Virus of the genus iridovirus isolated from the cultured fish of the genus lateolabrax

Abstract

The application discloses a large yellow croaker iridovirus double antigen subunit vaccine and a preparation method thereof. The application can significantly improve the immunogenicity, stability and expression amount of baculovirus of MCP protein and ATPase protein sequences of the large yellow croaker iridovirus, and the two kinds of recombinant proteins are expressed by using a baculovirus expression system, so that the large-scale production of the two kinds of recombinant proteins can be realized by large-scale serum-free suspension culture of a bioreactor, the production batch is stable, the protein expression amount is high, and the production cost is low. The large yellow croaker iridovirus double antigen subunit vaccine prepared by mixing the two kinds of recombinant proteins has high antibody level of the immunized large yellow croaker, is far superior to the existing single antigen vaccine and inactivated vaccine, has strong protection effect, and only a small amount of immunization is needed to obtain a protection effect of more than 78.1%, and the large yellow croaker iridovirus double antigen subunit vaccine is non-pathogenic to the large yellow croaker and has high safety.

Description

Technical Field

[0001] This invention relates to a double-antigen subunit vaccine for iridovirus in large yellow croaker, its preparation method and application, belonging to the field of animal immunization drug technology. Background Technology

[0002] Large yellow croaker iridovirus (LYCIV) disease is one of the most serious diseases threatening large yellow croaker aquaculture in recent years. It is a highly lethal infectious disease characterized by "white gill disease" as its main symptom. LYCIV was first isolated from farmed large yellow croaker in marine environments in 2001. It is a lethal virus and is currently one of the most serious pathogens affecting large yellow croaker. Large yellow croaker at all growth stages can be infected, but 10-15cm juveniles are more susceptible and have a higher mortality rate. Clinical symptoms of LYCIV disease often include slow swimming, emaciation, and darkening of the body color. Some infected fish show congestion and redness in the lower jaw. Dissection reveals pale gills, enlarged spleen and kidneys, and congestion at the liver margins, often with mottled lesions on the liver. LYCIV is difficult to control; once infected, it is difficult to treat effectively, causing huge economic losses and seriously affecting the healthy aquaculture of large yellow croaker.

[0003] LYCIV belongs to the genus Megalovirus and is a type of iridovirus that primarily infects fish. This genus of iridoviruses mainly infects nearly a hundred species of freshwater and saltwater fish, including those in the orders Perciformes, Pleuronectiformes, and Gadiformes. LYCIV is a cytoplasmic double-stranded DNA virus with a particle size between 100 and 200 nm. The virus particles are spherical, with an icosahedral nucleocapsid structure and an outer lipid envelope.

[0004] Based on the major capsid protein (MCP) or adenosine triphosphatase (ATPase) gene, iridoviruses of the genus *Iridovirus* are classified into three genotypes: ISKNV, RSIV, and TRBIV. MCP is the major protein produced after viral infection. The MCP protein is approximately 50 kDa in size and is highly conserved among iridoviruses. ATPase is an ATPase-related gene involved in DNA replication and repair.

[0005] Active prevention is the most effective way to control the occurrence and spread of this disease, but currently there is a lack of vaccines that can effectively prevent LYCIV infection. For example, CN119499362A discloses an inactivated vaccine against large yellow croaker iridovirus. Although its preparation method is simple, its relative immunoprotective rate is low. While researchers have also proposed various subunit vaccine patents for other fish iridoviruses, CN116375814A discloses a method for preparing a largemouth bass iridovirus subunit vaccine using a prokaryotic expression system to express MCP protein. However, this technical solution mainly targets specific truncated sequences to construct recombinant proteins, resulting in a relatively simple antigen design, and its immunoprotective effect still has room for further improvement. CN108079287B discloses a method for preparing a grouper iridovirus subunit vaccine using a prokaryotic expression system. However, this technical solution mainly relies on a single structural protein as an antigen, resulting in a relatively simple antigen composition, and still has certain limitations in inducing broader and more durable immunoprotection. In particular, existing reported subunit vaccines mainly produce immunoprotective effects against other fish iridoviruses, rather than large yellow croaker iridovirus. In addition, existing methods mostly use prokaryotic expression systems to express MCP proteins, which may affect the protein's native conformation and immunogenicity, thus requiring further improvement in the immunoprotective effect. Summary of the Invention

