Application of avian multi-killer pasteurella lpxM gene deletion strain in preparation of avian cholera vaccine

Abstract

This invention discloses avian Pasteurella multocida. lpxM A gene-deleted strain was used in the preparation of fowl cholera vaccine. This strain, with accession number CCTCC NO: 2026575, was named PD021Δ. lpxM This strain can produce MVs with low LPS content, and these MVs have good immunogenicity. Using nitrogen cavitation technology, the gene-deleted strain PD021Δ can be produced. lpxM Large-scale preparation of MVs. The prepared MVs can be used to develop fowl cholera subunit vaccines or immune enhancers, showing ideal effects in immunization and effectively resisting infection by Pasteurella multocida. This invention utilizes the gene-deleted strain PD021Δ lpxM The low-load MVs produced by LPS can be used to prepare subunit vaccines, which have immunoprotective effects against infection by different serotypes of Pasteurella multocida strains in birds and have broad application prospects in the market.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to *Pasteurella multocida*. lpxM Gene-deleted strains are used in the preparation of fowl cholera vaccines. Background Technology

[0002] Jiangsu Province, as a core breeding area for Hy-Line Brown laying hens in my country, faces significant challenges in preventing and controlling bacterial diseases due to the long laying cycle. Among these challenges is the prevalence of Pasteurella multocida (Pasteurella multocida). Pasteurella multocida Folverine cholera (FCC) caused by antibiotics (Pm) is a major infectious disease affecting laying hens, easily leading to increased mortality and a sharp drop in egg production, causing serious economic losses. Under the current environment of "banning or restricting antibiotics," the traditional antibiotic-dependent prevention and control model is unsustainable. Developing safe and efficient immunization methods has become an urgent need to ensure the safe production of poultry farming.

[0003] Currently, commercially available fowl cholera vaccines are mainly whole-cell inactivated vaccines and live attenuated vaccines, but both have significant drawbacks: inactivated vaccines have limited cross-protection against different serotypes of Pm strains, making them difficult to cope with the spread of complex serotypes in the field; while live attenuated vaccines can induce cellular immunity, there is a risk of virulence reversion, and safety concerns limit their application. Subunit vaccines (especially multi-epitope vaccines) are considered an important research direction due to their potential broad-spectrum cross-protection advantage, but how to efficiently present multi-epitope antigens and elicit a strong immune response remains a technical challenge.

[0004] Recent studies have found that nanoscale membrane vesicles (MVs) naturally released by bacteria during their growth process possess multiple advantages for vaccine development: (1) they naturally carry multiple pathogenic epitopes, which can stimulate a broad-spectrum immune response; (2) they have self-adjuvant activity, which can efficiently activate the host's innate immunity; and (3) they have no replication ability, making them significantly safer than live vaccines. These characteristics make MVs a highly promising new vaccine vector, especially suitable for the control of multi-serotype pathogens such as Pasteurella multocida. However, the high lipopolysaccharide (LPS) content in natural bacterial MVs may cause excessive inflammatory responses, limiting their safety as direct vaccines.

[0005] Therefore, developing a novel vaccine that can retain the advantages of natural multi-antigenic epitopes of MVs while reducing their LPS toxicity to enhance safety has important application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides avian Pasteurella multocida. lpxM Gene-deleted strains are used in the preparation of fowl cholera vaccines. lpxMThe gene results in a relatively low production of lipopolysaccharide (LPS) in Pasteurella multocida. The membrane vesicles (MVs) produced by the gene-deleted strain have a low LPS content, and therefore the membrane vesicles prepared from this deletion strain have good immunogenicity.

[0007] This invention is achieved through the following technical solution:

[0008] avian Pasteurella multocida lpxM The gene-deleted strain is used in the preparation of fowl cholera vaccine. The preservation number of the gene-deleted strain is CCTCC NO: 2026575.

