A vaccine against ichthyophthirius multifiliis and its preparation method and application

Abstract

The application provides a vaccine against ichthyophthiriasis and a preparation method and application thereof, and belongs to the technical field of fish disease prevention and control. The application uses inactivated whole worms of in-vitro large-scale cultured Amphilina alata as a cross antigen, and fish complement C5a protein as an immunoadjuvant, and the two synergistically play a high-efficiency immune protection role. The application mixes and emulsifies the C5a adjuvant and the antigen at an effective dose of 1x10 4 6x10 4 per tail to prepare the vaccine. After intraperitoneal injection and secondary boosting, the vaccine produces significant cross protection against the infection of the ichthyophthirius, and the protection effect is significantly better than that of a traditional Freund's adjuvant vaccine. The application breaks the bottleneck that the ichthyophthirius cannot be cultured in vitro and thus a large number of worm bodies cannot be obtained for large-scale preparation of whole worm vaccines, and also solves the problem of poor safety of traditional adjuvants, has the advantages of low cost, good effect, easy large-scale production and the like, and is suitable for green immune prevention and control of ichthyophthiriasis of freshwater fish.

Description

Technical Field

[0001] This invention relates to the field of fish disease prevention and control technology, and in particular to a vaccine against fish ichthyophthirius multifiliis disease, its preparation method, and its application. Background Technology

[0002] Ichthyophthiriasis is the most serious parasitic ciliate disease affecting farmed freshwater fish worldwide. Also known as "white spot disease," it is caused by the parasite *Ichthyophthirius multifiliis*, which infects the skin, gills, and fins of almost all freshwater bony fish, directly disrupting their respiratory and water-salt metabolism, leading to respiratory obstruction, osmotic imbalance, and death. *Ichthyophthirius multifiliis* is widely distributed globally, posing a significant threat to freshwater aquaculture, ornamental fisheries, and the conservation of wild fish resources. Outbreaks are more likely to occur and cause substantial economic losses in high-density aquaculture environments.

[0003] Due to the significant limitations of chemical drug control, vaccination is widely recognized as the most effective strategy to replace chemical drugs and achieve green and sustainable control of aquatic diseases. In recent years, research on vaccines against Ichthyophthirius multifiliis (white spot disease), including whole-worm vaccines, DNA vaccines, and subunit vaccines, has made some progress. However, Ichthyophthirius multifiliis vaccines have not yet been commercially available. The core bottleneck lies in the complexity of parasite antigens. Different serotypes of Ichthyophthirius multifiliis may express different specific antigens, posing challenges to the screening and design of vaccine targets. For example, the inhibitory antigen is a frequently studied target for Ichthyophthirius multifiliis vaccines. However, as research has progressed, it has been found that the inhibitory antigen of Ichthyophthirius multifiliis exhibits serotype specificity. That is, vaccines using the inhibitory antigen of the same strain have varying protective effects against infection in different strains, and may even offer no protection at all. Therefore, the development of DNA or subunit vaccines requires the search for other effective antigens. Compared to DNA vaccines and subunit vaccines, whole-worm vaccines use the complete worm as the antigen, thus fully preserving the natural antigenic composition of the pathogen. This allows the host's immune system to recognize multi-stage, multi-target antigenic epitopes, thereby inducing a more comprehensive and efficient protective immune response. This makes it an ideal direction for the development of vaccines against fish parasitic ciliates. However, the preparation of whole-worm vaccines requires a large number of worms. Currently, there is no in vitro culture method for *Ichthyophthirius multifiliis*, and worm collection still requires isolation from artificially infected live fish. This results in extremely low worm yield, high acquisition costs, and poor batch-to-batch quality stability, severely restricting the large-scale preparation and industrial application of whole-worm vaccines.