[0006] The main objective of this invention is to provide a double antigen subunit vaccine for large yellow croaker iridovirus and its preparation method, so as to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned objectives, the present invention employs the following technical solution.

[0008] According to a first aspect of the present invention, a recombinant antigen of large yellow croaker iridovirus is provided, comprising:

[0009] Recombinant MCP protein, encoded by the first nucleic acid molecule;

[0010] Recombinant ATPase protein, which is encoded by a second nucleic acid molecule.

[0011] In one embodiment, the mass ratio of the recombinant MCP protein to the recombinant ATPase protein is approximately 1:1.

[0012] According to a second aspect of the present invention, a gene encoding a recombinant antigen of a large yellow croaker iridovirus is provided, comprising:

[0013] The first nucleic acid molecule encodes the recombinant MCP protein;

[0014] The second nucleic acid molecule encodes the recombinant ATPase protein.

[0015] According to a third aspect of the present invention, a recombinant vector for preparing recombinant antigens of large yellow croaker iridovirus is provided, comprising:

[0016] The first carrier contains the first nucleic acid molecule;

[0017] The second carrier contains a second nucleic acid molecule.

[0018] In one embodiment, the first or second carrier includes, but is not limited to, pFastBac 1, pVL1393, pFastBac dual, or pDEST8, with pFastBac 1 being preferred.

[0019] According to a fourth aspect of the present invention, a host cell for preparing recombinant antigen of large yellow croaker iridovirus is provided, comprising:

[0020] The first host cell contains the first nucleic acid molecule;

[0021] The second host cell contains a second nucleic acid molecule.

[0022] In one embodiment, the first or second host cell includes, but is not limited to, Sf9, HighFive, or Sf21 cells, preferably Sf9 cells.

[0023] The first nucleic acid molecule in this invention may have the sequence shown in SEQ ID NO:1 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:1. More preferably, the sequence of the first nucleic acid molecule is as shown in SEQ ID NO:1.

[0024] The second nucleic acid molecule of this invention may have the sequence shown in SEQ ID NO:2 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:2. More preferably, the sequence of the second nucleic acid molecule is as shown in SEQ ID NO:2.

[0025] According to a fifth aspect of the invention, an immune composition is provided, comprising: the large yellow croaker iridovirus recombinant antigen; and a pharmaceutically acceptable carrier.

[0026] In one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, any one or a combination of two or more of MONTANIDE ISA 206VG, ​​MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, and plant cell lectins, preferably one or two of MONTANIDE ISA 206VG and MONTANIDE ISA 201VG.

[0027] In one embodiment, the recombinant antigen of large yellow croaker iridovirus comprises recombinant MCP protein and recombinant ATPase protein in a mass ratio of approximately 1:1.

[0028] According to a sixth aspect of the present invention, a method for preparing recombinant antigen of large yellow croaker iridovirus is provided, comprising:

[0029] Provide the combination of the host cells;

[0030] First host cells and second host cells were cultured under suitable conditions, and then recombinant MCP protein and recombinant ATPase protein were isolated.

[0031] In one embodiment, the preparation method specifically includes:

[0032] S1. Prepare nucleic acid molecules encoding recombinant MCP protein and recombinant ATPase protein respectively;

[0033] S2. Construct a recombinant vector by cloning the nucleic acid molecules described in step S1 into a shuttle vector to obtain a recombinant shuttle vector containing the target gene.

[0034] S3. Transform the recombinant shuttle vector into competent cells to obtain recombinant plasmids, and then transfect host cells to obtain recombinant baculoviruses.

