Pasteurella multocida capsule mutant strain and application thereof

Abstract

The invention discloses a pasteurella multocida capsule mutant strain and application thereof, and belongs to the field of microorganisms, the pasteurella multocida capsule mutant strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M 20242234. A mouse immune challenge test and a detection test of a Pm strain antibody detection indirect ELISA method show that the RPmF2019s has a better immune effect and has more advantages as a coating antigen of the Pm detection ELISA method.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a Pasteurella multocida capsule mutant strain. Background Art

[0002] Pasteurella multocida (Pm) is a small Gram-negative bacillus that can cause a variety of animal infections, and there are also reports of human infection. The Pm capsule is an important virulence factor, which mainly plays a role in bacterial protection and immune evasion. According to the different capsules, Pm is divided into five serotypes, A, B, D, E and F. The immune cross-protection between serotypes is weak. In addition, the immune protection effect of bacterial vaccines is generally inferior to that of vaccines for viral diseases. Sepsis and latent infections caused by Pm infection have always threatened the healthy development of my country's animal husbandry industry, especially in the rabbit industry. At present, the vaccine used for the prevention of this disease is still mainly bacterial inactivated vaccine. Many reports show that the immune protection effect of self-made vaccines is higher than that of commercial bacterial inactivated vaccines. The specific reason is still unclear, and it is likely related to many factors such as Pm variation, strain differences, and serotype. Early studies reported that different animals have a preference for the capsule serotype of Pm infected, but in recent years, different serotypes of Pm have been isolated and identified in the same livestock and poultry. Chinese scholars have isolated type A, D and F Pm strains in infected rabbits. In the context of strict control of drug abuse and reduction of antibiotic use in my country, with the development of animal husbandry, the research and development of new and efficient vaccines has always been a research hotspot, but the progress is relatively slow. Rapid and scientific serological monitoring technology research has important practical significance in Pm infection control and purification.

[0003] ELISA detection technology has broad prospects due to its good specificity and sensitivity, high throughput and commercialization. However, in the current research and application of ELISA detection technology for Pm, Pm bacterial antigens are used as coating sources or recombinant proteins of artificially expressed bacterial proteins are used as coating sources for detection. A certain protein as a coating source cannot fully reflect the situation of all bacterial protein antibodies in the test sample. Whether the bacterial antigen is intact or broken, it cannot solve the effect of capsule components on the coating effect and the biosafety risks caused by virulence during the storage and cultivation of strains before coating. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Pasteurella multocida capsule mutant strain RPmF2019s and application thereof.

[0005] The technical solution of the present invention is: a Pasteurella multocida capsule mutant strain RPmF2019s, which is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20242234.

[0006] The capsule mutant strain RPmF2019s described above is used as a coating antigen for ELISA detection of Pasteurella antibodies.

[0007] The application of the capsule mutant strain RPmF2019s described above in the preparation of Pasteurella multocida vaccine.

[0008] A kit comprising the capsule mutant strain RPmF2019s described above.

[0009] A Pasteurella multocida vaccine contains the inactivated capsule mutant strain RPmF2019s described above.

[0010] Through more than 50 continuous passages over a year, the capsule mutant strain RPmF2019s (strain collection number CCTCC M 20242234) of the wild strain PMF0 of Pasteurella multocida (Pm) was obtained. After nearly two years of continuous passage, the culture characteristics of the mutant strain RPmF2019s were stable. Compared with PMF0, the colony of RPmF2019s became smaller and the viscosity of the colony surface decreased; no obvious capsule was observed on the surface of the bacteria under transmission electron microscopy, and the quantitative results of the capsule showed that the capsule production was reduced; its growth rate slowed down, its resistance to salt, acid and alkali, and penetration was weakened, it was more sensitive to temperature, and its sensitivity to multiple drugs increased to varying degrees, and its virulence was greatly reduced. The attack test showed that the LD of PMF0 50 7.5×10 6 CFU, and up to 2.42×10 13 The RPmF2019s strain liquid with CFU still did not kill the experimental mice. This shows that the virulence of the strain is greatly weakened, the culture characteristics are relatively stable, and the biosafety risk is low. It is an ideal candidate strain for attenuated immunization and an antigen candidate for immunological detection methods.

