Divalent recombinant protein egg yolk antibody for resisting trichomonas pigeonae infection and application of divalent recombinant protein egg yolk antibody

Abstract

The invention discloses a divalent recombinant protein egg yolk antibody for resisting trichomonas pigeonae infection and an application of the divalent recombinant protein egg yolk antibody. The egg yolk antibody is obtained by separating and purifying egg yolk of poultry eggs produced by laying fowls immunized by subunit vaccines, and the subunit vaccines comprise trichomonad recombinant AP33 protein and recombinant AP65 protein. The purified antigen and the adjuvant are compatible to prepare the vaccine, the vaccine is used for immunizing laying fowls to induce high-titer specific antibody response, and finally the target egg yolk antibody is extracted from egg yolk and purified to obtain the target egg yolk antibody. The egg yolk antibody shows a remarkable effect in prevention and treatment of trichomonas pigeon infection, and can effectively improve the survival rate of infected meat pigeons and reduce the oral lesion degree. Compared with existing chemical drugs and the like, the egg yolk antibody provided by the invention has the comprehensive advantages of being high in safety, not easy to generate drug resistance, free of drug residues, environment-friendly, suitable for large-scale production and the like, and shows a good application prospect in the aspect of green prevention and control of trichomonad of pigeons.

Description

Technical Field

[0001] This application relates to a bivalent recombinant protein yolk antibody against Trichomonas vaginalis infection in pigeons and its application, belonging to the field of biological veterinary drug technology. Background Technology

[0002] Pigeon trichomoniasis is a common parasitic disease in pigeons. It is a parasitic infectious disease caused by Trichomonas gallinae, a parasitic parasite that is characterized by emaciation, diarrhea, and decreased growth performance. Infected pigeons usually show lesions in the digestive tract, with necrotic ulcers appearing in the mouth, crop, esophagus, and even other parts of the body. Death often results from obstruction of the digestive and respiratory tracts, hindering feeding and normal breathing.

[0003] Trichomoniasis in pigeons has been found and reported worldwide. Pigeons of all ages are infected to varying degrees, with young pigeons having the highest infection rate; in one pigeon farm, the positive rate for trichomoniasis in young pigeons reached as high as 90.0%. Young pigeons are next, with the highest infection rate reaching 62.5%. The infection rate in breeding pigeons is lower than that in young pigeons, approximately 23%, while the infection rate in young pigeons is higher than that in young pigeons and breeding pigeons. The main mode of transmission for pigeon trichomoniasis is oral transmission. Pigeons become infected by ingesting feed or water contaminated with Trichomonas vaginalis. Because the Trichomonas vaginalis accumulates in large numbers in the oral ulcer lesions of infected pigeons, their saliva contains a large number of Trichomonas vaginalis, so susceptible pigeons can also become infected by kissing infected or carrier pigeons. In addition to infected pigeons, asymptomatic carrier pigeons are also an important source of infection, making it difficult to eradicate pigeon trichomoniasis in pigeon farms and causing serious economic losses to the pigeon breeding industry.

[0004] Currently, the commonly used chemical drugs for treating trichomoniasis in pigeons are nitroimidazole drugs such as metronidazole and dimetronidazole. On the one hand, metronidazole has a very clear effect in killing trichomoniasis, but the resistance of pigeon trichomoniasis to it has increased significantly, and the cure rate of pigeons treated with metronidazole has gradually decreased, while the recurrence rate has also increased. On the other hand, long-term use of metronidazole has significant side effects (such as carcinogenicity and teratogenicity), drug residues, and other potential risks, and is subject to strict regulation, especially in food animals where its use is restricted. Therefore, under the severe situation of drug use, a healthy and environmentally friendly antibody drug is urgently needed for pigeon farming. Summary of the Invention

[0005] The main objective of this application is to provide a bivalent recombinant protein yolk antibody against Trichomonas vaginalis infection in pigeons and its application, in order to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, this application employs the following technical solution.

[0007] According to a first aspect of this application, a bivalent recombinant protein yolk antibody against pigeon trichomoniasis infection is provided. This bivalent recombinant protein yolk antibody is isolated from the yolk of a poultry egg produced by laying hens immunized with a recombinant subunit vaccine. The vaccine comprises a recombinant protein and a pharmaceutically acceptable carrier.

