Application of Hnf4alpha or biological material containing Hnf4alpha in preparation of product for inhibiting bacteria and / or viruses

Abstract

The invention provides application of Hnf4alpha or a biological material containing Hnf4alpha in preparation of a product for inhibiting bacteria and / or viruses, and belongs to the technical field of biological medicines. In-vivo and in-vitro experiments show that overexpression of the Hnf4alpha not only can effectively prevent infection of aeromonas salmonicida and grass carp hemorrhagic disease viruses, but also can remarkably inhibit co-infection of the aeromonas salmonicida and the grass carp hemorrhagic disease viruses. The bacterial or virus load after infection is obviously reduced, and the activity of cells and the survival rate of juvenile fish are improved. The invention provides a molecular target for prevention and control of bacterial infectious diseases, virus infectious diseases and mixed infectious diseases of cyprinid fishes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of Hnf4α or a biomaterial containing Hnf4α in the preparation of products for inhibiting bacteria and / or viruses. Background Art

[0002] In recent years, with the rapid development of modern fishery, the aquaculture scale has advanced by leaps and bounds, and the world fishery production has increased rapidly, continuously providing high-quality protein for human tables. However, due to the large variety and scale of aquaculture and the continuous deterioration of the aquaculture environment, various fish diseases occur frequently and are complex, causing significant economic losses to aquaculture and seriously affecting the development of the aquaculture industry.

[0003] Aeromonas salmonicida belongs to Proteobacteria, Aeromonadaceae, and Aeromonas. It is a Gram-negative short bacillus. In addition to infecting salmonid fish, it can also infect carp, perch, mullet, catfish, etc. Among them, grass carp and zebrafish are also infected by Aeromonas salmonicida. Infection with Aeromonas salmonicida can cause fish to develop scabies or ulcers, which 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 virus in the genus Aquareovirus and causes grass carp hemorrhage disease, which can lead to severe hemorrhage and death of grass carp, with a high mortality rate. GCRV exists in three genotypes: GCRV-I, GCRV-II, and GCRV-III, and their representative strains are GCRV-873 (type I), GCRV-HZ08 (type II), and GCRV104 (type III). In aquaculture, due to the special growth environment of fish, the situation of mixed infection with multiple pathogens occurs frequently, seriously endangering the aquaculture benefits.

[0004] Hepatocyte nuclear factors (HNFs) are a class of transcription factors that regulate the specific expression of genes in the liver. The complex regulatory network formed by these transcription factors and their DNA or protein interactions with other genes precisely controls liver development and hepatocyte function. Hnf4 belongs to the nuclear receptor superfamily subfamily 2A (NR2A), including three members: Hnf4α, Hnf4β, and Hnf4γ. Among them, Hnf4α and Hnf4γ are widely distributed in mammals, while Hnf4β is only present in fish, amphibians, and birds. Hnf4α is closely related to type I maturity-onset diabetes of the young (MODY1) and is also involved in the occurrence of diseases such as liver fibrosis, cirrhosis, hepatocellular carcinoma, and fatty liver. Some studies have shown that the expression of HNF4α decreases during hepatocellular carcinoma, and overexpression of HNF4α inhibits the invasiveness of hepatocellular carcinoma cells and reduces the proliferation rate. HNF4α has a certain anti-tumor effect, but there are few reports on HNF4α in fish diseases, and the connection between HNF4α and apoptosis-related molecules has not been mentioned much in other studies. In addition, some research results have shown that while certain genes enhance protection against bacterial diseases, they increase the susceptibility to viral diseases; vice versa. Therefore, screening target molecules that can resist both bacterial and viral diseases is crucial for the prevention and control of fish infectious diseases, especially for the prevention and control of bacterial-viral co-infections. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of Hnf4α or a biological material containing Hnf4α in the preparation of products for inhibiting bacteria and / or viruses.

[0006] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in the preparation of products for inhibiting bacteria and / or viruses.

[0008] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in the preparation of at least one of the following (1) to (3) products,

[0009] (1) Products for preventing and treating diseases caused by bacterial infections;

[0010] (2) Products for preventing and treating diseases caused by viral infections;

[0011] (3) Products for preventing and treating diseases caused by bacterial and viral co-infections.

[0012] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in at least one of the following I to III,

[0013] Ⅰ. Promote the growth and reproduction of fish;

[0014] Ⅱ. Prepare products for promoting the growth and reproduction of fish;

[0015] Ⅲ. Prepare products for improving the disease resistance of fish.

[0016] Preferably, the bacterium is Aeromonas salmonicida; the virus is Grass carp reovirus.

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

[0018] a. An expression vector containing Hnf4α;

[0019] b. A host bacterium containing Hnf4α;

[0020] c. A host cell containing Hnf4α.

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

[0022] Preferably, the fish is a Cyprinidae fish.

