Micropterus salmoides rhabdovirus MSRV-M recombinant protein, polyclonal antibody, preparation method and application

Abstract

The invention discloses micropterus salmoides rhabdovirus MSRV-M recombinant protein, a polyclonal antibody, a preparation method and application, belongs to the technical field of biology, and particularly relates to the technical field of biology. The amino acid sequence of the MSRV-M recombinant protein is as shown in SEQ ID No: 1. According to the invention, a strain of MSRV (MSRV-JN01) is separated and identified from juvenile largemouth micropterus salmoides, and the homology of G and N protein gene sequences of the MSRV and other MSRVs is 98% or above; rT-PCR and RT-qPCR methods for detecting the MSRV are established, the specificity is high, the sensitivity is high, and the method can be repeated; the MSRV-M protein is expressed, the polyclonal antibody is prepared, the highest titer is 1: 256000, and the antibody can also be used for detecting the MSRV-M protein and viruses. The research supplements research data for the MSRV family and provides a new thought for immune prevention and treatment of the MSRV disease.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant protein of largemouth bass rhabdovirus MSRV-M, a polyclonal antibody, its preparation method, and its application. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Largemouth bass is currently one of the main freshwater aquaculture species in my country. With the expansion of aquaculture scale, invasive diseases caused by bacteria, viruses, and parasites frequently occur. Among these, diseases caused by largemouth bass rhabdovirus (LHV-11) are particularly prevalent. Micropterus salmoides Diseases caused by rhabdovirus (MSRV) infection frequently break out in the fry stage of largemouth bass. It is highly contagious and has a high mortality rate, making it difficult to cultivate high-quality fry.

[0003] Rhabdoviruses possess simple negative-sense single-stranded RNA, and their genomes are typically (but not always) single RNA molecules with partially complementary ends. Almost all rhabdovirus genomes include five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). However, many rhabdovirus genomes encode other proteins in alternative open reading frames (ORFs) within other genes or structural protein genes. Furthermore, all rhabdoviruses possess non-coding 3' leader sequences and 5' tail sequences.

[0004] The rhabdoviral particle consists of two structural units: an internal helical ribonucleoprotein complex (RNP) and a lipid envelope derived from the host cell membrane during budding. The RNP is composed of a closely associated RNA genome and nucleoprotein (N). A highly phosphorylated phosphoprotein (P) and RNA-dependent RNA polymerase (L) also bind to the RNP. The exact location of the matrix protein (M) remains controversial; it may be contained within the central channel of the RNP or embedded in the inner layer of the viral particle membrane. A spike of a segmental glycoprotein (G) used for viral particle binding to host cell receptors protrudes through the viral particle membrane.

[0005] Currently, basic research on various diseases of largemouth bass is insufficient, prevention and control measures are lacking, and there are few effective drugs for treating fish diseases, which seriously hinders the development of largemouth bass aquaculture. Therefore, establishing effective disease detection technology is particularly important. With convenient and rapid detection technology, disease conditions can be monitored and problems addressed in a timely manner during the aquaculture process, enabling the industry to adapt to market changes and challenges and promote the sustainable and healthy development of largemouth bass aquaculture.

[0006] Currently, there are no industry or national standards for the diagnosis of largemouth bass rhabdovirus disease (MSRV), so there is an urgent need to establish an effective and convenient MSRV detection method. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to propose a recombinant protein of largemouth bass rhabdovirus MSRV-M, a polyclonal antibody, and its preparation method and application.

[0008] The present invention solves the above-mentioned technical problems through the following technical means:

[0009] The first aspect of the present invention provides a recombinant protein of largemouth bass rhabdovirus MSRV-M, the amino acid sequence of which is shown in SEQ ID No: 1.

[0010] Preferably, the MSRV-M recombinant protein has also undergone purification or purification-renaturation treatment.

[0011] The purification method includes the following steps: first, washing the MSRV-M recombinant protein with lysis buffer, then eluting with imidazole washing buffer, and centrifuging to obtain the purified MSRV-M recombinant protein. The concentration of the imidazole washing buffer is 10-60 mM, and the volume is 50-150 mL.

[0012] The purification and refolding method includes the following steps: dialysis of the purified MSRV-M recombinant protein in a urea solution. The concentration of the urea solution is 1-4 mM.

[0013] The concentration of purified MSRV-M recombinant protein was 0.386 µg / µL, and the concentration of purified and refolded MSRV-M recombinant protein was 3.026 µg / µL.

[0014] A second aspect of the present invention provides a gene encoding the above-mentioned MSRV-M recombinant protein, the sequence of which is shown in SEQ ID No: 2 or SEQ ID No: 3.

[0015] A third aspect of the present invention provides a recombinant expression vector containing a nucleotide sequence as shown in SEQ ID No: 3.

[0016] Preferably, the recombinant expression vector is pET32a-MSRV-M, which is an expression vector constructed based on pET-32a.

[0017] A fourth aspect of the present invention provides a genetically engineered strain for preparing the above-mentioned MSRV-M recombinant protein, wherein the recombinant expression vector pET32a-MSRV-M is transformed into... E. coli Recombinant strains obtained from DH5α competent cells.

[0018] The fifth aspect of the present invention provides a polyclonal antibody against largemouth bass rhabdovirus, derived from serum produced after immunizing rabbits with the aforementioned MSRV-M recombinant protein.

[0019] Preferably, the MSRV-M recombinant protein is any one or more of the following: purified MSRV-M recombinant protein and purified and refolded MSRV-M recombinant protein.

[0020] Preferably, when the purified MSRV-M recombinant protein is used as the antigen, the titer of the prepared polyclonal antibody is 1:128,000.

[0021] Preferably, when the purified and refolded MSRV-M recombinant protein is used as the antigen, the titer of the prepared polyclonal antibody is 1:256,000.

[0022] A sixth aspect of the present invention provides a method for preparing the above-mentioned polyclonal antibody, comprising the following steps: Using MSRV-M recombinant protein as an antigen, New Zealand white rabbits were immunized three times, and serum was collected to prepare polyclonal antibodies.