[0008] Furthermore, the recombinant protein includes: The recombinant Trichomonas pigeonis AP33 protein has the sequence shown in SEQ ID NO:7 or its conserved variant sequence, for example, a sequence that is more than 95% identical to the sequence in SEQ ID NO:7; The recombinant Trichomonas pigeonis AP65 protein has a sequence as shown in SEQ ID NO:8 or a conserved variant thereof, such as a sequence that is more than 95% identical to the sequence in SEQ ID NO:8.

[0009] Furthermore, the egg-laying poultry includes laying hens, such as Hainan Brown hens, Hy-Line White, Lohmann Brown, or Isa Brown, which are commercially available high-yielding egg-laying breeds, with Hainan Brown hens being the preferred choice.

[0010] According to a second aspect of this application, a method for preparing a bivalent recombinant protein yolk antibody against Trichomonas vaginalis infection is provided, comprising: Egg-laying poultry were immunized with a recombinant subunit vaccine containing recombinant proteins against Trichomonas pigeon infection, including the recombinant Trichomonas pigeon AP33 protein and the recombinant Trichomonas pigeon AP65 protein. The bivalent recombinant protein yolk antibody was isolated from the yolk of eggs produced by the aforementioned poultry.

[0011] Furthermore, the recombinant protein is purified and has a purity of over 85%.

[0012] Furthermore, the recombinant subunit vaccine also includes a pharmaceutically acceptable carrier. For example, the adjuvant may include, but is not limited to, any one or more combinations of white oil, Span, aluminum stearate, or Tween.

[0013] In one embodiment, the preparation method of the divalent recombinant protein yolk antibody specifically includes the following steps: S1. Prepare nucleic acid molecules for encoding recombinant Trichomonas guinea AP33 protein and recombinant Trichomonas guinea AP65 protein, respectively.

[0014] 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).

[0015] 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.

[0016] S4. After purifying and measuring the titer of the recombinant baculovirus, inoculate it into Sf9 cells to obtain the expression products recombinant Trichomonas vaginalis AP33 protein and recombinant Trichomonas vaginalis AP65 protein. This step may also include the separation and purification of recombinant Trichomonas vaginalis AP33 protein and recombinant Trichomonas vaginalis AP65 protein, wherein the optional separation and purification methods include, but are not limited to, chromatography, dialysis, or other methods known in the art.

[0017] S5. The purified recombinant Trichomonas vaginalis AP33 protein and recombinant Trichomonas vaginalis AP65 protein are mixed with a pharmaceutically acceptable carrier to prepare a recombinant subunit vaccine. For example, the purified recombinant Trichomonas vaginalis AP33 protein and recombinant Trichomonas vaginalis AP65 protein can be diluted and mixed in an appropriate ratio, and then formulated with an oil adjuvant at a ratio of 2:3 to prepare an oil emulsion subunit vaccine, so that the vaccine contains 100 μg / bird dose / 0.2 ml of recombinant Trichomonas vaginalis AP33 protein and recombinant Trichomonas vaginalis AP65 protein.

[0018] S6. Immunization: Immunize laying hens with the above-mentioned recombinant subunit vaccine according to the immunization schedule: 0.2 ml / hen subcutaneously injected into the chest; two weeks after the first immunization, administer a second immunization at the same dose and route; two weeks after the second immunization, administer a third immunization at the same dose and route.

[0019] S7. Obtaining the bivalent recombinant protein yolk antibody: Sterilize the eggs produced by the immunized hens, separate the egg white and yolk, thoroughly remove the egg white and yolk mesembryolk membranes, and mix the yolk with sterile water for injection to form the first mixture. Incubate at 60–65°C for 8–15 minutes. Add five times the original yolk volume of acetate buffer (0.12 mol / L, pH 5.0) to the first mixture, stir well, and incubate at 2–8°C for 12–15 hours. Centrifuge and filter the supernatant to obtain the second mixture. Slowly add the second mixture to caprylic acid to a final concentration of 0.5% (V / V), and incubate at 20–25°C for 2–4 hours to obtain the third mixture. The third mixture was filtered through a 0.45 μm pore size filter membrane to clarify it. The clear liquid was then concentrated through a 50 kDa ultrafiltration membrane. Formaldehyde solution was added to the concentrated clear liquid to inactivate it, and then it was filtered through a 0.22 μm microporous filter to remove bacteria, thus obtaining the bivalent recombinant protein yolk antibody against Trichomonas vaginalis.

[0020] According to a third aspect of this application, a pharmaceutical composition is provided comprising the aforementioned bivalent recombinant protein yolk antibody against Trichomonas vaginalis infection.

