A trivalent vaccine against avibacterium paragallinarum and a method for its preparation

Abstract

The application discloses a kind of paragallinarievaccines and preparation method thereof, the paragallinarievaccines include A type paragallinarie, B type paragallinarie and C type paragallinarie HMTp210 hypervariable region protein and avian pathogenic escherichia coli ghost.The application uses three serotypes HMTp210 hypervariable region recombinant protein and avian pathogenic escherichia coli ghost (especially O78 type avian pathogenic escherichia coli ghost) in combination, synergistically enhances humoral and cellular immunity, provides more broad-spectrum protection, while reaching the protection effect equivalent to white oil adjuvant, significantly improves injection safety, avoids the granuloma and other side reactions of white oil adjuvant, realizes the technical goal of "equivalent protection, better safety".

Description

Technical Field

[0001] This invention relates to the fields of veterinary biological products and genetic engineering technology, specifically to a trivalent avian bacillus vaccine and its preparation method. Background Technology

[0002] Infectious coryza (IC) is an acute respiratory disease in chickens caused by *Avianobacter paragallinarum*, which is widespread worldwide, leading to decreased egg production and stunted growth, causing significant economic losses. The pathogen mainly has three serotypes: A, B, and C, with weak cross-protection between different serotypes. Currently, most commercially available vaccines are inactivated vaccines (such as aluminum gel inactivated vaccines and oil emulsion inactivated vaccines) or subunit vaccines based on hemagglutinin proteins, but they suffer from insufficient immunogenicity, significant side effects, and the need for potent adjuvants. While traditional white oil adjuvants can enhance the immune response, they easily form granulomas at the injection site and are difficult to induce mucosal immunity, becoming a major bottleneck restricting vaccine safety. Bacterial ghosts are empty bacterial envelopes formed after Gram-negative bacteria are lysed by the PhiX174 lysis gene E. They retain an intact outer membrane structure, possess natural adjuvant activity, can be efficiently taken up by antigen-presenting cells, and have good safety profiles. However, although there are reports in the existing technology of using Escherichia coli molluscs as a vaccine immunogen (such as CN105861404A), there is no technical inspiration for using it as an adjuvant for heterologous antigens (Avian bacillus paragallinarum HMTp210 protein), especially for trivalent subunit vaccines against Avian bacillus paragallinarum.

[0003] Therefore, providing a trivalent vaccine for avian bacillus that can provide broader-spectrum protection against the three serotypes of avian bacillus paragallinarum and overcome the side effects of white oil adjuvant is a problem that this invention urgently needs to solve. Summary of the Invention

[0004] In view of the problems of weak cross-protection of infectious coryza vaccines in chickens and the side effects of traditional adjuvants in the existing technology, the purpose of this invention is to provide a subunit vaccine composition with strong immunogenicity, good safety and ability to simultaneously resist avian paraguine bacilli of types A, B and C, and its preparation method.

[0005] To achieve the above objectives, this invention provides a trivalent *Avianella paragallinarum* vaccine based on HMTp210 protein and avian pathogenic *Escherichia coli* slough adjuvant. The trivalent *Avianella paragallinarum* vaccine comprises recombinant protein of *Avianella paragallinarum* type A, recombinant protein of *Avianella paragallinarum* type B, recombinant protein of *Avianella paragallinarum* type C, and slough. The recombinant proteins of type A, type B, and type C of *Paragonimus westermani* each include the HMTp210 hypervariable region protein of *Paragonimus westermani*, *Paragonimus westermani*, and *Paragonimus westermani*, respectively. The bacterial molting was selected from the molting of pathogenic avian Escherichia coli.

[0006] Preferably, the amino acid sequence of the recombinant protein of *Avianobacter paragallinarum* type A is shown in SEQ ID No:3, and its corresponding coding sequence is shown in SEQ ID No:6; and / or, The amino acid sequence of the recombinant protein from *Avianobacter pylori* type B is shown in SEQ ID No:4, and its corresponding coding sequence is shown in SEQ ID No:7; and / or, The amino acid sequence of the recombinant protein of *Avianobacter pylori* type C is shown in SEQ ID No:5, and its corresponding coding sequence is shown in SEQ ID No:8.

[0007] SEQ ID No:3: ; SEQ ID No:6: SEQ ID No:4: ; SEQ ID No:7: SEQ ID No:5: ; SEQ ID No:8:

[0008] Preferably, the avian pathogenic Escherichia coli spores are O78 serotype avian pathogenic Escherichia coli spores.

[0009] Preferably, in 1 mL of the trivalent avian parafossa vaccine, the total content of the recombinant protein of avian parafossa type A, avian parafossa type B, and avian parafossa type C is 150-400 μg, and the content of the avian pathogenic Escherichia coli spores is 1×10⁻⁶. 7 -1×10 8 CFU equivalent.