[0023] Preferably, the initial immunization dose is 250-350µg, and the second and third immunization doses are 150-250µg.

[0024] The seventh aspect of the present invention provides a specific primer set for detecting largemouth bass rhabdovirus, comprising MSRV-03-F / R or MSRV-11-F / R; the sequence of MSRV-03-F / R is shown in SEQ ID No: 8~9; the sequence of MSRV-11-F / R is shown in SEQ ID No: 10~11.

[0025] An eighth aspect of the present invention discloses the application of the aforementioned MSRV-M recombinant protein, recombinant expression vector, genetically engineered strain, and polyclonal antibody in the preparation of a product for detecting largemouth bass rhabdovirus. The product includes a detection kit.

[0026] A ninth aspect of the present invention provides a kit for detecting largemouth bass rhabdovirus, comprising the aforementioned polyclonal antibody or specific primer set.

[0027] The beneficial effects of this invention are as follows: 1. This invention isolated and identified an MSRV strain (MSRV-JN01) from juvenile largemouth bass. The G and N protein gene sequences of this strain showed over 98% homology with other MSRV strains. RT-PCR and RT-qPCR methods for detecting MSRV were established, exhibiting high specificity, sensitivity, and reproducibility. MSRV-M protein was expressed, and a polyclonal antibody was prepared with a maximum titer of 1:256,000. This antibody can also be used for the detection of MSRV-M protein and the virus itself. This study supplements research data on the MSRV family and provides new insights into the immunomodulatory prevention and control of MSRV disease.

[0028] 2. This invention establishes RT-PCR and RT-qPCR detection methods for MSRV, which are highly specific, reproducible, and have sensitivities reaching 10-1. 2 copies / µL and 10 1 The copies / µL data provide data support for the clinical diagnostic criteria of MSRV disease.

[0029] 3. This invention establishes a complete detection method by isolating and identifying MSRV and preparing polyclonal antibodies through RT-PCR, RT-qPCR, and other methods to monitor largemouth bass rhabdovirus disease. It is also expected to provide new ideas and theoretical basis for the prevention and control of MSRV.

[0030] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0031] Figure 1 The images shown are of the examination results of diseased fish in Embodiment 1 of the present invention, where a and b are images of the surface symptoms of diseased largemouth bass; and c is an image of the observation results of the gills of the diseased fish. Figure 2 The images shown are pathological sections of brain tissue from largemouth bass in Embodiment 1 of the present invention, where a and c are pathological sections of normal fish brain tissue; and b and d are pathological sections of brain tissue from diseased fish (where a and b are magnified 40 times, and c and d are magnified 200 times). Figure 3 The images shown are pathological sections of the head kidney tissue of a largemouth bass in Embodiment 1 of the present invention, where a and c are pathological sections of normal fish head kidney tissue; and b and d are pathological sections of diseased fish head kidney tissue (where a and b are magnified 40 times, and c and d are magnified 200 times). Figure 4 The images shown are pathological sections of liver tissue from largemouth bass in Embodiment 1 of the present invention, where a and c are pathological sections of normal fish liver tissue; and b and d are pathological sections of liver tissue from diseased fish (where a and b are magnified 40 times, and c and d are magnified 200 times). Figure 5The results are RT-PCR detection results of samples with different primers in Example 1 of this invention. (M: 2000 marker; 1-4: primer SCRV-F / R; 5-8: primer SHRV-F / R; 9-12: primer MSRV-1-F / R; 13-16: primer MSRV-2-F / R; 17-20: primer MSRV-F / R) Figure 6 Agarose gel images of MSRV-G and MSRV-N gene amplification in Example 1 of this invention (M: 2000 marker; 1, 2: primers MNF / R; 3, 4: primers MGF / R; 5: negative control). Figure 7 This is a phylogenetic tree diagram of the MSRV-JN01 G nucleotide sequence in Example 1 of the present invention; Figure 8 This is a phylogenetic tree diagram of the MSRV-JN01 N nucleotide sequence in Example 1 of the present invention; Figure 9 The images show the pathological phenomena of FHM cells after challenge with MSRV in Example 1 of this invention: a: normal cells; b: cells infected with MSRV for 12 h; c: cells infected with MSRV for 24 h; d: cells infected with MSRV for 36 h. Figure 10 This is the RT-qPCR amplification curve of the MSRV-JN01 nucleic acid sample in Example 2 of the present invention; Figure 11 This is a graph showing the statistical analysis results of RT-qPCR for the MSRV-JN01 nucleic acid sample in Example 2 of this invention; Figure 12 This is a diagram of the RT-qPCR specificity test in Example 2 of the present invention, where ①: cDNA RT-qPCR amplification curve of MSRV; Figure 13 This is a graph showing the RT-PCR amplification results using MSRV-03-F / R primers in Example 2 of this invention. M: 2000 marker; Lane 1: 1×10 5 copies / μL; Lane 2: 1×10 4 copies / μL; Lane 3: 1×10 3 copies / μL; Lane 4: 1×10 2 copies / μL; Lane 5: 1×10 1 copies / μL; Figure 14 This is a graph showing the RT-qPCR amplification results using MSRV-03-F / R primers in Example 2 of this invention; ①: 1×10 5copies / μL; ②: 1×10 4 copies / μL; ③: 1×10 3 copies / μL; ④: 1×10 2 copies / μL; ⑤: 1×10 1 copies / μL; Figure 15 The image shows the results of the RT-qPCR repeatability test in Example 2 of this invention; ①: 1×10 5 copies / μL; ②: 1×10 3 copies / μL; Figure 16 This is a graph showing the specific band intensity results of largemouth bass rhabdovirus against different primers in Example 2 of the present invention, where (a): M: 2000 marker; 1: primer is MSRV-2-F / R; 2: primer is MSRV-01-F / R; 3: primer is MSRV-02-F / R; 4: primer is MSRV-03-F / R; 5: primer is MSRV-04-F / R; where (b): M: 2000 marker; 1: Primer is MSRV-02-F / R; 2: Primer is MSRV-NF / R; 3: Primer is MSRV-GF / R; 4: Primer is MSRV-05-F / R; 5: Primer is MSRV-06-F / R; 6: Primer is MSRV-07-F / R; 7: Primer is MSRV-08-F / R; 8: Primer is MSRV-09-F / R; 9: Primer is MSRV-10-F / R; 10: Primer is MSRV-11-F / R; 11: Primer is MSRV-12-F / R; Figure 17 This image shows the amplification results of different viral nucleic acids using primers MSRV-11-F / R in Example 2 of this invention. M: 2000 marker; 1: MSRV viral nucleic acid; 2: LMBV viral nucleic acid; 3: OSHV viral nucleic acid; 4: SVCV viral nucleic acid; 5: IHNV viral nucleic acid; 6: ISKNV viral nucleic acid; 7: NNV viral nucleic acid; 8: CEV viral nucleic acid; 9: KHV viral nucleic acid; 10: GCRV viral nucleic acid; 11: negative control. Figure 18 This is a graph showing the results of an RT-PCR amplification sensitivity test using MSRV-11-F / R primers in Example 2 of this invention. M: 2000 marker; Lane 1: 1×10 6 copies / μL; Lane 2: 1×10 5 copies / μL; Lane 3: 1×10 4 copies / μL; Lane 4: 1×10 3copies / μL; Lane 5: 1×10 2 copies / μL; Lane 6: 1×10 1 copies / μL; Figure 19 This is a diagram showing the codon optimization results of the MSRV-M gene in Example 3 of the present invention; Figure 20 Figure 3 shows the results of the MSRV-M protein purification condition exploration in Example 3 of this invention. (a) compares the results of washing solutions containing different concentrations of imidazole; M: 180kDa marker; 1: washing solution containing 10mM imidazole; 2: washing solution containing 20mM imidazole; 3: washing solution containing 30mM imidazole; 4: washing solution containing 40mM imidazole; 5: washing solution containing 50mM imidazole; 6: washing solution containing 60mM imidazole; (b) compares the results of different washing solution volumes; M: 180kDa marker; 1: washing solution volume is 50mL; 2: washing solution volume is 75mL; 3: washing solution volume is 100mL; 4: washing solution volume is 50mL; 5: washing solution volume is 50mL. Figure 21 The figures shown are for the purification and refolding verification of MSRV-M protein in Example 3 of this invention, where (a): protein purification result; M: 180kDA marker; 1-5: verification of protein purification after different large-scale expression; where (b): protein refolding result; M: 180kDA marker; 1: verification of purified protein after combination; 2: verification of protein after refolding. Figure 22 The following is a chromatogram of the Western blot analysis results of the polyclonal antibody in Example 3 of the present invention: M: 180kDA marker; (a): polyclonal antibody prepared from purified protein; (b): polyclonal antibody prepared from refolded protein; Figure 23 This is a diagram showing the results of the specificity verification of the polyclonal antibody IFA in Example 3 of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0033] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0034] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0035] Sources of diseased fish, viruses, and cell lines: The diseased fish originated from a fish farm in Jining, Shandong.

