Application of micropterus salmoides neuropeptide in preparation of product for preventing and treating aeromonas hydrophila infection

Abstract

The invention discloses an application of micropterus salmoides neuropeptide in preparation of a product for preventing and treating aeromonas hydrophila infection. The amino acid sequence of the micropterus salmoides neuropeptide is as shown in SEQ ID NO. 1. The micropterus salmoides neuropeptide provided by the invention is a novel antibacterial peptide, can obviously enhance the aeromonas hydrophila infection resistance of micropterus salmoides when being applied in vivo and in vitro, and provides a new technical choice for preventing and treating aeromonas hydrophila related diseases and realizing'antibiotic-free culture 'of micropterus salmoides; the survival rate of the largemouth bass in the largemouth bass fry stage can be increased, green breeding is promoted, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptides, and in particular to application of a largemouth bass neuropeptide in preparing a product for preventing and treating Aeromonas hydrophila infection. Background Art

[0002] Largemouth bass (Micropterus salmoides), also known as largemouth bass, belongs to the order Perciformes, family Centrarchide, genus Micropterus. It is a famous sport fish with tender meat, rich nutrition, strong disease resistance and rapid growth. It is known as "freshwater yellow croaker" and has important commercial value. In recent years, with the continuous increase in breeding density and breeding scale, the breeding environment of largemouth bass has deteriorated, and bacterial diseases have occurred frequently, resulting in serious problems such as precocious maturity, decreased growth rate, poor food conversion efficiency and poor disease resistance in the breeding industry of largemouth bass. In particular, the disease problem in the early development stage of largemouth bass is particularly serious, which hinders the healthy development of its breeding industry and causes heavy economic losses.

[0003] Aeromonas hydrophila is a pathogen widely distributed in freshwater and soil. It is a pathogenic bacterium that is shared by humans, livestock and fish. It can cause pathogenic diseases (such as gastroenteritis, sepsis, etc.) in fish, birds, reptiles and mammals. It can also infect humans through fish or aquatic products, causing vomiting, abdominal pain, diarrhea and other symptoms. Aeromonas hydrophila is a Gram-negative bacterium. Its virulence factors are exotoxins (such as enterotoxins, hemolysins, etc.), extracellular proteases (such as ribozymes, phospholipases, phosphatases, etc.), structural proteins (such as lipopolysaccharides, S proteins, etc.) and signal-related proteins (such as porins and secretory system proteins, etc.). In aquatic animals, when Aeromonas hydrophila causes disease, its multiple virulence factors work together to cause aquaculture species to become sick, and even cause large-scale deaths, which brings serious harm to the aquaculture industry and causes large economic losses to fishery farmers. Aeromonas hydrophila can infect a variety of fish, such as catfish, goldfish, salmon, etc. When fish are infected by Aeromonas hydrophila, the main symptoms are enteritis, scurvy, blackening of the spleen and kidneys, etc., and the infected fish die within about 3 days. In the past, the treatment of Aeromonas hydrophila infection mainly used broad-spectrum antibiotics (such as quinolones, etc.), but excessive use of antibiotics increases bacterial resistance to antibiotics, thereby limiting its therapeutic effect. Therefore, it is urgent to explore other methods for preventing and treating Aeromonas hydrophila.

[0004] At present, those skilled in the art usually use antibiotics to prevent and control bacterial diseases of largemouth bass, but long-term or excessive use of antibiotics leads to problems of drug residues and increased bacterial resistance. Antimicrobial peptides are a general term for a class of short cationic peptides with antimicrobial activity. They are also an important component of the innate immune system of organisms and are ideal substitutes for antibiotics. In the prevention and treatment of Aeromonas hydrophila infections, the antimicrobial peptide Piscidin can effectively and quickly kill bacteria by inducing membrane permeability and interaction with genomic DNA. Antimicrobial peptides are divided into natural AMPs and synthetic AMPs, but previous studies have found that natural AMPs have problems such as low stability, high cost, and host cell toxicity, so more stable, shorter, and more efficient synthetic peptides are currently commonly used. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides the use of a largemouth bass neuropeptide in preparing a product for preventing and treating Aeromonas hydrophila infection.

[0006] The first object of the present invention is to provide a use of a largemouth bass neuropeptide in preparing a product for preventing Aeromonas hydrophila infection.

