Micropterus salmoides iridovirus mcp-2 recombinant protein and application thereof

Abstract

The application can obtain the core antigen epitope of the virus antigen by truncating the MCP-2 protein fragment expressing the MCP protein, improves the immune protection performance. Further, the MCP-2 protein is made into a subunit vaccine injection preparation, reduces the stress reaction symptoms after injection, improves the healthy rate of injection immunity, and high-intensity challenge test shows that the relative immune protection rate of the injection preparation is the highest. The application has a wide application prospect in the development of high-efficiency bigmouth bass iridovirus disease vaccine and its prevention and control.

Description

Technical fields:

[0001] This invention belongs to the field of biotechnology, specifically relating to the recombinant protein of largemouth bass iridovirus MCP-2 and its applications. Background technology:

[0002] Largemouth bass (Micropterus salmoides), native to freshwater rivers and lakes of North America, also known as California bass or black bass, belongs to the order Perciformes, suborder Porcoidei, family Cehtrachidae, and genus Micropterus. It is a eurythermal fish, characterized by its rapid growth, short farming cycle, tender and succulent flesh, and high price, making it highly sought after by fishermen. Largemouth bass farming has proven economically beneficial in China and has rapidly developed into a major aquaculture species in the past decade. However, largemouth bass are susceptible to various viral diseases during farming, with largemouth bass virus disease (LMBV) being the most serious.

[0003] To date, there is still a lack of effective treatments for diseases caused by LMBV. Vaccination is the most effective method for preventing infectious diseases in aquaculture. Therefore, the development of LMBV-related vaccines remains a top priority in addressing this viral disease. Currently, there are no commercially available largemouth bass iridovirus vaccines on the domestic or international market, and vaccine development for LMBV is still in the exploratory stage, mainly including nucleic acid vaccines and subunit vaccines. Researchers have found that LMBV DNA vaccines have good immunogenicity, with an immunoprotective rate of up to 63% (Yi et al. 2020). Although DNA vaccines can provide good immunoprotective effects, they pose potential risks to the body. Regarding subunit vaccines, Ma Dongmei et al. (2016) found that a subunit vaccine encoding the full-length MCP gene of largemouth bass iridovirus could achieve a protection rate of 67.7%. Jia et al. (2020) constructed a subunit vaccine that, through immersion immunization, was used to prevent infection caused by largemouth bass iridovirus, achieving an immunoprotective rate of over 80%.

[0004] Currently, fish vaccines are mainly administered through three methods: intraperitoneal or intramuscular injection, immersion or spraying, and oral immunization via feeding with vaccine-containing feed. Injection immunization involves injecting antigens into the fish, typically into the abdomen, muscles, or base of the pelvic fins. Injection immunization allows for precise control of the antigen dosage, resulting in high serum antibody levels and long-lasting immunity, providing high immune protection (generally 40%-90%). However, injection immunization is cumbersome, requiring highly skilled personnel and advanced equipment, consuming significant manpower, financial resources, and time. It is unsuitable for immunizing fry or small fish, and can easily cause stress in the fish. The accompanying physical damage can also lead to bacterial infections, resulting in decreased survival rates and reduced immunization effectiveness. Furthermore, vaccines alone are insufficient for effective immune protection during injection immunization; therefore, adjuvants are usually added to enhance vaccine efficacy. Traditional adjuvants (such as mineral oil) are widely used in fish bacterial vaccines, but these adjuvants are difficult to inject, can cause strong tissue inflammation, and produce significant side effects. Current research has also explored the use of carbon nanotube particles, β-glucan, plant polysaccharides, cytokines, and traditional Chinese medicine ingredients as adjuvants in fish vaccines. In the preparation of LMBV vaccines, reducing stress responses in fish, minimizing stress-induced mortality, and improving immune efficacy are pressing technical challenges that need to be addressed.

[0005] In addition, screening for dominant antigenic epitopes of viruses is a common method to improve vaccine efficacy. Dominant antigenic epitope screening generally aims to enhance vaccine immunoprotective effects by identifying core antigens with strong immunogenicity. Some antigenic epitopes on antigens can induce lymphocyte activation, thereby generating humoral and cellular immunity. However, lymphocytes can only recognize some small antigenic molecules, while an antigen usually contains a large number of antigenic epitopes. Therefore, discovering antigenic epitopes with strong immunogenicity is key to developing more effective subunit vaccines. The major capsid protein (MCP) is a widely recognized and feasible viral vaccine antigen. Although the LMBV MCP gene sequence of largemouth bass is conserved among different strains, previous experiments have shown that sequence analysis is insufficient to accurately obtain the distribution information of dominant antigenic epitopes in largemouth bass LMBV MCP. Currently, there are no reports of using the dominant antigenic epitopes of largemouth bass LMBV MCP for vaccine product development. Summary of the Invention:

