Use of largemouth bass neuropeptide in preparation of products for preventing and treating aeromonas hydrophila infection

Abstract

This invention discloses the application of largemouth bass neuropeptide in the preparation of products for preventing and treating Aeromonas hydrophila infection. The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1. The largemouth bass neuropeptide provided by this invention is a novel antimicrobial peptide that can significantly enhance the ability of largemouth bass to resist Aeromonas hydrophila infection both in vivo and in vitro. It provides a new technical option for the prevention and treatment of Aeromonas hydrophila-related diseases and for achieving "antibiotic-free aquaculture" of largemouth bass. It helps to improve the survival rate of largemouth bass fry, promotes green aquaculture, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and more specifically, to the application of largemouth bass neuropeptide in the preparation of products for the prevention and treatment of Aeromonas hydrophila infection. Background Technology

[0002] Largemouth bass (Micropterus salmoides), also known as bigmouth perch, belongs to the order Perciformes, family Centrarchide, and genus Micropterus. It is a well-known recreational fish, prized for its tender flesh, rich nutrition, strong disease resistance, and rapid growth, earning it the nickname "freshwater yellow croaker" and significant commercial value. In recent years, however, with the continuous increase in farming density and scale, the farming environment for largemouth perch has deteriorated, leading to frequent bacterial diseases. This has resulted in serious problems in the largemouth perch aquaculture industry, including premature maturity, decreased growth rate, poor food conversion efficiency, and weak disease resistance. Diseases occurring particularly severely during the early developmental stages of the largemouth perch hinder the healthy development of the aquaculture industry and cause significant economic losses.

[0003] Aeromonas hydrophila is a pathogen widely distributed in freshwater and soil. It is a zoonotic bacterium that can cause diseases in fish, birds, reptiles, and mammals (such as gastroenteritis and septicemia). It can also infect humans through fish or aquatic products, causing symptoms such as vomiting, abdominal pain, and diarrhea. Aeromonas hydrophila is a Gram-negative bacterium. Its virulence factors include exotoxins (such as enterotoxins and hemolysins), extracellular proteases (such as ribozymes, phospholipases, and phosphatases), structural proteins (such as lipopolysaccharides and S proteins), and signal-related proteins (such as porins and secretion system proteins). In aquatic animals, when Aeromonas hydrophila causes disease, the combined action of its multiple virulence factors leads to disease in farmed species, even causing large-scale mortality. This poses a serious threat to the aquaculture industry and causes significant economic losses to fish farmers. Aeromonas hydrophila can infect various fish species, such as catfish, goldfish, and salmon. When fish are infected with Aeromonas hydrophila, the main symptoms include enteritis, scurvy, and blackening of the spleen and kidneys, with infected fish typically dying within three days. Previous treatments for Aeromonas hydrophila infection primarily involved broad-spectrum antibiotics (such as quinolones), but excessive antibiotic use has increased bacterial resistance, thus limiting treatment effectiveness. Therefore, there is an urgent need to explore alternative methods for the prevention and treatment of Aeromonas hydrophila.

[0004] Currently, those skilled in the art typically use antibiotics to prevent and control bacterial diseases in largemouth bass. However, long-term or excessive use of antibiotics leads to drug residues and increased bacterial resistance. Antimicrobial peptides are a general term for a class of short cationic peptides with antimicrobial activity and are also an important component of the organism's innate immune system, making them ideal alternatives to antibiotics. In the prevention and treatment of Aeromonas hydrophila infection, the antimicrobial peptide Piscidin can effectively and rapidly exert its bactericidal effect by inducing membrane permeability and interaction with genomic DNA. Antimicrobial peptides are divided into natural AMPs and synthetic AMPs. However, previous studies have found that natural AMPs have problems such as low stability, high cost, and host cell toxicity. Therefore, synthetic peptides, which are more stable, shorter, and more efficient, are currently widely used. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides the application of largemouth bass neuropeptide in the preparation of products for preventing and treating Aeromonas hydrophila infection.

[0006] The first objective of this invention is to provide the use of largemouth bass neuropeptide in the preparation of products for the prevention of Aeromonas hydrophila infection.

[0007] A second objective of this invention is to provide the use of largemouth bass neuropeptide in the preparation of products for treating Aeromonas hydrophila infections.

