Application of a grass carp FGF1 recombinant protein in the preparation of products against Aeromonas hydrophila infection

Abstract

This invention belongs to the field of aquaculture biotechnology and discloses the application of a grass carp FGF1 recombinant protein in the preparation of products against Aeromonas hydrophila infection. The amino acid sequence of the recombinant protein is shown in SEQ ID NO:2. This invention is the first to discover that this known protein effectively reduces the pathogen load in tissues such as the liver and spleen by repairing the intestinal mucosal physical barrier (significantly increasing the number of intestinal goblet cells, promoting epithelial cell proliferation, and inhibiting apoptosis) and strengthening the immune barrier (upregulating key antibacterial factors such as IL-22, MUC2, Spla2, and HB-EGFa), and significantly increases the 14-day survival rate of grass carp infected with Aeromonas hydrophila from 10% to 56.67%. This invention overcomes the shortcomings of existing technologies that rely solely on in vitro sterilization or are limited to non-infectious factors, providing a green and efficient biological product solution based on host barrier repair for bacterial diseases in grass carp.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture biotechnology, and in particular relates to the application of a grass carp FGF1 recombinant protein in the preparation of products resistant to Aeromonas hydrophila infection. Background Technology

[0002] grass carp( Ctenopharyngodon idella Aeromonas hydrophila is one of the important economic fish species in my country's freshwater aquaculture and a core species for ensuring the stability of my country's "vegetable basket." Aeromonas hydrophila Aeromonas hydrophila is a common pathogen in freshwater aquaculture in my country. Grass carp infected with this bacterium suffer from serious diseases such as septicemia, enteritis, and liver damage. In recent years, the diseases caused by this bacterium have resulted in significant economic losses for the grass carp industry. Currently, the prevention and control of this pathogen still relies excessively on antibiotics, but this method easily leads to problems such as antibiotic resistance, drug residues in fish, and disruption of the aquatic microbial balance. Therefore, developing safe, efficient, and environmentally friendly products to combat Aeromonas hydrophila infection is of great importance.

[0003] Fibroblast growth factor 1 (FGF1) Fibroblast growth factor 1 FGF1 (FGF1) is a member of the FGF family and is widely involved in core life processes such as cell proliferation, differentiation, migration, and organogenesis. Existing research has identified FGF1 in grass carp, rockfish, tilapia, zebrafish, and mirror carp, and its transcriptional level is regulated by starvation and growth hormone. Furthermore, FGF1 can effectively alleviate ectopic lipid deposition, glucose metabolism disorders, and liver damage in rainbow trout induced by high-fat, high-carbohydrate diets, indicating that FGF1 has significant application potential in aquaculture. However, current research on FGF1 in fish is limited to non-infectious factors such as growth and metabolism; the direct control of bacterial infections by fish FGF1 has never been disclosed, and there are no reports on its use in the control of aquatic pathogens such as Aeromonas hydrophila.

[0004] In Chinese patent document CN119185514A, a recombinant protein of FGF8a, a member of the grass carp FGF family, exhibits direct, broad-spectrum bactericidal activity in vitro and can be used to prepare antibacterial agents. Amino acid sequence homology comparison revealed that grass carp FGF8 and FGF1 share only 31.7% amino acid homology, suggesting that the direct in vitro bactericidal function of FGF8 cannot be based on this. Therefore, it is expected that FGF1, with its extremely low sequence homology, possesses related functions against Aeromonas hydrophila infection. Furthermore, the existing technology still has significant limitations: First, the patent only focuses on FGF8 and does not cover the antibacterial / anti-infective functions of other FGF family members such as FGF1; second, it only verifies the direct bactericidal effect of FGF8 protein in vitro and does not involve the repair effect of FGF family proteins on the intestinal mucosal barrier of fish hosts or the regulatory effect on the host's own immune defense system, thus failing to address the core pain point of fish intestinal mucosal barrier damage after Aeromonas hydrophila infection; third, it only completes the verification of in vitro antibacterial activity and does not verify the effect on improving the survival rate after Aeromonas hydrophila infection in live fish, lacking application data and effect support in aquaculture scenarios.