[0006] To address the aforementioned technical challenges, based on previous research on MCP subunit vaccines, this invention further explores other possibilities for largemouth bass iris subunit vaccines to enrich the variety of largemouth bass iris subunit vaccines and explore more effective immunization strategies. This invention aims to provide a recombinant MCP-2 protein containing the major MCP antigenic epitopes and its applications. This recombinant protein exhibits good immunogenicity and is of positive significance for the preparation of subunit vaccines.

[0007] To solve the above technical problems, the present invention adopts the following technical solution:

[0008] A recombinant protein MCP-2 based on largemouth bass iridovirus antigen, characterized in that the amino acid sequence of the recombinant protein MCP-2 is shown in SEQ ID No: 5.

[0009] A gene encoding the recombinant protein MCP-2, characterized in that the nucleotide sequence of the gene is shown in SEQ ID No: 2.

[0010] An expression vector, said expression vector being pET32a-MCP-2, is an expression vector constructed based on the pET-32a expression vector and contains the nucleotide sequence shown in SEQ ID No: 2.

[0011] A genetically engineered strain for preparing the recombinant protein MCP-2 is a recombinant strain obtained by transforming the recombinant plasmid pET32a-MCP-2 into E. coli BL21(DE3).

[0012] A method for preparing the recombinant protein MCP-2, characterized by comprising the following steps:

[0013] Step 1: Construction of recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain; The recombinant nucleotide sequence with the sequence SEQ ID No: 2 was synthesized and cloned into the corresponding restriction sites on the pET-32a expression vector to obtain the recombinant plasmid pET32a-MCP-2. The recombinant plasmid was then transformed into E. coli BL21(DE3) to obtain the recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain;

[0014] Step 2: Induction and expression of recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain: A small amount of the production strain E. coli BL21 / pET32a-MCP-2 was picked up using an inoculation loop and streaked onto an LB solid medium plate. After static incubation at 37°C for 16-18 hours, a single colony was picked and inoculated into LB liquid medium. The culture was then incubated at 37°C at 160-180 rpm for 12-16 hours as the primary seed culture. The primary seed culture was then inoculated into LB liquid medium at a 1% (v / v) concentration and incubated at 37°C at 160-180 rpm for 14-16 hours as the secondary seed culture. The secondary seed culture was then inoculated into LB medium at a 1% (v / v) concentration, with ampicillin added to a final concentration of 100 μg / ml. Fermentation was carried out at 37°C with aeration for 5-7 hours at dissolved oxygen levels of 30-40%, until the OD500 of the bacterial culture was reached. 600 When the value is 1.1-1.3, add IPTG to a final concentration of 0.001 mol / L, induce culture at 37℃ for 6 hours, and then stop fermentation;

[0015] Step 3: Bacterial solution treatment and ultrasonic disruption;

[0016] Step 4: Protein purification: using Ni 2+ The protein was obtained by affinity chromatography using a chelate affinity chromatography column, and then purified by dialysis.

[0017] A subunit vaccine formulation for largemouth bass iridovirus MCP-2 is characterized in that the subunit vaccine formulation comprises the following components: 100 μg / mL recombinant MCP-2 protein, 5 mg / mL astragalus polysaccharide, 2 mg / mL taurine, 50 μg / mL sodium selenite, 20 μg / mL zinc sulfate, 2 mg / mL inosine, 500 μg / mL sodium ascorbate, and 50 mg / mL span-80; the amino acid sequence of the recombinant protein MCP-2 is shown in SEQ ID No: 5.

[0018] The nucleotide sequence encoding the recombinant protein MCP-2 is shown in SEQ ID No: 2.

