An oral vaccine containing a largemouth bass iridovirus ATPase and its preparation method and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIMEI UNIV
- Filing Date
- 2026-02-26
- Publication Date
- 2026-05-29
Smart Images
Figure CN121714685B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fish molecular immunology, specifically relating to an oral vaccine containing largemouth bass iridovirus ATPase and its preparation method. Background Technology
[0002] Largemouth bass ( Micropterus salmoides Also known as California bass, it belongs to the order Perciformes, family Centrarchidae, and genus Perch. Micropterus Since its introduction to my country from North America in 1983, the largemouth bass has seen continuous expansion in aquaculture due to its rapid growth, strong environmental adaptability, and delicious meat. However, diseases caused by largemouth bass virus (LMBV) infection have become a key constraint on the development of the largemouth bass aquaculture industry.
[0003] LMBV belongs to the family Iridoviridae and the genus Frogvirus. Ranavirus Fish infected with LMBV exhibit typical symptoms such as blackening of the body surface, abdominal swelling, and unbalanced swimming. Dissection reveals pale liver, congestion and inflammation of the spleen and intestines. It can lead to large-scale fish deaths in a short period of time, causing serious economic losses to fish farmers.
[0004] Currently, control measures against LMBV still have many limitations. Traditional aquaculture management and water quality control measures are insufficient to fundamentally block virus transmission, while chemical drugs and antibiotics are not very effective against viral diseases. In the field of aquatic animal disease control, vaccination is the most effective means of prevention and control, and existing immunization routes mainly include injection, immersion, and oral administration. Among them, while injection vaccines can achieve a high level of immune protection, the procedure is complex, can cause significant stress to the fish, and is limited by fish size, making it difficult to promote and apply in large-scale aquaculture. Immersion vaccines, on the other hand, suffer from high production costs and short duration of immunity.
[0005] Oral vaccines, with their significant advantages such as ease of administration, lack of stress, no size restrictions, and low cost, are more suitable for large-scale aquaculture applications and are considered an ideal solution for large-scale control of LMBV. Currently, only a few reports exist on LMBV vaccine research, including DNA vaccines based on the major capsid protein (MCP) (administered via injection, with an RPS of 63.0%), oral vaccines based on yeast-expressed MCP (RPS of 52.1%), and oral vaccines based on Bacillus subtilis-expressed MCP (RPS of 45%). Existing oral vaccine research largely focuses on the development and application of MCP monoantigens, and no studies have been conducted using LMBV ATPases to prepare LMBV vaccines. Summary of the Invention
[0006] Our study found that oral administration of ATPase to largemouth bass iridovirus in fish can induce immunity against the virus.
[0007] Based on the above findings, this invention provides the application of ATPase of largemouth bass iridovirus in the preparation of an oral vaccine against largemouth bass iridovirus.
[0008] In one specific embodiment, the amino acid sequence of the ATPase is shown in SEQ ID NO:3.
[0009] The present invention also provides an oral vaccine against largemouth bass iridovirus, comprising the ATPase of largemouth bass iridovirus.
[0010] In one specific embodiment, the ATPase is displayed on the surface of Bacillus subtilis spores, and the amino acid sequence of the ATPase is shown in SEQ ID NO:3.
[0011] This invention also provides a method for preparing an oral vaccine against largemouth bass iridovirus, comprising the following steps:
[0012] S1: Construct a fusion protein expression plasmid of ATPase of largemouth bass iridovirus and Bacillus subtilis surface anchoring protein;
[0013] S2: The plasmid is transferred into Bacillus subtilis to obtain a recombinant strain that can express the fusion protein;
[0014] S3: Cultivate the recombinant strain to obtain spores, thus obtaining the oral vaccine.
[0015] In one specific implementation, in S1, the Bacillus subtilis surface anchoring protein is the spore capsid protein CotC, and its amino acid sequence is shown in SEQ ID NO:1;
[0016] The amino acid sequence of the ATPase is shown in SEQ ID NO:3.
[0017] In one specific implementation, in S1, the expression of the fusion protein is initiated using the amyE promoter.
