Use of hepatocyte nuclear factor HNF4β in the preparation of fish disease prevention and / or treatment biological products for fishery

Abstract

The application provides an application of hepatocyte nuclear factor HNF4beta in preparation of a fish disease preventing and / or treating fish biological product, and belongs to the technical field of biotechnology. Through in-vivo experiments, it is found that overexpression of hepatocyte nuclear factor HNF4beta in a fish body can effectively prevent and / or inhibit pathogenic bacteria, especially Edwardsiella tarda, spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Meanwhile, the hepatocyte nuclear factor HNF4beta can effectively regulate the immune system, control the occurrence of hepatobiliary syndrome and significantly improve the survival rate of juvenile fish. In addition, the application first proves that HNF4beta can effectively control the hepatobiliary syndrome induced by GCRV infection. The application provides a molecular target for prevention and control of infectious diseases, metabolic diseases and metabolic disease-induced infectious diseases of the Cyprinidae family.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of hepatocyte nuclear factor HNF4β in the preparation of aquatic biological products for the prevention and / or treatment of fish diseases. Background Technology

[0002] In recent years, with the application of intensive aquaculture technology, the scale of aquaculture has expanded rapidly, and the world's fishery output has increased dramatically. However, due to the large number of aquaculture species, the large scale, and the complexity of diseases, various diseases have caused significant economic losses to aquaculture. Edwardsiella piscicida is a pathogen that can infect a variety of fish species, causing hemorrhagic septicemia in many important economic fish species. Spring viremia of carp is caused by spring carp virus (SVCV), which mainly affects carp and catfish, and is listed as a Class II disease in the list of imported animal quarantine diseases. Grass carp hemorrhagic disease is caused by grass carp hemorrhagic disease virus (GCRV). Grass carp hemorrhagic disease is a viral infectious disease with a wide prevalence, long epidemic season, high morbidity and mortality rates, and is highly destructive. It is most harmful to fry in the seedling stage, with a mortality rate as high as 90%. Hepatobiliary syndrome is a metabolic disease in fish, with fatty liver, enlarged liver and gallbladder, and discoloration as typical symptoms. This disease has a long course (the epidemic season lasts from June to October) and a high mortality rate; in recent years, it has posed a huge threat to the farming of major freshwater economic fish such as carp, crucian carp, grass carp, and black carp. The "new three diseases" that have emerged in recent years, namely hepatobiliary syndrome, viral hemorrhagic disease, and bacterial complications, have caused huge losses to the local grass carp and black carp farming industry.

[0003] Hepatocyte nuclear factors (HNFs) are a class of transcription factors (mainly including HNF1, HNF3, HNF4, and HNF6) that regulate gene-specific expression in the liver, playing a crucial role in hepatocyte differentiation and metabolism. HNF4 is a zinc finger protein with two isoforms in mammals, encoded by the genes HNF4A (HNF4α) and HNF4G (HNF4γ), respectively; and three isoforms in fish, amphibians, and birds, encoded by the genes HNF4A (HNF4α), HNF4B (HNF4β), and HNF4G (HNF4γ), respectively. Current research mainly focuses on HNF4α, with only a few reports on HNF4β and HNF4γ; reports on HNF4β are particularly rare. HNF4α is closely associated with type 1 juvenile diabetes mellitus (MODY1) and is also involved in the development of liver fibrosis, cirrhosis, hepatocellular carcinoma, and fatty liver disease. Besides regulating glucose transport and glycolysis, HNF4α also protects the gut against dextran sulfate-induced colitis, playing a crucial role in inflammatory diseases. To date, the function of HNF4β in infectious and / or metabolic diseases in fish has not been reported. Furthermore, some studies have shown 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 essential for the prevention and control of infectious diseases in fish. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of hepatocyte nuclear factor HNF4β in the preparation of aquatic biological products for the prevention and / or treatment of fish diseases.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention also provides the application of hepatocyte nuclear factor HNF4β in the preparation of aquatic biological products for the prevention and / or treatment of fish diseases.

[0007] Preferably, the fish diseases include one or both of infectious diseases and metabolic diseases.

[0008] Preferably, the metabolic disease includes one or both of hepatobiliary syndrome and grass carp hemorrhagic disease virus complicated by hepatobiliary syndrome.

[0009] Preferably, the infectious disease includes viral diseases and / or bacterial diseases.

[0010] Preferably, the virus includes one or both of carp spring virus and grass carp hemorrhagic disease virus; the bacteria includes Edwardsiella tarda.

[0011] Preferably, the hepatocyte nuclear factor HNF4β is fish HNF4β.