[0019] The preparation method of the recombinant protein MCP-2 includes the following steps:

[0020] Step 1: Construction of recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain; The recombinant nucleotide sequence with the sequence SEQ ID No: 2 was synthesized and cloned into the corresponding restriction sites on the pET-32a expression vector to obtain the recombinant plasmid pET32a-MCP-2. The recombinant plasmid was then transformed into E. coli BL21(DE3) to obtain the recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain;

[0021] Step 2: Induction and expression of recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain: A small amount of the production strain E. coli BL21 / pET32a-MCP-2 was picked up using an inoculation loop and streaked onto an LB solid medium plate. After static incubation at 37°C for 16-18 hours, a single colony was picked and inoculated into LB liquid medium. The culture was then incubated at 37°C at 160-180 rpm for 12-16 hours as the primary seed culture. The primary seed culture was then inoculated into LB liquid medium at a 1% (v / v) concentration and incubated at 37°C at 160-180 rpm for 14-16 hours as the secondary seed culture. The secondary seed culture was then inoculated into LB medium at a 1% (v / v) concentration, with ampicillin added to a final concentration of 100 μg / ml. Fermentation was carried out at 37°C with aeration for 5-7 hours at dissolved oxygen levels of 30-40%, until the OD500 of the bacterial culture was reached. 600 When the value is 1.1-1.3, add IPTG to a final concentration of 0.001 mol / L, induce culture at 37℃ for 6 hours, and then stop fermentation;

[0022] Step 3: Bacterial solution treatment and ultrasonic disruption;

[0023] Step 4: Protein purification using Ni 2+ The protein was obtained by affinity chromatography using a chelate affinity chromatography column, and then purified by dialysis.

[0024] The present invention also claims protection for the use of the recombinant protein of largemouth bass iridovirus MCP-2 in the preparation of an iridovirus subunit vaccine, wherein the recombinant protein of MCP-2 can be used for injection immunization of largemouth bass and can stimulate the body to produce stronger corresponding antibodies compared to the MCP protein.

[0025] Based on the above technical solutions, the present invention has the following advantages and beneficial effects:

[0026] Firstly, based on the company's prior research, this invention, by truncating the MCP-2 protein fragment expressing the effective immunogenic protein MCP, can obtain the core antigenic epitopes of the virus, thereby improving immunoprotective performance. Animal experiments show that immunization with the MCP-2 recombinant protein produces higher titers of neutralizing antibodies compared to immunization with the MCP recombinant protein alone. On day 7 post-immunization, the highest dose of the MCP-2 recombinant protein group showed significantly higher immune-related indicators than the MCP group. Based on these experimental results, this invention, by truncating the MCP-2 protein fragment expressing the MCP protein, exhibits better immunogenicity than using the MCP protein fragment alone, stimulating the body to produce higher levels of neutralizing antibodies, which is of positive significance for the prevention of LMBV.

[0027] Secondly, this invention addresses the problems of fish mortality caused by stress reactions and wound infections during aquatic injection immunization by optimizing and adjusting the formulation of the injection preparation. Taurine, sodium selenite, zinc sulfate, and inosine are added to the injection preparation, and these components work synergistically, significantly improving the stress resistance of sea bass, reducing post-injection stress symptoms, and increasing the survival rate of immunized fish. High-intensity challenge tests show that the injection preparation of this invention has the highest relative immunoprotection rate, reaching 89.2%, which is nearly 13.5 percentage points higher than the 75.7% of MCP-2 protein alone, achieving unexpected technical results.

[0028] In summary, this invention, by truncating the MCP-2 protein fragment expressing the MCP protein, can obtain the core antigenic epitope of the virus, thereby improving immunoprotective performance. Furthermore, by formulating the MCP-2 protein into a subunit vaccine injectable formulation, post-injection stress symptoms are reduced, and the survival rate of injected immunizations is improved. High-intensity challenge experiments show that the injectable formulation of this invention has the highest relative immunoprotective rate. This invention has broad application prospects in the development of highly effective vaccines against largemouth bass iridovirus disease and in the prevention and control of largemouth bass iridovirus disease. Attached image description:

[0029] Figure 1 This was used for the identification of truncated recombinant plasmids. M: Maker DL 5000; 1-4: double digestion products of MCP-1, MCP-2, MCP-3, and MCP-4; labeled as the target gene.

[0030] Figure 2 SDS-PAGE was used to identify the expression of recombinant LMBV-MCP-2 protein. Wherein, M: Marker; 1: E. coli BL21 / pET32a-MCP-2 culture after induction.

[0031] Figure 3Western blot identification of recombinant LMBV-MCP-2 protein. Wherein, M: Marker; 1: Uninduced E. coli BL21 / pET32a-MCP-2 culture; 2: Induced E. coli BL21 / pET32a-MCP-2 culture.

[0032] Figure 4 This is the result of serum antibody titer determination after immunization. Specific implementation examples:

[0033] Example 1:

[0034] Construction and identification of recombinant Escherichia coli strain E. coli BL21 / pET32a-MCP-2.

