An immune enhancer for largemouth bass

Abstract

The present application provides an immune enhancer for largemouth bass, comprising astragalus polysaccharide and nucleotides with improved immune effect; further, the immune enhancer further comprises forsythia suspensa polysaccharide and / or largeleaf figleaf ginseng polysaccharide; preferably, the immune enhancer further comprises vitamin C; further, the immune enhancer further comprises protein peptide. The immune enhancer for largemouth bass provided by the present application has the effect of resisting largemouth bass rhabdovirus MSRV, can inhibit the replication of largemouth bass rhabdovirus MSRV; and the protein peptide used has antibacterial activity, thereby effectively controlling the reproduction of pathogenic bacteria of largemouth bass.

Description

Technical Field

[0001] This invention belongs to the field of fish immunology technology, specifically relating to an immunostimulant for largemouth bass. Background Technology

[0002] Largemouth bass is an important economically important freshwater aquaculture species in my country. However, with the gradual expansion of aquaculture scale, diseases are becoming increasingly frequent during the farming period. Largemouth bass diseases mainly include bacterial, parasitic, and viral diseases, with viruses being a key factor hindering the development of the largemouth bass industry. For example, largemouth bass rhabdovirus primarily affects largemouth bass fry, spreading rapidly, affecting a wide area, and causing high mortality. Currently, there is no specific drug developed for largemouth bass rhabdovirus, and research on antiviral drugs is relatively lacking. Therefore, screening or developing drugs against largemouth bass rhabdovirus is of great significance. Summary of the Invention

[0003] The purpose of this invention is to provide an immune enhancer for largemouth bass, thereby effectively increasing the antiviral and antibacterial effects of largemouth bass.

[0004] The immune enhancer provided by this invention comprises astragalus polysaccharide and nucleotides that enhance immune function;

[0005] Furthermore, the immune enhancer also contains Forsythia polysaccharide and / or Isatis indigotica polysaccharide;

[0006] Preferably, the immune enhancer also contains vitamin C;

[0007] As a specific example, the immune enhancer has the following mass percentage composition:

[0008] Nucleotides 10-20%, Astragalus polysaccharide 10-20%, Forsythia polysaccharide 30-40%, Isatis indigotica polysaccharide 5-20%, Vitamin C 5-20%;

[0009] The nucleotides mentioned can be free nucleotides commonly used in the field of fish immunity, such as oligodeoxyribonucleotides;

[0010] Furthermore, the immune enhancer also contains protein peptides.

[0011] The protein peptides mentioned are antibacterial peptides isolated from largemouth bass.

[0012] The protein peptide described herein has the following amino acid sequence:

[0013] DVTADQDGLKYGGWSSMGFHRAAEIDSNREENYRTISNEDQHAGRQECDVDPVVIAGIISSESRAHTLLKGALEEYNKGEKVVESYA (SEQ ID NO: 1).

[0014] The present invention also provides a feed for largemouth bass, wherein the above-mentioned immune enhancer is added.

[0015] The largemouth bass immune enhancer provided by this invention has an anti-largemouth bass rhabdovirus (MSRV) effect, which can inhibit the replication of MSRV; and the protein peptides used have antibacterial activity, thereby effectively controlling the reproduction of pathogenic bacteria in largemouth bass. Attached Figure Description

[0016] Figure 1 : Transcriptional expression diagram of N and M genes in head tissue, where a: N gene, b: M gene, group B: challenge group, group C: single agent + challenge group, group D: compound agent + challenge group.

[0017] Figure 2 : Transcriptional expression diagram of N and M genes in visceral tissues, where a: N gene, b: M gene, group B: challenge group, group C: single agent + challenge group, group D: compound agent + challenge group.

[0018] Figure 3 : Transcriptional expression diagram of N and M genes in muscle tissue, where a: N gene, b: M gene, group B: challenge group, group C: single agent + challenge group, group D: compound agent + challenge group.

[0019] Figure 4 : Transcriptional expression diagram of N and M genes in caudal fin tissue, where a: N gene, b: M gene, group B: challenge group, group C: single agent + challenge group, group D: compound agent + challenge group.

[0020] Figure 5 Figure 1: Results of inhibition zone assay. A: MS-AP protein at 100 μg / ml; B: MS-AP protein at 50 μg / ml; C: Negative control PBS solution. Detailed Implementation

[0021] The largemouth bass used in this embodiment of the invention were purchased from an aquatic product company in Huzhou, with a body length of 1.82±0.05cm and a weight of 0.12±0.01g. Healthy largemouth bass were selected for the experiment after being temporarily held for 7 days. Trizol reagent, reverse transcription kit, and SYBR Premix ExTaq were purchased from Takara; other reagents were from Sinopharm Reagent. The real-time PCR instrument (Mx3005P) was from Stratagene.

