Sheep-derived A-type pasteurella multocida strain, vaccine and application

Abstract

The invention relates to the technical field of pathogenic bacteria and vaccines thereof, in particular to a sheep-derived A-type pasteurella multocida strain, a vaccine and application. The invention discloses a sheep-derived A-type pasteurella multocida strain, and the preservation number of the sheep-derived A-type pasteurella multocida strain is The Pm-ALa strain is subjected to frame shift mutation on the Lon gene, and finally the truncated Lon protein is formed. The Pm-ALa strain is not only suitable for being used as a candidate strain for vaccine production, but also can be used for carrying out specificity test and vaccine immune effect evaluation by using truncated protein. According to the scheme, an efficient and stable Pm-ALa strain vaccine preparation method is established through optimized culture and fermentation process research. According to the technical scheme, the technical problem that the A-type pasteurella multocida vaccine for sheep is lacked in the prior art can be solved. The Pm-ALa strain and the vaccine product derived from the Pm-ALa strain show remarkable application potential, have important significance for preventing and controlling diseases caused by the pathogen, and open up a new way for future vaccine research and development.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic bacteria and vaccines thereof, and in particular to a sheep-derived type A Pasteurella multocida strain, a vaccine and an application thereof. Background Art

[0002] Pasteurella multocida (Pm) is a zoonotic pathogen that can colonize the upper respiratory tract of healthy animals as a symbiotic bacterium. Under various stressful environmental conditions (such as long-distance transportation, environmental changes, reduced immunity, etc.), Pm proliferates in large quantities, spreads to the lower respiratory tract and enters the lungs or invades other parts of the body through the blood circulation, causing diseases such as pneumonia and hemorrhagic sepsis, and in severe cases, can lead to the death of animals.

[0003] Based on the difference of capsule antigen, Pm can be divided into five capsule serotypes: A, B, D, E, and F. Sheep are one of the susceptible animals to Pasteurella multocida. Sheep fibrinous pneumonia caused by Pasteurella multocida type A is one of the main serotypes of Pasteurella multocida in sheep, which is a serious threat to the sheep breeding industry.

[0004] Although there are reports of natural isolates of Pasteurella multocida type A, there are differences in virulence and immunogenicity between different strains, and they need to be strictly screened and verified before they can be used in the production and application of sheep vaccines for Pasteurella multocida type A. At present, there is no successful sheep Pasteurella multocida type A strain to complete this process, and there is no related commercial vaccine on the market. Therefore, it is urgent to further explore and study sheep-derived Pasteurella multocida type A strains that can be used for vaccine preparation to meet market demand.

[0005] Therefore, research on sheep-derived type A P. multocida strains is particularly urgent, aiming to explore and develop effective vaccines that meet market demand. In order to meet this challenge, the biological characteristics, pathogenic mechanisms, and interactions between Pm and the host need to be further studied, and the technical means of vaccine development need to be continuously improved. Further identification and selection of strains most suitable as vaccine components and optimization of vaccine preparation processes, extensive laboratory tests and clinical trials should be conducted to ensure the safety and effectiveness of new vaccines. In addition, the cost-effectiveness ratio of the vaccine needs to be considered so that it can be mass-produced and promoted, ultimately achieving effective prevention and control of P. multocida infection and protecting the healthy development of the sheep farming industry. Summary of the invention

[0006] The present invention aims to provide a sheep-derived type A Pasteurella multocida strain to solve the technical problem of the lack of a sheep-use type A Pasteurella multocida vaccine in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A sheep-derived type A Pasteurella multocida strain, whose deposit number is CGMCC NO.32900.

[0009] Furthermore, the Lon gene in the strain mutated, and the nucleotide sequence of the mutated Lon gene is as shown in SEQ ID NO.11.

[0010] Furthermore, the mutated Lon gene encodes a truncated protein with an amino acid sequence as shown in SEQ ID NO.12.

[0011] Furthermore, the strain is isolated from sick sheep with goat bronchial symptoms; the samples collected from the sick sheep are first smeared on Martin agar containing 4.0% calf serum, and shake-cultured in a 37°C constant temperature incubator for 16 to 18 hours, and then a single colony is picked, and PCR is performed using Pasteurella multocida serotype A specific primers to screen positive strains; the sequences of the Pasteurella multocida serotype A specific primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] Furthermore, its LD for mice 50 5×10 2 CFU.

[0013] The technical solution also provides a use of a sheep-derived type A Pasteurella multocida strain in the preparation of a vaccine.

[0014] The technical solution also provides a Pasteurella multocida vaccine containing a sheep-derived type A Pasteurella multocida strain. The sheep-derived type A Pasteurella multocida is inactivated with formaldehyde and then mixed with an aluminum hydroxide gel adjuvant to obtain an inactivated vaccine.

[0015] Furthermore, the number of Pasteurella multocida type A from sheep in the inactivated vaccine is ≥ 1.0 × 10 10 CFU / ml;

[0016] Mice were immunized with 0.1 ml of inactivated vaccine and then 5.0 × 10 3 The survival rate of mice challenged with CFU sheep-derived type A Pasteurella multocida strain was ≥ 90%;

[0017] Sheep were immunized with 2.0 ml of inactivated vaccine and then 1.0 × 10 10 When sheep were challenged with CFU sheep-derived type A Pasteurella multocida strain, the sheep protection rate was ≥80%.

