Avibacterium paragallinarum gene deletion strain, construction method thereof and application thereof in preparation of chicken infectious rhinitis live vaccine

Abstract

The invention provides an application of deletion of genes cdtA, hctA, acbD, ccbF1 and / or aroA in reduction of pathogenicity of avibacterium paragallinarum. The nucleotide sequences of the genes cdtA, hctA, acbD, ccbF1 and aroA are shown as SEQ ID No.1-5. The invention further provides an application of the deletion of the genes cdtA, hctA, acbD, ccbF1 and aroA in reduction of pathogenicity of avibacterium paragallinarum. The invention also provides the Avibacterium paragallinarum Apg double gene deletion strain A type GZ acb-aroA, and the preservation number of the Avibacterium paragallinarum Apg double gene deletion strain A type GZ acb-aroA is CGMCC (China General Microbiological The invention further provides a chicken infectious rhinitis live vaccine containing the GZ acb-aroA. According to the invention, a capsular gene natural deletion strain is screened, and the aroA gene is deleted by adopting a natural conversion method, so that the feasibility and high efficiency of the natural conversion method are verified. The GZ acb-aroA strain disclosed by the invention has the advantages that the pathogenicity of chicken is obviously reduced, the safety is good, an aluminum hydroxide adjuvant is added to prepare a vaccine product, and an ideal protection effect can be achieved after leg intramuscular injection.

Description

Technical Field

[0001] The invention relates to a gene-deficient strain of Avibacterium paragallinarum, a construction method thereof, and application thereof in preparing a live vaccine of infectious coryza in chickens, and belongs to the field of veterinary biological products. Background Art

[0002] Infectious rhinitis in chickens is an acute upper respiratory tract infectious disease caused by Avibacterium paragallinarum. It can cause swelling of the infraorbital sinus and surrounding areas, runny nose, tearing, poor growth in growing chickens, and a 10% to 40% decrease in egg production in laying hens, causing serious economic losses to farmers.

[0003] According to Page's agglutination test typing scheme, Avian bacillus paragallinarum can be divided into three serotypes: A, B, and C. Types A, B, and C all have different degrees of pathogenicity, and the cross-protection between different serotypes is weak. Therefore, it is necessary to develop vaccines containing serotype strains in epidemic areas based on epidemiological characteristics. The three serotypes of infectious coryza A, B, and C are all prevalent in my country, and have shown a high incidence trend in recent years. Although the inactivated vaccine for infectious coryza has been widely used in laying hen farms, outbreaks still occur from time to time. This may be related to the short incubation period of infectious coryza, the decline in the inherent immunity of chickens due to cold stress, the antigenic differences between vaccine strains and wild strains, and the fact that some immunized chickens do not show clinical symptoms but are carriers.

[0004] The live vaccine for infectious coryza in chickens is expected to become one of the effective methods for preventing and controlling infectious coryza in chickens because it can simulate natural infection, activate cellular immunity and mucosal immunity, and provide cross-protection.

[0005] For bacterial gene-deleted live vaccines, the target genes are mainly divided into: deletion of related virulence genes, such as toxins and adhesins; or deletion of "housekeeping" genes in bacterial metabolic pathways or adaptation to environmental regulation. The virulence factors of Avian Bacillus paragallinarum that have been identified include capsular polysaccharides, cilia, hemagglutinin, lipopolysaccharide, bacterial lethal distending toxin (CDT), RTX toxin, etc., and there are also reports of vesicles as virulence factors. The "housekeeping" genes in metabolic pathways or environmental regulation are contained in every bacterium. By deleting such genes through molecular biological methods, the wild strains can be weakened, which has better universality.

[0006] In addition to the incomplete research on virulence factors and pathogenic mechanisms, the inefficiency of gene deletion methods is also a key factor that restricts the development of live vaccines for infectious coryza in chickens. Currently, the widely used gene deletion methods include the TargeTron insertion and deletion system; introducing suicide plasmids into the target strain by electroporation, or introducing suicide plasmids by natural competence, and then obtaining gene-deficient strains by homologous recombination; or introducing transposons into the target strain by natural competence, randomly inserting them into the genome, and then screening for deletion strains with significantly reduced virulence. However, each of these methods has different defects. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art, by analyzing the virulence factor-related genes and "housekeeping" genes of Avibacterium paragallinarum, wherein the virulence factor-related genes include cytolethal swelling toxin-related genes cdtABC, repeat-containing toxin RTX-related genes avxIABCD, LPS synthesis-related genes (including but not limited to lpxABCDHKLM), capsule synthesis and transport-related genes (including genes involved in capsule polysaccharide transport (hctABCD gene cluster)), genes involved in capsule polysaccharide component synthesis (chondroitin synthesis gene cluster acbEDGHCB, heparin synthesis gene cluster ccbEF1F2CB), bacterial secretion system-related genes and or IgA enzyme-related genes, etc. "Housekeeping" genes include aroA, a gene related to catalyzing aromatic amino acid synthesis, luxS, a gene related to bacterial quorum sensing and signal molecule synthesis, and natural competent genes related to nucleic acid uptake and recombination from the outside (including but not limited to CRP, Sxy, comEFM, pilABCDMW), etc. By comparing the effects of different related genes or "housekeeping" genes on the pathogenicity of A. paragallinarum, we aim to obtain a deletion strain of A. paragallinarum with attenuated virulence and excellent immunogenicity for the development of a live vaccine for infectious coryza in chickens.

[0008] Based on this, the present invention provides the use of the cytolethal distending toxin gene cdtA deletion in reducing the pathogenicity of Avibacterium paragallinarum. The nucleic acid sequence of the gene cdtA is shown in SEQ ID No.1.

[0009] The present invention also provides application of capsule synthesis transport gene hctA deletion in reducing pathogenicity of Avibacterium paragallinarum. The nucleic acid sequence of the gene hctA is shown in SEQ ID No.2.

[0010] The present invention also provides the use of the deletion of chondroitin synthesis gene acbD and heparin synthesis gene ccbF1 in reducing the pathogenicity of Avibacterium paragallinarum. The nucleic acid sequences of the gene acbD and the gene ccbF1 are shown in SEQ ID No.3-4.