[0035] 1. Materials and Methods

[0036] 1.1 Materials

[0037] 1.1.1 Plasmids and Strains

[0038] The pET-32a-MCP plasmid and the E. coli BL-21 / pET-32a-MCP prokaryotic expression strain were preserved by the Aquatic Animal Disease Laboratory of the College of Animal Science and Technology, Northwest A&F University.

[0039] 1.1.2 Reagents

[0040] Sodium chloride, anhydrous ethanol, and glycerol were purchased from Sinopharm Chemical Reagent Co., Ltd.; positive serum for LMB / VMCP protein immunization of largemouth bass was preserved by the Aquatic Disease Laboratory of the College of Animal Science and Technology, Northwest A&F University; TMB enzyme colorimetric kit, BCA protein concentration assay kit, and PBST were purchased from Beijing Solarbio Science & Technology Co., Ltd.; dialysis bags, mouse-derived 6×histidine (His)-tagged monoclonal antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG monoclonal antibody, and His-tagged protein purification kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.; primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; protein markers were purchased from Tiangen Biotech (Beijing) Co., Ltd.; tryptone and yeast extract were purchased from Sigma-Aldrich (USA); IPTG and ampicillin were purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0041] 1.1.3 Test Instruments

[0042] Electronic balance ALC-1100.2, Beijing Sartorius Instrument Systems Co., Ltd.; Clean bench YT-CJ-2ND, Beijing Yatai Kelon Instrument Technology Co., Ltd.; T100 PCR instrument, Bio-Rad, USA; H1650-W benchtop high-speed microcentrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; KQ-500DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; 1-15K high-speed refrigerated centrifuge, Sigma, USA; Ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.; DYCZ-24DN vertical electrophoresis apparatus, Beijing Liuyi Instrument Factory; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA.

[0043] 1.2 Test Methods and Results

[0044] 1.2.1 Gene identification and expression plasmid construction of each truncated form of MCP

[0045] Based on the MCP protein gene sequence of LMBV FS001 strain (Accession no.: OM319463), the gene sequence was truncated into four segments and named MCP-1 (SEQ ID No: 1), MCP-2 (SEQ ID No: 2), MCP-3 (SEQ ID No: 3), and MCP-4 (SEQ ID No: 4). There is a 30 bp overlap between the two segments. The gene sequence was sent to a biotechnology company to synthesize an expression vector ligated to pET-32a.

[0046] The constructed recombinant plasmids pET-32a-MCP-1, pET-32a-MCP-2, pET-32a-MCP-3, and pET-32a-MCP-4 were identified by double enzyme digestion and sequencing, respectively. Electrophoresis was performed after enzyme digestion (e.g.,...). Figure 1 As shown in the figure, combined with the plasmid sequencing results, it is shown that each plasmid was constructed correctly. Among them, the band size corresponding to the MCP gene is 1392bp, and the band sizes corresponding to the segmented genes MCP-1, MCP-2, MCP-3 and MCP-4 are 375, 378, 378 and 369bp, respectively.

[0047] 1.2.2 Construction of prokaryotic expression strains for each truncated MCP gene

[0048] The recombinant plasmids of each gene segment were named pET-32a-MCP-1, pET-32a-MCP-2, pET-32a-MCP-3, and pET-32a-MCP-4, respectively, thus obtaining the corresponding prokaryotic expression strains E. coli BL-21 / pET-32a-MCP-1, E. coli BL-21 / pET-32a-MCP-2, E. coli BL-21 / pET-32a-MCP-3, and E. coli BL-21 / pET-32a-MCP-4.

[0049] 1.2.3 Prokaryotic expression of MCP and its truncated form

[0050] The four truncated prokaryotic expression strains constructed in the previous step and the expression strain containing the untruncated MCP gene were inoculated separately into 100 mL of LB liquid medium containing ampicillin (100 μg / mL) and cultured in a shaker at 37°C and 180 rpm until the bacteria reached the logarithmic growth phase (OD50). 600 =1.1-1.5), add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.001 mol / L in the culture medium, and continue to shake and incubate for 6-8 h to end the induction.

[0051] 1.2.4 Isolation and purification of MCP and its truncated form

[0052] Collect the bacterial culture induced in the previous step, centrifuge at 12000g and 4℃ for 10 min, and discard the supernatant. Resuspend the precipitate in an equal volume of phosphate-buffered saline (PBS), and then sonicate the cells using an ultrasonic cell disruptor (300W, 2s sonication, 3s interval) until the culture is clear and transparent (the reaction is performed on ice). Then, pass the culture through a His-tagged Ni... 2+ The obtained protein liquid was purified by chelate affinity chromatography column, and the purified protein solution was freeze-dried to make lyophilized powder, which was stored at -20℃ and dissolved in sterile water before use.