[0004] Recent studies by our team have confirmed that *Ichthyophthirius multifiliis*, as a facultative parasitic ciliate, is highly similar to *Ichthyophthirius multifiliis* in terms of nutritional mode, parasitic sites (gills, skin, fins), and surface antigen characteristics. More importantly, this parasite can be cultured in vitro on a large scale using plant-based culture media such as wheat grains, offering advantages such as short generation time (approximately 2.5 hours) and rapid reproduction, enabling convenient and low-cost acquisition of sufficient antigens. Although we have previously developed a technology for preparing vaccines against *Ichthyophthirius multifiliis* infection in fish using *Ichthyophthirius multifiliis*, the protective effect against *Ichthyophthirius multifiliis* infection remains unsatisfactory; only at high doses can a relatively ideal protective effect be achieved. Summary of the Invention

[0005] In view of this, the present invention provides a safe, efficient, and easily scalable cross-protective vaccine against Ichthyophthirius multifiliis (white spot disease) in fish, along with its preparation method and application. The invention aims to develop a dual-effect vaccine using *Ichthyophthirius multifiliis*, which is easily cultured in vitro, as the core antigen, and complement C5a as a novel and highly effective adjuvant, utilizing a cross-protective mechanism to control Ichthyophthirius multifiliis. This invention overcomes the bottleneck of antigen source in Ichthyophthirius multifiliis vaccine development and solves the problems of high toxicity and poor safety of traditional Freund's adjuvant, providing a novel technical solution for the green and efficient control of ciliate diseases in freshwater fish. It has significant theoretical research value and practical application value.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a vaccine against ichthyophthirius multifiliis disease in fish, wherein the vaccine uses inactivated whole worms of *Ichthyophthirius multifiliis* as antigen and complement C5a protein as an immunizing adjuvant;

[0008] The inactivated *Ichthyophthirius multifiliis* was *Ichthyophthirius multifiliis* that had been cultured and inactivated in vitro.

[0009] The amino acid sequence of the complement C5a protein is shown in SEQ ID NO. 6;

[0010] The vaccine contains 1×10 per 0.1 mL. 4 ~6×10 4 One inactivated insect body.

[0011] Preferably, the in vitro culture of the hook-spining *Ichthyophthirius multifiliis* uses grain culture medium, and the culture medium is prepared by:

[0012] (1) Mix wheat grains and water at a mass-volume ratio of 10g:180~220mL and sterilize;

[0013] (2) Centrifuge the above mixture to obtain the supernatant, and mix the supernatant with water at a volume ratio of 1:3~5 to obtain the mixture.

[0014] Preferably, the adjuvant content in the vaccine is 0.010~0.015μg / 0.1mL.

[0015] Preferably, the method for preparing the complement C5a protein includes the following steps:

[0016] (1) Total RNA was extracted from the spleen and head kidney tissues of fish and reverse transcribed into cDNA;

[0017] (2) The target C5a fragment was obtained by PCR amplification using a cDNA template and specific primers;

[0018] (3) The C5a fragment was ligated to the pET-28a vector to construct the recombinant plasmid pET-28a-C5a;

[0019] (4) The recombinant plasmid was transformed into Rosetta Escherichia coli, and expression was induced by IPTG. The complement C5a protein was obtained by purification by Ni-NTA chromatography column.

[0020] The present invention also provides a method for preparing the vaccine, comprising the following steps:

[0021] S1. In vitro culture and purification of *Ichthyophthirius multifiliis*: *Ichthyophthirius multifiliis* was cultured in the wheat grain medium described above, and purified worms were obtained by drainage method and gradient centrifugation and washing.

[0022] S2. Inactivation of parasites: Pure parasites were inactivated with 0.8-1.2 v / v% formaldehyde for 28-32 min, washed with PBS, and the inactivated whole parasite antigen was obtained.

[0023] S3. Preparation of complement C5a protein: Fish complement C5a protein was prepared according to the method described above;

[0024] S4. Vaccine emulsification: The inactivated Hooked Ichthyophthirius antigen is thoroughly mixed with complement C5a protein and emulsified to obtain the vaccine.

[0025] Preferably, the gradient centrifugation washing conditions are: centrifugation at 1400~1600 rpm for 2.5~3.5 min, centrifugation at 1800~2200 rpm for 32.5~3.5 min, and centrifugation at 2800~3200 rpm for 2.5~3.5 min, with the supernatant discarded after each step and resuspended and mixed with sterile water.

[0026] The present invention also provides the use of the vaccine in the preparation of drugs or preparations for preventing Ichthyophthirius multifiliis infection in fish.