[0010] Preferably, in the trivalent avian bacillus vaccine, the mass ratio of the recombinant protein of type A avian bacillus, the recombinant protein of type B avian bacillus, and the recombinant protein of type C avian bacillus is 1:0.5-2:0.5-2.

[0011] For example, in one specific embodiment, the trivalent avian bacillus vaccine of the present invention is a composition comprising an active ingredient and an adjuvant. The active ingredient is recombinant protein of avian bacillus type A (hereinafter referred to as A or type A), recombinant protein of avian bacillus type B (hereinafter referred to as B or type B), and recombinant protein of avian bacillus type C (hereinafter referred to as C or type C), with a preferred mass ratio of 1:1:1; the adjuvant is serotype O78 avian pathogenic Escherichia coli (hereinafter referred to as O78 serotype). The total amount of the three recombinant proteins in each dose of vaccine is 50 μg (i.e., approximately 16.7 μg each of A, B, and C), and the O78 serotype content is 1 × 10⁻⁶. 7 CFU equivalent (bacterial count before lysis). The vaccine formulation is an injectable suspension prepared with sterile PBS. It should be noted that the numerical values ​​provided here are specific limitations for illustrative purposes only; in actual operation, those skilled in the art can make reasonable choices within the ranges given above, which will not be elaborated upon further here.

[0012] The present invention also provides a method for preparing the above-described trivalent avian bacillus vaccine, the method comprising: Preparation of S100, recombinant proteins of type A, type B, and type C of *Paragonimus westermani*: After amplifying the hypervariable region of the HMTp210 gene of *Paragonimus westermani*, type B, and type C, the recombinant proteins of type A, type B, and type C of *Paragonimus westermani* were expressed and purified to obtain the corresponding recombinant proteins of type A, type B, and type C of *Paragonimus westermani*. S200, Preparation of avian pathogenic Escherichia coli molluscs: After activation, culture and induced lysis of avian pathogenic Escherichia coli, the precipitate is collected and purified to obtain avian pathogenic Escherichia coli molluscs; S300 Assembly of the trivalent avian bacillus vaccine: The recombinant proteins of type A, type B, and type C of avian bacillus obtained in step S100, and the avian pathogenic Escherichia coli slough obtained in step S200, are mixed to obtain the trivalent avian bacillus vaccine.

[0013] Preferably, in step S100, the amplification process specifically includes: extracting genomic DNA from type A avian bacillus, type B avian bacillus and type C avian bacillus, and then amplifying them respectively using amplification primer pairs as shown in SEQ ID No:1 and SEQ ID No:2.

[0014] Preferably, the type A avian bacillus is selected from the Apg JS28 strain; and / or, The B-type avian bacillus was selected from the Apg JS40 strain; and / or; The C-type avian bacillus was selected from the Apg JS44 strain.

[0015] Similarly, a specific embodiment is described in detail here. It should be noted that the scope of protection of this invention is based on the foregoing description, and is not limited to the specific values ​​and strain selections in the following specific embodiments. The operating methods (e.g., preparation of recombinant proteins and preparation of bacterial erosions) can also be carried out in a manner that is understandable and conventionally used by those skilled in the art, and will not be elaborated upon here. In a more specific embodiment, the preparation method is as follows: Preparation of HMTp210-A / B / C recombinant protein: Genomic DNA was extracted from Apg JS28 (type A), JS40 (type B), and JS44 (type C), respectively. The hypervariable region of the HMTp210 gene (approximately 1500 bp) was amplified using primer pairs shown in SEQ ID No:1 and SEQ ID No:2, with BamHI and SalI restriction sites introduced into the primers. The amplified products and pET-32a(+) vector were double-digested with BamHI / SalI, ligated, transformed into Trans1-T1 cells, and positive clones were screened. Recombinant plasmids pET-32a(+)-HMTp210-A, pET-32a(+)-HMTp210-B, and pET-32a(+)-HMTp210-C were obtained after successful sequencing. These plasmids were transformed into BL21(DE3) competent cells and induced with IPTG (final concentration 1.0 mM) at 37℃ for 5 h. The cells were lysed by sonication, and SDS-PAGE and Western spectroscopy were performed. Blot analysis identified the expression forms (HMTp210-A was mainly found in inclusion bodies, while HMTp210-B and HMTp210-C were found in the supernatant); inclusion body proteins were dissolved in 6M urea, and supernatant proteins were directly purified by Ni-NTA affinity chromatography; the purified proteins were refolded by gradient dialysis (6M→4M→2M urea, PBS dialysis), concentrated with sucrose, and the concentration was determined by the BCA method (type A 1.1 μg / μL, type B 2.5 μg / μL, type C 0.8 μg / μL), and stored at -80℃ for later use.

