Application of overexpression of opPGRP-SC2 gene in prevention and treatment of bacterial infection in Oplegnathus punctatus

Abstract

This invention provides overexpression OpPGRP-SC2 The application of genes in the prevention and control of bacterial infections in spotted sea bream falls under the field of genetic engineering technology. OpPGRP-SC2 The gene open reading frame sequence is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2. This invention is the first to demonstrate... OpPGRP - SC2 This gene is a key to the innate immunity of spotted rockfish, providing a novel target for the prevention and control of bacterial diseases in fish. Overexpression of this gene can activate the NF-κB signaling pathway and upregulate the expression of various immune effector molecules, providing a new molecular target and technical solution for the prevention and control of diseases in spotted rockfish, and promoting the development of green aquaculture of spotted rockfish.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of overexpression of the OpPGRP-SC2 gene in the prevention and control of bacterial infection in spotted sea bream. Background Technology

[0002] Spotted rock seabream (Oplegnathus punctatus), an important marine economic fish species in my country, is widely distributed in coastal waters. Due to its delicious flesh and high nutritional value, it has become a key species for development in the marine aquaculture industry. However, during large-scale aquaculture, factors such as excessively high stocking density, deteriorating water quality, and environmental stress make spotted rock seabream susceptible to infection by various pathogenic bacteria in seawater, leading to outbreaks of disease, a significant decrease in survival rate, and reduced product quality. This severely restricts the healthy and sustainable development of the spotted rock seabream aquaculture industry, causing huge economic losses to farmers. Therefore, developing efficient and green technologies for the prevention and control of bacterial infections in spotted rock seabream has significant industrial value and application prospects.

[0003] Among the pathogenic bacteria infecting spotted sea bream, Edwardsiella tarda and Vibrio anguillarum are the two most serious Gram-negative bacteria. Both have a wide host range and high pathogenicity, making them major pathogens of bacterial diseases in marine aquaculture fish. Edwardsiella tarda was first isolated and identified by Hoshina in Japanese eels in 1959. This short rod-shaped bacterium proliferates rapidly in core visceral tissues such as the liver and spleen after infecting fish, forming characteristic white nodules, which then lead to tissue necrosis, organ failure, and ultimately death. Its hosts include many important marine and freshwater aquaculture fish such as eels, tilapia, and flounder, often causing large-scale outbreaks with persistently high mortality rates, resulting in severe losses to the aquaculture industry.

[0004] Vibrio anguillarum was first discovered in 1909 by Bergman in diseased eels in Japan. This bacterium is arc-shaped or comma-shaped, possesses single-terminal polar flagella, and is highly motile. It forms characteristic colonies on selective culture media, making it easily identifiable. It primarily infects marine fish and some crustaceans, including eels, flounders, sea bass, and spotted sea bream, among other commercially important aquaculture species. It can cause explosive hemorrhagic septicemia in fish, characterized by rapid onset, rapid spread, and extremely high mortality. It is one of the most serious pathogens in marine aquaculture, severely threatening the aquaculture safety of marine fish such as spotted sea bream.

[0005] Innate immunity is the first line of defense for fish against invading pathogens. Pattern recognition receptors (PRRs), as core molecules of the innate immune response, can specifically recognize pathogen-associated molecular patterns (PAMPs), initiating downstream immune signaling pathways and activating the body's immune defense response. Peptidoglycan recognition protein SC2 (PGRP-SC2) is a highly conserved pattern recognition receptor that can specifically recognize peptidoglycan (PGN), a key component of bacterial cell walls, thereby mediating the body's immune response to bacterial infection and playing an irreplaceable and crucial role in innate immune defense.

[0006] Currently, the PGRP-SC2 gene has been gradually identified in bony fishes, and multiple studies have confirmed its involvement in the immune process against bacterial infections. For example, the AcPGRP-SC2 gene identified in the double-banded clownfish (Amphiprion clarkii) encodes a product with broad bacterial binding capacity and agglutination activity, effectively binding and agglutinating various pathogenic bacteria. Simultaneously, it significantly upregulates the expression of immune-related genes such as NF-κB1, NF-κB2, and stat3 in Fat Head Minnow cells, indicating that it can regulate the body's immune response by activating NF-κB and related signaling pathways, thus playing an important role in combating bacterial infections.