[0053] 1.2.5 Immunogenicity evaluation of the truncated MCP

[0054] Immunogenicity was evaluated using an enzyme-linked immunosorbent assay (ELISA). The specific procedure was as follows: First, largemouth bass positive serum was diluted 1:1000 with ELISA coating buffer. 100 μL of the diluted serum was added to each well of the ELISA plate as antigen, and the plate was coated at 4°C for 24 h. After discarding the liquid in the wells, 200 μL of 1% bovine serum albumin was added to each well, and the plate was blocked at 37°C for 1 h. After blocking, the plate was washed three times with phosphate-buffered saline with Tween-20 (PBST) for 3 min each time. Then, 100 μL of the protein solution after separation and purification of different recombinant plasmid expression products was added to each well, and the plate was incubated at 37°C for 1 h. After washing three times with PBST, the plate was incubated at 37°C for 1 h each time with a mouse 6×His-tagged monoclonal antibody as the primary antibody and an HRP-labeled goat anti-mouse IgG monoclonal antibody as the secondary antibody. After rinsing three times with PBST, TMB chromogenic solution was added, and the reaction was carried out at 37°C in the dark for 3-5 minutes. Then, 50 μL of stop solution was added to each well, and the absorbance (OD) of each well was measured at 450 nm using a microplate reader within 20 minutes. 450 The results are shown in Table 1.

[0055] Table 1. Immunogenicity evaluation of MCP truncated derivatives

[0056]

[0057] Under the same target protein content, Table 1 shows that the OD value of MCP-2 is significantly higher than that of MCP and other segments of MCP, proving that the immunogenicity of MCP-2 is significantly higher than that of MCP and other segments of MCP. This result provides a basis for the final screening to obtain the dominant antigenic epitope of the main capsid protein of largemouth bass iridovirus.

[0058] 1.2.6 Identification of MCP-2, the dominant antigenic epitope protein of MCP

[0059] The truncated somatic protein MCP-2, which yielded the best results in the previous step, was selected for SDS-PAGE detection and identification. The specific procedure is as follows: The sample was denatured using 5×SDS-PAGE loading buffer. A lower separating gel and an upper stacking gel were prepared. After the gel solidified, they were immersed in electrophoresis buffer. The denatured sample was added to the gel wells, and electrophoresis was performed at 80V and 120V for 20 and 80 min, respectively. The electrophoresis results were observed by Coomassie brilliant blue staining and destaining, and Western blotting analysis was performed using a mouse His-tagged monoclonal antibody as the primary antibody and an HRP-labeled goat anti-mouse IgG monoclonal antibody as the secondary antibody.

[0060] SDS-PAGE analysis was performed on the protein product expressed by the recombinant plasmid containing the dominant MCP-2 epitope. The results showed that the expressed MCP-2-containing target protein was approximately 35 kDa in size. Figure 2 The results were consistent with expectations; in the Western blotting analysis, a clear identification band consistent with the design was observed at 35 kDa. Figure 3 These results indicate that the recombinant plasmid pET-32a-MCP-2 can effectively express the truncated MCP-2 protein in Escherichia coli.

[0061] Example 2

[0062] Immunogenicity of MCP-2 subunit vaccine against dominant antigenic epitope of largemouth bass iridovirus primary capsid protein.

[0063] 2 Materials and Methods

[0064] 2.1 Materials

[0065] 2.1.1 Experimental animals and viruses

[0066] Healthy largemouth bass (1.0±0.5g, 4-5cm) were purchased from a fish farm in Foshan City, Guangdong Province. The water temperature was maintained at 25±1℃, and dissolved oxygen was kept above 6mg / L. Fish were fed twice daily, at 8:00 AM and 5:00 PM, with uneaten food and feces promptly removed from the bottom of the tank. Half the water volume was changed every two days. The experiment was conducted after a 14-day period of temporary rearing. The largemouth bass iridovirus used was preserved in the Aquatic Animal Disease Laboratory of Northwest A&F University.

[0067] 2.1.2 Reagents

[0068] The truncated MCP variant (specifically MCP-2) was prepared in the Aquatic Animal Disease Laboratory of the College of Animal Science and Technology, Northwest A&F University, following the method described in Example 1 above. Mouse-derived His-tagged monoclonal antibody and HRP-labeled goat anti-mouse IgG monoclonal antibody were purchased from Sangon Biotech (Shanghai) Co., Ltd. All other reagents were of analytical grade.

