Recombinant subunit vaccine against Trichomonas vaginalis infection in pigeons and its preparation method

Abstract

This invention discloses a recombinant subunit vaccine against Trichomonas vaginalis infection in pigeons and its preparation method. The vaccine comprises two recombinant proteins having sequences shown in SEQ ID NO:7 and SEQ ID NO:8, respectively, and a pharmaceutically acceptable carrier. This invention uses Sf9 cells to express Trichomonas vaginalis adhesion protein 33 and adhesion protein 65, respectively. The resulting recombinant proteins exhibit immunogenicity similar to the natural proteins, high expression levels, and strong immunogenicity. Even very small amounts can provide good immune protection, and the vaccines are non-pathogenic to pigeons. Furthermore, large-scale serum-free suspension culture can be performed using a bioreactor, significantly reducing the cost of vaccine production.

Description

Technical Field

[0001] This invention specifically relates to a recombinant subunit vaccine against Trichomonas vaginalis infection in pigeons and its preparation method, belonging to the field of animal immunotherapy technology. Background Technology

[0002] Trichomoniasis in pigeons is a parasitic disease with a high incidence in pigeons. Affected pigeons exhibit symptoms such as difficulty breathing, loss of appetite, swallowing difficulties, weight loss, increased thirst, depression, diarrhea, vomiting, ruffled feathers, unsteady gait, and drooping crop. Young pigeons are highly susceptible to death, and surviving pigeons may remain latently infected and carry the parasite for a long time. Therefore, trichomoniasis severely impacts pigeon breeding and growth performance, hindering the development of pigeon farming. Currently, nitroimidazole drugs such as metronidazole and dimetronidazole are commonly used to treat trichomoniasis in pigeons. However, nitroimidazole drugs have side effects such as carcinogenicity and teratogenicity, as well as problems with drug resistance and drug residues. Furthermore, these drugs are prohibited from use in animal-derived food production. In addition, high-density pigeon housing and poor hygiene conditions further exacerbate the spread and infection of trichomoniasis. Therefore, there is an urgent need to develop safer and more effective alternative treatments.

[0003] Infection with *Trichomonas gallinae* is the causative agent of trichomoniasis in pigeons. *Trichomonas gallinae* belongs to the phylum Protozoa, class Flagellates, subclass Caryotaea, order Polymastia, and primarily parasitizes the mucosal surface of the digestive tract of pigeons. Current research has identified several immunogenic *Trichomonas* proteins, such as adhesion protein 33 (AP33), adhesion protein 65 (AP65), and G3. Among these, the *Trichomonas gallinae* adhesion protein family plays a crucial role in infection. For example, AP33 and AP65 are important functional proteins of *Trichomonas gallinae*, synthesized and secreted onto the surface of the parasite. They primarily play an adhesive role in the adhesion of *Trichomonas gallinae* to host cells. Higher levels of AP33 and AP65 expression indicate stronger infectivity. Furthermore, AP33 and AP65 also play irreplaceable roles in the immune evasion and immunosuppression processes of *Trichomonas gallinae*.

[0004] In recent years, genetically engineered vaccines against Trichomonas vaginalis using systems such as Escherichia coli and Pichia pastoris to express related recombinant proteins have been reported. However, the expression levels of AP33 and AP65 proteins expressed in E. coli are low, and endotoxins are difficult to remove. Furthermore, the method of recombinantly expressing AP65 in Pichia pastoris requires methanol as an inducer, which poses certain risks, and the glycosylation level of the Pichia pastoris expression system is relatively low. Summary of the Invention

[0005] The main objective of this invention is to provide a recombinant subunit vaccine against Trichomonas vaginalis infection and its preparation method, so as to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the present invention employs the following technical solution.

[0007] A first aspect of the present invention provides a recombinant protein composition comprising:

[0008] The first recombinant protein has the sequence shown in SEQ ID NO:7 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:7;

[0009] The second recombinant protein has the sequence shown in SEQ ID NO:8 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:8.

[0010] In one embodiment, the mass ratio of the first recombinant protein to the second recombinant protein is 1:1.

[0011] A second aspect of the present invention provides a gene composition comprising:

[0012] The first gene encodes the first recombinant protein;

[0013] The second gene encodes the second recombinant protein.

[0014] A third aspect of the present invention provides a gene composition comprising:

[0015] The first gene has the sequence shown in SEQ ID NO:1 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:1;

[0016] The second gene has the sequence shown in SEQ ID NO:2 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:2.