[0027] The present invention also provides an immunization method for improving the resistance of fish to Ichthyophthirius multifiliis infection, comprising the following steps: mixing and emulsifying the antigen with complement C5a protein adjuvant to obtain a vaccine; and then immunizing the fish, wherein the adjuvant content in the vaccine is 0.010~0.015μg / 0.1mL.

[0028] Preferably, the vaccine is administered via intraperitoneal injection, and the immunization schedule is a booster immunization on days 13-15 after the initial immunization, with an injection dose of 0.08-0.12 mL / tail.

[0029] Preferably, the fish species include freshwater bony fish, and the freshwater bony fish species include koi.

[0030] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention uses inactivated whole *Ichthyophthirius multifiliis* cultured on a large scale in vitro as a cross-antigen and fish complement C5a protein as an immune adjuvant; the two work synergistically to exert a highly effective immunoprotective effect. The present invention mixes the C5a adjuvant with the antigen at an effective dose of 1×10-1 4 ~6×10 4 The vaccine is prepared by emulsifying individual cells / tails. After intraperitoneal injection and a second booster immunization, this vaccine provides significant cross-protection against Ichthyophthirius multifiliis infection, with a protective effect significantly superior to traditional Freund's adjuvant vaccine. This invention overcomes the bottleneck of obtaining large quantities of Ichthyophthirius multifiliis for mass production of whole-worm vaccines due to the inability to culture the parasite in vitro. It also solves the problem of poor safety of traditional adjuvants, offering advantages such as low cost, high efficacy, and ease of large-scale production. It is suitable for green immunization control of Ichthyophthirius multifiliis disease in freshwater fish. Attached Figure Description

[0031] Figure 1 To culture *Ichthyophthirius multifiliis* for 4 days using an optimized culture medium.

[0032] Figure 2 The protective efficacy of different ciliate whole-worm vaccines against Ichthyophthirius multifiliis infection in koi carp was evaluated, where A was 1×10⁻⁶. 4 Survival curves of koi carp challenged with *Ichthyophthirius multifiliis* (IM), *Chilodonella lanceolata* (CU), or *Tetrahymena thermophila* (TT) after immunization with inactivated *Ichthyophthirius multifiliis* (IM), *Chilodonella lanceolata* (CU), or *Tetrahymena thermophila* (TT); BD represents different doses (2×10⁻¹⁰). 4 4×10 4 6×10 4 The relative survival rate of koi carp challenged with Ichthyophthirius multifiliis after immunization with IM vaccine (B), CU vaccine (C), and TT vaccine (D) (inactivated ciliates / tail).

[0033] Figure 3 1×10 4 Survival curves of koi carp immunized with inactivated ciliate vaccine against challenge by Ichthyophthirius multifiliis.

[0034] Figure 4 Survival curves of koi carp immunized with complement C5a adjuvant in combination with different doses of inactivated ciliates against Ichthyophthirius multifiliis challenge. Detailed Implementation

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1. Preparation of antigen and adjuvant

[0037] 1. In vitro culture and collection of *Ichthyophthirius multifiliis*

[0038] (1) Optimize the preparation of culture medium

[0039] a. Weigh 10g of wheat grains, add 200mL of sterile water, sterilize under high temperature and high pressure, and store at 4℃;

[0040] b. After centrifuging the culture medium from step a at 6000 rpm for 3 min, take the supernatant, add 1 mL of supernatant to each culture dish, and then add 4 mL of sterile water to obtain the optimized in vitro culture medium for *Ichthyophthirius multifiliis*.

[0041] (2) In vitro culture and amplification of Hooked Ichthyophthirius multifiliis: Naturally infected worms were isolated and purified, inoculated into wheat grain medium, and cultured at 20-25℃ in the dark for 4 days until the logarithmic growth phase. The worms were then collected by filtration.

[0042] (3) Collection of *Ichthyophthirius multifiliis*

[0043] Collection of *Ichthyophthirius multifiliis* includes the following steps:

[0044] a. Culture *Ichthyophthirius multifiliis* using the optimized grain culture medium described above to obtain cultured worms;

[0045] b. Collect the worms obtained in step a into a 2mL centrifuge tube using the drainage method, centrifuge at 1500rpm for 3min, remove the supernatant, add 1mL of sterile water, and mix by pipetting.