[0007] However, to date, no research has been reported on the PGRP-SC2 gene (OpPGRP-SC2) in spotted sea bream. Its mechanism of action in the sea bream's resistance to pathogenic bacteria such as Edwardsiella tarda and Vibrio anguillarum remains unclear, and no applications of expressing this gene for the prevention and control of bacterial infections in spotted sea bream have been found. This leaves a gap in the development of green control technologies for bacterial infections in spotted sea bream. Therefore, in-depth research into the function of the OpPGRP-SC2 gene, clarifying its role in the resistance of spotted sea bream to bacterial infections, and developing prevention and control technologies based on this gene are of great significance for solving bacterial disease problems in spotted sea bream farming and promoting the high-quality development of the aquaculture industry. Summary of the Invention

[0008] In view of this, the present invention provides the application of overexpression of the OpPGRP-SC2 gene in the prevention and control of bacterial infection in spotted sea bream, in order to solve the above problems.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides the application of a functional product overexpressing the OpPGRP-SC2 gene in the preparation of a treatment for bacterial infection in spotted sea bream.

[0011] Preferably, the bacteria include Edwardsiella tarda and / or Vibrio anguillarum.

[0012] Preferably, the functional product has the function of overexpressing, transcribing, or expressing the OpPGRP-SC2 gene or its expression product.

[0013] Preferably, the application is achieved by upregulating the expression of the OpPGRP-SC2 gene or overexpressing the OpPGRP-SC2 protein in the spotted sea bream, thereby activating the NF-κB signaling pathway, upregulating the expression of immune effector molecules, and enhancing the antibacterial immune response of the spotted sea bream.

[0014] Preferably, the immune effector molecules include hepcidin2, morocidin, TNF-α, and β-defensin.

[0015] The present invention also provides a recombinant expression vector comprising the aforementioned opPGRP-SC2 gene of the spotted sea bream and a base vector, wherein the base vector is a pcDNA3.1 plasmid.

[0016] This invention also provides a method for enhancing the antibacterial immunity of spotted sea bream by introducing the recombinant expression vector into the spotted sea bream, overexpressing the spotted sea bream OpPGRP-SC2 gene, activating the NF-κB signaling pathway, and enhancing the spotted sea bream's defense against Edwardsiella tarda and Vibrio anguillarum.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: The open reading frame sequence of the OpPGRP-SC2 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2. This invention is the first to confirm that OpPGRP-SC2 is a key gene for the innate immunity of spotted sea bream, providing a novel target for the prevention and control of bacterial diseases in fish; overexpression of this gene can activate the NF-κB signaling pathway and upregulate the expression of various immune effector molecules, providing a novel molecular target and technical solution for the prevention and control of diseases in spotted sea bream, and promoting the development of green aquaculture of spotted sea bream. Attached Figure Description

[0018] Figure 1 Phylogenetic tree analysis of the OpPGRP-SC2 gene in spotted sea bream and its PGRP-SC2 gene in other species.

[0019] Figure 2 The relative expression level of OpPGRP-SC2 in healthy tissues of spotted sea bream; Note: *P<0.05, **P<0.01.

[0020] Figure 3 To slow down the expression level of OpPGRP-SC2 in the kidneys of spotted sea bream at different time points after Edwardsiella tarda infection; Note: *P<0.05.

[0021] Figure 4The expression level of OpPGRP-SC2 in the kidneys of spotted sea bream at different time points after Vibrio anguillarum infection; Note: *P<0.05.

[0022] Figure 5 Overexpression of OpPGRP-SC2 affected the expression levels of five disease resistance-related genes after immune stimulation; Note: *P<0.05, **P<0.01.

[0023] Figure 6 Overexpression of the OpPGRP-SC2 gene increases the expression level of NF-κB protein. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] The healthy spotted sea bream used in this experiment were all purchased from Shandong Laizhou Mingbo Aquatic Products Co., Ltd., and weighed about 120 g. They were temporarily kept in a water tank for 5 days before the experiment to ensure sufficient dissolved oxygen.