[0012] Preferably, the amino acid sequence of the fish HNF4β is shown in SEQ ID No. 1; the nucleotide sequence of the fish HNF4β is shown in SEQ ID No. 2.

[0013] Preferably, the fishery biological product includes any one of the following:

[0014] (1) Hepatocyte nuclear factor HNF4β;

[0015] (2) An expression vector containing the above-mentioned hepatocyte nuclear factor HNF4β;

[0016] (3) Host bacteria containing the aforementioned hepatocyte nuclear factor HNF4β;

[0017] (4) Host cells containing the aforementioned hepatocyte nuclear factor HNF4β.

[0018] Preferably, the fishery biological product further includes a molecularly acceptable carrier.

[0019] Preferably, the sole active ingredient of the fishery biological product is hepatocyte nuclear factor HNF4β.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides the application of hepatocyte nuclear factor 4β in the preparation of aquatic biological products for the prevention and / or treatment of fish diseases. Through in vivo experiments, this invention has found that overexpression of hepatocyte nuclear factor 4β in fish can effectively prevent and / or inhibit pathogens, especially Edwardsiella faecium, carp spring virus SVCV, and grass carp reovirus GCRV. Simultaneously, the hepatocyte nuclear factor 4β of this invention can effectively regulate the immune system, control the occurrence of hepatobiliary syndrome, and significantly improve the survival rate of juvenile fish. Furthermore, this invention is the first to demonstrate that HNF4β can effectively control GCRV infection induced by hepatobiliary syndrome. This invention provides a molecular target for the prevention and control of infectious diseases, metabolic diseases, and metabolic diseases-induced infectious diseases in cyprinid fish. Attached Figure Description

[0022] Figure 1 Electrophoresis diagram of the protein from the zebrafish HNF4β-FLAG recombinant expression vector;

[0023] Figure 2In Figure A, the effect of overexpression of FLAG and zfHNF4β-FLAG in zebrafish on the survival of infected zebrafish juveniles is shown. In Figure B, the effect of overexpression of FLAG and zfHNF4β-FLAG in zebrafish on the proliferation of Edwardsiella tumefaciens in zebrafish juveniles is shown.

[0024] Figure 3 The effect of overexpression of FLAG and zfHNF4β-FLAG in zebrafish on the survival rate of zebrafish juveniles infected with SVCV;

[0025] Figure 4 The study investigated the intervention effect of zfHNF4β-FLAG overexpression in zebrafish on dexamethasone-induced hepatobiliary syndrome. A represents the effect of FLAG and zfHNF4β-FLAG overexpression on the survival rate of juvenile zebrafish in the absence or presence of dexamethasone. B represents the effect of FLAG and zfHNF4β-FLAG overexpression on hepatomegaly in juvenile zebrafish in the absence or presence of dexamethasone. C represents the effect of FLAG and zfHNF4β-FLAG overexpression on lipid deposition in the liver of juvenile zebrafish in the absence or presence of dexamethasone. D represents the statistical results of the effect of FLAG and zfHNF4β-FLAG overexpression on lipid deposition in the liver of juvenile zebrafish in the absence or presence of dexamethasone.

[0026] Figure 5 To illustrate the synergistic damaging effect of zfHNF4β knockout in zebrafish on dexamethasone-induced hepatobiliary syndrome, A represents the difference between wild-type and zfHNF4β knockout in the absence or presence of dexamethasone. - / - Survival rate of gene knockout zebrafish, where B represents the survival rate of wild-type and zfHNF4β zebrafish in the absence or presence of dexamethasone. - / - Results of the effects of gene knockout on hepatomegaly in zebrafish, C represents the effect of dexamethasone in the absence or presence of wild-type and zfHNF4β. - / - Statistical results of the effect of gene knockout on hepatomegaly in zebrafish, where D represents the effect of dexamethasone in the absence or presence of wild-type and zfHNF4β. - / - Results of the effects of gene knockout on hepatic lipid deposition in zebrafish, E represents the effect of dexamethasone in the absence or presence of wild-type and zfHNF4β. - / - Statistical results on the effects of gene knockout on hepatic lipid deposition in zebrafish;

[0027] Figure 6 The immune signaling pathway and differentially expressed genes that were significantly enriched during the suppression of dexamethasone-induced hepatobiliary syndrome by overexpression of zfHNF4β in zebrafish;

[0028] Figure 7 In Figure A, the survival rate of wild-type zebrafish infected with or without GCRV was shown in the presence or absence of dexamethasone. In Figure B, the effect of overexpression of FLAG and zfHNF4β in zebrafish on the survival rate of juvenile fish with hepatobiliary syndrome complicated by GCRV infection was shown. Detailed Implementation

[0029] This invention also provides the application of hepatocyte nuclear factor HNF4β in the preparation of aquatic biological products for the prevention and / or treatment of fish diseases.

