Micropterus salmoides iridovirus polyclonal antibody, MCP subunit vaccine as well as preparation method and application of micropterus salmoides iridovirus polyclonal antibody and MCP subunit vaccine

Abstract

The invention discloses a micropterus salmoides iridovirus polyclonal antibody, an MCP subunit vaccine as well as a preparation method and application thereof, and belongs to the technical field of biology. The micropterus salmoides iridovirus polyclonal antibody is derived from serum generated after a white rabbit is immunized by MCP recombinant protein and MCP372 recombinant protein, and the gene sequence of the MCP recombinant protein is shown as SEQ ID No: 1; the gene sequence of the MCP372 recombinant protein is as shown in SEQ ID No: 2. According to the present invention, the MCP full length and the MCP main protection region 372 fragment are subjected to recombinant expression, the polyclonal antibody is prepared, and the highest titers obtained by detecting the specificity are 1: 128,000 and 1: 64,000 respectively. The LMBV inactivated vaccine and the MCP subunit vaccine are successfully constructed, the immune effect is evaluated, and it is found that the immune protection effect of the MCP372 medium-concentration subunit vaccine is the best.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polyclonal antibody against largemouth bass iridovirus, an MCP subunit vaccine, and their preparation methods and applications. Background Technology

[0002] Largemouth bass (Micropterus salmoides) are fast-growing and highly adaptable, making them one of the important freshwater aquaculture species in my country. In recent years, largemouth bass farming production has steadily increased. However, with the continuous increase in high-density intensive farming, disease problems occur frequently during the farming process, seriously affecting the healthy development of the largemouth bass aquaculture industry. Among these, the most damaging is largemouth bass ranavirus (LMBV) infection, which causes large-scale mortality of adult largemouth bass and results in huge economic losses for the industry. Currently, there is no specific treatment for this disease; therefore, effective immunization is crucial. Research reports that the major capsid protein (MCP) of LMBV has good immunogenicity and can be used as a candidate subunit vaccine.

[0003] Subunit vaccines are a type of genetically engineered vaccine. They are prepared by transferring protective genes from bacteria or viruses into expression vectors for antigen protection, then mixing the vectors with adjuvants. These vaccines are non-pathogenic, highly immunogenic, and have a single, highly effective composition. There are many types of expression vectors, with E. coli expression vectors being the most widely used, but yeast and lactic acid bacteria expression vectors are also increasingly being developed. The grouper iridovirus immersion subunit vaccine generates an immersion subunit vaccine by binding the novel adjuvant NE to the MCP protein. MCP-specific antibodies can be detected two weeks after immunization, improving the survival rate of infected fish in a dose-dependent manner. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to propose a polyclonal antibody against largemouth bass iridovirus, an MCP subunit vaccine, and its preparation method and application.

[0005] The present invention solves the above-mentioned technical problems through the following technical means: The first aspect of the present invention provides a polyclonal antibody against largemouth bass iridovirus, derived from serum produced after immunizing rabbits with MCP372 recombinant protein, the sequence of which is shown in SEQ ID No: 5.

[0006] The MCP372 recombinant protein can also be replaced with the MCP recombinant protein, the sequence of which is shown in SEQ ID No: 3.

[0007] Preferably, the MCP372 recombinant protein is obtained by purifying Escherichia coli transformed with a recombinant expression vector after induction of expression.

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned polyclonal antibody, comprising the following steps: preparing the above-mentioned MCP372 recombinant protein by prokaryotic expression, purifying and refolding it, immunizing New Zealand white rabbits, and then collecting serum to prepare polyclonal antibodies.

[0009] Preferably, the purification and refolding method includes the following steps: passing the recombinant protein through a column, washing it with imidazole washing solution, eluting it with elution solution to obtain purified protein, and dialyzing the purified protein in urea solution to obtain the final product.

[0010] A third aspect of the present invention provides a recombinant MCP372 protein, the sequence of which is shown in SEQ ID No: 5.

[0011] A fourth aspect of the present invention provides a recombinant expression vector containing a nucleotide sequence as shown in SEQ ID No: 5.

[0012] Preferably, the recombinant expression vector is pET-28a-MCP372, which is an expression vector constructed based on pET-28a.

[0013] The fifth aspect of this invention provides a genetically engineered strain for preparing the above-mentioned recombinant MCP372 protein, wherein the recombinant plasmid pET-28a-MCP372 is transformed into... E. coli Recombinant strains obtained from DH5α competent cells.

[0014] A sixth aspect of the present invention discloses the application of the above-mentioned MCP372 recombinant protein, recombinant expression vector, genetically engineered strain, and polyclonal antibody in the preparation of a product for detecting largemouth bass iridovirus. The product includes a detection kit.

[0015] A seventh aspect of the present invention provides a kit for detecting largemouth bass iridovirus, comprising the aforementioned polyclonal antibody.

[0016] The eighth aspect of the present invention provides a largemouth bass iridovirus MCP subunit vaccine, the active ingredient of which includes MCP recombinant protein or MCP372 recombinant protein; the sequence of the MCP recombinant protein is shown in SEQ ID No: 3; the sequence of the MCP372 recombinant protein is shown in SEQ ID No: 5.

[0017] Preferably, the concentration of the MCP recombinant protein or MCP372 recombinant protein is 0.1~0.4 μg / μL, more preferably 0.2 μg / μL or 0.4 μg / μL.

[0018] The ninth aspect of the present invention provides a method for preventing and controlling largemouth bass iridovirus, comprising the following steps: injecting the above-mentioned MCP subunit vaccine into the abdominal cavity of largemouth bass.