[0016] Preparation of O78 avian pathogenic Escherichia coli bacterial slough: Glyceryl bacillus O78 (APEC O78), containing the temperature-controlled lysis plasmid pBV221-E, was inoculated into LB (Amp+) medium and cultured at 28°C with shaking until A... 600 ≈0.5; rapidly transfer to a 42℃ shaker (220 r / min) to induce pyrolysis, measuring A every 30 min. 600 The value was increased until it stopped decreasing; the lysis efficiency was calculated by plating (lysis rate > 99.9%); β-propiolactone was added to the bacterial molting solution to a final concentration of 0.025% (v / v), and the solution was incubated at 42°C for 1 h for initial inactivation; the precipitate was collected by centrifugation, washed 3 times with PBS, resuspended, and then β-propiolactone was added to a final concentration of 0.05% (v / v) and incubated at 42°C for 2 h for complete inactivation; after passing the sterility test, the solution was stored at 4°C for later use.

[0017] Vaccine assembly: The three recombinant proteins (types A, B, and C) obtained above were diluted to the same concentration with sterile PBS and mixed at a volume ratio of 1:1:1, so that the concentration of each protein in the mixture was 167 μg / mL (total protein 500 μg / mL). An appropriate amount of O78 bacterial suspension (calculated as 1 × 10⁻⁶ cells / mL) was taken. 8The CFU equivalents / mL are mixed with an equal volume of protein to obtain the vaccine (each 0.2 mL contains 50 μg of total protein and 1 × 10⁻⁶ molluscs). 7 CFU equivalent).

[0018] The specific method of using the trivalent avian bacillus vaccine of the present invention is as follows: subcutaneous immunization is performed in the neck, followed by a booster immunization 14 days after the first immunization, with 0.2 mL per bird.

[0019] This invention combines recombinant HMTp210 hypervariable regions of three serotypes with avian pathogenic Escherichia coli molluscs (especially O78 avian pathogenic Escherichia coli molluscs). Specifically, the hypervariable regions of HMTp210 proteins of types A, B, and C are expressed, purified, and then mixed. Avian pathogenic Escherichia coli molluscs is used as an adjuvant and delivery carrier to synergistically enhance humoral and cellular immunity, providing broader-spectrum protection. While achieving a protective effect comparable to that of white oil adjuvant, it significantly improves injection safety and avoids side effects such as granulomas associated with white oil adjuvant, thus achieving the technical goal of "equivalent protection with superior safety". Attached Figure Description

[0020] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is an SDS-PAGE image of the recombinant proteins HMTp210-A, HMTp210-B, and HMTp210-C prepared in Preparation Example 1. Figure 2 This is a Western blot diagram of the recombinant proteins HMTp210-A, HMTp210-B and HMTp210-C obtained in Preparation Example 1; Figure 3 This is an SDS-PAGE image of the purified recombinant proteins HMTp210-A, HMTp210-B, and HMTp210-C from Preparation Example 1. Figure 4 This is a graph showing the serum antibody titer detected by indirect ELISA two weeks after the second immunization in Example 1. Figure 5 This is a line graph showing the clinical symptom scores after challenge in Case 2. Figure 6 This is a line graph showing the weight change after challenge in Case 2. Figure 7 This is the result of qPCR detection of bacterial shedding from the pharyngeal swab in Case 2; Figure 8 These are comparative photographs of the infraorbital sinus lesions examined in Case 2; Figure 9These are comparative photos of tracheal necropsy lesions from Case 2. Detailed Implementation

[0021] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0022] The Apg JS28 strain (Avianobacterium paragallinarum type A), Apg JS40 strain (Avianobacterium paragallinarum type B), and Apg JS44 strain (Avianobacterium paragallinarum type C) were obtained from the College of Veterinary Medicine, Yangzhou University; the pET-32a(+) vector was a commercially available product from EMD Biosciences; Trans1-T1 was a commercially available product from Beijing TransGen Biotech Co., Ltd.; BL21(DE3) was a commercially available product from Beijing TransGen Biotech Co., Ltd.; the APEC O78 strain was obtained from the College of Veterinary Medicine and Public Health, Jiangsu Vocational College of Agriculture and Animal Husbandry Science and Technology; the HB64 strain was obtained from the College of Veterinary Medicine, Yangzhou University; the JS31 strain was obtained from the College of Veterinary Medicine, Yangzhou University; and the JS80 strain was obtained from the College of Veterinary Medicine, Yangzhou University.