[0007] The second object of the present invention is to provide the use of the largemouth bass neuropeptide in preparing a product for treating Aeromonas hydrophila infection.

[0008] The third object of the present invention is to provide the use of the neuropeptide of largemouth bass in preparing a product for enhancing the ability of largemouth bass to resist infection by Aeromonas hydrophila.

[0009] The fourth object of the present invention is to provide the use of the largemouth bass neuropeptide in the preparation of a method for alleviating tissue damage caused by the largemouth bass infected with Aeromonas hydrophila.

[0010] A fifth object of the present invention is to provide a preparation for preventing and treating Aeromonas hydrophila infection.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] The present invention first discovered that injecting or soaking largemouth bass juveniles with largemouth bass neuropeptide (MSNP) can improve the immune response of largemouth bass to Aeromonas hydrophila, alleviate cell apoptosis caused by infection, and improve intestinal damage caused by infection. MSNP improves the innate immunity level and immune regulation ability of largemouth bass, regulates antibacterial response, and achieves the effect of preventing immune stress and body damage caused by Aeromonas hydrophila.

[0013] Therefore, the present invention requests protection for the following:

[0014] Application of a largemouth bass neuropeptide in preparing a product 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 infected by Aeromonas hydrophila includes largemouth bass.

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

[0017] Application of a largemouth bass neuropeptide in preparing a product 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 infected by Aeromonas hydrophila includes largemouth bass.

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

[0020] The invention discloses an application of a largemouth bass neuropeptide in preparing a product for enhancing the ability of largemouth bass to resist infection by Aeromonas hydrophila. 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 invention discloses an application of a largemouth bass neuropeptide in preparing a product for enhancing the immune response ability of largemouth bass after being infected by Aeromonas hydrophila. 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] Application of a largemouth bass neuropeptide in the preparation of a drug for alleviating tissue damage caused by largemouth bass infection with Aeromonas hydrophila. The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.1.

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

[0026] A preparation for preventing and treating Aeromonas hydrophila infection, wherein the active ingredient comprises a 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 the present invention is a new type of antibacterial peptide, which 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 preventing and treating Aeromonas hydrophila-related diseases and realizing "antibiotic-free farming" of largemouth bass, helps to improve the survival rate of largemouth bass in the seedling stage, promotes green farming, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Heat map of the changes in mRNA expression levels of Y8b, IL-1β, IL-10, IFN-γ, and TGF-β in different tissues of largemouth bass fry after injection of different concentrations of MSNPs, using one-way ANOVA with Duncan's multiple range method for multiple comparisons.

[0030] Figure 2 The effects of different doses of MSNP on serum immune-related indicators of largemouth black bass fry after injection. A is the serum alkaline phosphatase level of largemouth black bass fry after injection of different doses of MSNP; B is the serum glucose level of largemouth black bass fry after injection of different doses of MSNP; C is the serum alkaline phosphatase level of largemouth black bass fry at different times after injection of MSNP; D is the serum glucose level of largemouth black bass fry at different times after injection of MSNP; All data are expressed as mean ± standard error, and one-way analysis of variance was used for multiple comparisons with Duncan's multiple range method. Different letters in the same row indicate significant differences between groups (p<0.05).

[0031] Figure 3 Figure 2 Heat map of the changes in gene expression levels in different tissues at different times after injection of largemouth bass fry with MSNPs, using one-way ANOVA with Duncan's multiple range method for multiple comparisons.

[0032] Figure 4 The expression levels of IL-10, IL-1β and TNF-α genes in different tissues of largemouth bass fry infected with Aeromonas hydrophila 24 hours after injection with MSNP were analyzed. The data were expressed as mean ± SE. One-way ANOVA and Duncan's multiple range method were used for multiple comparisons. Different letters in the same row indicated significant differences among the groups (p < 0.05).

[0033] Figure 5 The expression levels of ifn-γ, npy and y8b genes in different tissues of largemouth bass fry infected with Aeromonas hydrophila 24 hours after injection of MSNP were analyzed. The data were expressed as mean ± standard error. One-way ANOVA and Duncan's multiple range method were used for multiple comparisons. Different letters in the same row indicated significant differences among the groups (p < 0.05).