[0021] According to a fourth aspect of this application, the use of the aforementioned bivalent recombinant protein yolk antibody against Trichomonas pigeon infection is provided in the production of a pharmaceutical agent for inducing an immune response against Trichomonas pigeon antigens in test animals. The test animals include, but are not limited to, pigeons.

[0022] According to a fifth aspect of this application, the use of the aforementioned bivalent recombinant protein yolk antibody against pigeon trichomoniasis infection in the production of a medicament for the prevention or treatment of pigeon trichomoniasis infection in animals is provided. The animals include, but are not limited to, pigeons.

[0023] For example, the bivalent recombinant protein yolk antibody against Trichomonas vaginalis infection can be prepared into various dosage forms, such as: Oral solution: Antibody aqueous solution, which can be taken directly by dripping or added to drinking water.

[0024] Freeze-dried powder: Improves stability, facilitates transportation and storage, and can be reconstituted before use.

[0025] Spray: Used for local treatment of oral mucosa.

[0026] Microcapsules / granules: These are administered after coating to protect antibodies from digestive enzymes and enable targeted release into the intestines.

[0027] Feed premix: Mix evenly with feed for group prevention.

[0028] Furthermore, the aforementioned bivalent recombinant protein yolk antibody against pigeon trichomoniasis infection can be applied to domestic pigeon breeding and racing pigeon health care, biosecurity control in breeding pigeon farms, prevention and control of other poultry trichomoniasis diseases, diagnostic auxiliary reagents for trichomoniasis infection, and the development of functional feeds or additives.

[0029] For example, the bivalent recombinant protein yolk antibody can be administered regularly as a feed additive or oral preparation to prevent trichomoniasis infection in pigeon flocks; it can also be used to treat individuals that have already shown clinical symptoms, reducing morbidity and mortality.

[0030] Alternatively, the aforementioned divalent recombinant protein yolk antibody can be used to preventively administer the vaccine to imported breeding pigeons and young pigeons to block the vertical and horizontal transmission of Trichomonas vaginalis.

[0031] Alternatively, through adaptive studies, the bivalent recombinant protein yolk antibody can be extended to susceptible poultry such as chickens, turkeys, and quails, serving as a highly specific and safe biological control product.

[0032] Alternatively, the aforementioned bivalent recombinant protein yolk antibody can be used as a core raw material for immunoassay reagents (such as ELISA and immunochromatographic test strips) for rapid screening and epidemiological monitoring of pigeon trichomoniasis.

[0033] Compared with the prior art, this application has at least the following beneficial effects: (1) This application uses purified, highly immunogenic recombinant pigeon trichomonas adhesion protein 33 (AP33) and adhesion protein 65 (AP65) expressed by Sf9 cells to make a vaccine. After immunizing laying hens, it can rapidly induce the production of high-level specific yolk antibodies. The antibodies produced have high titers and long duration of action, and can inhibit the invasion, adhesion to cells and growth and reproduction of pigeon trichomonas, thereby providing immune protection for the pigeon flock.

[0034] (2) The bivalent recombinant protein yolk antibody against Trichomonas gypsum of the present application can effectively prevent and treat Trichomonas gypsum infection. Compared with vaccines, there is no need to wait for antibody production time and the effect can be produced immediately. Compared with chemical drugs, it has both therapeutic effects and no side effects such as carcinogenicity, teratogenicity, or drug residues. It can replace the use of human drug metronidazole, reduce the abuse of antibiotics, and avoid the development of drug resistance.

[0035] (3) The bivalent recombinant protein yolk antibody against pigeon trichomoniasis of this application has advantages such as low production cost, high safety, good stability, batch stability, no drug residue, and environmental friendliness, which helps to alleviate many problems in the current prevention and control of pigeon trichomoniasis. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0037] Figures 1A-1B The diagrams show the transfer vectors pFastBac1-AP33 and pFastBac1-AP65 containing the target gene constructed in Example 1, respectively.

[0038] 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.

[0039] 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.

[0040] Figures 4A-4BThe images show gel electrophoresis images of the PCR products after PCR amplification of the blue-white screening colony samples following transposition of pFastBac1-AP33 and pFastBac1-AP65 in Example 2.

[0041] 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.

[0042] Figures 6A-6B The images show the Western blot results of the two sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) products from Example 4.