[0005] In summary, there are currently no reports of using grass carp FGF1 recombinant protein for anti-Aeromonas hydrophila infection, and it is unclear whether FGF1 recombinant protein can be developed into a novel candidate product for anti-Aeromonas hydrophila infection. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide an application of grass carp FGF1 recombinant protein in the preparation of products against Aeromonas hydrophila infection.

[0007] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0008] This invention provides a recombinant grass carp FGF1 protein for the preparation of anti-Aeromonas hydrophila (Aeromonas hydrophila) Aeromonas hydrophila The application of the grass carp FGF1 recombinant protein in infected products, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0009] The above-mentioned application, further, the method for preparing the grass carp FGF1 recombinant protein includes the following steps: (1) The grass carp FGF1 gene was amplified using primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4; (2) Construct a recombinant expression vector containing the gene obtained in step (1); (3) Transform the recombinant expression vector into host cells and induce expression; (4) Purify and remove endotoxins to obtain grass carp FGF1 recombinant protein.

[0010] Furthermore, the product for treating Aeromonas hydrophila infection is a product for repairing intestinal mucosal barrier damage caused by Aeromonas hydrophila infection; the repair of intestinal mucosal barrier damage caused by Aeromonas hydrophila infection is manifested in the following ways: the sharp decrease in the number of goblet cells in the intestine of grass carp caused by Aeromonas hydrophila infection is reversed and improved, the proliferative activity of intestinal epithelial cells is enhanced, and the programmed death of intestinal mucosal epithelial cells is inhibited.

[0011] Furthermore, the product for combating Aeromonas hydrophila infection is a product that upregulates the expression level of intestinal antimicrobial factors, wherein the intestinal antimicrobial factors are one or more of IL-22, MUC2, Spla2, and HB-EGFa.

[0012] Furthermore, the product for combating Aeromonas hydrophila infection is a product that reduces the Aeromonas hydrophila load in fish tissues after infection, wherein the tissues are the liver and / or spleen.

[0013] Furthermore, the dosage form of the product for treating Aeromonas hydrophila infection is one or more of the following: injection, oral preparation, soaking solution, or feed additive.

[0014] Furthermore, the product for treating Aeromonas hydrophila infection is an intraperitoneal injection.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to propose the use of grass carp FGF1 recombinant protein as an anti-infective agent for fish, for the repair of the intestinal mucosal barrier and for the application of Aeromonas hydrophila infection. It provides important data for the development of it into a green, safe and efficient antibacterial biological product for aquaculture, and fills the technical gap of FGF1 in the field of bacterial infection prevention and control in fish.

[0016] 2. Unlike existing technologies that directly kill bacteria in vitro, this invention improves the host's anti-infection ability by repairing the host's intestinal mucosal physical barrier (promoting proliferation, inhibiting apoptosis, and increasing goblet cells) and strengthening the immune barrier (upregulating multiple antibacterial factors), thus solving the pain point of intestinal damage caused by pathogen infection in aquaculture. This invention also provides experimental data at the live grass carp level, proving that the recombinant protein can significantly increase the survival rate of Aeromonas hydrophila 14 days after challenge from 10% to 56.67%, which has direct industrial application value.

[0017] 3. The grass carp FGF1 recombinant protein product of the present invention is a biological product that can replace or reduce the use of antibiotics and meets the requirements of green aquaculture. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a graph showing the results of SDS-PAGE electrophoresis detection of the purification effect of grass carp FGF1 recombinant protein in an embodiment of the present invention.

[0020] Figure 2 Figure A shows the results of AB-PAS staining to detect changes in the number of goblet cells in the intestine of grass carp after treatment with recombinant FGF1 protein in Experiment 1 of this invention; Figure B shows the AB-PAS staining results of each group and the statistical results of the number of goblet cells in each group.