[0017] A fourth aspect of the present invention provides a combination of recombinant vectors, comprising:

[0018] The first recombinant vector contains the first gene with the sequence shown in SEQ ID NO:1;

[0019] The second recombinant vector contains a second gene with the sequence shown in SEQ ID NO:2.

[0020] In one embodiment, the first or second recombinant vector includes, but is not limited to, pFastBac 1, pVL1393, pFastBac dual, or pDEST8, with pFastBac 1 being preferred.

[0021] A fifth aspect of the invention provides a combination of host cells, comprising:

[0022] The first host cell contains the first gene;

[0023] The second host cell contains the second gene.

[0024] In one embodiment, the first or second host cell includes, but is not limited to, Sf9, HighFive, or Sf21 cells, preferably Sf9 cells.

[0025] A sixth aspect of the present invention provides an immune composition comprising: the recombinant protein composition; and a pharmaceutically acceptable carrier.

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

[0027] In one embodiment, the recombinant protein composition comprises a first recombinant protein and a second recombinant protein in a mass ratio of 1:1.

[0028] In one embodiment, the content of the first recombinant protein and the second recombinant protein in the immune composition may be 50 μg to 250 μg / ml.

[0029] A seventh aspect of the present invention provides a method for preparing a recombinant protein, comprising:

[0030] Provide the combination of the host cells;

[0031] First host cells and second host cells were cultured under suitable conditions, and then the first recombinant protein and the second recombinant protein were isolated.

[0032] In one embodiment, the preparation method specifically includes:

[0033] S1. Prepare nucleic acid molecules encoding the first recombinant protein and the second recombinant protein, respectively;

[0034] S2. Construct a recombinant vector by cloning the nucleic acid molecules described in step S1 into a shuttle vector to obtain a recombinant shuttle vector containing the target gene.

[0035] S3. Transform the recombinant shuttle vector into competent cells to obtain recombinant plasmids, and then transfect host cells to obtain recombinant baculoviruses.

[0036] S4. The recombinant baculovirus is inoculated into host cells to obtain expression products, namely the first recombinant protein and the second recombinant protein.

[0037] For example, the preparation method includes:

[0038] S1. Prepare nucleic acid molecules encoding recombinant AP33 protein and recombinant AP65 protein respectively;

[0039] S2. Construct recombinant vectors by cloning the nucleic acid molecules from step S1 into shuttle vectors (e.g., pFastBac1) to obtain recombinant shuttle vectors containing the target gene (e.g., pFastBac1-AP33 and pFastBac1-AP65).

[0040] S3. Transform the recombinant shuttle vector into DH10Bac bacteria, and obtain recombinant plasmids (such as Re-Bacmid-AP33 recombinant plasmid and Re-Bacmid-AP65 recombinant plasmid) by blue-white screening and PCR identification. Then, transfect Sf9 cells to obtain recombinant baculovirus.

[0041] S4. After purifying the recombinant baculovirus and determining its titer, the virus is inoculated into Sf9 cells to obtain the expression products recombinant AP33 protein and recombinant AP65 protein, namely the first recombinant protein and the second recombinant protein.

[0042] Furthermore, the preparation method may also include the steps of separating and purifying the first recombinant protein and the second recombinant protein, wherein the optional separation and purification methods include, but are not limited to, chromatography, dialysis, or other methods known in the art.

[0043] In one embodiment, the preparation method may further include:

[0044] S5. Mix the first recombinant protein and the second recombinant protein with a pharmaceutically acceptable carrier.

[0045] Furthermore, the first recombinant protein and the second recombinant protein can be diluted to 100 μg / ml to 500 μg / ml respectively, and mixed at a 1:1 mass ratio, so that the content of each recombinant protein in the obtained mixed liquid is 50 μg / ml to 250 μg / ml, and then mixed with a pharmaceutically acceptable carrier.

[0046] For example, recombinant AP33 protein and recombinant AP65 protein can be diluted to 100 μg / ml respectively, mixed at a mass ratio of 1:1, and then mixed with white oil adjuvant. After emulsification at an antigen:adjuvant ratio of 2:3, one dose of vaccine contains 20 μg each of recombinant AP33 and AP65 protein.

[0047] An eighth aspect of the invention provides the use of the recombinant protein composition, the gene composition, or the immune composition in the preparation of an agent for inducing an immune response against Trichomonas giardiasis in test animals or for preventing Trichomonas giardiasis in animals.

[0048] A ninth aspect of the invention provides the use of the recombinant protein composition or the immune composition in the preparation of a recombinant subunit vaccine against Trichomonas vaginalis infection.