[0046] c. Centrifuge the insect solution obtained in step b at 2000 rpm for 3 min, remove the supernatant, add 1 mL of sterile water, and mix by pipetting.

[0047] d. Centrifuge the insect solution obtained in step c at 3000 rpm for 3 min; after removing the supernatant, the bottom of the centrifuge tube contains the cleaned insects.

[0048] Using optimized culture media and collection methods, high-density, high-vitality *Ichthyophthirius multifiliis* can be obtained after 4 days of culture. Figure 1 The culture medium contamination rate was significantly reduced, and the insect purity was high, providing high-quality antigen materials for vaccine preparation.

[0049] 2. Inactivation of *Ichthyophthirius multifiliis* worms

[0050] (1) Inactivation treatment: The collected and washed insect bodies were suspended in a formaldehyde solution with a final concentration of 1% (v / v) and inactivated at room temperature for 30 min;

[0051] (2) Purification and preservation: Centrifuge the inactivated insect fluid at 7000g for 10 min, collect the insect body precipitate, wash it at least 3 times with sterile PBS buffer (pH 7.4) to remove residual formaldehyde, and finally resuspend the insect body in sterile PBS buffer and store it at -80℃ for later use, thus obtaining the inactivated whole insect antigen of Hooked Thylocorus spp.

[0052] 3. Preparation of complement C5a protein

[0053] (1) Template preparation and target fragment amplification

[0054] Spleen and head kidney tissues of healthy koi carp were collected, and total RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed with specific primers C5aF / C5aR, and the target C5a fragment was recovered and purified. Then, using the purified product as a template, a second PCR was performed with primers containing homologous arms and BamHI and XhoI restriction sites, and the fragment was recovered for later use.

[0055] C5aF: 5'-TCAGCTGACCTGGCACAAATGTTTG-3' (SEQ ID NO. 1);

[0056] C5aR: 5'-TCAGCTGACCTGGCACAAATGTTTG-3' (SEQ ID NO. 2);

[0057] C5a-2-BamHI-F:5'-CGCGGATCCATGTCAGCTGACCTGGCACAAATGTTTG-3' (SEQ ID NO. 3);

[0058] C5a-2-XhoI-R:

[0059] 5'-CCGCTCGAGTTAGCGACTTAGTATGAATTTATCCTTA-3' (SEQ ID NO. 4).

[0060] (2) Construction and identification of recombinant plasmids

[0061] The pET-28a vector was purified by double digestion with BamHI and XhoI, and then ligated with the C5a fragment with homologous arms via homologous recombination to construct the recombinant plasmid pET-28a-C5a. This plasmid was transformed into competent Rosetta coli cells, and after ampicillin resistance screening, colony PCR, and sequencing identification, it was cultured and maintained. The plasmid was purified using an endotoxin-free plasmid extraction kit, quantified using Nanodrop 8000, and then used for further processing.

[0062] (3) Expression and purification of recombinant proteins

[0063] The recombinant plasmid was transformed into Rosetta strain, positive clones were screened with ampicillin, and the culture was expanded; bacterial culture OD... 600 When the concentration reaches 0.4-0.6, IPTG is added and induced at 28℃ for 6 h. The cells are collected by centrifugation and lysed. SDS-PAGE is used to analyze protein expression and solubility. At 4℃, the supernatant is filtered and loaded onto a Ni-NTA chromatography column. After washing and imidazole elution, the target protein is obtained. After dialysis to remove imidazole, the protein is aliquoted and stored at -80℃. The protein concentration is determined by BCA method.