[0021] Figure 3 Figure 2 shows the results of fluorescence microscopy detection of changes in BrdU fluorescence signal in the intestine of grass carp after treatment with recombinant FGF1 protein in Experiment 2 of this invention; Figure A is a schematic diagram of BrdU staining in each group; Figure B is a statistical result of BrdU fluorescence intensity in each group.

[0022] Figure 4 Figure 2 shows the results of fluorescence microscopy detection of changes in TUNEL fluorescence signal in the intestine of grass carp after treatment with recombinant FGF1 protein in Experiment 2 of this invention; Figure A is a schematic diagram of TUNEL staining in each group; Figure B is a statistical result of TUNEL fluorescence intensity in each group.

[0023] Figure 5 The figures shown are from Experiment 3 of this invention, where qRT-PCR was used to detect the expression levels of antibacterial molecules in the intestinal tract of grass carp after treatment with recombinant FGF1 protein. Figure A shows the expression level of IL-22; Figure B shows the expression level of MUC2; Figure C shows the expression level of Spla2; and Figure D shows the expression level of HB-EGFa.

[0024] Figure 6 The figure shows the results of qRT-PCR detection of the expression level of Aerolysin, a virulence factor of Aeromonas hydrophila, in the intestine of grass carp after treatment with recombinant FGF1 protein in Experiment 4 of this embodiment of the invention.

[0025] Figure 7 The figure shows the detection results of Aeromonas hydrophila in the liver and spleen of grass carp after treatment with recombinant FGF1 protein in Experiment 4 of this invention using the plate counting method.

[0026] Figure 8 The figure shows the detection results of the effect of grass carp FGF1 recombinant protein on the survival rate of grass carp after Aeromonas hydrophila infection in Experiment 5 of the present invention. Detailed Implementation

[0027] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] The experimental reagents, sequencing materials, and laboratory animals involved in this invention were provided by the following companies: Gene sequencing and primer synthesis were completed by Beijing Qingke Biotechnology Co., Ltd.

[0031] LB medium, Escherichia coli DH5α and BL21(DE3) competent cells, Ni-NTA His-tagged protein purification kit (containing Lysis Buffer, Wash Buffer I / II, and Elution Buffer), ampicillin, BCA protein concentration assay kit, antigen retrieval solution, immunostaining blocking solution, and cell permeabilizer were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0032] Mouse anti-BrdU monoclonal antibody, Cy3-labeled goat anti-mouse IgG fluorescent secondary antibody, Alixin Blue-Periodic Acid Schiff (AB-PAS) staining kit, and 4% paraformaldehyde fixative were purchased from Wuhan Saiwei Biotechnology Co., Ltd.

[0033] The total RNA extraction kit, cDNA reverse transcription kit, qRT-PCR SYBR Mix, and TUNEL staining kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0034] The anti-fluorescence quenching mounting solution (containing DAPI) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0035] Endotoxin removal kit and BrdU were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0036] BamH I and Xho I restriction endonucleases were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0037] The healthy grass carp were purchased from "Hunan Yuelushan Aquatic Breeding Technology Co., Ltd."

[0038] Example This invention provides a recombinant grass carp FGF1 protein for the preparation of anti-Aeromonas hydrophila (Aeromonas hydrophila) Aeromonas hydrophila The application of the grass carp FGF1 recombinant protein in the infected product is described in SEQ ID NO: 2, and the nucleotide sequence of its encoding gene is described in SEQ ID NO: 1.

[0039] The preparation method of grass carp FGF1 recombinant protein includes the following steps: (1) Total RNA extraction and cDNA synthesis Take 30 mg of intestinal tissue from healthy grass carp, extract total RNA according to the kit instructions, and synthesize cDNA using a reverse transcription kit after passing the test. Store at -20℃ for later use.