[0049] A tenth aspect of the invention provides a recombinant subunit vaccine against Trichomonas vaginalis infection in pigeons, the vaccine comprising the recombinant protein composition. Further, the vaccine may also comprise a pharmaceutically acceptable carrier.

[0050] Furthermore, the vaccine is a bivalent genetically engineered subunit vaccine.

[0051] The recombinant subunit vaccine against Trichomonas vaginalis infection provided in this invention only requires inoculation of animals with an effective dose. The "effective dose" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0052] The eleventh aspect of the present invention provides a method for inducing an immune response against Trichomonas giardiasis or for protecting a test animal from Trichomonas giardiasis infection, the method comprising administering the test animal a recombinant subunit vaccine against Trichomonas giardiasis infection.

[0053] A twelfth aspect of the invention also provides a vaccine suitable for inducing an immune response against Trichomonas vaginalis infection in test animals, the vaccine comprising: the recombinant protein composition and an adjuvant. As used in this specification, "adjuvant" means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen encoded by the gene. Further, the adjuvant may preferably be an adjuvant produced by Suzhou Womei Biotechnology Co., Ltd., which can improve vaccine efficacy.

[0054] A thirteenth aspect of the present invention also provides a kit comprising the recombinant protein composition, the gene composition, a combination of the recombinant vectors, a combination of the host cells, or the immune composition.

[0055] Furthermore, the kit may also include containers or instruments, such as syringes, for packaging or administering the recombinant protein composition, the gene composition, the combination of recombinant vectors, the combination of host cells, or the immune composition to animals.

[0056] Compared with the prior art, the technical solution provided by the embodiments of the present invention has at least the following advantages:

[0057] (1) Based on the transmission pathway and life activities of Trichomonas pigeonis, the adhesion proteins AP33 and AP65 of Trichomonas pigeonis were screened through bioinformatics analysis. The sequences were optimized by insect cell codons, and the Kozak sequence and signal peptide sequence were added to increase the expression level. The obtained recombinant AP33 and AP65 proteins are similar to natural proteins, with high expression levels, strong stability, and strong immunogenicity. A small amount can provide good immune protection, and they are non-pathogenic to pigeons and have high safety.

[0058] (2) By using the baculovirus expression system to express recombinant AP33 and AP65 proteins, large-scale production can be achieved in bioreactors with high protein expression levels; quality control is easy and batch-to-batch quality is stable; the produced proteins have strong stability and immunogenicity, meeting the needs of large-scale production.

[0059] (3) Recombinant AP33 protein and AP65 protein were mixed at a mass ratio of 1:1 and emulsified with a vaccine adjuvant to prepare a multivalent subunit vaccine against Trichomonas vaginalis. The prepared vaccine produced high antibody levels in immunized pigeons, and the antibodies produced could inhibit Trichomonas vaginalis's invasion, cell adhesion, growth, reproduction, and other vital activities, thereby providing immune protection for pigeons. What is particularly surprising is that the vaccine prepared in this application can provide pigeons with immune protection against infection by multiple genotypes of Trichomonas vaginalis, and a strong immune protection effect can be achieved with a single immunization. In comparison, existing vaccines of the same type require multiple immunizations, and the vaccine reduces the stress response of pigeons during the immunization process, greatly reducing costs. Attached Figure Description

[0060] Figure 1A-Figure 1B The diagrams show the transfer vectors pFastBac1-AP33 and pFastBac1-AP65 containing the target gene constructed in Example 1, respectively.

[0061] Figures 2A-2B These are gel electrophoresis images of the PCR products after PCR amplification of the codon-optimized Trichomonas vaginalis AP33 and AP65 genes in Example 1.

[0062] Figures 3A-3B The images show gel electrophoresis diagrams of the PCR products after PCR amplification of colony samples transformed with the AP33 and AP65 genes, respectively, in Example 1.

[0063] Figures 4A-4B The images show gel electrophoresis images of the PCR products after PCR amplification of the blue-white screening colony samples after transposition of pFastBac1-AP33 and pFastBac1-AP65 in Example 2.

[0064] Figures 5A-5B The images are SDS-PAGE gel electrophoresis images of the cell culture supernatant containing recombinant AP33 protein and recombinant AP65 protein harvested in Example 4.

[0065] Figures 6A-6B The images shown are Western blotting images of the two SDS-PAGE electrophoresis products from Example 4.