[0011] Whole genome sequencing revealed that the RPmF2019s strain lacked a 4577bp fragment (including complete phyA and phyB genes and part of the fcbE fragment) in the phyA-phyB-fcbE segment compared with PMF0. qRT-PCR detection of virulence genes showed that the expression of virulence genes such as hexA, hexC, and PM0442 was significantly downregulated compared with PMF0, the expression of hexD and wzzE did not change significantly, and the expression of cya and oxyR was significantly upregulated. In RPmF2019s, the expression of outer membrane protein-related genes such as lolB, pal, and hemR was significantly downregulated compared with PMF0, the expression of metQ, plpE, and bam was significantly downregulated, the expression of lolA, lppB, and bamD decreased, and the expression of ompA did not change significantly. It was confirmed at the genetic level that the RPmF2019s strain was a capsule structure deletion mutant with significantly reduced virulence.

[0012] PMF0 and RPmF2019s inactivated vaccines were prepared for mouse immunization experiments. PMF0 and RPmF2019s were used as coating sources. The test results of anti-Escherichia coli mouse positive serum and anti-Salmonella mouse positive serum were negative, while the results of mouse anti-RPmF2019s serum and mouse anti-PMF0 serum were positive. The OD values ​​of mouse anti-RPmF2019s serum detected by ELISA with PMF0 as coating source and RPmF2019s as coating source were higher than the OD values ​​of corresponding mouse anti-PMF0 serum, indicating that RPmF2019s has a better immune effect. The OD values ​​of mouse anti-RPmF2019s serum and mouse anti-PMF0 serum detected by RPmF2019s as coating source were higher than the detection values ​​with PMF0 as coating source, indicating that RPmF2019s is more advantageous as coating source.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention obtains a Pm capsule-deficient variant strain through long-term artificial culture. The virulence of the strain is greatly weakened, the culture characteristics are relatively stable, and the biosafety risk is low. The strain is a candidate strain for an ideal attenuated strain for immunization and an antigen candidate for an immunological detection method.

[0015] Collection information:

[0016] The Pasteurella multocida capsule mutant strain RPmF2019s was deposited in the China Center for Type Culture Collection on October 17, 2024, with the deposit number CCTCC NO: M 20242234. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The colony morphology of PMF0 and RPmF2019s, PMF0 on the left and RPmF2019s on the right.

[0018] Figure 2 These are the staining microscopic images of PMF0 and RPmF2019s (1000×), A.PMF0 B.RPmF2019s.

[0019] Figure 3 Transmission electron micrographs of PMF0 and RPmF2019s (A, B, C are PMF0, D, E, F are RPmF2019s, A, D 6000×, B, E 12000×, C, F 40000×).

[0020] Figure 4 This is the growth curve of PMF0 and RPmF2019s.

[0021] Figure 5 Scatter plot for the linear regression equation.

[0022] Figure 6 Quantification of PMF0 and RPmF2019s capsule.

[0023] Figure 7 Differences in biofilm formation between PMF0 and RPmF2019s, A. Crystal violet staining, B. Biofilm quantification.

[0024] Figure 8 Transcriptional expression analysis of virulence-related genes in PMF0 and RPmF2019s, A-C. Transcriptional expression of genes related to capsule synthesis; D, E transcriptional expression of genes related to biofilm formation; F, G. Transcriptional expression of virulence-related genes Pm0442 and plp4; H, I. Transcriptional expression of genes related to iron transport; J-M. Transcriptional expression of genes related to LPS synthesis and transport; N. Transcriptional expression of genes related to outer membrane proteins.

[0025] Fig. 9 These are the salt resistance test results of PMF0 and RPmF2019s.

[0026] Fig.10 These are the anti-penetration test results of PMF0 and RPmF2019s.

[0027] Fig.11 These are the acid-base sensitivity test results of PMF0 and RPmF2019s.

[0028] Fig.12 These are the temperature sensitivity test results of PMF0 and RPmF2019s.

[0029] Fig.13This is the immune protection diagram of PMF0 and RPmF2019s.

[0030] Fig.14 Collinearity analysis of PMF0 and RPmF2019s, A. Whole-gene collinearity analysis, B. phyA-phyB-fcbE deletion segment, C. Non-coding region deletion segment.