[0026] The OPK cell line used in the immune stimulation experiment was obtained from the Aquatic Biotechnology and Genomics Laboratory of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0027] Example 1. Sequence analysis and phylogenetic tree of OpPGRP-SC2 of the spotted sea bream.

[0028] The peptidoglycan recognition protein gene PGRP-SC2 used in this study is assigned the mRNA sequence number Female_Chr7_Gene00725. The sequence information was obtained from the CNGBdb reference genome of Opregnatus (Project ID: CNP0001488), and its chromosomal location in the female reference genome is Chr7:22113496~22114206 (negative strand). The cDNA of the Opregnatus OpPGRP-SC2 gene was analyzed, and the amino acid sequence was predicted using DNAman software. The molecular weight and theoretical isoelectric point of the OpPGRP-SC2 gene were predicted using SWISS-PROT (https: / / www.expasy.org / ). The results showed that the open reading frame of the OpPGRP-SC2 cDNA is 330 bp (SEQ ID NO.1), encoding 109 amino acids (SEQ ID NO.2), with a predicted protein molecular weight of 11.5 kDa and a theoretical isoelectric point of 5.42.

[0029] CDS sequence of the OpPGRP-SC2 gene:

[0030] ATGACCAAAAAAGGCTTTGATGACATTGGATATAATTTCCTAGTCGGAGGAGATGGTACCGTGTACGAGGGCCGTGGCTGGGGTGTGGTAGGAGCACATGCCAAGGGCAACAATCATGATTCAGTGGGGGTTGCCTTCATGGGCAATTTCAACGATGACACACCAAG CGCAGAGGCAATATCATCAGTCAAACAGCTGCTGCAGGCTGGAGTCTCTCAGGGCTTTTTAGAGCAACCGTTCGCCCTGTTTTGGGCACAGAGATGTGGGATCCACCGAATGTCCAGGAAAAAATCTTTATGCTGCGCTGCCACAACTGAGGGGCACCACATAA (SEQ ID NO.1).

[0031] The amino acid sequence encoded by the OpPGRP-SC2 gene:

[0032] MTKKGFDDIGYNFLVGGDGTVYEGRGWGVVGAHAKGNNHDSVGVAFMGNFNDDTPSAEAISSVKQLLQAGVSQGFLEQPFALFGHRDVGSTECPGKNLYAALPQLRGTT (SEQ ID NO. 2).

[0033] SMART software prediction showed that the gene has a PGRP domain (amino acids 1-89).

[0034] Homologous conserved functional domains were predicted using the SMART online website (https: / / smart.embl.de / smart / set_mode.cgi?NORMAL=1). PGRP-SC2 amino acid sequences from different animals were downloaded from NCBI and homology alignment was performed using BLAST. Multiple alignments of PGRP-SC2 amino acid sequences between species were performed using MEGA6. BLAST alignment revealed that the amino acid sequence encoded by OpPGRP-SC2 showed high similarity to the amino acid sequences encoded by PGRP-SC2 in other bony fishes, with similarities of 94.50%, 84.40%, and 84.26% with *Oplegnathus fasciatus*, *Pampus punctatissimus*, and *Sinipercachuatsi*, respectively. The similarity to *Danio rerio* was lower, at 43.14%.

[0035] To investigate the role of OpPGRP-SC2 in animal evolution, a phylogenetic tree of PGRP from mammals, reptiles, and bony fishes was constructed using MEGA 6 software via the Neighbor-joining method. The phylogenetic analysis showed that OpPGRP-SC2 clustered with bony fishes, with the closest relationship to the striped rockfish. Meanwhile, human and mouse PGRP clustered into four other clades, all of which were more distantly related to OpPGRP-SC2.

[0036] Example 2. Expression of OpPGRP-SC2 in various tissues of spotted sea bream

[0037] To analyze the expression distribution of the OpPGRP-SC2 gene in various tissues of spotted rock seabream, four healthy individuals were randomly selected. After anesthesia with an MS-222 syringe, tissue samples were collected from muscle, liver, skin, intestine, gills, spleen, brain, kidneys, and blood, totaling nine types of tissues. All collected tissues were immediately flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for storage.

