A subunit vaccine of Ornithobacter avianus (type A) in chickens, its preparation method and application

Abstract

This invention belongs to the field of genetic engineering vaccine technology, specifically relating to a subunit vaccine against *Avibacterium avium* (type A) in chickens, its preparation method, and its application. The subunit vaccine comprises the OMA87 protein, with a size of 97.15 kDa, the gene sequence of which is shown in SEQ.ID.NO.4, and the amino acid sequence in SEQ.ID.NO.5. After induction of expression, the OMA87 protein was purified and detoxified to obtain a semi-finished antigen with a purity of over 50% and an endotoxin content below 2500 EU / mL. Using white oil adjuvant, a qualified OMA87 protein subunit vaccine was prepared through emulsification, thus becoming the *Avibacterium avium* subunit vaccine. This vaccine demonstrates good safety in broilers and laying hens, producing high and long-lasting antibody levels, providing a method for preventing *Avibacterium avium* infection in broilers and laying hens.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering vaccine technology, specifically relating to a subunit vaccine of Avibacterium avium (type A) in chickens, its preparation method, and its application. Background Technology

[0002] ORT can occur year-round, and studies have shown that it can occur during the brooding, growing, and laying periods. Laying flocks are most susceptible, with the highest infection rate, followed by growing flocks, while brooding flocks show the lowest incidence. Therefore, ORT can occur at different growth stages of chickens, and the incidence increases with age. Why is the incidence of ORT rising rapidly in my country? Several factors may be at play: firstly, the pathogenicity of ORT is increasing; secondly, reduced drug use allows bacterial diseases to proliferate easily; and thirdly, there is currently no corresponding vaccine available in China. While whole-cell inactivated vaccines are available abroad, ORT has weak reproductive capacity, requires stringent culture conditions, grows slowly, and requires a culture time exceeding 24-48 hours, resulting in high fermentation costs. Therefore, based on whole-cell inactivated vaccines, prokaryotic expression of immunogenic proteins using genetic engineering technology could be a promising research direction.

[0003] Subunit vaccines are vaccines in which the protective antigen gene encoding a pathogenic microorganism is introduced into a receptor, such as bacteria, yeast, or animal cells, enabling its efficient expression. After the protective antigen is isolated and purified, it is combined with an adjuvant to create a type of vaccine that induces antibody production and immune protection, exhibiting high safety, good stability, requiring no inactivation, and containing no nucleic acid. Currently, increasingly stringent food safety requirements, stricter national policies and regulations, industry consolidation and restructuring, the need to improve market quality competitiveness, the demand for precision immunization, and rapid upstream development all underscore the growing importance of veterinary subunit vaccines. Summary of the Invention

[0004] The purpose of this invention is to provide a subunit vaccine for Avibacterium avium (type A) in chickens, its preparation method and application, which has low endotoxin, low protein content, and high safety, and is applicable to broilers and laying hens.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a subunit vaccine against Avibacterium avium (type A), characterized in that the subunit vaccine comprises OMA87 protein, which has a size of 97.15 kD, and its gene sequence is shown in SEQ.ID.NO.4, and its amino acid sequence is shown in SEQ.ID.NO.5.

[0007] Furthermore, the OMA87 protein is expressed in prokaryotes using pET28a as the expression vector, IPTG as the prokaryote expression inducer, at a temperature of 37°C, and for 5 hours.

[0008] The present invention also provides a method for preparing the *Animal Rosobacterium avium* (type A) subunit vaccine, comprising the following steps:

[0009] (1) ORT isolates were obtained and serotypes were identified. Primers were obtained as shown in SEQ.ID.NO.1: ATTTTTAGATATTCAAA and SEQ.ID.NO.2: GACGGATAATTAATTC. The gene sequence of the ORT isolates was shown in SEQ.ID.NO.3. The ORT isolates were deposited at the China Center for Type Culture Collection on November 15, 2023. The accession number was CCTCC No.: M20232159.

[0010] (2) Using the genome of the isolate as a template, PCR reaction was performed to amplify the OMA87 gene, and the gene was inserted into the pET28a prokaryotic expression plasmid through gene recombination. The OMA87 protein prokaryotic expression strain was obtained by transforming BL(21) competent cells.

[0011] (3) The OMA87 protein was expressed in the prokaryotic strain of OMA87 protein in step (2), its reactivity was identified by Western blot, and semi-finished antigen with endotoxin less than 2500 EU / mL and protein purity of more than 50% was obtained by ammonium sulfate precipitation and Triton-114 detoxification.

[0012] (4) Mix the semi-finished antigen with Tween-80 as the aqueous phase, and emulsify it with white oil adjuvant to obtain the chicken nasal tracheobacterium (type A) subunit vaccine.