[0030] In this invention, a hepatocyte nuclear factor HNF4β is provided, preferably fish HNF4β. More preferably, the amino acid sequence of the fish HNF4β is shown in SEQ ID No. 1.

[0031] In this invention, a gene encoding hepatocyte nuclear factor HNF4β is also provided, wherein the preferred nucleotide sequence of the fish HNF4β is shown in SEQ ID No. 2.

[0032] In this invention, the fish diseases preferably include one or two of infectious diseases and metabolic diseases. The metabolic diseases preferably include one or two of hepatobiliary syndrome and grass carp hemorrhagic disease virus complicated by hepatobiliary syndrome. The infectious diseases preferably include viral diseases and / or bacterial diseases. The viruses preferably include one or two of carp spring virus and grass carp hemorrhagic disease virus; the bacteria preferably include Edwardsiella tarda. As a preferred embodiment, this invention also provides the application of hepatocyte nuclear factor HNF4β in the preparation of a product inhibiting Edwardsiella tarda infection. This invention also provides the application of hepatocyte nuclear factor HNF4β in the preparation of a product inhibiting carp spring virus infection. The hepatocyte nuclear factor HNF4β is zebrafish HNF4β, the amino acid sequence of which is shown in SEQ ID No. 1, and the nucleotide sequence of which is shown in SEQ ID No. 2.

[0033] In this invention, the fishery biological product preferably includes molecularly acceptable excipients. These excipients include immune adjuvants, diluents, excipients, preservatives, buffers, dispersants, or surfactants. In this invention, the fishery biological product includes any one of the following:

[0034] (1) Hepatocyte nuclear factor HNF4β;

[0035] (2) An expression vector containing hepatocyte nuclear factor HNF4β;

[0036] (3) Host bacteria containing hepatocyte nuclear factor HNF4β;

[0037] (4) Host cells containing hepatocyte nuclear factor HNF4β.

[0038] In the above-described biological material of the present invention, the hepatocyte nuclear factor HNF4β is fish HNF4β. The fish HNF4β includes zebrafish HNF4β. The amino acid sequence of the zebrafish HNF4β is shown in SEQ ID No. 1, and the nucleotide sequence of the zebrafish HNF4β is shown in SEQ ID No. 2. The backbone of the expression vector is preferably p3×FLAG-CMV. TM -14, the host bacterium is preferably Escherichia coli, such as Escherichia coli Top 10. The host cell is preferably EPC cell. The sole active ingredient of the fishery biological product is preferably hepatocyte nuclear factor HNF4β.

[0039] 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.

[0040] In the following embodiments, the empty FLAG plasmid is p3×FLAG-CMV. TM -14.

[0041] Example 1

[0042] Construction of zebrafish HNF4β eukaryotic expression plasmid and validation of its protein expression

[0043] This embodiment uses the zebrafish HNF4β nucleotide sequence 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 HNF4β ORF region was amplified using wild-type zebrafish cDNA as a template, and after double enzyme digestion, 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 validation. 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 UV spectrophotometer. The zebrafish HNF4β plasmid was named zfHNF4β-FLAG. zfHNF4β-FLAG was transfected into EPC cells, and cells were collected 48 hours after transfection. Cell proteins were extracted using RIPA lysis buffer, and expression was validated by Western blotting.

[0044] Experimental results are as follows Figure 1As shown, the protein size detected by Western blotting matches the predicted HNF4β-FLAG protein size, and the band is single, approximately 50 kDa. Sequencing results show that the amino acid sequence of zebrafish HNF4β (zfHNF4β) is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2. This indicates that the zebrafish HNF4β eukaryotic expression plasmid was successfully constructed.

[0045] Example 2

[0046] Effects of HNF4β overexpression in fish on juvenile fish infection survival and proliferation of *E. piscicida*

[0047] In this embodiment, the empty FLAG plasmid and the zfHNF4β-FLAG plasmid prepared in Example 1 were diluted to the required concentration of 200 ng / μL and microinjected into zebrafish fertilized eggs at the single-cell or two-cell stage, with an injection volume of 2 nL. The injected embryos were then reared in culture water at 28°C. At 4 days post-feeding (dpf), the hatched fry were randomly divided into groups of 30 fish each, and 5 mL of a 2×10⁻⁶ nitrate diluted in culture water was added quantitatively. 8 Zebrafish were infected by immersion in E. piscicida at CFU / mL. 20 mL of culture water was added 6 hours after infection. Mortality of zebrafish juveniles was recorded daily, and the survival difference between zebrafish juveniles overexpressing empty plasmid FLAG and zfHNF4β-FLAG after E. piscicida infection was compared using the log-rank test for survival curve analysis.