[0023] SPF chicken is a commercially available product from Boehringer Ingelheim.

[0024] Unless otherwise stated, the reagents used in the specific embodiments of the present invention are reagents that are conventionally available and usable by those skilled in the art.

[0025] In this invention, statistical analysis data are expressed as mean ± SD. GraphPad Prism 8.0 one-way ANOVA is used, with p < 0.05 considered significant (*) and p < 0.01 considered highly significant (**).

[0026] Preparation Example 1: Preparation of recombinant proteins of *Avianobacter paragallinarum* type A (denoted as HMTp210-A), *Avianobacter paragallinarum* type B (denoted as HMTp210-B), and *Avianobacter paragallinarum* type C (denoted as HMTp210-C). 1. Construction of prokaryotic expression vectors Frozen bacterial cultures of Apg JS28, Apg JS40, and Apg JS44 were streaked onto TSA plates and anaerobic incubated for 24 h. Single colonies were picked and inoculated onto TSB plates, and cultured at 37°C for 16 h. Genomic DNA was extracted. PCR amplification was performed using the genomic DNA extracted from the three strains as templates, as shown in the primers below: Upstream (HMTp210-GB-F, SEQ ID No:1): 5'-CTCGGATCCGATGGCACAATTACATTTACA-3' (BamHI) Downstream (HMTp210-GB-R, SEQ ID No:2): 5'-CGCGTCGACGGACTAACTTCATCACCATAC-3' (SalI) PCR reaction system (20 μL): 13.4 μL ultrapure water, 2.0 μL 10×Taq Buffer, 1.6 μL dNTPs (2.5 mmol / L), 0.4 μL each of forward and reverse primers (10 μmol / L), 2.0 μL DNA template, and 0.2 μL EasyTaq DNA Polymerase (5 U / μL). Cycling parameters: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min. The amplified fragment was approximately 1500 bp. After gel recovery, the PCR product was double-digested with BamHI / SalI (37℃, 2 h), ligated into the similarly double-digested pET-32a(+) vector, transformed into Trans1-T1, and plated on Amp plates. Clones were picked, plasmids were extracted, double-digested for identification, and sequenced. The correctly sequenced recombinant plasmids were named pET-32a(+)-HMTp210-A (prepared using genomic DNA extracted from Apg JS28 as a template), pET-32a(+)-HMTp210-B (prepared using genomic DNA extracted from Apg JS40 as a template), and pET-32a(+)-HMTp210-C (prepared using genomic DNA extracted from Apg JS44 as a template).