[0013] Furthermore, in step (2), the specific steps for amplifying the OMA87 gene are as follows:

[0014] S1. The obtained ORT isolates were revived, the plates were washed with purified water and the bacterial cells were collected. After boiling for 5 minutes, the plates were centrifuged at 7000 rpm for 5 minutes and the supernatant was separated, which is the DNA template.

[0015] S2. PCR amplification was performed using the PCR amplification primers shown in SEQ.ID.NO.6 and SEQ.ID.NO.7 to obtain the OMA87 gene with the nucleotide sequence shown in SEQ.ID.NO.4 and the amino acid sequence shown in SEQ.ID.NO.5.

[0016] Furthermore, in step S2, the amplification system during the PCR amplification process is as follows:

[0017]

[0018] The amplification procedure is as follows:

[0019]

[0020]

[0021] Furthermore, in step (2), the specific construction method of the OMA87 protein prokaryotic expression strain includes the following steps:

[0022] S1. The PCR reaction product was subjected to agarose gel electrophoresis (1%). The bands were recovered using an agarose gel gene recovery kit. The product was double-digested with endonucleases XhoI and HindIII and incubated at 37°C for 4 hours. The digestion products were recovered. The pET-28a plasmid was also double-digested and recovered in the same way to obtain the OMA87 gene double-digestion recovery product and the pET-28a plasmid double-digestion recovery product.

[0023] S2. Take the two enzyme digestion products mentioned above and ligate them using T4 DNA Ligase. Incubate overnight at 16°C to obtain the ligation product.

[0024] S3. The ligation product was heat-transformed into DH5α competent cells and plated on LA + 100 μg / mL AMP plates, then incubated overnight at 37°C. Single clones were picked and transferred to 5 mL LB + 100 μg / mL AMP medium, and incubated at 37°C and 180 rpm for 8 h. Positive clones were screened by bacterial PCR. The identified positive bacterial cultures were used to extract plasmids, which were then sequenced and verified by double enzyme digestion. The results were correct, and the plasmids were kept for later use.

[0025] S4. The method for preparing the *Animal Rosobacterium avium* (type A) subunit vaccine according to claim 6, characterized in that, in step S2, the linking system is:

[0026]

[0027] Further, in step (3), the ammonium sulfate precipitation and Triton-114 detoxification steps are as follows: After centrifuging the OMA87 protein-induced expression cells, add physiological saline at a ratio of 1:10 (W / V) to resuspend the cells, sonicate them, add 8% (W / V) ammonium sulfate to fully dissolve them, let them stand at 4°C for 4 hours, centrifuge at 4°C and 7000 rpm for 40 minutes, discard the precipitate, add 12% (W / V) ammonium sulfate to the supernatant to fully dissolve it, let it stand at 4°C for 4 hours, centrifuge at 4°C and 7000 rpm for 40 minutes, discard the supernatant, resuspend the precipitate with the original volume of physiological saline, and the resuspended solution is the semi-finished antigen.

[0028] Furthermore, in step (3), Tween80 is added to the semi-finished antigen at 4% (V / V) as the aqueous phase, and the aqueous phase is emulsified with white oil at a ratio of 1:3 (V / V). The formulation of the avian bronchiolitis (type A) subunit vaccine is water-in-oil.

[0029] The present invention also provides the application of the aforementioned Ornithobacter avianus (type A) subunit vaccine in the prevention of Ornithobacter avianus type A.

[0030] The present invention discloses the following technical effects:

[0031] (1) This invention provides a feasible approach for the prevention of avian influenza. A subunit vaccine is formulated by expressing the protein in prokaryotes, adding a white oil adjuvant, and emulsifying it. This vaccine can provide chickens with high and long-lasting ORT antibody levels.

[0032] (2) The present invention uses the ORT conserved membrane protein OMA87, which provides a possibility for cross-protection between different ORT serotypes.

[0033] (3) The subunit vaccine formulated in this invention has low endotoxin content, low protein content, and high safety, and can be applied to broilers and laying hens.

[0034] (4) The prokaryotic expression protein of the present invention can be formulated into a subunit vaccine, which can be easily formulated with other vaccines to form a bivalent or trivalent vaccine, so as to achieve multiple protections with one vaccine. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 SDS-PAGE images of OMA87 protein expression identification, purification, and detoxification; Lane 1: supernatant after centrifugation after disruption; Lane 2: precipitate after centrifugation after disruption; Lane 3: supernatant after purification with ammonium sulfate precipitation; Lane 4: precipitate after ammonium sulfate precipitation; Lane 5: sample after detoxification.

[0037] Figure 2 The image shows the Western blot identification of the reactivity of OMA87 protein; lane 1: negative control with empty vector, lane 2: supernatant after disruption, lane 3: purified by ammonium sulfate precipitation, and lane 4: detoxified sample.