[0019] The beneficial effects of this invention are as follows: 1. This invention successfully constructed an efficient E. coli expression system for expressing both full-length MCP protein and truncated MCP372 protein, and purified the target proteins with single and bright bands. Two rabbit polyclonal antibodies were prepared by immunizing rabbits, and their titers were detected by ELISA. The results showed that the titer of the MCP372 polyclonal antibody was 1:128,000, and the titer of the MCP polyclonal antibody was 1:64,000. The antibody titer produced by the MCP372 protein was higher than that of the MCP protein. Indirect immunofluorescence detection showed that the antigen and antibody specifically bound, and both exhibited bright green fluorescence. The MCP protein produced more green fluorescence than the MCP372 protein, indicating that the full-length MCP protein had more binding sites, and that both polyclonal antibodies specifically bound to it. Subsequent Western blot analysis showed that the target bands were detected at the expected positions of 54 kDa and 17 kDa, indicating that both prepared polyclonal antibodies could specifically recognize the MCP protein.

[0020] 2. This invention successfully constructed an LMBV inactivated vaccine and an MCP subunit vaccine. The immunization effects were evaluated, and it was found that the medium-concentration MCP372 subunit vaccine (100 μL of 15 g largemouth bass containing 10 μg MCP372 protein) had the best immunoprotective effect.

[0021] 3. This invention developed two types of MCP subunit vaccines and an inactivated vaccine, and evaluated the immunogenicity of the vaccines through animal experiments. Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0022] Figure 1 These are partial pathological materials and anatomical diagrams from Embodiment 1 of the present invention; Figure 2 This is a PCR detection image of suspected LMBV samples in Example 1 of the present invention; Figure 3 The images show the pathological phenomena of FHM cells after challenge with the virus in Example 1 of this invention, where (a) represents normal cells; (b) represents cells infected with the virus for 12 hours; (c) represents cells infected with the virus for 24 hours; and (d) represents cells infected with the virus for 36 hours. Figure 4This is a diagram showing the amplification results of the MCP gene in Example 2 of the present invention; where M: 2000 marker; lanes 1-2: positive iridovirus DNA; Figure 5 The diagram shows the transformation results of the recombinant plasmid in Example 2 of this invention; where (a): verification of DH5α-pET-28a-MCP colonies; M: 2000 marker; lane N: negative control water; lane P: pET-28a plasmid; lanes 1-3: colonies with failed ligation; lanes 4-5: positive colonies with successful ligation; (b): verification of BL21-pET-28a-MCP colonies; M: 2000 marker; lane N: pET-28a plasmid; lanes 1-3: colonies with failed transformation; lane 4: positive colonies with successful transformation; Figure 6 This is a diagram showing the amplification results of the MCP372 gene in Example 2 of the present invention; M: 2000 marker; lanes 1-2: positive iridovirus DNA; Figure 7 This is a diagram showing the transformation results of the recombinant plasmid in Example 2 of the present invention; where (a): verification of DH5α-pET-28a-MCP372 colonies; M: 2000 marker; lanes 1 and 3: primers MCP372-F / R; lanes 2 and 4: primers T7-F / R; (b): verification of BL21-pET-28a-MCP colonies; M: 2000 marker; lane N: pET-28a plasmid; lanes 1-2: successfully transformed positive colonies; Figure 8 This is a graph showing the SDS-PAGE gel electrophoresis results in Example 2 of the present invention; where M: 180kDa marker; lanes 1-2: pET-28a-MCP372 protein supernatant; lanes 3-4: pET-28a-MCP372 protein inclusion bodies; lanes 5-6: pET-28a-MCP protein supernatant; lanes 7-8: pET-28a-MCP protein inclusion bodies; Figure 9 The purification of the recombinant protein in Example 2 of this invention; wherein (a): pET-28a-MCP372 protein purification result; M: 180kDa marker; lanes 4-6: pET-28a-MCP372 protein purification; (b): pET-28a-MCP protein purification result; M: 180kDa marker; lanes 6-7: pET-28a-MCP protein purification; Figure 10The following are the Western Blot detection results in Example 2 of this invention; where (a): Western Blot verification results of pET-28a-MCP372 protein; M: 180kDa marker; lanes 1-2: pET-28a-MCP372 protein; (b): Western Blot verification results of pET-28a-MCP protein; M: 180kDa marker; lanes 1-2: pET-28a-MCP protein; Figure 11 The image shows the results of the indirect immunofluorescence assay of FHM cells infected with LMBV in Example 2 of this invention (10×). Figure 12 This is a graph showing the Western blot specificity verification results of the polyclonal antibody in Example 2 of the present invention; where M: 180kDa marker; (a): polyclonal antibody prepared from MCP372 protein; (b): polyclonal antibody prepared from MCP protein; Figure 13 This is a graph showing the sampling and detection results of LMBV in Example 3 of the present invention (partial sample); where M: Marker; P: Positive LMBV sample; 1-2: Samples tested; Figure 14 This is a graph showing the relative expression levels of immune-related genes in the spleen in Example 3 of the present invention; Note: * indicates P-value is less than 0.05, ** indicates P-value is less than 0.01, and *** indicates P-value is less than 0.001; Figure 15 This is a graph showing the effect of vaccine immunization on the activity of immune enzymes in Example 3 of the present invention; Note: * indicates P-value is less than 0.05, ** indicates P-value is less than 0.01, and *** indicates P-value is less than 0.001; Figure 16 This is a graph showing the change in antibody titer after immunization in Example 3 of the present invention; Figure 17 This is a cumulative mortality curve in Embodiment 3 of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0024] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0025] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0026] Pathogen and cell origin: Pathological samples: From October 2022 to February 2024, clinical samples were collected from largemouth bass farms in Linyi and Jining, Shandong Province, for testing. Clinical samples of the liver, kidneys, and spleen were collected from diseased fish exhibiting ulcers on their body surface, slight abdominal swelling, and spiral swimming backwards. A total of 50 clinical samples were collected, and all samples were labeled, classified, and stored at -80℃.