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

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

[0025] Preferably, the disease - resistant fish can resist diseases caused by at least one of Aeromonas salmonicida infection, Grass carp reovirus infection, and Aeromonas salmonicida - 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] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in the preparation of products for inhibiting bacteria and / or viruses. Through in - vivo and in - vitro experiments, the present invention finds that the over - expression of Hnf4α can not only effectively prevent the infection of Aeromonas salmonicida and Grass carp hemorrhagic disease virus, but also significantly inhibit the co - infection of Aeromonas salmonicida and Grass carp hemorrhagic disease virus. It significantly reduces the bacterial or viral load after infection, improves the activity of cells and the survival rate of juvenile fish. The present invention provides a molecular target for the prevention and control of bacterial infectious diseases, viral infectious diseases, and mixed infectious diseases of Cyprinidae fish. Description of the Drawings

[0029] Figure 1 Protein electrophoresis pattern of the gcHnf4α-FLAG recombinant expression vector;

[0030] Figure 2 Expression and subcellular localization of gcHnf4α. A: Constitutive expression of gcHnf4α in healthy grass carp. B: Inducible expression of gcHnf4α at 6 hours post-infection (6hpi) and 24 hours post-infection (24hpi) after grass carp were infected with Aeromonas salmonicida and / or grass carp hemorrhage virus. C: Subcellular localization after overexpressing FLAG and gcHnf4α-FLAG in CIK cells with or without Aeromonas salmonicida infection. D: Subcellular localization after overexpressing FLAG and gcHnf4α-FLAG in CIK cells and infecting with grass carp hemorrhage virus;

[0031] Figure 3 Effect of overexpression of gcHnf4α in CIK cells on the proliferation of Aeromonas salmonicida and cell survival. A: Effect of overexpressing FLAG and gcHnf4α-FLAG in CIK cells on bacterial proliferation at 3 hours post-infection (3hpi) and 6 hours post-infection (6hpi) after Aeromonas salmonicida infection. B: Effect of overexpressing FLAG and gcHnf4α-FLAG in CIK cells on cell mortality after Aeromonas salmonicida infection. C: Effect of overexpressing FLAG and gcHnf4α-FLAG on cell viability was evaluated at 6, 24, and 48 hours in CIK cells with or without Aeromonas salmonicida infection;

[0032] Figure 4 Effect of overexpression of gcHnf4α in CIK cells on GCRV replication and cell survival. A: Crystal violet staining after overexpressing FLAG and gcHnf4α-FLAG in CIK cells after GCRV-I infection. B: Detection of GCRV-I titer after overexpressing FLAG and gcHnf4α-FLAG in CIK cells;

[0033] Figure 5To evaluate the effect of overexpression of gcHnf4α in CIK cells on Aeromonas salmonicida and GCRV-I co-infection. A is the crystal violet staining of overexpression of FLAG and gcHnf4α-FLAG in CIK cells after infection with Aeromonas salmonicida and / or GCRV-I. B is the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells on cell viability in the case of Aeromonas salmonicida and / or GCRV-I infection. C is 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. D is to verify the overexpression of gcHnf4α after single pathogen infection with GCRV-I or co-infection with Aeromonas salmonicida and GCRV-I after overexpression of FLAG and gcHnf4α-FLAG in CIK cells. E is to evaluate the change in relative expression level of the key molecule NS38 of GCRV-I after overexpression of FLAG and gcHnf4α-FLAG in CIK cells and single pathogen infection with GCRV-I or co-infection with Aeromonas salmonicida and GCRV-I. F is to evaluate the change in relative expression level of the key molecule NS80 of GCRV-I after overexpression of FLAG and gcHnf4α-FLAG in CIK cells and single pathogen infection with GCRV-I or co-infection with Aeromonas salmonicida and GCRV-I;

[0034] Figure 6 To evaluate the effect of overexpression of gcHnf4α in CIK cells on Aeromonas salmonicida and GCRV-I co-infection in the case of caspase 3 inhibition. A is the crystal violet staining of overexpression of FLAG and gcHnf4α-FLAG in CIK cells after infection with Aeromonas salmonicida and / or GCRV-I after inhibitor treatment. B is the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells on cell viability after Aeromonas salmonicida and / or GCRV-I infection after caspase 3 inhibition. C is 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 DEVD. D is to verify the overexpression of gcHnf4α and evaluate the change in relative expression levels of the key molecules NS38 and NS80 of GCRV-I after overexpression of FLAG and gcHnf4α-FLAG in CIK cells and caspase 3 inhibition, followed by single pathogen infection with GCRV-I or co-infection with Aeromonas salmonicida and GCRV-I;

[0035] Figure 7To evaluate the effect of overexpression of gcHnf4α in CIK cells on the co-infection of Aeromonas salmonicida and GCRV-I under the condition of caspase 9 inhibition. A shows the crystal violet staining after overexpression of FLAG and gcHnf4α-FLAG in CIK cells and then infecting with Aeromonas salmonicida and / or GCRV-I after LEHD treatment. B shows the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells on cell viability after infecting with Aeromonas salmonicida and / or GCRV-I after inhibiting caspase 9. C shows the effect of overexpression of FLAG and gcHnf4α-FLAG in CIK cells on bacterial proliferation after single infection with Aeromonas salmonicida or co-infection with Aeromonas salmonicida and GCRV-I in the presence of LEHD. D shows inhibiting caspase 9 after overexpression of FLAG and gcHnf4α-FLAG in CIK cells, then conducting single-pathogen infection with GCRV-I or co-infection with Aeromonas salmonicida and GCRV-I, verifying the overexpression of gcHnf4α and evaluating the changes in the relative expression levels of key molecules NS38 and NS80 of GCRV-I;