[0018] The technical scheme also provides a method for preparing a Pasteurella multocida vaccine containing a sheep-derived type A Pasteurella multocida strain, comprising the following steps performed in sequence:

[0019] S1: performing three-step fermentation culture on the sheep-derived type A Pasteurella multocida strain to obtain a fermentation liquid;

[0020] S2: The fermented bacterial liquid is concentrated and inactivated with formaldehyde to obtain an inactivated antigen bacterial liquid;

[0021] S3: The inactivated antigen bacterial solution and the aluminum hydroxide gel adjuvant are mixed in a volume ratio of 5:1 to prepare an inactivated vaccine.

[0022] Further, in S1, the three-step fermentation culture is:

[0023] First-level seed propagation: inoculate the sheep-derived type A Pasteurella multocida strain into Martin broth medium, and culture it at 37°C and 120r / min for 10 hours; then streak inoculate it on Martin agar plate and culture it at 37°C for 18 hours; then inoculate the formed colony on Martin agar slant and culture it at 37°C for 10 hours to form first-level seeds through culture; preferably, the first-level seeds are stored at 2-8°C, and the storage period of the first-level seeds does not exceed 7 days;

[0024] Secondary seed propagation: rinse the primary seeds on the slant with Martin broth medium containing 0.2% bovine lysed blood, and then culture the bacteria with Martin broth medium containing 0.2% bovine lysed blood at 37°C, 120r / min, and 10h; after the bacteria pass the purity test, obtain the secondary seed solution; preferably, the secondary seed solution is stored at 2-8°C, and the storage period of the secondary seed solution does not exceed 4 days;

[0025] Preparation of fermentation liquid: using Martin broth medium containing 0.2% bovine lysed blood cells to culture the secondary seed liquid at 37°C, 120r / min, for 10h to obtain fermentation liquid; preferably, the volume percentage of the secondary seed liquid added to the Martin broth medium is 1-2%;

[0026] In S2, the fermented bacterial solution is centrifuged at 9000 r / min and 4° C. for 20 min, and the obtained bacterial precipitate is resuspended with PBS at pH 7.2-7.4 to obtain a concentrated bacterial solution; formaldehyde is added at 0.15% of the volume of the concentrated bacteria, and the solution is inactivated at 37° C. for 18-24 h; during the inactivation treatment, the solution is intermittently shaken 10-12 times, and the shaking treatment is performed once at 120 r / min and 1 min / time every 2 h to prepare an inactivated antigen bacterial solution;

[0027] In S3, the inactivated antigen and bacteria solution is first stirred at 50-100 r / min for 5 minutes, and then the aluminum hydroxide gel adjuvant is added and stirred for 15-20 minutes to obtain the inactivated vaccine.

[0028] In summary, the principle and beneficial effects of this technical solution are:

[0029] In view of the current shortage of Pasteurella multocida type A vaccine for sheep, the applicant successfully isolated and identified a Pasteurella multocida type A strain from sheep (named Pm-ALa) from clinical samples. Whole genome sequencing and in-depth characteristic analysis were performed on the strain, and the results showed that it has unique genetic characteristics, which are different from all strain records in the NCBI database. Specifically, the Pm-ALa strain lacks two deoxyribonucleotide GG bases at positions 1315 and 1316 of the Lon gene, resulting in a gene frameshift mutation and premature termination, ultimately forming a truncated Lon protease. This mutation gives the Pm-ALa strain a unique identification mark.

[0030] As an ATP-dependent proteolytic enzyme, Lon protease is involved in the degradation of abnormal proteins in cells and the specific regulation of various regulatory proteins, playing a key role in the pathogenic mechanism of pathogens. Therefore, the Pm-ALa strain is not only suitable as a candidate strain for vaccine production, but also has important research value due to its unique genetic characteristics.

[0031] Based on the above findings, this technical solution has established an efficient and stable method for preparing Pm-ALa strain vaccines through a series of optimized culture and fermentation process studies. At the same time, based on a comprehensive understanding of the characteristics of the strain, a series of quality control test methods suitable for the product have been developed. In addition, through rigorous safety and efficacy tests, it has been confirmed that the vaccine exhibits excellent safety and immune protection effects in both alternative animal models and target host animals.

[0032] In summary, the Pm-ALa strain and its derivative vaccine products show significant application potential, which is expected to alleviate the current problem of insufficient vaccine supply and provide valuable resources for the study of Pasteurella multocida. This research result is of great significance for the prevention and control of diseases caused by this pathogen and opens up a new path for future vaccine research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of PCR identification of Pasteurella multocida serotype A in Example 1 (M.DL-2000 Marker; -. negative control; +. positive control; 1-6. isolated strains).

[0034] Figure 2 This is the biochemical identification result of the sheep-derived Pasteurella multocida Pm-ALa strain in Example 1.

[0035] Figure 3 This is a diagram of pathological changes in the lungs of sheep that became ill after the virus attack in Example 5.

[0036] Figure 4This is the PCR identification result of the blank control diseased sheep after the virus attack in Example 5 (M.DL-2000Marker; -. negative control; +. positive control; 1-5: strains isolated from the lungs of 5 blank control sheep). DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0038] Example 1: Isolation and Identification of Pasteurella multocida Pm-ALa Strain from Sheep

[0039] (1) Reagents and equipment

[0040] Martin broth and Martin agar were purchased from Zhonghai Biotechnology Co., Ltd.; bovine whole blood with lysed blood cells was purchased from Hohhot Caoyuan Lvye Bioengineering Materials Co., Ltd.; calf serum was purchased from Inner Mongolia Aopsay Biotechnology Co., Ltd.; DNA Marker was purchased from Beijing Solebow Technology Co., Ltd.; agarose was purchased from Oxoid Company; bacterial DNA extraction kit and 2×Taq PCR MasterMixⅡ were purchased from Tiangen Biochemical Technology (Beijing Co., Ltd.).