[0038] Using β-actin as an internal reference gene, the expression level of OpPGRP-SC2 in the aforementioned tissues was detected by qRT-PCR. The experiment included a control group and an experimental group, each containing four biological replicates (N=4). The specific steps are as follows:

[0039] 1. Total RNA extraction from spotted sea bream

[0040] Total RNA was extracted from the tissue of *Sinocyclocheilus maculatus* using the Trizol method. The specific procedure was as follows: Approximately 30 mg of tissue sample was placed in a 1.5 mL RNase-free centrifuge tube containing 4 sterile steel beads and 500 μL of Trizol. The sample was homogenized at 60 Hz / min for 2 min. Then, 500 μL of pre-chilled Trizol was added, and the mixture was incubated on ice for 10 min, with manual shaking every 3 min. Subsequently, the sample was centrifuged at 12000 rpm for 5 min at 4 °C. The supernatant was transferred to a new tube, and 200 μL of pre-chilled chloroform was added. The mixture was vortexed for 15 s and incubated on ice for 10 min. The sample was then centrifuged again at 12000 rpm for 15 min at 4 °C. The upper aqueous phase was carefully aspirated to a new tube, and 500 μL of pre-chilled isopropanol was added. The mixture was gently mixed and incubated at -20 °C for 1 h to precipitate. The RNA precipitate was collected by centrifugation at 12,000 rpm for 10 min at 4 °C. The supernatant was discarded, and the precipitate was gently washed three times with 1 mL of pre-cooled 75% ethanol (prepared with enzyme-free water). Finally, the precipitate was centrifuged for 5 min under the same conditions to remove as much residual ethanol as possible. The precipitate was then allowed to evaporate in a clean bench for 5 min with the lid open. 20 μL of RNase-free water was added to dissolve the precipitate. RNA concentration and purity were determined using a DNA / Protein Analyzer P100, and integrity was assessed by 1% agarose gel electrophoresis. RNA samples with acceptable purity (A260 / A280 ratio between 1.8 and 2.0) and clear electrophoretic bands were selected and stored at -80 °C for later use.

[0041] 2. cDNA Synthesis

[0042] (1) Removal of genomic DNA

[0043] Prepare the following system in a 200 μL enzyme-free PCR tube: 1 μL gDNA Eraser, 1 μL 5× gDNA Eraser Buffer, 1 ng RNA, and RNase-free ddH2O to a final volume of 10 μL. Incubate at 42 °C for 2 min to remove genomic DNA.

[0044] (2) Reverse transcription reaction

[0045] Using extracted sea bream RNA as a template, the mRNA was reverse transcribed into cDNA using a first-strand synthesis kit and a reverse transcription kit. The reaction system is shown in Table 1.

[0046] Table 1 RT-PCR reaction system

[0047] reagents Dosage (μL) Spotted sea bream RNA template solution 10 <![CDATA[RNase Free ddH2O]]> 4 5× PrimeScript Buffer 2 4 RT Primer Mix 1 PrimeScript RT Enzyme Mix I 1

[0048] RT-PCR reaction procedure: 37 ℃ for 15 min, 85 ℃ for 5 s.

[0049] Using the reverse transcription product as a template, the reliability of the cDNA was verified by PCR reaction using the β-actin F / R internal reference primer from the spotted sea bream. The PCR reaction system is shown in Table 2.

[0050] β-actin F: 5'-GCTGTGCTGTCCCTGTA-3' (SEQ ID NO.3);

[0051] β-actin R: 5'-GAGTAGCCACGCTCTGTC-3' (SEQ ID NO. 4).

[0052] Table 2 PCR reaction system

[0053] reagents Dosage (μL) cDNA of spotted rock seabream 1 2× Taq Plus PCR Master Mix 10 <![CDATA[RNase Free ddH2O]]> 7 β-actin F 1 β-actin R 1

[0054] PCR program: 95 ℃ for 5 min, (95 ℃ for 30 s, 59 ℃ for 30 s, 72 ℃ for 30 s) × 34 cycles, 72 ℃ for 10 min.