[0064] The nucleotide sequence encoding complement C5a is as follows:

[0065] TCAGCTGACCTGGCACAAATGTTTCGAACAAAAGGCCAAAAAGTATGGGAAGTTTTACAGTTGCTGCACATTCGGGATAGCGAGTAGCCCAACGCTTGAAGCCTGTCAGCAGCGCTCAGAGAGACTGTCTTATCCTGCGAAGACAATGTGTCAGCAAGCTTTCCAGGAATGCTGTGGCTTTGCTATGAAGCAGCGCCAAGAGAATAAGGATAAATTCATACTAAGTCGC (SEQ ID NO.5);

[0066] The amino acid sequence of complement C5a is as follows:

[0067] SADLAQMFEQKAKKYGKFYSCCTFGIASSPTLEACQQRSERLSYPAKTMCQQAFQECCGFAMKQRQENKDKFILSR (SEQ ID NO. 6).

[0068] Example 2. Vaccine Preparation

[0069] 1. Preparation of complement C5a adjuvant vaccines

[0070] Primary immunization vaccine: Mix inactivated parasites with complement C5a protein molecule adjuvant (0.012 μg per fish) and emulsify thoroughly using a hand-held homogenizer.

[0071] The preparation method for booster vaccines is the same as that for primary immunization vaccines.

[0072] Stability was verified by a water droplet diffusion test: a few drops of the emulsion sample were added to sterile water. If no diffusion occurred within 10 minutes, the emulsion was deemed stable and could be used for subsequent immunization experiments.

[0073] 2. The preparation method of Freund's adjuvanted vaccine is as follows:

[0074] Primary immunization vaccine: Inactivated parasites are mixed with an equal volume of Freund's complete adjuvant (FCA) at a 1:1 (v / v) antigen-adjuvant ratio and thoroughly emulsified using a hand-held homogenizer to form a stable oil-in-water emulsion. The stability is verified by a water droplet diffusion test. The emulsion is stable and can be used for subsequent immunization.

[0075] Boosting immunization vaccine: Inactivated parasites are mixed with an equal volume of Freund's incomplete adjuvant (FIA) at a 1:1 (v / v) antigen-adjuvant ratio and emulsified using the same method.

[0076] 3. The control group vaccine was prepared using the following method:

[0077] For primary immunization: Sterile PBS buffer and Freund's complete adjuvant (FCA) are mixed at a ratio of 1:1 (v / v) and thoroughly emulsified using a hand-held homogenizer to form a stable oil-in-water emulsion. The stability is verified by a water droplet diffusion test. The emulsion is stable and can be used for subsequent immunization.

[0078] Boosting immunization vaccine: Mix sterile PBS buffer with Freund's incomplete adjuvant (FIA) at a ratio of 1:1 (v / v), emulsify using the same method, and prepare an adjuvant buffer emulsion.

[0079] Example 3. Verification of the immunoprotective and cross-protective effects of inactivated ciliate vaccine combined with Freund's adjuvant

[0080] This embodiment uses koi carp as experimental subjects to verify the protective effect of inactivated whole-worm vaccines against Ichthyophthirius multifiliis infection against three ciliates: *Ichthyophthirius multifiliis* (CU), *Ichthyophthirius multifiliis* (IM), and *Tetrahymena thermophila* (TT). An immunization regimen of initial Freund's complete adjuvant followed by a second Freund's incomplete adjuvant was used, with 1×10⁻⁶ doses administered. 4 2×10 4 4×10 4 6×10 4 A dose gradient of 1 / tail was used, with PBS + Freund's adjuvant as a control. To evaluate the protective efficacy of the three inactivated vaccines, lethal doses of Ichthyophthirius multifiliis (3 × 10⁻⁶) were administered. 4 The predator (body / tail) was used to conduct a virus challenge experiment.

[0081] Immunization dose: The vaccine contains an effective dose of inactivated whole ciliates, with an effective dose range of 1 × 10⁻⁶ per 0.1 mL. 4 ~6×10 4 One inactivated insect body.

[0082] Immunization route: Intraperitoneal injection was used, with each fish injected with 0.1 mL of the above-mentioned emulsified stable vaccine or control emulsion.

[0083] Immunization program: A two-stage immunization program was adopted, that is, on the 14th day after the initial immunization, the experimental fish were given a booster immunization, and the injection dose was the same as that of the initial immunization.