[0038] Figure 3 Figure showing the effect of OMA87 protein subunit vaccine on broiler feed intake;

[0039] Figure 4 Figure showing the effect of OMA87 protein subunit vaccine on broiler weight gain;

[0040] Figure 5 A graph showing the duration of antibody production in broiler chickens immunized with the OMA87 protein subunit vaccine.

[0041] Figure 6 This image shows the duration of antibody production in laying hens immunized with the OMA87 protein subunit vaccine. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] The strains, instruments, and reagents used in the following examples are as follows:

[0045] 1. This invention uses *Animal Bacillus nasolacrimal* strain as the clinical isolate, serotype A;

[0046] 2. The identification serum used in this invention was preserved by Shandong Binzhou Wohua Bioengineering Co., Ltd.;

[0047] 3. The ELISA antibody level detection method used in this invention was established by Shandong Binzhou Wohua Bioengineering Co., Ltd.

[0048] 4. Primer synthesis and gene sequencing for this experiment were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0049] 5. The white oil adjuvant used in the preparation of the vaccine in this invention was purchased from SEPPIC, France;

[0050] 6. In this invention, the broiler chickens used are Merial broilers, and the laying hens are Hy-Line Brown breeder chickens.

[0051] 7. All reagents used in this invention are of analytical grade from Sinopharm.

[0052] 8. The HRP-rabbit anti-chicken IgG and TMB chromogenic solution used in this invention were both purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0053] The following embodiments are for further illustration of the present invention and do not constitute a limitation thereof.

[0054] Example 1: Preparation of DE3 prokaryotic expression strain

[0055] In 2021, the applicant obtained a strain of Ornithobacterium nasalicum from swabs taken from the infraorbital sinus, brain, and oral cavity of broiler chickens from a chicken farm in Jiangsu Province.

[0056] The method for separating, purifying, and identifying ORT is as follows:

[0057] a. Take the head of the diseased chicken and cut open the infraorbital sinus and skull in a clean bench to expose the internal infraorbital sinus and brain tissue;

[0058] b. Wipe the infraorbital sinus, brain, and oral cavity with a sterile cotton swab, then spread the swab onto the isolation culture medium and incubate in a carbon dioxide incubator for 48 hours.

[0059] c. The isolation and purification medium was tryptic soybean agar (TSA), with the following formula: 15.0 g tryptic; 5.0 g soybean papain hydrolysate; 5.0 g sodium chloride; 15.0 g agar; 1000 ml purified water;

[0060] d. Take ORT-like colonies in sterile purified water, boil for 5 min, centrifuge at 7000 rpm for 5 min, and use the supernatant as a template for PCR identification;

[0061] e. After correct identification, use an inoculation loop to pick up the isolated bacteria and streak them in the four zones of a new culture medium plate. Once the isolated bacteria are pure and free of contaminants, preserve the strain.

[0062] The ORT serological identification method is an agarose gel diffusion test, and the specific steps are as follows:

[0063] a. Weigh 1g agarose and 8g NaCl, add 100mL PBS, heat to dissolve, then take 19mL and pour it into a culture dish. After cooling, the agarose gel plate is obtained.

[0064] b. Use a punch to make holes in an agarose gel plate. Add the resuspension of the isolated bacteria to the central well, and add ORTA-type, B-type, C-type, D-type, E-type, and G-type hyperimmune sera to the surrounding 6 wells, respectively. Incubate at 37°C for 48 hours. After the time is up, observe the position of the sedimentation line to confirm that the sera type of the isolated strain is A, and name it ORT Q9 strain. This strain, *Ornithobacterium rhinotracheale* (ORT) Q9, was deposited at the China Center for Type Culture Collection (CCTCC) on November 15, 2023; accession number: CCTCC No.: M20232159. Freeze-dry the ORT Q9 strain for later use. The ORT hyperimmune sera were prepared and preserved by Shandong Binzhou Wohua Biotechnology Co., Ltd.

[0065] ORT-PCR identification primers such as

[0066] SEQ.ID.NO.1:ATTTTTAGATATTCAAA,

[0067] SEQ.ID.NO.2: shown in GAACGGATAATTAATTC;

[0068] The gene sequence is shown in SEQ.ID.NO.3:

[0069] SEQ.ID.NO.3:

[0070]

[0071] The correct purified ORT strain was cultured and identified, a DNA template was prepared, the ORT outer membrane protein OMA87 was amplified, the prokaryotic expression plasmid pET28a-OMA87 was constructed, transformed into BL21(DE3) competent cells, and positive monoclonal strains were screened and preserved. The specific methods are as follows:

[0072] a. DNA template preparation: Resuscitate the preserved bacterial strain, wash the plates with purified water and collect the bacterial cells, boil in water for 5 minutes and centrifuge at 7000 rpm for 5 minutes, and separate the supernatant, which is the DNA template;

[0073] b. Primer selection: The OMA87 gene is shown in SEQ.ID.NO.4.

