Grass carp reovirus (gcrv-Ⅱ type) subunit recombinant vaccine, preparation method and application

Abstract

The application belongs to the technical field of biology, and particularly relates to a grass carp reovirus (GCRV-Ⅱ type) subunit recombinant vaccine, a preparation method and application. 274‑367 , VP5 105‑336 , NS38 143‑338 sequence fragments, and the three sequence fragments are recombined and expressed in a prokaryotic expression vector to prepare the subunit recombinant vaccine. The recombinant vaccine has high safety, strong activity and broad spectrum, and has good application potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular, it relates to a grass carp reovirus (GCRV-II) subunit recombinant vaccine, its preparation method, and its application. Background Technology

[0002] Grass carp (Ctenopharyngodon idella), one of the "four major freshwater fish" in China, plays a crucial role in global freshwater fisheries production. However, grass carp hemorrhagic disease caused by grass carp reovirus is one of the most serious infectious diseases hindering the healthy and sustainable development of grass carp aquaculture. Grass carpreovirus (GCRV) belongs to the family Reoviridae and the genus Aquareovirus. Its genome includes 11 segmented double-stranded RNAs encoding seven structural polypeptides (VP1-VP7) and five non-structural polypeptides (NS80, NS38, NS31, NS26, and NS16). It has a double capsid and an icosahedral symmetry structure. Typical symptoms of GCRV include hemorrhages and congestion in certain areas of the mouth, upper jaw, top of the head, around the eye socket, abalone cap, abalone, and the base of the fin rays. GCRV is mainly divided into three genotypes (GCRV-I, II, and III), among which GCRV-II has the widest prevalence, the strongest virulence, and the greatest harm. Therefore, conducting prevention and control research on GCRV-II is of extremely important and urgent practical significance.

[0003] Currently, the prevention and control of grass carp hemorrhagic disease mainly relies on vaccines. However, the number of commercially available vaccines is limited and cannot meet the current needs of aquaculture. Traditional grass carp reovirus vaccines include inactivated vaccines, live attenuated vaccines, and subunit vaccines. Compared to the safety concerns of traditional inactivated or live attenuated vaccines, subunit vaccines retain only some immunogenic components of the pathogen, such as polypeptides, sugars, and proteins, without containing viral genetic material. They offer high safety and good stability, representing a cutting-edge trend in vaccine development. CN201711023099.7 discloses a grass carp reovirus VP56 protein subunit vaccine, which can stimulate the proliferation of immune cells in fish, upregulate the expression of antiviral-related genes, and produce antibodies. However, its protective efficiency and broad-spectrum efficacy need further improvement. CN201711022026.6 discloses a grass carp reovirus VP35 protein subunit vaccine, which improves the survival rate of grass carp infected with grass carp reovirus, but its protective efficiency also has room for improvement. CN202510785323.4 discloses a fusion gene, recombinant protein, composition, and application of grass carp NPM1a and type II reovirus VP38. This patent constructs a fusion protein by combining nucleolar phosphatase 1 (NPM1) with enterovirus VP38 and adds indole-3-lactic acid as an immune enhancer, thereby increasing the vaccine's protective rate and repairing the intestinal mucosal barrier. While this technology offers high protective efficiency, it also has drawbacks, including significant impact on grass carp and limited broad-spectrum efficacy. Due to the high mutation rate of GCRV virus, further research is needed to develop vaccines with strong immunogenicity and broad-spectrum efficacy. Summary of the Invention

[0004] In response to the problems of high production costs, insufficient immunogenicity, or biosafety risks associated with existing traditional inactivated or attenuated vaccines, and the low immunogenicity and limited broad-spectrum protection of existing subunit vaccines, this invention, through long-term and in-depth research, has developed a GCRV-II type subunit recombinant vaccine with high broad-spectrum protection, strong immunogenicity, and good protective effect.

[0005] Specifically, the technical solution of the present invention is as follows: In one aspect, the present invention discloses a GCRV-II grass carp reovirus subunit recombinant vaccine, wherein the antigen protein of the subunit recombinant vaccine is composed of the grass carp reovirus capsid protein recombinant sequence VP4. 274-367 -VP5 105-336 -NS38 143-338 The nucleotide sequence is shown in SEQ ID NO:37, and the amino acid sequence is shown in SEQ ID NO:38.