[0027] 2. Induced Expression and Identification The recombinant plasmids obtained above were transformed into BL21(DE3), and single colonies were picked and cultured in LB(Amp) cells. + Incubate at 37℃ until A 600 ≈0.5, add IPTG to 1.0 mM, and induce at 37℃ for 5 h to obtain the induced recombinant proteins HMTp210-A, HMTp210-B, and HMTp210-C, respectively. Collect bacterial cells by centrifugation, wash with PBS, and sonicate (60W, 2 s working / 3 s intermittent, 30 min). Perform 12% SDS-PAGE on whole cells, supernatant, and precipitate. The SDS-PAGE results are shown below. Figure 1 As shown. Among them, Figure 1 Figure A shows the SDS-PAGE identification results of the recombinant protein HMTp210-A. Figure 1 Figure B shows the SDS-PAGE identification results of the recombinant protein HMTp210-B. Figure 1Figure C shows the SDS-PAGE identification results of recombinant protein HMTp210-C. Band M is the protein molecular standard marker. Band 1 is the whole cell of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-A without induced lysis; Band 2 is the whole cell of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-A induced lysis; Band 3 is the supernatant of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-A induced lysis; Band 4 is the precipitate of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-A induced lysis; Band 5 is the precipitate of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis; Band 6 ...7 is the precipitate of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis; Band 8 is the precipitate of recombinant bacterium BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis; Band 9 is the precipitate of recombinant bacterium BL21(DE3)-pET Band 7 represents the whole bacterial culture lysed by BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis. Band 8 represents the whole bacterial culture lysed by BL21(DE3)-pET-32a(+)-HMTp210-B without induced lysis. Band 9 represents the whole bacterial culture lysed by BL21(DE3)-pET-32a(+)-HMTp210-B without induced lysis. At 0-C, lysis and precipitation were induced. Band 10 represents the supernatant lysed by recombinant BL21(DE3)-pET-32a(+)-HMTp210-C induced lysis; band 11 represents the whole bacterial lysed by recombinant BL21(DE3)-pET-32a(+)-HMTp210-C induced lysis; and band 12 represents the whole bacterial lysed by recombinant BL21(DE3)-pET-32a(+)-HMTp210-C without induced lysis. Coomassie brilliant blue staining showed that HMTp210-A was mainly found in the precipitate (inclusion bodies), while HMTp210-B and HMTp210-C were mainly found in the supernatant. Western blot identification results are as follows: Figure 2 As shown. Among them, Figure 2 Figure A shows the Western blot identification results of the recombinant protein HMTp210-A. Figure 2 Figure B shows the Western blot identification results of the recombinant protein HMTp210-B. Figure 2Figure C shows the Western blot identification results of recombinant protein HMTp210-C. Band M is the protein molecular standard marker. Band 1 is the lysis and precipitation of recombinant bacteria BL21(DE3)-pET-32a(+)-HMTp210-A induced by recombinant bacteria; Band 2 is the supernatant of lysis induced by recombinant bacteria BL21(DE3)-pET-32a(+)-HMTp210-A induced by recombinant bacteria; Band 3 is the whole cell lysis of recombinant bacteria BL21(DE3)-pET-32a(+)-HMTp210-A induced by recombinant bacteria; Band 4 is the whole cell lysis of recombinant bacteria BL21(DE3)-pET-32a(+)-HMTp210-A without induced lysis; Band 5 is the lysis and precipitation of recombinant bacteria BL21(DE3)-pET-32a(+)-HMTp210-B induced by recombinant bacteria; Band 6 is the whole cell lysis of recombinant bacteria BL21(DE3)-pET-32a(+)-HMTp210-B induced by ... Band 7 represents the recombinant BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis supernatant; band 8 represents the recombinant BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis whole cell; band 9 represents the recombinant BL21(DE3)-pET-32a(+)-HMTp210-B uninduced lysis whole cell; and band 9 represents the recombinant BL21(DE3)-pET-32a(+)-HMTp210-B induced lysis supernatant. 10-C induced lysis and precipitation: Band 10 represents the supernatant from the 10-C induced lysis of recombinant BL21(DE3)-pET-32a(+)-HMTp2; Band 11 represents the whole-cell lysis of recombinant BL21(DE3)-pET-32a(+)-HMTp2; and Band 12 represents the whole-cell lysis of recombinant BL21(DE3)-pET-32a(+)-HMTp2 without induced lysis. Western blot (anti-His tag) confirmed specific bands, each approximately 68 kDa (including the tag), consistent with expectations.

[0028] 3. Purification and Renaturation HMTp210-A inclusion bodies were dissolved overnight in Lysis equilibration buffer (containing 6M urea), and the supernatant was collected by centrifugation. The supernatants of HMTp210-B and HMTp210-C were directly loaded onto a Ni-NTA column, respectively. The cells were washed with LE buffer, followed by washing with 20mM imidazole to remove impurities, and finally eluted with a gradient of imidazoles (100-250mM). The eluent was collected, and the purified recombinant proteins HMTp210-A, HMTp210-B, and HMTp210-C were analyzed by SDS-PAGE. The results are shown below. Figure 3 As shown, where, Figure 3 Figure A shows the SDS-PAGE identification results of the recombinant protein HMTp210-A. Figure 3 Figure B shows the SDS-PAGE identification results of the recombinant protein HMTp210-B. Figure 3Figure C shows the SDS-PAGE identification results of recombinant protein HMTp210-C. Band M is the protein molecular standard marker, bands 1 and 2 are the filtrate, band 3 is the equilibration effluent, band 4 is the washing effluent, and bands 5-7 are the elution effluent. The SDS-PAGE purity was >90%. The HMTp210-A eluent was dialyzed sequentially against 6M, 4M, and 2M urea-PBS (6 hours each time, 3 times with buffer changes), and finally dialyzed against PBS to remove urea. All proteins were concentrated with PEG or sucrose, and the concentration was determined by the BCA method: the concentration of recombinant protein from *A. paragallinarum* type A was 1.1 μg / μL, the concentration of recombinant protein from *A. paragallinarum* type B was 2.5 μg / μL, and the concentration of recombinant protein from *A. paragallinarum* type C was 0.8 μg / μL. Aliquots were stored at -80℃.