[0027] The fathead bream muscle cells (FHM) were kindly donated by Researcher Wang Qing of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0028] Main reagents: Table 1. Names and sources of reagents used

[0029] Main instruments Table 2. Instruments used in the experiment

[0030] Solution and reagent preparation: (1) M199 medium with 10% FBS: Add 10 mL of FBS and 100 μL of triple antibodies aseptically to 90 mL of M199 medium, mix thoroughly, and store at 4°C.

[0031] (2) M199 medium with 2% FBS: Add 2 mL of FBS and 100 μL of triple antibodies aseptically to 98 mL of M199 medium, mix thoroughly, and store at 4°C. (3) Amp / LB liquid medium: After the LB liquid medium is cooled by autoclaving, add an appropriate amount of Amp.

[0032] (4) Kan / LB liquid medium: After the LB liquid medium is cooled by autoclaving, add an appropriate amount of Kan.

[0033] (5) Amp / LB solid medium: After autoclaving, LB solid medium was placed at room temperature for 15 min, Amp was added aseptically, mixed well, and then quickly poured into a petri dish, shaken and spread evenly. After cooling, it was stored in a refrigerator at 4℃ for later use.

[0034] (6) Kan / LB solid medium: After autoclaving, LB solid medium is placed at room temperature for 15 min, Kan is added aseptically, mixed well, and then quickly poured into a petri dish, shaken and spread evenly. After cooling, it is stored in a refrigerator at 4℃ for later use.

[0035] (7) 50 mg / mL IPTG: Weigh 1 g of IPTG powder and dissolve it in 20 mL of sterile water. After complete dissolution, filter it through a 0.22 μm sterile filter, dispense it into aliquots, and store it at -20℃.

[0036] Example 1: Isolation and Identification of the Virus Strain 1.1 Treatment of Pathological Materials (1) Place the diseased fish on a sterile operating table and dissect it using sterilized scissors. (Some diseased materials and dissection diagrams are shown below.) Figure 1 (As shown) (2) Take the kidney, liver and spleen and put them into sterile 1.5 mL centrifuge tubes. Use scissors and tweezers one at a time.

[0037] (3) DNA extraction was performed immediately after the procedure, and the remaining tissue samples were stored in a -80°C refrigerator.

[0038] 1.2 Extraction of DNA from Pathological Tissue Using the DNA extraction kit from Tiangen Biotech, extract samples (less than 30 mg each) from the kidney, liver, and spleen using scissors, following these steps: (1) Add the collected sample tissues into a labeled sterile 1.5 ml centrifuge tube, add 200 μL of GA buffer to each tube, and vortex for 15 s.

[0039] (2) Add 20 μL of proteinase K (20 mg / mL) to the centrifuge tube after shaking, vortex again, mix well, and then centrifuge briefly to remove liquid droplets on the inner wall and cap of the centrifuge tube. Then place the centrifuge tube on a 56°C constant temperature metal bath to allow the tissue to lyse. Shake once every 20 min for 15 s each time, for about 1.5 h until the tissue is completely dissolved and there is no obvious solid. Vortex to mix it well, and then centrifuge briefly.

[0040] (3) Add 200 μL of GB buffer to the centrifuge tube, invert and mix thoroughly. White flocculent precipitate may appear in some centrifuge tubes. Place the centrifuge tube in a 70°C constant temperature metal bath for 10 min. At this time, the white flocculent precipitate will disappear and the solution will become clear. Centrifuge briefly again.

[0041] (4) Add 200 μL of anhydrous ethanol to the centrifuge tube, and mix thoroughly by inverting the tube several times. Flocculent precipitate may appear. Perform a short centrifugation.

[0042] (5) Add the mixture of liquid and flocculent precipitate in the centrifuge tube to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid in the tube, and put the adsorption column CB3 back into the collection tube.

[0043] (6) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid in the tube, and put the adsorption column CB3 back into the collection tube.

[0044] (7) Add 600 μL of washing solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid in the tube, and put the adsorption column CB3 back into the collection tube.

[0045] (8) Repeat step (7).

[0046] (9) Place the adsorption column back into the collection tube, put it into a centrifuge at 12,000 rpm for 2 min, discard the waste liquid in the tube, place the adsorption column CB3 at room temperature for 5 min, and wait for it to dry completely to remove the residual rinsing liquid in the adsorption column.

[0047] (10) Place the adsorption column CB3 into a sterile 1.5 mL centrifuge tube, add 100 μL of elution buffer dropwise to the middle of the adsorption membrane, place at room temperature for 5 min, then centrifuge at 12,000 rpm for 2 min, finally collect the solution into the centrifuge tube, label it, and store it in a -20°C refrigerator.

[0048] 1.3 PCR detection of LMBV in pathogen samples Using nucleic acids extracted from pathogen samples as DNA templates, specific primers were designed in the CDS region based on the gene sequence of largemouth bass iridovirus FR682503 in GenBank to identify the full length of the major capsid protein MCP gene. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 3. Samples were added according to the reaction system in Table 4.

[0049] Table 3: Primers for virus identification

[0050] Table 4: PCR amplification reaction system I

[0051] After adding the reaction mixture, mix it thoroughly and centrifuge briefly. Place it in a PCR instrument and set the reaction program as follows: (1) pre-denaturation at 95℃ for 5 min, (2) denaturation at 95℃ for 30 s, (3) annealing at 52℃ for 30 s, (4) extension at 72℃ for 1 min, and then extension at 72℃ for 5 min. Repeat this process for 33 cycles and then terminate the reaction at 4℃. The PCR products are then identified by agarose gel electrophoresis.

[0052] Test results: Of the 50 clinical samples collected, 32 were confirmed to be LMBV positive by PCR testing, resulting in an LMBV positivity rate of 64% in the collected samples. Figure 2 (As shown) 1.4 Cell Culture and Virus Isolation (a) FHM cell culture (1) Take out a frozen FHM cell from the liquid nitrogen tank, revive the cells, and place them in a 28°C, 5% CO2 incubator for static culture. Observe the cell growth status at regular intervals.