[0048] In the bacterial proliferation experiment, 10 juvenile fish were collected from each group at 24 h and 48 h post-infection. After rinsing with PBS, 1 mL of PBS was added and the fish were thoroughly homogenized using a glass homogenizer. The homogenized samples were then thoroughly mixed on a vortex mixer and serially diluted with PBS. 100 μL of the suspension was transferred to TSB agar plates and incubated upside down at 28 °C for 24 h before colony counting.

[0049] The results are as follows Figure 2 As shown, the survival rate of zebrafish juveniles microinjected with the empty FLAG plasmid was 10%, while the survival rate of zebrafish juveniles overexpressing zfHNF4β-FLAG was 56.67%. Compared with zebrafish juveniles microinjected with FLAG, the survival rate of zebrafish juveniles overexpressing zfHNF4β-FLAG was increased by 46.67% (see...). Figure 2(A) At 24 h post-infection with *E. piscicida*, the bacterial load in zebrafish in the microinjected FLAG group was 3833.33 ± 1065.62 CFU / 10 fish, and the bacterial load in the microinjected zfHNF4β-FLAG group was 933.33 ± 262.47 CFU / 10 fish; at 48 h post-infection with *E. piscicida*, the bacterial load in zebrafish in the microinjected FLAG group was 298666.67 ± 13199.33 CFU / 10 fish, and the bacterial load in the microinjected zfHNF4β-FLAG group was 20000 ± 8640.99 CFU / 10 fish (see A). Figure 2 (B in the text). This demonstrates that overexpression of zfHNF4β significantly inhibits the proliferation of E. piscicida in juvenile fish and improves the survival rate of zebrafish juveniles infected with E. piscicida.

[0050] Example 3

[0051] Effect of HNF4β overexpression in fish on survival rate of juvenile fish infected with carp spring virus (SVCV)

[0052] In this embodiment, FLAG empty plasmid at a concentration of 200 ng / μL or zfHNF4β-FLAG plasmid prepared in Example 1 was microinjected into wild-type zebrafish embryos at the 1-2 cell stage. On day 4 of embryonic development, hatched juvenile fish were randomly divided into groups of 30, and each group was given 5 mL of a virus mixture diluted with culture water. The final infection concentration of SVCV was 2 × 10⁻⁶. 6 CFU / mL. 20 mL of culture water was added 24 hours after infection. Three replicates were set up for each group. Mortality of zebrafish juveniles in each group after SVCV infection was recorded daily, and dead juveniles were removed promptly. The survival difference between zebrafish juveniles overexpressing empty plasmid FLAG and zfHNF4β-FLAG after SVCV infection was compared using the log-rank test for survival curve analysis.

[0053] The results are as follows Figure 3 As shown, under SVCV infection, the survival rate of zebrafish juveniles microinjected with the empty FLAG plasmid was 47.78%, while the survival rate of zebrafish juveniles overexpressing zfHNF4β-FLAG was 64.44%. Compared with zebrafish juveniles microinjected with FLAG, the survival rate of zebrafish juveniles overexpressing zfHNF4β-FLAG was increased by 16.66%.

[0054] Example 4

[0055] The intervention effect of HNF4β overexpression in fish on dexamethasone-induced hepatobiliary syndrome

[0056] Hepatobiliary syndrome is characterized by fatty liver and hepatomegaly. There are many contributing factors to hepatobiliary syndrome, such as overcrowding, deteriorated aquatic environment, spoiled feed, and vitamin deficiency. These external factors hinder the breakdown and metabolism of fat in the fish liver, leading to a gradual loss of hepatobiliary function and ultimately fish mortality. Researchers have established a dexamethasone-induced hepatobiliary syndrome model in zebrafish. This example studies the effects of HNF4β overexpression in zebrafish on dexamethasone-induced fish survival, hepatomegaly, and hepatic lipid deposition, revealing the possible regulatory role of HNF4β in the development of hepatobiliary syndrome. The specific procedures are as follows:

[0057] (1) Weigh an appropriate amount of dexamethasone (Sigma-Aldrich, #D1756), add DMSO to dissolve it, prepare a 100mM stock solution, and dispense it into 1.5mL EP tubes. Store the solution in a -20℃ refrigerator. When treating juvenile fish, take an appropriate amount of the stock solution and dilute it with clean culture water to prepare dexamethasone working solutions of different concentrations.