[0043] Figure 7 These are SDS-PAGE electrophoresis images of the cell culture supernatant of recombinant AP33 protein before and after purification in Example 7; Figure 8 These are SDS-PAGE electrophoresis images of the cell culture supernatant of recombinant AP65 protein before and after purification in Example 7; Figure 9 This is a graph showing the changes in the level of specific egg yolk antibody IgY after immunization in laying hens in Example 9. Detailed Implementation

[0044] The present application is further illustrated below by way of examples. All reagents and raw materials used in the following examples are commercially available, and experimental methods without specific conditions 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 application 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.

[0045] Example 1: Construction and Identification of Transfer Vectors pFastBac1-AP33 and pFastBac1-AP65 1. Amplification and purification of AP33 and AP65 genes 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.

[0046] Table 1 Gene Primer Sequences Table 2 Gene amplification system 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.

[0047] 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.

[0048] 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 was observed 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.

[0049] 2. Enzyme digestion and purification 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.

[0050] 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.

[0051] Table 3 Gene Enzyme Digestion Reaction System Table 4 pFastBac 1 plasmid digestion reaction system 3. Connection 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 1B As shown.

[0052] Table 5. Ligation system of gene digestion products and pFastBac 1 plasmid 4. Transformation 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.

[0053] 5. Colony PCR and sequencing identification 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.

[0054] The bacterial cultures that test positive are sent to a sequencing company for sequencing, and the cultures that are correctly sequenced are stored.

[0055] Example 2: Construction of plasmids Re-Bacmid-AP33 and Re-Bacmid-AP65 1. DH10Bac transformation 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.

[0056] Figure 4AThe 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.

[0057] 2. Selecting monoclonal antibodies 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.

[0058] Example 3: Recombinant Baculovirus Transfection and Purification 1. Recombinant virus transfection In a six-well plate, each well is inoculated with 0.8 × 10⁸ g of seed. 6 Sf9 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.

[0059] 2. Purification of recombinant viruses (plaque purification) 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.

[0060] 3. Recombinant virus titer determination 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. 6 pfu / ml was used to amplify recombinant baculovirus rBac-AP65 as a seed virus for later use.

[0061] Example 4: SDS-PAGE Detection of Toxic Expression Products 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.

[0062] 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.

[0063] Figure 5BThe 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.

[0064] The sequences of recombinant AP33 protein and recombinant AP65 protein are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0065] Example 5: Identification of the Toxic Expression Products by Western Blot 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 of 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.

[0066] 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.

[0067] Figure 6B Another method for detecting the product by Western blotting 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.

[0068] 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.

[0069] Example 6: Serum-free suspension culture of insect cells in a bioreactor and quantification of AP33 and AP65 expression. 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. 6Cells / 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.

[0070] Example 7 Recombinant Protein Purification The cell culture supernatant prepared in Example 6 was filtered through a 0.22 μm filter membrane and loaded onto a pre-equilibrated nickel column at a flow rate of 1 ml / min. The loading solution was allowed to flow out naturally under gravity, and the effluent was collected. This loading process was repeated three times to improve binding efficiency. After loading, the solution was washed with 20 mM Tris-500 eluent. The column was washed with HCl, 100 mM NaCl, 10 mM imidazole (pH 7.4), followed by elution with 20 mM Tris-HCl. The protein was eluted using a column containing HCl, 100 mM NaCl, and 200 mM imidazole (pH 7.4), and the eluent was collected to obtain the target protein. The obtained protein was then dialyzed, concentrated, and stored at 4°C for later use.

[0071] See Figure 7 The results of SDS-PAGE gel electrophoresis of cell culture supernatant before and after purification of recombinant AP33 protein are shown, with the target band appearing around 33 kDa.

[0072] See Figure 8 The results of SDS-PAGE gel electrophoresis of cell culture supernatant containing recombinant AP65 protein before and after purification are shown, with the target band appearing around 65 kDa.

[0073] The purified proteins were quantified using BCA total protein assay, and then the purity of the target proteins was determined by grayscale scanning. The concentration of recombinant AP33 protein was 1320 μg / mL, with a purity of 87%. The concentration of recombinant AP65 protein was 1720 μg / mL, with a purity of 92%.

[0074] Example 8: Preparation of Subunit Vaccine The two purified antigen solutions from Example 7 were diluted and mixed with PBS solution, and then formulated into an oil emulsion vaccine with oil adjuvant at a ratio of 2:3, so that the vaccine contained 100 μg / dose / 0.2 ml 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 mixed antigen stock solution. Then, the mixture was emulsified using an emulsifier to prepare the oil emulsion subunit vaccine 1.