[0053] (2) When the cells are in good growth condition and the density reaches more than 80%, the cells are passaged. The culture medium in the bottle is poured out, 1 mL of PBS buffer is added for rinsing, and the cells are discarded after rinsing. 1 mL of trypsin is added to the wall of the cell, and the cells are gently shaken. After digestion for 1 min, the cell condition is observed under a microscope and the trypsin is poured out.

[0054] (3) Add 10 mL of 10% FBS M199 medium, pipette the adherent cells off and mix well, then transfer 5 mL into a new cell culture flask, i.e., one cell to two cells.

[0055] (ii) Preparation of virus suspension (1) Take the positive tissue samples out of the -80°C freezer, put them into a sterilized mortar, add an appropriate amount of liquid nitrogen, grind them thoroughly with a grinding rod, and collect the ground tissue powder into a sterile centrifuge tube.

[0056] (2) Add an appropriate amount of sterile PBS buffer, shake well to mix thoroughly, and freeze and thaw three times at -80°C and room temperature.

[0057] (3) Place the supernatant in a centrifuge at 12,000 rpm and 4°C for 10 min. Filter the supernatant through 0.45 μm and 0.22 μm filters to remove bacteria. Aliquot the obtained virus suspension, label it, and store it in a -80°C freezer.

[0058] (III) FHM cell challenge and virus isolation (1) Select FHM cells in good growth condition and grow them to a density of about 80% for later use.

[0059] (2) Thaw the virus suspension quickly, discard the culture medium in the cell culture bottle, add 2 mL of PBS buffer and shake slowly to wash, so as to prevent the residual serum in the culture medium from affecting virus adsorption.

[0060] (3) Add 5 mL of virus suspension, shake well using the figure-eight method, and incubate in a constant temperature cell culture incubator at 28℃ for 2 h.

[0061] (4) Discard the virus suspension, add 5 mL of M199 medium containing 2% FBS, and continue to culture in a 28℃ cell culture incubator, and continue to observe changes in cell morphology.

[0062] (5) After three consecutive blind passages, obvious cytopathic effects were observed in the cells. (Continuously observe changes in cells before and after imaging, save images for comparison and analysis. Results showed that after challenge with the virus and replacement with maintenance medium for continued culture, the normal cells of the negative control (e.g.) Figure 3 As shown in Figure a), the cells are dense and compact; cells infected with LMBV 12 hours later are as shown in Figure a. Figure 3 As shown in Figure b), some cells begin to shrink and clump together, creating a cavity; cells 24 hours after infection show (as shown in Figure b) Figure 3 As shown in Figure c), cell cavities gradually increase, and a small number of dead cells float; after 36 hours of cell infection, as shown in Figure c. Figure 3 (As shown in d), a large number of cells have died, resulting in numerous cell cavities, and a large number of dead cells float in the supernatant. At this point, the lesion degree reaches 80%, and the cells can be collected and the cell suspension preserved. The supernatant from the cell culture flasks was collected, and after three freeze-thaw cycles, it was centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was then stored. Subsequently, the virus was detected by PCR, and the obtained virus was quantified by fluorescence quantitative PCR. The purified and isolated virus was named LMBV-JN23.6.8 virus strain.

[0063] Example 2: Results of protein expression and polyclonal antibody preparation 2.1 Bioinformatics analysis of MCP genes and proteins MCP, as the main capsid protein, has multiple biological functions. The full-length MCP gene of the LMBV-JN23.6.8 virus strain obtained in Example 1 was amplified and sequenced. Based on the sequencing results, the protein sequence encoded by the MCP gene was predicted and analyzed. The software and websites used are shown in Table 5. (The full-length MCP gene sequence is shown in SEQ ID No: 1, the CDS sequence of the MCP gene is shown in SEQ ID No: 2, and the protein sequence encoded by the MCP gene is shown in SEQ ID No: 3.) Table 5 Information on Bioinformatics-Related Software

[0064] Bioinformatics analysis showed that the MCP protein has no signal peptide and no transmembrane domain, making it a relatively stable hydrophobic protein.

[0065] 2.2 Construction of recombinant prokaryotic expression plasmid pET-28a-MCP Using positive largemouth bass iridovirus DNA as a template, amplification was performed using the mqMCP-F / R enzyme digestion primer. The results showed a single, bright band after electrophoresis, consistent with expectations. The obtained DNA fragment was 1,392 bp in size (e.g., ...). Figure 4 (As shown).

[0066] The MCP gene fragment was ligated into the pET28a vector and transformed into... E. coli DH5α competent cells were cultured overnight, and single colonies were selected for verification using primers T7-F / R (e.g., ...). Figure 5 As shown in Figure a), electrophoresis results revealed successfully linked positive colonies.

[0067] Successfully ligated DH5α-pET-28a-MCP was transformed into BL21(DE3) competent cells, and positive colonies were screened and verified using primers T7-F / R (e.g., ...). Figure 5 (As shown in b).

[0068] 2.3 Construction of recombinant prokaryotic expression plasmid pET-28a-MCP372 The construction of the pET-28a-MCP372 recombinant prokaryotic expression plasmid was largely consistent with the construction of pET-28a-MCP. The pET-28a(+) vector and the target fragment were double-digested, ligated, and transformed using the same restriction endonucleases Xho I and EcoRI. The difference was that MCP372 was a truncated version of MCP, and new primers were designed and synthesized. The successfully ligated bacterial cultures were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. (The sequence of the MCP372 gene is shown in SEQ ID No: 4, and the protein sequence encoded by the MCP372 gene is shown in SEQ ID No: 5.) Using positive largemouth bass iridovirus DNA as a template, amplification was performed using mqMCP-F / R enzyme digestion primers. The results showed a single, bright band after electrophoresis, consistent with expectations. The obtained DNA fragment was 372 bp in size. Figure 6 ).