[0008] A third objective of this invention is to provide the use of largemouth bass neuropeptides in the preparation of products that enhance the ability of largemouth bass to resist Aeromonas hydrophila infection.

[0009] A fourth objective of this invention is to provide the application of largemouth bass neuropeptides in the preparation of products that alleviate tissue damage caused by largemouth bass infection with Aeromonas hydrophila.

[0010] The fifth object of the present invention is to provide an agent for preventing and treating Aeromonas hydrophila infection.

[0011] To achieve the above objectives, the present invention is implemented through the following solution:

[0012] This invention is the first to discover that injecting or immersing juvenile largemouth bass with neuropeptide (MSNP) can enhance their immune response to Aeromonas hydrophila, alleviate infection-induced apoptosis, and improve intestinal damage caused by the infection. MSNP improves the innate immune level and immunomodulatory capacity of largemouth bass, regulates antibacterial responses, and thus prevents immune stress and bodily damage caused by Aeromonas hydrophila.

[0013] Therefore, this invention seeks protection for the following:

[0014] The application of largemouth bass neuropeptide in the preparation of products for preventing Aeromonas hydrophila infection, wherein the amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1.

[0015] Preferably, the host of the *Aeromonas hydrophila* includes largemouth bass.

[0016] More preferably, the largemouth bass is a juvenile fish.

[0017] The application of largemouth bass neuropeptide in the preparation of products for treating Aeromonas hydrophila infection, wherein the amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1.

[0018] Preferably, the host of the *Aeromonas hydrophila* includes largemouth bass.

[0019] More preferably, the largemouth bass is a juvenile fish.

[0020] The application of largemouth bass neuropeptide in the preparation of products that enhance the ability of largemouth bass to resist Aeromonas hydrophila infection, wherein the amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1.

[0021] Preferably, the largemouth bass is a juvenile fish.

[0022] The application of largemouth bass neuropeptide in the preparation of products that enhance the immune response of largemouth bass after infection with Aeromonas hydrophila, wherein the amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1.

[0023] Preferably, the largemouth bass is a juvenile fish.

[0024] The application of largemouth bass neuropeptide in the preparation of a treatment to alleviate tissue damage caused by Aeromonas hydrophila infection in largemouth bass, the amino acid sequence of which is shown in SEQ ID NO.1.

[0025] Preferably, the largemouth bass is a juvenile fish.

[0026] An agent for the prevention and treatment of Aeromonas hydrophila infection, the active ingredient of which includes largemouth bass neuropeptide with an amino acid sequence as shown in SEQ ID NO.1.

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

[0028] The largemouth bass neuropeptide provided by this invention is a novel antimicrobial peptide that can significantly enhance the ability of largemouth bass to resist Aeromonas hydrophila infection both in vivo and in vitro. It provides a new technical option for the prevention and treatment of Aeromonas hydrophila-related diseases and the realization of "antibiotic-free aquaculture" of largemouth bass. It helps to improve the survival rate of largemouth bass fry, promotes green aquaculture, and has broad application prospects. Attached Figure Description

[0029] Figure 1 A heatmap showing the changes in mRNA expression levels of Y8b, IL-1β, IL-10, IFN-γ, and TGF-β in different tissues after injection of different concentrations of MSNP into largemouth bass fry was generated. One-way ANOVA and Duncan's multiple range method were used for multiple comparisons.

[0030] Figure 2 The effects of different doses of MSNP on serum immune-related parameters in largemouth bass fry were investigated. A represents serum alkaline phosphatase levels after injection of different doses of MSNP into largemouth bass fry; B represents serum glucose levels after injection of different doses of MSNP into largemouth bass fry; C represents serum alkaline phosphatase levels at different time points after MSNP injection; and D represents serum glucose levels at different time points after MSNP injection. All data are expressed as mean ± standard error. One-way ANOVA was used for multiple comparisons, with Duncan's multiple range test. Different letters in the same row indicate significant differences between groups (p < 0.05).

[0031] Figure 3 To generate a heatmap of gene expression levels in different tissues at different time points after MSNP injection into largemouth bass fry, one-way ANOVA and Duncan's multiple range method were used for multiple comparisons.