[0035] S4. The recombinant baculovirus is inoculated into host cells to obtain expression products, namely recombinant MCP protein and recombinant ATPase protein.

[0036] Furthermore, the preparation method may also include the steps of separating and purifying the recombinant MCP protein and the recombinant ATPase protein, wherein the optional separation and purification methods include, but are not limited to, chromatography, dialysis, or other methods known in the art.

[0037] In one embodiment, the preparation method may further include:

[0038] S5. The purified recombinant MCP protein and recombinant ATPase protein are mixed in an appropriate ratio (e.g., a molar ratio of about 1:1) to obtain the recombinant antigen of large yellow croaker iridovirus.

[0039] In one embodiment, the preparation method may further include:

[0040] S6. Mix the recombinant antigen of large yellow croaker iridovirus with a pharmaceutically acceptable carrier.

[0041] According to a seventh aspect of the invention, the use of the recombinant antigen of the large yellow croaker iridovirus, the encoding gene of the recombinant antigen of the large yellow croaker iridovirus, or the immune composition is provided in the production of an agent for inducing an immune response against large yellow croaker iridovirus infection in test animals or for preventing animals from being infected with large yellow croaker iridovirus.

[0042] Furthermore, the animals include marine or freshwater fish, such as fish belonging to the orders Perciformes, Pleuronectiformes, or Gadiformes, preferably large yellow croaker.

[0043] According to an eighth aspect of the invention, the use of the large yellow croaker iridovirus recombinant antigen or the immune composition in the preparation of a large yellow croaker iridovirus dual antigen subunit vaccine is provided.

[0044] According to a ninth aspect of the present invention, a large yellow croaker iridovirus dual-antigen subunit vaccine is provided, the vaccine comprising the recombinant antigen of the large yellow croaker iridovirus. Further, the vaccine may also comprise a pharmaceutically acceptable carrier.

[0045] Furthermore, when applying the large yellow croaker iridovirus dual-antigen subunit vaccine, only the effective amount needs to be administered to the animals. The "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.

[0046] According to a tenth aspect of the present invention, a method for preparing the large yellow croaker iridovirus dual antigen subunit vaccine is provided. In one embodiment, the preparation method includes the following steps:

[0047] S1. Prepare nucleic acid molecules for encoding recombinant MCP and ATPase proteins of large yellow croaker iridovirus, respectively.

[0048] S2. Construct a recombinant vector and clone the nucleic acid molecule from step 1 into the plasmid vector pFastBac 1 to obtain recombinant plasmids pFastBac 1-MCP and pFastBac 1-ATPase containing the target gene.

[0049] S3. Transform DH10Bac bacteria with recombinant plasmids pFastBac 1-MCP and pFastBac 1-ATPase, respectively. Select recombinant bacteria, extract the genome, and transfect Sf9 cells to obtain recombinant baculovirus.

[0050] S4. Cultivate the Sf9 cells and then recombinantly express recombinant MCP and ATPase proteins respectively.

[0051] S5. The recombinant MCP protein and ATPase protein are added to the adjuvant to obtain the vaccine.

[0052] According to an eleventh aspect of the present invention, a method is provided for inducing an immune response against large yellow croaker iridovirus infection or for protecting a test animal from large yellow croaker iridovirus infection, the method comprising administering the large yellow croaker iridovirus dual antigen subunit vaccine to the test animal.

[0053] According to a twelfth aspect of the invention, a vaccine suitable for inducing an immune response against large yellow croaker iridovirus infection in test animals is also provided, the vaccine comprising: the large yellow croaker iridovirus recombinant antigen and an adjuvant. As used in this specification, "adjuvant" means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen encoded by the gene.