[0069] The MCP372 gene fragment was ligated into the pET28a vector and transformed into... E. coli DH5α competent cells were cultured overnight, and single colonies were selected for verification using primers T7-F / R (e.g., ...). Figure 7 As shown in Figure a), electrophoresis results revealed successfully linked positive colonies.

[0070] Successfully ligated DH5α-pET-28a-MCP372 cells were transformed into BL21(DE3) competent cells, and positive colonies were screened and verified using primers T7-F / R (e.g., ...). Figure 7 (As shown in b).

[0071] 2.4 Results of induction and identification of recombinant protein The sequencing-positive bacterial culture was added to an IPTG induction solution at a concentration of 1 mmol / L and induced at 30°C and 220 rpm for 6 h. The supernatant and precipitate were collected for SDS-PAGE electrophoresis to verify expression levels. The results showed that recombinant pET-28a-MCP372 and pET-28a-MCP proteins exhibited bands of the expected size at 17 kDa and 54 kDa, respectively. This indicated successful expression of the recombinant proteins under IPTG induction, with the protein primarily in the form of inclusion body precipitates, and a relatively small amount in the supernatant. Figure 8 ).

[0072] After high-level protein expression, protein purification was performed. The protein was passed through a column, washed with denaturing wash buffers of different imidazole concentrations, and finally eluted with denaturing elution buffer to obtain the purified protein. SDS-PAGE validation results showed single target bands at 17 kDa and 54 kDa, indicating successful purification of the recombinant protein. Figure 9 ).

[0073] Western blotting was performed using the purified recombinant protein as a sample. The results showed a single, specific protein band, consistent with the expected sizes of 17 kDa and 54 kDa. Figure 10 This indicates that the target protein was successfully expressed.

[0074] 2.5 Preparation and Specificity Detection Results of Polyclonal Antibodies (I) Preparation of polyclonal antibodies The two purified recombinant proteins were refolded as follows: (1) Prepare PBS buffer solutions with urea solutions of 6 mM, 4 mM, 2 mM and 1 mM for later use.

[0075] (2) Cut the dialysis membrane into small segments of appropriate length, boil them in boiling water 3 times, 10 min each time, and cool them in deionized water each time. Clamp one end of the prepared dialysis bag with a dialysis clamp, add 10 mL of purified protein, leaving a suitable gap (to prevent the dialysis bag from bursting), and clamp the other end with a dialysis clamp.

[0076] (3) Place the dialysis bag containing protein into the highest concentration of urea buffer solution, dialyze at 4°C for 10 h, and gently stir with a magnetic stirrer.

[0077] (4) Replace the urea solution with a decreasing concentration every 10 h. After the last concentration of renaturation is completed, recover the renatured protein and perform SDS-PAGE verification.

[0078] Two refolded recombinant proteins were used as antigens to immunize New Zealand white rabbits with a body weight of (1 ± 0.100) kg / rabbit. The initial immunization dose was 500 mg, and the doses for the second and third immunizations were 300 mg (when the antigen concentration was high, it was diluted to 0.5 mL with sterile physiological saline).

[0079] Before the first immunization, the protein and Freund's complete adjuvant were thoroughly mixed in a 1:1 ratio using an adjuvant emulsifier (emulsified until the vaccine forms a stable drop in water without spreading), and then injected subcutaneously at multiple sites on the back of the rabbit's neck. For the second immunization, two weeks after the first immunization, an equal volume of protein and Freund's incomplete adjuvant were emulsified and administered at the same site. For the third immunization, four weeks after the first immunization, the antigen protein was dissolved in physiological saline and administered to the rabbits at the same site.

[0080] One week after the third immunization, blood was collected from the marginal ear vein of rabbits using a disposable lancet. The blood was incubated at 37°C for 2 hours, then at 4°C overnight. The coagulated serum was centrifuged at 8,000 r / min for 5 minutes, and the supernatant was collected as serum, stored at -80°C for later use. (Serve from unvaccinated rabbits was collected before the initial immunization as a negative control.) (II) ELISA detection of antibody titer Pretreatment: Using the two purified MCP proteins as antigens, the concentration was adjusted to 10 μg / mL with coating solution; the serum to be tested was serially diluted at ratios of 1:1,000 to 1:512,000 (1:1,000, 1:2,000, 1:4,000, 1:8,000, 1:16,000, 1:32,000, 1:64,000, 1:128,000, 1:256,000, 1:512,000). (1) Antigen coating: Coat 96-well plates with diluted protein antigen, perform 3 replicates for each gradient according to the serum antibody dilution, 100 μL / well, incubate overnight at 4°C (at least 16 hours), remove the liquid in the wells on the second day, wash 3 times with washing buffer, and pat dry. (2) Add 200 μL of blocking solution to each well, block at 37℃ for 2 h, wash 3 times with washing solution, and pat dry. (3) Primary antibody incubation: Add 100 μL of serially diluted serum to each well, with negative serum as a control, incubate at 37°C for 2 h, wash 3 times with washing buffer, and pat dry. (4) Secondary antibody incubation: Add 100 μL / well of HRP-labeled goat anti-rabbit IgG antibody (1:5,000 dilution, diluent is 5% skim milk powder), incubate at 37℃ for 1 h, wash 3 times with washing buffer, and pat dry; (5) Color development: Add 100 μL / well of soluble single-component TMB substrate solution and develop color at 37°C in the dark for 20 min; (6) Termination: Add 50 μL of stop solution to terminate the reaction, and measure the absorbance of each well at 450 nm within 30 min using an enzyme-linked immunosorbent assay (ELISA) reader. Results: The titers of LMBV-MCP372 and LMBV-MCP polyclonal antibodies were detected by ELISA, with the average value taken from three parallel replicates. Positive serum OD 450 nm With negative serum OD 450 nm A serum dilution with a ratio greater than 2.1 is considered the titer of the serum to be tested.