[0058] (2) On the fourth day after embryo fertilization, the hatched zebrafish juveniles overexpressing the empty FLAG plasmid or the zfHNF4β described in Example 1 were randomly and evenly divided into groups of 25 juveniles each, with three replicates per group. Subsequently, 10 mL of 100 μM dexamethasone mixture or DMSO diluted with culture water was added to each replicate in each group, and the mixture was gently shaken to mix. The culture dishes were placed in a 28°C incubator.

[0059] (3) Statistical analysis of the survival curve of juvenile zebrafish within one week after dexamethasone treatment. Daily mortality of zebrafish juveniles was recorded and dead juveniles were removed. The number of survivors per day was calculated and the survival curve was plotted.

[0060] (4) Zebrafish juveniles overexpressing FLAG or zfHNF4β were treated with 100 μM dexamethasone or DMSO for 24 and 48 hours, respectively. Ten to fifteen juveniles were collected from each group. The zebrafish were fixed on 6% carboxymethyl cellulose plates and photographed in a lateral position under a stereomicroscope. The images were then imported into ImageJ, the liver region was plotted, and the pixel value of the target region was defined as the liver size. Ten fish from each group were counted, and the units were measured in square millimeters. Finally, a 150 μm scale bar was added to each image.

[0061] (5) After 72 hours of treatment with 100 μM dexamethasone or DMSO, zebrafish juveniles overexpressing FLAG or zfHNF4β were collected from each group and placed in a six-well plate. 1 mL of pre-cooled 4% paraformaldehyde was added to each well and the plate was fixed overnight at 4°C.

[0062] (6) After fixation, aspirate 4% paraformaldehyde, add 1 mL of PBST to each well and wash 3 times, 3-5 minutes each time. Then soak in 1 mL of 60% isopropanol for 20 minutes to remove background staining. During this period, prepare 0.3% Oil Red O working solution, dilute 0.5% Oil Red O dye (Sigma-Aldrich, #O1391) with sterile water, and filter through a 0.22 μM microporous filter.

[0063] (7) After waiting 20 min, remove 60% isopropanol, add 500 μL of freshly filtered Oil Red O working solution to each well, and stain at room temperature in the dark for 30 min. After staining, wash each well twice with 1 mL of 60% isopropanol for 3 minutes each time. Then wash twice with 1 mL of PBST for 3 minutes each time. Finally, fix with 1 mL of 4% paraformaldehyde.

[0064] (8) The juvenile fish were fixed on a 6% carboxymethyl cellulose plate and placed in a lateral position under a stereomicroscope for photographing. The images were then imported into ImageJ to measure the IOD (integrated optical density) of the liver region in each group. Finally, a 300 μm scale bar was added to each image.

[0065] The results are as follows Figure 4 As shown, for the group microinjected with the FLAG empty plasmid, the survival rate of zebrafish juveniles in the absence of dexamethasone was 85.33%; however, in the presence of dexamethasone-induced hepatobiliary syndrome, the survival rate of zebrafish juveniles with the hepatobiliary syndrome model was only 5.33%, indicating that 100 μM dexamethasone-induced hepatobiliary syndrome severely impaired the survival of zebrafish juveniles. In the absence of dexamethasone, compared with the control fish microinjected with the FLAG empty plasmid, zebrafish HNF4β overexpression had no significant effect on juvenile survival (p = 0.5108). However, in the presence of dexamethasone-induced hepatobiliary syndrome, compared with the control fish microinjected with the FLAG empty plasmid (5.33% survival rate), zebrafish HNF4β overexpression significantly improved the survival rate of juveniles with induced hepatobiliary syndrome (50.67% survival rate), an increase of 45.34% (see [link to relevant documentation]). Figure 4 (A) In the microinjection of the FLAG empty plasmid group, dexamethasone treatment at both 24 h and 48 h significantly induced hepatomegaly. At 24 h of dexamethasone treatment, zebrafish HNF4β overexpression, while inhibiting dexamethasone-induced hepatomegaly, showed no statistically significant difference; however, at 48 h of dexamethasone treatment, zebrafish HNF4β overexpression completely blocked dexamethasone-induced hepatomegaly (see A). Figure 4(B in the text). Consistent with the results of dexamethasone-induced hepatomegaly, dexamethasone treatment significantly increased lipid deposition in the livers of zebrafish in the FLAG empty plasmid microinjection group; overexpression of zebrafish HNF4β inhibited dexamethasone-induced hepatic lipid deposition (see [reference missing]). Figure 4 (C and D in the text). In summary, overexpression of HNF4β can inhibit dexamethasone-induced hepatomegaly, hepatic lipid deposition, and mortality, suggesting that HNF4β has potential applications in the prevention or treatment of hepatobiliary syndromes.