[0055] The PCR products were detected by 1% agarose gel electrophoresis. The cDNA with a single and bright band was selected and stored at -20 ℃ for subsequent real-time quantitative PCR experiments.

[0056] 3. Real-time quantitative PCR

[0057] Using OpPGRP-SC2-F / R as primers, the cDNA of *Striker spp.* obtained by reverse transcription was used as a template, and β-actin was used as an internal reference gene. SYBR Green was employed. ® The expression level of the OpPGRP-SC2 gene was determined using the Green Pro Taq HS premixed qPCR kit (AG, China) strictly following the instructions on a 7500FAST real-time PCR instrument (ABI, USA). The real-time PCR reaction system is shown in Table 3.

[0058] Upstream primer OpPGRP-SC2-F: 5'-GTTGCCTTCATGGGCAATTT-3' (SEQ ID NO.5);

[0059] Downstream primer OpPGRP-SC2-R: 5'-GGCGAACGGTTGCTCTAA-3' (SEQ ID NO.6).

[0060] Table 3. Real-time PCR reaction system

[0061] reagents Dosage (μL) 2× SYBR Green Pro Taq HS Premix 10 cDNA 1 PGRP-SC2-RT-F 0.8 PGRP-SC2-RT-R 0.8 ROX Reference Dye 0.4 <![CDATA[RNase Free ddH2O]]> 7

[0062] The quantitative PCR reaction program was: 95 ℃ for 30 s, followed by (95 ℃ for 5 s, 60 ℃ for 34 s) × 40 cycles. Two [units / items] were used. –ΔΔCt The relative expression levels of the gene were calculated. Excel was used to statistically analyze the relative expression levels of the PGRP-SC2 gene in various tissues of the spotted sea bream. Graphpad Prism software was used to perform significance tests on gene expression levels and to visualize the results of quantitative real-time PCR. The results are as follows: Figure 2 As shown.

[0063] The results showed that OpPGRP-SC2 was expressed in all nine tissues of the spotted sea bream, with relatively high expression levels in blood and kidneys, and slightly lower expression levels in muscle, liver, skin, intestines, gills, spleen, and brain.

[0064] Example 3. Expression analysis of OpPGRP-SC2 in kidney immune tissues after Edwardsiella tarda infection.

[0065] Fifty healthy, well-grown spotted sea bream were randomly divided into two groups. Each fish in the experimental group received an intraperitoneal injection of 100 μL of Edwardsiella tarda (1×10⁻⁶). 8 (CFU / mL), while the control group received an equal volume of 1×PBS intraperitoneally. Five spotted sea bream were randomly selected at five time points (0h, 3h, 6h, 12h, and 24h) after injection. After anesthesia, they were dissected, and kidney immunological tissue was collected. The tissue was quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0066] The relative expression level of OpPGRP-SC2 in the kidneys at different time points after intraperitoneal injection of Edwardsiella tarda was detected using qRT-PCR. Four replicates (N=4) were performed in the PBS group, and five replicates (N=5) were performed in the Edwardsiella tarda group. The detection method was the same as in Example 2. The results are as follows: Figure 3 As shown.

[0067] The results showed that OpPGRP-SC2 responded rapidly to Edwardsiella tarda, and the expression levels of OpPGRP-SC2 in kidney tissue were significantly increased at 12 h and 24 h, respectively, which were 2.6 times and 1.8 times that of the control group.

[0068] Example 4. Expression analysis of OpPGRP-SC2 in kidney tissues after Vibrio anguillarum infection

[0069] Fifty healthy spotted sea bream in good condition were selected and randomly divided into two groups. Each fish in the experimental group was injected intraperitoneally with 100 μL of Vibrio anguillarum (1×10⁻⁶). 7(CFU / mL), the control group was injected with an equal volume of 1×PBS, and then 5 spotted sea bream were randomly selected at 0 h, 3 h, 6 h, 12 h and 24 h after injection, anesthetized and dissected, and their kidney tissue was taken. The tissue was quickly immersed in liquid nitrogen for flash freezing and then stored in a -80℃ freezer for later use.