[0074] SEQ.ID.NO.4:

[0075]

[0076] The translated amino acid sequence is shown in SEQ.ID.NO.5 attached:

[0077] SEQ.ID.NO.5:

[0078] MCSLTKKLNKNEYYLRSNSFKFEGKKAFKSDLEDYVSQKPNARMLGILPLQDWMYNLVPAKFDSTFEAYYSYSRKERNQKLLDSLYIKFGLKEYVGKNNFLYRQFYNWGAEPVILDTTSSYSSARNLKQMFFERGYFEAEVDPTFKIDTAAQKARVTYNIKLNEPSFIKDYNQVITNTDMEDLYKENEDKSAVKVGERFDVRNFELERDRLTRIFKNNGYFNFNEFGEELIFKVDSTNSKELGITMRIAKPKGDSIQNFIKYRWGTIDIFTNNTDSNIKHIREYKGYTLKSNEEFQFKPRVFTDAITITEGDVYSEKAIDETRTLIFDRENFSLTSMVPEKNEKDSLINFKIFLQPKPKYDLQLSFEGMYSQFLNFGISPGLRLLNRNIFRGGENLEFNLKGTVGTVNKAGHENHFFNAYELSFNTEMTFPRWLLPFNTEDLFPKSYNLKSSIGLGLSGQKNIGLGSRSYVAYMDYKFQPGVSEVTIEPLSFQYIRNTEKDKYYRVFTLDNEIREKTFNAFFHYRPHIQKLYDEGKLSEPHLERLIYQDEKFAETLPNAQGKGYNFQDYTDFRNMVFRKRSITQDVFIQPISVAWHYNENKRVDKENPWNIYTRVAVSGAILRLADLILNFEKEDNFFGNKTSLIGGVPYSEYLRFDLDVRKTFNVSEKSAIALRGLFGIAYPYGNSNTIPFSRSYFAGGSNDVRAWKAYELSPAPLRPNDQGTYVDDMKITVNAEYRFPISGIFHGATFVDAGNIWSVKNTNERTSFKINQFYKQLGVGGGFGARFVFPFVVARLDLAYKLHDPAYPEGDRWFKNFNFLKPRIQFGINYPF,

[0079] The PCR amplification primers are:

[0080] SEQ.ID.NO.6:CGCGGATCCATGTGTAGCTTGACCAAAAAAATT;

[0081] SEQ.ID.NO.7:CCGCTCGAGTCAGAACGGATAATTAATTC;

[0082] c. PCR amplification of the target gene: The PCR amplification system is shown in Table 1:

[0083] Table 1 PCR amplification system

[0084]

[0085] The PCR procedure is shown in Table 2:

[0086] Table 2 PCR Procedure

[0087]

[0088]

[0089] d. Double digestion and ligation: PCR products were subjected to agarose gel electrophoresis (1%). The bands were recovered using an agarose gel gene recovery kit and double-digested with restriction endonucleases XhoI and HindIII. The mixture was incubated at 37°C for 4 hours. The digested products were then recovered as PCR products. The pET-28a plasmid was also double-digested and recovered in the same manner. The recovered products from the double digestion of the OMA87 gene and the pET-28a plasmid were then ligated using T4 DNALigase and incubated overnight at 16°C. The ligation system is as follows:

[0090] Table 3 Connection System

[0091]

[0092] e. Transformation: The ligation product was heat-shocked into DH5α competent cells, plated on LA + ampicillin (AMP, 100 μg / mL) plates, and incubated overnight at 37°C. Single colonies were picked and transferred to 5 mL of LB + AMP (100 μg / mL) medium, and incubated at 37°C and 180 rpm for 8 h. Positive colonies were screened by bacterial PCR. Plasmids were extracted from the identified positive bacterial cultures, and sequenced and double-enzyme digested for verification. Plasmids with correct results were kept for later use.

[0093] f. Screening for positive strains. The correctly constructed plasmid was transformed into BL21(DE3) competent cells using a heat shock transformation method, and the culture process was the same as for DH5α competent cells. Positive strains were screened by colony PCR to obtain the pET28a-OMA87-BL21(DE3) prokaryotic expression strain. The positive strains were stored for later use.

[0094] Example 2: Preparation of Subunit Vaccine Semi-finished Product

[0095] 1. Inoculate the pET28a-OMA87-BL21(DE3) prokaryotic expression strain into TB medium (100 μg / mL ampicillin) and incubate overnight at 37°C and 180 rpm. The TB medium is prepared by weighing 11.8 g of tryptone and 23.6 g of yeast extract, dissolving them in 900 mL of deionized water, and adding 5 mL of 80% glycerol (pre-prepared). Autoclave at 115°C for 20 minutes.