[0032] Figure 4 To investigate the changes in the expression levels of il-10, il-1β, and tnf-α genes in different tissues of largemouth bass fry 24 hours after MSNP injection, data are presented as mean ± standard error. One-way ANOVA and Duncan's multiple range method were used for multiple comparisons. Different letters in the same row indicate significant differences between groups (p < 0.05).

[0033] Figure 5 To investigate the changes in the expression levels of the ifn-γ, npy, and y8b genes in different tissues of largemouth bass fry 24 hours after MSNP injection, data are presented as mean ± standard error. One-way ANOVA and Duncan's multiple range method were used for multiple comparisons. Different letters in the same row indicate significant differences between groups (p < 0.05).

[0034] Figure 6 The changes in intestinal morphology of largemouth bass fry infected with Aeromonas hydrophila after MSNP injection.

[0035] Figure 7 Changes in apoptosis of cells in the head kidney of largemouth bass fry infected with Aeromonas hydrophila after MSNP injection.

[0036] Figure 8Changes in apoptosis in the spleen of largemouth bass fry infected with Aeromonas hydrophila after MSNP injection.

[0037] Figure 9 This study investigated the changes in gene expression levels in different tissues of largemouth bass fry after immersion treatment in different water bodies following infection with Aeromonas hydrophila. All data are expressed as mean ± standard error. One-way ANOVA and Duncan's multiple range test were used for multiple comparisons. Different letters in the same row indicate significant differences between groups (p < 0.05).

[0038] Figure 10 To investigate the changes in spleen cell apoptosis in largemouth bass fry after immersion in different water bodies following infection with Aeromonas hydrophila.

[0039] Figure 11 To investigate the changes in apoptosis of cells in the head kidneys of largemouth bass fry after immersion in different bodies of water following infection with Aeromonas hydrophila.

[0040] Figure 12 The changes in gene expression levels in different tissues of largemouth bass fry infected with different bacteria 24 hours later were investigated. All data are expressed as mean ± standard error. One-way ANOVA and Duncan's multiple range test were used for multiple comparisons. Different letters in the same row indicate significant differences between groups (p<0.05).

[0041] Figure 13 The effects of MSNP injection on apoptosis in the head kidneys of largemouth bass fry infected with different bacteria 24 hours later were investigated.

[0042] Figure 14 The effects of MSNP injection on apoptosis in the spleen of largemouth bass fry infected with different bacteria 24 hours later were investigated. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0044] Example 1: Effects of injection dosage and timing of largemouth bass neuropeptide (MSNP) on largemouth bass fry.

[0045] 1. Preparation of MSNP injection solution

[0046] The amino acid sequence of MSNP from N-terminus to C-terminus is YPVKPENPGEDAPADELAKYYSALRHYINLITRQR (SEQ ID NO.1). The peptide was synthesized by Shanghai Qiangyao Company, and amidation modification was performed at the C-terminus to obtain MSNP lyophilized powder with a purity greater than 95%.

[0047] MSNP lyophilized powder was dissolved in 1×PBS to obtain an MSNP stock solution with a concentration of 10 μg / μl, which was then stored at -20℃. For use, the MSNP stock solution was diluted with PBS according to the injection dosage to obtain the MSNP injection solution. The injection dosage of MSNP was 1 ng / g BW, 10 ng / g BW, or 100 ng / g BW, where ng refers to the mass of the MSNP dry powder and g BW refers to the weight of the fish fry.

[0048] 2. Injection Experiment

[0049] (1) Effect of injection dosage on largemouth bass fry

[0050] Largemouth bass fry (30-60 days old, weighing 2-4g) were randomly divided into 4 groups (12 fish in each group), designated as control group, treatment group 1, treatment group 2 and treatment group 3, and raised for one week.

[0051] The control group was injected intraperitoneally with 1×PBS, while treatment groups 1–3 were injected intraperitoneally with MSNP at doses of 1 ng / g BW, 10 ng / g BW, and 100 ng / g BW, respectively. The corresponding injection volume was calculated based on the body weight of each largemouth bass juvenile. Twelve hours after injection, tissue samples (head, kidney, spleen, brain, and intestine) and serum samples were collected from each group of largemouth bass juveniles for subsequent testing.