[0009] Preferably, the vaccine is based on the avian Pasteurella multocida. lpxM Subunit vaccines, adjuvants, or immune enhancers prepared from lipopolysaccharide-laden membrane vesicles produced by gene-deleted strains.

[0010] Preferably, the vaccine is a broad-spectrum subunit vaccine.

[0011] Preferably, the subunit vaccine contains Pasteurella multocida. lpxM The effective concentration of membrane vesicles from the gene-deleted strain was 5.0 × 10⁻⁶. 6 ~1.0×10 7 CFU / 0.1 mL.

[0012] A method for preparing a fowl cholera subunit vaccine includes the following steps:

[0013] Step 1) Preparation of bacterial solution: Add avian Pasteurella multocida to the bacterial solution. lpxM Gene-deleted strains were streaked on BHI solid medium, and single colonies were picked and inoculated into BHI liquid medium until the bacterial count reached 1.0 × 10⁻⁶. 9 CFU / mL; the preservation number of the gene-deleted strain is CCTCC NO: 2026575;

[0014] Step 2) Prepare avian Pasteurella multocida using nitrogen cavitation technology, ultracentrifugation, and density gradient centrifugation. lpxM Membrane vesicles of gene-deleted strains;

[0015] Step 3) Mix the membrane vesicles obtained in Step 2) with ISA 201 VG adjuvant at a volume ratio of 1:1 to make the final concentration of membrane vesicle protein 2 mg / mL, and store at -20℃ to obtain the final product.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) This invention targets the missing parts lpxMGenetic modification of Pasteurella multocida in birds significantly reduces the content of lipopolysaccharide (LPS) in bacterial cells and membrane vesicles (MVs), weakens endotoxin toxicity, eliminates the risk of virulence reversion from the source, overcomes the safety hazards of traditional attenuated live vaccines, and provides a stable engineered strain basis for the preparation of highly safe subunit vaccines.

[0018] (2) The MVs vaccine prepared based on the modified strain of the present invention has multiple immune advantages: ① Natural enrichment of multiple antigenic epitopes: It retains cross-protective antigens such as Pasteurella outer membrane proteins and lipoproteins, overcoming the defect of insufficient protection against heterologous serotypes by inactivated vaccines; ② Self-adjuvant effect: The nanoscale structure of MVs can efficiently activate dendritic cells and T / B cells immune responses, inducing the production of high-level neutralizing antibodies and Th1-type cell immunity; ③ Verified by animal experiments: It provides significant cross-protection against heterologous strains of fowl cholera, effectively reducing morbidity and mortality, and achieving the broad-spectrum prevention and control goal of "one vaccine against multiple types".

[0019] (3) The present invention uses nitrogen cavitation method to prepare MVs on a large scale. The process is stable and low cost. The yield per batch is significantly improved compared with the traditional ultracentrifugation method, which meets the needs of commercial production. The obtained MVs have uniform particle size and intact membrane, which meets the standards of nano-vaccine preparation. In the trial application in large-scale egg-laying chicken farms in Jiangsu Province, the challenge test results showed that compared with the negative control group with a mortality rate of 100%, the immunized group maintained a 100% survival rate throughout the observation period, and the cross-immune protection was higher than 60%, which proved its practical application value.

[0020] (4) This invention uses MVs produced by the lpxM gene deletion strain of Pasteurella multocida to prepare a broad-spectrum subunit vaccine, which has an immune protective effect against infection by different serotypes of Pasteurella multocida strains. Its application can significantly reduce the use of antibiotics in the breeding process, provide a safe, broad-spectrum and efficient original solution for the prevention and control of bacterial diseases in poultry, and help the poultry breeding industry to transform towards green and sustainable development, which has significant social and economic value. Attached Figure Description

[0021] Figure 1 Pasteurella multocida in Example 2 lpxM Gene-deleted strain PD021Δ lpxM Transmission electron microscopy (TEM) observation of the prepared membrane vesicles (MVs);