[0096] 2. Take the revived bacterial culture and add it to fresh TB medium (100 μg / mL ampicillin) at a ratio of 1:100 (V / V). Incubate until the OD600 value is approximately 0.6-0.8. Add 1 mm of isopropyl thiogalactoside (IPTG) and induce expression at 37℃ and 180 rpm for 5 h. Centrifuge at 4℃ and 10000 rpm for 10 min to obtain bacterial cells, weigh them, and store them at -80℃ for later use.

[0097] 3. Take the bacterial cells stored at -80℃, resuspend them in physiological saline at a ratio of 1:10 (w / v), and sonicate them. Dissolve the cells completely in 8% (w / v) ammonium sulfate, and let them stand at 4℃ for 4 hours. Centrifuge at 4℃ and 7000 rpm for 40 minutes, discard the precipitate, add 12% (w / v) ammonium sulfate to the supernatant, dissolve it completely, let it stand at 4℃ for 4 hours, and centrifuge at 4℃ and 7000 rpm for 40 minutes, discard the supernatant, and resuspend the precipitate in the original volume of physiological saline. The resuspended solution is the protein purification solution.

[0098] 4. Take the protein purification solution, add 1.3% (V / V) Triton-114, place on a shaker, and treat at 4℃ and 90 rpm for 8 h. After warming in a 30℃ water bath, centrifuge at 30℃ and 10000 rpm for 30 min. After the protein purification solution separates into layers, carefully aspirate the upper protein solution, being careful not to aspirate the lower layer solution. Then, treat twice with 1.2% (V / V) Triton-114 following the above steps for detoxification. The endotoxin content in the upper protein solution was determined using a Limulus Amebocyte Lysate (LAL) reagent endotoxin detection kit and found to be below 2500 EU / mL (Table 4).

[0099] Table 4. Results of endotoxin determination in detoxified protein solutions

[0100]

[0101] 5. The reactivity of the detoxified protein from step 4 was identified by Western blot, and the specific method is as follows:

[0102] a. Perform SDS-PAGE electrophoresis on OMA87 protein. After electrophoresis, carefully remove the gel;

[0103] b. Wash twice with distilled water, then transfer to transfer buffer and incubate on a shaker for 20 min;

[0104] c. Cut two 3mm thick filter papers and one PVDF membrane;

[0105] e. Soak the PVDF membrane in anhydrous methanol for 1 min, then change to transfer buffer and incubate on a shaker for 5 min;

[0106] f. Soak the filter paper in the transfer buffer solution. After preparation, assemble the semi-dry electrotransfer apparatus: anode base - filter paper - PVDF membrane - gel - filter paper - cathode cover (from bottom to top), remove air bubbles, and maintain a constant voltage of 24V for 1 hour.

[0107] g. After the transfer is complete, disassemble the electrotransfer instrument, place the PVDF membrane in a clean culture dish containing TBST, and gently shake it on a shaker for 2×5 min.

[0108] h. Add PBST (blocking solution) containing 5% skim milk, incubate on a shaker for 2 hours or overnight at 4°C to block the membrane;

[0109] i. Wash membrane with PBST for 3 × 5 min;

[0110] j. Incubate overnight at 4°C with chicken-derived ORT hyperimmune serum (primary antibody, blocked solution diluted 1:2000);

[0111] wash membrane with k.PBST for 3 × 5 min;

[0112] l. Wash with HRP-rabbit anti-chicken IgG (TBST1:10000 dilution) on a shaker for 1.5-2 hours;

[0113] Wash membrane with m.PBST for 3 × 5 min;

[0114] n. Prepare the colorimetric solution at a 1:1 ratio, expose and photograph the solution using a gel imaging system (Shanghai Tianneng Tanon 4800), and save the image.

[0115] The SDS-PAGE results showed that the OMA87 protein expression size was correct, at 97.15 kDa. Figure 1 After purification by ammonium phosphate precipitation, the protein purity reached over 50%. Figure 1 Furthermore, Western blot analysis confirmed that the OMA87 protein exhibited good reactivity with ORT whole-cell hyperimmune serum. Figure 2 SDS-PAGE results showed no significant protein loss after detoxification. Figure 1 ).

[0116] 6. Take the detoxified protein solution, add 7 / 10000 (V / V) formaldehyde, inactivate at 37℃ for 5-7 days, and pass the sterility test. The OMA87 protein semi-finished product is now ready. Store at 4℃ for later use.

[0117] Example 2: Preparation and morphological detection of subunit vaccine product

[0118] The following steps are all performed under aseptic conditions:

[0119] 1. Dilute the purified and detoxified OMA87 protein to 250 μg / mL with PBS buffer, add 4% (V / V) Tween-80 to prepare the aqueous phase of the vaccine, and use white oil adjuvant as the oil phase. Emulsify thoroughly at a ratio of aqueous phase: oil phase = 1:3 (V / V). Take a drop of the emulsified vaccine and gently drop it onto the water surface. If it does not spread, the emulsification is qualified, and the subunit vaccine product is obtained.