[0052] (2) Effect of injection timing on largemouth bass fry

[0053] Largemouth bass fry (30-60 days old, weighing 2-4g) were randomly divided into 6 groups (12 fish per group), designated as control-12h group, treatment-12h group, control-24h group, treatment-24h group, control-72h group, and treatment-72h group, and were raised for one week.

[0054] The control-12h group, control-24h group, and control-72h group were injected intraperitoneally with 0 ng / g BW MSNP injection solution. Tissue and serum samples were collected at 12h, 24h, and 72h after injection for subsequent testing.

[0055] The treatment-12h, treatment-24h, and treatment-72h groups were intraperitoneally injected with 10 ng / g BW MSNP injection solution. Tissue samples (head kidney, spleen, brain, and intestine) and serum samples were collected at 12h, 24h, and 72h after injection for subsequent testing.

[0056] 3. qPCR detection

[0057] Total RNA was extracted from tissue samples of largemouth bass fry from each group, and cDNA was obtained by reverse transcription. Using cDNA as a template, the mRNA expression levels of neuropeptide receptor gene (Y8b, which may be involved in immune regulation in immune organs) and inflammatory factors (IL-1β, IL-10, IFN-γ, and TGF-β) were detected by qPCR. β-actin was used as an internal control. The primers used are shown in Table 1. A negative control was set up, and each sample was tested in duplicate.

[0058] Table 1 qPCR primers

[0059]

[0060] The qPCR reaction system consisted of: 1 μL template, 8.2 μL ddH2O, 0.4 μL upstream primer, and 0.4 μL downstream primer. Green Mix 10μL.

[0061] The qPCR reaction program is as follows: (1) 95℃, 5min; (2) 95℃, 5sec; 60℃, 30sec; 72℃, 30sec; 40 cycles; (3) 95℃, 15sec; anneal at 65-95℃ at an increment of 0.5℃ per sec to prepare a melting curve.

[0062] 4. Detection of immune-related indicators in serum

[0063] The levels of Akp and Glu in serum samples from largemouth bass fry of each group were detected using an alkaline phosphatase (AKP) test kit (Nanjing Jiancheng, A154-1-1) and a glucose (GLU) test kit (Nanjing Jiancheng, A059-2).

[0064] 5. Test Results

[0065] like Figure 1As shown, compared with MSNP (i.e., PBS) with 0 ng / g BW, the expression of pro-inflammatory factors ifn-γ and il-1β in the head kidney and spleen increased after injection of MSNP with 1 ng / g BW, 10 ng / g BW, and 100 ng / g BW, with the most significant increase after injection of MSNP with 10 ng / g BW. In spleen tissue, the expression level of anti-inflammatory factor il-10 decreased after intraperitoneal injection of MSNP. Compared with the control group, the expression levels of intestinal ifn-γ, il-1β, and y8b genes showed a trend of first increasing and then decreasing with increasing MSNP injection dose, with y8b significantly increasing after injection of 1 ng / g BW. In addition, after injection of MSNP with 10 ng / g BW, the expression of ifn-γ and il-10 in the brain region increased significantly.

[0066] like Figure 2 As shown in Figures A through B, intraperitoneal injection of MSNP at concentrations of 10 ng / g and 100 ng / g BW significantly increased serum alkaline phosphatase activity in largemouth bass, and also resulted in higher serum glucose levels; Figure 2 As shown in C-D, at the three time points after MSNP injection, the serum alkaline phosphatase level of largemouth bass was higher than that of the control group (i.e., injected with PBS), while the serum glucose level increased 12h and 24h after MSNP injection, with a more significant increase at 24h, but decreased after 72h.

[0067] The above results indicate that the injection dose of MSNP is significantly dose-dependent on the expression levels of each gene and serum enzyme activity, and 10 ng / g BW is the optimal injection dose of MSNP.