[0031] Fig.15 The results of qRT-PCR identification of the missing gene. DETAILED DESCRIPTION

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0033] 1. Screening and identification of colony variants of rabbit-derived Pm isolate (PMF0)

[0034] A nutrient agar solid medium (containing 5% New Zealand newborn calf serum) was prepared, and a rabbit-derived Pasteurella multocida strain PMF0 was continuously subcultured in vitro according to the conventional plate streak method, and subcultured once every 5-12 days. After about 50 generations of continuous subculture, colonies smaller than PMF0 appeared on the solid medium, and translucent and tiny needle-shaped colonies appeared. They were identified as Pasteurella multocida by 16S rDNA PCR and named RPmF2019s. In addition, the continuous subculture characteristics of RPmF2019s were stable.

[0035] 2. Growth morphology and Gram staining microscopy of PMF0 and RPmF2019s

[0036] The PMF0 and RPmF2019s strains were streaked onto nutrient agar solid medium (containing 5% New Zealand calf serum) and incubated inverted in a 37°C constant temperature incubator for 24 h. The morphology of the two strains was observed and the purified single colonies were picked for Gram staining observation.

[0037] The colony morphology of PMF0 and RPmF2019s is as follows Figure 1 As shown in the figure, the colonies of PMF0 are translucent dew droplets, smooth and sticky. Although the colonies of PMF0 are also translucent, they are only the size of a needle tip and the surface is slightly dry. The Gram staining microscopy of PMF0 and RPmF2019s are shown in the figure. Figure 2 shown.

[0038] 3. Transmission electron microscopy observation of PMF0 and RPmF2019s

[0039] The negative staining method of phosphotungstic acid was adopted. The purified bacteria enrichment droplets were placed on a copper mesh covered with a carbon film, and allowed to stand for 10 minutes. The excess liquid was absorbed with filter paper, and 2% phosphotungstic acid (PTA, pH = 7.0) was added for staining for 10 minutes. After standing and drying, the bacterial morphology was observed using a transmission electron microscope (TEM). This step was completed by Chengdu Lilai Medical Experiment Center.

[0040] Transmission electron microscopy images of PMF0 and RPmF2019s are shown below: Figure 3 As shown, from the transmission electron microscopy image, it can be found that there are significant differences in the bacteria between PMF0 and RPmF2019s. The bacteria of PMF0 are relatively small, rough, and have grooves, while the bacteria of RPmF2019s are relatively large and smooth.

[0041] 4. Detection of biological characteristics of PMF0 and RPmF2019s

[0042] (1) Biochemical test

[0043] Inoculate the bacteria into the biochemical fermentation tube, seal it and insert it into the foam plate, culture it at 37℃ for 24h, observe the test results and record and analyze them according to the identification manual.

[0044] After the two strains were cultured in the logarithmic growth period, they were inoculated in microbiochemical identification tubes to identify the differences in biochemical reactions. Both strains were consistent with the biochemical characteristics of Pasteurella multocida. The results showed that the biochemical characteristics of RPmF2019s did not change relative to PMF0 after mutation.

[0045] (2) Drug sensitivity test

[0046] The Kirby-Barer disk diffusion method was used to screen the drug sensitivity of the isolated bacteria with reference to the CLSI Identification Manual (2021 edition).

[0047] The results of the drug sensitivity test are shown in Table 1. After the relative mutation of PMF0, RPmF2019s had mutations in resistance to amikacin, sulfamethoxazole, and tobramycin. Although the drug sensitivity results for cefotaxime, bacitracin, amoxicillin, penicillin, midecamycin, florfenicol, enrofloxacin, tetracycline, and gentamicin did not change, the diameter of the drug sensitivity zone changed significantly. There was no significant change in resistance to cefoperazone, norfloxacin, doxycycline, polymyxin B, co-trimoxazole, ceftriaxone, and chloramphenicol.

[0048] Table 1 Drug sensitivity results of PMF0 and RPmF2019s strains

[0049]

[0050] Note: R: resistant; I: intermediate; S: sensitive

[0051] 3. Growth curve determination

[0052] PMF0 and RPmF2019s were selected and cultured overnight in BHI culture medium (containing 5% New Zealand newborn calf serum), and the concentration of the bacterial solution was adjusted to 10 in PBS buffer. 7 CFU / mL were transferred to 100mL BHI (containing 5% New Zealand calf serum) liquid culture medium at a ratio of 1:100, and cultured at 37°C in a constant temperature shaking incubator at 120r / min. Every 1h, 3mL of bacterial solution was taken into a cuvette and the OD was measured by a spectrophotometer. 600 At the same time, 100 μL of bacterial solution was taken for gradient dilution and plate counting, and the in vitro growth curves of the three bacteria were recorded and drawn in real time.