[0075] Another antigen solution purified in Example 7 was diluted and mixed with PBS solution, and then formulated into an oil emulsion vaccine with an oil adjuvant at a ratio of 2:3, so that the vaccine contained 200 μg / dose / 0.2 ml of recombinant AP33 protein. 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 antigen stock solution. Then, the mixture was emulsified using an emulsifier to prepare an oil emulsion subunit vaccine 2.

[0076] Take one of the purified antigen solutions from Example 7, dilute and mix it with PBS solution, and then prepare an oil emulsion vaccine with oil adjuvant at a ratio of 2:3, so that the vaccine contains 200 μg / dose / 0.2 ml of recombinant AP65 protein. Specifically, add 1429 g of white oil, 70.2 g of Span, 8.43 g of aluminum stearate, and 53.3 g of Tween to every 1 L of antigen stock solution. Then, use an emulsifier to break down and emulsify the solution to prepare an oil emulsion subunit vaccine 3.

[0077] Example 9: Monitoring of Specific Egg Yolk Antibody Levels 1. Preparation of whole-worm emulsion vaccine for Trichomonas vaginalis in pigeons Take the culture medium of Trichomonas vaginalis, centrifuge at 5000 rpm for 10 min, discard the supernatant, add an appropriate amount of PBS (pH 7.2~7.4), sonicate to disrupt the protein, and dilute to 2.5 mg / ml for later use. Prepare a whole-parasite emulsion vaccine by mixing whole-parasite antigen and oil adjuvant at a ratio of 2:3, so that the vaccine contains approximately 200 μg of whole-parasite antigen per dose / 0.2 ml.

[0078] 2. Immunization program Hainan Brown breed hens aged 160-200 days were purchased and randomly divided into 5 groups of 10 each. Four weeks before the start of egg production, the hens received their first vaccination using the oil-emulsion subunit vaccine prepared in Example 8, administered subcutaneously to the chest (0.2 ml / hen). Two weeks after the first vaccination, a second vaccination was administered using the same dose and route. Two weeks after the second vaccination, a third vaccination was administered using the same dose and route. The whole-parasite antigen immunization group, the blank control group, and the recombinant protein immunization group underwent the same treatment. See Table 6.

[0079] Table 6. Immunization Dosage and Schedule for Hens 3. Preparation of egg yolk antibodies Gently scrub the eggshell surface with a soft-bristled brush under running water to remove visible dirt such as feces and feathers, then wipe dry with a clean paper towel or cloth. Disinfect each group of eggs in a 0.1% benzalkonium chloride solution at 42℃ for 15–20 minutes. After removal, wipe the eggshell surface evenly with 95% alcohol under aseptic conditions until completely dry. Separate the egg white and yolk, thoroughly removing the egg white and yolk mesentery. Collect the obtained yolk in a beaker and stir thoroughly until it becomes a homogeneous paste. Add an equal volume of sterile water for injection, stir well, and incubate at 60–65℃ for 8–15 minutes, then cool to room temperature. Add five times the original volume of the yolk dilution to acetate buffer (0.12 mol / L, pH 5.0), stir well, and then cool to 2–8℃ and let stand for 12–15 hours. Centrifuge and filter the supernatant, and store for later use. While stirring, slowly add the filtrate to caprylic acid to a final concentration of 0.5% (V / V), and incubate at 20–25°C for 2–4 hours. Collect the supernatant and filter it through a 0.45 μm pore size membrane until clear. Concentrate the supernatant using a 50 kDa ultrafiltration membrane. Add formaldehyde solution to the concentrated supernatant to a final concentration of 0.05%, and inactivate the yolk antibody at 20–25°C for 2–4 hours with intermittent stirring. Adjust the pH of the inactivation solution to 6.0–7.0, filter using a 0.22 μm microporous filter for sterilization, and store the filtered egg yolk antibody at 4°C for later use.

[0080] 4. Indirect ELISA detection of egg yolk antibodies Egg yolk antibody titers were measured before immunization, 2 weeks after the first immunization, 2 weeks after the second immunization, and 2, 4, 6, and 8 weeks after the third immunization. Ten eggs were randomly selected from each group for egg yolk antibody preparation. The prepared egg yolk antibodies were then detected by indirect ELISA. The specific method is as follows: 1) Coating: Dilute the whole worm antigen of Trichomonas vaginalis to 20 μg / ml using CBS buffer, add 100 μl to each well of the microplate, and incubate overnight at 4°C.

[0081] 2) Washing: Discard the liquid in the plate and wash 3 times with 0.5% PBST (pH 7.4).