[0068] like Figure 3 As shown, 12 hours after MSNP injection, the levels of ifn-γ mRNA increased in the head kidney, spleen, and brain. 24 hours after MSNP injection, the expression of ifnγ, il-1β, and il-10 significantly decreased in the head kidney, spleen, and brain, and this phenomenon persisted until 72 hours after MSNP injection. Notably, compared to the control group, the expression of all four genes in the gut increased 24 hours after MSNP injection, with ifn-γ expression being particularly significant. In serum tests, we found that serum alkaline phosphatase levels in largemouth bass were higher than in the control group at all three time points after MSNP injection. Serum glucose levels increased at 12 and 24 hours after MSNP injection, with a more pronounced increase at 24 hours, but decreased after 72 hours.

[0069] The above results indicate that intraperitoneal injection of MSNP into juvenile largemouth bass has a regulatory effect on inflammatory factors and related biochemical indicators at different time points.

[0070] Example 2: Effects of injection of largemouth bass neuropeptide (MSNP) into largemouth bass fry infected with Aeromonas hydrophila.

[0071] 1. Preparation of MSNP injection solution

[0072] Same as Example 1.

[0073] 2. Infection experiment

[0074] Largemouth bass fry (30-60 days old, weighing 2-4g) were randomly divided into 4 groups (12 fish in each group), designated as group 1 to group 4, and raised for one week.

[0075] After anesthetizing largemouth bass fry in each group with MS222, group 1 (i.e., PBS + A. hydrophila (12h)) was injected intraperitoneally with 0 ng / g BW MSNP. 12 hours later, 100 μL of Aeromonas hydrophila (1×10⁻⁶) was injected. 6 Group 2 (i.e., PBS + A. hydrophila (24h)) received an intraperitoneal injection of 0 ng / g BW MSNP for 24 hours, followed by an injection of 100 μL of Aeromonas hydrophila (1×10⁻⁶ CFU / mL); Group 2 received PBS + A. hydrophila (24h) and then 100 μL of A. hydrophila (1×10⁻⁶ CFU / mL). 6 Group 3 (i.e., MSNP + A. hydrophila (12h)) received an intraperitoneal injection of 10 ng / g BW MSNP. Twelve hours later, they were injected with 100 μL of Aeromonas hydrophila (1×10⁻⁶ CFU / mL). 6 Group 4 (i.e., MSNP + A. hydrophila (24h)) received an intraperitoneal injection of 10 ng / g BW MSNP, followed by an injection of 100 μL of Aeromonas hydrophila (1×10⁻⁶ CFU / mL); Group 4 received MSNP + A. hydrophila (24h) and 100 μL of A. hydrophila (1×10⁻⁶ CFU / mL) 24 6 (CFU / mL)

[0076] After all injections were completed, the Aeromonas hydrophila in each group were returned to the aquarium for continued rearing, with the water temperature maintained at 30°C. After 24 hours of rearing, serum and tissue samples (whole brain, head kidney, spleen, and intestines) were collected by tail docking.

[0077] 3. qPCR detection

[0078] qPCR detection was performed according to the method in Example 1. The sequence of the upstream primer for detecting the MSNP gene was 5'-AGAAGCCTCATAAACACC-3', and the sequence of the downstream primer was 5'-TTCACTACAAAGCCACAAC-3'.

[0079] 4. TUNEL cell apoptosis experiment

[0080] The tissue damage of largemouth bass fry in each group was detected using the TUNEL luciferin apoptosis detection kit (Novizan, A111).

[0081] 5. Test Results

[0082] like Figure 4 and Figure 5 As shown, the elevated expression of the il-10 gene in immune tissues of the group infected with Aeromonas hydrophila for 12 hours without MSNP injection could be reduced by the injection of exogenous MSNP. Similarly, this phenomenon was also observed in the gene expression of tnf-α and ifn-γ in the head kidney tissue. The levels of MSNP and y8b mRNA in the spleen significantly increased after MSNP injection, while the expression of y8b in the intestine significantly decreased. In largemouth bass fry reinfected with Aeromonas hydrophila after MSNP injection, the levels of ifn-γ and il-1β in the brain, intestine, head kidney, and spleen were significantly lower than those without MSNP injection. This indicates that MSNP injection can reduce the inflammatory response induced by Aeromonas hydrophila infection in largemouth bass juveniles and alleviate the immune stress response in the body.