[0081] As can be seen from the data in Table 6, OD 450 (Antiserum:Negative Serum) = 2.51 > 2.1, indicating that the titer of the MCP372 polyclonal antibody is 1:128,000; As shown in Table 7, OD... 450 (Antiserum:Negative serum) = 2.66 > 2.1, indicating that the titer of the MCP polyclonal antibody is 1: 64,000.

[0082] Table 6: Titer of MCP372 polyclonal antibody (the refolded protein is the antigen)

[0083] Table 7: Titer of MCP polyclonal antibodies (the refolded protein is the antigen)

[0084] (III) Western blot detection of antibody specificity FHM cells were challenged and maintained for one day. The culture medium was discarded, and the cells were washed twice with 2 mL of PBS each time, then the PBS was discarded. 1 mL of trypsin was added to the culture flask, and the cells were digested at 37°C for 2 minutes. After terminating digestion, twice the volume of culture medium (5% serum medium) was added, and the solution was transferred to an EP tube. The tube was capped and centrifuged at 2,000 rpm for 5 minutes. The supernatant was then discarded, and 1 mL of PBS was added to disperse the cell clumps. The cells were centrifuged at 2,000 rpm for 5 minutes at 4°C. The supernatant was discarded again, and an appropriate amount of lysis buffer (based on the amount of cells in the centrifuge tube) was added and mixed. The cells were lysed at 4°C for half an hour, followed by centrifugation at 12,000 rpm for 10 minutes at 4°C. After centrifugation, the supernatant was transferred and stored at -20°C.

[0085] Proteins extracted from challenged cells were subjected to SDS-PAGE electrophoresis, followed by Western blotting for identification. The primary antibody used for incubation was the prepared polyclonal antibody serum, and the secondary antibody was HPR-labeled goat anti-rabbit IgG.

[0086] Proteins were extracted from challenged FHM cells, and Western blot analysis was performed using two types of rabbit antiserum as primary antibodies and HPR-labeled goat anti-rabbit IgG as secondary antibody. Results (e.g.) Figure 12 As shown, the target bands were detected at the expected locations of 54 kDa and 17 kDa.

[0087] (iv) IFA for identifying the specificity of polyclonal antibodies (1) Cell preparation: First, resuscitate FHM cells and pass them three times to observe their growth and stability. Select an appropriate gradient for plating based on the number of cells. Plate the cells onto a 12-well cell plate. The cells should not be too densely packed. After about 24 hours, the cells will grow into a monolayer. After inoculating with the virus, continue culturing for another 24 hours.

[0088] (2) Discard the old growth medium and wash three times with PBS.

[0089] (3) Fix with 4% paraformaldehyde, just enough to cover the bottom of the hole, and incubate at 28°C for 10 min.

[0090] (4) Discard the fixative and wash three times with PBS.

[0091] (5) Add 500 μL of primary antibody (two polyclonal antibody serums prepared) and incubate at 28°C for 1 h.

[0092] (6) Discard the primary antibody and wash three times with PBS.

[0093] (7) Add an equal amount of secondary antibody (FITC-labeled goat anti-rabbit IgG) in the dark and incubate at 28°C in the dark for 1 h.

[0094] (8) Discard the secondary antibody and wash three times with PBS.

[0095] (9) Add 50 μL of DAPI for staining in the dark, and place at 28°C in the dark for 5 min.

[0096] (10) Discard the DAPI and wash three times with PBS. Then observe with a fluorescence microscope and retain the images.

[0097] Indirect immunofluorescence detection was performed on LMBV-infected FHM cells using two rabbit polyclonal antibody sera at a 1:200 dilution. Unchallenged cells served as a negative control. After staining the cell nuclei with DAPI, bright blue fluorescence was observed in all groups (challenged cells showed limited cell count due to extensive shrinkage and death caused by cytopathic effects). After incubation with FITC-labeled fluorescent secondary antibodies, the LMBV-challenged group showed specific binding of the antigen and antibody, resulting in bright green fluorescence. No fluorescence was observed in the control group. MCP cells produced more green fluorescence than MCP372 cells, indicating that MCP has more full-length binding sites, suggesting that both polyclonal antibodies specifically bound to the cells. (e.g.) Figure 11 (As shown) Example 3: Subunit vaccine preparation and evaluation of its immunization effect 3.1 Preparation of inactivated vaccines (a) Selection of virus inactivation conditions After the expanded culture of the virus suspension was thawed from the -80°C freezer, it was aliquoted into 15 tubes. Formaldehyde was selected as the inactivating agent. Inactivation was performed at 37°C on a shaker for 24 h, 36 h, 48 h, 60 h, and 72 h at formaldehyde concentrations of 1%, 2%, and 4%, respectively. After inactivation, the results were tested by infecting cells with the 15 vaccine groups via inoculation. Normal cells were used as negative controls, and uninactivated virus suspension was used as a positive control. The cells were maintained in M199 medium with 2% FBS for three days. The pathological changes in the cells were observed. The cells were blindly passaged three times consecutively to observe whether the cell state was stable and whether cytopathic effects occurred. If no cytopathic effects were observed in all three passages, the inactivation effect was considered satisfactory.

[0098] Table 8. Blind passage effect of cells after inactivation

[0099] Note: + indicates that CPE was detected in the cells, - indicates that CPE was not detected in the cells. As shown in Table 8, the formaldehyde group with a final concentration of 2% did not produce cytopathic effects after three blind passages at 37℃ for 60 h, and the quantitative fluorescence detection was negative, indicating that the inactivation was complete.

[0100] (ii) Safety testing of vaccines The inactivated virus suspension was emulsified with Freund's adjuvant at a 1:1 ratio using an adjuvant emulsifier. After complete emulsification, a safety test was performed.

[0101] (1) Appearance inspection: Check whether there are impurities or sediment in the emulsified vaccine, and whether the vaccine is milky white.