[0011] The present invention also provides application of the nutrient metabolism gene aroA deletion in reducing the pathogenicity of Avibacterium paragallinarum. The nucleic acid sequence of the gene aroA is shown in SEQ ID No.5.

[0012] On the other hand, the present invention provides Avibacterium paragallinarum Apg double gene deletion strain A type GZ△acb-aroA, and the Avibacterium paragallinarum was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 16, 2024, with the deposit number CGMCC No.31708.

[0013] Furthermore, the present invention provides the use of the Avibacterium paragallinarum Apg double gene deletion strain A type GZ△acb-aroA in the preparation of chicken infectious coryza immune products.

[0014] On the other hand, the present invention provides a live vaccine for infectious coryza in chickens, which contains the Avibacterium paragallinarum Apg double gene deletion strain A type GZ△acb-aroA.

[0015] According to a preferred embodiment, the dose of the vaccine is 1.0×10 8 CFU / mouse, the vaccine also contains 10% aluminum hydroxide adjuvant based on the total volume of the vaccine, and the aluminum ion content in the vaccine is not higher than 2 mg / ml.

[0016] Particularly preferably, the vaccine is administered by injection into the leg muscle.

[0017] The present invention also provides a method for constructing the Avibacterium paragallinarum GZ△acb-aroA, which comprises the following steps:

[0018] (1) After the natural capsule gene deletion strain GZ△acb of Avibacterium paragallinarum was revived, it was cultured in a nutrient-poor medium, and cyclic adenosine monophosphate was added to the cultured bacterial solution to induce the formation of a natural competent state. The naturally competent bacteria were mixed with plasmids containing the upstream and downstream homologous arms of the aroA gene and incubated, and inoculated on a plate containing kanamycin for culture. Single colonies growing on a plate containing kanamycin resistance were selected and amplified using kana gene primers kana-F / R. The positive clones that amplified a 977bp band were deletion strains with a single exchange insertion of the aroA gene. The single crossover insertion strains were verified using primers aroA-up-BamHI-F / aroA-dn-EcoRI-R;

[0019] Among them, the Avibacterium paragallinarum Apg capsule gene naturally deleted strain A type GZ△acb was deposited at the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 16, 2024, with a deposit number of CGMCC No.33091;

[0020] (2) The positive clones screened in step (1) were continuously passaged in chicken broth medium without NaCl, and the double exchange deletion strains were amplified and screened using primers aroA-up-BamHI-F / aroA-dn-EcoRI-R. As a result, a 2003 bp fragment was amplified from the capsule natural deletion strain, and a 1359 bp fragment was amplified from the double exchange deletion strain, indicating that the plasmid was eliminated in the deletion strain. The aroA gene double exchange deletion strain was named GZ△acb-aroA, which is a double gene deletion strain of the capsule gene and aroA.

[0021] Preferably, the method of inducing the strain to form a natural competent state is to add cyclic adenosine monophosphate after culturing Avibacterium paragallinarum in a nutrient-poor medium. Preferably, a chicken broth medium without NaCl can be used as the nutrient-poor medium, and the cyclic adenosine monophosphate is added to a concentration of 1 to 200 mmol / L, and particularly preferably a final concentration of 50 mmol / L.

[0022] Specifically, the present invention screened a natural capsule gene deletion strain GZ△acb of Avibacterium paragallinarum, and then used the natural transformation method to construct the Avibacterium paragallinarum Apg double gene deletion strain A type GZ△acb-aroA. The strain was preserved at the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on November 8, 2024, with the preservation number CGMCC No.31708.

[0023] Among them, the Avibacterium paragallinarum Apg capsule gene naturally deleted strain type A GZ△acb strain was isolated from the infraorbital sinus of chickens with infectious rhinitis in a farm in Quzhou, Zhejiang. It is a capsule gene naturally deleted strain. The strain was sent to the General Microbiology Center of China Microorganism Culture Collection, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 11, 2024, and the preservation number is CGMCC No. 33091.

[0024] The invention also constructs a cdtA gene deletion strain of A. paragallinarum cell lethal swelling toxin with serotype C. The nutrient-poor culture medium used is a chicken broth culture medium without NaCl, and cyclic adenosine monophosphate (cAMP) is added to improve the transformation efficiency of the natural competent state method, the amount added is no more than 200uL cAMP, and the final concentration of cAMP is 1-200mmol / L. A total of 6 clinically isolated A. paragallinarum strains were cultured in a nutrient-poor culture medium containing cyclic adenosine monophosphate, and finally two strains achieved single exchange insertion and deletion, and one of the two strains achieved double exchange substitution deletion, which is better than the currently reported gene deletion method of A. paragallinarum.

[0025] Some bacteria can form a natural competent state, absorb exogenous DNA from the environment, and undergo homologous recombination with their own genome to allow the bacteria to acquire new genes. The characteristics of the exogenous DNA fragments themselves will affect natural transformation. Bacteria will preferentially recognize and ingest DNA carrying a certain short fragment, which is called the uptake signal sequence (USS). Literature reports that the USS sequence of infectious coryza in chickens is ACCGCACTT (Donghui Liu, Yantao Wu, Basic Characterization of Natural Transformation in Avibacterium paragallinarum, Microbiology Spectrum, May / June 2023 Volume 11 Issue 3).

[0026] Cytolethal distending toxin (CDT) is a protein toxin secreted by various Gram-negative pathogens. The complete CDT is a heterotrimer composed of three subunits: CdtA, CdtB and CdtC. The main function of CDT discovered so far is to destroy the chromosomal DNA of eukaryotic cells, thereby disrupting the normal division cycle of cells and causing cell apoptosis. The present invention also verifies that the constructed cdtA gene-deficient Avibacterium paragallinarum has lower pathogenicity to chickens than the wild strain.

[0027] As an embodiment of the present invention, the present invention uses a natural competent state method to construct a serotype C Avibacterium paragallinarum capsule synthesis transport gene hctA deletion strain, and verifies that the constructed hctA gene deletion strain has lower pathogenicity to chickens than the wild strain.