[0083] like Figure 6 As shown, largemouth bass fry that were not injected with MSNP showed severe intestinal tissue damage after infection with Aeromonas hydrophila, a phenomenon particularly pronounced in the intestines of uninjected fish 24 hours post-infection. In contrast, the MSNP-injected group showed significantly reduced intestinal damage, primarily manifested as vacuolation and a decrease in the area of ​​damage to intestinal villi. This indicates that MSNP injection can reduce intestinal damage induced by Aeromonas hydrophila infection in largemouth bass fry.

[0084] like Figure 7 and Figure 8 As shown, largemouth bass fry that were not injected with MSNP showed a higher number of apoptotic cells in the head kidney and spleen after 24 hours of infection with Aeromonas hydrophila; while largemouth bass fry injected with MSNP showed a significantly reduced number of apoptotic cells in the head kidney and spleen after 24 hours of infection with Aeromonas hydrophila. This indicates that MSNP can reduce apoptosis induced by Aeromonas hydrophila infection and alleviate tissue damage caused by Aeromonas hydrophila to the head kidney and spleen.

[0085] Example 3: Comparison of the effects of MSNP and gallnut on treating Aeromonas hydrophila infection in largemouth bass fry.

[0086] 1. Infection experiment

[0087] Largemouth bass fry (30-60 days old, weighing 2-4g) were randomly divided into 4 groups (8 fish per group), designated as Group 1 to Group 4, and raised for one week.

[0088] After anesthetizing largemouth bass fry with MS222, group 1 (control) was injected intraperitoneally with 100 μL of 1×PBS and then immersed in culture water for 24 hours; group 2 (A. hydrophila) was injected intraperitoneally with 100 μL of Aeromonas hydrophila (1×10⁻⁶). 6 After being placed in culture water for 24 hours (CFU / mL), group 3 (i.e., A. hydrophila + MSNP) was treated with intraperitoneal injection of 100 μL of Aeromonas hydrophila (1 × 10⁻⁶ CFU / mL). 6 After being placed in culture water containing 200 μg / L MSNP, the bacteria were soaked for 24 hours; Group 4 (i.e., A. hydrophila + Galla) was injected intraperitoneally with 100 μL of Aeromonas hydrophila (1×10⁻⁶ CFU / mL). 6 After being placed in culture water containing 200 μg / L gallnut (CFU / mL), the gallnuts were cultured for 24 hours.

[0089] After soaking, serum and tissue samples (whole brain, head kidney, spleen, and intestines) were collected from the severed tail.

[0090] 2. qPCR detection

[0091] qPCR detection was performed according to the method in Example 2.

[0092] 3. TUNEL cell apoptosis experiment

[0093] The tissue damage of each group of largemouth bass fry was detected using the method described in Example 2.

[0094] 4. Test Results

[0095] like Figure 9 As shown, in the head kidney tissue, the TNF-α mRNA level of largemouth bass infected with Aeromonas hydrophila for 24 hours followed by MSNP immersion was significantly different from that of largemouth bass infected with Aeromonas hydrophila and immersed in culture water. Furthermore, TNF-α levels recovered to normal after MSNP immersion compared to the control group. The IL-10 gene expression trends in the head kidney and spleen were similar compared to the control group, suggesting that MSNP may play a similar role in the signaling pathway in immune tissues. In addition, IFN-γ expression in the spleen of largemouth bass infected with Aeromonas hydrophila was significantly upregulated after gallnut treatment, while the IFN-γ level in the spleen of the MSNP-treated group did not show this dramatic response. There were no differences in the gene changes of Y8b in the spleen tissue or IL1β in the intestine.

[0096] like Figure 10 and Figure 11 As shown, both MSNP and gallnut can reduce cell apoptosis in the head kidney and spleen of largemouth bass caused by Aeromonas hydrophila infection.

[0097] The above results indicate that both MSNP and gallnut can reduce immune tissue damage caused by Aeromonas hydrophila infection in largemouth bass, with MSNP showing better efficacy.

[0098] Example 4: Comparison of the effects of MSNP injection on the treatment of largemouth bass fry infected with two different bacteria.

[0099] 1. Preparation of MSNP injection solution

[0100] Same as Example 1.

[0101] 2. Infection experiment

[0102] Largemouth bass fry (30-60 days old, 2-4g in weight) were randomly divided into 4 groups (8 fish in each group), designated as group 1 to group 4, and raised for one week.