[0082] 3) Sealing: Use 5% skim milk for sealing, 200 μL / well, place in a 37°C incubator for 2 hours, and then wash the plate as above.

[0083] 4) Primary antibody incubation: Dilute the prepared egg yolk antibody 1:100 and add it to the enzyme-labeled reaction plate, 100 μL / well, and incubate at 37℃ for 1 hour. Wash the plate as above.

[0084] 5) Secondary antibody incubation: Dilute the enzyme-labeled rabbit anti-pigeon IgY-HRP secondary antibody 1:17000, add 100 μL / well to the enzyme-labeled reaction plate, and incubate at 37℃ for 30-60 minutes. Wash the plate as above. 6) Color development: Under light-protected conditions, add the substrate solution to the enzyme-labeled reaction plate at 100 μL / well and react at room temperature for 15 minutes. 7) Termination: Add 2M H2SO4 to terminate the reaction, 50μL / well.

[0085] 8) Read the OD value: Use an ELISA reader to read the absorbance value at a wavelength of 450nm.

[0086] Indirect ELISA results show ( Figure 9 Compared to the whole-worm antigen and either single protein (AP33 or AP65) immunization groups, the egg yolk antibody levels of laying hens immunized with recombinant AP33+AP65 protein increased faster, reached higher peaks, and remained for a longer period.

[0087] Example 10: Evaluation of the therapeutic effect of egg yolk antibody 1 Experimental Design 150 squabs were divided into 15 groups of 10 each. Groups 1-14 were infected with Trichomonas vaginalis, and each time they were orally inoculated with 1×10⁻⁶ mmol / L. 7 After 5 consecutive days of infection, pigeons were administered recombinant bivalent protein yolk antibody (0.2 ml / bird, 0.5 ml / bird, 1.0 ml / bird, respectively) orally in groups 1-3; recombinant AP33 protein yolk antibody (0.2 ml / bird, 0.5 ml / bird, 1.0 ml / bird, respectively) in groups 4-6; recombinant AP65 protein yolk antibody (0.2 ml / bird, 0.5 ml / bird, 1.0 ml / bird, respectively) in groups 10-12; whole-parasite antigen yolk antibody (0.2 ml / bird, 0.5 ml / bird, 1.0 ml / bird, respectively) in group 13; metronidazole (0.1 g / bird) in group 13; group 14 served as the infection control; and group 15 served as the blank control. These antibodies or drugs were administered continuously for 5 days. On day 7 post-infection, oral swabs were collected to determine the Trichomonas vaginalis content. The pigeons were observed for 14 days, with oral lesions observed and mortality recorded.

[0088] 2 Results As shown in Table 7, under artificial infection with Trichomonas vaginalis, the bivalent recombinant protein yolk antibody has a significant therapeutic effect on oral lesions.

[0089] Oral pathogen load was measured on day 7 post-infection. High-dose (1.0 ml / infant) and medium-dose (0.5 ml / infant) recombinant protein yolk antibody groups, high-dose (1.0 ml / infant) and high-dose (1.0 ml / infant) recombinant AP33 protein yolk antibody groups, and high-dose (1.0 ml / infant) recombinant AP65 protein yolk antibody groups, along with metronidazole, significantly reduced the pathogen load, although the differences were not statistically significant. Low-dose (0.2 ml / infant) and medium-dose (0.2 ml / infant) recombinant AP33 protein yolk antibody groups also showed significant reductions in pathogen load. The recombinant AP65 protein yolk antibody (0.5 ml / bird), the medium-dose recombinant AP65 protein yolk antibody (0.5 ml / bird), and the high-dose whole-parasite antigen (1.0 ml / bird) all significantly reduced the pathogen load, but the differences were not significant. Similarly, the recombinant AP33 protein yolk antibody (0.2 ml / bird), the recombinant AP65 protein yolk antibody (0.2 ml / bird), the medium-dose whole-parasite antigen yolk antibody (0.5 ml / bird), and the low-dose whole-parasite antigen yolk antibody (0.2 ml / bird) all significantly reduced the pathogen load, but the differences were not significant. No Trichomonas vaginalis was detected in the blank control group, thus the control was valid.