[0036] The fathead bream muscle cells (FHM) were kindly donated by Researcher Wang Qing of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0037] Largemouth bass iridovirus (LMBV), oyster herpesvirus (OSHV), and koi herpesvirus (KHV) are preserved in our laboratory. Infectious hematopoietic necrosis virus (IHNV), infectious spleen and kidney necrosis virus (ISKNV), carp spring viremia virus (SVCV), carp edema virus (CEV), grass carp reovirus (GCRV), and nerve necrosis virus (NNV) were kindly donated by Researcher Pan Xiaoyi of the Zhejiang Freshwater Fisheries Research Institute.

[0038] Reagents and kits used in the examples: Table 1: Reagents Used

[0039] Reagent kits used: RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit and FastKing cDNA First-Strand Synthesis Kit were purchased from Tiangen Biotech Co., Ltd.; FastPure® GeI DNA Extraction Mini Kit and FastPure® Plasmid Mini Kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; HIS Tag Protein Purification Kit was purchased from MDBio, Inc.; BCA Protein Quantification Kit was purchased from Kangwei Century Co., Ltd.; Ultrasensitive ECL Chemiluminescence Kit was purchased from Beyotime Biotechnology Co., Ltd.; One-Step PAGE Gel Rapid Preparation Kit was purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; and Total Protein Extraction Kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0040] Example 1: Identification and Separation of MSRV Largemouth bass fry at a fish farm in Jining, Shandong Province, developed a disease. The main symptoms included slow swimming, hovering at the surface, loss of balance, and spinning in a spiral. Dead, diseased fish were collected and stored at -80°C. Liver, spleen, and kidney tissues were collected aseptically from diseased fish. Part of the tissues were used for RNA extraction, and the rest were fixed overnight in 4% paraformaldehyde and sent to Wuhan Saiwei Biotechnology Co., Ltd. for pathological testing.

[0041] (1) Observation results of the symptoms of the diseased fish (largemouth bass): A simple surface observation revealed that most of the diseased fish swam slowly, lacked vitality, floated on the surface, stopped feeding, and exhibited body imbalance, bending, and spiraling movements. There were no other obvious abnormal symptoms on their surface (such as...). Figure 1 (As shown in a and b). Dissection revealed severe enlargement and congestion of the liver, spleen, and kidneys of the diseased fish, with a few petechiae on the gills (e.g., ...). Figure 1 As shown in (c), no abnormalities were observed in other tissues and organs. No parasites were found in the diseased fish, no dominant pathogens were isolated, and IHN and SVC were not detected according to current national standards.

[0042] (2) Pathological and histological changes in infected largemouth bass After collecting fresh brain, head, kidney, and liver tissues from diseased fish and preparing histopathological sections, observations revealed: Compared with the control group (e.g.) Figure 2 Compared to (a) and (c), the brain tissue of the diseased fish had a richer number of neurons, with neatly arranged and regularly shaped nerve cells, and no obvious pathological changes (such as...). Figure 2 (As shown in b and d).