[0103] After anesthetizing largemouth bass fry with MS222, group 1 (control) was injected intraperitoneally with 100 μL of 1×PBS solution for 24 hours, followed by another injection of 1×PBS solution; group 2 (PBS + MSNP) was injected intraperitoneally with 100 μL of 1×PBS solution for 24 hours, followed by an injection of 10 ng / g BW MSNP; group 3 (A. hydrophila + MSNP) was injected intraperitoneally with 100 μL of Aeromonas hydrophila (1×10⁻⁶). 6 24 hours after receiving CFU / mL, another 10 ng / g BW MSNP was injected; Group 4 (i.e., S. agalactiae + MSNP) received an intraperitoneal injection of 100 μL of agalactiae streptococci (1×10⁻⁶ CFU / mL). 6 24 hours later, inject 10 ng / g BW MSNP (CFU / mL).

[0104] After all injections were completed, the largemouth bass fry were returned to the tank for further rearing, with the water temperature maintained at 30°C. After 24 hours of rearing, serum and tissue samples (whole brain, head kidney, spleen, and intestines) were collected by tail cutting.

[0105] 3. qPCR detection

[0106] qPCR detection was performed according to the method in Example 2.

[0107] 4. TUNEL cell apoptosis experiment

[0108] The tissue damage of each group of largemouth bass fry was detected using the method described in Example 2.

[0109] 5. Test Results

[0110] like Figure 12As shown, in the head kidney and intestinal tissues of largemouth bass infected with Streptococcus agalactiae and intraperitoneally injected with MSNP (Group 4), TNF-α gene expression was consistent with that of MSNP injection alone (Group 2). However, in largemouth bass infected with Aeromonas hydrophila and intraperitoneally injected with MSNP (Group 3), the TNF-α mRNA level returned to the level of the control group (Group 1). Furthermore, the IFN-γ mRNA level was significantly increased in the head kidney, spleen, and intestinal tissues of largemouth bass fry infected with Streptococcus agalactiae and injected with MSNP (Group 4). This indicates that... Streptococcus agalactiae had a significant immunostimulatory effect on largemouth bass fry, while MSNP may not be able to inhibit this effect. IL-10 levels in the head kidney and spleen increased significantly after intraperitoneal injection of MSNP, with the head kidney of largemouth bass infected with Streptococcus agalactiae also showing this upward trend. However, the IL-10 levels of largemouth bass infected with Aeromonas hydrophila returned to normal after intraperitoneal injection of MSNP, indicating that MSNP is more effective in immunotherapy against Aeromonas hydrophila. Compared with the control group, there was no significant difference in Y8b and IL-1β levels.

[0111] like Figure 13 and Figure 14 As shown, compared with Streptococcus agalactiae infection, intraperitoneal injection of MSNP into the head kidney and spleen of largemouth bass after Aeromonas hydrophila infection significantly reduced the number of apoptotic cells and the fluorescence intensity.

[0112] The above results indicate that MSNP has a significant therapeutic effect on largemouth bass infected with Aeromonas hydrophila, but no significant effect on Streptococcus agalactiae infection. MSNP's antibacterial effect may be genus-specific.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of largemouth bass neuropeptide in the preparation of products for preventing Aeromonas hydrophila infection in largemouth bass, characterized in that, The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1, and the largemouth bass is a juvenile fish.

2. The application of largemouth bass neuropeptide in the preparation of products for treating largemouth bass infected with Aeromonas hydrophila, characterized in that, The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1, and the largemouth bass is a juvenile fish.

3. The application of largemouth bass neuropeptides in the preparation of products that enhance the resistance of largemouth bass to Aeromonas hydrophila infection, characterized in that, The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1, and the largemouth bass is a juvenile fish.

4. The application of largemouth bass neuropeptides in the preparation of products that enhance the immune response of largemouth bass after infection with Aeromonas hydrophila, characterized in that, The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1, and the largemouth bass is a juvenile fish.

5. The application of largemouth bass neuropeptides in the preparation of products that alleviate tissue damage caused by Aeromonas hydrophila infection in largemouth bass, characterized in that, The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1, and the largemouth bass is a juvenile fish.

Images