[0018] In one specific implementation, in S3, the spores are obtained by the despore-forming method.
[0019] In one specific implementation, the Bacillus subtilis is B. subtilis -WB600 strain.
[0020] This invention uses Bacillus subtilis B. subtilis- The spores of strain WB600 serve as antigen delivery vectors, utilizing spore coat protein C (CotC) as an anchoring protein. Key proteins of the largemouth bass iridovirus are then introduced via gene fusion technology. ATPase Genes and CotC Gene fusion was used to construct a recombinant spore surface display system, achieving stable display of ATPase antigen on the spore surface and preparing an oral vaccine. After oral administration to fish, the vaccine developed tolerance to the gastrointestinal environment and was precisely delivered to the gut-associated lymphoid tissue, stimulating the fish's immunity and ultimately achieving control of LMBV. Attached Figure Description
[0021] Figure 1 PCR amplification of gene fragments encoding the CotC gene fragment (A), the ATPase gene fragment (B), and the CotC-ATPase-HA (C) fusion protein.
[0022] Figure 2 PCR amplification and validation of the plasmid pDG1730-CotC-ATPase-HA fragment. Lane M: DL2000; Lanes 1-3: CotC-ATPase-HA.
[0023] Figure 3 PCR validation of the recombinant strain BS-WB600-CotC-ATPase-HA. Lane M: DL5000; Lanes 1-4: BS-WB600-CotC-ATPase-HA; Lane 5: BS-WB600.
[0024] Figure 4 The strains were positive for amylase activity verification. Among them, colony 1: after 48 h of culture, the starch around the WB600-CotC-ATPase-HA colony was not degraded; colony 2: after 48 h of culture, the starch around the WB600 colony was degraded.
[0025] Figure 5 The expression of the fusion protein CotC-ATPase-HA in the spores of the recombinant strain was detected by Western blotting.
[0026] Figure 6 To detect the expression of the fusion protein CotC-ATPase-HA on the surface of recombinant strain spores using immunofluorescence in situ.
[0027] Figure 7 This study investigated the expression of immune-related genes in the livers of largemouth bass after oral vaccination.
[0028] Figure 8 This study investigated the expression of immune-related genes in the spleen of largemouth bass after oral vaccination.
[0029] Figure 9This study describes the expression of immune-related genes in the head kidney of largemouth bass after oral vaccination.
[0030] Figure 10 This study investigated the expression of immune-related genes in the gut of largemouth bass after oral vaccination.
[0031] Figure 11 This is a statistical chart showing the survival rate in virus challenge experiments.
[0032] Figure 12 A photomicrograph of a typical section of the liver of a largemouth bass used in a challenge experiment.
[0033] Figure 13 A micrograph of a typical section of the spleen of a largemouth bass used in a challenge experiment.
[0034] Figure 14 A photomicrograph of a typical section of the head kidney of a largemouth bass used in a challenge experiment.
[0035] Figure 15 A photomicrograph of a typical section of the intestine of a largemouth bass used in a challenge experiment. Detailed Implementation
[0036] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.
[0037] 1. Construct a recombinant plasmid expressing LMBV-ATPase protein.
[0038] Based on the gene sequence of spore cap protein CotC (Amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as shown in SEQ ID NO:2), LMBV ATPase Gene (amino acid sequence as shown in SEQ ID NO:3, nucleotide sequence as shown in SEQ ID NO:4) sequence, and specific primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0039] Using previously stored LMBV DNA samples as templates, PCR amplification was performed to obtain... ATPase Gene( Figure 1 B); Using Bacillus subtilis WB600 bacterial culture as a template, PCR amplification was performed to obtain... CotC Gene( Figure 1 A).
[0040] Combine the target fragment DNA with pMD TM The 19-T Vector uses Solution I for connection, and the connection yields a result containing... ATPase, CotCRecombinant plasmids of gene fragments. Positive strains were screened by colony PCR and then sequenced for verification.