[0070] Using β-actin as an internal control, the expression level of OpPGRP-SC2 in the kidneys of the experimental and control groups of spotted sea bream was detected by qRT-PCR. Four replicates (N=4) were performed in the PBS group, and five replicates (N=5) were performed in the Vibrio anguillarum group. The detection method was the same as in Example 2. The results are as follows: Figure 4 As shown.

[0071] The results showed that after stimulation by Vibrio anguillarum, the expression level of OpPGRP-SC2 in the kidneys showed an up-regulating trend, and the expression level was significantly increased at 6 h, 12 h and 24 h, which were 5.1 times, 8.6 times and 7.3 times that of the control group, respectively.

[0072] Example 5. Effects of OpPGRP-SC2 overexpression on immune regulation after immune stimulation

[0073] To further investigate the role of OpPGRP-SC2 in the antibacterial infection of spotted sea bream, this experiment designed an OpPGRP-SC2 overexpression and in vitro immune stimulation experiment using spotted sea bream kidney cell lines.

[0074] 1. Construction of pcDNA3.1(+)-OpPGRP-SC2 overexpression vector

[0075] (1) Primers were designed to obtain the cDNA sequence of the spotted sea bream. Then, using the cDNA sequence of the spotted sea bream as a template, the CDS sequence of OpPGRP-SC2 was amplified using PGRP-SC2F / PGRP-SC2R primers. Then, using the CDS sequence of the OpPGRP-SC2 gene as a template, specific primers PGRP-SC2-EcoRI-F / PGRP-SC2-HindIII-R were designed and synthesized. EcoRI and HindIII restriction endonuclease recognition sequences were introduced at the 5' end of the primers, respectively.

[0076] PGRP-SC2F: 5'-ATGACCAAAAAAGGCTTTG-3' (SEQ ID NO. 7);

[0077] PGRP-SC2R: 5'-TTATGTGGTGCCCCTCAGTT-3' (SEQ ID NO. 8);

[0078] PGRP-SC2-EcoRI-F:

[0079] 5'-tatcggatccgaattcATGACCAAAAAAGGCTTTGATGACAT-3' (SEQ ID NO. 9);

[0080] PGRP-SC2-HindIII-R: 5'-gtgcggccgcaagcttTGTGGTGCCCCTCAGTTGT-3' (SEQ ID NO. 10);

[0081] (2) Obtaining the overexpression vector

[0082] The OpPGRP-SC2 gene fragment was obtained by polymerase chain reaction (PCR). The PCR product and the empty pcDNA3.1 vector digested with EcoRI and HindIII were ligated using the ClonExpressUltraOneStepCloningKit V3 (Vazyme) homologous recombination method to obtain the ligation product.

[0083] The homologous recombination system was as follows: 5 μL of 2 × CE Mix V3; 2.5 μL of empty pcDNA3.1 vector digested with EcoRI and HindIII; and 2.5 μL of the OpPGRP-SC2 gene fragment (containing homologous arms). All components were mixed and incubated at 50°C for 20 min to obtain the ligation product.

[0084] (3) Extract large quantities of recombinant overexpression vectors

[0085] The ligation product was transformed into Escherichia coli DH5α competent cells. After screening and amplification, the plasmid was extracted and verified by enzyme digestion and amplification with primers CMV-F and BGH-R for pcDNA3.1(+) insertion detection, followed by sequencing. The recombinant overexpression vector with the correct sequence was obtained and named pcDNA3.1(+)-OpPGRP-SC2.

[0086] CMV-F: 5'-CTAGAGAACCCACTGCTTAC-3' (SEQ ID NO. 11);

[0087] BGH-R: 5'-TAGAAGGCACAGTCGAGG-3' (SEQ ID NO. 12);

[0088] 2. In vitro immune simulation stimulation experiment

[0089] This experiment constructed a blank control group, an LPS-stimulated control group, and an OpPGRP-SC2 overexpression + LPS stimulation group, set up biological replicates and a unified transfection system, and used qRT-PCR to detect changes in the expression of OpPGRP-SC2 and related immune molecules after LPS induction stimulation, to explore the role of OpPGRP-SC2 in LPS-mediated immune response.