[0043] Compared with the control group (e.g.) Figure 3 Compared to (as shown in a and c), the kidney capsule of the diseased fish showed severe hemorrhage (green arrow), unclear tissue structure, extensive cell necrosis, fragmented or dissolved nuclei, increased eosinophilicity of the cytoplasm (black arrow), significantly reduced number of lymphocytes, and less dense arrangement than normal cells. Blood sinuses showed congestion (orange arrow). Figure 3 (As shown in b and d).

[0044] Compared with the control group (e.g.) Figure 4 Compared to (as shown in a and c), the hepatocytes of the diseased fish were arranged in double rows as plates with large, round nuclei. The hepatic plates were distributed in a radiating pattern, with multiple sinusoidal congestion and dilation (orange arrows) centered on the central vein. No obvious inflammatory changes were observed (e.g., Figure 4 (As shown in b and d).

[0045] Pathological sections showed that the brain tissue of the diseased fish did not show obvious pathological changes, while the head kidneys and liver cells showed lesions such as cell necrosis and hemorrhage.

[0046] (3) Pathogen detection results PCR amplification results Liver, spleen, and kidney of dead diseased fish were aseptically collected and homogenized. RNA was extracted from the homogenate, and the resulting cDNA was used as a template. Several specific detection primers were used to amplify the cDNA, and the results showed (e.g.) Figure 5 As shown in the figure, only one specific primer for MSRV amplified the corresponding specific band at 353 bp, while neither the detection primers for SCRV nor SHRV amplified the specific band.

[0047] The product showing a specific band was sequenced and identified by NCBI-Blast as largemouth bass rhabdovirus (MSRV).

[0048] (4) Results of phylogenetic tree construction Using the cDNA obtained in (3) as a template, the MSRV-G and MSRV-N genes were amplified by electrophoresis using specific primers MGF / R and MNF / R, yielding specific bands at approximately 770bp and 1290bp, consistent with the expected results (e.g., Figure 6 As shown in the figure, products exhibiting specific bands will be sequenced and identified, and then compared with NCBI-Blast.

[0049] Comparison revealed that the G nucleotide sequence of this virus showed 98.05%, 98.70%, 98.96%, and 98.70% homology with the G nucleotides of MSRV-YH01, MSRV-HZ01, MSRV-2021, and MSRV-J985, respectively; while the N nucleotide sequence of this virus showed 97.83%, 99.22%, 99.07%, and 99.07% homology with the N nucleotides of the four largemouth bass rhabdoviruses, MSRV-YH01, MSRV-HZ01, MSRV-2021, and MSRV-J985, respectively. A phylogenetic tree was constructed (e.g., Figure 7 , 8 As shown in the figure, the pathogen was found to be a new MSRV strain, which was named MSRV-JN01 based on its geographical origin.

[0050] (5) Results of cell challenge test The viral suspension that was identified as positive was inoculated into a monolayer of fathead mullet muscle cells (FHM) and observed for 3 days, paying attention to any pathological changes that occurred.

[0051] The results showed that, compared with normal cells (such as...), Figure 9 Compared to (as shown in Figure a), cells infected with MSRV 12 h later (as shown in Figure a) showed a significant improvement. Figure 9 As shown in Figure b), a small number of cells begin to shrink, become smaller, and clump together. Cells infected with MSRV 24 hours later (as shown in Figure b) Figure 9As shown in c), cells detached, resulting in numerous cavities (red arrows), and the cells shrank together, with a small number of dead cells floating in the supernatant. Cells were infected with MSRV 36 h later (as shown in c). Figure 9 As shown in d), the cell pathology is severe, with a large number of cells dying and detaching, and a large number of dead cells floating in the supernatant.

[0052] Uninfected cells have normal morphology and adhere well to the culture vessel.

[0053] The CPE (cytopathic effect) observed in FHM cell lines infected with this virus exhibits a typical "broken fishnet-like structure." Electron microscopy sections of infected FHM cells revealed numerous bullet-shaped viral particles within the cytoplasm. Therefore, the virus was further identified as largemouth bass rhabdovirus (MSRV).

[0054] Example 2: Establishment of RT-qPCR and RT-PCR detection methods I. Establishment of RT-qPCR (1) Establishment of the standard curve The concentration of MSRV-JN01 viral DNA in the sample was measured to be 544.338 ng / L, which translates to a copy number of 1.07 × 10⁻⁶. 8 The samples were then serially diluted. After dilution, RT-qPCR amplification was performed, with three replicates of the same dilution. This yielded the real-time quantitative PCR amplification curves for the tested samples (e.g., [image of RT-qPCR amplification curves]). Figure 10 (As shown).

[0055] Statistical analysis of the test results was performed using R software (e.g., Figure 11 As shown), the curve equation is Y = -3.4831X + 30.2627, and the correlation coefficient R0 is... 2 =0.9953, this equation laid the foundation for subsequent virus quantification.

[0056] (2) Specificity test results Nucleic acids of LMBV, OSHV, SVCV, IHNV, ISKNV, NNV, CEV, KHV, and GCRV were used as templates, cDNA of positive MSRV was used as a positive control, and RNase-free ddH2O was used as a negative control for RT-qPCR amplification.

[0057] The results obtained are as follows Figure 12 As shown, it was found that, except for the positive control which had a typical amplification curve and high fluorescence signal value, the other control group samples did not have specific amplification signals.

[0058] (3) Sensitivity test results RT-qPCR amplification of the same MSRV-JN01 viral nucleic acid plasmid (e.g.) Figure 13(as shown) and RT-PCR amplification (as shown) Figure 14 After (as shown), it was found that for the same primer MSRV-03-F / R (see Table 2), the lowest detectable value for RT-PCR was 10. 3 copies / µL, while RT-qPCR can detect as low as 10 copies / µL. 1 The copies / µL indicates that the sensitivity of the RT-qPCR method is much higher than that of the conventional PCR method.

[0059] (4) Results of repeatability tests After performing quantitative real-time amplification (qLA) on positive viral nucleic acids of different copy numbers, the amplification curves largely overlapped near the threshold line, with essentially the same fluorescence signal intensity (e.g., ...). Figure 15 (As shown).

[0060] This demonstrates that the established RT-qPCR detection method is highly reproducible and readily available.