[0041] The gel imaging system was purchased from Bio-Rad Laboratories-Segart; the conventional gradient PCR instrument and nucleic acid electrophoresis instrument were both purchased from Bio-Rad; the ultracentrifuge was purchased from Eppendorf, Germany; the vortex mixer (model XW-80A) was purchased from Shanghai Qingpu Huxi Instrument Factory; the ultrapure water instrument (model Milli-Q) was purchased from Millipore, France; and the bacterial DNA extraction kit was purchased from Tiangen.

[0042] (2) Sample collection and isolation of Pasteurella multocida in sheep

[0043] Six cotton swabs were collected from the deep nasal cavity of sick goats with bronchial symptoms in a goat farm in Rongchang District, Chongqing. A nasal swab was collected from the deep nasal cavity of each sick goat with a sterile cotton swab and placed in a 5ml centrifuge tube with physiological saline for later pathogen molecular detection and bacterial isolation and identification. All collected samples were placed in a refrigerated sampling box and transported to this laboratory.

[0044] The collected samples were plated on Martin agar containing 4.0% calf serum and cultured in a 37°C constant temperature incubator for 18 to 24 hours. Then a single colony was picked and inoculated into Martin broth containing 4.0% calf serum and cultured at 37°C constant temperature shaking (160r / min) for 16 to 18 hours for bacterial growth. Pinpoint-sized grayish white, transparent or translucent dew-shaped colonies grew on Martin agar medium containing 4.0% calf serum. The purified colonies were gram-stained as single-morphological gram-negative coccobacilli.

[0045] (3) Extraction and identification of bacterial genomes

[0046] Take the above-mentioned enriched bacterial solution and use the Tiangen Bacterial DNA Extraction Kit to extract the bacterial genome. Take the above-extracted bacterial genomic DNA as the template for PCR amplification, and use the specific primers for identifying the serotype of Pasteurella multocida for PCR amplification. The specific primer information is shown in Table 1. The primers are synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. Amplification system (20.0μl): MasterMix premixed enzyme 10.0μl, upstream and downstream primers 1.0μl each, DNA template 2.0μl, sterile water 6.0μl. PCR amplification program: 95℃ pre-denaturation for 5min; 94℃ denaturation for 1min, 58℃ annealing for 45s, 72℃ extension for 1min, a total of 35 cycles, and finally 72℃ final extension for 10min. Take the PCR product and electrophoresed it in 1.5% agarose gel, and observe the results with a gel imaging system. Take the extracted bacterial genomic DNA as a template and amplify it using the specific PCR detection method for identifying the capsule serotype of Pasteurella multocida. The results showed that among the 6 strains of Pasteurella multocida, 3 were identified as type A. Figure 1 .

[0047] Table 1: Primer sequence information for identification of different serotypes of Pasteurella multocida

[0048]

[0049]

[0050] (4) Pathogenicity test

[0051] The three A-type isolated strains were inoculated into Martin broth containing 4.0% calf serum and cultured at 37°C with shaking (160 r / min) for 16 h. The bacteria were counted and diluted in 10-fold gradients to prepare 5.0×10 6 ~5.0×10 1CFU / ml of bacterial solution, 0.5ml of each dilution was taken and inoculated into 5 Kunming mice by intraperitoneal injection, and sterilized Martin broth was inoculated as a control. After inoculation, the mice were isolated and raised, and the incidence and death of the mice were observed. The median lethal dose was calculated according to the Reed-Muench method. The dead mice were dissected, the gross lesions were observed, and Pasteurella multocida was isolated.

[0052] Healthy Kunming mice were challenged with three isolates of Pasteurella multocida. The number of mice that died at each dilution was counted, and the LD of each isolate to mice was calculated according to the Reed-Muench method. 50 , respectively 5×10 5 CFU / 0.5mL, 5×10 2 CFU / 0.5mL, 5×10 3 CFU / 0.5mL. Immediate autopsy of mice after death showed that the liver of mice was enlarged and had bleeding spots, the intestinal mucosa had bleeding spots, and the lung lesions were not obvious. Bacterial isolation obtained Pasteurella multocida. No mice were morbid or died in Martin broth control, and no lesions were found in the dissected organs, and no bacteria were isolated from the organs. The results of the median lethal dose showed that the second strain had the strongest virulence, LD 50 5×10 2 CFU can be used as a subsequent vaccine candidate strain and named Pm-ALa strain.

[0053] Relevant data showed that Pasteurella multocida can decompose and utilize sucrose, mannitol, sorbitol, maltose, galactose, glucose, lactose, xylose, raffinose and fructose, but cannot decompose and utilize lactose, dulcitol and simon's citrate. OF test was fermentation type, hydrogen sulfide was negative, and VP test was negative. The results of biochemical culture test showed that the culture characteristics of the isolated bacteria Pm-ALa strain were consistent with those of Pasteurella multocida (such as Figure 2 , as shown in Table 2).

[0054] Table 2: Biochemical identification of Pasteurella multocida Pm-ALa strain in sheep

[0055]

[0056]

[0057] (5) Colony count and seed preservation

[0058] Based on the above bacterial isolation, serotype PCR identification and pathogenicity studies, the isolated strain with strong proliferation ability and virulence effect was selected as the vaccine strain and named Pm-ALa strain. The Pm-ALa strain was inoculated into 100 ml of Martin broth containing 4.0% calf serum at a ratio of 2.0%, and cultured overnight at 120 r / min at 37°C for about 18 hours. The colony count was counted on a Martin agar plate containing 4.0% calf serum, and the CFU was about 3.0×10 9 CFU / ml, and the Pm-ALa strain was packaged and freeze-dried to preserve it as the original strain. The Pm-ALa strain is now preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration.