[0066] Figure 7 This is a graph showing the antibody detection results on day 14 of the immunization in different immunization groups in Example 9. Detailed Implementation

[0067] The present invention is further illustrated below by way of examples. All reagents and raw materials used in the following examples are commercially available, and experimental methods not specifically described are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0068] Example 1: Construction and Identification of Transfer Vectors pFastBac1-AP33 and pFastBac1-AP65

[0069] 1. Amplification and purification of AP33 and AP65 genes

[0070] The codon-optimized AP33 gene (SEQ ID NO:1) and AP65 gene (SEQ ID NO:2) were synthesized at Suzhou Genewiz Biotechnology Co., Ltd., and cloned into the pUC17 vector to obtain pUC17-AP33 and pUC17-AP65 plasmid vectors, respectively. PCR amplification was performed using pUC17-AP33 and pUC17-AP65 plasmids as templates, respectively. The primer sequences and amplification systems are shown in Tables 1 and 2.

[0071] Table 1 Gene Primer Sequences

[0072]

[0073] Table 2 Gene amplification system

[0074] Reactive components Unit (μl) plasmid 1 F primer 2 R primer 2 2×PCR MasterMix 25 <![CDATA[ddH2O]]> 20

[0075] The reaction conditions were: 95℃ pre-denaturation for 5 minutes; 94℃ denaturation for 45 seconds, 60℃ annealing for 45 seconds, 72℃ extension for 1 minute, 35 cycles; 72℃ extension for 10 minutes.

[0076] The PCR products were subjected to gel electrophoresis to verify the size of the target gene. After successful amplification of the target gene, it was recovered and purified using a gel recovery and purification kit.

[0077] Specifically, such as Figure 2A As shown, the PCR product of the codon-optimized Trichomonas vaginalis AP33 gene was amplified by PCR and then subjected to gel electrophoresis. A band appeared around 1014 bp. Lane M is the DNA marker, lane 1 is the negative control, and lane 2 is the PCR amplification product of the AP33 gene. Figure 2B As shown, the PCR product of the codon-optimized Trichomonas vaginalis AP65 gene was amplified by PCR and then subjected to gel electrophoresis. A band was observed around 1785 bp. Lane M is the DNA marker, lane 1 is the negative control, and lane 2 is the PCR amplification product of the AP65 gene.

[0078] 2. Enzyme digestion and purification

[0079] The plasmids and PCR amplification products of the AP33 and AP65 gene expression frames were digested with EcoR I and Xba I enzymes at 37°C for 3 hours. The specific enzyme digestion reaction system is shown in Tables 3 and 4.

[0080] The enzyme digestion products were subjected to gel electrophoresis, and the digested pFastBac 1 plasmid, AP33, and AP65 gene fragments were purified using a gel recovery and purification kit.

[0081] Table 3 Gene digestion reaction system

[0082] Reactive components Unit (μl) PCR products 20 reaction buffer 5 EcoR I enzyme 2 Xba I enzyme 2 <![CDATA[ddH2O]]> 21

[0083] Table 4 pFastBac 1 plasmid digestion reaction system

[0084] Reactive components Unit (μl) plasmid 10 reaction buffer 5 EcoR I enzyme 2 Xba I enzyme 2 <![CDATA[ddH2O]]> 31

[0085] 3. Connection

[0086] The double-digested pFastBac1 plasmid and the digestion products of AP33 and AP65 genes were ligated using T4 DNA ligase at 16°C overnight. The specific ligation reaction system is shown in Table 5. The resulting transfer vectors pFastBac1-AP33 and pFastBac1-AP65, containing the target genes, have 5746 and 6514 base pairs, respectively. Figure 1A , Figure 1BAs shown.

[0087] Table 5. Ligation system of gene digestion products with pFastBac 1 plasmid

[0088] Reactive components Unit (μl) Gene digestion products 6 Enzyme digestion plasmid 2 reaction buffer 1 T4 DNA ligase 1

[0089] 4. Transformation

[0090] Add 10 μl of the ligation product to 100 μl of DH5α competent cells and mix well. Incubate on ice for 30 minutes, then subject to heat shock at 42°C for 90 seconds, followed by an ice incubation for 2 minutes. Add 900 μl of Amp-free LB liquid medium and incubate at 37°C for 1 hour. Take 1.0 ml of the bacterial culture, concentrate it to 100 μl, and spread it onto LB solid medium containing Amp. Incubate at 37°C for 16 hours.