[0120] 2. Physical properties test: The inactivated vaccine is visible to the naked eye as a white emulsion with a uniform texture. The vaccine can be seen floating on the surface of distilled water in a round shape, indicating that the vaccine formulation is water-in-oil.

[0121] 3. Vaccine stability test: Take an appropriate amount of the prepared vaccine and centrifuge it at 3000 rpm / min for 15 min. The results showed that no oil phase or water phase precipitation occurred, so the vaccine was judged to be in good stability.

[0122] 4. Sterility Test: First, an appropriate amount of bacterial seed culture was cultured on a blood agar plate to observe for the growth of contaminating bacteria. Next, appropriate amounts of inactivated bacterial solution, white oil adjuvant, and finished vaccine were inoculated into broth culture medium and cultured at 37℃ with shaking at 120 rpm / min. 5% sheep blood agar plates and LB agar plates were incubated upside down at 37℃. After 3-5 days of incubation, purity tests showed that the bacterial seed culture medium was free of contamination. Sterility tests showed that the broth culture medium was not turbid, and no colonies grew on the sheep blood agar and LB agar plates, proving that both the finished and semi-finished vaccine products were free of bacterial contamination.

[0123] Example 3: Safety and efficacy testing of subunit vaccines in broilers

[0124] 3.1. Vaccine safety testing

[0125] Thirty 40-50 day old Merial broilers were divided into three groups. The first group received a subcutaneous injection of 0.5 mL / bird in the neck, the second group received a 0.5 mL / bird injection in the pectoral muscle, and the third group served as a healthy control group, receiving a subcutaneous injection of sterile saline in the neck. After immunization, the chickens were observed for 8 hours. There was no significant difference in mental state between the immunized and control groups. Within 8 hours after the subcutaneous and pectoral muscle immunizations, no significant stress was observed in any group of chickens, and their mental state was consistent with the control group. Initial feed intake was measured, and feed intake in each group was measured at 2h, 4h, 6h, 8h, 24h, 36h, 48h, and 60h. The results showed that at 24h, the feed intake in the immunized groups returned to the same level as the control group. Figure 3 The initial weight of chickens in each group and their weights at 21 and 28 days were measured. The results showed no significant difference in weight gain between the immunized group and the control group. Figure 4 This indicates that immunization with the vaccine does not affect the growth of chickens. Dissection of 5 chickens in each group at 21 and 28 days post-immunization showed that at 21 days, the subcutaneous 5 / 5 group in the neck and the 5 / 5 group immunized in the pectoral muscle were well absorbed. Dissection at 28 days showed that the subcutaneous 5 / 5 group in the neck and the 5 / 5 group immunized in the pectoral muscle were well absorbed. These results indicate that the formulated vaccine has good safety.

[0126] 3.2. Detection of antibody levels produced by the vaccine

[0127] 3.2.1 Forty 42-day-old Merial broilers were randomly divided into two groups of 20 each, labeled as the immunization group and the healthy control group. Five chickens from each group were randomly selected to collect blood from the wing vein to separate serum for later use. The chickens were then raised in separate isolators according to their respective groups.

[0128] 3.2.2 The immunized group was subcutaneously injected with 0.5 mL of the prepared subunit vaccine in the neck, while the control group was subcutaneously injected with 0.5 mL of physiological saline as a control. Five chickens were randomly selected from each group to collect blood and separate serum for later use. Blood was collected from the immunized group and the control group every week after immunization until the antibody level declined. The serum collected weekly was used to determine the antibody level and antibody duration according to the ELISA antibody detection method in the invention content 4. The results showed that the antibody level reached a high level 21 days after immunization, which was significantly different from that of the healthy control group (Table 5). Therefore, the subunit vaccine can induce the body to produce a high antibody level. As shown in Figure (5), the antibody level of the immunized broilers gradually increased, and the antibody level reached the highest value in the 4th week after immunization. The OD value in the 24th week was 0.5 mL. 450 The value still reached 0.58, higher than the positive cutoff of 0.35. These results indicate that the immunity duration of this subunit vaccine can reach 24 weeks.

[0129] Table 5. Detection of antibody levels in immunized broilers.

[0130]

[0131] There were no significant differences in the superscripts of the same letters in different columns of the table (P > 0.05), but significant differences were found in the superscripts of different letters (P < 0.05).