[0036] Figure 8 To investigate the effect of overexpression of gcHnf4α in zebrafish on the survival of juvenile fish under bacterial and viral infections. A shows the survival rate of wild-type zebrafish after overexpression of FLAG and gcHnf4α-FLAG without infection. B shows the effect of overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish on the survival rate of juvenile fish after single-pathogen infection with Aeromonas salmonicida. C shows the effect of overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish on the survival rate of juvenile fish after single-pathogen infection with GCRV-II. D shows the effect of overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish on the survival rate of juvenile fish after co-infection with Aeromonas salmonicida and GCRV-II;

[0037] Figure 9To inhibit the effect of caspase 3 on the survival of larvae after overexpression of gcHnf4α in zebrafish followed by bacterial and viral infections, A shows the survival rate of uninfected larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish in the presence of DEVD, B shows the effect of single-pathogen infection with Aeromonas salmonicida on the survival rate of larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish under the condition of inhibiting caspase 3, C shows the effect of single-pathogen infection with GCRV-II on the survival rate of larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish under the condition of inhibiting caspase 3, and D shows the effect of co-infection with Aeromonas salmonicida and GCRV-II on the survival rate of larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish under the condition of caspase 3 inhibition.

[0038] Figure 10 To inhibit the effect of caspase 9 on the survival of larvae after overexpression of gcHnf4α in zebrafish followed by bacterial and viral infections, A shows the survival rate of uninfected larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish in the presence of LEHD, B shows the effect of single-pathogen infection with Aeromonas salmonicida on the survival rate of larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish under the condition of inhibiting caspase 9, C shows the effect of single-pathogen infection with GCRV-II on the survival rate of larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish under the condition of inhibiting caspase 9, and D shows the effect of co-infection with Aeromonas salmonicida and GCRV-II on the survival rate of larvae after overexpression of FLAG and gcHnf4α-FLAG in wild-type zebrafish under the condition of inhibiting caspase 9. Specific implementation manners

[0039] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in the preparation of products for inhibiting bacteria and / or viruses.

[0040] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in the preparation of at least one of the following products (1) to (3),

[0041] (1) Products for preventing and treating diseases caused by bacterial infections;

[0042] (2) Products for preventing and treating diseases caused by viral infections;

[0043] (3) Products for preventing and treating diseases caused by co-infections of bacteria and viruses.

[0044] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in at least one of the following I to III:

[0045] I. Promoting the growth and reproduction of fish;

[0046] II. Preparing a product for promoting the growth and reproduction of fish;

[0047] III. Preparing a product for improving the disease resistance of fish.

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

[0049] In the present invention, the bacterium is Aeromonas salmonicida, and the virus is Grass carp reovirus. In III, the product can improve the disease resistance of fish caused by at least one of Aeromonas salmonicida infection, Grass carp reovirus infection, and Aeromonas salmonicida - Grass carp reovirus co - infection.

[0050] In the present invention, the biological material containing Hnf4α includes any one of the following a to c:

[0051] a. An expression vector containing Hnf4α;

[0052] b. A host bacterium containing Hnf4α;

[0053] c. A host cell containing Hnf4α.

[0054] In the present invention, an existing mammalian cell expression vector can be used to construct an expression vector containing Hnf4α. The empty vector of the mammalian cell expression vector is preferably p3×FLAG - CMV TM -14; a prokaryotic system can be used to construct a host bacterium containing Hnf4α. The prokaryotic system is preferably Escherichia coli, such as Escherichia coli Top 10; the host cell is preferably CIK cell.

[0055] In the above - mentioned biological material, as a preferred method, the preparation of the expression vector containing Hnf4α in the present invention includes the step of inserting Hnf4α into the p3×FLAG - CMV TM -14 vector to obtain an expression vector containing Hnf4α. The insertion of Hnf4α into p3×FLAG - CMV TMThe restriction enzyme of the vector is between Hind III and BamHI. The preparation of the host bacterium containing Hnf4α includes the step of transforming the expression vector containing Hnf4α into Escherichia coli Top 10 competent cells. The preparation of the host cell containing Hnf4α includes the step of transfecting the expression vector containing Hnf4α into CIK cells. The present invention can ensure the overexpression of Hnf4α and also ensure the activity of Hnf4α by using the above-mentioned expression vector, host bacterium or host cell.

[0056] The present invention provides an application of Hnf4α or a biological material containing Hnf4α in the preparation of a product for improving the disease resistance of fish, and the product can improve the resistance of fish to diseases caused by at least one of Aeromonas salmonicida infection, grass carp reovirus infection, and Aeromonas salmonicida-grass carp reovirus co-infection.

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

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

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

[0060] In the present invention, the way to increase the expression level and / or activity of Hnf4α in recipient fish is achieved by introducing the above-mentioned biological material into the recipient fish, such as injecting the above-mentioned expression vector containing Hnf4α into the recipient fish. The above-mentioned biological material has been described above and will not be elaborated here one by one.

[0061] In the present invention, the disease-resistant fish can resist diseases caused by at least one of Aeromonas salmonicida infection, grass carp reovirus infection, and Aeromonas salmonicida-grass carp reovirus co-infection. The recipient fish is a Cyprinidae fish, such as grass carp and / or zebrafish.