[0084] First, administer whole-cell vaccines against Ichthyophthirius multifiliis (CU), Ichthyophthirius multifiliis (IM), and Tetrahymena thermophila (TT) at a dose of 1×10⁻⁶. 4 Koi were immunized with a dose of 100 cells / tail, with PBS + Freund's adjuvant as a control. 28 days after immunization, a lethal dose of Ichthyophthirius multifiliis (3 × 10⁻⁶ cells / tail) was administered. 4 The koi carp were challenged with a virus (the carp's body / tail) and monitored for 14 consecutive days after the challenge. The survival rate of the koi carp was then recorded.

[0085] The protective efficacy of various vaccines against Ichthyophthirius multifiliis infection in koi carp after challenge was as follows: Figure 2 As shown in A in the figure. The results showed that the IM group had the highest final survival rate of 60%, with the best protective effect; the CU group had a final survival rate of 6.67%, with a weak protective effect; the TT group had a final survival rate of 3.33%, with almost no protection; and the control group had all deaths, with a survival rate of 0%.

[0086] Then, whole-parasite vaccines against Ichthyophthirius multifiliis (CU), Ichthyophthirius multifiliis (IM), and Tetrahymena thermophila (TT) were administered at a dose of 2×10⁻⁶. 4 (Low-dose group), 4×10 4 (Medium-dose group), 6×10 4 Koi were immunized with a dose gradient of 100 individuals / tail (high-dose group), with PBS + Freund's adjuvant as a control. The protective efficacy of each vaccine against Ichthyophthirius multifiliis infection in koi was as follows: Figure 2 As shown in BD.

[0087] The protective effects of different doses of IM (Ichthyophthirius multifiliis) whole worm vaccine are as follows: Figure 2 As shown in B, when IM is 6×10 4 / Tail and 4×10 4 When the tail is / , the final survival rate is 100%; when IM is 2×10 4 When the tail is shaped, the final survival rate is 80%.

[0088] The protective effects of different doses of CU (Chilodonella uncinata) whole worm vaccine, such as Figure 2 As shown in C, when CU is 6×10 4 When the tail is at 1 / 2, the overall survival rate is 100%; when the CU is 4×10 4 When the tail is / , the final survival rate is 70%; when the CU is 2×10 4 At the end of the period, the final survival rate was 33.33%.

[0089] The protective effects of different doses of TT (Tetrahymena thermophila) whole worm vaccine are as follows: Figure 2As shown in D, when TT is 6×10 4 At the tail position, the final survival rate was 53.33%; when TT was 4×10 4 When the tail is / , the final survival rate is 33.33%; when TT is 2×10 4 When the tail is shaped, the final survival rate is only 10%.

[0090] In summary, 6×10 4 High-dose Ichthyophthirius multifiliis vaccine per koi carp can effectively protect koi carp from death caused by Ichthyophthirius multifiliis infection. There is significant cross-protection between Ichthyophthirius multifiliis (CU) and Ichthyophthirius multifiliis (IM) whole worm vaccines.

[0091] It is evident that, due to the bottleneck of not being able to obtain a large number of Ichthyophthirius multifiliis (Ich) cells for mass production of whole-worm vaccines because Ich cannot be cultured in vitro, Cu can serve as an alternative antigen for IM. High-dose Cu vaccines can achieve the same protective effect as IM homologous vaccines.

[0092] Example 4. Verification of the synergistic effect of complement C5a as an immune adjuvant

[0093] Using PBS + Freund's adjuvant as a blank control, 1×10 4 The *Ichthyophthirius multifiliis* vaccine (CU-F) and *Ichthyophthirius multifiliis* vaccine (IM-F), formulated with inactivated parasites and Freund's adjuvant, served as positive controls. Complement C5a was mixed with 1×10⁻⁶ parasites and 1×10⁻⁶ adjuvant. 4 One inactivated Hooked Ichthyophthirius multifiliis antigen, 1×10 4 Two inactivated Ichthyophthirius multifiliis antigens were combined to form experimental groups (CU-C5a, IM-C5a), as shown in Table 1. Each group followed the secondary immunization protocol of Example 3, and 28 days after immunization, they were administered a lethal dose of Ichthyophthirius multifiliis (3 × 10⁻⁶). 4 The predator (body / tail) was challenged with the virus, and the koi were monitored continuously for 14 days after challenge to determine the survival rate. Survival curves of koi from different immune groups after challenge with *Ichthyophthirius multifiliis* are shown below. Figure 3 As shown.