[0091] 5. Colony PCR and sequencing identification

[0092] Single colonies from the plates were inoculated into LB liquid medium and incubated at 37°C for 2 hours. Using the bacterial culture as a template, colony PCR was performed using the primers in Table 1. The PCR products were then subjected to gel electrophoresis to verify the size of the target gene. Figure 3A As shown, after PCR amplification of the AP33 gene-transformed colony samples and gel electrophoresis, a positive sample appeared near the 1014bp band. Lane M is the DNA marker, lane 1 is the negative control, and lanes 2, 3, 4, and 5 contain the AP33 colony PCR amplification products. Figure 3B As shown, after PCR amplification of the AP65 gene-transformed colony samples and gel electrophoresis, a positive sample appeared near the 1785bp band. Lane M is the DNA marker, lane 1 is the negative control, and lanes 2, 3, 4, and 5 are the AP65 colony PCR amplification products.

[0093] The bacterial cultures that test positive are sent to a sequencing company for sequencing, and the cultures that are correctly sequenced are stored.

[0094] Example 2: Construction of plasmids Re-Bacmid-AP33 and Re-Bacmid-AP65

[0095] 1. DH10Bac transformation

[0096] Take 1 μl of pFastBac 1-AP33 plasmid from Example 1 and add it to 100 μl of DH10Bac competent cells. Mix well, incubate on ice for 30 minutes, subject to heat shock at 42°C for 90 seconds, then incubate on ice for 2 minutes. Add 900 μl of LB liquid medium without Amp and incubate at 37°C for 5 hours. Take 100 μl of the bacterial culture, dilute it 100-fold, and spread 100 μl of the diluted bacterial culture onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG. Incubate at 37°C for 48 hours. Perform the same procedure on the pFastBac 1-AP65 plasmid from Example 1.

[0097] Figure 4A The results of gel electrophoresis of PCR products from blue-white screening colonies after pFastBac1-AP33 transposition are shown. Positive samples appeared near the 3314bp band. Lane M is the DNA marker, lane 1 is the blank control, lane 2 is the PCR amplification product of AP33 colonies selected by blue-white screening, and lane 3 is the negative control. Figure 4B The results of gel electrophoresis of PCR products from blue-white screening colonies after pFastBac1-AP65 transposition are shown. Positive samples appeared near the 4085bp band. Lane M is the DNA marker, lane 1 is the blank control, lane 2 is the PCR amplification product of AP65 colonies selected by blue-white screening, and lane 3 is the negative control.

[0098] 2. Selecting monoclonal antibodies

[0099] Large white colonies were picked using an inoculation needle and streaked onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG. The cultures were incubated at 37°C for 48 hours. Single colonies were then picked and inoculated onto LB liquid medium containing gentamicin, kanamycin, and tetracycline. After identification using universal primers M13F and M13R, correctly identified strains were preserved, and plasmids were extracted. Plasmids Re-Bacmid-AP33 and Re-Bacmid-AP65 were obtained.

[0100] Example 3: Transfection and purification of recombinant baculovirus

[0101] 1. Recombinant virus transfection

[0102] In a six-well plate, each well is inoculated with 0.8 × 10⁸ g of seed. 6Sf9 cells were collected, with a cell confluence of 50-70%. For each well, the following complex was prepared: 4 μl of PEI transfection reagent was diluted with 200 μl of SF-SFM medium and briefly vortexed; 4 μg of Re-Bacmid-AP33 plasmid from Example 4 was diluted with 200 μl of SF-SFM medium. The diluted transfection reagent and plasmid were mixed and gently blown to prepare the transfection mixture. After cell attachment, the above transfection complex was added, and the cells were incubated at 27°C for 5 hours. The supernatant was removed, and 2 ml of fresh SF-SFM medium containing 10% FBS was added. The cells were incubated at 27°C for 4-5 days, and the supernatant was harvested. Recombinant baculovirus rBac-AP33 was obtained. Re-Bacmid-AP65 plasmid was transfected using the same method to obtain recombinant baculovirus rBac-AP65.