[0059] The specific deposit information of Pm-ALa strain is as follows:

[0060] Deposit number: CGMCC NO.32900;

[0061] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;

[0062] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; Tel: 010-64807355);

[0063] Classification and nomenclature: Pasteurella multocida;

[0064] Collection date: 2024.12.02.

[0065] The Pm-ALa strain was sent to a gene company for whole genome sequencing. The sequencing results showed that the Pm-ALa strain had unique properties (different from all strains reported by NCBI). Two deoxyribonucleotide GG bases were missing at positions 1315 and 1316 of its Lon gene, and the gene frameshifted and terminated prematurely. The full length of the Lon gene of the Pm-ALa strain discovered in this scheme was 1323bp, and the nucleic acid sequence was SEQ ID NO.11. The insertion of the above nucleotides resulted in the truncation of the Lon protease, with a total of 440 amino acid residues, and the protein sequence was SEQ ID NO.12. The protein sequence expressed by the Lon gene of the wild-type Pasteurella multocida can be found in the database (NCBI, ID: WP_064969000.1), with a total of 596 amino acid residues. Compared with WP_064969000.1, only the first 438 amino acid residues of the Lon protease in this scheme are consistent with the wild-type protein, and the sequence after the 440 amino acid residues of the Lon protease in this scheme is completely missing, that is, the protein sequence of 440-596 is completely missing in the Lon protein of the Pm-ALa strain in this scheme. The mutation of the Lon gene of the Pm-ALa strain can be used for the specific identification of the strain.

[0066] SEQ ID NO.11:

[0067]

[0068] SEQ ID NO.12:

[0069] VNPSFLAEKALSWQELRPELQLTELPHSSLDFFALQQRASAAVNQFLHNPHRHLCVLKADEQGEYAHLLEQFVARQQQHDDKVHGVQYFVEQGDSFSFPRIFMEVAQNQN DNFAAKRKVASALYVDQFQLLGSIRIHPSSQEIQLNPGLVHQLNGGVLILGVGSLINQFDLWIRLKQLLLTQRFDWYSAHPFKALPCDIPSYPLSLKVILLGNREELATL AELDADLYDLADYSEIESYYAIESAEQRMKWAEYVQSMASYHQLPQLTLDGLNKLYQLLVRESEDRAFVSISPLKLKAILSGSAILKQKDTNALLSAVDFEQFFQQKEMQ QSFLKEHTYSDILHEQVYVATEGDIVGQINGLSVIEYPGTPMAFGEPSRISCIVQFGDGEVVDVERKSELAGNIHGKSIMIAQACLAHTLGFPSQLPFSASLVFEQSYRN.

[0070] Lon protease is an ATP-dependent protease that can degrade abnormal proteins and specifically regulate many regulatory proteins, and is closely related to the pathogenic mechanism of pathogens. The results of this study show that in the absence of the Lon protease protein sequence, Pasteurella can still maintain virulence and immunogenicity. Subsequent experimental studies of the present invention have proved that the strain can be used for vaccine production and produce good immune effects. In addition, this specific natural gene mutation can also be used for the specific identification of the strain.

[0071] In addition, in the prevention and control strategy based on immunization, traditional vaccines have played an important role in blocking the transmission and spread of Pasteurella multocida-related diseases within and between animal groups. However, in practice, it was found that vaccinated animals and naturally infected animals could not be distinguished. Both vaccination and natural infection will produce antibodies. Researchers cannot distinguish whether the production of antibodies is due to vaccination or natural infection, and thus cannot determine the effectiveness of vaccine immunity. In addition, animals in the same group contain both immunized animals and susceptible and infected animals. If they cannot be effectively distinguished, it will be difficult to achieve isolation and prevention of animals. In order to effectively detect whether antibodies are produced by vaccine immunity or natural infection, strains with gene deletion characteristics are usually prepared for vaccine preparation, which is a conventional operation method in the prior art. For example, the document "Zhang Fuxian, Development of colloidal gold test strips for distinguishing animals naturally infected with brucellosis from animals immunized with vaccines, Chinese Animal Husbandry and Veterinary Medicine, 2009" mentioned: BP26 protein was used as a detection antigen (T line) to develop a colloidal gold rapid test strip; the established test strip was used to detect the Brucella infection serum of mice, and mice infected with bovine Brucella S19 and immunized with the BP26-deficient vaccine strain YZ_2 could be successfully identified. Since the BP26-deficient vaccine strain YZ_2 does not contain BP26 protein, the mice immunized with the vaccine do not contain anti-BP26 antibodies. However, BP26 protein exists in natural bovine Brucella, and the antibodies produced by natural immunity contain anti-BP26 antibodies. Therefore, the identification of natural immunity and specific vaccine immunity can be achieved by the missing gene (protein). This technical solution discovered a Lon gene-deficient strain with high virulence that can be used for animal immunization. At the same time, due to the natural absence of this gene (protein), it is possible to distinguish between natural immunity and specific vaccine immunity based on this point, which can more effectively evaluate the effectiveness of vaccines and achieve effective distinction and isolation of immune animals, susceptible animals and infected animals.