[0069] 2.1.3 Test Instruments

[0070] ALC-1100.2 electronic balance, Beijing Sartorius Instrument Systems Co., Ltd.; HH-4 digital display constant temperature water bath, Shanghai Techeng Machinery Equipment Co., Ltd.; H1650-W benchtop high-speed microcentrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; 1-15K high-speed refrigerated centrifuge, Sigma-Aldrich, USA; ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.; DYCZ-24DN vertical electrophoresis apparatus, Beijing Liuyi Instrument Factory; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA; T100 PCR instrument, Bio-Rad Laboratories, USA.

[0071] 2.2 Test Methods

[0072] 2.2.1 Immunity

[0073] Healthy largemouth bass were anesthetized and immunized via intramuscular injection at the base of the dorsal fin after being held for 14 days, with an injection volume of 10 μL per fish. The prepared MCP and MCP-2 were dissolved in sterile PBS at their respective concentrations to serve as vaccines, and diluted to different concentration gradients to evaluate the efficacy of the vaccines at different immunization doses. The treatment dose for the MCP group was 10.0 μg / fish, and the treatment doses for the MCP-2 groups were 1.0, 5.0, and 10.0 μg / fish (see Table 2 for specific groupings). Each vaccine treatment group contained 60 fish, and each group consisted of three replicates.

[0074] Table 2. Immunization Grouping

[0075]

[0076] 2.2.2 Serum immune antibody titer determination

[0077] Blood samples from largemouth bass were collected at 7, 14, 21, and 28 days post-immunization, with three fish sampled each time for antibody titer determination. The collected blood samples were allowed to stand at room temperature for 2 hours, then incubated overnight at 4°C to allow for natural coagulation. Finally, the samples were centrifuged at 5000g for 10 minutes using a refrigerated centrifuge, and the supernatant serum was collected and stored at -20°C for subsequent antibody titer determination. For antibody titer determination, purified MCP was used as the antigen, largemouth bass serum was used as the test serum, mouse-His-tagged monoclonal antibody was used as the primary antibody, and HRP goat anti-mouse IgG monoclonal antibody was used as the secondary antibody, all diluted 1:1000. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antibody titer in the serum. After color development, the absorbance was measured at 450nm using a microplate reader.

[0078] The results of serum antibody titer determination of largemouth bass after injection immunization are as follows: Figure 4As shown, starting 14 days post-immunization, antibody titers in both the MCP and MCP-2 vaccine treatment groups gradually increased with the extension of immunization time. At each detection time point, antibody titers were significantly higher than those in the control group, reaching their peak at 28 days post-immunization. At 28 days post-immunization, antibody titers in all MCP-2 vaccine treatment groups were significantly higher than those in the control group, with the highest dose (10 μg / tail) showing significantly higher antibody titers, approximately three times higher than the control group. Furthermore, at 28 days post-immunization, the antibody titers in the 5 μg / tail MCP-2 vaccine treatment group were already higher than those in the 10 μg / tail MCP vaccine treatment group.

[0079] 2.2.3 Challenge Test

[0080] Twenty-eight days after immunization, 37 largemouth bass were randomly selected from each group and placed in new tanks with identical rearing conditions for a challenge experiment. Each fish was injected intraperitoneally with 3.50 × 10⁻⁶ ppm. 6 TCID 50 / mL LMBV virus solution (50μL). Observe continuously for 14 days, regularly check and record the morbidity, and finally calculate the mortality rate and relative immune protection rate:

[0081] Relative immunization protection rate = (1 - mortality rate of vaccine treatment group / mortality rate of control group) × 100%.

[0082] Largemouth bass were challenged with LMBV virus 28 days after immunization. Morbidity and mortality were recorded daily. After 14 days of challenge, the largemouth bass in each group reached a relatively stable state with little population change. Table 3 shows the statistical results of mortality and relative immunoprotection rates for each group during these 14 days. The results indicate that the mortality rate in the control group was 100%, and the mortality rates in all vaccine-treated groups were lower than those in the control group, while the relative immunoprotection rates were higher in all groups. 14 days after challenge, the MCP-2 group (specifically the highest dose vaccine-treated group) had the highest relative immunoprotection rate at 73.0%, while the relative immunoprotection rate in the MCP vaccine-treated group was only 45.9%.

[0083] Table 3. Mortality and relative immunoprotection rate after immunization and challenge.

[0084]

[0085] Based on the above experimental results, it can be seen that the MCP-2 recombinant protein has relatively better immunogenicity than the MCP recombinant protein. It can stimulate the body to produce higher antibody levels in the early stage of immunity, thereby achieving immune protection in the early stage of infection. This has positive significance for the prevention of disease in fish populations in the early stage of the disease. It can reach an appropriate antibody level in a shorter time, which is also of positive significance for preventing the mass mortality of fish fry in the early stage of infection.