[0094] Table 1. Types and dosages of antigens and adjuvants

[0095] Group vaccine type Adjuvant type Dosage (per animal / tail) PBS-F Blank control Freund's adjuvant <![CDATA[1×10 4 ]]> CU-F Pilocheilus fasciatus Freund's adjuvant <![CDATA[1×10 4 ]]> CU-C5a Pilocheilus fasciatus C5a adjuvant <![CDATA[1×10 4 ]]> IM-F Ichthyophthirius multifiliis Freund's adjuvant <![CDATA[1×10 4 ]]> IM-C5a Ichthyophthirius multifiliis C5a adjuvant <![CDATA[1×10 4 ]]>

[0096] Figure 3 The effects of each group on infection with Ichthyophthirius multifiliis are shown as follows:

[0097] PBS-F control group: All died on day 8, with a final survival rate of 0%.

[0098] CU-C5a: The final survival rate was 60%, which was significantly better than the Freund's adjuvant group of the same vaccine (CU-F: 23.33%).

[0099] IM-C5a: The final survival rate was 83.33%, which was significantly better than the Freund's adjuvant group of the same vaccine (IM-F: 66.67%).

[0100] CU-F: Final survival rate 23.33%, the worst protection effect.

[0101] IM-F: Final survival rate 66.67%.

[0102] The results show that C5a adjuvant is significantly superior to traditional Freund's adjuvant in both vaccines and both challenge models, not only greatly improving the protective effect of the vaccines, but also enhancing the cross-protective effect of the vaccines.

[0103] The above results show that at 1×10 4 Under low-dose conditions per person / tail, complement C5a significantly enhances vaccine protective efficacy and cross-protective efficacy compared to Freund's adjuvant.

[0104] Example 5. Dosage optimization of whole-worm vaccine with added complement C5a adjuvant

[0105] Set 2×10 4 3×10 4 The protective effects of different doses of IM and CU vaccines combined with C5a adjuvant were compared using individual / tail-based dose gradients. The experimental groups are shown in Table 2.

[0106] Table 2. Types and dosages of antigens and adjuvants

[0107] Group antigen type Adjuvant type Dosage (per animal / tail) PBS-C5a Blank control C5a -- IM-C5a low-dose group Ichthyophthirius multifiliis C5a <![CDATA[2×10 4 ]]> IM-C5a high-dose group Ichthyophthirius multifiliis C5a <![CDATA[3×10 4 ]]> CU-C5a low-dose group Pilocheilus fasciatus C5a <![CDATA[2×10 4 ]]> CU-C5a high-dose group Pilocheilus fasciatus C5a <![CDATA[3×10 4 ]]>

[0108] The immunization route and procedure were the same as in Example 3. A booster immunization was administered 28 days later with a lethal dose of Ichthyophthirius multifiliis (3 × 10⁻⁶). 4 The carp were challenged with predators (body / tail) and monitored for 14 days after challenge. The survival rate of the carp was then recorded. Survival curves of koi immunized with complement C5a adjuvant combined with different doses of inactivated ciliates against Ichthyophthirius multifiliis challenge are shown below. Figure 4 As shown.

[0109] Figure 4 The results showed that mortality began to occur in the PBS-C5a blank control group on day 3, with a final survival rate of only 6.67%. The high-dose IM-C5a group had no mortality throughout the treatment, achieving a final survival rate of 100%, providing complete protection; the low-dose IM-C5a group had only a few mortalitys, with a final survival rate of 90%, demonstrating excellent protective efficacy. The high-dose CU-C5a group had very few mortalitys, with a final survival rate of 96.67%, showing cross-protective efficacy close to that of homologous vaccines; the low-dose CU-C5a group had a final survival rate of 73.33%, significantly better than the control group. This indicates that using C5a as an adjuvant at 3×10 4At a dose of one individual per tail, *Ichthyophthirius multifiliis* can achieve high cross-protection against *Ichthyophthirius multifiliis*. *Ichthyophthirius multifiliis* can serve as a highly efficient alternative antigen, providing a feasible and excellent solution to overcome the technical bottleneck of the difficulty in in vitro culture of *Ichthyophthirius multifiliis*.