[0103] 2. Purification of recombinant viruses (plaque purification)

[0104] 2.0 × 10⁻⁶ cells were seeded in a 6 cm diameter tissue culture dish. 6 ~2.5×10 6 100 Sf9 cells were incubated at room temperature for 5 minutes. Serial dilutions were then performed (dilution ratio: 10-1). -4 ~10 -7 The recombinant baculovirus rBac-AP33 was used as follows: 1.0 ml of diluted virus solution was added to each culture dish, and the dish was incubated at room temperature for 1 hour, with gentle shaking every 15 minutes to ensure complete viral infection. A 2% low-concentration agarose solution was prepared in sterile water and microwaved to 60°C until fully melted. The dish was then placed in a 42°C water bath, and 1 volume of 2×Grace medium was added and mixed thoroughly. The supernatant of the culture medium in the cell culture dish was discarded, and 4.0 ml of 1% agarose was applied to the cell surface, while simultaneously removing all air bubbles. After the agarose solidified, the culture dish was placed in a humid environment and incubated at 27°C for 7 days, observing the plaques. A single plaque was picked from the culture dish and inoculated into SF-SFM medium, and incubated overnight at 4°C with shaking. The culture was harvested, inoculated into Sf9 cells, and incubated at 27°C for 72 hours. The cell culture was then harvested and designated as the recombinant baculovirus rBac-AP33-P0 generation. Similarly, the rBac-AP65-P0 generation was prepared.

[0105] 3. Recombinant virus titer determination

[0106] The purified rBac-AP33-P0 and rBac-AP65-P0 virus strains were analyzed for viral titers using indirect immunofluorescence assays. The titer of the rBac-AP33-P0 virus strain was 8.2 × 10⁻⁶. 6 PFU / ml was used to amplify recombinant baculovirus rBac-AP33 as a seed virus. The viral titer of rBac-AP65-P0 seed virus was 8.9 × 10⁻⁶. 6pfu / ml was used to amplify recombinant baculovirus rBac-AP65 as a seed virus for later use.

[0107] Example 4: SDS-PAGE detection of the seed virus expression product

[0108] The cell cultures harvested in Example 3 were subjected to SDS-PAGE analysis, with Sf9 cells infected with empty baculovirus used as a negative control. The specific procedure was as follows: 40 μl of the harvested cell culture was added to 10 μl of 5× loading buffer, incubated in boiling water for 5 minutes, centrifuged at 12000 rpm for 1 minute, and the supernatant was collected for SDS-PAGE gel electrophoresis (12% concentration gel). After electrophoresis, the gel was stained, destained, and the target bands were observed.

[0109] Figure 5A The results of SDS-PAGE gel electrophoresis of cell culture supernatant containing recombinant AP33 protein are shown. The target band appears near the molecular weight of approximately 33 kDa, while the negative control shows no band at the corresponding position. Lane M is the protein marker, lane 1 is the baculovirus empty vector control, and lane 2 is the recombinant AP33 protein.

[0110] Figure 5B The results of SDS-PAGE gel electrophoresis of cell culture supernatant containing recombinant AP65 protein are shown. The target band appears near the molecular weight of approximately 65 kDa, while the negative control shows no band at the corresponding position. Lane M is the protein marker, lane 1 is the baculovirus empty vector control, and lane 2 is the recombinant AP65 protein.

[0111] The sequences of recombinant AP33 protein and recombinant AP65 protein are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0112] Example 5: Western Blot Identification of Seed Virus Expression Products

[0113] The products from the two SDS-PAGE electrophoresis methods in Example 4 were transferred onto a PVDF (polyvinylidene fluoride) membrane, blocked with 5% skim milk for 2 hours, incubated with positive serum for trichomoniasis in pigeons for 2 hours, rinsed, incubated with HRP-labeled rabbit anti-pigeon polyclonal antibody secondary antibody for 2 hours, rinsed, and then an enhanced chemiluminescent fluorescent substrate was added. The images were taken using a chemiluminescence imaging system.

[0114] Figure 6A The results of Western blotting analysis of the products after SDS-PAGE electrophoresis are shown. Lane M is the protein marker, lane 1 is the baculovirus empty vector control, and lane 2 is the recombinant AP33 protein.

[0115] Figure 6B Another result of Western blotting analysis of the product after SDS-PAGE electrophoresis is shown. Lane M is the protein marker, lane 1 is the baculovirus empty vector control, and lane 2 is the recombinant AP65 protein.

[0116] As can be seen, all recombinant baculovirus expression samples showed the target band, while the negative controls did not show the target band, indicating that the target antigen protein was correctly expressed in Sf9 cells.

[0117] Example 6: Serum-free suspension culture of insect cells in a bioreactor and quantification of AP33 and AP65 expression.