[0028] Capsule is an important virulence factor of Avibacterium paragallinarum. Capsule can protect bacteria from phagocytosis and complement-mediated bactericidal effects, thereby increasing the pathogenicity of bacteria. Studies by Jin-Ru Wu et al. showed that the genome of Avibacterium paragallinarum contains two sets of capsule polysaccharide synthesis genes (type I and type II), which are involved in the synthesis, modification and transport of chondroitin and heparin respectively (Jin-Ru Wu, Ping-Yi Chen, Poa-Chun Chang, etc. Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum, AVIAN DISEASES 59: 87–93, 2015). The capsule biosynthesis gene clusters of many Gram-negative bacteria are composed of three functionally unique regions, I, II and III. The genes in regions I and III contain genes involved in the transport and phospholipid replacement of capsule polysaccharides, and region II contains genes involved in the biosynthesis of capsule polysaccharides. The capsule synthesis gene region I of Avibacterium paragallinarum contains 4 genes involved in the transport of capsular polysaccharides (hctABCD gene cluster), and the region II contains 4 to 6 genes required for the biosynthesis of capsular polysaccharides (chondroitin synthesis gene cluster acbEDGHCB, heparin synthesis gene cluster ccbEF1F2CB). Poa-Chun Chang et al. showed that after the loss of the capsule transport-related gene hctA, the hemagglutination titer of Avibacterium paragallinarum increased, but the pathogenicity to chickens decreased significantly. (Tzu-Yi Tu, Poa-Chun Chang, Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum, AVIAN DISEASES 59:87–93, 2015).

[0029] As an embodiment of the present invention, a natural capsule gene deletion strain was screened from 5 strains of Avibacterium paragallinarum isolated from clinical sites, and the serotype was identified as type A. A 644 bp DNA fragment in the middle of the nutrient metabolism gene aroA of the strain was deleted by natural transformation, thereby constructing a new GZ△acb-aroA double gene deletion strain.

[0030] A good live bacterial vaccine needs to have both low toxicity and better immune protection, and be easily cleared by the body. Deleting the aroA gene is a widely studied and successful bacterial attenuation method. The aroA gene encodes 5-enolpyruvylshikimate-3-phosphate synthase, which mainly catalyzes the synthesis of aromatic amino acids. The aromatic biosynthesis pathway is a common pathway for Gram-negative and Gram-positive bacteria, but mammals do not have this synthesis pathway. Bacteria that lack the aroA gene cannot synthesize aromatic amino acids, so that the bacteria can only reproduce in a limited manner in the host body and grow relatively slowly, resulting in a significant reduction in the virulence of the bacteria, and thus have high safety.

[0031] The invention provides the constructed Avibacterium paragallinarum GZ△acb-aroA which can be used for the development of a live vaccine for infectious rhinitis in chickens, and the vaccine can be used for preventing and treating infectious rhinitis in chickens caused by infection with Avibacterium paragallinarum type A, type B and type C. Symptoms of infectious rhinitis in chickens include swelling on one or both sides of the chicken's face, or runny nose, or tearing, which leads to a decrease in egg production, growth stunting of growing chickens, delayed laying period of laying hens, decreased weight gain of broilers, etc.

[0032] The present invention verifies that the deletion of the gene cdtA related to the cell lethal swelling toxin of Avibacterium paragallinarum, the gene hctA related to capsule transport, the genes acbD and ccbF1 related to capsule synthesis, and the gene aroA related to nutrient metabolism can all reduce the pathogenicity of the strain. Among them, the most preferred is the naturally deleted strain of capsule gene GZ△acb, on the basis of which the aroA gene deleted strain GZ△acb-aroA of the strain is constructed, and it is verified that the virulence of the double gene deleted strain is significantly reduced and the immunogenicity is excellent. On this basis, the present invention uses the constructed GZ△acb-aroA double gene deleted strain to study the live vaccine of infectious coryza in chickens. Animal experiments have confirmed that GZ△acb-aroA has a 1.0×10 8 CFU / each was injected into the infraorbital sinus of 8-week-old SPF chickens, and none of the infected chickens showed typical symptoms of infectious coryza in chickens. The live vaccine of the missing strain was immunized by intranasal drops, infraorbital sinus, and intramuscular injection, and the virus was challenged 21 days after immunization. The result showed that the intramuscular injection group was 100% protected. The results show that Avibacterium paragallinarum GZ△acb-aroA, which lacks both the capsule gene and the aroA gene, has the potential to be developed into a live attenuated vaccine.

[0033] Microbiological information

[0034] Avibacterium paragallinarum Apg double gene deletion strain type A GZ△acb-aroA, the strain was deposited on November 8, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.31708.

[0035] The Avibacterium paragallinarum Apg capsule gene naturally deleted strain A type GZ△acb was deposited at the General Microbiology Center of China Microorganism Culture Collection, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 11, 2024, with the deposit number CGMCC No.33091. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The PCR results for the construction and identification of the cdtA gene single crossover insertion strain in Example 1; wherein, M: DL2000 DNA marker; 1: cdtA gene upstream and downstream primers (wild strain); 2: cdtA gene upstream and downstream primers (single crossover insertion strain); 3: cdtA gene upstream and downstream primers (negative control); 4: kanamycin primers (negative control); 5: kanamycin primers (single crossover insertion strain);

[0037] Figure 2 The capsule gene amplification results of the clinical isolated strain of Avibacterium paragallinarum in Example 5, wherein M: DL2000DNA marker; 1-5, the genome of the clinical isolated strain

[0038] FIG3 is a capsule staining photograph of the strain of Example 5, wherein: Figure 3A This is a photo of the capsule staining of the GZ△acb strain. Figure 3B This is a photo of capsule staining of type A Q3 strain;

[0039] Figure 4 The upstream and downstream fragments of the aroA gene of Avibacterium paragallinarum, the kana gene fragment and the tandem amplification results of Example 6, wherein M: DL2000 DNA marker; 1: kana resistance expression cassette (977 bp); 2: aroA-up upstream homology arm (723 bp); 3: aroA-down downstream homology arm (636 bp); 4: recombinant fragment (2336 bp)

[0040] Figure 5 This is the PCR identification result of the aroA gene deletion strain of Avibacterium paragallinarum in Example 6, wherein M: DL2000 DNA marker; 1: double exchange deletion strain; 2: aroA gene insertion deletion strain. DETAILED DESCRIPTION

[0041] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0042] In the present invention, unless otherwise specified, "%" used to explain concentrations refers to weight percentage, and ":" refers to weight ratio.