[0054] In addition, some embodiments of the present invention may provide a kit comprising the recombinant antigen of the large yellow croaker iridovirus, the encoding gene of the recombinant antigen of the large yellow croaker iridovirus, the recombinant vector for preparing the recombinant antigen of the large yellow croaker iridovirus, the host cell for preparing the recombinant antigen of the large yellow croaker iridovirus, or the immune composition.

[0055] Furthermore, the kit may also include containers or instruments, such as syringes, for packaging or administering the large yellow croaker iridovirus recombinant antigen or the immune composition to animals.

[0056] Compared with the prior art, the present invention has at least the following beneficial effects:

[0057] (1) This invention optimizes the sequences of MCP protein and ATPase protein of large yellow croaker iridovirus, which significantly improves its immunogenicity, stability and expression level in baculovirus. The recombinant MCP protein and ATPase protein are expressed simultaneously in baculovirus expression system and Sf9 cells, respectively. The antigenicity and immunogenicity of the obtained protein are similar to those of the natural protein. The expression level is high and the immunogenicity is strong. A very small amount can provide good immune protection. It is non-pathogenic to large yellow croaker. At the same time, it can be prepared by large-scale serum-free suspension culture in bioreactor, which greatly reduces the cost of vaccine production. It is easy to control the quality, has high safety and batch-to-batch stability.

[0058] (2) The present invention prepares a large yellow croaker iridovirus vaccine by mixing the aforementioned recombinant MCP protein and ATPase protein. The antibody level produced by the vaccine immunizing large yellow croaker is high, which is far superior to existing single antigen vaccines and inactivated vaccines. It can generate strong humoral immunity in large yellow croaker. After immunization, large yellow croaker can resist strong virus attack and has a strong protective effect. Only one immunization is needed to achieve a protective effect of 78.1%. It is non-pathogenic to large yellow croaker and has high safety. Attached Figure Description

[0059] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0060] Figure 1 This is a schematic diagram of the transfer vector pFastBac 1-MCP containing the target gene constructed in Example 1.

[0061] Figure 2 This is a schematic diagram of the transfer vector pFastBac 1-ATPase containing the target gene constructed in Example 1.

[0062] Figure 3 This is a gel electrophoresis image of the product of PCR amplification of the large yellow croaker iridovirus MCP gene with optimized codons in Example 1.

[0063] Figure 4 This is a gel electrophoresis image of the product of PCR amplification of the large yellow croaker iridovirus ATPase gene with optimized codons in Example 1.

[0064] Figure 5 This is a gel electrophoresis image of the PCR product after PCR amplification of the colony sample transformed with the MCP gene in Example 1.

[0065] Figure 6 This is a gel electrophoresis image of the PCR product after PCR amplification of the colony sample transformed with the ATPase gene in Example 1.

[0066] Figure 7 This is a gel electrophoresis image of the PCR products after PCR amplification of the blue-white screening colony samples after pFastBac1-MCP transposition in Example 2.

[0067] Figure 8 This is a gel electrophoresis image of the PCR products after PCR amplification of the blue-white screening colony samples after pFastBac1-ATPase transposition in Example 2.

[0068] Figure 9 This is an SDS-PAGE gel electrophoresis image of the cell culture supernatant containing recombinant MCP protein harvested in Example 3.

[0069] Figure 10 This is an SDS-PAGE gel electrophoresis image of the cell culture supernatant containing recombinant ATPase protein harvested in Example 3.

[0070] Figure 11This is a Western blot image of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) product corresponding to the recombinant MCP protein in Example 4.

[0071] Figure 12 This is an immunoblotting image of the SDS-PAGE electrophoresis product corresponding to the recombinant ATPase protein in Example 4.