[0034] Figure 6 Changes in intestinal morphology of largemouth bass fry infected with Aeromonas hydrophila after injection with MSNPs.

[0035] Figure 7 Figure 2 Changes of apoptosis in the head kidney of largemouth bass fry infected with Aeromonas hydrophila after injection with MSNPs.

[0036] Figure 8Figure 2 Changes of apoptosis in the spleen of largemouth bass fry infected with Aeromonas hydrophila after injection with MSNPs.

[0037] Fig. 9 This is the change of gene expression levels in different tissues of largemouth bass fry infected with Aeromonas hydrophila and then immersed in different water bodies for treatment. Data are expressed as mean ± standard error. One-way analysis of variance and Duncan's multiple range method were used for multiple comparisons. Different letters in the same row indicate significant differences among the groups (p<0.05).

[0038] Fig.10 To investigate the changes of apoptosis in spleen of largemouth bass fry infected with Aeromonas hydrophila and then immersed in different water bodies.

[0039] Fig.11 To investigate the changes of apoptosis in the head kidney of largemouth bass fry infected with Aeromonas hydrophila and then immersed in different water bodies.

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

[0041] Fig.13 Changes of apoptosis in the head kidney of largemouth bass fry infected with different bacteria and injected with MSNP 24 hours later.

[0042] Fig.14 Changes of apoptosis in the spleen of largemouth bass fry infected with different bacteria and injected with MSNP 24 hours later. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0044] Example 1 Effects of injection dose and injection time of largemouth bass neuropeptide (MSNP) on largemouth bass fry

[0045] 1. Preparation of MSNP injection

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

[0047] Dissolve the MSNP freeze-dried powder in 1×PBS to obtain a MSNP mother solution with a concentration of 10 μg / μl, and store it at -20°C. When used, dilute the MSNP mother solution with PBS according to the injection dose to obtain the MSNP injection solution. The injection dose of MSNP is 1 ng / g BW, 10 ng / g BW or 100 ng / g BW, where ng refers to the mass of MSNP dry powder and g BW refers to the weight of the fry.

[0048] 2. Injection experiment

[0049] (1) Effects of injection dose on largemouth bass fry

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

[0051] The control group was intraperitoneally injected with 1×PBS, and the treatment groups 1 to 3 were intraperitoneally injected with MSNP injections at doses of 1 ng / g BW, 10 ng / g BW, and 100 ng / g BW, respectively. The corresponding injection volume was calculated according to the weight of each largemouth bass fry. At 12 h after injection, tissue samples (head kidney, spleen, brain, and intestine) and serum samples of largemouth bass fry in each group were collected for subsequent testing.

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

[0053] Largemouth black bass fry (30-60 days old, weighing 2-4 g) were randomly divided into 6 groups (12 in each group), namely control-12h group, treatment-12h group, control-24h group, treatment-24h group, control-72h group and treatment-72h group, and domesticated for one week.

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

[0055] The treated-12h group, treated-24h group, and treated-72h group were intraperitoneally injected with 10 ng / g BW of MSNP injection, and tissue samples (head kidney, spleen, brain, and intestine) and serum samples were collected at 12h, 24h, and 72h after injection for subsequent detection.

[0056] 3. qPCR detection

[0057] Total RNA was extracted from tissue samples of largemouth bass fry in each group, and cDNA was obtained by reverse transcription. Using cDNA as a template, the mRNA expression levels of neuropeptide receptor genes (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 reference, and the primers used are shown in Table 1. A negative control was set, and each sample was repeated twice.

[0058] Table 1 qPCR primers

[0059]

[0060] The qPCR reaction system was as follows: template 1 μL, ddH2O 8.2 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, Green Mix 10μL.

[0061] The qPCR reaction program was as follows: (1) 95°C, 5 min; (2) 95°C, 5 sec; 60°C, 30 sec; 72°C, 30 sec; 40 cycles; (3) 95°C, 15 sec; annealing at 65-95°C at an increasing rate of 0.5°C per sec to generate a melting curve.