[0029] Preparation Example 2: Preparation and inactivation of O78 type avian pathogenic Escherichia coli spores APEC O78 strain (laboratory name D78) containing pBV221-E, stored at -80℃, was inoculated with LB (Amp+) and activated overnight at 28℃. The next day, 200 mL of LB (Amp+) was transferred at a 1:100 ratio and cultured at 28℃ until A… 600 =0.5. Immediately transfer to a 42℃ water bath shaker, induce pyrolysis at 220 r / min, and measure A every 30 min. 600 Approximately 3 hours later, A 600 The bacterial count dropped to a plateau (approximately below 5% of the initial value). Dilute the pre-induction bacterial culture and plate it on a plate (10 μL). -7 ~10 -9 ) count; dilution of bacterial culture after induction (10) -4 ~10 -6 Spread the bacteria onto LB agar plates and incubate at 37°C for 12 hours for counting. The lysis rate = (1 - CFU after induction / CFU before induction) × 100% > 99.9%. Collect the bacterial suspension, add β-propiolactone to 0.025% (v / v), incubate at 42°C for 1 hour; centrifuge, discard the supernatant, wash three times with PBS, resuspend in PBS, add β-propiolactone again to 0.05%, and incubate at 42°C for 2 hours. After passing the sterility test (spreading on LB agar plates and inoculating with LB liquid, sterile growth at 37°C for 72 hours), O78 avian pathogenic Escherichia coli bacterial slough (referred to as O78 slough) is obtained and stored at 4°C for later use. When using, adjust the concentration of the prepared slough to 1 × 10⁻⁶. 8 CFU equivalents / mL.

[0030] Example: Preparation of trivalent vaccine The three recombinant proteins (HMTp210-A, HMTp210-B, and HMTp210-C) obtained in Preparation Example 1 were diluted to the same concentration with sterile PBS and mixed at a volume ratio of 1:1:1 to obtain a mixed protein solution with a protein concentration of 167 μg / mL (total protein 500 μg / mL). The O78 bacterial suspension (1×10⁻⁶) obtained in Preparation Example 2 was then used. 8 The CFU equivalent / mL) is mixed with an equal volume of the above-mentioned mixed protein solution to obtain the trivalent vaccine (i.e., each 0.2 mL of vaccine contains 50 μg of total protein and 1 × 10⁻⁶ molluscs). 7 CFU equivalent).

[0031] Preparation of comparative and white oil adjuvant vaccines The mixed protein solution from Example 1 was emulsified with ISA 71 VG adjuvant at a 1:1 ratio to obtain a white oil adjuvant vaccine. That is, each 0.2 mL of the obtained white oil adjuvant vaccine contains 50 μg of total protein.

[0032] Preparation Example 3: Animal Immunization Grouping and Antibody Collection Twenty 21-day-old SPF chickens were randomly divided into 4 groups (5 chickens per group) and immunized with the following reagents: Group 1 (spore molting adjuvant group): HMTp210 trivalent protein + spore molting (i.e., the trivalent vaccine prepared in the examples); Group 2 (white oil adjuvant group): HMTp210 trivalent protein + white oil adjuvant (i.e., the white oil adjuvant vaccine prepared in the comparative ratio). Group 3 (control group): PBS; Group 4 (blank group): No immunization, PBS was given during challenge.

[0033] Immunization route: 0.2 mL / animal subcutaneously injected into the neck. A second immunization is administered in the same manner 14 days after the first immunization.

[0034] Blood was collected from the wing vein of SPF chickens 14 days after the second immunization, serum was separated, and the obtained test antibodies were stored at -20℃.

[0035] The serum samples mentioned in the following test examples are the four groups of test antibodies obtained after the second immunization with the four immunoassay reagents prepared and stored here.

[0036] Example 1: Indirect ELISA detection of antibodies The purified HMTp210-A, HMTp210-B, and HMTp210-C proteins (2 μg / mL) obtained in Preparation Example 1 were coated onto ELISA plates, 100 μL / well, and incubated overnight at 4°C. After blocking, serially diluted test serum (i.e., the antibody from Preparation Example 3, starting at 1:1000) was added and incubated. HRP-induced reaction with goat anti-chicken IgG (1:5000) was performed. TMB color development was then performed, and OD450 was measured. The serum antibody titer was detected by indirect ELISA two weeks after the second immunization, as shown in the figure. Figure 4 As shown, where, Figure 4 Figure A shows the antibody levels in the serum sample tested against HMTp210 protein type A (HMTp210-A). Figure 4 Figure B shows the antibody levels in the serum sample tested against HMTp210 protein type B (HMTp210-B). Figure 4 The middle figure (C) shows the antibody levels produced in the serum sample against HMTp210 protein type C (HMTp210-C).

[0037] pass Figure 4 The results showed that the antibody titers against recombinant proteins of *Avianobacterium paragallinarum* type A, *Avianobacterium paragallinarum* type B, and *Avianobacterium paragallinarum* type C were all above 1:105 in both the bacterial spore adjuvant group and the white oil adjuvant group, with no significant difference between the groups (p>0.05). The control group was negative. This indicates that the bacterial spore adjuvant can effectively enhance protein-specific humoral immunity, with an effect comparable to that of the white oil adjuvant.