[0072] Figure 13 The graph shows the results of specific antibody levels in the MCP-ATPase group and PBS group at different time points after immunization in Example 7. Detailed Implementation

[0073] The present invention is further illustrated below by way of examples. All reagents and raw materials used in the following examples are commercially available, and experimental methods not specifically described are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0074] Example 1: Construction and identification of recombinant plasmids rBacmid-MCP and rBacmid-ATPase

[0075] 1. Construction and identification of recombinant plasmid pFastBac 1-MCP

[0076] 1.1 Amplification and purification of MCP and ATPase genes

[0077] The codon-optimized MCP gene (SEQ ID NO:1) and ATPase gene (SEQ ID NO:2) were synthesized at Suzhou Genewiz Biotechnology Co., Ltd., and cloned into the pUC17 vector to obtain pUC17-MCP and pUC17-ATPase plasmid vectors, respectively. PCR amplification was performed using pUC17-MCP and pUC17-ATPase plasmids as templates, respectively. The primer sequences and amplification systems are shown in Tables 1 and 2.

[0078] Table 1 Gene Primer Sequences

[0079]

[0080] Table 2 Gene Amplification Procedure

[0081]

[0082] The reaction conditions were: 95℃ pre-denaturation for 5 minutes; 94℃ denaturation for 45 seconds, 60℃ annealing for 45 seconds, 72℃ extension for 1 minute, 35 cycles; 72℃ extension for 10 minutes.

[0083] The size of the target gene was verified by gel electrophoresis of the PCR products. For example... Figure 3 As shown, the target band of the MCP gene appears around 1455bp, where lane M is the DNA marker, lane 1 is the negative control, and lane 2 is the PCR amplification product of the MCP gene. Figure 4 As shown, the target band of the ATPase gene appeared around 813 bp, with lane M representing the DNA marker, lane 1 representing the negative control, and lane 2 representing the PCR amplification product of the ATPase gene. These results confirm successful amplification of the target gene, which was then purified using a gel extraction kit.

[0084] 1.2 Enzyme digestion

[0085] The pFastBac 1 plasmid and the PCR product recovered and purified in step 1.1 were digested with EcoRI and XbaI enzymes at 37°C for 3 hours. The specific digestion reaction system is shown in Tables 3 and 4.

[0086] Table 3 Gene digestion reaction system

[0087]

[0088] Table 4 pFastBac 1 plasmid digestion reaction system

[0089]

[0090] 1.3 Connection

[0091] The double-digested pFastBac 1 plasmid and the MCP and ATPase gene digestion products were ligated using T4 DNA ligase in a 16°C water bath overnight. The specific ligation reaction system is shown in Table 5.

[0092] Table 5. Ligation system of gene digestion products with pFastBac 1 plasmid

[0093]

[0094] 1.4 Transformation

[0095] Add 10 μl of the ligation product to 100 μl of DH5α competent cells and mix well. Incubate on ice for 30 minutes, then subject to heat shock at 42°C for 90 seconds, followed by an ice incubation for 2 minutes. Add 900 μl of Amp-free LB liquid medium and incubate at 37°C for 1 hour. Take 1.0 ml of the bacterial culture, concentrate it to 100 μl, and spread it onto LB solid medium containing Amp. Incubate at 37°C for 16 hours.

[0096] 1.5 Colony PCR and Sequencing Identification

[0097] Single colonies from the plates were inoculated into LB liquid medium and incubated at 37°C for 2 hours. Using the bacterial culture as a template, colony PCR was performed using the primers in Table 1. The PCR products were then subjected to gel electrophoresis to verify the size of the target gene. Figure 5 As shown, the MCP gene-positive sample exhibited a 1455bp band, where lane M represents the DNA marker, lane 1 is the negative control, and lanes 2, 3, 4, and 5 contain MCP colony PCR amplification products. Figure 6 As shown, the ATPase gene-positive samples exhibited an 813bp band, with lane 1 serving as the negative control and lanes 2, 3, 4, and 5 containing ATPase colony PCR amplification products. The identified positive bacterial cultures were sent to a sequencing company for sequencing. Cultures with correctly sequenced cultures were preserved, and plasmids were extracted to obtain the recombinant plasmids pFastBac 1-MCP and pFastBac 1-ATPase, whose structures are shown below. Figure 1 As shown in Figure 2.