[0061] II. Establishment of RT-PCR RT-PCR establishment results (1) Primer screening results: Redesigned and synthesized primers were used to observe the intensity of specific bands amplified by different primers (e.g., Figure 16 As shown in the figure, after comparison and analysis, the best primer was MSRV-11-F / R, which has high specificity and can be used as a specific primer for subsequent RT-PCR detection methods.

[0062] Table 2 Primer Screening Collection

[0063] (2) Specificity test results: Using the nucleic acids of several common aquatic viruses, including LMBV, OSHV, SVCV, IHNV, ISKNV, NNV, CEV, KHV, and GCRV, as templates, the reverse-transcribed MSRV cDNA served as a positive control, and RNase-free ddH2O served as a negative control. The selected specific primers MSRV-11-F / R were used to amplify the RT-PCR reaction according to the established system.

[0064] Observation of amplification results revealed (e.g.) Figure 17 As shown in the figure): Except for the positive control, no specific bands were observed in other viral nucleic acids, proving that the primer has high specificity and can be used for subsequent experiments.

[0065] (3) Sensitivity test results: The copy number of positive viral nucleic acid was calculated, and after serial dilution and RT-PCR amplification, the intensity of its specific bands was observed (e.g., Figure 18(As shown). Comparison and analysis revealed that the established RT-PCR method can detect as low as 1×10⁻⁶. 2 The number of copies / µL indicates that this method is highly sensitive, laying a good foundation for subsequent virus detection.

[0066] Clinical sample validation results Twenty clinical samples from a farm in Shandong were tested. RT-PCR detected 17 positive samples (85%), while RT-qPCR detected 19 positive samples (95%).

[0067] This demonstrates that the two detection methods established in this study have good repeatability and high detection performance.

[0068] The experimental results showed that RT-qPCR and RT-PCR detection methods have high specificity, exhibiting specific fluorescent amplification signals and bands only for MSRV, while showing no amplification signals or bands for LMBV, OSHV, SVCV, IHNV, ISKNV, NNV, CEV, KHV, and GCRV; they also have high sensitivity, with RT-PCR capable of detecting as low as 10. 2 copies / µL, while RT-qPCR can detect as low as 10 copies / µL. 1 The results showed high reproducibility, with the fluorescence signal intensity and specific bands remaining essentially the same in multiple repeated experiments. Furthermore, clinical sample validation revealed a positive rate of 85% for RT-PCR and 95% for RT-qPCR, demonstrating the high performance of the two nucleic acid detection methods established in this study and their potential application in clinical diagnosis.

[0069] Example 3: Prokaryotic expression, purification, and refolding of MSRV-M protein (1) Bioinformatics analysis of M protein Design specific primers, M627-F: 5′-ATGCCTCTGTTTAAGAAGGGCAACA-3′ (SEQ ID No: 12), M627-R: 5′-TTAATGCCAGCTATGACCAGGATCT-3′ (SEQ ID No: 13), primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0070] Using the specific primers M627-F and M627-R, the MSRV-JN01 virus strain isolated in Example 1 was amplified and sequenced using the M gene (denoted as MSRV-M gene, sequence as shown in SEQ ID No: 2). Bioinformatics analysis was performed on the protein sequence encoded by the M gene (denoted as MSRV-M protein, sequence as shown in SEQ ID No: 1) to explore its biological characteristics and provide a reference for subsequent protein function studies.

[0071] Bioinformatics analysis results show that the MSRV-M protein has a high hydrophilicity index, no signal peptide, and no transmembrane domain, and is highly stable and can be induced to express.

[0072] (2) Optimization of MSRV-M gene and expression of protein 1. Optimization of the MSRV-M gene Due to limitations of the vector and the presence of rare codons, our initial attempts to induce MSRV-M protein expression failed. We used the Codon Optimization Tool to obtain codon optimization results (e.g., ...). Figure 19 As shown in the figure, after codon optimization, the gene CAI value was increased from 0.41 to 0.76 and the GC content was increased from 48.80% to 51.28%, resulting in the optimized MSRV-M gene (denoted as MSRV-M youhua gene, sequence shown in SEQ ID No: 3), and the induction expression of the M protein was successfully completed.

[0073] To compare the changes in the optimized gene sequence with the original sequence, DNAMAN software was used to compare the sequences before and after optimization. It was found that the sequence after codon optimization matched the original sequence by 78.27%.

[0074] The sequences before and after optimization were translated into amino acids and then compared using DNAMAN software. The results showed that there was no difference in the amino acid sequences corresponding to the M gene before and after optimization, with a consistency of 100%.

[0075] 2. Expression of MSRV-M recombinant protein First, construct the recombinant expression vector pET32a-MSRV-M Primers Myouhua-F / R were redesigned and synthesized targeting the MSRV-Myouhua gene (see Table 4). The target fragment MSRV-M youhua gene was ligated with the enzyme-digested vector fragment pET32a, and the following reaction system was prepared in a sterile microcentrifuge tube (Table 3): Table 3 Connection Reaction System

[0076] Mix the above reaction system thoroughly and react at 16°C overnight.

[0077] Then, construct the recombinant expression strain: The recombinant expression vector was transformed into DH5α competent cells. After transformation, the bacterial culture was plated on LB agar medium containing Amp and incubated overnight at 37°C. The next day, single colonies were selected and verified by PCR using Myouhua-F / R primers and T7-F / R primers. The primer sequences are shown in Table 4. Then, agarose gel electrophoresis was performed for identification, and samples that were confirmed to be correct were sequenced.

[0078] Table 4 Primer sequences

[0079] Culture a large number of positive colonies that have been correctly sequenced.

[0080] Finally, the induced expression of the MSRV-M recombinant protein: 1) Incubate the positive colonies with correct sequencing results overnight.

[0081] 2) The following day, the bacterial culture was inoculated into LB liquid medium containing Amp and cultured until OD600 = 0.5. Then, 1 mmol / L IPTG was added, and the culture was incubated at 28°C with shaking at 220 rpm for 12 h (a control group was set up without IPTG). After the culture was completed, a portion of the whole bacterial solution was taken for subsequent validation.