[0006] In one aspect, the present invention discloses the recombinant sequence VP4 of the grass carp reovirus capsid protein. 274-367 -VP5 105-336 -NS38 143-338 Application of VP4 in the preparation of grass carp reovirus subunit recombinant vaccine 274-367 -VP5 105-336 -NS38 143-338 The nucleotide sequence is shown in SEQ ID NO:37, and the encoded amino acid sequence is shown in SEQ ID NO:38.

[0007] In some embodiments, the VP4 274-367 -VP5 105-336 -NS38 143-338 Recombinant expression vectors are prepared by operatively linking nucleotide sequences to expression vectors.

[0008] A recombinant expression vector containing the recombinant sequence VP4 of grass carp reovirus capsid protein. 274-367 -VP5 105-336 -NS38 143-338 .

[0009] In some embodiments, the expression vector is a prokaryotic expression vector, a eukaryotic expression vector, a plant expression vector, or an insect expression vector.

[0010] In some embodiments, the expression vector is a prokaryotic expression vector; preferably, the prokaryotic expression vector is selected from pET series expression vectors, pGEX series expression vectors, and pMAL series expression vectors. More preferably, the expression vector is the pET32a expression vector.

[0011] In one aspect, the present invention discloses the application of the aforementioned recombinant expression vector in the preparation of a grass carp reovirus subunit recombinant vaccine.

[0012] In one aspect, this invention discloses a method for preparing a recombinant GCRV-II grass carp reovirus subunit vaccine, comprising the following steps: (1) Obtain the specific immunogen sequence VP4 274-367 -VP5 105-336 -NS38 143-338 .

[0013] (2) Constructing VP4 containing the recombinant immunogen sequence 274-367 -VP5 105-336 -NS38 143-338 The carrier of expression; (3) Introduce the expression vector into host cells, induce expression, and collect immune antigen proteins; (4) Mix the immunogenic protein with Freund's incomplete adjuvant to obtain the grass carp reovirus subunit recombinant vaccine.

[0014] In one aspect, the present invention discloses the application of the aforementioned grass carp reovirus subunit recombinant vaccine in the prevention and treatment of GCRV virus infection in grass carp.

[0015] In some embodiments, the grass carp reovirus (GCRV-II) subunit recombinant vaccine of the present invention is administered via intraperitoneal injection for the prevention and treatment of grass carp GCRV virus infection.

[0016] This invention first compares the major structural and non-structural proteins of different GCRV-II strains using homology sequences, and then uses bioinformatics techniques to predict their secondary and tertiary structures, antigenic epitopes, and transmembrane domains. From these, fragments with high conservation, predominantly non-helical secondary and tertiary structures, numerous antigenic epitopes, and non-transmembrane domains are screened, ultimately identifying VP4. 274-367 VP5 105-336 NS38 143-338 Sequence fragments. Then, the selected VP4... 274-367 VP5 105-336 NS38 143-338 Fragments were recombined to obtain a recombinant immunogen with multiple antigenic epitopes, strong immunogenicity, and the ability to target GCRV-II variants. Furthermore, a recombinant subunit vaccine was prepared using these antigen sequences via E. coli. This subunit recombinant vaccine uses highly conserved antigens with multiple antigenic epitopes and strong immunogenicity. Compared to other live attenuated or inactivated vaccines, it contains no natural viral components and has high safety. Simultaneously, the antigens of this invention were obtained by screening and comparing highly conserved regions from multiple different strains. Therefore, this subunit recombinant vaccine can also address the highly variable nature of GCRV-II virus and is broadly applicable to all GCRV-II virus strains.

[0017] Beneficial effects The grass carp reovirus (GCRV-II) subunit recombinant vaccine prepared by this invention significantly activates related immune genes (IgM, IFN, MHC-IIa, CD4), significantly increases the level of specific antibodies in grass carp serum, significantly enhances the protective effect against grass carp, reduces viral load, and does not induce inflammatory responses in the fish. Furthermore, compared with Z-DNA vaccine (CN202210007548.3) (protection rate: 61.5%) and inactivated vaccine (CN201810472063.5) (protection rate: 76.9%), the vaccine prepared by this invention achieves a protection rate of 84.6%, indicating that the vaccine prepared by this invention has significant improvements, broad-spectrum activity, and great potential for industrial application. Attached Figure Description

[0018] Figure 1 VP4274-367 VP5 105-336 NS38 143-338 The image shows the PCR detection results of recombinant antigen construction into pET32a plasmid. Figure 1 The middle left image shows a single VP4. 274-367 VP5 105-336 NS38 143-338 Gene fragment, right image shows VP4 274-367 -VP5 105-336 -NS38 143-338 Recombinant sequence.