[0053] The results are as follows Figure 4 As shown, PMF0 reached the logarithmic growth phase at around 3h, while RPmF2019s reached the logarithmic growth phase at around 4h, and the growth rate of PMF0 in the early period of the logarithmic growth phase was much higher than that of RPmF2019s. In addition, PMF0 reached the stable phase at around 9h, while RPmF2019s reached the stable phase at around 10h. In the end, the OD600 of PMF0 was larger than that of RPmF2019s. This indicates that the growth rate of RPmF2019s slowed down after the mutation compared to PMF0.

[0054] 4. Capsule quantitative test

[0055] (1) Pick a single colony of the three strains and transfer it to Martin liquid medium at 30°C to the logarithmic growth phase. Centrifuge at 10,000 r / min for 5 min, discard the supernatant, add sterile PBS for washing, centrifuge under the same conditions, repeat this step 2-3 times, aspirate 1 mL of PBS to resuspend the bacteria, and then plate and count after gradient dilution.

[0056] (2) Place the resuspended cells in a 42°C metal bath and incubate for 1-2 h. Dilutions are plated and counted.

[0057] (3) The incubated bacterial solution was centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was transferred to a sterile 1.5 mL EP tube. 100 μL of the supernatant was mixed with 900 μL of the capsule staining solution.

[0058] (4) Prepare the standard: Mix 0, 1, 2, 3, 4, 5, and 10 μg / 100 μL of hyaluronic acid with 900 μL of capsule stain. Add the sample and standard at a dose of 200 μg / well into a 96-well plate and measure the OD value by spectrophotometer. 640 The standard curve was drawn by measuring the absorbance of the cells and the OD value of the supernatant produced by the lysis of dead cells after treatment at 42℃ for 1h. 640The absorbance.

[0059] The results are as follows Figure 5 and Figure 6 As shown, according to the OD of standard hyaluronic acid (HA) solution 640 The capsule content and OD 640 The linear regression relationship is Y = 0.04598*X + 0.1561 (R 2 =0.9599), and the OD value was measured after the supernatant of PMF0 and RPmF2019s lysis and capsule staining solution after treatment at 42°C was mixed. 640 The value can be used to calculate the capsule content, and then the concentration of the bacterial solution obtained by coating the plate can be used to calculate the percentage of each 10 8 The amount of capsule contained in CFU bacteria. The average capsule content of PMF0 is 2.9882μg / CFU·10 -8 The average capsule content of RPmF2019s was only 0.2869 μg / CFU·10 -8 The capsule content of RPmF2019s was significantly decreased compared with that of PMF0.

[0060] 5. Biofilm quantitative test

[0061] (1) Single colonies of the three strains were picked and inoculated into 5 mL of Martin liquid medium at 30°C and 220 rpm for 12 h. After gradient dilution, the colonies were plated and counted.

[0062] (2) After counting, use BHI liquid medium to adjust the colony count to 1×10 8 CFU / mL, the diluted bacterial solution was added to a 48-well cell culture plate at a dose of 400 μL / well in a clean bench, and each strain was repeated three times. 400 μL / well of BHI liquid culture medium was added to the negative control group, and the plates were cultured in a constant temperature incubator at 30°C for 48 hours.

[0063] (3) Take out the cell culture plate, remove the solution from each well, add PBS and gently wash three times, invert and dry at room temperature, and add 200 μL of anhydrous methanol to each well for 30 min.

[0064] (4) Methanol was removed, and the cells were gently washed three times with PBS. The cells were dried at room temperature, and 200 μL of 1% crystal violet staining solution was added to each well for staining at room temperature for 30 min.

[0065] (5) Aspirate the staining solution, wash with PBS three times, add 200 μL / well of 33% glacial acetic acid, let stand at 37°C for 30 min, take out and place in a spectrophotometer to measure OD 640 The absorbance value at the place where the test was taken was recorded and analyzed.