[0040] (2) Primer design and PCR amplification Specific amplification primers (FGF1-F and FGF1-R, see Table 1) were designed based on the conserved sequence of the grass carp FGF1 gene coding region. Primer design avoided hairpins, dimers, and mismatches. The coding base sequence of grass carp FGF1 is shown in SEQ ID NO: 1, and the corresponding amino acid sequence is shown in SEQ ID NO: 2. The primer length was 28 bp. The upstream primer introduced a BamHI restriction site, and the downstream primer introduced an XhoI restriction site. PCR amplification was performed using cDNA as a template under standard reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 40 s, 62℃ annealing for 40 s, 72℃ extension for 40 s, 30 cycles; final extension at 72℃ for 10 min. The PCR products were identified by 1%–2% agarose gel electrophoresis and then recovered by gel excision.

[0041] (3) Double enzyme digestion and vector construction The target fragment and the pET32a vector were double-digested using BamHI and XhoI restriction endonucleases at 37°C for 3 h. After recovery of the digestion products, ligation was performed overnight at 16°C using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α and plated on LB agar plates containing ampicillin (100 μg / mL). Positive clones were screened by colony PCR, and plasmids were extracted from correctly sequenced positive clones to obtain the recombinant plasmid pET32a-FGF1.

[0042] (4) Recombinant protein induced expression The recombinant plasmid was transformed into *E. coli* BL21(DE3) and inoculated into LB liquid medium containing ampicillin, and cultured with shaking for 12 h. 200 μL of the culture medium after 12 h of shaking was added to 200 mL of fresh LB liquid medium containing ampicillin, and cultured at 37°C and 220 rpm for 4 h with shaking until OD was reached. 600 = 0.6, add IPTG to a final concentration of 0.4 mM, and induce for 12 h at 16℃ and 150 rpm. Centrifuge at 5000 rpm for 5 min at 4℃ to collect bacterial cells, add 5 mL of lysis buffer to dissolve the cells, and incubate at -80℃ overnight. Remove the cells from the -80℃ freezer and place them on ice to thaw. Sonicate the cells at 4℃ until clear and free of precipitate. Sonication program: 200 W, 3 s on, 8 s off, 30 min total. Centrifuge at 12000 rpm for 20 min at 4℃, collect the supernatant, which contains recombinant FGF1 protein (the target protein is expressed in a soluble manner).

[0043] (5) Protein purification and endotoxin removal Protein purification was performed using an affinity chromatography column. First, the supernatant was added to a pre-equilibrated nickel column and incubated at 4°C with gentle shaking for 30 min to allow the target protein to bind to the Ni-NTA layer. After binding, the sample was removed from the column, and 3 mL of Wash Buffer I (containing 30 mM imidazole) was added. The column was incubated at 4°C with gentle shaking for 20 min, and the solution was drained. Then, 3 mL of Wash Buffer II (containing 100 mM imidazole) was added and incubated at 4°C with gentle shaking for 20 min. Finally, 3 mL of Elution Buffer (containing 250 mM imidazole) was added and incubated at 4°C with gentle shaking for 30 min. The eluent was collected, and the collected sample was subjected to SDS-PAGE electrophoresis to verify the purification effect. The eluted protein was dialyzed against pre-cooled sterile PBS for 12 h, with a molecular weight cutoff of 10 kDa in the dialysis bag. An endotoxin removal kit was used to treat the protein to ensure that the endotoxin content was below 0.1 EU / μg protein.

[0044] After BCA quantification, the protein was aliquoted and stored. The purification results of the grass carp FGF1 recombinant protein are shown below. Figure 1 .Depend on Figure 1 It can be seen that the grass carp FGF1 recombinant protein was successfully prepared. The target protein mainly exists in the supernatant of the cell lysis in a soluble form. After purification by Ni-NTA affinity chromatography, a high-purity recombinant protein with a single band was obtained with no obvious contamination from other proteins, which fully meets the requirements of subsequent in vivo and in vitro functional experiments.