[0022] The immune enhancer provided by this invention uses astragalus polysaccharide and nucleotides that enhance immunity. The astragalus polysaccharide can be the astragalus polysaccharide commonly used in the aquaculture industry; while the nucleotides are free nucleotides commonly used in the aquaculture industry that enhance immunity.

[0023] Previous studies have shown that MSRV (monovirus rotavirus) is pathogenic to largemouth bass, with a high mortality rate. Infection can cause significant lesions in the brain, liver, intestines, and muscle tissues of largemouth bass. After MSRV infection, the expression levels of N and M genes are increased in the head, internal organs, muscles, and tail fin tissues.

[0024] 1. Feeding management and challenge experiments

[0025] Healthy largemouth bass juveniles were randomly divided into four groups of 750 fish each: Group A (blank control group), Group B (virus-challenged group), Group C (single-ingredient + virus-challenged group), and Group D (compound + virus-challenged group). Groups A and B were fed regular feed; Groups C and D were fed an immune enhancer mixed with their feed for 5 days at a dosage of 0.4% of their body weight, twice daily (morning and evening). The water temperature was maintained at 28℃ to ensure efficient feeding. The single-ingredient immune enhancer consisted only of nucleotides; the compound immune enhancer was a combination of nucleotides and polysaccharides, with one specific composition as follows: 15% free nucleotides, 20% Astragalus polysaccharide, 40% Forsythia polysaccharide, 20% Isatis indigotica polysaccharide, and 5% Vitamin C.

[0026] Feed was withheld for 24 hours before artificial infection. The prepared virus suspension was diluted 1:100, and largemouth bass were immersed in the diluted virus solution for 30 minutes before being transferred to 120L rearing tanks. The control group was immersed in PBS solution. During this period, the water temperature was maintained at 22–24℃, and appropriate feed (without immune enhancers) was provided. Head, viscera, muscle, and tail fin tissues were collected at different time points after challenge (0, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours). Tissue extraction was performed on ice, and the tissues were immediately placed in liquid nitrogen and then transferred to -80℃ for later use. Nucleic acid and protein extraction were performed later to study the differential expression of largemouth bass rhabdovirus (MSRV) in largemouth bass tissues and the proliferation effect of MSRV under the action of immune enhancers.

[0027] 2. Effects of immune enhancers on N and M gene expression

[0028] Tissue samples were collected at 0h, 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h, and 168h. RNA was extracted using the Trizol method, and its concentration was determined. cDNA was synthesized using random primers and M-MLV transcriptase. Based on the MSRV N and M gene sequences and the largemouth bass β-actin gene sequence, real-time quantitative PCR primers were designed (Table 1). Using largemouth bass β-actin as an internal reference gene, specific primers were used to perform real-time quantitative PCR analysis of the N and M genes.

[0029] Table 1: Primer list for real-time quantitative PCR amplification

[0030]

[0031] 3. Experimental Results

[0032] 3.1 Anti-MSRV effect of immune enhancers in head tissue

[0033] Immunostimulants were mixed into feed and fed to largemouth bass. The anti-MSRV effects of different immunostimulant formulations were analyzed and evaluated using real-time quantitative PCR. The transcriptional expression of MSRV's N and M genes in the head tissue is shown below. Figure 1 As shown. Figure 1 As shown, the transcriptional expression of the N gene in group B was significantly higher than that in groups C and D. From 6 hpi to 168 hpi, the relative expression levels of the N gene in groups C and D remained at a low level, except at 120 hpi. At 120 hpi, the relative expression level of the N gene in group D was lower than that in group B, but higher than that in group C.

[0034] For the M gene, except at 12 hpi, the relative expression level of the M gene in group B was higher than that in groups C and D at all other time points. At 12 hpi, the expression level of the M gene in group D was higher than that in groups B and C. At 36 hpi, the relative expression levels of the M gene, from highest to lowest, were group B, group C, and group D. At 72 hpi and 96 hpi, the M gene expression showed consistency, with group B having the highest M gene expression level, followed by group D, and group C having the lowest; this is similar to the expression of the N gene at 120 hpi.

[0035] 3.2 Anti-MSRV effect of immune enhancers in visceral tissues

[0036] like Figure 2 As shown, in visceral tissues, the transcriptional expression of N and M genes in group B was significantly higher than that in groups C and D. From 6 hpi to 168 hpi, the expression levels of N and M genes in group B were relatively high, while the relative expression levels of N and M genes in groups C and D remained at relatively low levels.

[0037] 3.3 Anti-MSRV effect of immune enhancers in muscle tissue

[0038] like Figure 3 As shown, at 6 hpi and 12 hpi, the transcriptional expression of the N gene in group B was lower than that in groups C and D, with the expression levels decreasing in the order of D, C, and B. At 24 hpi, the N gene expression level in group B increased, exceeding that in groups C and D. At 36 hpi, the N gene expression levels in groups B, C, and D all reached their peak, exceeding the N gene expression levels at other time points. At 48 hpi, the N gene expression level in group B was significantly higher than that in groups C and D; after 48 hpi, the N gene expression levels in all groups remained at a relatively low level relative to 36 hpi; however, for the M gene, the transcriptional expression in group B was much higher than that in groups C and D.