[0090] The experiment consisted of three groups: a control group, a control stimulation group (Control+LPS), and a control overexpression stimulation group (OpPGRPSC2-OE+LPS), each with 5 wells. The transfection system and treatment conditions for each well are shown in Table 5. Each group received 3 μL Lipo8000 and 125 μL Opti-MEM Medium. The OpPGRPSC2-OE+LPS group received an additional 2.5 μg pcDNA3.1(+)-OpPGRP-SC2 plasmid, while the Control+LPS and Control groups received no recombinant plasmid. 24 h after transfection, the OpPGRPSC2-OE+LPS and Control+LPS groups were simultaneously stimulated with 100 μg / mL LPS. The Control group received an equal volume of PBS as a blank control. Cells from each group were collected 12 h after stimulation. Total RNA was extracted and reverse transcribed to prepare qRT-PCR templates. Real-time quantitative PCR was used to detect the expression levels of OpPGRP-SC2 and other immune-related molecules after immune stimulation. The real-time quantitative PCR primers are shown in Table 4. The detection results are shown in [Table 4]. Figure 5 .

[0091] Table 4 Primer sequences used in PGRP-SC2 overexpression and LPS stimulation experiments

[0092] Sequence / Primer Sequence (5′-3′) Serial Number OpPGRP-SC2-F GTTGCCTTCATGGGCAATTT SEQ ID NO.5 OpPGRP-SC2-R GGCGAACGGTTGCTCTAA SEQ ID NO.6 Optnf-αF GCCCTTCTAGTTATGGCTCTTT SEQ ID NO.13 Optnf-αR GCTTCTGTTTGGCCTGATTG SEQ ID NO.14 Opβ-defensinF GGAGGGCAATGATCCAGAAA SEQ ID NO.15 Opβ-defensinR CCGTGATGACCAACGATGTA SEQ ID NO.16 Ophepcidin1F ACTCGTGCTCGCCTTTATTT SEQ ID NO.17 Ophepcidin1R CTGGAGTGTCATTGCTCTCTG SEQ ID NO.18 Ophepcidin2F CCGTCGTGCTCACCTTTATT SEQ ID NO.19 Ophepcidin2R TCATTGCTCATTGGCTCCTC SEQ ID NO.20 OpmoronecidinF TTCACCATGTCTCCTCCTATCT SEQ ID NO.21 OpmoronecidinR CTTCACTGCCTCTCTCTTTCTG SEQ ID NO.22

[0093] Table 5. Components added to each well in different groups of PGRP-SC2 overexpression and LPS stimulation experiments

[0094] Group Lipo8000 / μL plasmid pcDNA3.1(+)- OpPGRP-SC2 / μg Opti-MEM Medium / μL Is LPS processing used? OpPGRP-SC2-OE+LPS 3 2.5 125 yes Control+LPS 3 \ 125 yes Control 3 \ 125 no

[0095] The results showed that in the Control+LPS group, the expression level of OpPGRP-SC2 was only slightly higher than that in the Control group, indicating that LPS stimulation itself slightly induced the expression of this gene. In the OpPGRP-SC2-OE+LPS group, the expression level of OpPGRP-SC2 was significantly higher than that in the first two groups. Figure 5 ).

[0096] Analysis of the remaining five immune genes revealed that, compared to the Control group, LPS treatment alone significantly upregulated the expression of hepcidin2, moroonecidin, TNF-α, and β-defensin genes, with β-defensin showing the largest upregulation, reaching 4.15 times the expression level of the Control group; while hepcidin1 showed downregulation. In the OpPGRP-SC2-OE+LPS group, the expression trends of each immune gene were consistent with those of the Control+LPS group, but the overall upregulation was significantly increased. Figure 5 ).