[0041] The purified two gene fragments were mixed in an equimolar ratio and used as templates for amplification. CotC-ATPase-HA Fusion fragments ( Figure 1 C).
[0042] Recycle CotC-ATPase-HA The gene was ligated into the linearized pMD19-T vector, and the positive plasmid was named pMD19-T-CotC-ATPase-HA. Positive strains were screened by colony PCR and sequenced for verification. Glycerol was added to the correctly sequenced bacterial cultures for preservation.
[0043] Plasmid pDG1730 and plasmid pMD19-T-CotC-ATPase-HA were double-digested with HindIII and EcoRI, respectively. The digested linear pDG1730 plasmid and CotC-ATPase-HA fragment were recovered, ligated, and transformed into [a specific technology / organization]. E. coli Recombinant plasmid pDG1730-CotC-ATPase-HA was constructed from DH5α. Transformants were selected and identified by PCR using BS-CotC-ATPase F1 / BS-CotC-ATPase R2 primers. Agarose gel electrophoresis results are shown below. Figure 2 As shown, after PCR detection, the transformed bacteria showed a single band of ATPase at approximately 1100 bp, consistent with the expected size, indicating that the recombinant integrative plasmid pDG1730-CotC-ATPase-HA was successfully constructed.
[0044] Table 1 Primer sequences used in plasmid construction
[0045]
[0046] 2. Construction of recombinant Bacillus subtilis
[0047] The recombinant plasmid pDG1730-CotC-ATPase-HA was transformed into Bacillus subtilis WB600 using electroporation. Single colonies grew on selection plates, and six single colonies were randomly selected and cultured in liquid medium with shaking. Colony PCR was performed using BS-CotC-ATPase F1 / BS-CotC-ATPase R2 and amyEF / amyEF R as upstream and downstream primers, respectively. The results are as follows: Figure 3 As shown, all four plasmids were amplified to the target band, and their sizes were consistent with the target fragment, indicating that the recombinant plasmid was successfully transformed into Bacillus subtilis.
[0048] The recombinant Bacillus subtilis, correctly identified by PCR, was added to 1% soluble starch plates and incubated at 37°C for 18-24 hours. Iodine-potassium iodide solution was then applied around the colonies to observe for the presence of a clear hydrolysis zone. Results are as follows: Figure 4 As shown, after the addition of iodine solution, no transparent hydrolysis zone was observed around the colonies except for the wild-type strain WB600. This indicates that the amylase gene in the transformant was damaged, preventing the secretion of amylase to decompose the starch around the colony. Therefore, after the addition of iodine solution, the starch around the transformant reacted with iodine and changed color, while the starch around the WB600 colony was decomposed by its secreted amylase, resulting in a transparent hydrolysis zone. The results demonstrate that the recombinant plasmid was successfully transformed into Bacillus subtilis, and homologous recombination double crossover occurred.
[0049] 3. Expression and identification of recombinant strains
[0050] (1) Western blot verification of recombinant strain protein expression
[0051] After culturing the recombinant strain, 1% (1:100) was inoculated into 50 mL of DSM medium, and spores were obtained using the spore-forming method. The spores were then washed with 1 mol / L KCl, 1 mol / L NaCl, and ultrapure water to obtain pure spores. 10 μL of the purified spores were collected. 10 The spores were suspended in SDS-DTT solution and treated at 65°C for 10 min, followed by a water bath at 37°C for 2 h, and centrifuged at 4°C for 10 min. The cells were washed three times with 50 mmol / L Tris-HCl (pH 7.5) and resuspended in 5 mL of lysis buffer. The bacterial suspension was placed on ice and sonicated to disrupt the cells (300 W, 4 s on, 6 s off). The disrupted solution was centrifuged at 4°C to precipitate the spores; the supernatant was the spore capsid protein.