[0132] Example 4: Effect of subunit vaccine on egg production rate in laying hens

[0133] 4.1 To evaluate the effects of the OMA87 subunit vaccine on the egg production rate and antibody titer of parent layer breeder hens, 360 stable 55-week-old layer breeder hens were selected from a Hy-Line Brown layer breeder farm in Shandong Province and divided into 3 groups with 4 replicates per group and 30 hens per replicate. The experimental groups were injected subcutaneously into the neck with 0.5 mL / hen of the OMA87 subunit vaccine. A 0.5 mL saline group and a non-immunized blank control group were also included. The egg production rate of each group was recorded before immunization and 1-4 weeks after immunization. Blood samples were collected before immunization and at 3, 6, 10, and 14 weeks after immunization to detect antibody levels.

[0134] 4.2 Results showed that two weeks after immunization, the egg production rate in the immunized group decreased slightly, but the difference was not significant compared with the control group (P>0.05) (Table 6). Three weeks after immunization, the antibody level in the immunized group was significantly higher than before immunization. Figure 6 The antibody levels in the immunized group remained high 14 weeks post-immunization. These results indicate that the OMA87 subunit vaccine has minimal impact on egg production performance in laying hens, and induces high antibody levels and a long duration of antibody persistence. This trial provides a reliable option for preventing ORT infection during the egg-laying period in chicken farms.

[0135] Table 6. Effects of OMA87 protein subunit vaccine on egg production rate in laying hens.

[0136]

[0137] The results showed that immunization with the OMA87 protein subunit vaccine resulted in high antibody levels against the whole ORT bacteria in both broilers and laying hens, with no stress response, no impact on broiler weight gain or diet, and an acceptable impact on laying hen egg production. Therefore, this vaccine is suitable for widespread use in production to prevent ORT infection.

[0138] Of course, the above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments and drawings are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. This invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A subunit vaccine of Avibacterium subsp. columnaris type A of chickens, characterized in that, The subunit vaccine comprises OMA87 protein with a size of 97.15 kD, a gene sequence as shown in SEQ ID NO. 4, and an amino acid sequence as shown in SEQ ID NO. 5; The OMA87 protein is expressed in a prokaryotic expression manner, the expression vector is pET28a, the prokaryotic expression inducer is IPTG, the temperature is 37 DEG C, and the induction time is 5 hours; The chicken avibacterium paragallinarum type A subunit vaccine is prepared through the following steps: (1) An ORT isolate is obtained, and a serotype identification is performed, so that the obtained ORT isolate contains a gene sequence as shown in SEQ ID NO. 3, and a primer sequence for amplifying the gene sequence as shown in SEQ ID NO. 3 is as shown in SEQ ID NO. 1 and SEQ ID NO. 2; the ORT isolate is preserved in the China Center for Type Culture Collection on November 15, 2023, and the preservation number is CCTCC No: M20232159; (2) The genome of the ORT isolate is used as a template to perform a PCR reaction, and an OMA87 gene is amplified, and the gene is inserted into a pET28a prokaryotic expression plasmid through gene recombination, and BL21 competent cells are transformed to obtain an OMA87 protein prokaryotic expression strain; (3) The OMA87 protein prokaryotic expression strain obtained in step (2) is used to express the OMA87 protein, western blot is used to identify the reaction originality of the protein, and ammonium sulfate precipitation and Triton-114 detoxification are combined to obtain semi-finished antigen with an endotoxin content less than 2500 EU / mL and a protein purity of more than 50%; (4) The semi-finished antigen is mixed with Tween-80 as an aqueous phase, and then emulsified by mixing with white oil adjuvant, so that a chicken avibacterium paragallinarum type A subunit vaccine is obtained; In step (2), the specific steps for amplifying the OMA87 gene are as follows: S1, the obtained ORT isolate is recovered, the plate is washed with purified water and the bacterial body is collected, boiled water is boiled for 5 min, and then centrifuged at 7000 rpm for 5 min, and the supernatant is separated, which is the DNA template; S2, PCR amplification is performed by using PCR amplification primers with sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 7, so that an OMA87 gene with a nucleotide sequence as shown in SEQ ID NO. 4 and an amino acid sequence as shown in SEQ ID NO. 5 is obtained.

2. The process for preparing the Avibacterium subunit vaccine of type A of chicken nasal trachea according to claim 1, characterized in that, The following steps are included: (1) An ORT isolate is obtained, and a serotype identification is performed, so that the obtained ORT isolate contains a gene sequence as shown in SEQ ID NO. 3, and a primer sequence for amplifying the gene sequence as shown in SEQ ID NO. 3 is as shown in SEQ ID NO. 1 and SEQ ID NO. 2; the ORT isolate is preserved in the China Center for Type Culture Collection on November 15, 2023, and the preservation number is CCTCC No: M20232159; (2) Taking the genome of the ORT strain as a template, a PCR reaction is performed to amplify the OMA87 gene, and the gene is inserted into a pET28a prokaryotic expression plasmid through gene recombination, and BL21 competent cells are transformed to obtain an OMA87 protein prokaryotic expression strain; (3) The OMA87 protein prokaryotic expression strain obtained in step (2) is used to express the OMA87 protein, western blot is used to identify the reactogenicity of the protein, and ammonium sulfate precipitation combined with Triton-114 detoxification is used to obtain a semi-finished antigen with an endotoxin content of less than 2500 EU / mL and a protein purity of more than 50%; (4) The semi-finished antigen is mixed with Tween-80 as an aqueous phase, and then emulsified with white oil adjuvant to obtain a chicken nasal airway Ornithobacterium type A subunit vaccine.