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

[0063] By overexpressing Hnf4α in recipient fish, the present invention can improve the resistance of recipient fish to diseases caused by at least one of Aeromonas salmonicida infection, grass carp reovirus infection, and Aeromonas salmonicida-grass carp reovirus co-infection, thereby promoting the survival rate and growth of recipient fish.

[0064] In the present 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] In the following embodiments, the Aeromonas salmonicida is ATCC 33658; the GCRV-I is GCRV-873; 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] In this example, the nucleotide sequence of grass carp Hnf4α was downloaded from the NCBI database, combined with the multiple cloning sites of the eukaryotic expression plasmid p3×FLAG-CMV TM -14, and primers were designed using SnapGene software. The ORF region of Hnf4α was amplified using grass carp cDNA as a template. After double digestion with Hind III and BamHI enzymes, it was inserted into the p3×FLAG-CMV TM -14 expression vector, transformed with Escherichia coli Top 10 competent cells, and monoclonal colonies were picked for verification. According to the sequencing results, the strain with correct sequencing was selected to extract the plasmid using the OMEGA kit, and the concentration and purity were detected using a micro-spectrophotometer. The grass carp Hnf4α plasmid was named gcHnf4α-FLAG. The gcHnf4α-FLAG was transfected into CIK cells. After 48 hours of transfection, the cells were collected, and the cell proteins were extracted using RIPA lysis buffer and verified by Western Blot.

[0070] The experimental results are as Figure 1 shown. The size of the protein detected by Western Blot was consistent with the predicted size of the gcHnf4α-FLAG protein and the band was single, with a size of approximately 50 kDa. From the sequencing results, the amino acid sequence of grass carp Hnf4α (gcHnf4α) is shown in SEQ ID No.1, and the nucleotide sequence of grass carp Hnf4α (gcHnf4α) is shown in SEQ ID No.2. This indicates that the grass carp Hnf4α plasmid was successfully constructed.

[0071] 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 example, healthy grass carp (average weight 10 ± 1 g) were cultured in aerated water at a temperature maintained at 25 ± 2 °C and fed commercial pellet feed at 3% of their body weight daily.

[0078] For constitutive expression analysis, 3 grass carp were randomly selected, and 7 tissues including liver, heart, intestine, spleen, brain, gill, and kidney were collected respectively. All samples were stored at -80 °C and used for qRT-PCR detection after RNA extraction and reverse transcription.

[0079] For the inducible expression analysis experiment, grass carp were randomly divided into 4 groups. By intraperitoneal injection, PBS (as the control group) was injected at a dose of 15 μL / g, Aeromonas salmonicida (A. salmonicida) at 1×10 5 CFU / mL, GCRV-II virus solution at 1.38×10 9 copies / uL, and a mixture of A. salmonicida bacterial solution and GCRV-II virus solution were used for infection (at the concentrations in the first two groups of experiments). At 6 and 24 h after infection, the livers of 3 fish were collected, RNA was extracted, and used for qRT-PCR detection.

[0080] For the subcellular localization experiment, CIK cells were seeded in 24-well plates. The next day, 800 ng of FLAG empty plasmid and the gcHnf4α-FLAG plasmid prepared in Example 1 were transfected into each well respectively. After 36 h of transfection, cells were either not infected or infected with A. salmonicida (MOI = 0.5) or GCRV-I (MOI = 0.5) respectively. After lesions appeared, the cells were rinsed with PBS, and an appropriate amount of 4% paraformaldehyde was added to each well and fixed at room temperature for 1 h. The fixing solution was removed, and after rinsing 3 times with PBS, 0.1% Triton X-100 was added for permeabilization treatment for 10 min, and then rinsed 3 times with PBS. Next, each well was added with PBS solution containing 5% BSA and blocked at room temperature for 1 h, and then rinsed 3 times with PBS. Anti-FLAG antibody (1:1000) was added and incubated overnight (for samples after GCRV-I infection, NS38 antibody of GCRV-I needed to be additionally added), and then rinsed 3 times with PBST. Then, the secondary antibody of fluorescein 488-conjugated anti-mouse IgG (1:500) and fluorescein 594-conjugated anti-rabbit IgG (1:500) were diluted with PBST solution containing 2% BSA, incubated in the dark for 2 h, and rinsed 3 times with PBST at room temperature for 5 min each time. After that, the slides were taken out, and each slide was sealed with 10 μL of mounting medium containing DAPI, taking care to avoid air bubbles. After incubating overnight at 4 °C, photographs were taken and observed with a fluorescence inverted microscope.

[0081] The results are as Figure 2 shown. In grass carp, the expression level of Hnf4α was the highest in the liver, followed by the kidney and intestine, and the least distribution was in the gill (see Figure 2 A in). When grass carp were infected with Aeromonas salmonicida alone, GCRV-II alone, or co-infected with Aeromonas salmonicida and GCRV-II respectively, it was found that at 6 h and 24 h after different pathogen infections, the expression level of grass carp Hnf4α was significantly up-regulated compared with the control group injected with PBS (see Figure 2 B in). At the same time, the subcellular localization of grass carp HNF4α was detected in CIK cells, and it was found that grass carp HNF4α was mainly localized in the nucleus when not infected, and still localized in the nucleus after Aeromonas salmonicida infection or GCRV-I infection, and there was no change in subcellular localization (see Figure 2 C and D in).