[0019] Figure 2 VP4 274-367 VP5 105-336 NS38 143-338 The image shows the PCR results of recombinant antigen plasmid transformed into E. coli. Figure 2 The middle left image shows a single VP4. 274-367 VP5 105-336 NS38 143-338 Gene fragment, right image shows VP4 274 -367 -VP5 105-336 -NS38 143-338 Recombinant sequence.

[0020] Figure 3 For His tag, VP4 274-367 VP5 105-336 NS38 143-338 PAGE image of purified recombinant antigen protein.

[0021] Figure 4 The results of screening for the optimal antigen epitopes in enzyme-linked immunosorbent assay (ELISA) are shown in the figure.

[0022] Figure 5 Flowchart for the injection immunization and challenge experiment.

[0023] Figure 6 The graph shows the serum neutralizing antibody levels on day 14 after immunization.

[0024] Figure 7 This is a graph showing the viral load results in various tissues on day 14 after the challenge experiment.

[0025] Figure 8 This is a graph showing the survival rate of grass carp within 14 days after the virus challenge experiment.

[0026] Figure 9A-F represents the relative expression levels of IL1β, TNFα inflammation-related genes, and IFN, MHC-IIa, IgM, and CD4 immune-related genes in the intestine, liver, spleen, and kidney tissues of grass carp 7 days after immunization with different vaccines.

[0027] Figure 10 A graph showing the survival rate of grass carp 14 days after challenge experiments with different vaccines. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. Example 1: Preparation of Grass Carp Reovirus (GCRV-II) Subunit Recombinant Vaccine 1.1 Recombinant Antigen Screening Analysis Nucleotide and amino acid sequences of all GCRV-II strains were obtained from the Nucleotide database on the NCBI website. Homology sequence alignment of the GCRV-II capsid protein was performed using MEGA11 software. Simultaneously, antigenic epitopes of each protein were analyzed using the online IEDB database, secondary structure was analyzed using PSIPRED, tertiary structure was predicted using Alphafold3, and antigenicity was predicted using VaxiJen2.0. Based on the combined analysis of homology alignment sequences and immunogenicity, candidate antigen fragments with high homology, numerous antigenic epitopes, predominantly non-helical secondary and tertiary structures, and non-transmembrane domains were screened. Finally, secondary and tertiary structures, transmembrane structures, antigenicity, and physicochemical properties were predicted for the candidate recombinant antigens. Ultimately, partial sequences of VP4, VP5, and NS38 proteins were predicted to have high potential for immunological applications.

[0029] 1.2 Construction of relevant expression vectors

[0030] Based on the known sequences, primers were designed, and cDNA templates of VP4 (SEQ ID NO:31), VP5 (SEQ ID NO:32), and NS38 (SEQ ID NO:33) were extracted and amplified by PCR to obtain VP4. 274-367 (SEQ ID NO:34), VP5 105-336 (SEQ ID NO:35), NS38 143-338 (SEQ ID NO:36) gene sequence, according to VP4 274-367 -VP5 105-336 -NS38 143-338The recombinant sequence (SEQ ID NO:37) was obtained by sequential tandem cloning, and TA cloning (e.g.) was performed. Figure 1 ).

[0031] The PCR amplification reaction system is as follows: 2 µl of cDNA templates for VP4, VP5, and NS38, 1 µl each of forward and reverse primers, 0.5 µl of LA Taq DNA polymerase, 5 µl of 10×LA PCR Buffer, 4 µl of dNTPs, and ddH2O to bring the total to 50 µl.

[0032] VP4 after rubber cutting and recycling 274-367 VP5 105-336 NS38 143-338 1 µl each of cDNA template and VP4 274-367 upstream primer NS38 143-338 1 µl each of downstream primers, 0.5 µl of LA Taq DNA polymerase, 5 µl of 10×LA PCR Buffer, 4 µl of dNTPs, and ddH2O to bring the total to 50 µl.

[0033] Table 1. Primer sequences for PCR gene acquisition

[0034] Based on the restriction enzyme sites selected according to the pET32a plasmid sequence, EcoRI and HindIII were used to design homologous recombination primers. VP4 was then recombinated using homologous recombination. 274-367 VP5 105-336 NS38 143-338 VP4 274-367 -VP5 105-336 -NS38 143-338 The sequence was appended with a homologous arm, and the pET32a plasmid was double-digested with restriction endonucleases at the corresponding restriction sites. The product was then recovered.