[0022] Figure 2 For the nanoparticle tracking analysis (NTA) measurement and detection of gene-deleted strain PD021Δ in Example 2 lpxM The average particle size and concentration of bacterial membrane vesicles (MVs) produced by natural culture;

[0023] Figure 3The gene-deleted strain PD021Δ prepared by nitrogen cavitation was observed by transmission electron microscopy in Example 3. lpxM MVs;

[0024] Figure 4 For the preparation of the gene-deleted strain PD021Δ by nanoparticle tracking analysis (NTA) in Example 3 to detect nitrogen cavitation lpxM The average particle size and concentration of MVs;

[0025] Figure 5 For the comparative detection of gene-deleted strain PD021Δ in Example 4 lpxM Differences in lipopolysaccharide (LPS) between wild-type PD021 and other strains;

[0026] Figure 6 The gene-deleted strain PD021Δ in Example 4 lpxM Comparison of LPS content in MVs produced by wild-type PD021;

[0027] Figure 7 The gene-deleted strain PD021Δ in Example 6 lpxM Detection of antibody levels after immunization with MVs subunit vaccine. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Weak LPS expression in dominant serotype strains of Pasteurella multocida in avian birds lpxM Construction of gene deletion strains

[0032] A dominant and prevalent serotype A:1 strain of Pasteurella multocida (wild-type strain PD021, which was isolated and preserved from diseased laying hens in a chicken farm in Jiangsu Province in July 2021 by our laboratory, but not preserved) was selected to construct a strain with weak LPS expression.

[0033] Lipopolysaccharide (LPS) is a key virulence factor in Gram-negative bacteria, and its modifying genes lpxM Encodes lipid A acyltransferase, involved in regulating the immunogenicity and toxicity of LPS. (Deficit) lpxM This can significantly weaken bacterial virulence, reduce host inflammatory damage, and potentially optimize the immune recognition properties of membrane vesicles (MVs). Based on this, the following construction scheme was designed:

[0034] First, competent cells were prepared from candidate strains, and genes targeting LPS formation were designed. lpxM Homologous recombination primers were used. The constructed homologous recombination thermosensitive plasmid was then transformed into competent Pasteurella multocida cells for gene knockout. The recombinant thermosensitive plasmid was added to the competent Pasteurella multocida cells and transferred to the bottom of a pre-cooled electroporation cuvette. The electroporation parameters were set as follows: 2.5 KV / 2cm, resistance 500Ω, capacitance 25 μF. After electroporation, 1.0 mL of antibiotic-free BHI liquid medium was added, mixed well, and transferred to an EP tube. The tube was placed in a bacterial shaking incubator and cultured at 30℃ and 180 rpm for 2 h. After rejuvenation, the cells were centrifuged at 4000 rpm for 5 min, 200 μL of supernatant was reserved, and the rest was discarded. The cells were resuspended and distributed on BHI solid medium containing kanamycin and cultured statically at 37℃ for 24–36 h. Single colonies were picked and cultured in BHI medium containing kanamycin at 30℃ and 180 rpm for 16 hours. h, incubate on ice, and record as one generation; then after each generation, PCR is used to identify gene deletion and recombination. After the appearance of exchange bands, the culture with gene exchange is transferred 1:100 to antibiotic-free BHI liquid medium and cultured at 37℃ and 180 rpm for 12 h as one generation. This is repeated for 3-5 generations to eliminate plasmids. Then, pick the culture onto media with and without kanamycin resistance, and after overnight culture, pick single colonies that grow on antibiotic-free plates but not on kanamycin-resistant plates. Verify again with identification primers, perform PCR identification, and sequence alignment to finally obtain the LPS weakly expressing strain, which is *Pasteurella multocida* avianis. lpxM The gene-deleted strain is designated PD021Δ lpxM .

[0035] Using the above method, multiple independent construction experiments were successfully conducted and obtained. lpxM One of the gene-deleted strains was deposited on April 2, 2026, at the China Center for Type Culture Collection (CCTCC), address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: 2026575, and classified as Pasteurella multocida PD021. Pasteurella multocida PD021).