[0082] Example 3

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

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

[0085] Figure 3 Result A in [reference] shows that gcHnf4α has antibacterial effects in CIK cells, and overexpression of gcHnf4α can inhibit bacterial proliferation at 3 h and 6 h after A. salmonicida infection. At 3 h of infection, the average A. salmonicida load in the gcHnf4α overexpression group was 4.45×10 4 CFU / mL, while the average bacterial load in the FLAG group was 1.82×10 5 CFU / mL. At 6 h of infection, the average A. salmonicida load in the gcHnf4α overexpression group was 2.95×10 6 CFU / mL, while the average bacterial load in the FLAG group was 4.58×10 6 CFU / mL. Overexpression of gcHnf4α can significantly inhibit the proliferation of A. salmonicida in cells.

[0086] In the cell mortality detection experiment, the FLAG empty plasmid and the gcHnf4α-FLAG plasmid prepared in Example 1 were transfected into CIK cells respectively. After 36 h of transfection, A. salmonicida infection was carried out. At 6 hpi, the cell supernatant was collected and centrifuged at 600×g for 5 min. 50 μL of the centrifuged clear supernatant was transferred to a 96-well plate (each sample was repeated 3 times), and then an equal volume of CytoTox reagent was added to each well. Incubate in the dark at room temperature for 30 min, and then add 50 μL of reaction termination solution. After 1 h, the absorbance signal was detected at 490 nm with an enzyme-linked immunosorbent assay reader. Calculate the percentage of cell death according to the formula in the instruction manual: percentage of cell death = (A490 nm of sample - A490 nm of uninfected cells) / (A490 nm of cells treated with lysis solution - A490 nm of uninfected cells) × 100.

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

[0088] In the cell viability detection experiment, well-growing CIK cells were inoculated into 48-well plates at a density of 1.5×10 5 / well. The next day, 400 ng of FLAG empty plasmid and the gcHnf4α-FLAG plasmid prepared in Example 1 were transfected into each well respectively. 36 h after transfection, the medium was discarded, and 200 μL of A. salmonicida with an MOI of 0.5 diluted with MEM medium was added to each well as the infected 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 to continue the infection. At 6, 24, and 48 h after infection respectively, the medium was discarded and the cells were rinsed twice with MEM medium. Subsequently, 200 μL of CCK-8 solution diluted with MEM medium (medium:CCK-8 solution = 100:1) was added to each well. After incubating in a 37 °C cell culture incubator for 4 h, the absorbance signal was detected at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader for cell viability analysis.

[0089] The results are as Figure 3 shown in C in . Overexpression of gcHnf4α can improve the viability of uninfected and infected cells. In the absence of infection, overexpression of gcHnf4α can improve the viability of CIK cells. At 6 h, 24 h, and 48 h, compared with the corresponding control groups, the cell viabilities of the gcHnf4α group increased by 17.53%, 25.88%, and 20.15% respectively; at 24 h and 48 h after A. salmonicida infection, the cell viabilities of the cells overexpressing gcHnf4α were increased by 32.24% and 25.82% respectively compared with the cell viabilities of the FLAG control group.

[0090] In summary, gcHnf4α can inhibit bacterial proliferation, reduce cell mortality, and improve cell viability after bacterial infection.

[0091] Example 4

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

[0093] In the cell virus infection experiment, first, CIK cells in good growth state were evenly inoculated into 24-well plates at a density of 3×10 5 cells / well. After 18 h, 800 ng of FLAG or the gcHnf4α-FLAG plasmid prepared in Example 1 was transfected. After 36 h of transfection, the cell culture medium was discarded, and 500 μL of GCRV-I virus diluted to different gradients (multiplicity of infection was 0, 0.02, 0.2, 2 respectively) with serum-free MEM medium was added to each well. It was placed in an incubator at 25 °C. After 1 h, the virus dilution was removed, and it was replaced with MEM medium containing 2% FBS for continued culture. After cytopathic effect appeared in the cells, the supernatant was collected for titer determination, and the cells were fixed with 4% paraformaldehyde. After 1 h, the fixing solution was removed, 1% crystal violet solution was added, and it was stained overnight at room temperature. After removing the crystal violet solution, it was air-dried and photographed.

[0094] The results are as Figure 4 shown. From the crystal violet staining results after virus infection, it can be seen that when cells overexpressing gcHnf4α were infected with GCRV-I at different multiplicities of infection, gcHnf4α could resist the infection of GCRV-I, and the number of remaining cells in the well plate was more than that in the control group (see Figure 4 A in it). The supernatant of the medium of this group of cells infected with MOI = 0.2 was collected, and the TCID 50 method was used to detect the virus titer. It can be seen that overexpression of gcHnf4α could significantly inhibit the replication of GCRV-I, and the viral load was reduced by 18.1 times compared with the control group (see Figure 4 B in it). It shows that gcHnf4α has an antiviral effect in CIK cells.