[0035] The two products were recovered and purified, and pET32a-VP4 was ligated using a ligase. 274-367 pET32a-VP5 105-338 NS38 143-338 pET32a-VP4 274-367 -VP5 105-336 -NS38 143-338 The sequence was ligated into the pET32a plasmid, transformed into *E. coli* DH5α for large-scale amplification, and a portion of the bacterial culture was sent for testing and verification. After extracting the plasmid and determining its concentration, the recombinant plasmid was transformed into *E. coli* BL21, and a portion of the bacterial culture was sent for testing and verification (e.g., ...). Figure 2), and then a large number of proteins were induced (gaattc is the EcoRI restriction site, aagctt is the HindIII restriction site).

[0036] Table 2 Primer sequences for PCR acquisition of homologous recombination genes

[0037] 1.3 Protein purification

[0038] Add the bacterial culture to 200 ml-300 ml LB Amp. + In resistant medium, cultured at 37°C and 220 rpm until OD 600 When the concentration is 0.8-1.0, add IPTG to a final concentration of 0.5 mM, induce overnight, and collect the precipitate by centrifugation at 5000 rpm for 5 min. Resuspend the precipitate in 10 ml of PBS, sonicate on ice at 20-25% energy, 15 s pulse, 10 s pause, 8 min, centrifuge at 8000 rpm for 5 min. Discard the supernatant, resuspend the precipitate in 10 ml of inclusion body washing buffer A (sonication: 20-25% energy, 15 s pulse, 10 s pause, 1 min), centrifuge at 8000 rpm for 5 min. Discard the supernatant, then resuspend the precipitate in 10 ml of inclusion body washing buffer B and 10 ml of ddH2O, centrifuge at 8000 rpm for 5 min, and discard the supernatant. Resuspend the precipitate in 4 ml of PBS, add the appropriate amount of loading buffer, heat in a 95°C water bath for 5 min; place on ice to cool. Prepare 10% SDS-PAGE gels (2 cm stacking gel, without comb). Load 700-800 µl of sample onto each gel. Electrophoresis: 80-110 V for 25-30 min, 105-145 V for 50-80 min. After electrophoresis, place the separating gel in 1M KCl solution for 20 min to determine the location of the target protein. Cut off the corresponding gel containing the target protein with a blade. Place the cut gel into a dialysis bag (10 mm, molecular weight <6000). Electrophore in SDS-free Laemmli electrophoresis buffer at 150 V in a chromatography freezer, with a current not exceeding 100 mA. After electroelution for 3-4 h, reverse electrophoresis for 5-6 min. Remove the gel. Place the dialysis bag containing the antigen solution in equilibration buffer at 4℃ overnight. The next day, concentrate the antigen solution with polyethylene glycol, collect it in centrifuge tubes, and store at -80℃.

[0039] To validate the purified protein, a 10% SDS-PAGE gel was prepared and electrophoresed at 80-110 V for 25-30 min, then at 105-145 V for 50-80 min. The gel was then stained with Coomassie Brilliant Blue solution for 1 h, followed by overnight destaining to determine the location of the target protein (e.g., ...).Figure 3 ).

[0040] 1.4 Enzyme-linked immunosorbent assay (ELISA)

[0041] Grass carp anti-GCRV-II positive serum was diluted to 1:500, mixed with coating buffer, and coated onto 96-well plates. The plates were incubated overnight at 4°C. The antigen protein was diluted to the same concentration using sample dilution buffer, and PBS, His tag, and VP4 were added. 274-367 VP5 105-336 NS38 143-338 VP4 274-367 -VP5 105-336 -NS38 143-338 Protein was added to the wells and incubated at 37°C for 4 h. The liquid was discarded, and the cells were washed three times with washing buffer for 5 min each time. Anti-His-Tag mouse monoclonal antibody was diluted 1:2000 and added to the wells, incubated at 37°C for 2 h. The liquid was discarded, and the cells were washed three times with washing buffer for 5 min each time. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody was diluted 1:2000 and added to the wells, incubated at 37°C for 2 h. The liquid was discarded, and the cells were washed three times with washing buffer for 5 min each time. After adding chromogenic buffer and developing at 37°C for 5 min, stop buffer was added, and absorbance was read at 450 nm. The results showed that the recombinant protein had the highest absorbance, indicating that the recombinant protein has more antigenic epitopes (such as...) compared to the fragment protein. Figure 4 ).