[0043] The present invention relates to the following microbial materials:

[0044] Avibacterium paragallinarum A type Q3 strain, formerly known as Avibacterium paragallinarum A type QL-Apg-3 strain, is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No. 18173; Avibacterium paragallinarum B type Q26 strain, formerly known as Avibacterium paragallinarum B type QL-Apg-26 strain, is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No. 18174; Avibacterium paragallinarum C type Q15 strain, formerly known as Avibacterium paragallinarum C type QL Apg-15 strain, is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No. 18175. The above microorganisms are disclosed in Chinese invention patent application CN 110448690A.

[0045] The present invention relates to the following culture medium:

[0046] Haemophilus parahaemolyticus plate: Use 32 g of Haemophilus parahaemolyticus dry powder culture medium (commercial product) and 15 g of agar powder, add into 1000 ml of purified water, heat to dissolve, adjust the pH value to 7.4±0.1, sterilize by high pressure at 116°C for 40 minutes, when the temperature drops to about 50°C, add filter-sterilized NADH (coenzyme I) to a final concentration of 60 μg / ml and healthy chicken serum to a final concentration of 10%, and cast the plate.

[0047] Liquid medium for Haemophilus parahaemophilus: 32g of dry powder medium for Haemophilus parahaemophilus was added to 1000ml of purified water, heated to dissolve, adjusted the pH value to 7.4±0.1, and sterilized by high pressure at 116℃ for 40 minutes. As a culture medium for bacterial enrichment, add NADH (coenzyme I) that has been filtered and sterilized to a final concentration of 15μg / ml and add healthy chicken serum to a final concentration of 2.5% before use. As a nutrient-poor culture medium, reduce the final concentration of NADH (coenzyme I) to a minimum of 0μg / ml and the final concentration of chicken serum to a minimum of 0.

[0048] Chicken broth medium: The manufacturing method is in the "Regulations of the People's Republic of China on Veterinary Biological Products" (2000 edition). As a nutrient-poor medium, reduce the final concentration of sodium chloride to a minimum of 0g.

[0049] The present invention relates to the following primers, all of which were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.:

[0050] Table 1. Primer sequences used in the present invention

[0051]

[0052] Note: The italic bases with dotted lines are restriction sites (BamHI, EcoRI), the bases with horizontal lines are USS series, and the shaded bases are KanR overlapping regions.

[0053] Example 1 Construction of a cdtA gene deletion strain of the cell-lethal swelling toxin of Avibacterium paragallinarum

[0054] 1. Primer design

[0055] The method was constructed according to the literature (Yang Jun, Yan He, Construction of lpxM gene deletion strain of Haemophilus parasuis and analysis of biological characteristics, Chinese Agricultural Science, 2020, 53). The nucleic acid was extracted from A. paragallinarum type Q3 strain, B type Q26 strain, and C type Q15 strain and sent to BGI for whole genome sequencing and genome annotation. The gene sequences of the capsule synthesis transport gene cdtA of the three strains were compared, and the upstream and downstream primers were designed (see Table 1).

[0056] 2. Cloning of upstream and downstream fragments of cdtA gene and kana gene fragment of Avian bacterium paragallinarum

[0057] The genomic nucleic acid of Avibacterium paragallinarum type A Q3 strain was extracted, and the upstream and downstream fragments of the cdtA gene were amplified, respectively. The sizes of the amplified fragments were 490 bp and 503 bp, respectively. The upstream sequence contained the restriction site BamHI, USS sequence, and kana gene primers; the downstream sequence contained the restriction site EcoRI, USS sequence, and kana gene primers. The kana gene sequence was amplified from the cloning plasmid pET28a. Using the amplified three fragments as templates, the three gene segments were connected in series by overlap extension PCR to obtain the homologous recombinant fragment cdtA-up-kana-down.

[0058] 3. Construction of transfer plasmid pUC-cdtA

[0059] The homologous recombination fragment cdtA-up-kana-down and pUC plasmid were double-digested, purified, and ligated overnight with T4 DNA ligase. The ligated plasmid was heat-shocked into E. coli DH5α competent cells and spread on nutrient agar plates containing kanamycin resistance (50 ng·μL -1 , the same below), and the pUC-cdtA plasmid was obtained after PCR identification.

[0060] 4. Screening of naturally competent strains and construction of cdtA gene deletion strain of Avian Bacterium paragallinarum by natural transformation

[0061] 4.1 Construction and identification of single crossover insertion strains

[0062] Six strains of Avibacterium paragallinarum were isolated from clinical sites between 2021 and 2022, and the serotypes were identified as 2 strains of type A and 4 strains of type C. They were inoculated on Haemophilus paragallinarum plates containing NADH (coenzyme I) and healthy chicken serum for recovery. They were cultured in an incubator at 37°C and 5% CO2 for 18 to 24 hours, and the overnight cultured bacteria were washed with 3 mL of Haemophilus paragallinarum liquid culture medium, inoculated in chicken broth medium without NaCl, and cultured statically at 37°C for 6 to 10 hours. The culture was centrifuged and the OD was adjusted. 600 0.9~1.0. Take 20μL bacterial solution in a 1.5mL EP tube, add cAMP to a final concentration of 50mmol / L, then add 10μL (about 1μg) pUC-cdtA plasmid, mix well and act for 10min; at the same time, mix 10μL sterile water with bacteria as a negative control. Transfer the bacteria and plasmid mixture after the action to a Haemophilus parahaemolyticus plate containing NADH (coenzyme I) and healthy chicken serum, with a diameter of about 1cm. Place the plate upright at 37℃ for 5h, then wash it with 200μL Haemophilus parahaemolyticus liquid culture medium, and spread it on a Haemophilus parahaemolyticus plate containing kanamycin resistance, and invert it at 37℃ for 24~48 hours. Pick the positive clones and use kanamycin gene primers for PCR amplification. The positive clones are those where a single exchange insertion of the cdtA gene has occurred. The single-crossover insertion strain was amplified by PCR using the upstream primer of the upstream fragment of the cdtA gene and the downstream primer of the downstream fragment. The wild-type strain amplified a 1863 bp fragment, and the single-crossover insertion strain amplified a 1983 bp fragment. Figure 1 Among the 6 clinically isolated A. paragallinarum strains, one strain was able to form a natural competent state. A positive clone was selected from this strain to achieve single exchange insertion and deletion of the cdtA gene. The original wild strain was named GD23708 strain, which was serotype C.