[0095] Example 5

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

[0097] In the single pathogen infection experiment of Aeromonas salmonicida, CIK cells were inoculated into 24-well plates at 3×10 5 cells per well and cultured overnight, and 800 ng of FLAG or gcHnf4α-FLAG was transfected. After 36 h of transfection, 250 μL of A. salmonicida with a multiplicity of infection of 0.5 diluted with MEM medium was added to each well for infection. At 24 hpi, the cells and supernatant were collected for crystal violet staining, colony counting, CCK8 cell viability detection, and qRT-PCR.

[0098] In the single pathogen infection experiment of GCRV-I, CIK cells were inoculated into 24-well plates at 3×10 5Cells were seeded in 24-well plates and cultured overnight, and then transfected with 800 ng of FLAG or gcHnf4α-FLAG. After 36 h of transfection, 250 μL of GCRV-I with a multiplicity of infection of 0.5 diluted in MEM medium was added to each well for infection. After 1 h, the virus dilution was removed and replaced with MEM medium containing 2% FBS for continued culture. At 24 h post-infection (hpi), cells and supernatants were collected for crystal violet staining, colony counting, CCK8 cell viability assay, and qRT-PCR.

[0099] In the experiment of co-infection of cells, CIK cells were seeded at 3×10 5 cells per well in 24-well plates and cultured overnight, and then transfected with 800 ng of FLAG or gcHnf4α-FLAG. After 36 h of transfection, 250 μL of A. salmonicida with a multiplicity of infection of 0.5 diluted in MEM medium was added to each well for infection. After culturing at 25 °C for 1.5 h, an equal volume of MEM medium containing 4% fetal bovine serum was added for continued infection. After 6 h, the medium was carefully removed to retain A. salmonicida adsorbed on the cells, 500 μL of MEM medium containing GCRV-I (MOI = 0.5) was added and cultured for 1 h, the medium was discarded, and cultured in MEM containing 2% FBS at 25 °C for 18 h. Then cells and supernatants were collected for crystal violet staining, colony counting, CCK8 cell viability assay, and qRT-PCR.

[0100] The results are as Figure 5 shown. After overexpression of gcHnf4α and single-pathogen infection with GCRV-I and A. salmonicida, as well as co-infection with GCRV-I and A. salmonicida, crystal violet staining and cell viability assays were performed. It can be seen that the cell viability of gcHnf4α was higher than that of the control group in different infection modes, and the cell viability increased by 13.30%, 24.36%, and 13.17% respectively (see Figure 5 A and B). And in the single-pathogen infection of A. salmonicida and co-infection with GCRV-I, the bacterial loads of gcHnf4α were 1.4×10 3 and 1.7×10 3 , respectively, which were much lower than those of the control group (see Figure 5 C). After single-pathogen infection with GCRV-I and co-infection of bacteria and virus, overexpression of gcHnf4α was verified by fluorescence quantitative PCR and the relative expression levels of non-structural proteins NS38 and NS80 of GCRV-I were detected (the expression levels of viral proteins NS38 and NS80 are positively correlated with the load of GCRV-I). It can be seen that gcHnf4α significantly reduced the expression levels of NS38 and NS80 (see Figure 5Among D, E, and F), it shows that gcHnf4α plays a role in resisting single or co-infection of bacteria and / or viruses.

[0101] Example 6

[0102] Effect of inhibiting caspase 3 on the disease resistance ability of overexpressed gcHnf4α in CIK cells

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

[0104] The results are as Figure 6 shown. After caspase 3 was inhibited by the inhibitor, the role of gcHnf4α in resisting single-pathogen infection of bacteria or viruses and co-infection shown in Example 5 was inhibited. From the experimental results of crystal violet staining, it can be seen that there was no significant difference in gcHnf4α in different infection modes compared with the control group (see Figure 6 A); in the presence of caspase 3 inhibitor Z-DEVD-FMK, there was no difference in the cell viability of gcHnf4α in different infection modes compared with the control group (see Figure 6 B); when caspase 3 was inhibited, there was no difference in the colony counting of gcHnf4α after single-pathogen infection of bacteria, while in the bacteria-virus co-infection, the bacterial load of the gcHnf4α-FLAG overexpression group (2.54×10 4 CFU / mL) even exceeded that of the FLAG control group (1.96×10 4 CFU / mL) (see Figure 6 C); for NS38 of GCRV-I, there was no difference in the expression level in single-pathogen infection of the virus, and the relative expression level of NS38 was higher than that of the control group in the bacteria-virus co-infection, upregulated by 2.67 times, while the relative expression levels of NS80 were higher than those of the corresponding control groups in both single-pathogen infection and co-infection of the virus, upregulated by 4.07 times and 3.03 times respectively (see Figure 6 D), indicating that when caspase 3 was inhibited, gcHnf4α could not play its original role in resisting single-pathogen infection of bacteria or viruses or bacteria-virus co-infection.

[0105] Example 7

[0106] Effect of inhibiting caspase 9 on the disease resistance ability of overexpressed gcHnf4α in CIK cells

[0107] Based on the single infection and co-infection experimental methods in Example 5, 1 h before infection, cells were treated with 50 μM caspase 9 inhibitor Z-LEHD-FMK. Then, cell infection was carried out, and samples were collected after lesions appeared to evaluate the role of gcHnf4α in this process by crystal violet staining, CCK8 cell viability detection, colony counting, and qRT-PCR.