[0110] As can be seen from the above embodiments, the present invention provides a vaccine against ichthyophthirius multifiliis disease in fish, its preparation method, and its application. The present invention uses *Ichthyophthirius multifiliis* as a highly effective substitute antigen and C5a as an adjuvant, achieving highly effective protection against ichthyophthirius multifiliis disease.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A vaccine against ichthyophthirius multifiliis disease in fish, characterized in that, The vaccine uses inactivated whole worms of *Ichthyophthirius multifiliis* as antigen and complement C5a protein as an immunizing adjuvant. The inactivated *Ichthyophthirius multifiliis* was *Ichthyophthirius multifiliis* that had been cultured and inactivated in vitro. The amino acid sequence of the complement C5a protein is shown in SEQ ID NO. 6; The vaccine contains 1×10 4 ~6×10 4 One inactivated insect body / 0.1 mL.

2. The vaccine according to claim 1, characterized in that, The in vitro culture of *Ichthyophthirius multifiliis* was performed using grain culture medium, and the culture medium was prepared as follows: (1) Mix wheat grains and water at a mass-volume ratio of 10g:180~220mL and sterilize; (2) Centrifuge the above mixture to obtain the supernatant, and mix the supernatant with water at a volume ratio of 1:3~5 to obtain the mixture.

3. The vaccine according to claim 1, characterized in that, The adjuvant content in the vaccine is 0.010~0.015μg / 0.1mL.

4. The vaccine according to claim 1, characterized in that, The preparation method of the complement C5a protein includes the following steps: (1) Total RNA was extracted from the spleen and head kidney tissues of fish and reverse transcribed into cDNA; (2) The target C5a fragment was obtained by PCR amplification using a cDNA template and specific primers; (3) The C5a fragment was ligated to the pET-28a vector to construct the recombinant plasmid pET-28a-C5a; (4) The recombinant plasmid was transformed into Rosetta Escherichia coli, and expression was induced by IPTG. The complement C5a protein was obtained by purification by Ni-NTA chromatography column.

5. The method for preparing the vaccine according to claim 1, characterized in that, Includes the following steps: S1. In vitro culture and purification of *Ichthyophthirius multifiliis*: *Ichthyophthirius multifiliis* was cultured in the grain culture medium described in claim 2, and purified worms were obtained by drainage method, gradient centrifugation, and washing. S2. Inactivation of parasites: Pure parasites were inactivated with 0.8-1.2 v / v% formaldehyde for 28-32 min, washed with PBS, and the inactivated whole parasite antigen was obtained. S3. Preparation of complement C5a protein: Fish complement C5a protein is prepared according to the method described in claim 5; S4. Vaccine emulsification: Inactivated chilodonella antigen is mixed with complement C5a protein and emulsified to obtain the vaccine.

6. The preparation method according to claim 5, characterized in that, The gradient centrifugation is as follows: centrifuge at 1400~1600 rpm for 2.5~3.5 min, centrifuge at 1800~2200 rpm for 2.5~3.5 min, and centrifuge at 2800~3200 rpm for 2.5~3.5 min. After each step, discard the supernatant and resuspend and mix with sterile water.

7. The use of the vaccine according to any one of claims 1 to 4 in the preparation of a drug or preparation for the prevention of ichthyophthirius multifiliis disease in fish.

8. An immunization method for improving the resistance of fish to Ichthyophthirius multifiliis infection, characterized in that, The method includes the following steps: mixing and emulsifying the antigen described in claim 1 with complement C5a protein adjuvant to obtain a vaccine; and then immunizing fish with the vaccine, wherein the adjuvant content in the vaccine is 0.010~0.015μg / 0.1mL.

9. The immunization method according to claim 8, characterized in that, The vaccine is administered via intraperitoneal injection. The immunization schedule is a booster immunization on days 13-15 after the initial immunization, with an injection dose of 0.08-0.12 mL per tail.

10. The immunization method according to claim 8, characterized in that, The fish species include freshwater bony fish, and the freshwater bony fish include koi.

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