[0118] Sf9 insect cells were aseptically cultured in 1000ml shake flasks for 3–4 days until the concentration reached 3–5 × 10⁻⁶. 6 When the cell count is 3-8 × 10⁶ cells / ml and the cell viability is greater than 95%, the cells are seeded into a 5L bioreactor at a seeding concentration of 3-8 × 10⁶ cells / ml. 5 cells / ml. When the cell concentration reaches 3~8×10⁻⁶ 6 When the cell concentration reaches 3~8×10⁶ cells / mL, the cells are seeded into a 50L bioreactor and allowed to grow to a concentration of 3~8×10⁶ cells / mL. 6 Cells / ml were seeded into a 500L bioreactor, and the cell concentration was increased to 2×10⁻⁶ cells / ml. 6 At a cell / ml concentration, recombinant baculoviruses AP33 and AP65 were inoculated separately. The reactor culture conditions were pH 7.2, temperature 25–27°C, dissolved oxygen 30–80%, and stirring speed 100–180 rpm. Considering the optimal conditions for cell culture, the preferred settings were pH 7.2, cell culture temperature 27°C, dissolved oxygen 50%, and stirring speed 100–180 rpm. After culturing for 5–9 days post-infection, a final concentration of BEI (1 / 1000) was added, and the mixture was incubated at 37°C for 48 hours. Then, a final concentration of Na2S2O3 (2 / 1000) was added to terminate the inactivation. The cell culture supernatant was harvested by centrifugation or hollow fiber filtration and stored as vaccine stock at 2–8°C.

[0119] Example 7: Vaccine Preparation

[0120] The vaccine stock solution expressed in Example 6 was taken and diluted with PBS solution. The mixed vaccine stock solution was then formulated with white oil adjuvant to prepare an oil-emulsion vaccine, ensuring that each dose (0.2 ml) contained 20 μg each of recombinant AP33 and AP65 proteins. Specifically, 1429 g of white oil, 70.2 g of Span, 8.43 g of aluminum stearate, and 53.3 g of Tween were added to every 1 L of the mixed vaccine stock solution. The mixture was then emulsified using an emulsifier to prepare an oil-emulsion adjuvant subunit vaccine.

[0121] Example 8 ELISA Detection Method

[0122] Trichomonas pigeonis whole-worm antigen was prepared by ultrasonic disruption of the worm body. After concentration detection using BCA, it was diluted to 100 mg / ml for use. The whole-worm antigen was diluted to 20 μg / ml using coating buffer CBS, and 100 μl was added to each well of the ELISA plate and incubated overnight at 4°C. The plate was then removed, the liquid was discarded, and the plate was washed three times for 3 minutes each with washing buffer PBST (containing 0.05% Tween 20, pH 7.4). Then, 100 μl of blocking buffer was added to each well and the plate was incubated at 37°C for 2 hours. After washing the plate as above, add the sample to be tested (1:100 dilution) to the coated reaction wells (100 μl / well), incubate at 37°C for 60 minutes, then add enzyme-labeled rabbit anti-pigeon IgY-HRP (1:7000 dilution), incubate at 37°C for 30-60 minutes, wash the plate as above, and finally add 100 μl of substrate OPD / well, react for 10-15 minutes, add 50 μl of 2 mol / L H2SO4 to stop the reaction, and read the OD at 450 nm.

[0123] Example 9: Validity Test

[0124] The vaccine was prepared according to Example 7 and Table 6. Fifty one-month-old pigeons that were negative for Trichomonas vaginalis were injected with the prepared vaccine according to Table 6, with 10 pigeons injected per batch, 0.2 ml per pigeon; another 10 pigeons were injected with an equal volume of PBS solution as a control. Fourteen days after the first immunization, blood was collected from all immunized and control pigeons, and serum was separated for ELISA testing. Simultaneously, each pigeon was challenged with 1×10⁻⁶ ppm of the vaccine. 7 Type A trichomoniasis (viability greater than 90%) was administered to pigeons for 5 consecutive days using the same dosage and route of infection. The pigeons were observed for 14 days after the last administration, and the survival rate of each group was recorded. Serum antibodies were detected by ELISA, and the results are shown in Table 7 below. The antibody test results for each immunized group and the blank group 14 days after immunization are as follows: Figure 7 As shown.