[0072] More specifically, when distinguishing antibodies produced by natural infection and vaccine immunization, we can use Lon protein (preferably, a protein sequence fragment of 438-596) as an antigen, contact it with the serum of naturally infected animals or vaccine-immunized animals, and distinguish the two animals through the interaction of antigen and antibody. For naturally infected animals, since the wild-type strain has a complete Lon protein, the animal serum will contain a series of antibodies that bind to the complete Lon protein (especially antibodies that bind to the protein sequence fragment of 438-596); therefore, using Lon protein (preferably, a protein sequence fragment of 438-596) as an antigen will bind to the above antibodies. For vaccine-immunized animals (e.g., vaccines prepared by inactivating Pm-ALa strains), since Pm-ALa strains lack the 438-596 fragment of Lon protein, a series of antibodies that bind to the 438-596 fragment of Lon protein will not be produced in the animal serum; therefore, when the Lon protein (preferably, the 438-596 protein sequence fragment) is used as an antigen for detection, no relevant antibodies bind to the above antigen in the serum samples of vaccine-immunized animals. Due to the above differences, the production of antibodies by natural infection and vaccine immunization can be distinguished.

[0073] The Lon protein (preferably the missing 438-596 fragment) used as an antigen can be obtained by artificial synthesis or induced expression of corresponding transgenic engineering bacteria. Using strains with missing genes (proteins) to identify antibodies produced by natural infection and vaccine immunity is a conventional method in the prior art and is also one of the major application values ​​of gene-deficient bacterial strains. The main difficulty is how to obtain gene-deficient strains with good immune effects. The results of this technical solution solve the above difficulties.

[0074] In the prior art, a gene mutant strain lacking a certain protein is often obtained by artificial gene mutation. However, whether the gene mutant strain is suitable for vaccine preparation and related identification processes requires further study. For example, in the document "Wang Shuya, Construction and Biological Characteristics of Pasteurella multocida Lon Deficient Mutant, Northeast Agricultural University, Master's Thesis", a Pasteurella multocida Lon Deficient Mutant was constructed.

[0075] First, the main purpose of this technical solution is to study the effect of the Lon gene on Pasteurella multocida, and the Lon deletion mutant was not used to prepare the vaccine. From this technical solution, the skilled person in the art cannot know the immunogenicity of the Lon deletion mutant, nor whether it can be used for vaccine preparation.

[0076] Secondly, the process of constructing the Lon-deficient mutant of Pasteurella multocida is relatively complicated. The ΔLon mutant of Pasteurella multocida (deficient in Lon) was constructed using forward homologous recombination technology. The Lon-deficient strain Pm-ALa in this scheme was screened from naturally infected animals, not constructed by genetic engineering technology.

[0077] Again, in terms of safety, genetically engineered strains also have some disadvantages compared to natural strains. Genetic engineered strains may undergo gene reversion mutations during the passage process, resulting in the restoration of the original knocked-out gene function, which may increase safety risks. Although, in the study of ΔLon mutant bacteria, the stability of the bacteria after 20 generations of passage is acceptable, this is far from enough for the Pasteurella multocida strains used in vaccine production, and the stability of genetically engineered strains is far inferior to that of natural strains obtained by natural screening. At present, there is limited understanding of the long-term health effects of vaccines prepared by genetically engineered strains, while natural strains isolated from diseased animals do not have the above problems. Genetically engineered strains are obtained by artificially manipulating one or more genes, and there are complex interactions and regulatory relationships between genes in many pathogens. Simply manipulating one or more genes may not accurately predict the overall effect, because other compensatory mechanisms may be activated, affecting the safety and effectiveness of the final product. The natural strains obtained by screening are strains selected by nature, and the probability of the above problems is relatively small.

[0078] Finally, it is more difficult to prepare genetically engineered strains into vaccine products than natural strains. In the transition from laboratory scale to industrial production, the performance of genetically engineered strains may change, including growth characteristics, antigen production, and sensitivity to environmental conditions, which increases the difficulty of large-scale production. Compared with genetically engineered strains, the natural strains obtained by screening are relatively less difficult to achieve large-scale production. Since genetically engineered strains involve gene editing technology, such vaccines need to undergo strict regulatory review to ensure their safety and effectiveness, and meet ethical standards. Different countries and regions have different regulatory policies for genetically modified biological products, which also increases the product development cycle and cost. The application of gene editing technology may cause public concern about safety and ethical issues, affecting the acceptance of vaccines.

[0079] In summary, compared with artificially prepared gene-deficient strains, natural gene-deficient bacterial strains have advantages in preparation difficulty (lower difficulty and lower cost) because they avoid complex transgenic operations and the introduction of exogenous nucleic acid vectors. In addition, natural gene-deficient strains have better genetic stability and better strain safety (transgenic operations have safety issues), and are more suitable for the preparation of corresponding vaccines. In the prior art, there is no natural Pasteurella multocida with Lon deficiency, and the Pm-ALa strain discovered in this technical solution has good immunogenicity, and the deficiency of Lon can be used for specific detection.

[0080] Example 2: Preparation of sheep-derived Pasteurella multocida Pm-ALa strain vaccine

[0081] In this embodiment, the sheep Pasteurella multocida strain Pm-ALa isolated in the above embodiment 1 is used to prepare an inactivated sheep Pasteurella multocida vaccine, which specifically includes the following steps:

[0082] (1) Preparation of inactivated vaccines

[0083] (1.1) Fermenting and culturing the sheep-derived type A Pasteurella multocida Pm-ALa strain to obtain a fermentation solution, specifically:

[0084] First-level seed propagation: Inoculate 200 μL of freeze-dried strains of Pasteurella multocida Pm-ALa strain from sheep in 10 ml of Martin broth medium, incubate at 37°C, 120 r / min for 10 hours, then streak inoculate on Martin agar, incubate at 37°C for 18 hours, select typical colonies under a microscope, inoculate several Martin agar slopes, incubate at 37°C for 10 hours, and use as first-level seeds. Store at 2-8°C, and the shelf life shall not exceed 7 days.