[0066] Example 5

[0067] Synergistic damaging effects of HNF4β gene knockout in fish on dexamethasone-induced hepatobiliary syndrome

[0068] To further verify the role of HNF4β in regulating the occurrence of hepatobiliary syndrome, homozygous strains with HNF4β gene knockout in zebrafish were obtained using gene editing technology. The effects of HNF4β gene knockout on dexamethasone-induced hepatomegaly, hepatic lipid deposition, and mortality were investigated. The specific procedures are as follows:

[0069] (1) On the fourth day after embryo fertilization, the hatched wild-type zebrafish or zfHNF4β - / - Homozygous zebrafish juveniles were randomly and evenly divided into groups of 25 juveniles each, placed in 70 mm culture dishes, with three replicates. Then, 10 mL of 100 μM dexamethasone solution or DMSO diluted in culture water was added to each group, and the mixture was gently shaken to mix. The culture dishes were placed in a 28°C incubator.

[0070] (2) Calculate the survival rate of juvenile zebrafish within one week after dexamethasone treatment. Record the mortality of zebrafish juveniles daily and remove dead juveniles. Calculate the number of survivors each day and plot the survival curve.

[0071] (3) WT or zfHNF4β - / - Homozygous zebrafish juveniles were treated with 100 μM dexamethasone or DMSO for 48 hours, and 10–15 juveniles were collected from each group. The zebrafish were fixed on 6% carboxymethyl cellulose plates and photographed in a lateral recumbent position under a stereomicroscope. The images were then imported into ImageJ, and the liver region was plotted, with the pixel values ​​of the target region defined as the liver size. Ten fish from each group were counted, and the units were measured in square millimeters (mm). 2 Finally, a 150μm scale bar is added to each image.

[0072] (4) WT or zfHNF4β - / -Homozygous zebrafish juveniles were treated with 100 μM dexamethasone or DMSO for 72 hours (i.e., juveniles on the seventh day after fertilization). 10-15 juveniles were collected from each group and placed in a six-well plate. 1 mL of pre-cooled 4% paraformaldehyde was added to each well and the plate was fixed overnight at 4°C.

[0073] (5) After fixation, aspirate 4% paraformaldehyde, add 1 mL of PBST to each well and wash 3 times, 3-5 minutes each time. Then soak in 1 mL of 60% isopropanol for 20 minutes to remove background staining. During this period, prepare 0.3% Oil Red O working solution, dilute 0.5% Oil Red O dye with sterile water, and filter through a 0.22 μM microporous filter.

[0074] (6) After waiting 20 minutes, remove 60% isopropanol and add 500 μL of freshly filtered Oil Red O working solution to each well. Stain at room temperature in the dark for 30 minutes. After staining, wash each well twice with 1 mL of 60% isopropanol for 3 minutes each time. Then wash twice with 1 mL of PBST for 3 minutes each time. Finally, fix with 1 mL of 4% paraformaldehyde.

[0075] (7) The juvenile fish were fixed on a 6% carboxymethyl cellulose plate and photographed in a lateral position under a stereomicroscope. The images were then imported into ImageJ. Ten fish were counted in each group, and the IOD of the liver region in each group was measured. Finally, a 300 μm scale bar was added to each image.

[0076] The results are as follows Figure 5 As shown, for wild type and zfHNF4β - / - In gene knockout zebrafish, dexamethasone treatment significantly reduced the survival rate of juvenile zebrafish. In the absence of dexamethasone, the survival rate of wild-type zebrafish was 97.18%; in the presence of dexamethasone, the survival rate was 54.80%, a decrease of 42.38%. In the absence of dexamethasone, zfHNF4β... - / - The survival rate of zebrafish was 80.56%; in the presence of dexamethasone, zfHNF4β - / - The survival rate of zebrafish was 29.73%, a decrease of 50.83%. In the absence of dexamethasone, compared to wild-type zebrafish, zfHNF4β... - / - The survival rate of gene knockout fish was reduced by 16.63%; in the presence of dexamethasone, the survival rate of zfHNF4β- / - gene knockout fish was reduced by 25.07% compared with the corresponding wild-type zebrafish (see...). Figure 5 (A in the text). This demonstrates that HNF4β gene knockout further exacerbates dexamethasone-mediated death. Similarly, HNF4β gene knockout further increases dexamethasone-induced hepatomegaly (see A in the text). Figure 5(B and C) and hepatic lipid deposition (see B ... Figure 5 (D and E in the text). Thus, data from both overexpression and gene knockout confirm that HNF4β can intervene in the occurrence and development of hepatobiliary syndrome.