[0102] (2) Stability test: Drop the prepared vaccine into water and observe whether the vaccine spreads. At the same time, take 10 mL and add it to a sterile centrifuge tube. Place the tube in a centrifuge at 3,000 rpm for 10 min and observe the changes in the vaccine before and after centrifugation, and whether there is any layering or demulsification. (3) Sterility test: 100 µL of the emulsified vaccine was taken and inoculated into LB solid medium and LB liquid medium, and placed in an incubator at 37°C for one week to observe whether there was bacterial growth. At the same time, a negative control group was set up for comparison.

[0103] (4) Safety test: Healthy largemouth bass were temporarily held for one week and then divided into two groups of 15 fish each. The first group was injected intraperitoneally with 0.1 mL of inactivated vaccine, while the second group served as a negative control and was injected with 0.1 mL of sterile PBS buffer. After the injection, the largemouth bass were observed to determine the health status of the fish after vaccination and whether any adverse reactions occurred.

[0104] The test results are as follows: (1) Appearance inspection qualified: The emulsified vaccine is milky white and no sediment or impurities are produced.

[0105] (2) Stability test passed: After emulsification, the vaccine was dropped into water and remained stable on the water surface without diffusion. After centrifugation for 10 minutes, there was no significant change in the vaccine before and after centrifugation, and no stratification or demulsification occurred.

[0106] (3) Sterility test passed: After one week of culture in the emulsified vaccine culture medium, no bacterial growth was observed, consistent with the negative control.

[0107] (4) Safety test passed: After vaccination, the largemouth bass ate normally and swam normally, which was consistent with the negative control.

[0108] 3.2 Preparation of subunit vaccines The purified recombinant protein concentration was measured again, and the concentrations of MCP protein and MCP372 protein were adjusted to a medium concentration of 0.2 μg / μL and a high concentration of 0.4 μg / μL, respectively. The proteins of different concentrations were emulsified with an equal volume of Freund's adjuvant, resulting in 10 μg of target protein per 100 μL of medium-concentration vaccine and 15 μg of target protein per 100 μL of high-concentration vaccine. The four prepared vaccines were then subjected to safety testing.

[0109] 2.2.3.3 Evaluation of Immunization Efficacy (a) Vaccination Six hundred largemouth bass were temporarily held in a 26°C recirculating aquaculture system and fed three times a day (morning, noon, and evening). After the fish stabilized, largemouth bass of about 10 cm in length and 15 g in weight were selected and divided into six groups of 100 fish each. At the same time, three fish were randomly sampled to test for largemouth bass iridovirus.

[0110] Largemouth bass were divided into groups and administered 100 μL of vaccine intraperitoneally. A PBS group served as the control group, receiving an equal volume of sterile PBS. Five subgroups were established: Group 1 received the inactivated vaccine; Group 2 received the medium-concentration MCP subunit vaccine; Group 3 received the high-concentration MCP subunit vaccine; Group 4 received the medium-concentration MCP372 subunit vaccine; and Group 5 received the high-concentration MCP372 subunit vaccine. The first immunization was emulsified with Freund's complete adjuvant, and two weeks later, it was emulsified with Freund's incomplete adjuvant. After each injection, the fishpond was disinfected with iodine to prevent stress from improper vaccination. The growth of the largemouth bass was observed after vaccination, and they were fed normally.

[0111] Sampling test results of LMBV (e.g.) Figure 13 (as shown) Three healthy largemouth bass were randomly selected, their morphological characteristics were observed, and they were dissected. DNA was extracted from tissues such as the kidneys, spleen, and liver for PCR testing. The results showed that the largemouth bass were not infected with iridovirus.

[0112] (II) Measurement of spleen immune gene expression levels Sample collection: Select sufficient sterile, enzyme-free 1.5 mL centrifuge tubes and fill them with RNA Keeper buffer for later use. On days 1, 7, 14, 21, and 28 post-vaccination, randomly select 3 largemouth bass from each group. After observing that the fish are in normal condition, dissect them on a sterile laminar flow hood. Light an alcohol lamp, remove the spleen with sterile scissors, and immediately place it into the prepared centrifuge tubes. Invert the tubes to completely immerse the spleen in the buffer and store them at 4°C to better protect the integrity of the spleen RNA.

[0113] Results of immune gene expression level detection: At 14, 21, and 28 days post-immunization, three fish were randomly selected for dissection. Spleen RNA was extracted, and relative quantification of mRNA levels of immune-related genes IgM, IL-8, IL-1β, TNF-α, and IFN was performed. β-actin was used as an internal control, and the mRNA levels of these genes in the PBS group served as a blank control. The data analysis results are as follows: Figure 14At days 14 and 21, the high concentration of IgM in the MCP1392 group was significantly higher than that in the control group, and at day 28, the medium concentration in the MCP372 group was significantly higher than that in the control group. The overall concentration of IL-8 in the immune gene increased over time, and at days 21 and 28, the medium concentration in the MCP372 group was significantly higher than that in the control group. There was no significant difference in IL-1β at day 14, but at days 21 and 28, most immune groups showed significant differences compared to the control group. At day 14, the high concentration of TNF-α in the MCP372 group was significantly higher than that in the control group, and at days 21 and 28, the medium concentration in the MCP372 group was significantly higher than that in the control group. At days 14, 21, and 28, the high concentration of IFN in the MCP372 group was significantly higher than that in the control group, and at days 21 and 28, the medium concentration in the MCP372 group was significantly higher than that in the control group.

[0114] (III) Immunoenzyme activity assay On days 14, 21, and 28 post-immunization, three bass were randomly selected from each group, blood was collected from their tail veins, serum was separated, and the enzyme activities were tested using an acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) kit according to the manufacturer's instructions.