[0063] 4.2 Construction and identification of double-crossover deletion strains

[0064] The screened positive clones were spread on plates containing kanamycin-resistant Haemophilus pararesistant bacteria, cultured at 37°C for 24 hours, and the bacterial moss was washed and inoculated into chicken broth medium without NaCl. It was statically cultured at 37°C for 12 to 16 hours and subcultured once. After 5 consecutive subcultures, equal amounts of diluted bacterial liquid were spread on kanamycin-resistant and non-resistant plates respectively, and cultured at 37°C for 24 hours. When the number of colonies on the resistant plate was significantly less than that on the non-resistant plate, it indicated that double exchange had occurred. The colonies on the non-resistant plate were picked and streaked on the resistant and non-resistant plates respectively, and cultured at 37°C for 24 hours. The colonies grown only on the non-resistant plate were selected for colony PCR verification. The deletion strain was amplified by PCR using the upstream primer of the upstream fragment of the cdtA gene and the downstream primer of the downstream fragment. The amplified product was sent to the company for sequencing. The results showed that the wild strain amplified a 1863bp fragment, the cdtA gene deletion strain amplified a 1013bp fragment, and the 850bp gene fragment was missing in the middle. The cdtA gene deletion strain was named GD23708△cdtA.

[0065] Example 2 Determination of the toxicity of the cell-lethal swelling toxin gene deletion strain GD23708ΔcdtA of Avibacterium paragallinarum

[0066] 30 SPF chickens aged 35 days or more were divided into 6 groups, with 5 chickens in each group. The cytolethal swelling toxin gene deletion strain GD23708△cdtA and the wild strain GD23708 were streaked onto non-resistant Haemophilus parasitica plates. The bacterial lawn was washed and the OD was calculated. 600 Dilute to 5 × 10 5 CFU / ml, 5×10 4 CFU / ml, 5×10 3 CFU / ml, live bacteria count to confirm the actual number of challenged bacteria. The challenge was injected into the infraorbital sinus, the challenge volume was 0.2 ml / mouse, and the animals were observed for 7 days after the challenge. The observation results are shown in Table 2.

[0067] Table 2 Comparative test results of toxicity of GD23708△cdtA and GD23708 to SPF chickens

[0068]

[0069] The results showed that the wild-type GD23708 strain had a challenge dose of 1.0×10 3 CFU / bird, all 5 chickens became sick; the cdtA gene-deficient strain GD23708△cdtA was infected with the same challenge dose of 1.0×10 3 CFU / , the incidence rate was only 20%; the challenge dose was increased to 1.0×10 4 CFU / chicken, the incidence rate reached 80%. The results showed that the deletion of cdtA gene of cytolethal swelling toxin of A. paragallinarum could reduce the pathogenicity of the strain to chickens.

[0070] Example 3 Construction of a strain of Avibacterium paragallinarum capsule synthesis and transport hctA gene deletion

[0071] 1. Primer design

[0072] By comparing the hctA gene sequences related to capsule synthesis and transport in the whole genome sequences of Avibacterium paragallinarum type A Q3 strain, type B Q26 strain, and type C Q15 strain, upstream and downstream primers were designed (see Table 1).

[0073] 2. Cloning of upstream and downstream fragments of hctA gene and kana gene fragment of Avian bacterium paragallinarum

[0074] The genome of Avibacterium paragallinarum type A Q3 strain was extracted and used as a template to amplify the upstream and downstream fragments of the hctA gene. The sizes of the amplified fragments were 824 bp and 444 bp, respectively. The upstream sequence contained the restriction site BamHI, USS sequence, and kana gene primers; the downstream sequence contained the restriction site EcoRI, USS sequence, and kana gene primers. The kana gene sequence was amplified from the cloning plasmid pET28a. Using the amplified three fragments as templates, the three genes were connected in series by overlap extension PCR to obtain the homologous recombinant fragment hctA-up-kana-down.

[0075] 3. Construction of transfer plasmid pUC-hctA

[0076] The homologous recombination fragment hctA-up-kana-down and pUC plasmid were double-digested, purified, and connected overnight with T4 DNA ligase. The connected plasmid was heat-shocked and transformed into Escherichia coli DH5α competent cells, and spread on a nutrient agar plate containing kanamycin resistance. The pUC-hctA plasmid was obtained after PCR identification.

[0077] 4. Construction of hctA gene deletion strain of Avian Bacillus paragallinarum by natural transformation

[0078] The method in Example 1 was referred to. Using A. paragallinarum C type GD23708 strain as the starting strain, an A. paragallinarum hctA gene deletion strain was constructed by natural transformation, and PCR amplification was performed using kanamycin primers to screen for a single crossover insertion strain of the hctA gene.

[0079] The single exchange insertion strain of the hctA gene was continuously propagated in a chicken broth medium without NaCl to screen for double exchange substitution deletion strains. The deletion strain was PCR amplified using the upstream primer of the upstream fragment of the hctA gene and the downstream primer of the downstream fragment. The amplified product was sent to the company for sequencing. The results showed that the wild strain amplified a fragment of about 1770bp, the deletion strain amplified a fragment of about 1270bp, and a gene fragment of about 500bp was missing in the middle. The hctA gene deletion strain was named GD23708△hctA.

[0080] Example 4 Virulence determination of the capsule synthesis and transport gene deletion strain GD23708ΔhctA of Avibacterium paragallinarum

[0081] The same procedure as in Example 2 was performed, except that the GD23708ΔhctA strain and the wild strain GD23708 strain were streaked onto non-resistant Haemophilus pararesistant plates, the bacterial lawn was washed off before challenge, 0.2 ml / head was injected into the infraorbital sinus, and the animals were observed for 7 days after challenge. The results are shown in Table 3.