3. The process for the preparation of Avibacterium subunit vaccine type A of chicken nasal trachea according to claim 2, characterized by that, In step (2), the specific steps for amplifying the OMA87 gene are as follows: S1, the obtained ORT strain is resuscitated, the plate is washed with purified water and the bacterial body is collected, boiled water is boiled for 5 min, and then centrifuged at 7000 rpm for 5 min, and the supernatant is separated, which is the DNA template; S2, PCR amplification is performed using PCR amplification primers with sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 7 to obtain an OMA87 gene with a nucleotide sequence as shown in SEQ ID NO. 4 and an amino acid sequence as shown in SEQ ID NO.

5.

4. The process for the preparation of Avibacterium subunit vaccine type A of chicken nasal trachea according to claim 3, characterized by, In step S2, the amplification system of the PCR amplification process is as follows: ； The amplification program is as follows: 。 5. The process for the preparation of Avibacterium subunit vaccine type A of chicken nasal trachea according to claim 2, characterized by, In step (2), the specific construction method of the OMA87 protein prokaryotic expression strain includes the following steps: S1, the product of the PCR reaction is subjected to agarose gel electrophoresis at a concentration of 1%, the target band is recovered using an agarose gel gene recovery kit, endonuclease XhoI and HindIII are used to double-enzyme cut the recovered product, which is placed at 37°C for 4 h, and the enzyme cutting product is recovered; the pET-28a plasmid is subjected to the same double-enzyme cutting and recovery operation to obtain OMA87 gene double-enzyme cutting recovery product and pET-28a plasmid double-enzyme cutting recovery product; S2, the two enzyme cutting recovery products are taken and connected using T4 DNA Ligase, 16°C overnight, to obtain a connection product; S3, the connection product is transformed into DH5α competent cells by heat shock, and is coated on an LA plate containing 100 μg / mL ampicillin and cultured at 37°C overnight; a single colony obtained by culture is inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin, and is cultured at 37°C and 180 rpm for 8 h; positive single colonies are selected by bacterial liquid PCR, and the plasmid of the identified positive bacterial liquid is extracted, sequenced and double-enzyme cut verified, and the plasmid is reserved after the verification result is correct.

6. The process for the preparation of Avibacterium subunit vaccine type A of chicken nasal trachea according to claim 5, characterized by that, In step S2, the connection system is as follows: 。 7. The process for the preparation of Avibacterium subunit vaccine type A of chicken nasal trachea according to claim 2, characterized by that, In step (3), the step of ammonium sulfate precipitation combined with Triton-114 detoxification is as follows: the bacteria collected by centrifugation after OMA87 protein induction expression is resuspended with physiological saline at a mass-volume ratio of 1:10, and then broken by using an ultrasonic disrupter; 8% ammonium sulfate is added to the broken solution at a mass-volume ratio, and then the solution is fully dissolved and placed at 4°C for 4 hours; the precipitate is discarded by using a high-speed centrifuge at 4°C, 7000 rpm for 40 min; 12% ammonium sulfate is added to the supernatant at a mass-volume ratio, and then the solution is fully dissolved and placed at 4°C for 4 hours; the supernatant is discarded by using a high-speed centrifuge at 4°C, 7000 rpm for 40 min; the precipitate is resuspended with physiological saline at the original volume to obtain a protein purification solution; 1.3% Triton-114 is added to the protein purification solution at a volume ratio, and then the solution is treated on a 4°C, 90 rpm shaker for 8 hours; after being warmed in a 30°C water bath, the solution is centrifuged at 30°C, 10,000 rpm for 30 min, and then the upper protein solution is collected; the above steps are repeated twice using 1.2% Triton-114 at a volume ratio, and then the treated protein solution obtained is the semi-finished antigen.

8. The process for the preparation of Avibacterium subunit vaccine type A of chicken nasal trachea according to claim 2, characterized by, In step (4), 4% Tween 80 is added to the semi-finished antigen as an aqueous phase, and then the aqueous phase is mixed and emulsified with white oil adjuvant at a volume ratio of 1:3, and the dosage form of the Avibacterium A type subunit vaccine is water-in-oil.

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