[0045] The base sequence of grass carp FGF1 (NCBI accession number: XM_051859865.1) is shown in SEQ ID NO: 1: ATGACTGAGGCCGATATTGCAGTGAAATTCAACCCGCCCGACTATAAGAAGCTCACGCGGCTGTACTGTATGAATGGAGGATATCACCTTCAGATCCTGGCGGACGGGACAGTGGCTGGAGCTCGCGAGGAAAACATCTACAGCATACTGCGCATAAAAGCAACAAGTCCAGGAGTGGTGGTCATTGAAGGAACAGAGGCAGGACTTTACCTCTCGATGAAT GAAGATGGCAAGCTGTATGCGTCAGCATTAGTAACGGATGAAAGTTATTTCTTGGAGAAGATGGAGGAGAACCACTACAACACATATCAGTCTCAGAAGTATGGTGAAAACTGGTATGTCGGAATAAAAAAGAATGGAAAAATGAAACGAGGCCCAAGGACACACATTGGACAGAAGGCTATTTTCTTTCTCCCGCGACAGGTGGCCCAGGCCAAGGACTAA.

[0046] The amino acid sequence of grass carp FGF1 (NCBI accession number: XP_051715825.1) is shown in SEQ ID NO: 2: MTEADIAVKFNPPDYKKLTRLYCMNGGYHLQILADGTVAGAREENIYSILRIKATSPGVVVIEGTEAGLYLSMNEDGKLYASALVTDESYFLEKMEENHYNTYQSQKYGENWYVGIKKNGKMKRGPRTHIGQKAIFFLPRQVAQAKD.

[0047] Table 1. Primers used in this embodiment to amplify the coding sequence of grass carp FGF1.

[0048] Experiment 1: Effects of recombinant FGF1 protein from grass carp on goblet cells in the grass carp intestine The experimental steps are as follows: (1) Injection of grass carp FGF1 recombinant protein Healthy grass carp weighing approximately 20 g were randomly divided into two groups (PBS group and grass carp FGF1 recombinant protein treatment group). Each fish in the PBS group was intraperitoneally injected with 100 μL of sterile PBS buffer, while each fish in the grass carp FGF1 recombinant protein treatment group was injected with 100 μL of a 200 μg / mL grass carp FGF1 recombinant protein solution. The grass carp FGF1 recombinant protein was prepared in this embodiment and dissolved in sterile PBS. At least nine biological replicates were established for each group. The recirculating aquaculture system was maintained at a water temperature of 25°C, with continuous aeration by an oxygen pump, and fish were fed normally.

[0049] (2) Infection with Aeromonas hydrophila Aeromonas hydrophila (Latin name) Aeromonas hydrophila The strain was purchased from the China Center for Type Culture Collection (CCTCC), strain accession number: CCTCC AB 2016038. It was inoculated into LB liquid medium and activated overnight at 28°C. The next day, 200 μL of the bacterial culture was transferred to 200 mL of fresh LB medium and incubated at 28°C until OD500 reached. 600 The concentration was set to 0.6, and then the cells were collected by centrifugation at 10,000 rpm for 5 min at room temperature. The cells were then resuspended in sterile PBS to a final concentration of 1×10⁶. 7 CFU / mL indicates a successfully prepared Aeromonas hydrophila suspension. Four hours after injection of the grass carp FGF1 recombinant protein, each fish in both the PBS group and the grass carp FGF1 recombinant protein treatment group was intraperitoneally injected with 100 μL of the prepared Aeromonas hydrophila suspension (infectious dose 1×10⁻⁶). 6 (cfu / tail). After infection by challenge, the animals were continued to be cultured under the same conditions.

[0050] (3) AB-PAS staining of intestinal tissue Intestinal tissue was harvested 48 h after infection, fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin and sectioned (section thickness 4 μm). The sections were stained using the AB-PAS staining kit, observed under a microscope, and the number of goblet cells was counted. Results are shown below. Figure 2 ( p <0.0001, Mann-Whitney U test). Experimental results showed that the number of intestinal goblet cells in the grass carp FGF1 recombinant protein treatment group (20.56 ± 2.79 cells) was significantly higher than that in the PBS group (6.22 ± 1.64 cells). See the results below. Figure 2 ( p <0.0001 (Mann-Whitney U test). This indicates that treatment with grass carp FGF1 recombinant protein can significantly reverse the sharp decrease in the number of goblet cells in the grass carp intestine caused by Aeromonas hydrophila infection, and effectively restore the number and function of intestinal mucus secretory cells.