[0085] Secondary seed propagation: Take a primary seed, use 4.0ml of Martin broth medium containing 0.2% bovine lysed blood to wash off the bacterial moss on the Martin agar slope, inoculate it into 200ml of Martin broth medium containing 0.2% bovine lysed blood, place it at 37℃, 120r / min, culture for 10h, and after passing the purity test, store it at 2-8℃ as secondary seed.

[0086] Preparation of fermentation liquid: The liquid culture is cultured in a fermenter to culture Pasteurella multocida liquid. Martin broth medium is sterilized at 116°C for 30 minutes by high pressure. When the temperature of the medium is cooled to about 37°C, bovine lysed blood is added to make the bovine lysed blood content in the medium reach 0.2% (v / v). Secondary seed liquid is added at 1% to 2% (v / v) of the total amount of the medium, mixed evenly, and cultured at 37°C, 120r / min for 10 hours.

[0087] After the bacterial culture is completed, samples are taken for purity inspection according to the appendix of the current "Pharmacopoeia of the People's Republic of China". It should be pure, and samples are taken for live bacteria count at the same time.

[0088] (1.2) Concentrating and inactivating the fermented bacterial solution obtained in step (1.1) to obtain an inactivated antigen bacterial solution, specifically:

[0089] Concentration of fermentation broth: The fermentation broth was concentrated by centrifugation at 9000 r / min, 4°C, for 20 min. The bacterial precipitate was then collected and resuspended in sterile PBS (phosphate buffer) at pH 7.2-7.4 to prepare a 10-fold concentrated broth.

[0090] Inactivation of fermented bacterial solution: add formaldehyde solution at 0.15% (v / v) of the concentrated bacterial solution, inactivate at 37°C for 18-24h, shake intermittently 10-12 times (shake once every 2h, 1min / time, 120r / min), and prepare inactivated antigenic bacterial solution. Inactivation test is carried out according to the appendix of the current "Pharmacopoeia of the People's Republic of China", and there should be no sterile growth.

[0091] (1.3) The inactivated antigenic bacteria solution obtained in step (1.2) was mixed with the aluminum hydroxide gel adjuvant in a volume ratio of 5:1 to prepare an inactivated vaccine, specifically:

[0092] First add the inactivated antigen solution to the emulsification tank, stir at low speed (50-100r / min) for 5 minutes, then slowly add aluminum hydroxide gel adjuvant (in accordance with the quality standard of aluminum hydroxide gel 3603 of the 2020 edition of the Pharmacopoeia of Veterinary Medicine, Part III), stir for 15-20 minutes to fully mix. The final vaccine contains no less than 1.0×10 Pm-ALa strain antigen. 10 CFU / ml. According to the current appendix of the "Pharmacopoeia of Veterinary Medicine of the People's Republic of China", sterile growth should be absent.

[0093] (1.4) Packaging

[0094] After passing the above sterility test, quantitative packaging should be carried out. Stirring should be done at any time during packaging to ensure uniform mixing. Stoppering and sealing should be done. Labeling should be applied. Store at 2-8℃.

[0095] According to the above method steps, this embodiment uses the Pm-ALa strain to prepare three batches of inactivated sheep Pasteurella multocida vaccines (vaccine batch numbers: 2401, 2402, 2403).

[0096] Example 3: Safety and efficacy of the vaccine in KM mice

[0097] (1) Safety test on mice

[0098] Five KM mice weighing 18-22 g were subcutaneously injected with 0.2 ml of the vaccine and observed for 10 days. All mice were alive and well.

[0099] (2) Immune efficacy test on mice

[0100] Thirty healthy KM mice were randomly divided into three groups, each with 10 mice. The three batches of vaccines (2401, 2402, and 2403) prepared in Example 2 were inoculated subcutaneously on the back of the KM mice at a dose of 0.1 ml. Ten control KM mice with the same conditions were inoculated with sterilized PBS in the same way as controls. 21 days after inoculation, the 30 immunized KM mice and the 10 control KM mice were challenged with Pasteurella multocida (Pm-ALa strain, challenge dose of 5.0×10 3 CFU), and then observed for 7 consecutive days to observe the death of KM mice and determine the protection status. The test results are shown in Table 3 below.

[0101] Table 3: Results of efficacy test on KM mice

[0102] Vaccine batch number Immunization dose (ml) Number of KM mice The number of challenge bacteria (CFU) Protection 2401 0.1ml 10 <![CDATA[5.0×10 3 ]]> 9 / 10 2402 0.1ml 10 <![CDATA[5.0×10 3 ]]> 9 / 10 2403 0.1ml 10 <![CDATA[5.0×10 3 ]]> 10 / 10 Control group 0.1ml 10 <![CDATA[5.0×10 3 ]]> 0 / 10

[0103] From the results in Table 3 above, it can be seen that after the KM mice were challenged with the Pasteurella multocida Pm-ALa strain, the 2401 and 2402 batches of vaccines showed an immune protection effect of 9 / 10, and 2403 showed an immune protection effect of 10 / 10. After the control KM mice were challenged with Pasteurella multocida, all 10 KM mice in the group died. The above results show that the inactivated Pasteurella multocida vaccine prepared by the present invention can effectively protect the immune KM mice against the challenge strain after inoculation of KM mice, and the immune effect of each batch of inactivated vaccine is stable.

[0104] Example 4: Safety test of inactivated sheep Pasteurella multocida (Pm-ALa strain) vaccine

[0105] In this example, three batches of inactivated Pasteurella multocida vaccines prepared in Example 2 (vaccine batch numbers: 2401, 2402, 2403) were tested for safety on healthy susceptible sheep (about 3 months old). The specific operation is as follows.