[0077] Example 6

[0078] Fish HNF4β overexpression significantly enriched immune signaling pathways and differentially expressed genes during the suppression of dexamethasone-induced hepatobiliary syndrome.

[0079] Transcriptome sequencing was used to analyze the signaling pathway by which HNF4β overexpression regulates the occurrence of hepatobiliary syndrome. The specific procedures are as follows:

[0080] (1) Zebrafish juveniles overexpressing FLAG or zfHNF4β as described in Example 1 were treated with 100 μM dexamethasone or DMSO for 48 hours, and 40 juveniles were collected from each group. After washing three times with RNase-free water, the fish were dried and flash-frozen in liquid nitrogen. Total RNA extraction and high-throughput transcriptome sequencing of the zebrafish juveniles were performed by BioMed Biotechnology Co., Ltd.

[0081] (2) The company uses an efficient RNA extraction method to extract total RNA from the test samples and tests the purity, concentration and integrity of the RNA to ensure that the total amount of RNA in each sample is more than 1 μg.

[0082] (3) After enriching eukaryotic mRNA with oligo(dT) magnetic beads, the mRNA was randomly fragmented using fragmentation buffer. cDNA was then synthesized using the mRNA as a template and purified. Next, the purified double-stranded cDNA underwent end repair, A-tailing, and ligation with sequencing adapters. Fragment size selection was then performed using AMPure XPbeads. Finally, a cDNA library was obtained through PCR enrichment. The inserted fragments in the library were detected using a Qsep400 high-throughput analysis system. If the inserted fragments met expectations, the effective concentration of the library was accurately quantified using Q-PCR. The effective concentration of the library must be greater than 2 nM to ensure library quality. After passing quality control, the library was sequenced in PE150 mode using an Illumina NovaSeq 6000 sequencing platform.

[0083] (3) The raw sequencing data were quality controlled using the bioinformatics analysis platform BMKCloud (www.biocloud.net). A total of 80.68 Gb of valid sequencing data was obtained from the samples in this project. The valid data of each sample reached 6.14 Gb, and the Q30 base percentage was 97.19% or higher, which met the requirements for subsequent analysis.

[0084] (4) The effective data were quickly and accurately aligned with the reference genome using the HISAT2 software to obtain the location information of the expressed transcript sequence fragments (reads) on the reference genome. The alignment efficiency of the reads for each sample was between 92.17% and 93.44%. Then, the aligned reads were assembled using the StringTie software to reconstruct the transcriptome for subsequent analysis.

[0085] (5) The gene expression level is calculated based on the number of sequences aligned to each transcript (Count). In this project, StringTie is used with the maximum flow algorithm and FPKM is employed for standardization as an indicator of transcript or gene expression level. The FPKM calculation formula is as follows: = {number of fragments aligned to a transcript\over{total number of fragments aligned to the transcript, in units of 10^6 * transcript length, in units of 10^3 bases}}. Gene expression level is quantified by estimating the number of sequences mapped per million per thousand bases of transcripts.

[0086] (6) Differentially expressed genes were screened using differential expression analysis software based on the Count value of genes in each sample. For differentially expressed groups with biological repetitions, DESeq2 software was used for differential analysis. During the differentially expressed gene detection process, Fold Change ≥ 2 and FDR < 0.01 were used as the screening criteria.

[0087] (7) Using the KEGG (Kyoto encyclopedia of genes and genomes) database, genes were classified according to the pathways they participated in or the functions they performed. Pathway significant enrichment analysis was performed using pathways in the KEGG database as units, applying hypergeometric tests to identify pathways that were significantly enriched among differentially expressed genes compared to the overall genomic background. Pathway significant enrichment can determine the most important biochemical metabolic pathways and signal transduction pathways involved by genes. The enrichment results were visualized using bubble charts, bar charts, and network diagrams using ClusterProfiler.

[0088] The results are as follows Figure 6As shown, under conditions where qvalue ≤ 0.05 and in the presence of dexamethasone, the signaling pathway significantly enriched by zfHNF4β is the C-type lectin receptor signaling pathway. The differentially expressed genes significantly regulated by this pathway include: NewGene_6567; LOC100331996; LOC100332081; LOC101884586; LOC103911530; cyldb; egr2a; egr3; il1b; pik3r3b; pik3r3b-4; ppp3cca-3; ptgs2a; ptgs2b; si_ch211-191a16.5; si_dkey-88n24.11.