[0051] Experiment 2: Effects of grass carp FGF1 recombinant protein on intestinal cell proliferation and apoptosis The experimental steps are as follows: (1) Injection of grass carp FGF1 recombinant protein Following the same steps as in Experiment 1 (1), each group should have at least 9 biological replicates.

[0052] (2) Infection with Aeromonas hydrophila Same as step (2) in Experiment 1.

[0053] (3) BrdU staining of intestinal tissue Forty-four hours after infection in step (2) above, each group was intraperitoneally injected with 100 μL of BrdU (100 μg / tail). Four hours later, intestinal tissue was collected, fixed in 4% paraformaldehyde for 24 hours, and then embedded in paraffin and sectioned (section thickness 4 μm). The tissue sections were treated with antigen retrieval solution and incubated at 95℃ for 20 min. Cell permeabilizer was added and incubated at room temperature for 10 min. After washing with PBS, antibody blocking solution was added and blocked for 1-2 h. 50 μL of mouse anti-BrdU primary antibody (diluted 1:500-1:1000) was added and incubated overnight at 4℃. Washed 3 times with PBS, 3 min each time. 50 μL of Cy3-labeled goat anti-mouse IgG fluorescent secondary antibody (1:200) was added and incubated at 37℃ in the dark for 1 h. 50 μL of anti-fluorescence quenching mounting solution (containing DAPI) was added for mounting, and the fluorescence intensity was observed and counted under a fluorescence microscope. Experimental results showed that the intestinal BrdU fluorescence intensity in the PBS group (1.59% ± 0.43%) was significantly lower than that in the grass carp FGF1 recombinant protein treatment group (12.30% ± 2.08%). (See attached figures). Figure 3 ( p <0.0001 (Mann-Whitney U test). This indicates that treatment with grass carp FGF1 recombinant protein can significantly enhance the proliferative activity of grass carp intestinal epithelial cells after Aeromonas hydrophila infection, and accelerate the structural reconstruction and restoration of the integrity of the damaged intestinal mucosal epithelial layer.

[0054] (4) TUNEL staining of intestinal tissue Forty-eight hours after infection in step (2) above, the tissue was fixed in 4% paraformaldehyde for 24 hours, followed by paraffin embedding and sectioning (section thickness 6 μm). The sections were stained using a TUNEL staining kit, and the TUNEL fluorescence intensity of the intestinal tissue was observed and counted under a microscope. The experimental results showed that the intestinal TUNEL fluorescence intensity in the PBS group (15.12% ± 2.31%) was significantly higher than that in the grass carp FGF1 recombinant protein treatment group (2.94% ± 0.85%). (See attached table). Figure 4 ( p<0.0001 (Mann-Whitney Utest). This indicates that treatment with grass carp FGF1 recombinant protein can significantly reduce programmed cell death of intestinal mucosal epithelial cells caused by pathogen invasion and maintain the cell number and structural integrity of the intestinal mucosal epithelial layer.

[0055] Experiment 3: Effects of grass carp FGF1 recombinant protein on the expression of intestinal antibacterial factors The experimental steps are as follows: (1) Injection of grass carp FGF1 recombinant protein Following the same steps as in Experiment 1 (1), each group should have at least 9 biological replicates.

[0056] (2) Infection with Aeromonas hydrophila Same as step (2) in Experiment 1.