[0125] Table 6 Vaccine Preparation

[0126] Vaccine preparation group The protein content of each dose of vaccine (0.2ml) Immunogroup 1 40 μg of recombinant AP33 protein Immunogroup 2 40 μg of recombinant AP65 protein Immunogroup 3 20 μg each of recombinant AP33 and AP65 proteins Whole insect antigen group <![CDATA[Whole worm antigen (Trichomonas gallinae 1×10 9 individuals)]]> Blank group PBS

[0127] Table 7. Results of Immunization and Challenge

[0128]

[0129] Example 10 Immunoprotection Experiment

[0130] The vaccine was prepared according to Example 7 and Table 8. 150 one-month-old pigeons that were negative for Trichomonas vaginalis were injected with the prepared vaccine according to Table 8, with 10 pigeons injected per batch, 0.2 ml per pigeon; another 10 pigeons were injected with an equal volume of PBS solution as a control. The pigeons were then challenged according to Table 8, with each pigeon challenged with 1×10⁻⁶ PBS. 7 One group of pigeons was infected with Trichomonas vaginalis (survival rate greater than 90%) and was continuously infected for 5 days with the same amount of worms and route of infection. After the last infection, the pigeons were observed for 14 days. The survival rate of each group of pigeons was calculated and the results are shown in Table 9.

[0131] Table 8. Vaccine preparation and insect challenge genotypes

[0132]

[0133] Table 9. Survival rate of pigeons in each immunized group 14 days after parasite attack.

[0134] Type A Type B T-shaped AP33 50% 60% 60% AP65 60% 70% 70% AP33+ AP65 100% 100% 100% Whole insect antigen 30% 40% 50% Blank group 10% 20% 20%

[0135] Based on the above test results, it can be seen that the vaccine prepared in the embodiments of the present invention has a good immunization effect, which is superior to existing whole worm vaccines and monovalent vaccines.

[0136] It should be understood that the above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. The use of the recombinant protein composition in the preparation of a medicament for inducing an immune response against Trichomonas vaginalis infection in test animals, characterized in that, The recombinant protein composition comprises: The first recombinant protein, the sequence of which is shown in SEQ ID NO:

7. The second recombinant protein has the sequence shown in SEQ ID NO:8; The mass ratio of the first recombinant protein to the second recombinant protein is 1:1; The animal in question is a pigeon.

2. The use of the recombinant protein composition in the preparation of a medicament for the prevention of infection in animals by Trichomonas vaginalis, characterized in that, The recombinant protein composition comprises: The first recombinant protein, the sequence of which is shown in SEQ ID NO:

7. The second recombinant protein has the sequence shown in SEQ ID NO:8; The mass ratio of the first recombinant protein to the second recombinant protein is 1:1; The animal in question is a pigeon.

3. The use of the recombinant protein composition in the preparation of a recombinant subunit vaccine against Trichomonas vaginalis infection, characterized in that, The recombinant protein composition comprises: The first recombinant protein, the sequence of which is shown in SEQ ID NO:

7. The second recombinant protein has the sequence shown in SEQ ID NO:8; The mass ratio of the first recombinant protein to the second recombinant protein is 1:

1.

4. Use of the immune composition in the preparation of a pharmaceutical agent for inducing an immune response against Trichomonas vaginalis infection in test animals, said immune composition comprising a recombinant protein composition and a pharmaceutically acceptable carrier; characterized in that, The recombinant protein composition comprises: The first recombinant protein, the sequence of which is shown in SEQ ID NO:

7. The second recombinant protein has the sequence shown in SEQ ID NO:8; The mass ratio of the first recombinant protein to the second recombinant protein is 1:1; The animal in question is a pigeon.

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

6. Use of an immune composition in the preparation of a medicament for the prevention of infection in animals by Trichomonas vaginalis, said immune composition comprising a recombinant protein composition and a pharmaceutically acceptable carrier; characterized in that, The recombinant protein composition comprises: The first recombinant protein, the sequence of which is shown in SEQ ID NO:

7. The second recombinant protein has the sequence shown in SEQ ID NO:8; The mass ratio of the first recombinant protein to the second recombinant protein is 1:1; The animal in question is a pigeon.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable carriers include any one or a combination of two or more of MONTANIDE ISA 206VG, ​​MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, and plant cell lectins.

8. Use of an immune composition in the preparation of a recombinant subunit vaccine against Trichomonas vaginalis infection, said immune composition comprising a recombinant protein composition and a pharmaceutically acceptable carrier; characterized in that, The recombinant protein composition comprises: The first recombinant protein, the sequence of which is shown in SEQ ID NO:

7. The second recombinant protein has the sequence shown in SEQ ID NO:8; The mass ratio of the first recombinant protein to the second recombinant protein is 1:

1.

9. The use according to claim 8, characterized in that: The pharmaceutically acceptable carriers include any one or a combination of two or more of MONTANIDE ISA 206VG, ​​MONTANIDE ISA 201VG, liquid paraffin, camphor oil, white oil, and plant cell lectins.

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