, when *A. salmonicida* and GCRV-II were co-infected after caspase 3 inhibition, all juvenile fish in the Hnf4α-FLAG overexpression group died by day 5 post-infection, a 4.4% decrease in survival rate compared to the control (p<0.05). Figure 9 (D in the example). This demonstrates that inhibiting caspase 3 in zebrafish juveniles and overexpressing Hnf4α inhibits the original ability of Hnf4α to resist single pathogen infection of A. salmonicida and GCRV-II, as well as co-infection of A. salmonicida and GCRV-II. This further verifies the in vitro experimental results in Example 6, indicating that the functions of gcHnf4α in resisting bacterial single pathogen infection, viral single pathogen infection, and bacterial-viral co-infection depend on caspase 3.

[0117] Example 10

[0118] The effect of inhibiting caspase 9 on the overexpression of gcHnf4α in zebrafish on the survival of juvenile fish under bacterial and viral infections.

[0119] Following the method described in Example 8, the empty FLAG plasmid or the gcHnf4α-FLAG plasmid was overexpressed in zebrafish embryos. After hatching, the juveniles were treated with a 75 μM caspase 9 inhibitor, Z-LEHD-FMK, for 18 h. Subsequently, the juveniles were subjected to single pathogen and co-infection with bacteria and / or viruses. Daily mortality rates of the zebrafish juveniles were recorded, survival curves were plotted, and log-rank tests were used to compare survival differences between groups.

[0120] according to Figure 10 The survival curve statistics show that, after Z-LEHD-FMK treatment, there was no difference in the survival rate between the uninfected group overexpressing Hnf4α-FLAG (64.4%) and the group overexpressing the empty FLAG plasmid (73.3%) (see [link to data]). Figure 10 (A in the text). Following infection with *A. salmonicida* with caspase 9 inhibition, the survival rate of zebrafish juveniles overexpressing the empty FLAG plasmid was 15.6%, while the survival rate of zebrafish juveniles overexpressing Hnf4α-FLAG was 16.7%, with no significant difference between the two groups (see [link to original text]). Figure 10 (See section B). The survival rates of juvenile fish overexpressing gcHnf4α-FLAG or the FLAG empty plasmid group after GCRV-II infection were 12.2% and 29.2%, respectively. Inhibition of caspase 9 significantly reduced the survival of juvenile fish overexpressing gcHnf4α, decreasing the survival rate by 17.1% (p < 0.01). Figure 10 (C) In the case of co-infection with A. salmonicida and GCRV-II after caspase 3 inhibition, the survival rate of juvenile fish in the Hnf4α-FLAG overexpression group (12.2%) was 7.8% lower than that in the control group (20.0%), p<0.05 (see C). Figure 10 (D in the example). This demonstrates that inhibiting caspase 9 in zebrafish juveniles and overexpressing Hnf4α inhibits the original ability of Hnf4α to resist single pathogen infection of A. salmonicida and GCRV-II, as well as co-infection of A. salmonicida and GCRV-II. This further verifies the in vitro experimental results in Example 7, indicating that the functions of gcHnf4α in resisting bacterial single pathogen infection, viral single pathogen infection, and bacterial-viral co-infection also depend on caspase 9.

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Hnf4α-containing biomaterials in the preparation of at least one of the following products, characterized in that, (1) The product is used to prevent bacterial infections in cyprinid fish; (2) The product is used to prevent viral infections in cyprinid fish; (3) The product is used to prevent diseases caused by co-infection of bacteria and viruses in cyprinid fish; The bacteria is Aeromonas salmonidae, and the virus is grass carp reovirus; the grass carp reovirus is GCRV-I and / or GCRV-II. The Hnf4α-containing biological material is an Hnf4α-containing expression vector.

2. The application of Hnf4α-containing biomaterials in the preparation of products that enhance fish disease resistance, characterized in that, The fish is a cyprinid fish; the disease resistance refers to the ability to resist bacteria and / or viruses. The bacteria is Aeromonas salmonii, and the virus is grass carp reovirus; The grass carp reovirus is GCRV-I and / or GCRV-II; The Hnf4α-containing biological material is an Hnf4α-containing expression vector.

3. The application according to claim 1 or 2, characterized in that, The carp mentioned are grass carp and / or zebrafish.

4. A method for breeding disease-resistant fish, characterized in that, Includes the following steps: Increase the expression level of Hnf4α in recipient fish, wherein the disease resistance is against bacteria and / or viruses; wherein the bacteria is Aeromonas salmonidae, and the virus is grass carp reovirus; The grass carp reovirus is GCRV-I and / or GCRV-II; The recipient fish is a cyprinid fish.

5. The method according to claim 4, characterized in that, The carp mentioned are grass carp and / or zebrafish.

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