[0038] Example 2, Virus Challenge Protection Test Fourteen days after the second immunization (49 days old), each group of chickens (i.e., the groups treated according to the immunization grouping in Example 3, except for the blank group) was injected via the infraorbital sinus with 0.2 mL of a mixed virulent bacterial solution (containing 1×10⁻⁶ HB64 strain). 2 CFU, JS31 strain 1×10 1 CFU, JS80 strain 1×10 1 CFU). The control group was injected with PBS. Clinical symptoms were observed for 7 consecutive days post-challenge (facial swelling score: 0 points normal; 1 point mild swelling; 2 points moderate swelling, affecting eye opening and closing; 3 points severe swelling, eye closure), and weight changes were recorded. Pharyngeal swabs were collected on days 3, 5, and 7 post-challenge; DNA was extracted using the boiling method, and bacterial shedding was detected by qPCR (primers qApg-F / R, SYBR Green method). Two animals from each group were necropsy on day 5 post-challenge (the remaining animals were observed for up to 7 days) to observe infraorbital sinus and tracheal lesions.

[0039] The line graph of clinical symptom scores after viral challenge is shown below. Figure 5 As shown in the line graph of weight change after viral challenge, Figure 6 As shown, the qPCR test results for bacterial shedding from throat swabs are as follows: Figure 7As shown, the before-and-after photos of infraorbital sinus lesions obtained through necropsy are as follows: Figure 8 As shown (where, Figure 8 In the image, group A is the bacterial slough adjuvant group, group B is the white oil adjuvant group, group C is the control group, and group D is the blank group. (Red circles indicate hemorrhage points, blue circles indicate mucus, and yellow circles indicate caseous material.) Comparison photos of tracheal necropsy lesions are shown below. Figure 9 As shown, (where, Figure 9 In the diagram, A represents the bacterial slough adjuvant group, B represents the white oil adjuvant group, C represents the control group, and D represents the blank group. The red circles in the diagram indicate bleeding points.

[0040] pass Figures 5-9 The test results showed that the clinical symptom scores of both the bacterial slough adjuvant group and the white oil adjuvant group were significantly lower than those of the control group (p<0.01), with no statistically significant difference between the two groups; the weight loss was much smaller than that of the control group; the bacterial shedding volume on days 3 and 5 was significantly lower in both groups than in the control group (p<0.01 on day 5), and there was no significant difference between the bacterial slough group and the white oil group. Autopsy findings: the control group showed severe infraorbital sinus hemorrhage and caseous exudate (3 / 5), the bacterial slough adjuvant group had less caseous exudate (1 / 5), and the white oil group had no caseous exudate (0 / 5); the number of tracheal hemorrhage points was slightly higher in the bacterial slough group (3 / 5) than in the white oil group (1 / 5), but the severity of the lesions was milder in both groups than in the control group (5 / 5). Furthermore, mild granulomas were observed at the injection site in the white oil adjuvant group, while no gross abnormalities were visible at the injection site in the bacterial slough adjuvant group.

[0041] The above results indicate that the O78 bacterial slough adjuvant vaccine of the present invention can provide immune protection similar to that of the white oil adjuvant vaccine, and has higher safety and mild local injection reaction, thus achieving the invention objective of "equivalent protection and better safety".

[0042] As can be seen from the above specific embodiments, the present invention has at least the following advantages: 1. This invention combines the hypervariable region proteins of three serotypes HMTp210 to cover serotypes A, B, and C of Avian bacillus paragallinarum, overcoming the deficiency of weak cross-protection of single serotype vaccines.

[0043] 2. This study is the first to use avian pathogenic Escherichia coli O78 spores as an adjuvant (rather than an immunogen) for a subunit vaccine. The spores contain natural immunostimulants such as LPS and lipoproteins on their surface, effectively activating antigen-presenting cells and inducing a high level of humoral immune response. Animal experiments showed that the serum antibody titers of chickens immunized with the spores adjuvant group after a second immunization reached 1:105 or higher (ELISA), with no significant difference compared to the white oil adjuvant group (p>0.05), indicating that both are equally effective in inducing humoral immunity. However, in terms of safety, no visible granulomas were observed at the injection site in the spores adjuvant group, while obvious granulomas and vaccine residues were visible in the white oil adjuvant group. The spores adjuvant group showed significantly better safety than the white oil adjuvant group.