[0090] After 14 days of observation, the oral lesions and survival rates of the squabs in each group were as follows: No oral lesions were observed in the high-dose group (1.0 ml / squito) and medium-dose group (0.5 ml / squito) of the recombinant protein yolk antibody, and the survival rate reached 100%, comparable to the metronidazole treatment group; however, in the low-dose group (0.2 ml / squito), 20% of the squabs still had oral lesions, with a survival rate of 80%. In the high-dose group (1.0 ml / squito) of recombinant AP33 protein yolk antibody, only one squab had oral lesions, with a survival rate of 90%, while the survival rates in the high-dose group (0.5 ml / squito) and low-dose group (0.2 ml / squito) did not exceed 70%. In the high-dose group (1.0 ml / squito) of recombinant AP65 protein yolk antibody, two squabs had oral lesions, with a survival rate of 90%, while the survival rates in the high-dose group (0.5 ml / squito) and low-dose group (0.2 ml / squito) did not exceed 60%. The survival rate of squabs in the high, medium, and low dose groups of whole parasite antigen and egg yolk antibody did not exceed 70%. All squabs in the parasite-treated control group developed oral lesions, with a survival rate of only 10%, demonstrating the severe impact of infection on pigeon health. The blank control group showed no lesions and had a 100% survival rate, thus establishing the correct control outcome.

[0091] Table 7. Efficacy of different doses of antibodies against artificial infection with Trichomonas vaginalis. Note: In the same column, identical letters indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0092] Based on pathogen load, oral lesion rate, and survival rate of experimental pigeons, the prepared bivalent recombinant protein yolk antibody, administered orally at a medium dose (0.5 ml / pigeon) for 5 consecutive days, can effectively treat artificial infection of Trichomonas vaginalis in pigeons. It is more effective than whole-parasite antigen yolk antibody and any monovalent recombinant protein yolk antibody and can replace metronidazole.

[0093] Example 11 Evaluation of the preventive effect of egg yolk antibodies 1 Experimental Design One hundred and forty pigeons were divided into 14 groups of 10 each. Groups 1-3 were injected with 0.2ml, 0.5ml, and 1.0ml of recombinant bivalent protein yolk antibody, respectively; groups 4-6 were injected with 0.2ml, 0.5ml, and 1.0ml of recombinant AP33 protein yolk antibody, respectively; groups 7-9 were injected with 0.2ml, 0.5ml, and 1.0ml of recombinant AP65 protein yolk antibody, respectively; groups 10-12 were injected with 0.2ml, 0.5ml, and 1.0ml of whole-parasite antigen yolk antibody, respectively; group 13 served as the challenge control; and group 14 served as the blank control. Twenty-four hours after immunization, pigeons were orally infected with Trichomonas vaginalis at a dose of 1×10⁻⁶ for each oral inoculation. 7 Each pigeon was treated with the parasite for 5 consecutive days, and then observed for 14 days to observe oral lesions and record the mortality rate of the experimental pigeons.

[0094] 2 Results As shown in Table 8, under artificial infection conditions of *Trichomonas vaginalis*, the bivalent recombinant protein yolk antibody showed a significant protective effect against *Trichomonas vaginalis* infection. In groups 1-3 injected with different doses of the bivalent recombinant protein yolk antibody, no oral lesions were observed in the high-dose group (1.0 ml / bird) and the medium-dose group (0.5 ml / bird), with a survival rate of 100%. In the low-dose group (0.2 ml / bird), 20% of the squabs developed oral lesions, and one squab died, resulting in a survival rate of 90%. In groups 4-6 injected with different doses of recombinant AP33 protein yolk antibody, the survival rates in the medium and low-dose groups did not exceed 60%, while the oral lesion rate in the high-dose group (1.0 ml / bird) was 20%. The survival rate reached 100%; in groups 7-9 injected with different doses of recombinant AP65 protein yolk antibody, the survival rate in the medium and low dose groups did not exceed 60%, while the high dose group (1.0 ml / bird) had an oral lesion rate of 20% and a survival rate of 90%; in groups 10-12 injected with different doses of whole worm antigen yolk antibody, the survival rate in the high, medium, and low dose groups did not exceed 60%; the challenge control group received no treatment, and the lesion rate reached 100%, with a survival rate of 10%, demonstrating the serious impact of infection on pigeon health. The blank control group (uninfected) showed no lesions and had a survival rate of 100%.

[0095] Table 8. Preventive effects of different doses of antibodies against artificial infection with Trichomonas vaginalis in pigeons. Based on the oral lesion rate and the survival rate of experimental pigeons, the prepared bivalent recombinant protein egg yolk antibody, injected at a medium dose (0.5 ml / pigeon), can effectively prevent artificial infection of pigeon trichomoniasis.