[0066] The results are as follows Figure 7As shown, the biofilm production of RPmF2019s was significantly increased compared with PMF0.

[0067] 6. qRT-PCR detection of related virulence gene expression

[0068] The gene deletion of PMF0 and RPmF2019s led to changes in capsule content and virulence. To explore the primers for attenuated virulence at the transcriptional level, this experiment was based on the reported virulence factors such as capsule, LPS, outer membrane protein, biofilm formation, and iron transport of Pasteurella multocida. Primers were designed using Primer 5 according to the sequencing data. RNA of PMF0 and RPmF2019s was extracted and reversely transcribed into cDNA as a template for real-time fluorescence quantitative PCR detection. The primer sequences are shown in Table 2. Reaction system (25 μL): 2×SYBR Green HS Premix 12.5 μL, 0.5 μL of upstream and downstream primers, 2 μL of cDNA, and 9.5 μL of ultrapure water; reaction procedure: 95°C 5 min; 95°C 10 s followed by 60°C 30 s, 40 cycles; 95°C 15 sec, 60°C 60 sec, and 95°C 15 sec.

[0069] Table 2 Real-time fluorescence quantitative PCR primers

[0070]

[0071]

[0072]

[0073] The results are as follows Figure 8 As shown in the figure, in RPmF2019s, the expression levels of virulence genes such as hexA, hexC, PM0442, plp4, fecC, fecE, lpxD, kdsA and lptG were significantly down-regulated compared with PMF0, the expression levels of hexD and wzzE did not change significantly, and the expression levels of cya and oxyR were significantly up-regulated. In RPmF2019s, the expression levels of outer membrane protein-related genes lolB, pal, hemR, pcp, fbpC, fbpB, and ompW were significantly down-regulated compared with PMF0, the expression levels of metQ, plpE, bam, skp, bam, and ompR were significantly down-regulated, the expression levels of lolA, lppB, and bamD decreased, and the expression level of ompA did not change significantly.

[0074] 7. Median lethal dose (LD 50 ) determination

[0075] In the previous mouse pathogenicity pilot test, each mouse was injected with 2.42×10 13There was no mouse mortality in the CFU RPmF2019s group, indicating that RPmF2019s may cause latent infection or may not be lethal to mice. Therefore, only the PMF0 strain LD was tested. 50 Determination. The PMF0 strain culture was counted and diluted tenfold, and then the virulence test was carried out on experimental mice (Kunming mice). PMF0 grown to the logarithmic phase in BHI liquid medium was plated and counted. After counting, it was diluted 10 times into 5 concentration gradients corresponding to the colony content in Table 3. 60 Kunming mice (4 weeks old, 20-25g, half male and half female) were divided into 6 groups, and the virus was injected intraperitoneally at 100μL / dose. One group was injected with 100μL sterile PBS as a control. The mice were observed for one week, and the death symptoms and time of the mice were recorded. The LD was calculated according to the BLISS method. 50 .

[0076] Table 3 LD of PMF0 50 Test results

[0077]

[0078] The results are shown in Table 3. The LD of PMF0 strain 50 =7.5×10 6 CFU, indicating that the virulence of the mutant strain RPmF2019s is much lower than that of PMF0.

[0079] 8. Stress resistance test of PMF0 and RPmF2019s

[0080] (1) Salt resistance test

[0081] PMF0 and RPmF2019s were picked and cultured in BHI medium (containing 5% New Zealand newborn calf serum) overnight, and the concentration of the bacterial solution was adjusted to 10 in PBS buffer. 7 CFU / mL, inoculated into BHI culture medium containing 1, 1.5, 2, 2.5, 3, 3.5, and 4% NaCl at a ratio of 1:50, with the culture medium containing 0.5% NaCl as the control; after treatment, placed in a 37°C water bath, cultured overnight at 200 r / min for 12 hours, and the OD was measured 600 .

[0082] The results are as follows Fig. 9 As shown, the concentrations of both PMF0 and RPmF2019s decreased with increasing NaCl concentrations, and no bacteria survived in RPmF2019s when the NaCl concentration was 3% or above, indicating that the salt resistance of the RPmF2019s mutation was reduced compared to that of PMF0.