[0082] 3) Add the bacterial culture to a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of sterile PBS, mix well by pipetting, centrifuge, discard the supernatant, and repeat twice. Then add an appropriate amount of RIPA solution and protease inhibitor.

[0083] 4) The bacterial culture was then sonicated on ice at 200 W for 3 seconds, followed by a 2-second pause, until the culture was clear. It was then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant and precipitate were stored separately at -80°C. Success was confirmed by SDS-PAGE gel electrophoresis and Western blotting.

[0084] 3. Purification of MSRV-M recombinant protein The following are the specific steps for purifying proteins using a bioengineered protein purification kit: 1) Add the bacterial culture that induced the expression of recombinant MSRV-M protein to a 50 mL centrifuge tube, centrifuge, and discard the supernatant. Wash twice with PBS. Add an appropriate amount of lysis buffer, and then sonicate on ice at 300 W for 3 s, pause for 2 s, until the bacterial culture is clear. Centrifuge again, and filter the supernatant through a 0.45 μm microporous membrane.

[0085] 2) Pack 1 mL of a well-mixed HisPur Ni-IDA Agarose Gel into a column and equilibrate it with the lysis buffer from step 1). Then pour the bacterial lysis buffer prepared in step 1) into the column, incubate overnight at 4°C, collect the flow-through, and repeat the loading process 3-5 times to ensure adequate binding of the His tag protein.

[0086] 3) Based on previous verification, MSRV-M is known to be an inclusion body protein, so denaturing lysis buffer is used. Under these conditions, the column needs to be washed 5 times with 0.5-1 mL of lysis buffer first. Then, the column is washed with washing buffer (to investigate the effects of different concentrations of imidazole washing buffer (10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM) and different amounts of washing buffer (50 mL, 75 mL, 100 mL, 125 mL, 150 mL) to select the optimal concentration and amount).

[0087] 4) Elute 6-10 times with the corresponding 0.5 mL elution buffer to elute the target protein from the packing material. Transfer the target protein in the supernatant to a centrifuge tube and centrifuge at 5000 rpm for 2 min to obtain the purified MSRV-M recombinant protein.

[0088] Results: This study explored the purification effects of washing buffers containing different concentrations of imidazole and varying washing buffer volumes. The results were compared with those obtained using SDS-PAGE electrophoresis (e.g.,...). Figure 20 As shown in the figure, the optimal purification washing solution was determined to be 60 mM imidazole concentration, with a volume of 100 mL.

[0089] Using the above conditions as a guide, the protein was purified. After purification and verification by SDS-PAGE (…), Figure 21 (as shown in a).

[0090] 4. Refolding of MSRV-M recombinant protein Prepare urea solutions of different concentrations (6 mM, 4 mM, 2 mM, 1 mM) in advance, and add the corresponding amounts of GSH, GSSH, and L-arginine. Add 10 mL of the purified MSRV-M recombinant protein to the prepared dialysis bag. Place the dialysis bag containing the protein into the highest concentration of urea solution for renaturation at 4°C for 10 h, stirring gently with a magnetic stirrer. Change the urea solution every 10 h, successively at concentrations of 4 mM, 2 mM, and 1 mM. After renaturation, recover the protein and perform SDS-PAGE verification.

[0091] Results: The proteins that were successfully purified multiple times were combined, and the corresponding proteins were refolded in urea solutions of different concentrations (e.g., ...). Figure 21 (As shown in b).

[0092] 5. Determination of the concentration of purified and refolded proteins Follow the instructions for the Kangwei Century BCA Protein Quantitative Reagent Kit: Prepare the BCA working solution and mix thoroughly. Dilute the BSA standard: Serially dilute the BSA standard to 2 µg / µL, 1 µg / µL, 0.5 µg / µL, 0.25 µg / µL, 0.125 µg / µL, and 0.0625 µg / µL. Add 25 µL each of the diluted BSA standard and the purified and renatured protein to a 96-well plate. Add 200 µL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at 565 nm using a microplate reader. Plot a standard curve and calculate the protein concentration.

[0093] Protein concentration measurements showed that the purified protein concentration was 0.386 µg / µL, and the refolded protein concentration was 3.026 µg / µL. The concentration of the refolded protein was significantly higher than that of the purified protein alone.

[0094] Example 4: Preparation and Detection of Polyclonal Antibodies I. Preparation of Polyclonal Antibodies To investigate the effect of purified and refolded protein versus protein that has only undergone purification on the preparation of polyclonal antibodies, two control groups were established. Purified MSRV-M recombinant protein and purified and refolded MSRV-M recombinant protein were used as antigens, respectively, and injected into rabbits. The specific steps are as follows: New Zealand white rabbits were immunized with MSRV-M protein as an antigen. The initial immunization dose was 300 µg, and the doses for the second and third immunizations were 200 µg (if the antigen concentration was high, it was diluted to 0.5 mL with sterile physiological saline).

[0095] Before the first immunization, the antigen protein and Freund's complete adjuvant were added to a 50 mL centrifuge tube and thoroughly mixed using an emulsifier. After complete mixing, the mixture was injected subcutaneously at multiple sites on the back of the rabbit's neck. For the second immunization, Freund's incomplete adjuvant was used instead of the complete adjuvant, and the dosage was halved. The rabbits were inoculated again at the same sites. For the third immunization, the antigen protein was dissolved in physiological saline, and the dosage was the same as the second immunization. The rabbits were inoculated at the same sites. Seven days after the third immunization, blood was collected (negative serum was collected before the first immunization as a control).

[0096] II. Titer of Polyclonal Antibodies (1) Pretreatment: The purified and renatured MSRV-M protein was used as the antigen (two sets of variables were set, corresponding to the two sets of polyclonal antibodies prepared by immunization respectively), and it was diluted to 10 µg / mL with coating solution; in addition, the serum to be tested was serially diluted at the following ratios: 1:2,000, 1:4,000, 1:8,000, 1:16,000, 1:32,000, 1:64,000, 1:128,000, 1:256,000, 1:512,000, and 1:1,024,000.