[0036] Example 2: Avian Pasteurella multocida lpxM Extraction and nanoparticle tracking analysis (NTA) of naturally occurring MVs from gene-deleted strains

[0037] The gene-deleted strain PD021Δ was analyzed using ultracentrifugation and iodixanol density gradient centrifugation. lpxM Bacterial membrane vesicles (MVs) were extracted.

[0038] 1. Experimental Procedure

[0039] The gene-deleted strain PD021Δ was cultured overnight. lpxM Inoculated into 500 mL BHI broth medium and cultured at 37°C and 180 rpm until the mid-to-late logarithmic growth phase (OD2). 600 After centrifugation at 4°C and 8000×g for 10 min (≈1.0), the supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization. The filtrate was ultracentrifuged at 4°C and 200,000×g for 2 h. The precipitate was resuspended in 5 mL of pre-cooled 1×TE buffer (pH=7.4) and spread on the upper layer of a discontinuous iodixanol gradient (10%, 20%, 40% w / v). The precipitate was centrifuged at 4°C and 200,000×g for 2 h. The milky white band at the upper interface was collected, diluted with 10 times the volume of TE buffer, and centrifuged again at 4°C and 200,000×g for 2 h. The precipitate was resuspended in 5 mL of TE buffer and filtered twice through a 0.22 μm filter membrane. Then, it was concentrated to approximately 500 μL using a 100 kDa ultrafiltration tube, aliquoted, and stored at 4°C. 100 μL of the precipitate was taken. μL of sample was diluted 100-fold with PBS, and the particle size distribution and concentration of MVs were determined using an NTA300 system (Malvern). The detection conditions were: laser wavelength 488 nm, temperature 25℃. Each sample was analyzed three times, 60 s each time.

[0040] 2. Experimental Results

[0041] like Figure 1 As shown, transmission electron microscopy revealed the gene deletion strain PD021Δ lpxM Naturally grown MVs are approximately circular in shape.

[0042] like Figure 2 As shown, the concentration of MVs determined by nanoparticle tracking analysis (NTA) was approximately 1.61 × 10⁻⁶. 12 Particles / mL, gene-deleted strain PD021Δ lpxM The average particle size of naturally grown MVs is approximately 115 nm.

[0043] Example 3: Preparation of Avian Pasteurella multocida by Nitrogen Cavitation lpxM Detection of gene-deleted strains MVs and nanoparticle tracking analysis (NTA)

[0044] 1. Experimental Procedure

[0045] The gene-deleted strain PD021Δ was cultured overnight. lpxMTransferred to 50 mL centrifuge tubes and cultured at 37°C and 180 rpm for 12 h with shaking. Collected bacterial cells were then centrifuged at 4°C and 8000×g for 10 min. The supernatant was discarded, and the precipitate was resuspended in 15 mL of pre-chilled PBS and placed on ice. A nitrogen cavitation device (Parr Instruments) was pre-chilled to 4°C, connected to a nitrogen tank, and placed on a magnetic stirrer with a micro-magnetic stir bar added. The bacterial suspension was transferred to the device, the stirrer was turned on, and the pressure was gradually increased to 1500 PSI (approximately 10.3 MPa). After closing the valve, the device was incubated on ice for 25 min to allow the nitrogen to fully dissolve and equilibrate. The valve was wiped with sterile gauze, and the pressure was slowly released to collect the lysis buffer in pre-chilled centrifuge tubes. After sterilization via a 0.22 μm filter, the MVs were separated using ultracentrifugation (4°C, 200,000×g, 2 h) and iodixanol density gradient purification steps as described in Example 2. The final precipitate was resuspended in 500 μL of PBS and stored at 4°C. 100 μL of sample was diluted 100 times with PBS, and the particle size distribution and concentration of MVs were determined using an NTA300 system (Malvern). The detection parameters were set as follows: laser wavelength 488 nm, temperature 25℃. Each sample was detected 3 times, with each detection time being 60 s.