[0098] 2. Construction and identification of recombinant rBacmid-MCP and rBacmid-ATPase

[0099] 2.1 DH10Bac transformation

[0100] Add 1 μl of pFastBac 1-MCP recombinant plasmid to 100 μl of DH10Bac competent cells and mix well. Incubate on ice for 30 minutes, then subject to heat shock at 42°C for 45 seconds, followed by an ice incubation for 2 minutes. Add 900 μl of LB liquid medium without Amp and incubate at 37°C for 5 hours. Spread 100 μl of the diluted bacterial culture onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG, and incubate at 37°C for 48 hours. Transform DH10Bac cells with the pFastBac 1-ATPase plasmid using the same procedure.

[0101] 2.2 Selection of Monoclonal Clones

[0102] Large white colonies were picked using an inoculation needle and streaked onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG. The colonies were then incubated at 37°C for 48 hours. Single colonies were then picked and inoculated into LB liquid medium containing gentamicin, kanamycin, and tetracycline. After identification using universal primers M13F and M13R, the correctly identified strains were preserved, and plasmids were extracted to obtain recombinant plasmids rBacmid-MCP and rBacmid-ATPase, respectively.

[0103] Figure 7The results of gel electrophoresis of PCR products from blue-white screening colony samples after pFastBac1-MCP transposition are shown. Positive samples appeared near the 3740bp band. Lane M is the DNA marker, lane 1 is the blank control, lane 2 is the PCR amplification product of blue-white screening MCP colonies, and lane 3 is the negative control.

[0104] Figure 8 The results of gel electrophoresis of PCR products from blue-white screening colonies after pFastBac1-ATPase transposition are shown. Positive samples appear around 3110bp. Lane M is the DNA marker, lane 1 is the blank control, lane 2 is the PCR amplification product of blue-white screening ATPase colonies, and lane 3 is the negative control.

[0105] Example 2: Transfection with recombinant baculovirus

[0106] In a six-well plate, each well is inoculated with 0.8 × 10⁸ g of seed. 6 Sf9 cells were collected, with a confluence of 50-70%. For each well, the following complex was prepared: 4 μl of Cellfectin transfection reagent was diluted with 100 μl of transfection medium T1, and the mixture was briefly vortexed; 3 μg of recombinant Bacmid-MCP plasmid was diluted with 100 μl of transfection medium T1. The diluted transfection reagent and plasmid were mixed and gently blown to prepare the transfection mixture. After cell attachment, the above transfection complex was added, and the cells were incubated at 27°C for 5 hours. The supernatant was removed, and 2 mL of fresh SF-SFM medium was added. The cells were incubated at 27°C for 4-5 days, and the supernatant was harvested. P1 generation recombinant baculovirus rBac-MCP was obtained. The P1 generation recombinant virus was used to infect Sf9 cells. After three generations of amplification culture, the supernatant of the diseased cells was collected, and cells and debris were removed by low-speed centrifugation to obtain the recombinant virus rBac-MCP. The same procedure was used to transfect rBacmid-ATPase to obtain the recombinant virus rBac-ATPase.

[0107] Example 3: SDS-PAGE Detection

[0108] The cell culture harvested in Example 2 was subjected to reducing SDS-PAGE analysis, with Sf9 cells infected with empty baculovirus used as a negative control. The specific procedure was as follows: 40 μl of the harvested cell culture was added to 10 μl of 5× loading buffer, incubated in boiling water for 5 minutes, centrifuged at 12000 rpm for 1 minute, and the supernatant was used for SDS-PAGE gel electrophoresis (12% concentration gel). After electrophoresis, the gel was stained, destained, and the target bands were observed. Figure 9As shown, the recombinant baculovirus rBac-MCP showed the target band around a molecular weight of approximately 50 kDa, while the negative control showed no band at the corresponding position. Lane M represents the protein marker, lane 1 represents the baculovirus empty vector control, and lane 2 represents the recombinant MCP protein. Figure 10 As shown, the recombinant baculovirus rBac-ATPase showed the target band at a molecular weight of approximately 30 kDa, while the negative control showed no band at the corresponding position. Lane M is the protein marker, lane 1 is the baculovirus empty vector control, and lane 2 is the recombinant ATPase protein.