[0097] The above results preliminarily suggest that OpPGRP-SC2 may be involved in regulating the body's immune response to LPS stimulation. First, its expression is upregulated by LPS, and overexpression can further amplify this effect. Second, overexpression of OpPGRP-SC2 can significantly enhance the ability of LPS to induce the expression of most immune effector molecules (such as hepcidin2, morosiderin, tnf-α, and β-defensin), suggesting that it may act as an enhancing element in the innate immune signaling pathway, synergistically amplifying the host's antibacterial immune response with LPS stimulation.

[0098] Example 6. Activation of the NF-κB signaling pathway by OpPGRP-SC2 transcription in the kidney cells of spotted sea bream.

[0099] To investigate the immunoregulatory effect of the OpPGRP-SC2 gene on NF-κB, a dual-luciferase reporter assay was designed. The experiment used the constructed pcDNA3.1(+)-OpPGRP-SC2 overexpression plasmid and reporter plasmid pNF-κB-Luc, with pRL-TK as the internal control plasmid. OPK cells cultured to passage 20 were seeded into 48-well plates. When cell confluence reached 80%, plasmid co-transfection was performed according to the experimental groups. Forty-eight hours after transfection, the fluorescence activities of firefly luciferase and Renilla luciferase were detected using a dual-luciferase reporter gene assay kit from Shanghai Sangon Biotech Co., Ltd., and measured by microplate reader. Relative luciferase activities were calculated as the ratio of firefly luciferase fluorescence value to Renilla luciferase fluorescence value, and standardized analysis was performed.

[0100] Figure 6The results showed that, in the wild-type NF-κB reporter system, compared with the empty vector control group (pcDNA3.1 (+)-NC + pNF-κB-Luc), the relative luciferase activity of the OpPGRP-SC2 overexpression group (pcDNA3.1 (+)-OpPGRP-SC2 + pNF-κB-Luc) was significantly upregulated, approximately 2.1 times that of the control group; while there was no significant difference in relative luciferase activity between the mutant reporter plasmid groups (pcDNA3.1 (+)-OpPGRP-SC2 + pNF-κBm and pcDNA3.1 (+)-NC + pNF-κBm).

[0101] In summary, the experimental results confirm that OpPGRP-SC2 can significantly activate the NF-κB signaling pathway. It is speculated that by activating the NF-κB pathway, it can upregulate the expression of downstream immune effector molecules such as hepcidin2 and tnf-α, thereby positively regulating the body's antibacterial immune response.

[0102] As can be seen from the above embodiments, the present invention provides the application of overexpression of OpPGRP-SC2 gene in the prevention and control of bacterial infection in spotted sea bream, providing a novel target for the prevention and control of bacterial diseases in fish.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression OpPGRP-SC2 Use of a functional product of the gene in the preparation of a medicament for the prevention and treatment of bacterial infection in Oplegnathus punctatus.

2. Use according to claim 1, characterized in that, The bacteria include Edwardsiella tarda and / or Vibrio anguillarum.

3. The application according to claim 1 or 2, characterized in that, The functional product has overexpression OpPGRP- SC2 the expression of the gene, the transcription or the function of its expression product.

4. The application according to claim 3, characterized in that, The application increases the Op of the spotted sea bream. PGRP - SC2 Gene expression or overexpression of OpPGRP-SC2 protein is used to activate the NF-κB signaling pathway, upregulate the expression of immune effector molecules, and enhance the antibacterial immune response of spotted sea bream.

5. The application according to claim 4, characterized in that, The immune effector molecules include hepcidin2 , moronecidin , TNF - α , β - defensin .

6. A recombinant expression vector, characterized in that, Contains the spotted sea bream as described in claim 1 OpPGRP - SC2 Gene and basic vector, wherein the basic vector is pcDNA3.1 plasmid.

7. A method for enhancing the antibacterial immunity of spotted sea bream, characterized in that, By introducing the recombinant expression vector of claim 6 into the spotted sea bream, the OpPGRP-SC2 gene of claim 1 was overexpressed, activating the NF-κB signaling pathway and enhancing the spotted sea bream's defense against Edwardsiella tarda and Vibrio anguillarum.

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