[0086] Example 3.

[0087] Preparation of injectable formulation of MCP-2 subunit vaccine based on dominant antigenic epitope of largemouth bass iridovirus primary capsid protein.

[0088] This invention further investigates how to enhance the immunogenicity of the MCP-2 subunit vaccine injection formulation and reduce sea bass mortality caused by stress during the injection immunization process. The MCP-2 recombinant protein mentioned below is a purified protein (purity ≥60%); Astragalus polysaccharide (analytical grade) was purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd., CAS: 89250-26-0; Taurine, sodium selenite, zinc sulfate, inosine, sodium ascorbate, and Span-80 were all purchased from reagent companies and meet the relevant requirements for pharmacopoeia injections.

[0089] The MCP-2 subunit vaccine injection formulation of the present invention is composed of the following components: 100 μg / mL MCP-2 recombinant protein, 5 mg / mL Astragalus polysaccharide, 2 mg / mL taurine, 50 μg / mL sodium selenite, 20 μg / mL zinc sulfate, 2 mg / mL inosine, 500 μg / mL sodium ascorbate, and 50 mg / mL Span-80.

[0090] The preparation method of the MCP-2 subunit vaccine injection formulation includes the following steps:

[0091] First, weigh out the following ingredients according to their weight: MCP-2 recombinant protein, astragalus polysaccharide, taurine, sodium selenite, zinc sulfate, inosine, sodium ascorbate, and Span-80.

[0092] Second, the weighed astragalus polysaccharide, taurine, sodium selenite, zinc sulfate, inosine, and sodium ascorbate were dissolved in physiological saline. After stirring and dissolving, span-80 and MCP-2 recombinant protein were added, and physiological saline was used to bring the volume to a constant. After stirring quickly and evenly, a mixed emulsion was obtained.

[0093] Third, the mixed emulsion is filtered and sterilized, and then packaged to obtain the MCP-2 subunit vaccine injection formulation.

[0094] Example 4.

[0095] In the research process of the injectable formulation of this invention, a large number of experiments and studies were conducted on the selection of components and the determination of dosage in order to obtain the optimal injectable formulation. Specifically, the following experiments were also conducted during the research process of this invention, which are shown here for comparison:

[0096] Table 4 Composition and Proportioning Test of Injectable Formulations

[0097]

[0098]

[0099] The above-described Examples 3 and Control Groups 1-5 were prepared using the method described in Example 3, respectively, to obtain the subunit vaccine injection formulation of the present invention and the injection formulations of Control Groups 1-5.

[0100] Example 5.

[0101] Animal experiments on the injectable formulation of the MCP-2 subunit vaccine.

[0102] 5.1 Safety and post-immunization health:

[0103] 350 healthy largemouth bass (100-120g each) were collected and temporarily raised for 14 days. They were then divided into an immunization group and a control group, with 50 bass in each group. After anesthesia, immunization was administered via intramuscular injection at the base of the dorsal fin. Each bass in the immunization group received 0.1mL of the corresponding vaccine, while the control group received physiological saline instead of the vaccine. After injection, the bass were transferred to normal aquaculture water for recovery and feeding. They were observed continuously for 28 days, and their survival rate was recorded. The specific results are shown in Table 5 below.

[0104] Table 5. Liver status 28 days after immunization.

[0105]

[0106]

[0107] Based on the results in Table 5, it can be seen that within 28 days after injection, the stress response and trauma caused by the injection resulted in a certain degree of mortality in the bass. In the blank control group, 10 bass died, exhibiting a significant decrease in feed intake during feeding and showing obvious stress symptoms. The injection wounds of the dead bass also showed signs of infection, with a survival rate of 80%. In contrast, Example 3 and Control Groups 1-3 all showed higher survival rates. In particular, Example 3, with the optimal formula obtained in this invention, resulted in only one death, achieving a survival rate as high as 98%. Furthermore, the bass in this group showed milder stress symptoms, maintained normal feed intake as before immunization, and showed no difference in agility or other aspects compared to before immunization. In addition, the comparison between Control Groups 2 and 3 and Example 3 shows that the simultaneous addition of taurine, sodium selenite, zinc sulfate, and inosine in this invention significantly improved the bass's stress resistance, reduced post-injection stress symptoms, and increased the survival rate after injection immunization.