[0109] Example 4 Western Blot Identification

[0110] SDS-PAGE gel electrophoresis (12% concentration gel) was performed according to Example 3. The electrophoretic products were transferred onto a PVDF membrane, blocked with 5% skim milk for 2 hours, incubated with mouse-His-tagged monoclonal antibody for 2 hours, rinsed, incubated with HRP-labeled goat anti-mouse polyclonal antibody secondary antibody for 2 hours, rinsed, and then an enhanced chemiluminescent fluorescent substrate was added. The images were taken using a chemiluminescence imaging system. The results are as follows: Figure 11 and Figure 12 As shown. Figure 11 Lane M in the middle lane is a protein marker, lane 1 is a baculovirus empty vector control, and lane 2 is a recombinant MCP protein. Figure 12 Lane M in the middle of the swim bladder contains a protein marker, lane 1 contains a baculovirus empty vector control, and lane 2 contains recombinant ATPase protein. These results indicate that the target proteins (recombinant MCP protein and recombinant ATPase protein) are correctly expressed in Sf9 cells.

[0111] Example 5: Preparation of a dual-antigen subunit vaccine

[0112] The recombinant protein stock solution expressed in Example 2 was taken and diluted with PBS solution. The mixed vaccine stock solution was then formulated with white oil adjuvant to prepare an oil-emulsion vaccine, ensuring that each dose (0.2 ml) contained 40 μg each of recombinant MCP and ATPase protein. Specifically, 1429 g of white oil, 70.2 g of Span, 8.43 g of aluminum stearate, and 53.3 g of Tween were added to every 1 L of the mixed vaccine stock solution. The mixture was then emulsified using an emulsifier to prepare an oil-emulsion adjuvant subunit vaccine, i.e., a biantigen subunit vaccine.

[0113] Following the preparation method of the biantigen subunit vaccine, a recombinant MCP subunit vaccine was prepared, with each dose (0.2 ml) containing 80 μg of recombinant MCP protein.

[0114] Following the preparation method of the biantigen subunit vaccine, a recombinant ATPase subunit vaccine was prepared, with each dose (0.2 ml) containing 80 μg of recombinant ATPase protein.

[0115] Example 6: Immunization Application of Dual Antigen Subunit Vaccine

[0116] 1) 150 large yellow croakers were randomly divided into 3 groups of 50 each. In the first group, each fish was injected intraperitoneally with 100 μL of a mixture of recombinant protein and adjuvant (i.e., the double antigen subunit vaccine in Example 5). In the second group (control group), each fish was injected intraperitoneally with 100 μL of a mixture of PBS buffer and adjuvant (PBS group). In the third group, each fish was injected intraperitoneally with 100 μL of recombinant MCP subunit vaccine. In the fourth group, each fish was injected intraperitoneally with 100 μL of recombinant ATPase subunit vaccine.

[0117] 2) Detection of the immunoprotective effect of the dual-antigen subunit vaccine against LYCIV iridovirus in large yellow croaker

[0118] Thirty days after immunizing large yellow croakers with the vaccine, four groups of fish were intraperitoneally injected with 3.50 × 10⁻⁶ ppm. 6 TCID50 / mL LYCIV virus solution (50μL); within 15 days, the mortality of large yellow croaker was observed, and the vaccine protection rate was statistically analyzed, as shown in Table 6. The results showed that the relative protection rate of the double antigen subunit vaccine group was 78.1%.