[0042] 1.5 Vaccine Preparation

[0043] The antigen protein concentrate was diluted to 500 µg / ml with PBS buffer to prepare an antigen solution. Freund's incomplete adjuvant was mixed with the antigen solution at a volume ratio of 1:1 to obtain the grass carp reovirus (GCRV-II) subunit recombinant vaccine.

[0044] Test Example 1: Grass Carp Immunization Test

[0045] Prepare 6 groups of healthy grass carp of similar size, and set up a blank control group (PBS), a negative control group (His-labeled), and a test group (VP4). 274-367 Protein, test group VP5 105-336 Protein, test group NS38 143-338 Protein, Test group VP4 274-367 -VP5 105-336 -NS38 143-338 Recombinant vaccine, 30 doses per group. The flowchart for the injection immunization and challenge experiment follows... Figure 5As shown in the figure. Each group of grass carp was intraperitoneally injected with 400 µl of the mixture from each group. After immunization, the intestines, liver, spleen, kidneys and serum of grass carp were collected on days 1, 7 and 14 respectively. The relative expression levels of IL-1β, TNFα, IFN, CD4, MHC-IIa and IgM genes were detected by qRT-PCR. The results showed that on day 1 post-immunization, the expression of immune-related genes IFN, CD4, MHC-IIa, and IgM in some tissues of the recombinant vaccine group was significantly higher than that in other groups. The immune genes responded rapidly in the early stage and continued until day 14, proving that the recombinant vaccine could rapidly activate the innate immune defense of grass carp and maintain an immune-stimulated state for a long time. The expression of immune-related genes IFN, CD4, MHC-IIa, and IgM in some tissues of the recombinant vaccine group did not change significantly on day 1, but the expression level increased sharply on day 7 and remained significant on day 14, indicating that the adaptive immune response had been successfully established and maintained at a high level. The inflammatory factors TNFα and IL1β in some tissues of grass carp in the recombinant vaccine group did not change significantly within 14 days after injection immunization, and some tissues showed a trend of first increasing and then decreasing. The timely decrease of inflammatory factors on day 7 and day 14 avoided excessive and persistent inflammatory response, which is of great significance for maintaining the functional homeostasis of immune organs and preventing immunopathological damage, proving the safety of the vaccine. The level of neutralizing antibodies in the serum was also measured on day 14. The results showed that the OD values ​​of the grass carp serum immunized with the recombinant vaccine at different dilutions (1:100 to 1:2000) were significantly higher than those of other test groups, demonstrating that the recombinant vaccine group maintained a strong positive reaction at different dilutions. This result indicates that the recombinant vaccine can effectively stimulate grass carp to produce high-titer specific antibodies, and the level of humoral immunity induction is superior to that of other test groups (e.g., [missing data]). Figure 6 ).

[0046] Table 3. Primer sequences for grass carp qRT-PCR (5'-3')

[0047] Test Example 2: Grass Carp Challenge Experiment

[0048] A challenge experiment was conducted on grass carp 16 days after injection immunization. Each grass carp was intraperitoneally injected with 200 µl of GCRV-II virus. Intestinal, liver, spleen, and kidney tissues and serum were extracted from grass carp on days 16, 22, and 28. The relative expression levels of IL-1β, TNFα, IFN, CD4, MHC-IIa, and IgM genes were detected by qRT-PCR. The results showed that IFN, CD4, MHC-IIa, and IgM were significantly upregulated in the intestinal, liver, spleen, and kidney tissues of grass carp from day 16 to day 28 after challenge (p<0.05), indicating that it induced a durable and systemic immune response. However, the inflammatory factors TNFα and IL1β remained at low levels. The results indicate that the recombinant vaccine has effective immune protection while avoiding a severe inflammatory response. The GCRV-II viral load in the intestines, liver, spleen, and kidneys of grass carp was measured on day 28. The GCRV-II viral load in the intestinal, liver, spleen, and kidney tissues of the grass carp challenged with the recombinant vaccine on day 28 was lower than that in other experimental groups, indicating that the recombinant vaccine has a good virus clearance ability (e.g., Figure 7 The survival rate of grass carp challenged with the vaccine was recorded within two weeks. The survival rate of grass carp in the control group was only 6.7%, while the survival rate in the vaccine group was 66.7%, significantly higher than that of the control group and other test groups. These results indicate that the recombinant vaccine has a good immunoprotective effect (e.g., Figure 8 ).