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

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

[0064] 5. Test results

[0065] like Figure 1As shown in the figure, compared with the injection of 0 ng / g BW of MSNP (i.e. PBS), the expression of pro-inflammatory factors IFN-γ and IL-1β in the head kidney and spleen increased after the injection of 1 ng / g BW, 10 ng / g BW and 100 ng / g BW of MSNP, especially the most obvious increase after the injection of 10 ng / g BW of MSNP; in the spleen tissue, the expression level of the 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 the increase of MSNP injection dose, among which Y8B increased significantly after the injection of 1 ng / g BW; in addition, after the injection of 10 ng / g BW of MSNP, the expression of IFN-γ and IL-10 in the brain region increased significantly.

[0066] like Figure 2 As shown in A to B in Figure 3, intraperitoneal injection of 10 ng / g and 100 ng / g BW of MSNP significantly increased serum alkaline phosphatase activity in largemouth bass, and serum glucose levels were higher; Figure 2 As shown in Figures C to D, at the three time points after MSNP injection, the serum alkaline phosphatase levels of largemouth bass were higher than those of the control group (i.e., injected with PBS), and the serum glucose level increased 12h and 24h after MSNP injection, with a more obvious increase at 24h, but decreased after 72h.

[0067] The above results showed that the injection dose of MSNP was significantly dose-dependent with the expression level of each gene and serum enzyme activity, and 10 ng / g BW was the optimal injection dose of MSNP.

[0068] like Figure 3 As shown, 12h after MSNP injection, ifn-γ mRNA levels increased in the head kidney, spleen, and brain. 24h after MSNP injection, the expression of ifnγ, il-1β, and il-10 in the head kidney, spleen, and brain decreased significantly, and this phenomenon still existed 72h after MSNP injection. It is worth noting that compared with the control group, 24h after MSNP injection, the expression of the four genes in the intestine increased, among which the expression of ifn-γ was extremely significant. In serum testing, we found that at the three time points after MSNP injection, the serum alkaline phosphatase levels of largemouth bass were higher than those of the control group. Serum glucose levels increased 12h and 24h after MSNP injection, with a more obvious increase at 24h, but decreased after 72h.

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

[0070] Example 2 Effect of injection of largemouth bass neuropeptide (MSNP) on largemouth bass fry infected with Aeromonas hydrophila

[0071] 1. Preparation of MSNP injection

[0072] Same as Example 1.

[0073] 2. Infection Experiment

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

[0075] After the largemouth bass fry in each group were anesthetized with MS222, group 1 (i.e., PBS + A. hydrophila (12 h)) was intraperitoneally injected with 0 ng / g BW MSNP 12 h later, and then 100 μL Aeromonas hydrophila (1×10 6 CFU / mL); Group 2 (PBS+A.hydrophila (24h)) was injected intraperitoneally with 0 ng / g BW MSNP for 24 hours, and then injected with 100 μL Aeromonas hydrophila (1×10 6 CFU / mL); Group 3 (i.e., MSNP+A.hydrophila (12h)) was injected intraperitoneally with 10 ng / g BW MSNP for 12 hours, and then 100 μL Aeromonas hydrophila (1×10 6 CFU / mL); Group 4 (i.e., MSNP+A.hydrophila (24h)) was injected intraperitoneally with 10ng / gBW MSNP for 24 hours, and then 100μL Aeromonas hydrophila (1×10 6 CFU / mL).

[0076] After all the injections were completed, each group of Aeromonas hydrophila was returned to the water tank for continued feeding, and the water temperature was maintained at 30°C. After 24 hours of feeding, the tails were cut off to collect serum samples and tissue samples (whole brain, head kidney, spleen and intestine).

[0077] 3. qPCR detection

[0078] qPCR detection was performed according to the method of 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 assay

[0080] The tissue damage of largemouth bass fry in each group was detected by TUNEL fluorescence apoptosis detection kit (Novagen, A111).

[0081] 5. Test results

[0082] like Figure 4 and Figure 5 As shown in the figure, the increase of il-10 gene expression in immune tissues of the group without MSNP injection and infected with Aeromonas hydrophila for 12 hours can be reduced by injection of exogenous MSNP; similarly, this phenomenon also appeared in the gene expression of tnf-α and ifn-γ in head kidney tissue; the content of MSNP and y8b mRNA in spleen increased significantly after MSNP injection, while the expression of y8b in intestine decreased significantly; ifn-γ and il-1β in brain, intestine, head kidney and spleen of largemouth bass fry infected with Aeromonas hydrophila after MSNP injection were significantly lower than those without MSNP injection. This indicates that injection of MSNP can reduce the inflammatory response caused by Aeromonas hydrophila infection in largemouth bass fry and alleviate the immune stress response in vivo.