[0046] 2. Experimental Results

[0047] like Figure 3 As shown, transmission electron microscopy revealed that the gene-deleted strain PD021Δ prepared by nitrogen cavitation was... lpxM The MVs are approximately circular in shape.

[0048] like Figure 4 As shown, the NTA results indicate that the concentration of MVs is approximately 3.77 × 10⁻⁶. 13 Particles / mL, gene-deleted strain PD021Δ lpxM The average particle size of the MVs is approximately 124 nm.

[0049] Example 4: Wild-type strain of Pasteurella multocida PD021 and Pasteurella multocida lpxM Determination of LPS content in membrane vesicles (MVs) produced by gene-deleted strains and two strains

[0050] 1. Experimental Procedure

[0051] Cultivate to OD 600The bacterial culture with a pH of 1.0 was centrifuged at 8,000 × g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 500 μL of reagent A (TE buffer, pH=8.0), centrifuged again, and the supernatant was discarded. The precipitate was resuspended in 500 μL of reagent B (Proteinase K buffer), sonicated at 39 kHz for 15 min (ice-water bath), and centrifuged at 10,000 × g for 10 min to collect the supernatant. 500 μL of reagent C (Tris-saturated phenol, pH=6.8) was added to the supernatant, vortexed for 30 s, and incubated in a 68°C water bath for 30 min, inverting the mixture every 10 min during incubation. The mixture was then incubated overnight at 4°C. After centrifugation at 3,000 × g for 30 min, the upper aqueous phase was collected as the crude LPS extract. For MVs samples, LPS was extracted directly after resuspending in reagent B using the same procedure.

[0052] LPS was quantified using the PyroGene™ Recombinant Factor C Endotoxin Detection Kit: 100 μL of endotoxin standard (0.005~5 EU / mL gradient concentration), sample, and negative control were added to an ELISA plate. After pre-incubation at 37℃ for 10 min, 100 μL of working solution (containing recombinant factor C, fluorescent substrate, and reaction buffer) was added. After reacting in the dark for 25 min, the absorbance at 450 nm (excitation wavelength 380 nm) was measured using an ELISA reader. The LPS content was calculated using a standard curve and expressed in International Units (IU / mL).

[0053] 2. Experimental Results

[0054] like Figure 5 As shown, the test results indicate that the gene-deleted strain PD021Δ lpxM The LPS content in the medium was significantly lower than that in the wild-type PD021.

[0055] like Figure 6 As shown, the gene-deleted strain PD021Δ lpxM The LPS content in the generated MVs was also significantly lower than that in the MVs generated by the wild strain PD021.

[0056] Example 5: Avian Pasteurella multocida lpxM Preparation of subunit vaccines and safety evaluation of gene-deleted strain membrane vesicles (MVs)

[0057] 1. Preparation method of subunit vaccine

[0058] Gene-deleted strain PD021Δ was prepared by natural secretion (refer to Example 2) and nitrogen cavitation method (refer to Example 3). lpxM The MVs were inoculated onto BHI agar plates and incubated at 37°C for 48 h. No colonies were observed, confirming that the MVs formulation did not contain live bacteria. Subunit vaccines were then prepared based on this.

[0059] The concentration of MVs protein was determined using the BCA protein quantification kit (Thermo Scientific). The purified MVs were emulsified with ISA 201 VG adjuvant at a volume ratio of 1:1, and the final concentration of MVs protein was adjusted to 2 mg / mL. After aliquoting, the mixture was stored at -20℃ for later use.

[0060] 2. Safety evaluation

[0061] Sixty 15-week-old laying hens were randomly divided into four groups of 15 each. Before the experiment, the chicks were tested for fowl cholera to exclude individuals infected with Pasteurella multocida, and the hens that tested negative were selected as the experimental samples.