[0115] Results of immunoenzyme activity assay: Three fish were randomly selected for dissection at 14, 21, and 28 days post-immunization. Blood was collected from the tail vein to measure the activity of immune enzymes. The results are as follows: Figure 15 There was no significant difference in acid phosphatase (ACP) between the immunization groups and the control group. However, at day 28, the concentration of MCP372 in the immunization groups was significantly higher than that in the control group. At days 14, 21, and 28, the levels of alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) were significantly higher in all immunization groups than in the control group.

[0116] (iv) Neutralization test to detect serum antibody titer Neutralizing antibody detection follows the fixed virus diluted serum method, with the specific steps as follows: (1) Dilute the LMBV virus to 100 TCID 50 .

[0117] (2) Select FHM cells in good growth condition and plate them in 96-well cell culture plates for later use. (3) Dilute the serum sample to be tested two-fold with 2% FBS M199 medium, and then mix with 100 TCID50. 50 Mix the virus solution 1:1 and let it stand at 28°C for 60 minutes.

[0118] (4) Add the mixture to the 96-well plate, 100 µL per well, and perform 6 replicates for each gradient. At the same time, normal cells are set as negative controls and incubated in an incubator at 28°C for 3-7 days. (5) Observe the cytopathic effect every day. If the cytopathic effect exceeds 50%, it is recorded as positive. That is, the serum dilution factor of this well is the serum neutralization titer.

[0119] Serum antibody titer test results: Antibody titers in serum at 14, 21, and 28 days post-immunization were detected using the fixed-virus dilution serum method. Figure 16 It can be seen that the antibody titer of largemouth bass in the subunit immunization group generally increased with the increase of immunization days, while that in the inactivated group showed an initial increase followed by a decrease. The antibody titer in the control group gradually decreased with time. The maximum titer of MCP372 was 1:36 after 28 days of immunization, indicating that all vaccines can stimulate largemouth bass to produce neutralizing antibodies.

[0120] (v) Testing for immune protection rate using challenge experiments Four weeks after the first immunization, 30 largemouth bass were randomly selected from each group and injected intraperitoneally with LMBV virus, i.e., 10 5 At a 250% infection dose, the incidence and mortality of experimental fish were observed daily for 15 consecutive days, and the relative percent survival (RPS) of the vaccine was calculated. RPS = [1 - (mortality rate in the immunized group / mortality rate in the control group)] × 100%.

[0121] Relative immune protection rate test results: Twenty-eight days after immunization with largemouth bass, a challenge test was conducted to assess the immunoprotection rate. Figure 17 It can be seen that starting from the 5th day of the poisoning, largemouth bass began to die in each group. From the 7th to the 10th day, the mortality rate of fish in each group increased rapidly, and after the 11th day, it gradually entered a stable period.

[0122] As shown in Table 9, the cumulative mortality rate of the control group reached 90%, the mortality rate of the inactivated group reached 56.6%, and the lowest mortality rate was 16.6% in the medium concentration group of MCP372, with a relative protection rate of 81.48%.

[0123] Table 9. Relative Immunoprotection Rate of Vaccines

[0124] In summary, this invention used formaldehyde at final concentrations of 1%, 2%, and 4% to inactivate the target protein at 37°C for 24 h, 36 h, 48 h, 60 h, and 72 h, respectively. The results showed that the 2% formaldehyde final concentration group achieved the best inactivation effect at 37°C for 60 h, and can be used for preparing inactivated vaccines. To explore the immunogenicity of MCP protein and MCP372 protein subunits, the concentrations were adjusted to a medium concentration of 0.2 μg / μL and a high concentration of 0.4 μg / μL. After complete emulsification with an equal volume of Freund's adjuvant, each 100 μL of the medium-concentration vaccine contained 10 μg of the target protein, and each 100 μL of the high-concentration vaccine contained 15 μg of the target protein. Safety tests were conducted on the prepared inactivated and subunit vaccines. The results showed that the prepared vaccines exhibited no bacterial growth in culture, no cytotoxicity, and were safe for use.

[0125] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polyclonal antibody against largemouth bass iridovirus, characterized in that, The serum was derived from rabbits immunized with the MCP372 recombinant protein, the sequence of which is shown in SEQ ID No:

5.

2. The method for preparing the polyclonal antibody according to claim 1, characterized in that, The process includes the following steps: preparing the above-mentioned MCP372 recombinant protein through prokaryotic expression, purifying and refolding it, immunizing New Zealand white rabbits, and then collecting serum to prepare polyclonal antibodies.

3. A recombinant MCP372 protein, characterized in that, The gene sequence of the MCP372 recombinant protein is shown in SEQ ID No:

5.

4. A recombinant expression vector, characterized in that, It contains the nucleotide sequence shown in SEQ ID No:

5.

5. A genetically engineered strain for preparing the MCP372 recombinant protein of claim 3, characterized in that, The recombinant plasmid pET-28a-MCP372 was transformed into E. coli Recombinant strains obtained from DH5α competent cells.

6. The use of the MCP372 recombinant protein of claim 3, the recombinant expression vector of claim 4, the genetically engineered strain of claim 5, and the polyclonal antibody of claim 1 in the preparation of products for detecting largemouth bass iridovirus.

7. A kit for detecting largemouth bass iridovirus, characterized in that, Includes the polyclonal antibody as described in claim 1.

8. A largemouth bass iridovirus MCP subunit vaccine, characterized in that, Its active ingredients include MCP recombinant protein or MCP372 recombinant protein; the sequence of the MCP recombinant protein is shown in SEQ ID No: 3; the sequence of the MCP372 recombinant protein is shown in SEQ ID No:

5.

9. The largemouth bass iridovirus MCP subunit vaccine according to claim 8, characterized in that, The concentration of the MCP recombinant protein or MCP372 recombinant protein is 0.1~0.4 μg / μL.

10. A method for preventing and controlling largemouth bass iridovirus, characterized in that, The procedure includes the following steps: injecting the MCP subunit vaccine according to any one of claims 8 to 9 into the abdominal cavity of the largemouth bass.

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