[0057] (3) qRT-PCR analysis of antimicrobial-related genes in intestinal tissue Forty-eight hours after infection, intestinal tissue was harvested and rapidly frozen in liquid nitrogen. Total RNA was extracted from the intestinal tissue of each group according to the instructions of the total RNA extraction kit, and cDNA was synthesized by reverse transcription according to the instructions of the cDNA reverse transcription kit. Grass carp 18S rRNA was used as an internal reference gene, and specific qRT-PCR primers targeting four antibacterial related genes—IL-22, MUC2, Spla2, and HB-EGFa—were designed (see Table 2). A 20 μL reaction system was prepared using qRT-PCR SYBR Mix. The standard qRT-PCR reaction conditions were: 95℃ pre-denaturation for 40 s; 95℃ denaturation for 15 s, 62℃ annealing for 30 s, for 40 cycles. Melting curve analysis was used to verify amplification specificity. The instrument used was an ABI QuantStudio 5 real-time quantitative PCR system (purchased from Thermo Fisher Scientific, USA). Three technical replicates were set for each sample, using 2... -ΔΔCt The relative expression levels of each target gene were calculated using the method. Experimental results showed that the expression levels of IL-22, MUC2, Spla2, and HB-EGFa in the intestine of the grass carp FGF1 recombinant protein treatment group (1.60 ± 0.41; 2.65 ± 1.07; 2.49 ± 0.87; 1.67 ± 0.46, respectively) were significantly higher than those in the PBS group (0.97 ± 0.05; 1.03 ± 0.24; 1.04 ± 0.29; 1.14 ± 0.28, respectively). See the results below. Figure 5 ( p<0.001 (Mann-Whitney U test). This indicates that treatment with grass carp FGF1 recombinant protein can significantly upregulate the expression levels of four key antibacterial immune factors—IL-22, MUC2, Spla2, and HB-EGFa—in the intestine of grass carp infected with Aeromonas hydrophila, thereby strengthening the intestinal mucosal immune barrier and enhancing the grass carp's defense against Aeromonas hydrophila infection.

[0058] Table 2. Primers used in Experiment 3 to detect the expression of antibacterial factors in the intestinal tract of grass carp.

[0059] MUC2 is a mucin secreted by goblet cells in the intestine, which plays a role in protecting the intestinal mucosal barrier.

[0060] Experiment 4: Effect of grass carp FGF1 recombinant protein on Aeromonas hydrophila infection load in tissues The experimental steps are as follows: (1) Injection of grass carp FGF1 recombinant protein Following the same steps as in Experiment 1 (1), each group should have at least 9 biological replicates.

[0061] (2) Infection with Aeromonas hydrophila Same as step (2) in Experiment 1.

[0062] (3) qRT-PCR analysis of Aerolysin expression, a virulence factor of Aeromonas hydrophila, in intestinal tissue Using the cDNA from step (3) of Experiment 3 as a template, qRT-PCR was performed, with the same system as step (3) of Experiment 3. Specific qRT-PCR primers were designed for Aerolysin, the virulence factor of Aeromonas hydrophila (see Table 3), and the reaction was performed using qRT-PCR SYBRMix, with the same qRT-PCR procedure as step (3) of Experiment 3. Three technical replicates were set up for each sample, using 2... -ΔΔCt The relative expression levels of each target gene were calculated using the method. Experimental results showed that the expression of Aerolysin, a virulence factor of *Aeromonas hydrophila*, in the intestinal tissue of the grass carp FGF1 recombinant protein treatment group (0.38 ± 0.23) was significantly lower than that in the PBS group (1.14 ± 0.20), suggesting a significant reduction in the number of *Aeromonas hydrophila* in the intestinal tissue of the grass carp FGF1 recombinant protein treatment group. (See attached figures). Figure 6 ( p <0.001 (Mann-Whitney U test). This indicates that treatment with grass carp FGF1 recombinant protein can significantly inhibit the number of Aeromonas hydrophila in the intestine.

[0063] (4) Detection of Aeromonas hydrophila load in liver and spleen tissues Forty-eight hours after infection, liver and spleen tissues were harvested, weighed, and aseptically ground. The tissues were serially diluted with sterile PBS and spread onto LB agar plates. The plates were incubated at 28°C for 18 hours, and the colony count (CFU / mg) was calculated. Three technical replicates were performed for each sample. The results showed that the number of Aeromonas hydrophila in the liver and spleen tissues of the grass carp FGF1 recombinant protein treatment group (137.22 ± 21.23 CFU / mg; 189.67 ± 18.83 CFU / mg, respectively) was significantly lower than that in the PBS group (317.78 ± 32.19 CFU / mg; 338.00 ± 35.32 CFU / mg, respectively). (See attached table). Figure 7 ( p <0.001 (Mann-Whitney U test). This indicates that treatment with grass carp FGF1 recombinant protein can significantly reduce the pathogen load in the liver and spleen of grass carp infected with Aeromonas hydrophila.