[0049] Test Example 3: Comparative Test of Protective Efficacy of Different Vaccines

[0050] Four groups of healthy grass carp of similar size, 30 fish per group, were prepared. These were the control group (PBS), the Z-DNA vaccine group, the inactivated vaccine group, and the recombinant vaccine group. Each group of grass carp was intraperitoneally injected with 400 µl of a 1:1 mixture of the corresponding vaccine and Freund's incomplete adjuvant. On day 7 post-immunization, intestinal, liver, spleen, and kidney tissues were collected from the grass carp. The relative expression levels of IL-1β, TNFα, IFN, CD4, MHC-IIa, and IgM genes were detected using qRT-PCR. Compared with other vaccine groups, the recombinant vaccine group showed significantly enhanced expression levels of IFN, CD4, MHC-IIa, and IgM genes on day 7 after immunization. This indicates that the recombinant vaccine can activate the immune response more quickly. However, the expression of IL-1β and TNFα inflammatory factors was not significantly upregulated compared to other vaccine groups, indicating that the recombinant vaccine did not induce an excessive inflammatory response. These results demonstrate that the recombinant vaccine effectively activates specific immune responses (cellular immunity and early humoral immunity) without inducing an excessive inflammatory response, exhibiting a good balance between immunogenicity and safety (Figure 9). Eight days after immunization, a challenge experiment was conducted. Each grass carp was intraperitoneally injected with 200 µl of GCRV-II virus, and the survival rate of the grass carp was recorded over two weeks. The survival rate was 61.5% in the z-DNA vaccine group, 76.9% in the inactivated vaccine group, and as high as 84.6% in the recombinant vaccine group. These results indicate that the recombinant vaccine has better immunoprotective efficacy compared to other conventional vaccines (e.g., Figure 10 ).

[0051] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A recombinant GCRV-II grass carp reovirus subunit vaccine, characterized in that, The antigen protein of the subunit recombinant vaccine is derived from the recombinant sequence VP4 of the grass carp reovirus capsid protein. 274-367 -VP5 105-336 -NS38 143-338 The nucleotide sequence is shown in SEQ ID NO:37, and the amino acid sequence is shown in SEQ ID NO:

38.

2. Recombinant sequence VP4 of grass carp reovirus capsid protein 274-367 -VP5 105-336 -NS38 143-338 Its application in the preparation of grass carp reovirus subunit recombinant vaccine is characterized by, The VP4 274-367 -VP5 105-336 -NS38 143-338 The nucleotide sequence is shown in SEQ ID NO:37, and the encoded amino acid sequence is shown in SEQ ID NO:

38.

3. The application according to claim 2, characterized in that, The VP4 274-367 -VP5 105-336 -NS38 143-338 Recombinant expression vectors are prepared by operatively linking nucleotide sequences to expression vectors.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the recombinant sequence VP4 of grass carp reovirus capsid protein. 274-367 -VP5 105-336 -NS38 143-338 The nucleotide sequence is shown in SEQ ID NO:

37.

5. The recombinant expression vector according to claim 4, characterized in that, The expression vector is a prokaryotic expression vector, a eukaryotic expression vector, a plant expression vector, or an insect expression vector.

6. The recombinant expression vector according to claim 5, characterized in that, The expression vector is a prokaryotic expression vector; preferably, the prokaryotic expression vector is selected from pET series expression vectors, pGEX series expression vectors, and pMAL series expression vectors; more preferably, the expression vector is pET32a expression vector.

7. The use of a recombinant expression vector according to any one of claims 4-6 in the preparation of a grass carp reovirus subunit recombinant vaccine.

8. A method for preparing a recombinant GCRV-II grass carp reovirus subunit vaccine, characterized in that, The method includes the following steps: (1) Obtain the specific immunogen sequence VP4 274-367 -VP5 105-336 -NS38 143-338 ; (2) Constructing VP4 containing the recombinant immunogen sequence 274-367 -VP5 105-336 -NS38 143-338 The carrier of expression; (3) Introduce the expression vector into host cells, induce expression, and collect immune antigen proteins; (4) Mix the immunogenic protein with Freund's incomplete adjuvant to obtain the grass carp reovirus subunit recombinant vaccine.

9. The application of the grass carp reovirus subunit recombinant vaccine according to claim 1 in the prevention and control of GCRV virus infection in grass carp.

10. The application according to claim 9, characterized in that, Prevention and treatment of GCRV virus infection in grass carp by intraperitoneal injection.

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