[0096] Example 12 Safety test of egg yolk antibodies 1. Safety experiment in mice Twenty mice weighing 18-22g were used, divided into groups of 10. One group was injected with 0.5ml of bivalent recombinant protein egg yolk antibody, and the other group was injected with 1.0ml of bivalent recombinant protein egg yolk antibody. The mice were observed for 14 consecutive days.

[0097] The mice were in good spirits and all were healthy, indicating that the prepared bivalent recombinant protein yolk antibody had good safety.

[0098] 2. Safety experiments on target animals Twenty one-month-old squabs were used, divided into groups of 10. One group was given 1.0 ml of bivalent recombinant protein egg yolk antibody orally, while the other group was injected with 1.0 ml of bivalent recombinant protein egg yolk antibody. The groups were observed for 14 consecutive days.

[0099] Both groups of pigeons survived, indicating that the prepared bivalent recombinant protein yolk antibody has good safety.

[0100] As can be seen from Examples 7-12 above, this application is the first to use recombinant subunit antigens of Trichomonas vaginalis (including AP33 and AP65 proteins) expressed by Sf9 cells to immunize laying hens and obtain egg yolk antibodies. The antigen can be mass-produced using a bioreactor. The egg yolk antibodies prepared based on this standardized antigen are highly stable between batches, and quality control is simple. In particular, this application uses high-purity antigens to immunize laying hens, avoiding the safety risks that may be caused by whole-parasite antigens, and the obtained antibodies have high titers and strong specificity. In addition, the recombinant protein egg yolk antibodies prepared in this application can not only be used for the treatment of Trichomonas vaginalis infection, but also have outstanding preventive effects, realizing "prevention and treatment in one".

[0101] In summary, the egg yolk antibodies provided in the above embodiments of this application demonstrate significant effects in the prevention and treatment of trichomoniasis infection in pigeons, effectively improving the survival rate of infected pigeons and reducing the severity of oral lesions. Compared with existing chemical drugs, the egg yolk antibodies have comprehensive advantages such as high safety, low likelihood of drug resistance, no drug residues, environmental friendliness, and suitability for large-scale production, demonstrating their promising application prospects in the green prevention and control of trichomoniasis in pigeons.

[0102] It should be understood that the embodiments described above are only some, not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A bivalent recombinant protein yolk antibody against infection by Pigeon Blepharitis, characterized in that: The bivalent recombinant protein yolk antibody is isolated from the yolk of an avian egg produced by an egg-laying bird immunized with a recombinant subunit vaccine; The vaccine comprises a recombinant protein comprising a recombinant AP33 protein having a sequence as set forth in SEQ ID NO: 7 and a recombinant AP65 protein having a sequence as set forth in SEQ ID NO: 8, and a pharmaceutically acceptable carrier.

2. The bivalent recombinant protein yolk antibody against Infestation of Pigeon Threadworm according to claim 1, characterized by: The egg-laying bird comprises a chicken.

3. The bivalent recombinant protein yolk antibody against infestation of pigeon trichomonad according to claim 2, characterized by: The chicken comprises a Hainan Brown hen.

4. A process for the preparation of bivalent recombinant protein yolk antibody against infestation of pigeon feather mites, characterized by, The recombinant subunit vaccine comprises a recombinant protein comprising a recombinant AP33 protein having a sequence as set forth in SEQ ID NO: 7 and a recombinant AP65 protein having a sequence as set forth in SEQ ID NO:

8. The bivalent recombinant protein yolk antibody is isolated from the yolk of an avian egg produced by an egg-laying bird immunized with a recombinant subunit vaccine; The recombinant protein is purified and has a purity of greater than 85%.

5. The method of claim 4, wherein: The recombinant subunit vaccine further comprises a pharmaceutically acceptable carrier.

6. The method of claim 4, wherein: The adjuvant comprises any one or a combination of white oil, Span, aluminum stearate, or Tween.

7. The method of claim 6, wherein: The bivalent recombinant protein yolk antibody is isolated from the yolk of an avian egg produced by an egg-laying bird immunized with a recombinant subunit vaccine; 8. A pharmaceutical composition, characterized by, 9. Use of the bivalent recombinant protein yolk antibody against an infection with L. colubrum of any one of claims 1-2 in the manufacture of a medicament for inducing an immune response against an antigen of L. colubrum in a subject animal.

10. Use of the bivalent recombinant protein yolk antibody against an infection with L. colubrum of any one of claims 1-2 in the manufacture of a medicament for preventing or treating an infection with L. colubrum in an animal. ​

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