[0064] Table 3. Primers used in Experiment 4 to detect the expression of the Aerolysin virulence factor in Aeromonas hydrophila.

[0065] The mechanism by which the virulence factor Aerolysin of Aeromonas hydrophila disrupts the intestinal mucosal barrier has been confirmed.

[0066] Experiment 5: Effect of recombinant FGF1 protein on the survival rate of grass carp after infection The experimental steps are as follows: (1) Injection of grass carp FGF1 recombinant protein Following the same steps as in Experiment 1 (1), 30 biological replicates were set up for each group.

[0067] (2) Infection with Aeromonas hydrophila Same as step (2) in Experiment 1.

[0068] (3) Survival rate statistics The fish were observed continuously for 14 days, with the number of dead grass carp in each group recorded daily. Dead individuals were promptly removed, and the survival rate of each group was calculated and survival curves plotted. The results showed that the survival rate of the grass carp treated with the recombinant FGF1 protein (56.67%) was significantly higher than that of the PBS group (10.00%). (See attached table). Figure 8 ( p = 0.0002 (Log-rank test). This indicates that treatment with recombinant FGF1 protein can significantly improve the survival rate of grass carp infected with Aeromonas hydrophila.

[0069] In summary, unlike existing technologies that directly kill bacteria in vitro, this invention enhances the host's anti-infection ability by repairing the host's intestinal mucosal physical barrier (promoting proliferation, inhibiting apoptosis, and increasing goblet cells) and strengthening the immune barrier (upregulating multiple antibacterial factors), thus solving the pain point of intestinal damage caused by pathogen infection in aquaculture. This invention also provides experimental data at the live grass carp level, proving that the recombinant protein can significantly increase the survival rate of Aeromonas hydrophila 14 days after challenge from 10% to 56.67%, which has direct industrial application value.

Claims

1. The application of a grass carp FGF1 recombinant protein in the preparation of a product for treating Aeromonas hydrophila infection, characterized in that, The amino acid sequence of the grass carp FGF1 recombinant protein is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The method for preparing the grass carp FGF1 recombinant protein includes the following steps: (1) The grass carp FGF1 gene was amplified using primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4; (2) Construct a recombinant expression vector containing the gene obtained in step (1); (3) Transform the recombinant expression vector into host cells and induce expression; (4) Purify and remove endotoxins to obtain grass carp FGF1 recombinant protein.

3. The application according to claim 1, characterized in that, The product for treating Aeromonas hydrophila infection is a product for repairing intestinal mucosal barrier damage caused by Aeromonas hydrophila infection; the repair of intestinal mucosal barrier damage caused by Aeromonas hydrophila infection is manifested in the following ways: the sharp decrease in the number of goblet cells in the intestine of grass carp caused by Aeromonas hydrophila infection is reversed and improved, the proliferative activity of intestinal epithelial cells is enhanced, and the programmed death of intestinal mucosal epithelial cells is inhibited.

4. The application according to claim 1, characterized in that, The product for combating Aeromonas hydrophila infection is a product that upregulates the expression level of intestinal antimicrobial factors, which are one or more of IL-22, MUC2, Spla2, and HB-EGFa.

5. The application according to claim 1, characterized in that, The product for combating Aeromonas hydrophila infection is a product that reduces the Aeromonas hydrophila load in fish tissues after infection, wherein the tissues are the liver and / or spleen.

6. The application according to claim 1, characterized in that, The dosage form of the product for combating Aeromonas hydrophila infection is one or more of the following: injection, oral preparation, soaking solution, or feed additive.

7. The application according to claim 6, characterized in that, The product for treating Aeromonas hydrophila infection is an intraperitoneal injection.

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