[0119] Table 6. Protection against iridovirus infection in large yellow croaker with vaccines

[0120]

[0121] Example 7: Dynamic detection of serum antibody levels in large yellow croaker after immunization

[0122] At weeks 1, 2, 3, 4, and 5 following intraperitoneal immunization, five large yellow croakers were randomly selected from each group to collect blood from their tail veins. After standing at room temperature for 2 hours, the blood samples were transferred to 4°C and incubated overnight. The next day, the blood samples were centrifuged at 8000g for 10 minutes at 4°C, and the supernatant serum was collected and placed in a suitable environment. Store at 20℃ for later use. The levels of specific antibodies in the serum of large yellow croaker at different time points after immunization were determined using ELISA. Specifically, a 96-well microplate was coated with a mixture of recombinant MCP protein and recombinant ATPase protein. Large yellow croaker serum was used as the primary antibody, mouse anti-large yellow croaker IgM polyclonal antibody as the secondary antibody, and alkaline phosphatase-labeled goat anti-mouse IgG as the tertiary antibody. The content of specific anti-antibodies in the serum was evaluated.

[0123] The results are as follows Figure 13As shown, compared with the recombinant MCP subunit vaccine and recombinant ATPase subunit vaccine experimental groups, the double antigen subunit vaccine group was able to significantly induce specific antibody responses in large yellow croaker, and the antibody level gradually increased with the extension of immunization time, reaching the highest level in week 5.

[0124] It should be understood that the embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A double antigenic subunit vaccine of a rainbow virus of Larimichthys crocea, characterized in that, It contains recombinant antigen of large yellow croaker iridovirus and a pharmaceutically acceptable carrier; The recombinant antigen of the large yellow croaker iridovirus includes: The recombinant MCP protein is encoded by a first nucleic acid molecule with the sequence shown in SEQ ID NO:1; The recombinant ATPase protein is encoded by a second nucleic acid molecule with the sequence shown in SEQ ID NO:2; The mass ratio of the recombinant MCP protein to the recombinant ATPase protein is 1:1; Furthermore, the recombinant MCP protein and recombinant ATPase protein were obtained by expression in insect cells using a baculovirus expression system.

2. The PVM subunit vaccine of claim 1, wherein the PVM subunit vaccine comprises: a) a first PVM antigen comprising a PVM VP2 protein; and b) a second PVM antigen comprising a PVM VP3 protein. The pharmaceutically acceptable carriers include any one or a combination of two or more of MONTANIDE ISA 206VG, ​​MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, and plant cell lectins.

3. A preparation method of a double antigenic subunit vaccine of a mirror virus of Larimichthys crocea, characterized in that, include: Provided a first nucleic acid molecule encoding a recombinant MCP protein and a second nucleic acid molecule encoding a recombinant ATPase protein, the sequence of the first nucleic acid molecule being shown in SEQ ID NO:1 and the sequence of the second nucleic acid molecule being shown in SEQ ID NO:2; S2. Construct a recombinant vector by cloning the nucleic acid molecules described in step S1 into a shuttle vector to obtain a recombinant shuttle vector containing the target gene. The shuttle vector is selected from pFastBac 1, pVL1393, pFastBac dual, or pDEST8. S3. Transform the recombinant shuttle vector into competent cells to obtain recombinant plasmids, and then transfect host cells to obtain recombinant baculoviruses. S4. The recombinant baculovirus is inoculated into host cells to obtain expression products, namely recombinant MCP protein and recombinant ATPase protein, wherein the host cells are selected from Sf9, High Five or Sf21 cells. S5. The purified recombinant MCP protein and recombinant ATPase protein were mixed at a mass ratio of 1:1 to obtain the recombinant antigen of large yellow croaker iridovirus. S6. Mix the recombinant antigen of large yellow croaker iridovirus with a pharmaceutically acceptable carrier.

4. The method as described in claim 3, characterized in that: The pharmaceutically acceptable carriers include any one or a combination of two or more of MONTANIDE ISA 206VG, ​​MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, and plant cell lectins.

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