[0083] like Figure 6 As shown in the figure, after the largemouth bass fry that were not injected with MSNP were infected with Aeromonas hydrophila, severe tissue damage occurred in the intestine, which was particularly obvious in the intestine 24 hours after infection without MSNP injection. In the infection group after MSNP injection, intestinal damage was significantly inhibited, mainly manifested as vacuolization and reduced intestinal villus damage area. This shows that the injection of MSNP can reduce the intestinal damage caused by Aeromonas hydrophila infection in largemouth bass fry.

[0084] like Figure 7 and Figure 8 As shown in the figure, the number of apoptotic cells in the head kidney and spleen of largemouth bass fry that were not injected with MSNP was high after being infected with Aeromonas hydrophila for 24 hours; while the number of apoptotic cells in the head kidney and spleen of largemouth bass fry that were injected with MSNP was significantly reduced after being infected with Aeromonas hydrophila for 24 hours. This indicates that MSNP can reduce apoptosis caused 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 Chinese gallnut on treating Aeromonas hydrophila infection in largemouth bass fry

[0086] 1. Infection experiment

[0087] Largemouth black bass fry (30-60 days old, weighing 2-4 g) were randomly divided into 4 groups (8 fish in each group), respectively recorded as Group 1 to Group 4, and domesticated for one week.

[0088] After the largemouth bass fry in each group were anesthetized with MS222, group 1 (i.e., control) was intraperitoneally injected with 100 μL 1× PBS and then immersed in culture water for 24 h; group 2 (i.e., A. hydrophila) was intraperitoneally injected with 100 μL Aeromonas hydrophila (1×10 6 CFU / mL) and then immersed in culture water for 24 hours; Group 3 (i.e., A. hydrophila + MSNP) was injected intraperitoneally with 100 μL Aeromonas hydrophila (1×10 6 CFU / mL) and then immersed in aquaculture water containing 200 μg / L MSNP for 24 hours; Group 4 (i.e., A. hydrophila + Galla) was intraperitoneally injected with 100 μL Aeromonas hydrophila (1×10 6 CFU / mL) and then placed in culture water containing 200 μg / L gallnut for 24 hours.

[0089] After the soaking, the tails were cut off to collect serum samples and tissue samples (whole brain, head kidney, spleen and intestine).

[0090] 2. qPCR detection

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

[0092] 3. TUNEL cell apoptosis assay

[0093] The tissue damage of each group of largemouth sea bass fry was detected by the method of Example 2.

[0094] 4. Test results

[0095] like Fig. 9 As shown, in the head kidney tissue, the TNF-α mRNA level of largemouth bass infected with Aeromonas hydrophila and then immersed in MSNP for 24 hours was significantly different from that of the largemouth bass immersed in aquaculture water after infection with Aeromonas hydrophila, and the TNF-α after MSNP immersion could return to normal levels compared with the control group; compared with the control group, the gene expression trends of IL-10 in the head kidney and spleen were similar, indicating that the role of MSNP in the signaling pathway in immune tissues may be similar; in addition, the expression of IFN-γ in the spleen of largemouth bass infected with Aeromonas hydrophila was significantly upregulated after gallnut treatment, while the spleen IFN-γ level of the MSNP-treated group did not show such a drastic response; there was no difference in the genetic changes of Y8b in the spleen tissue and IL1β in the intestine.

[0096] like Fig.10 and Fig.11 As shown, both MSNP and Chinese gallnut could reduce apoptosis in the head kidney and spleen of largemouth bass induced by Aeromonas hydrophila infection.

[0097] The above results show that both MSNP and Chinese gall can reduce the immune tissue damage caused by Aeromonas hydrophila infection in largemouth bass, and MSNP has better therapeutic effect.

[0098] Example 4 Comparison of the effect of injecting MSNP on treating two different bacterial infections in largemouth bass fry

[0099] 1. Preparation of MSNP injection

[0100] Same as Example 1.