[0062] Among them, the first and second groups of laying hens were injected intramuscularly with 0.1 mL of MVs subunit vaccine with a protein concentration of 200 μg in the leg, which was derived from natural production and nitrogen cavitation preparation, respectively.

[0063] The third group received an intramuscular injection of 0.1 mL containing 1.0 × 10⁻⁶ ppm. 7 CFU viable count of wild-type PD021 strain;

[0064] The fourth group used PBS as a negative control.

[0065] 3. Experimental Results

[0066] Post-vaccination monitoring showed that no hens in the MVs vaccine group and the PBS control group died throughout the entire process, maintained good mental condition, normal appetite, and no abnormal pathological changes in organs, and Pasteurella antibodies could be detected in the serum; while hens inoculated with wild strains showed symptoms such as loss of appetite, lethargy, and rough back feathers from the first day of vaccination, and all died within 7 days, with necropsy showing typical Pasteurella infection lesions.

[0067] Example 6: Avian Pasteurella multocida lpxM Determination of antibody levels in serum of laying hens induced by gene-deleted strain MVs subunit vaccine

[0068] 1. Experimental Procedure

[0069] (1) Immunization program for laying hens

[0070] Sixty 15-week-old laying hens were divided into three groups and immunized with naturally produced (see Example 2), nitrogen-cavitation (see Example 3) MVs subunit vaccine and PBS (control group), respectively. After a 14-day interval, a second immunization was performed. One week after the second immunization, blood was collected from the laying hens, the serum was separated and stored at -20°C.

[0071] (2) To determine the antibody level in the serum of laying hens after secondary immunization, wild-type strain PD021 protein was selected as the detection antigen for Pasteurella multocida and detected by ELISA. The specific steps are as follows:

[0072] Wild-type strain PD021 was inoculated into BHI medium and cultured to the logarithmic growth phase. The bacterial cells were collected by centrifugation and washed with PBS. Subsequently, the bacterial cells were lysed using lysis buffer, and whole-cell protein was collected and its concentration determined using the BCA method. 0.5 μg of protein per well was coated onto an ELISA plate as an antigen at 4°C overnight. After coating, the plate was washed three times with 1×PBST, ensuring the plate was fully filled with washing buffer and then vigorously patted dry. Each wash lasted 3–5 min. Next, blocking buffer was added at 37°C for 2 h. After blocking, the plate was washed three more times with 1×PBST, each time for 3–5 min. Chicken serum samples were diluted, and 100 μL was added to each well, along with a negative control well. The plate was incubated at 37°C for 2 h before washing. HRP-labeled anti-chicken IgG diluted 1:5000 was added to each well as a secondary antibody, and the plate was incubated at 37°C for 1 h before washing. TMB chromogenic reagent was added to each well, and the reaction was incubated at room temperature in the dark for 15 min. The reaction was then terminated by adding 2 mol / L H₂SO₄. Finally, the OD value at 450 nm was read using a microplate reader to assess the antibody level in chicken serum.

[0073] 2. Experimental Results

[0074] like Figure 7 As shown, one week after the second immunization, the MVs subunit vaccines prepared by two different methods were tested. The results showed that both MVs subunit vaccines exhibited high antibody levels, and the antibody titer of the MVs subunit vaccine prepared by the nitrogen cavitation method was slightly higher than that of the subunit vaccine prepared by naturally cultured MVs.

[0075] Example 7: Avian Pasteurella multocida lpxM Immunoprotective efficacy of gene-deleted strain MVs subunit vaccine in laying hens

[0076] 1. Experimental Procedure

[0077] Ten days after the second immunization (see Example 6 for details), a challenge experiment was conducted on laying hens. Each hen was inoculated with the wild-type PD021 strain (5.0 × 10⁻⁶) via intramuscular injection. 8 (CFU / hen). 24 hours after challenge, 5 laying hens were randomly selected from each group, and the colonization level of PD021 in their lungs was determined using quantitative detection technology. The survival status of the laying hens in each group was closely observed and recorded in detail for the next 7 consecutive days as a key indicator for evaluating the vaccine's protective effect.