[0106] (1) Safety study of a single-dose vaccination

[0107] 20 healthy susceptible sheep were randomly divided into 4 groups as test sheep, 5 in each group, and the three batches of vaccines were inoculated into one group of test sheep through the neck muscle at a single dose of 2.0 ml (1 head), and 5 healthy susceptible sheep with the same conditions were inoculated with sterilized PBS in the same way as controls. The clinical manifestations of the test sheep and the control sheep were observed, and the body temperature was measured at the same time every day for 7 days. The observation results are shown in Table 4 below.

[0108] Table 4: Safety observations of a single dose vaccination of healthy susceptible sheep

[0109]

[0110] The results in Table 4 above show that after a single dose of three batches of vaccines prepared using the Pm-ALa strain, the body temperature, mental state, respiratory condition, appetite, etc. of the test sheep in each group during the observation period were normal, no redness, swelling or nodule formation was observed at the injection site, and the immunized sheep did not show clinical symptoms of Pasteurella multocida disease, and there was no significant difference from the control group.

[0111] (2) Safety study of repeated single-dose vaccination

[0112] 20 healthy susceptible sheep were randomly divided into 4 groups as test sheep, 5 in each group, and the three batches of vaccines were inoculated into one group of test sheep through the neck muscle with a single dose of 2.0 ml (1 serving), and 5 healthy susceptible sheep with the same conditions were inoculated with sterilized PBS in the same way as the control. 14 days after the first inoculation, the sheep were inoculated again in the same way and route. After repeated inoculation, the clinical manifestations of the immunized sheep and the control sheep were observed, and the body temperature was measured at the same time every day, and the observation was continued for 14 days. The observation results are shown in Table 5 below.

[0113] Table 5: Safety observation results of single-dose repeated vaccination

[0114]

[0115] The results in Table 5 above show that after repeated single-dose inoculation of three batches of vaccines prepared using the Pm-ALa strain, the body temperature, mental state, respiratory condition, appetite, etc. of the test sheep in each group during the observation period were normal, no redness, swelling or nodule formation was observed at the injection site, and the immunized sheep did not show clinical symptoms of Pasteurella multocida disease, with no significant difference from the control group.

[0116] (3) Safety test of a single overdose vaccination

[0117] 20 healthy susceptible sheep were randomly divided into 4 groups as test sheep, 5 in each group, and the three batches of vaccines were inoculated into one group of test sheep through the neck muscle with an overdose of 4.0 ml (2 doses), and 5 healthy susceptible sheep with the same conditions were inoculated with sterilized PBS in the same way as controls. The clinical manifestations of the immunized sheep and the control sheep were observed, and the body temperature was measured at the same time every day for 7 days. The observation results are shown in Table 6 below.

[0118] Table 6: Observation results of safety trial of one overdose vaccination

[0119]

[0120] The results in Table 6 above show that after one overdose inoculation of three batches of vaccines prepared using the Pm-ALa strain, the body temperature, respiratory condition, mental state, appetite, etc. of the immunized sheep in each group during the observation period were normal, no redness, swelling or nodule formation was observed at the injection site, and the immunized sheep did not show clinical symptoms of Pasteurella multocida, with no significant difference from the control group.

[0121] As shown in Tables 4 to 6 above, the results of a single-dose inoculation test, a single-dose repeated inoculation test, and an overdose inoculation test of three batches of sheep Pasteurella multocida inactivated vaccines (2401, 2402, and 2403) prepared by the present invention using the Pm-ALa strain on healthy susceptible sheep all show that during the observation period, the body temperature, mental state, respiratory condition, appetite, etc. of each group of test sheep were normal, no redness, swelling, or nodule formation was observed at the injection site, no clinical symptoms of sheep Pasteurella multocida disease appeared, and there was no significant difference from the control group. It can be seen that the inactivated vaccine prepared by the sheep Pasteurella multocida Pm-ALa strain of the present invention has good safety and can be further used in clinical applications.

[0122] Example 5: Immunization efficacy of inactivated Pasteurella multocida vaccine against sheep of this species

[0123] This example uses three batches of inactivated Pasteurella multocida vaccines prepared in Example 2 (vaccine batch numbers: 2401, 2402, 2403) to conduct efficacy tests on healthy susceptible sheep (about 3 months old).

[0124] 20 healthy susceptible sheep were randomly divided into 4 groups, 5 in each group, and the three batches of vaccines were respectively inoculated into one group of experimental sheep through the neck muscle at a dose of 1 dose (2.0 ml). Another 5 healthy susceptible sheep with the same conditions were inoculated with sterile PBS in the same way as the control. 21 days after inoculation, 15 immunized sheep and 5 control sheep were challenged with Pasteurella multocida (Pm-ALa strain, challenge dose was 1.0×10 10CFU), followed by 7 consecutive days of observation, observation of clinical symptoms of immunized sheep and control sheep, measurement of body temperature at the same time every day, and autopsy of immunized sheep and control sheep on the 7th day after challenge, observation and record of lung pathological changes, and collection of lung tissue for bacterial isolation and PCR identification, analysis and determination of the incidence and protection of sheep. The test results are shown in Table 7 below.

[0125] Table 7: Results of efficacy tests on test sheep

[0126]

[0127] Note: “-” indicates that the corresponding symptom does not occur for this item, and “+” indicates that the corresponding symptom occurs for this item.