[0082] Table 3 Comparative test results of toxicity of GD23708△hctA strain and wild strain GD23708 to SPF chickens

[0083]

[0084]

[0085] The results showed that the capsule synthesis and transport gene deletion strain GD23708△hctA challenge dose was 1.0×10 3 CFU / bird, none of the chickens showed typical symptoms of infectious coryza; when the challenge dose was increased to 1.0×10 4 CFU / chicken, 40% of the chickens became sick. The results showed that the capsule synthesis and transport gene deletion strain GD23708△hctA of A. paragallinarum could reduce the pathogenicity of the strain to chickens.

[0086] Example 5 Amplification of capsule synthetic genes of Avibacterium paragallinarum and analysis of characteristics of naturally deleted capsule genes

[0087] 1. Amplification of capsule synthesis genes

[0088] Two pairs of primers were synthesized according to the literature (Jin-Ru Wu, Ping-Yi Chen, Poa-Chun Chang, etc. Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum, AVIAN DISEASES 59:87–93, 2015), and the acbD gene and ccbF1 gene of Avibacterium paragallinarum were amplified using the genomes of Avibacterium paragallinarum type A C-Apg-8 strain (CVCC 254) and type A Q3 strain as templates, respectively. These two genes are involved in the synthesis of capsule polysaccharide components chondroitin and heparin, respectively.

[0089] From the 5 clinically isolated Avibacterium paragallinarum strains, 4 strains amplified either the chondroitin synthesis gene or the heparin synthesis gene, and 1 strain was negative for both genes. The strain with natural deletion of acbD gene and ccbF1 gene was named Avibacterium paragallinarum Apg capsule gene natural deletion strain A GZ△acb strain, and the serotype of the strain was confirmed to be type A.

[0090] 2. Observation of capsule morphology of natural capsule gene deletion strain

[0091] The capsule gene naturally deleted strain GZ△acb of A. paragallinarum and the A. paragallinarum type A Q3 strain were stained for capsule, and the capsule morphology was observed under an optical microscope, as shown in FIG3 .

[0092] 3. Virulence of the naturally deleted capsule gene strain on SPF chickens

[0093] Thirty SPF chickens aged more than 35 days were divided into 6 groups, with 5 chickens in each group. The capsule gene naturally deleted strain GZ△acb and Avibacterium paragallinarum type A Q3 strain were inoculated into 6-day-old SPF chicken embryos, and the yolk fluid of chicken embryos that died within 24 hours was harvested, quantitatively packaged, and stored at -80°C. Live bacteria were counted before the challenge, and the same batch of bacteria were diluted to 5×10 5 CFU / ml, 5×10 4 CFU / ml, 5×10 3 CFU / ml. Intraorbital sinus injection, 0.2 ml / animal, observation for 7 days after challenge, the results are shown in Table 4.

[0094] Table 4 Comparative test results of toxicity of GZ△acb strain and wild type to SPF chickens

[0095]

[0096]

[0097] The results showed that the minimum pathogenic dose of A. paragallinarum capsule-deficient strain GZ△acb to SPF chickens was less than 1.0×10 5 CFU / piece, the minimum pathogenic dose of type A Q3 strain for SPF chickens is 1.0×10 4 CFU / piece.

[0098] The results show that the deletion of the capsule synthesis and transport genes of A. paragallinarum (including the chondroitin synthesis gene acbD and the heparin synthesis gene ccbF1) can significantly reduce the pathogenicity of the strain to chickens, and its pathogenicity is lower than that of GD23708△cdtA obtained in Example 1 and GD23708△hctA obtained in Example 3.

[0099] Example 6 Construction and identification of the acb-aroA double gene deletion strain of Avibacterium paragallinarum

[0100] 1. Primer design

[0101] By comparing the nutrient metabolism gene aroA gene sequences in the whole genome sequences of Avibacterium paragallinarum type Q3 strain, type B Q26 strain, and type C Q15 strain, upstream and downstream primers were designed (see Table 1).

[0102] 2. Cloning of upstream and downstream fragments of the aroA gene and the kana gene fragment of Avibacterium paragallinarum

[0103] The genome of Avibacterium paragallinarum type A Q3 strain was extracted and used as a template to amplify the upstream and downstream fragments of the aroA gene. The sizes of the amplified fragments were 723 bp and 636 bp, respectively. The upstream sequence contained the restriction site BamHI, USS sequence, and kana gene primers; the downstream sequence contained the restriction site EcoRI, USS sequence, and kana gene primers. The kana gene sequence was amplified from the cloning plasmid pET28a. Using the amplified three fragments as templates, the three gene segments were connected in series by overlap extension PCR to obtain the homologous recombinant fragment aroA-up-kana-down.

[0104] 3. Construction of transfer plasmid pUC-aroA

[0105] The homologous recombinant fragment aroA-up-kana-down and pUC plasmid were double-digested, purified, and connected overnight with T4 DNA ligase. The connected plasmid was heat-shocked into E. coli DH5α competent cells and spread on a nutrient agar plate containing kanamycin resistance. Single colonies grown on the resistant plate were picked and shaken. The plasmid was extracted for PCR sequencing and double-digestion identification. PCR amplified a fragment of 2336bp in size. Double-digestion obtained a 2336bp recombinant fragment pUC and a 2686bp vector fragment (Figure). The PCR product was sequenced, and the sequencing result was compared with the target sequence to determine that the recombinant plasmid of the aroA gene was successfully constructed and named pUC-aroA.

[0106] 4. Construction of the acb-aroA double gene deletion strain of Avian Bacillus paragallinarum by natural transformation

[0107] Refer to the method in Example 1. The capsule gene natural deletion strain GZ△acb screened in Example 5 was inoculated on a Haemophilus parahaemolyticus plate containing NADH (coenzyme I) and healthy chicken serum for recovery. The revived bacteria were used to construct the aroA gene deletion strain by natural transformation. Positive clones were picked and PCR amplified using kanamycin gene primers. The positive clones were those in which a single exchange insertion of the aroA gene occurred. As a result, 5 single colonies grew on the plate containing kanamycin resistance. Positive clones were selected and amplified using primers kana-F / R. No band was amplified for the capsule natural deletion strain, while a 977bp band was amplified for the aroA gene insertion deletion strain, indicating that the kanamycin resistance expression cassette was integrated into the chromosome. The single cross-insertion strain was verified using primers aroA-up-BamHI-F / aroA-dn-EcoRI-R. The results showed that a 2003bp band was amplified from the natural capsule deletion strain, and a 2336bp band was amplified from the aroA gene insertion deletion strain, indicating that the 977bp kanamycin resistance expression cassette replaced the 644bp partial fragment of the aroA gene.