[0039] 3.4 Anti-MSRV effect of immune enhancers in caudal fin tissue

[0040] In the caudal fin tissue ( Figure 4 The transcriptional expression of N and M genes in group B was significantly higher than that in groups C and D. From 6 hpi to 168 hpi, the expression levels of N and M genes in group B were relatively high, while the relative expression levels of N and M genes in groups C and D remained at relatively low levels. This is similar to the transcriptional expression of the N gene in visceral tissues. Combined analysis of the results from various tissues revealed that after 12 hpi, the relative expression levels of N and M genes in group B were significantly higher than those in groups C and D, indicating that nucleotides and Astragalus polysaccharides possess antiviral effects.

[0041] 5. Adding protein peptides as feed additives to immune enhancers.

[0042] The immune enhancer also contains MS-AP, a protein peptide isolated from largemouth bass, which has antibacterial activity and an amino acid sequence of SEQ ID NO:1; it is added to the feed as a feed additive.

[0043] The determination steps and results of the minimum inhibitory concentration (MIC) of the protein peptide MS-AP are as follows:

[0044] The bacteria tested included Gram-negative Escherichia coli, Vibrio parahaemolyticus, Vibrio harbinii, and Aeromonas hydrophila; and Gram-positive Staphylococcus pasteurellii and Staphylococcus epidermidis.

[0045] 1) Preparation of bacterial culture: The experimental bacteria to be tested were inoculated into LB liquid medium and cultured at 25°C for 20 hours to bring the bacteria into the logarithmic growth phase (OD600 value of 1.2). The bacterial culture was then diluted with the corresponding sterile liquid medium and the optical density OD600 value was adjusted to 0.3.

[0046] 2) Gradient dilution method for determination

[0047] MS-AP protein was diluted using a serial dilution method. In a sterile 96-well plate, 20 μL of the diluted bacterial culture and 20 μL of the serially diluted MS-AP protein were mixed to achieve a final concentration range of 0.5 mg / mL. The strains were then incubated at their optimal temperature for 6–8 hours, and the absorbance was measured at 600 nm using a microplate reader.

[0048] Table 2: Minimum inhibitory concentration (MIC) of MS-AP protein against different bacterial strains (μg / ml)

[0049]

[0050] As shown in Table 2, the MS-AP protein has broad-spectrum bactericidal activity and good antibacterial effect against a variety of Gram-positive and Gram-negative bacteria.

[0051] 3) Oxford Cup Antibacterial Zone Experiment

[0052] Vibrio parahaemolyticus was added to LB liquid medium and incubated overnight at 25°C and 300 rpm for 10 h. The bacterial solution was then diluted to an OD600 of 0.2. 100 μL of the diluted bacterial solution was spread evenly on LB solid medium and placed in a sterile Oxford cup.

[0053] 100 μL of MS-AP protein (concentrations of 100 μg / ml and 50 μg / ml, respectively) was added to Oxford cups and incubated at 25℃ for 20 h. The size of the inhibition zone was observed and measured. The results showed that MS-AP protein had good antibacterial activity, and this activity was concentration-dependent. Figure 5 ).

[0054] The Vibrio parahaemolyticus solution immersion experiment showed that the incidence rate in the experimental group with MS-AP protein added (10 μg MS-AP protein / g feed) was much lower than that in the control group without MS-AP protein (3 / 20), indicating that MS-AP protein can effectively antagonize Vibrio parahaemolyticus. sequence list <110> Huzhou University <120> An immune enhancer for largemouth bass <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 87 <212> PRT <213> Artificial Sequence <400> 1 Asp Val Thr Ala Asp Gln Asp Gly Leu Lys Tyr Gly Gly Trp Ser Ser 1 5 10 15 Met Gly Phe His Arg Ala Ala Glu Ile Asp Ser Asn Arg Glu Glu Asn 20 25 30 Tyr Arg Thr Ile Ser Asn Glu Asp Gln His Ala Gly Arg Gln Glu Cys 35 40 45 Asp Val Asp Pro Val Val Ile Ala Gly Ile Ile Ser Ser Glu Ser Arg 50 55 60 Ala His Thr Leu Leu Lys Gly Ala Leu Glu Glu Tyr Asn Lys Gly Glu 65 70 75 80 Lys Val Val Glu Ser Tyr Ala 85

Claims

1. A protein peptide, characterized in that, The amino acid sequence of the protein peptide is SEQ ID NO:

1.

2. The use of the protein peptide according to claim 1 in the preparation of feed additives.

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