[0101] 2. Infection Experiment

[0102] Largemouth black bass fry (30-60 days old, weighing 2-4 g) were randomly divided into 4 groups (8 fish in each group), respectively recorded as Group 1 to Group 4, and domesticated for one week.

[0103] After the largemouth bass fry in each group were anesthetized with MS222, group 1 (i.e., control) was intraperitoneally injected with 100 μL 1×PBS solution 24 hours later, and then injected with 1×PBS solution; group 2 (i.e., PBS+MSNP) was intraperitoneally injected with 100 μL 1×PBS solution 24 hours later, and then injected with 10 ng / g BW MSNP; group 3 (i.e., A.hydrophila+MSNP) was intraperitoneally injected with 100 μL Aeromonas hydrophila (1×10 6 CFU / mL) for 24 hours, and then injected with 10 ng / g BW MSNP; Group 4 (i.e., S. agalactiae + MSNP) was intraperitoneally injected with 100 μL of Streptococcus agalactiae (1×10 6 CFU / mL) for 24 hours, and then 10 ng / g BW MSNPs were injected again.

[0104] After all the injections were completed, the largemouth black bass fry in each group were returned to the water tank for continued feeding. The water temperature was maintained at 30°C. After 24 hours of feeding, the tails were cut off to collect serum samples and tissue samples (whole brain, head kidney, spleen and intestine).

[0105] 3. qPCR detection

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

[0107] 4. TUNEL cell apoptosis assay

[0108] The tissue damage of each group of largemouth seabass fry was detected by the method of Example 2.

[0109] 5Test results

[0110] like Fig.12As shown in the figure, in the head kidney and intestinal tissues of largemouth bass injected with MSNP intraperitoneally after infection with Streptococcus agalactiae (i.e., Group 4), the gene expression of TNF-α was consistent with that of MSNP alone (i.e., Group 2), while in largemouth bass injected with MSNP intraperitoneally after infection with Aeromonas hydrophila (i.e., Group 3), the mRNA content of TNF-α was restored to the level of the control group (i.e., Group 1); and the IFN-γ mRNA level was significantly increased in the head kidney, spleen and intestinal tissues of largemouth bass fry injected with MSNP after infection with Streptococcus agalactiae (i.e., Group 4), which indicated that The results showed that Streptococcus agalactiae had a great immunostimulatory effect on largemouth black bass fry, while MSNP might not be able to inhibit this stimulatory effect; the IL-10 levels in the head kidney and spleen increased significantly after intraperitoneal injection of MSNP, and the IL-10 in the head kidney of largemouth black bass infected with Streptococcus agalactiae also showed this upward trend, but the largemouth black bass infected with Aeromonas hydrophila could return to normal levels after intraperitoneal injection of MSNP, which indicated that MSNP had a better immunotherapy effect on Aeromonas hydrophila; compared with the control group, there were no significant differences in Y8b and IL-1β levels.

[0111] like Fig.13 and Fig.14 As shown, the number and fluorescence intensity of apoptotic cells in the head kidney and spleen of largemouth bass injected intraperitoneally with MSNP after infection with Aeromonas hydrophila were significantly reduced compared with those infected with S. agalactiae.

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

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of a largemouth bass neuropeptide in the preparation of a product for preventing Aeromonas hydrophila infection, characterized in that: The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.

1.

2. The use of a largemouth bass neuropeptide in the preparation of a product for treating Aeromonas hydrophila infection, characterized in that: The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.

1.

3. Use of a largemouth bass neuropeptide in the preparation of a product that enhances the ability of largemouth bass to resist infection by Aeromonas hydrophila, characterized in that: The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.

1.

4. Use of a largemouth bass neuropeptide in the preparation of a product for enhancing the immune response of largemouth bass after being infected by Aeromonas hydrophila, characterized in that: The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.

1.

5. Use of a largemouth bass neuropeptide in preparing a method for alleviating tissue damage caused by largemouth bass infection with Aeromonas hydrophila, characterized in that: The amino acid sequence of the largemouth bass neuropeptide is shown in SEQ ID NO.

1.

6. The use according to any one of claims 3 to 5, characterized in that: The largemouth bass is a juvenile fish.

7. A preparation for preventing and treating Aeromonas hydrophila infection, characterized in that: The active ingredients include the largemouth bass neuropeptide with an amino acid sequence as shown in SEQ ID NO.1.

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