[0078] To further investigate the gene-deleted strain PD021Δ lpxM To assess the cross-protective ability of MVs subunit vaccines against different serotypes of Pasteurella multocida infection, subsequent challenge tests were conducted using strains of different serotypes, including PD075 (A:3), PD-NJ012 (A:4), and PD-NJ033 (F:3).

[0079] 2. Experimental Results

[0080] The results of the challenge test showed that, compared with the negative control group with a mortality rate of 100%, PD021Δ prepared by both methods had a significantly higher mortality rate. lpxM Laying hens immunized with the MVs subunit vaccine maintained a 100% survival rate throughout the observation period, while the colonization of PD021 in the lungs of chicks was significantly reduced.

[0081] Table 1. Protection rates against viral infection by different serotypes

[0082]

[0083] The results of challenge tests on different serotype strains are shown in Table 1. The PD021Δ strains prepared by the two methods... lpxM The cross-immunity of MVs subunit vaccines against pre-laying hens is higher than 60%.

[0084] The above experimental results indicate that *Pasteurella multocida* in birds... lpxM The gene-deleted strain MVs subunit vaccine has good immunogenicity and provides high immunoprotective efficacy and cross-immune protection for laying hens after vaccination.

[0085] In summary, through systematic verification via secondary immunization and challenge experiments, the gene-knockout strain PD021Δ of Pasteurella multocida, constructed with a gene deletion, has proven effective. lpxM The prepared subunit vaccine demonstrated significant immunoprotective efficacy in a laying hen model. Compared to the negative control group with a mortality rate as high as 100%, the vaccine-immunized laying hens not only achieved a 100% survival rate but also effectively inhibited the colonization of the wild-type PD021 strain in the lungs. Furthermore, challenge experiments against different serotypes further validated its potential cross-protective ability, indicating that the gene-deleted strain PD021Δ... lpxM It can serve as a high-quality candidate material for the development of highly effective subunit vaccines.

[0086] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. Pasteurella multocida in avian birds lpxM The application of gene-deleted strains in the preparation of fowl cholera vaccine is characterized by, The preservation number of the gene-deleted strain is CCTCC NO: 2026575.

2. The application according to claim 1, characterized in that, The vaccine is based on Pasteurella multocida. lpxM Subunit vaccines, adjuvants, or immune enhancers prepared from lipopolysaccharide-laden membrane vesicles produced by gene-deleted strains.

3. The application according to claim 2, characterized in that, The vaccine in question is a broad-spectrum subunit vaccine.

4. The application according to claim 2, characterized in that, The subunit vaccine contains Pasteurella multocida. lpxM The effective concentration of membrane vesicles from the gene-deleted strain was 5.0 × 10⁻⁶. 6 ~1.0×10 7 CFU / 0.1 mL.

5. A method for preparing a fowl cholera subunit vaccine, characterized in that, Includes the following steps: Step 1) Preparation of bacterial solution: Add avian Pasteurella multocida to the bacterial solution. lpxM Gene-deleted strains were streaked on BHI solid medium, and single colonies were picked and inoculated into BHI liquid medium until the bacterial count reached 1.0 × 10⁻⁶. 9 CFU / mL; the preservation number of the gene-deleted strain is CCTCC NO: 2026575; Step 2) Prepare avian Pasteurella multocida using nitrogen cavitation technology, ultracentrifugation, and density gradient centrifugation. lpxM Membrane vesicles of gene-deleted strains; Step 3) Mix the membrane vesicles obtained in Step 2) with ISA 201 VG adjuvant at a volume ratio of 1:1 to make the final concentration of membrane vesicle protein 2 mg / mL, and store at -20℃ to obtain the final product.

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