[0108] The results were as Figure 7 shown. After caspase 9 was inhibited by the inhibitor, the role of gcHnf4α that originally showed resistance to single pathogen infection of bacteria or virus and co-infection in Example 5 was inhibited. It can be seen from the experimental results of crystal violet staining that there was no significant difference in gcHnf4α in different infection modes compared with the control group (see Figure 7 A in); in the presence of caspase 9 inhibitor Z-LEHD-FMK, the cell viability of gcHnf4α gradually decreased in different modes of single pathogen infection of bacteria, single pathogen infection of virus, and bacteria-virus co-infection. In the case of GCRV-I infection, gcHnf4α could not increase the cell viability (75.71%), which was 9.41% lower than that of the control group (85.12%). After co-infection with GCRV-I and A. salmonicida, the cell viabilities of the gcHnf4α overexpression group and the FLAG group were 57.63% and 68.67% respectively, and the cell viability of the gcHnf4α group decreased by 11.04% (see Figure 7 B in); when caspase 9 was inhibited, the amount of bacterial proliferation increased after co-infection, but there was no difference in colony counting between the gcHnf4α overexpression group and the FLAG control group (see Figure 7 C in); inhibiting caspase 9 in the case of overexpressing gcHnf4α showed no difference in the expression levels of NS38 and NS80 of GCRV-I compared with the control group in single pathogen infection of virus and bacteria-virus co-infection (see Figure 7 D in). These results also indicate that in the case of inhibited caspase 9, gcHnf4α cannot play its original role in resisting single pathogen infection of bacteria or virus or bacteria-virus co-infection.

[0109] Example 8

[0110] Effect of overexpression of gcHnf4α in zebrafish on the survival of larvae under bacterial and viral infections.

[0111] Dilute the FLAG empty plasmid or gcHnf4α-FLAG plasmid to 200 ng / μL and perform microinjection at the single-cell stage or two-cell stage of zebrafish fertilized eggs at an injection volume of 2 nL. Incubate the injected embryos statically at 28 °C until 4 dpf, and divide the larvae into 4 groups: uninfected group, A. salmonicida infection group (bacterial infection group), GCRV-II infection group (viral infection group), and A. salmonicida and GCRV-II co-infection group (bacterial-viral co-infection group). Infect the larvae with A. salmonicida at a final concentration of 1×10 7 CFU / mL, GCRV-II at 2.76×10 10 copies / mL, or a bacterial and viral mixture containing 1×10 7 CFU / mL of A. salmonicida and 2.76×10 10 copies / mL of GCRV-II. Add 20 mL of culture water 6 h later. Regularly count the death of zebrafish larvae every day, and use the log-rank test to compare the survival differences between groups for survival curve analysis.

[0112] According to Figure 8 the statistical results of the survival curves, in the uninfected group, except for the normal death of larvae during the growth stage, the survival rates of the overexpressed Hnf4α-FLAG group and the overexpressed FLAG empty plasmid group were 84.4% and 81.1% respectively, and there was no difference between the two (see Figure 8 A therein). After A. salmonicida infection, the survival rate of the overexpressed FLAG empty plasmid group was 42.2%, while the survival rate of zebrafish larvae overexpressing Hnf4α-FLAG was 60.0%, which was 17.8% higher than that of the control group, p < 0.01 (see Figure 8 B therein). The survival rates of larvae in the overexpressed gcHnf4α-FLAG group or FLAG empty plasmid group after GCRV-II infection were 64.4% and 45.6% respectively. Overexpressing gcHnf4α could significantly increase the survival rate by 18.8% (see Figure 8 C therein). When co-infected with A. salmonicida and GCRV-II, the survival rate of larvae in the overexpressed Hnf4α-FLAG group was 44.4%, which was 23.3% higher than the survival rate of the control (21.1%), p < 0.001 (see Figure 8 D therein). It can be shown that overexpressing Hnf4α in wild-type zebrafish has the ability to resist single-pathogen infections of A. salmonicida and GCRV-II and bacterial-viral co-infections, which further verifies the results of the in vitro experiment in Example 5.

[0113] Example 9

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

[0115] According to the method in Example 8, the FLAG empty plasmid or the gcHnf4α-FLAG plasmid was overexpressed in zebrafish embryos respectively. After the juvenile fish hatched, the juvenile fish were soaked with the caspase 3 inhibitor Z-DEVD-FMK at a concentration of 75 μM for 18 h, and then the juvenile fish were subjected to single-pathogen and mixed infections of bacteria and / or viruses. The death of zebrafish juvenile fish was counted regularly every day, the survival curve was plotted, and the survival differences between groups were compared by log-rank test.