[0089] Example 7

[0090] Effect of HNF4β overexpression in fish on the survival rate of juvenile grass carp hemorrhagic virulence virus (GCRV) complicated with hepatobiliary syndrome

[0091] Hepatobiliary syndrome often leads to enlargement of immune organs (spleen and kidneys) in fish; the immune resistance of affected fish is significantly reduced. Therefore, hepatobiliary syndrome may induce susceptibility to other bacterial or viral diseases in fish. To verify this possibility, we investigated whether zebrafish with hepatobiliary syndrome had increased susceptibility to GCRV. Furthermore, we further investigated whether HNF4β could effectively prevent or treat GCRV infection concurrent with hepatobiliary syndrome. The specific procedures are as follows:

[0092] (1) On the third day after embryo fertilization, the hatched AB strain wild-type zebrafish fry were randomly divided into groups of 25 fry each and placed in 70 mm culture dishes, with three replicates per group. Subsequently, 5 mL of a mixture of 1, 10, and 100 μM dexamethasone or DMSO diluted in culture water was added to each replicate in each group and gently shaken to mix. The control group received no treatment.

[0093] (2) After treatment with dexamethasone or DMSO for 24 hours, add 250 μL of GCRV virus suspension to each replicate culture dish in each group and gently shake to mix. Place the culture dishes in a 28°C incubator. After 24 hours of high-concentration infection, add 20 mL of culture water to each culture dish.

[0094] (3) On the third day after embryo fertilization, the hatched zebrafish juveniles overexpressing FLAG or zfHNF4β as described in Example 1 were randomly and evenly divided into groups and placed in 70 mm culture dishes, with 25 juveniles in each group and three replicates in each group. Subsequently, 5 mL of 10 μM dexamethasone mixture diluted with culture water was added to each replicate in each group and gently shaken to mix.

[0095] (4) After 24 hours of dexamethasone treatment, add 250 μL of GCRV virus suspension or virus-free culture medium to each replicate culture dish in each group, and gently shake to mix. Place the culture dishes in a 28°C incubator. After 24 hours of high-concentration infection, add 20 mL of culture water to each culture dish.

[0096] (5) Calculate the survival rate of juvenile zebrafish within one week after GCRV infection. Record the mortality of zebrafish juveniles daily and remove dead juveniles. Calculate the number of survivors each day and plot the survival curve.

[0097] The results are as follows Figure 7 As shown, dexamethasone treatment increased susceptibility to GCRV. In the absence of dexamethasone, the survival rates of wild-type zebrafish after GCRV infection were 54.67% in the DMSO-treated group and 60% in the untreated group; the survival rate of wild-type zebrafish after GCRV infection was 33.33% in the 1 μM dexamethasone-treated group; and the survival rate of wild-type zebrafish after GCRV infection was 0% in the 10 and 100 μM dexamethasone-treated groups. Figure 7 (A) In zebrafish overexpressing the empty FLAG plasmid treated with 10 μM dexamethasone, the survival rate after GCRV infection was 0; however, the survival rate of zebrafish overexpressing zfHNF4β treated with 10 μM dexamethasone increased by 50.67%. Figure 7 (B in the text). This demonstrates that HNF4β can effectively prevent or treat hepatobiliary syndrome complicated by GCRV infection.

[0098] 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 hepatocyte nuclear factor HNF4β in the preparation of aquatic biological products for the prevention and / or treatment of fish diseases, characterized in that, The fish disease is one or both of infectious and metabolic diseases; The metabolic disease is one or both of hepatobiliary syndrome and grass carp hemorrhagic disease complicated by hepatobiliary syndrome; The infectious diseases mentioned are viral diseases and / or bacterial diseases; The virus is one or both of carp spring virus and grass carp hemorrhagic disease virus; the bacteria is Edwardsiella tectoris; The hepatocyte nuclear factor HNF4β is fish HNF4β; The amino acid sequence of HNF4β of the fish is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The nucleotide sequence of HNF4β of the fish is shown in SEQ ID No.

2.

3. The application according to claim 1, characterized in that, The fishery biological products include any one of the following: (1) An expression vector containing the hepatocyte nuclear factor HNF4β as described in claim 1 or 2; (2) A host bacterium containing the hepatocyte nuclear factor HNF4β as described in claim 1 or 2; (3) Host cells containing the hepatocyte nuclear factor HNF4β as described in claim 1 or 2.

4. The application according to claim 1, characterized in that, The fishery biological products include molecularly acceptable carriers.

5. The application according to claim 1, characterized in that, The sole active ingredient in the fishery biological product is hepatocyte nuclear factor HNF4β.

Images