[0108] 5.2 Virus Challenge Test

[0109] Twenty-eight days after immunization, 37 largemouth bass were randomly selected from each group and placed in new tanks with identical rearing conditions for a challenge experiment. Each fish was injected intraperitoneally with 3.50 × 10⁻⁶ ppm. 6 TCID 50 / mL LMBV virus solution (50μL). Observe continuously for 14 days, regularly check and record the morbidity, and finally calculate the mortality rate and relative immune protection rate:

[0110] Relative immune protection rate = (1 - mortality rate of vaccine treatment group / mortality rate of blank control group) × 100%.

[0111] Largemouth bass were challenged with LMBV virus 28 days after immunization. Morbidity and mortality were recorded daily. After 14 days of challenge, the largemouth bass in each group reached a relatively stable state with little population change. Table 6 shows the statistical results of mortality and relative immunity rates for each group during these 14 days.

[0112] Table 6. Mortality and relative immunoprotection rate after challenge with different injectable formulations.

[0113] Healthy last digit mortality rate(%) Relative immune protection rate (%) Example 3 33 10.8％ 89.2％ Blank control group 0 100.0％ 0.0％ Comparison 1 29 21.6％ 78.4％ Comparison 2 30 18.9％ 81.1％ Comparison 3 30 18.9％ 81.1％ Compare with 4 28 24.3％ 75.7％ Compare with 5 31 16.2％ 83.8％

[0114] Based on the results in Table 6, the mortality rate of the blank control group was 100%. The mortality rates of all vaccine treatment groups were lower than those of the blank control group, while the relative immune protection rates were higher in all vaccine treatment groups. After 14 days of challenge, Example 3 showed the highest relative immune protection rate, reaching 89.2%, which was nearly 13.5 percentage points higher than the 75.7% of Control Group 4. Moreover, compared with Control Groups 1-3, there was a certain synergistic effect among the components of the injectable formulation of the present invention. The lack of components such as Astragalus polysaccharide, zinc sulfate, and sodium selenite would reduce the immune effect. Furthermore, compared with Control Group 5, the specific ratio of the injectable formulation of the present invention also significantly improved the immune effect.

[0115] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A large-mouth bass iridovirus MCP-2 subunit vaccine injection preparation, characterized by, The subunit vaccine injection preparation is composed of the following components: MCP-2 recombinant protein 100 μg / mL, astragalus polysaccharide 5 mg / mL, taurine 2 mg / mL, sodium selenite 50 μg / mL, zinc sulfate 20 μg / mL, inosine 2 mg / mL, sodium ascorbate 500 μg / mL, span-80 50 mg / mL; the amino acid sequence of the MCP-2 recombinant protein is shown as SEQ ID No:

5.

2. The injection formulation of Micropterus salmoides iridovirus MCP-2 subunit vaccine according to claim 1, characterized in that, The preparation method of the MCP-2 recombinant protein comprises the following steps: Step 1: Recombinant Escherichia coli E. coli Construction of BL21 / pET32a-MCP-2 strain; a recombinant nucleotide sequence of SEQ ID No: 2 was synthesized, which was cloned into the corresponding enzyme cutting sites between the pET-32a expression vector to obtain a recombinant plasmid pET32a-MCP-2, and the recombinant plasmid was transformed into E. coli BL21 (DE3), i.e. a recombinant Escherichia coli E. coli BL21 / pET32a-MCP-2 strain; Step 2: Recombinant Escherichia coli E. coli Induction of expression of BL21 / pET32a-MCP-2 strain: The production strain E. coli Using an inoculation loop, a small amount of BL21 / pET32a-MCP-2 bacterial culture was streaked onto an LB solid medium plate and incubated statically at 37°C for 16-18 hours. A single colony was then inoculated into LB liquid medium and incubated at 37°C at 160-180 rpm for 12-16 hours as the primary seed culture. The primary seed culture was then inoculated at 1% (v / v) onto LB liquid medium and incubated at 37°C at 160-180 rpm for 14-16 hours as the secondary seed culture. The secondary seed culture was then inoculated into LB medium at 1% (v / v), with ampicillin added to a final concentration of 100 μg / ml. Fermentation was carried out at 37°C with aeration for 5-7 hours at dissolved oxygen levels of 30-40%, until the OD of the bacterial culture was reached. 600 When the value is 1.1-1.3, add IPTG to a final concentration of 0.001 mol / L, induce culture at 37℃ for 6 hours, and then stop fermentation; Step three: bacteria liquid treatment and ultrasonic crushing; Step four: Purification of the protein: The protein was isolated by affinity chromatography using a Ni 2＋ chelating affinity chromatography column and the purified protein was obtained by dialysis.

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