[0044] 3. The results of the challenge protection test (injection of a mixture of virulent strains HB64(A), JS31(B), and JS80(C) into the infraorbital sinus) showed that both the bacterial slough adjuvant group and the white oil adjuvant group significantly reduced clinical symptoms (lower facial swelling score), reduced bacterial shedding (pharyngeal swab qPCR detection showed significantly lower bacterial shedding 5 days after challenge compared to the control group, p<0.01), and reduced infraorbital sinus hemorrhage, caseous exudate, and tracheal hemorrhage. There were no statistically significant differences between the two groups in the core protective indicators (p>0.05), indicating that the bacterial slough adjuvant provided a protective effect comparable to that of the white oil adjuvant. The proportion of caseous exudate in the infraorbital sinus in the bacterial slough adjuvant group (1 / 5) was lower than that in the control group (3 / 5), and essentially the same as that in the white oil group (0 / 5), with milder tracheal hemorrhage. The weight loss was significantly less than that in the control group. In summary, this invention achieves equivalent protection to white oil adjuvant vaccines while completely eliminating the side effect of granulomas at the white oil adjuvant injection site, thus solving the technical problem of achieving both vaccine safety and efficacy in the prior art.

[0045] 4. The preparation process is simple, and the bacterial molts can be freeze-dried for long-term preservation, providing a safe and effective alternative for the development of new veterinary vaccine adjuvants.

[0046] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0047] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0048] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A trivalent avian bacillus vaccine, characterized in that, The trivalent avian parasitic bacillus vaccine comprises recombinant protein from avian parasitic bacillus type A, recombinant protein from avian parasitic bacillus type B, recombinant protein from avian parasitic bacillus type C, and bacterial slough; wherein... The recombinant proteins of type A, type B, and type C of *Paragonimus westermani* each include the HMTp210 hypervariable region protein of *Paragonimus westermani*, *Paragonimus westermani*, and *Paragonimus westermani*, respectively. The bacterial molting was selected from the molting of pathogenic avian Escherichia coli.

2. The trivalent avian bacillus vaccine according to claim 1, characterized in that, The amino acid sequence of the recombinant protein from *Avianobacter paragallinarum* type A is shown in SEQ ID No:3; and / or, The amino acid sequence of the recombinant protein from *Avianobacter pylori* type B is shown in SEQ ID No:4; and / or, The amino acid sequence of the recombinant protein of *Avianobacter pylori* type C is shown in SEQ ID No:

5.

3. The trivalent avian bacillus vaccine according to claim 1 or 2, characterized in that, The avian pathogenic Escherichia coli spores were O78 serotype avian pathogenic Escherichia coli spores.

4. The trivalent avian bacillus vaccine according to claim 1 or 2, characterized in that, In 1 mL of the aforementioned trivalent avian parafowl cholera vaccine, the total content of recombinant proteins from avian parafowl cholera type A, type B, and type C is 150-400 μg, and the content of avian pathogenic Escherichia coli spores is 1 × 10⁻⁶. 7 -1×10 8 CFU equivalent.

5. The trivalent avian bacillus vaccine according to claim 4, characterized in that, In the trivalent avian bacillus vaccine, the mass ratio of the recombinant protein of type A avian bacillus, the recombinant protein of type B avian bacillus, and the recombinant protein of type C avian bacillus is 1:0.5-2:0.5-2.

6. A method for preparing a trivalent avian bacillus vaccine as described in any one of claims 1-5, characterized in that, The preparation method includes: Preparation of S100, recombinant proteins of type A, type B, and type C of *Paragonimus westermani*: After amplifying the hypervariable region of the HMTp210 gene of *Paragonimus westermani*, type B, and type C, the recombinant proteins of type A, type B, and type C of *Paragonimus westermani* were expressed and purified to obtain the corresponding recombinant proteins of type A, type B, and type C of *Paragonimus westermani*. S200, Preparation of avian pathogenic Escherichia coli molluscs: After activation, culture and induced lysis of avian pathogenic Escherichia coli, the precipitate is collected and purified to obtain avian pathogenic Escherichia coli molluscs; S300 Assembly of the trivalent avian bacillus vaccine: The recombinant proteins of type A, type B, and type C of avian bacillus obtained in step S100, and the avian pathogenic Escherichia coli slough obtained in step S200, are mixed to obtain the trivalent avian bacillus vaccine.

7. The preparation method according to claim 6, characterized in that, In step S100, the amplification process specifically includes: extracting genomic DNA from type A, type B, and type C of *Paragonimus westermani*, and then amplifying them respectively using the amplification primer pairs shown in SEQ ID No:1 and SEQ ID No:

2.

8. The preparation method according to claim 7, characterized in that, The type A avian bacillus was selected from the Apg JS28 strain; and / or, The B-type avian bacillus was selected from the Apg JS40 strain; and / or; The C-type avian bacillus was selected from the Apg JS44 strain.

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