[0108] The positive clones screened were continuously subcultured in chicken broth medium without NaCl, and the double exchange substitution deletion strains were amplified and screened with primers aroA-up-BamHI-F / aroA-dn-EcoRI-R. As a result, a 2003bp fragment was amplified in the capsule natural deletion strain, and a 1359bp fragment was amplified in the double exchange deletion strain, indicating that the plasmid was eliminated in the deletion strain. The aroA gene double exchange deletion strain was named GZ△acb-aroA, which is a double gene deletion strain of the capsule gene and aroA.

[0109] Example 7 Virulence determination of Avibacterium paragallinarum double gene deletion strain GZ△acb-aroA

[0110] 30 SPF chickens aged 35 days or more were divided into 6 groups, with 5 chickens in each group. The double gene deletion strain GZ△acb-aroA and the capsule natural deletion strain GZ△acb were streaked on non-resistant Haemophilus pararesistant plates. 600 Dilute to 5 × 10 8 CFU / ml, 5×10 6 CFU / ml, 5×10 4 CFU / ml, live bacteria count to confirm the actual number of challenged bacteria. 0.2 ml / animal was injected into the infraorbital sinus and observed for 7 days after challenge. The results are shown in Table 5.

[0111] Table 5 Comparative test results of toxicity of GZ△acb and GZ△acb-aroA to SPF chickens

[0112]

[0113] The results showed that the challenge dose of the double gene deletion strain GZ△acb-aroA of Avibacterium paragallinarum capsule gene and aroA gene reached 1.0×10 8 CFU / bird, no typical symptoms of chicken infectious coryza were found, while the natural capsule-deficient strain GZ△aroA challenged 1.0×10 6 CFU / chicken, and all 5 chickens became ill. The results showed that the virulence of the double gene deletion strain GZ△acb-aroA was significantly reduced, at least 1.0×10 2 times, and was significantly lower than that of the recombinant bacteria lacking a single virulence factor-related gene.

[0114] Example 8 Safety Study of the Double-Gene Deletion Strain GZ△acb-aroA of Avibacterium paragallinarum on SPF Chickens

[0115] The bacterial solution obtained in Example 6 was mixed evenly with aluminum hydroxide adjuvant at a volume ratio of 9:1 as a vaccine product, and it was confirmed that the aluminum ion content in the vaccine product was not higher than 2 mg / ml. The vaccine product contained 1.0×10 viable bacteria of the GZ△acb-aroA strain. 8 CFU / ml.

[0116] 60 chickens were divided into 6 groups, 10 in each group; Group 1 was inoculated with 0.2 ml of the bacterial solution obtained in Example 6 (containing about 1×10 viable bacteria of GZ△acb-aroA) by nasal drops. 8 CFU, the same below); Group 2 was injected with 0.2 ml of bacterial solution into the infraorbital sinus; Group 3 was injected with 0.2 ml of bacterial solution into the nose and 1 ml of the vaccine product (containing 1.0×10 viable bacteria of GZ△acb-aroA strain) into the leg muscle. 8 CFU / ml), the 4th group was injected with 1ml vaccine product into the leg muscle, and the 5th group was not immunized as a control. Within 1 week after immunization, observe whether the immunized chickens have symptoms such as runny nose, tears, and facial swelling. All chickens were weighed before immunization and weighed 1 week after immunization. The feed was controlled for one night before weighing, and water was not controlled to evaluate the safety of immunization on chickens. One week after immunization, palate swabs were collected from each group of immunized chickens, and nucleic acids were extracted after mixing. The immunized chickens were tested for bacteria according to industry standards (NY / T 538-2015, Diagnostic Technology for Infectious Rhinitis in Chickens [S], Agricultural Industry Standards of the People's Republic of China). The 6th group of chickens were challenged with Avibacterium paragallinarum type A according to the virulence comparison test method, and were raised in different isolators. Swabs were collected in the same way for nucleic acid testing 1 week after the challenge as a positive control.

[0117] The results showed that SPF chickens were immunized with GZ△acb-aroA in different ways and observed for one week after immunization. None of the immunized chickens showed typical symptoms of infectious rhinitis in chickens, such as runny nose, tears, and facial swelling.

[0118] The safety of live vaccines, in addition to considering the reversion of their own virulence, also needs to consider the impact on other growth performance. Weighing was performed one week after immunization. Compared with the weight gain before immunization, the infraorbital sinus injection group had a significant effect on body weight (P < 0.05), while the bacterial solution plus aluminum hydroxide adjuvant group had no significant effect on body weight (P > 0.05). The evaluation method for chicken infectious coryza vaccine is injection into the infraorbital sinus. The results show that although the GZ△acb-aroA bacterial solution does not produce typical symptoms of chicken infectious coryza, it still has a certain residual virulence to chickens. Interestingly, when aluminum hydroxide adjuvant is added to mix into a vaccine product and injected into the leg muscle, the effect on chicken weight gain is not significant, and the injection site is well absorbed when anatomically observed one month after immunization.

[0119]

[0120] One week after immunization, palate swabs were collected from all chickens, and the same group of swabs were mixed and nucleic acid was extracted. The results showed that all immunized groups and non-immunized control groups were negative, while the nucleic acid test of the group challenged with Avibacterium paragallinarum type A Q3 strain was positive.

[0121] The results show that after the double gene deletion strain GZ△acb-aroA constructed by the present invention is prepared into a vaccine product with aluminum hydroxide adjuvant, its safety to chickens is further improved through intramuscular injection, and the immunized chickens do not excrete toxins.