[0052] Protein samples were separated by SDS-PAGE and then transferred to a PVDF membrane by electrotransfer. The membrane was washed once with PBS for 10 min each time. Blocking was performed with protein-free rapid blocking buffer at room temperature for 20 min. After blocking, the membrane was washed once with PBST and twice with PBS (10 min each time). The primary antibody was diluted with antibody dilution buffer (1:1000), incubated at room temperature for 30 min, and then overnight at 4°C. After overnight incubation, the membrane was incubated at room temperature for another 30 min before recovering the primary antibody and washing the membrane three times. The secondary antibody was diluted with PBS (1:5000), incubated at room temperature for 1 h 30 min, recovered, and the membrane washed three times. ECL chemiluminescent substrate was added for color development, and photographs were taken as needed. Results are shown below. Figure 5 As shown, fusion gene CotC-ATPase-HA The expressed protein is approximately 36 kDa; no band was observed in the WB600 control, indicating successful ATPase protein expression.
[0053] (2) Immunofluorescence detection of fusion protein expression on spore surface
[0054] ① Take 10 μL of spores and spread them evenly on a glass slide. Let them air dry naturally, then wash them three times with PBS, 5 min each time.
[0055] ② Fix with 4% paraformaldehyde solution at room temperature for 30 min, then wash 3 times;
[0056] ③ Block with 3% BSA-PBS blocking solution at 37℃ for 1 h, then wash 3 times;
[0057] ④ Add the diluted primary antibody to the spores on the glass slide, incubate at room temperature for 1 h, and wash 3 times;
[0058] ⑤ Add the FITC-labeled secondary antibody to a glass slide, cover the spores incubated with the primary antibody, incubate at room temperature in the dark for 45 min, and wash 3 times;
[0059] ⑥ Place a drop of fluorescent quenching mounting medium on top, cover with a coverslip, and observe and image under a fluorescence microscope.
[0060] Using WB600 as a control, immunofluorescence microscopy was performed on recombinant Bacillus subtilis spores using a specific antibody labeled with HA. Under fluorescence, the spores of WB600-CotC-ATPase-HA showed green fluorescence, while WB600 showed no fluorescence under the same conditions (e.g., Figure 6 A). After prolonged culture on the sporulation medium, the sporulation rate of WB600 was approximately 50%. Under an optical microscope, malachite green-stained spores appeared as dark green dots, while vegetative cells were difficult to stain and appeared transparent (e.g.). Figure 6 B). Fluorescence microscopy observation results showed that HA-tagged... CotC-ATPase-HA Gene sequence in amyE Fusion expression was successfully achieved under the induction of the gene promoter. The fusion protein was successfully displayed on the surface of Bacillus subtilis spores and was reactive, and could be recognized by the corresponding specific antibodies.
[0061] 4. Preparation and efficacy verification of oral vaccines
[0062] Fermentation medium B. subtilis WB600 (CotC-ATPase-HA) was cultured at 37℃ and 180 r / min for 48 h, then centrifuged at 8000×g for 10 min to collect spores. The spores were resuspended in PBS and sprayed evenly onto the surface of the formulated feed at a concentration of 1.6×10⁻⁶. 10 The oral vaccine was prepared at a CFU / g feed ratio, air-dried, and stored at 4°C for later use. Simultaneously, culturing... B. subtilis The WB600 strain was added to the feed at the same concentration as a control group.
[0063] 405 healthy largemouth bass were randomly divided into 3 groups of 45 each: a CotC-ATPase-HA vaccine group, a control group, and a control group. B. subtilis The WB600 group and the PBS group (Mock group) were used. Experiments were conducted after 7 days of temporary care. The oral administration experiment lasted for a total of 8 weeks. Three immunizations were administered at weeks 1, 3, and 5, each lasting one week.
[0064] Samples were collected at weeks 2, 4, and 6 following oral administration. Three fish were sampled from each group, and blood, liver, spleen, head kidney, and hindgut were collected. Changes in the expression of IgM, CD4-L, CD8-α, MHC I-α, TNF-α, Mx, and IL-1β were detected by qPCR.