[0116] According to Figure 9 the statistical results of the survival curve in, it can be seen that after treatment with Z-DEVD-FMK, in the uninfected group, the survival rate of the group overexpressing Hnf4α-FLAG (66.6%) was 17.8% lower than that of the group overexpressing the FLAG empty plasmid (84.4%), p < 0.01 (see Figure 9 A in). After infection with A. salmonicida when caspase 3 was inhibited, the survival rate of the group overexpressing the FLAG empty plasmid was 73.3%, while the survival rate of zebrafish juvenile fish overexpressing Hnf4α-FLAG was 43.3%, a decrease of 30.0% compared with the control group, p < 0.001 (see Figure 9 B in). The survival rates of juvenile fish in the group overexpressing gcHnf4α-FLAG or the FLAG empty plasmid after being infected with GCRV-II were 12.2% and 34.4% respectively. The inhibition of caspase 3 significantly reduced the survival of juvenile fish overexpressing gcHnf4α, and the survival rate decreased by 22.3%, p < 0.001 (see Figure 9 C in). When co-infected with A. salmonicida and GCRV-II after inhibiting caspase 3, all the juvenile fish in the group overexpressing Hnf4α-FLAG had died on the fifth day after infection, a decrease of 4.4% compared with the control survival rate, p < 0.05 (see Figure 9 D in). It can be thus shown that inhibiting caspase 3 in zebrafish juvenile fish blocked the originally possessed ability of overexpressing Hnf4α to resist single-pathogen infections of A. salmonicida and GCRV-II and co-infection of A. salmonicida and GCRV-II, which further verified the in vitro experimental results in Example 6, indicating that the functions of gcHnf4α in resisting single-pathogen bacterial infection, single-pathogen viral infection, and bacterial-viral co-infection depend on caspase 3.

[0117] Example 10

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

[0119] According to the method in Example 8, the FLAG empty plasmid or gcHnf4α-FLAG plasmid was overexpressed in zebrafish embryos respectively. After the juvenile fish hatched, the juvenile fish were soaked with the caspase 9 inhibitor Z-LEHD-FMK at a concentration of 75 μM for 18 h, and then the juvenile fish were subjected to single pathogen and co-infection of bacteria and / or virus. The death of zebrafish juvenile fish was counted regularly every day, the survival curve was drawn, and the survival differences between groups were compared by log-rank test.

[0120] According to Figure 10 the statistical results of the survival curve in, it can be seen that after treatment with Z-LEHD-FMK, in the uninfected group, there was no difference between the survival rate (64.4%) of the group overexpressing Hnf4α-FLAG and the survival rate (73.3%) of the group overexpressing the FLAG empty plasmid (see Figure 10 A in). After infection with A. salmonicida when caspase 9 was inhibited, the survival rate of the group overexpressing the FLAG empty plasmid was 15.6%, and the survival rate of the zebrafish juvenile fish overexpressing Hnf4α-FLAG was 16.7%, and there was no difference between the two (see Figure 10 B in). The survival rates of the juvenile fish overexpressing gcHnf4α-FLAG group or FLAG empty plasmid group after being infected with GCRV-II were 12.2% and 29.2% respectively. The inhibition of caspase 9 would significantly reduce the survival of the juvenile fish overexpressing gcHnf4α, and the survival rate decreased by 17.1%, p < 0.01 (see Figure 10 C in). When co-infected with A. salmonicida and GCRV-II after inhibiting caspase 3, the survival rate (12.2%) of the juvenile fish overexpressing Hnf4α-FLAG group was reduced by 7.8% compared with the survival rate (20.0%) of the control, p < 0.05 (see Figure 10 D in). It can be shown from this that inhibiting caspase 9 in zebrafish juvenile fish blocks the ability of overexpressed Hnf4α to resist single pathogen infections of A. salmonicida and GCRV-II and 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 single bacterial pathogen infection, single viral pathogen infection, and bacterial-viral co-infection also depend on caspase 9.

[0121] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Hnf4α or a biomaterial containing Hnf4α in the preparation of a product for inhibiting bacteria and / or viruses.

2. Use of Hnf4α or a biomaterial containing Hnf4α in the preparation of at least one of the following products (1) to (3), (1) Prevent and treat bacterial infections; (2) Prevent and treat viral infections; (3) Prevent and treat diseases caused by bacterial and viral co-infections.

3. Use of Hnf4α or a biomaterial containing Hnf4α in at least one of the following Ⅰ to Ⅲ, Ⅰ. Promote the growth and reproduction of fish; II. Preparation of products to promote fish growth and reproduction; III. Prepare products that improve fish's disease resistance.

4. The use according to claim 1 or 2, characterized in that: The bacteria is Aeromonas salmonicida; and the virus is grass carp reovirus.

5. The use according to claim 1 or 2, characterized in that: The Hnf4α-containing biological material includes any one of the following a to c: a, expression vector containing Hnf4α; b, host bacteria containing Hnf4α; c. Host cells containing Hnf4α.

6. The use according to claim 3, characterized in that: In III, the product can improve fish resistance to diseases caused by at least one of infection with Aeromonas salmonicida, infection with grass carp reovirus, and co-infection with Aeromonas salmonicida and grass carp reovirus.

7. The use according to claim 3, characterized in that: The fish is a carp.

8. The use according to claim 7, characterized in that: The carp fish is grass carp and / or zebrafish.

9. A method for breeding disease-resistant fish, characterized in that: The following steps are involved: The expression level and / or activity of Hnf4α in the recipient fish is increased.

10. The method according to claim 9, characterized in that The disease-resistant fish can resist diseases caused by at least one of infection with Aeromonas salmonicida, infection with grass carp reovirus and co-infection with Aeromonas salmonicida and grass carp reovirus.

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