[0097] (2) Antigen coating: Coat 96-well plates with diluted MSRV-M protein antigen. Perform 3 replicates for each gradient according to the serum antibody dilution, 100 µL / well, and incubate overnight at 4°C (at least 16 hours). On the second day, remove the liquid from the wells, wash 3 times with washing buffer, and pat dry. (3) Blocking solution: Add 200 µL to each well and react at 37℃ for 2 h. Wash 3 times and remove water.

[0098] (4) Primary antibody incubation: Add 100 μL of serially diluted primary antibody (using negative serum as a control) to each well. After adding all the primary antibody, incubate at 37°C for 2 h, gently agitate and wash 3 times, then blot dry with absorbent paper. Secondary antibody incubation: Add 100 µL of HRP-labeled goat anti-rabbit IgG antibody (pre-diluted with 5% skim milk powder) to each well. Incubate at 37°C for 1 h, gently agitate and wash 3 times, then blot dry with absorbent paper.

[0099] (5) Add 100 µL of soluble single-component TMB substrate solution to each well and place it at 37°C in the dark for 20 minutes for color development; add 50 µL of stop solution to each well and measure the absorbance of each well at 450 nm using an ELISA reader.

[0100] Results: After the MSRV-M polyclonal antibody was prepared, its titer was detected by ELISA. The results are shown in Tables 5 and 6. The average of the results from three repeated experiments was summarized.

[0101] The titer of the serum to be tested: the OD ratio of positive serum to negative serum at 450 nm is greater than 2.1.

[0102] Therefore, based on the summarized results, it can be seen that when the antiserum using purified MSRV-M protein as the antigen is diluted at a ratio of 1:128,000, the OD... 450The ratio = 3.37 > 2.1, indicating that the titer of the polyclonal antibody is 1:128,000 (Table 5); when the antiserum using the refolded MSRV-M protein as the antigen is diluted at a ratio of 1:256,000, the OD... 450 The ratio = 3.23 > 2.1, indicating that the titer of the polyclonal antibody is 1:256,000 (Table 6).

[0103] Table 5. Titer of MSRV-M polyclonal antibody (purified protein is the antigen)

[0104] Table 6. Titer of MSRV-M polyclonal antibody (the refolded protein is the antigen)

[0105] III. Polyclonal Antibody Western blot Analysis Cell protein extraction FHM cells were infected with the MSRV virus suspension obtained in Example 1 to allow the virus to enter the cells. Cell proteins were extracted 2 days later. The specific steps are as follows: (1) Add 10 µL of phosphatase inhibitor, 1 µL of protease inhibitor and 5 µL of 100 mM PMSF to each 1 mL of cold lysis buffer, and mix well. Store on ice for several minutes before use.

[0106] (2) After aspirating the culture medium from the cultured cells, wash twice with 10 mL of pre-cooled PBS buffer, treat the cells with trypsin, and pipette them off. Transfer the cell solution to a new ice-cold centrifuge tube, centrifuge at 2,000 rpm for 5 min, and aspirate the supernatant. Wash once with PBS buffer, and keep the cell pellet for later use.

[0107] (3) Add the prepared cold lysis working solution and place it on a shaking platform at 4℃. Gently shake for 15 min. Centrifuge at 12,000 rpm for 15 min, and take the supernatant as the whole protein extract. Quantify the protein using the BCA method.

[0108] The extracted cellular proteins were prepared and analyzed by Western blot. Serum obtained from rabbit immunization was used as the primary antibody, and HPR-labeled goat anti-rabbit IgG was used as the secondary antibody.

[0109] The extracted cellular proteins were subjected to SDS-PAGE electrophoresis, followed by membrane transfer. After transfer, the PVDF membrane was removed and rinsed three times with TBST solution. Then, an appropriate volume of rapid blocking buffer was added to completely submerge and cover the membrane. The membrane was then placed on a horizontal shaker and incubated at room temperature for 30 minutes.

[0110] Remove the sealed membrane and rinse it three times with TBST solution. Then, locate the corresponding bands on the PVDF membrane and cut it to the required size. Incubate with primary antibody (prepared polyclonal antibody serum) at 4°C overnight. After rinsing three times, incubate with secondary antibody (HPR-labeled goat anti-rabbit IgG) with shaking for 1 hour. Rinse three more times.

[0111] Afterwards, a digital gel imaging system is used for development, and different exposure times can be adjusted to obtain the best results.

[0112] Results: The results of the WB analysis are as follows Figure 22 As shown, both renatured and non-renatured proteins produced polyclonal antibodies that successfully showed the target band at around 39 kDa, consistent with the results of recombinant protein induction. This demonstrates that the prepared polyclonal antibodies can specifically recognize the MSRV-M protein in FHM cells and can be used for further research.

[0113] IV. Specificity of Polyclonal Antibodies FHM cells were seeded into 12-well cell culture plates. Once the cell density reached approximately 70%, the cells were infected with MSRV and cultured for another 36 hours. The old growth medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde, just enough to cover the bottom of the wells, and incubated at 28°C for 10 minutes. The fixative was then discarded, and the cells were washed three times with PBS.

[0114] Add primary antibody (prepared polyclonal antibody serum) and incubate at 28°C for 1 h. Discard the primary antibody and wash three times with PBS. Add secondary antibody (FITC-labeled goat anti-rabbit IgG) and incubate at 28°C for 1 h in the dark. Discard the secondary antibody and wash three times with PBS.

[0115] Add DAPI for staining, and incubate at 28°C in the dark for 5 minutes. Discard the DAPI and wash three times with PBS. Simultaneously, set up a negative control (PBS as primary antibody) and observe using a fluorescence microscope.

[0116] Result: As Figure 23 The results showed that the polyclonal antibody specifically recognizes the M protein in MSRV-infected FHM cells, demonstrating its good reactivity. Furthermore, comparisons at several different dilutions revealed that the polyclonal antibody exhibited the strongest specificity at a 1:200 dilution.