[0065] Liver, spleen, and head kidney of largemouth bass from each group were collected. Total RNA was extracted using Trizol reagent (Sangon Biotech (Shanghai) Co., Ltd.) and reverse transcribed to obtain cDNA. Real-time quantitative PCR was performed on the cDNA. The reaction system consisted of 5 μL of 2×SYBR Green Master Mix, 0.5 μL each of primers (forward and reverse primers), 3 μL of RNase-free water, and 1 μL of cDNA template, for a total of 10 μL. The reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ (extension), 82℃ (reading), for 40 cycles; 72℃ (extension) for 10 min, and 16℃ (hold). The expression of immune-related genes, including immunoglobulin (IgM), cluster 4-like (CD4-I), cluster 8 (CD8), major histocompatibility complex class I (MHCI-α), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and antiviral genes (Mx), was identified. Primer sequences are shown in Table 2. The results of the expression analysis of immune-related genes are as follows: Figure 7-10 As shown, immune-related genes were significantly upregulated in the oral vaccine group, resulting in an effective immune response.
[0066] Table 2 Primer sequences for PCR identification of immune-related gene expression in largemouth bass
[0067]
[0068] 5. Live animal testing for virus
[0069] Seven weeks post-vaccination, LMBV was injected intraperitoneally into largemouth bass (n = 30 per group). 200 μL of 1×10 6Infected individuals were challenged with copies / μL of virus, and each container was kept at 30°C using a heater. Mortality and clinical symptoms were monitored daily for 8 days. Mortality was recorded starting on day 1 post-challenge and continued until no further deaths occurred. Relative survival rate (RPS) was calculated using the following formula: RPS = [1 - (mortality rate % in vaccinated group / mortality rate % in control group)] × 100%. Results are as follows: Figure 11 As shown, the oral vaccine CotC-ATPase-HA group showed an immune protection rate of 36.03% against largemouth bass, which was significantly higher than that of the PBS group.
[0070] When largemouth bass are infected with LMBV, the affected fish exhibit mild congestion and ulceration on their body surface. During the infection period, they eat little or nothing, and some fish lose their balance. Dissection reveals waxy yellow secretions in the abdominal cavity, localized whitening or white spots on the liver tissue, and an enlarged spleen. Pathological examination of the liver, spleen, head, kidneys, and intestines is performed as follows: Figure 11-15 As shown in the figure. Histopathological examination revealed that the recombinant vaccine effectively stimulated an immune response and reduced histopathological damage caused by viral infection.
[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The application of a fusion protein of ATPase of largemouth bass iridovirus and Bacillus subtilis surface anchoring protein in the preparation of an oral vaccine against largemouth bass iridovirus, wherein the Bacillus subtilis surface anchoring protein is a spore capsid protein CotC, the amino acid sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of the ATPase is shown in SEQ ID NO:
3.
2. An oral vaccine against largemouth bass iridovirus, characterized in that, The invention comprises a fusion protein of ATPase from largemouth bass iridovirus and a surface anchoring protein of Bacillus subtilis, wherein the surface anchoring protein of Bacillus subtilis is a spore capsid protein CotC, the amino acid sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of the ATPase is shown in SEQ ID NO:
3.
3. The oral vaccine according to claim 2, characterized in that, The ATPase is displayed on the surface of Bacillus subtilis spores.
4. A method for preparing an oral vaccine against largemouth bass iridovirus, characterized in that, Includes the following steps: S1: Construct a fusion protein expression plasmid of ATPase of largemouth bass iridovirus and Bacillus subtilis surface anchoring protein, wherein the Bacillus subtilis surface anchoring protein is spore capsid protein CotC, and the amino acid sequence is shown in SEQ ID NO:1; the amino acid sequence of the ATPase is shown in SEQ ID NO:
3. S2: The plasmid is transferred into Bacillus subtilis to obtain a recombinant strain that can express the fusion protein; S3: Cultivate the recombinant strain to obtain spores, thus obtaining the oral vaccine.
5. The method according to claim 4, characterized in that, In S1, the expression cassette of the fusion protein is started using the amyE promoter.
6. The method according to claim 4, characterized in that, In S3, the spores are obtained by the despore method.
7. The method according to claim 4, characterized in that, The Bacillus subtilis is B. subtilis -WB600 strain.