[0128] From the results in Table 7 above, it can be seen that after the test sheep were challenged with Pasteurella multocida, the 2401 and 2402 batches of vaccines showed an immune protection effect of 5 / 5, and 2403 showed an immune protection effect of 4 / 5. The organs of the immune-protected test sheep did not undergo obvious pathological changes, and the lungs of the autopsy showed no abnormalities. However, after the sheep in the control group were challenged with Pasteurella multocida, all 5 sheep in the group became ill, and the lungs of the autopsy showed obvious Pasteurella multocida lesions (such as Figure 3 As shown), the lesions were isolated and identified by PCR (as shown Figure 4 ), where 1 to 5 are PCR amplification bands of strains isolated from lung lesions of 5 blank control sheep (primers: SEQ ID NO.1 and SEQ ID NO.2). The above results show that the sheep Pasteurella multocida inactivated vaccine prepared by the present invention can effectively protect the immunized sheep against the attacking strain after immunization, without potential safety risks, and the immune effect of each batch of inactivated vaccine is stable.

[0129] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A sheep-derived Pasteurella multocida type A strain, characterized in that: Its accession number is CGMCC NO.32900.

2. The sheep-derived Pasteurella multocida type A strain according to claim 1, characterized in that: The Lon gene in the strain is mutated, and the nucleotide sequence of the mutated Lon gene is as shown in SEQ ID NO.

11.

3. The sheep-derived Pasteurella multocida type A strain according to claim 2, characterized in that: The mutated Lon gene encodes a truncated protein with an amino acid sequence as shown in SEQ ID NO.

12.

4. The sheep-derived Pasteurella multocida type A strain according to claim 1, characterized in that: The strain is isolated from sick goats with goat bronchial symptoms; the samples collected from the sick goats are first smeared on Martin agar containing 4.0% calf serum, and shake-cultured in a constant temperature incubator at 37°C for 16 to 18 hours, and then a single colony is picked and PCR is performed using serotype A specific primers of Pasteurella multocida to screen positive strains; the sequences of the serotype A specific primers of Pasteurella multocida are shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. The sheep-derived Pasteurella multocida type A strain according to claim 1, characterized in that: Its LD in mice 50 5×10 2 CFU.

6. Use of a sheep-derived type A Pasteurella multocida strain according to any one of claims 1 to 5 in the preparation of a vaccine.

7. A Pasteurella multocida vaccine comprising a sheep-derived type A Pasteurella multocida strain according to any one of claims 1 to 5, characterized in that: The sheep-derived Pasteurella multocida type A was inactivated using formaldehyde and then mixed with aluminum hydroxide gel adjuvant to obtain an inactivated vaccine.

8. The Pasteurella multocida vaccine containing a sheep-derived type A Pasteurella multocida strain according to claim 7, characterized in that: The number of Pasteurella multocida type A from sheep in the inactivated vaccine is ≥ 1.0 × 10 10 CFU / ml; Mice were immunized with 0.1 ml of inactivated vaccine and then 5.0 × 10 3 The survival rate of mice challenged with CFU sheep-derived type A Pasteurella multocida strain was ≥ 90%; Sheep were immunized with 2.0 ml of inactivated vaccine and then 1.0 × 10 10 When sheep were challenged with CFU sheep-derived type A Pasteurella multocida strain, the sheep protection rate was ≥80%.

9. A method for preparing a Pasteurella multocida vaccine containing a sheep-derived type A Pasteurella multocida strain according to any one of claims 1 to 5, characterized in that: The method includes the following steps in sequence: S1: performing three-step fermentation culture on the sheep-derived type A Pasteurella multocida strain to obtain a fermentation liquid; S2: The fermented bacterial liquid is concentrated and inactivated with formaldehyde to obtain an inactivated antigen bacterial liquid; S3: The inactivated antigen bacterial solution and the aluminum hydroxide gel adjuvant are mixed in a volume ratio of 5:1 to prepare an inactivated vaccine.

10. The method for preparing a Pasteurella multocida vaccine containing a sheep-derived type A Pasteurella multocida strain according to claim 9, characterized in that: In S1, the three-step fermentation culture is: First-level seed propagation: inoculate the sheep-derived type A Pasteurella multocida strain into Martin broth medium, and culture it at 37°C and 120r / min for 10 hours; then streak inoculate it on Martin agar plate and culture it at 37°C for 18 hours; then inoculate the formed colony on Martin agar slant and culture it at 37°C for 10 hours to form first-level seeds through culture; preferably, the first-level seeds are stored at 2-8°C, and the storage period of the first-level seeds does not exceed 7 days; Secondary seed propagation: rinse the primary seeds on the slant with Martin broth medium containing 0.2% bovine lysed blood, and then culture the bacteria with Martin broth medium containing 0.2% bovine lysed blood at 37°C, 120r / min, and 10h; after the bacteria pass the purity test, obtain the secondary seed solution; preferably, the secondary seed solution is stored at 2-8°C, and the storage period of the secondary seed solution does not exceed 4 days; Preparation of fermentation liquid: using Martin broth medium containing 0.2% bovine lysed blood cells to culture the secondary seed liquid at 37°C, 120r / min, for 10h to obtain fermentation liquid; preferably, the volume percentage of the secondary seed liquid added to the Martin broth medium is 1-2%; In S2, the fermented bacterial solution is centrifuged at 9000 r / min and 4° C. for 20 min, and the obtained bacterial precipitate is resuspended with PBS at pH 7.2-7.4 to obtain a concentrated bacterial solution; formaldehyde is added at 0.15% of the volume of the concentrated bacteria, and the solution is inactivated at 37° C. for 18-24 h; during the inactivation treatment, the solution is intermittently shaken 10-12 times, and the shaking treatment is performed once at 120 r / min and 1 min / time every 2 h to prepare an inactivated antigen bacterial solution; In S3, the inactivated antigen and bacteria solution is first stirred at 50-100 r / min for 5 minutes, and then the aluminum hydroxide gel adjuvant is added and stirred for 15-20 minutes to obtain the inactivated vaccine.

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