[0117] Polyclonal antibodies were prepared by immunizing rabbits with purified and renatured MSRV-M protein. The titers of the polyclonal antibodies were then determined by ELISA. The results showed that the titer of the polyclonal antibody prepared using purified MSRV-M protein as the antigen was 1:128,000, while the titer of the polyclonal antibody prepared using renatured MSRV-M protein as the antigen was 1:256,000. This indicates that the titer of the polyclonal antibody prepared by renatured MSRV-M protein was significantly higher than that of the unrenatured one. Subsequent Western blot analysis and IFA validation both confirmed that the prepared polyclonal antibody could specifically recognize the MSRV-M protein in FHM cells. This demonstrates that the M protein polyclonal antibody prepared in this study and the established ELISA detection method can be used for subsequent virus detection.

[0118] MSRV-M protein sequence (SEQ ID No: 1) MPLFKKGNKKSAITPYQAPPPYSATALTPSAPMALPDSDYGIKTMMVELDFKIISSIELKTIGKIYQIAQYMLDEYTGPIRSKPLYMGLFLASCHNAVNPSMVHGKWHYGIQFRGPVGFNLANNTPLDWVCNPVAISYECNTPERSLVSYTCNMRPTKMTGSSFEKMFHGVLVHPAAAESVLGIFQIAEAEVRGEDIVFILKDPGHSWH MSRV-M gene sequence (SEQ ID No: 2) ATGCCTCTGTTTAAGAAGGGCAACAAGAAGTCGGCTATCACGCCATACCAAGCACCTCCGCCATACTCGGCAACCGCACTCACCCCGAGTGCTCCGATGGCCCTGCCAGACAGCGACTACGGAATCAAAACAATGATGGTGGAGTTGGACTTCAAGATCATATCCAGCATCGAGCTTAAAACGATTGGCAAGATTTACCAAATTGCGCAATATATGCTGGATGAGTATACAGGCCCTATTCGGAGTAAGCCTCTGTATATGGGCCTTTTCCTTGCATCATGCCACAATGCCGTTAATCCATCAATGGTCCATGGAAAGTGGCATTATGGGATACAGTTTAGGGGCCCAGTAGGATTCAATCTGGCAAACAACACACCCCTGGACTGGGTTTGTAACCCGGTTGCCATCTCTTATGAGTGCAACACTCCTGAACGTTCACTGGTCAGCTACACCTGCAATATGCGCCCCACCAAGATGACAGGATCATCTTTTGAGAAGATGTTCCACGGTGTGTTAGTGCACCCCGCAGCTGAGTCTGTTCTTGGAATCTTCCAGATAGCAGAAGCAGAGGTCCGAGGAGAAGACATTGTATTCATCCTGAAAGATCCTGGTCATAGCTGGCATTAA Sequence of the optimized gene of MSRV-M (MSRV-M optimized gene) (SEQ ID No: 3) ATGCCACTGTTCAAAAAAGGTAATAAAAAATCTGCAATTACCCCTTACCAGGCACCGCCTCCGTATTCCGCTACCGCTCTGACCCCATCTGCTCCGATGGCTCTGCCGGATTCCGACTATGGTATCAAAACCATGATGGTGGAGCTGGACTTTAAA ATCATTTCCTCCATCGAGCTGAAAACCATCGGCAAAATCTACCAAATTGCTCAGTACATGCTGGATGAATACACCGGCCCAATTCGTAGCAAACCTCTGTACATGGGTCTGTTTCTGGCATCTTGCCACAATGCGGTGAACCCTAGCATGGTTCACG GCAAATGGCACTACGGCATCCAGTTCCGTGGCCCGGTTGGTTTCAACCTGGCGAACAACACTCCGCTGGATTGGGTTTTGTAACCCGGTCGGCATCAGCTACGAGTGTAACACCCCTGAACGTTCCCTGGTGAGCTACACGTGCAACATGCGCCCTAC TAAGATGACCGGCTCTTCCTTCGAAAAAATGTTCCATGGCGTCCTGGTACACCCGGCTGCAGAATCTGTACTGGGCATCTTCCAGATTGCGGAAGCAGAAGTGCGCGGTGAGGACATTGTCTTCATTCTGAAAGACCCAGGTCATTCTTGGCACTAA The optimized protein sequence of MSRV-M is consistent with SEQ ID No: 1.

[0119] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant protein of largemouth bass rhabdovirus MSRV-M, characterized in that, Its amino acid sequence is shown in SEQ ID No:

1.

2. The gene encoding the MSRV-M recombinant protein of claim 1, characterized in that, The sequence of the gene is shown in SEQ ID No: 2 or SEQ ID No:

3.

3. A recombinant expression vector, characterized in that, It contains a nucleotide sequence as shown in SEQ ID No:

3.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is pET32a-MSRV-M, which is an expression vector constructed based on pET-32a.

5. A genetically engineered strain for preparing the MSRV-M recombinant protein of claim 1, characterized in that, The recombinant expression vector pET32a-MSRV-M is transformed into... E. coli Recombinant strains obtained from DH5α competent cells.

6. A polyclonal antibody against largemouth bass rhabdovirus, characterized in that, The serum derived from rabbits immunized with the MSRV-M recombinant protein of claim 1; the MSRV-M recombinant protein is any one or more of the following: purified MSRV-M recombinant protein and purified and renatured MSRV-M recombinant protein.

7. The method for preparing the polyclonal antibody according to claim 6, characterized in that, Includes the following steps: Using MSRV-M recombinant protein as an antigen, New Zealand white rabbits were immunized three times, and serum was collected to prepare polyclonal antibodies.

8. A specific primer set for detecting largemouth bass rhabdovirus, characterized in that, This includes MSRV-03-F / R or MSRV-11-F / R; the sequence of MSRV-03-F / R is shown in SEQ ID No: 8~9; the sequence of MSRV-11-F / R is shown in SEQ ID No: 10~11.

9. The use of the MSRV-M recombinant protein of claim 1, the recombinant expression vector of claim 3, the genetically engineered strain of claim 5, the polyclonal antibody of claim 6, and the specific primer set of claim 8 in the preparation of products for detecting largemouth bass rhabdovirus.

10. A kit for detecting largemouth bass rhabdovirus, characterized in that, Includes the polyclonal antibody of claim 6 or the specific primer set of claim 8.

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