[0122] Example 9 Study on the immunogenicity of Avibacterium paragallinarum double gene deletion strain GZ△acb-aroA in SPF chickens

[0123] 60 chickens were divided into 6 groups, 10 in each group; Group 1 was inoculated with 0.2 ml of bacterial solution (containing about 1×10 viable bacteria of GZ△acb-aroA) by intranasal drip 8 CFU, the same below); Group 2 injected 0.2 ml of bacterial solution into the infraorbital sinus; Group 3 respectively nasally dripped 0.2 ml of bacterial solution and injected 1 ml of the vaccine product of Example 8 into the leg muscle; Group 4 injected 1 ml of the vaccine product of Example 8 into the leg muscle; Group 5 was not immunized and raised in the same isolator as other immunized groups, as control one; Group 6 was not immunized and raised in a different isolator from other immunized groups, as control two. 21 days after inoculation, all chickens were challenged with the bacterial solution of Avian Bacillus type Q3 strain of chickens, injected into the infraorbital sinus, 0.2 ml / chicken (containing about 10,000 CFU / chicken). After the challenge, the chickens were observed for 7 days, and the results are shown in Table 6.

[0124] Table 6 Results of immunogenicity study of Avibacterium paragallinarum double gene deletion strain GZ△acb-aroA on SPF chickens

[0125]

[0126] The gene-deficient strain GZ△acb-aroA was immunized in SPF chickens in different ways, and the virus was challenged 21 days after immunization. The results showed that the protection rates of the nasal drop and infraorbital sinus injection groups were 20% and 0, respectively, and the protection rates of the nasal drop / leg muscle injection and leg muscle injection groups were 80% and 100%, respectively. The results showed that the vaccine product prepared by adding aluminum hydroxide adjuvant to the bacterial liquid of the double gene-deficient strain GZ△acb-aroA can provide the best immune protection through leg muscle injection.

[0127] In summary, the present invention verifies the feasibility and high efficiency of the natural transformation method by screening the natural deletion strain of the capsule gene and then using the natural transformation method to delete the aroA gene. Animal experiments show that the deletion strain GZ△acb-aroA constructed by the present invention has significantly reduced pathogenicity to chickens and has good safety for SPF chickens. The immunogenicity results show that after adding aluminum hydroxide adjuvant to the bacterial liquid to prepare a vaccine product and injecting it into the leg muscle, an ideal protective effect can be achieved, which can effectively resist the invasion of related pathogens and shows good application prospects as a live vaccine development.

[0128] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical content disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. Application of the cytolethal distending toxin gene cdtA deletion in reducing the pathogenicity of Avibacterium paragallinarum, wherein the nucleic acid sequence of the gene cdtA is shown in SEQ ID No.

1.

2. Application of capsule synthesis transport gene hctA deletion in reducing pathogenicity of Avibacterium paragallinarum, the nucleic acid sequence of the gene hctA is shown in SEQ ID No.

2.

3. Application of the deletion of the capsule component chondroitin synthesis gene acbD and the heparin synthesis gene ccbF1 in reducing the pathogenicity of Avibacterium paragallinarum, the nucleic acid sequences of the gene acbD and the gene ccbF1 are shown in SEQ ID No.3-4.

4. Application of the nutrient metabolism gene aroA deletion in reducing the pathogenicity of Avibacterium paragallinarum, the nucleic acid sequence of the gene aroA is shown in SEQ ID No.

5.

5. Avibacterium paragallinarum Apg double gene deletion strain type A GZ△acb-aroA, the Avibacterium paragallinarum was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 8, 2024, with the deposit number CGMCC No.31708.

6. Use of the Avibacterium paragallinarum Apg double gene deletion strain A type GZ△acb-aroA described in claim 5 in the preparation of chicken infectious rhinitis immune products.

7. A live vaccine for infectious coryza in chickens, comprising the Avibacterium paragallinarum Apg double gene deletion strain A type GZΔacb-aroA according to claim 6.

8. The chicken infectious coryza live vaccine according to claim 7, characterized in that The vaccine contains 10% aluminum hydroxide adjuvant based on the total volume, the aluminum ion content in the vaccine is not higher than 2 mg / ml, and the content of Avibacterium paragallinarum GZ△acb-aroA is 1.0×10 8 CFU / piece.

9. The chicken infectious coryza live vaccine according to claim 8, characterized in that The vaccine is administered by intramuscular injection into the leg.

10. The method for constructing the Avibacterium paragallinarum Apg double gene deletion strain A type GZ△acb-aroA according to claim 5, the method comprising the following steps: (1) After the natural deletion strain of Avibacterium paragallinarum Apg capsule gene A type GZ△acb was revived, it was cultured in a nutrient-poor medium, cyclic adenosine monophosphate was added to the cultured bacterial solution to induce the formation of natural competence, the natural competent bacteria were mixed and incubated with a plasmid containing the upstream and downstream homologous arms of the aroA gene, and inoculated on a plate containing kanamycin for culture; a single colony grown on a plate containing kanamycin resistance was selected, and amplified using kana gene primers kana-F / R, and the positive clone with a band of 977bp amplified was the deletion strain with a single exchange insertion of the aroA gene; the single crossover insertion strain was verified using primers aroA-up-BamHI-F / aroA-dn-EcoRI-R; Among them, the Avibacterium paragallinarum Apg capsule gene naturally deleted strain A type GZ△acb was deposited at the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 11, 2024, with a deposit number of CGMCC No.33091; (2) The positive clones screened in step (1) were continuously passaged in chicken broth medium without NaCl, and the double exchange deletion strains were amplified and screened using primers aroA-up-BamHI-F / aroA-dn-EcoRI-R. As a result, a 2003 bp fragment was amplified from the capsule natural deletion strain, and a 1359 bp fragment was amplified from the double exchange deletion strain, indicating that the plasmid was eliminated in the deletion strain. The aroA gene double exchange deletion strain was named GZ△acb-aroA, which is a double gene deletion strain of the capsule gene and aroA.

11. The construction method according to claim 10, characterized in that After Avibacterium paragallinarum is cultured in a nutrient-poor medium, cyclic adenosine monophosphate with a final concentration of 1 to 200 mmol / L is added to induce the strain to form a natural competent state.

12. The construction method according to claim 11, characterized in that The concentration of cyclic AMP in the culture medium was 50 mmol / L.

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