[0083] (2) Osmotic pressure test

[0084] PMF0 and RPmF2019s were selected and cultured overnight in BHI culture medium (containing 5% New Zealand newborn calf serum), and the concentration of the bacterial solution was adjusted to 10 in PBS buffer. 7 CFU / mL, inoculated into BHI culture medium containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mol / L KCl at a ratio of 1:50, with the culture medium containing 0 mol / L KCl as the control; after treatment, placed in a 37°C water bath shaker, cultured overnight at 200 r / min for 12 h, and the OD was measured 600 .

[0085] The results are as follows Fig.10 As shown in the figure, the number of bacteria surviving PMF0 and RPmF2019s decreased with the increase of KCl concentration, and at the same KCl concentration, PMF0 always had a higher bacterial concentration than RPmF2019s. And because no bacteria survived PMF0 at a KCl concentration of 0.5 mol / L, and no bacteria survived RPmF2019s at a KCl concentration of 0.4 mol / L, it was concluded that the mutation of RPmF2019s had a decreased ability to resist osmotic pressure compared to PMF0.

[0086] (3) pH sensitivity test

[0087] PMF0 and RPmF2019s were picked and cultured in BHI medium (containing 5% New Zealand newborn calf serum) overnight, and the concentration of the bacterial solution was adjusted to 10 in PBS buffer. 7 CFU / mL, inoculated into 5mL at pH 2, 3, 4, 5, 6, 7,

[0088] 8, 9, 10 BHI culture medium, of which the culture medium with pH 7 was used as the control; after treatment, the cells were placed in a 37°C water bath and cultured overnight at 200 r / min for 12 h, and the OD 600 .

[0089] The results are as follows Fig.11 As shown, the bacterial survival of PMF0 and RPmF2019s decreased with the increase of acidity or alkalinity, and at pH = 6, the bacterial survival of RPmF2019s was greater. At pH = 10, no bacteria survived in RPmF2019s while a small number of bacteria survived in PMF0, indicating that after the mutation, RPmF2019s had a slightly higher sensitivity to acid and alkali than PMF0.

[0090] (4) Temperature sensitivity experiment

[0091] PMF0 and RPmF2019s were picked and cultured in BHI medium (containing 5% New Zealand newborn calf serum) overnight, and the concentration of the bacterial solution was adjusted to 10 in PBS buffer. 7CFU / mL, inoculated into 5mL BHI culture medium at 1:50, and incubated in a water bath at 37, 40, 45, 50, 55, and 60℃ for 2h, with 37℃ for 2h as the control; after treatment, placed in a 37℃ water bath, cultured overnight at 200r / min, and the OD was measured 600 .

[0092] The results are as follows Fig.12 As shown, the bacterial survival of RPmF2019s after treatment at 50℃ for 2h was significantly reduced compared with that of PMF0, indicating that the mutation of RPmF2019s was more sensitive to temperature than that of PMF0. It is worth mentioning that the number of bacteria in RPmF2019s after treatment at 40℃ for 2h was increased compared with that at 37℃.

[0093] 9. Protective efficacy test of inactivated vaccines of PMF0 and RPmF2019s

[0094] (1) Preparation of inactivated vaccines

[0095] PMF0 and RPmF2019s cultured to the logarithmic growth phase were inoculated in 100mL of BHI liquid culture medium at a ratio of 1:50 for expansion culture. After dilution and counting, centrifuge at 4℃12000r / min for 10min, discard the culture medium, add appropriate PBS to resuspend the bacteria, add formaldehyde solution at a final concentration of 0.3%, place in a 37℃ constant temperature incubator for inactivation for 48h, and vortex mix every 2h to ensure complete inactivation of the bacteria. Then take 200μL of vaccine to coat the plate, and the inactivation is considered complete if no colonies grow. Aluminum hydroxide adjuvant is added at 1:60 and mixed at 4℃ for standby use. At the same time, Kunming mice are injected intraperitoneally, each inoculated with 0.3mL, and the reaction of the mice is observed for safety testing.

[0096] (2) Inactivated vaccine inoculation

[0097] Thirty Kunming mice (4 weeks old, 20-25 g, half male and half female) were vaccinated according to the dosage and strain in Table 4, and a second booster vaccination was performed 14 days after the first vaccination.

[0098] Table 4 Inactivated vaccine inoculation scheme

[0099]

[0100] (3) Serum antibody titer detection of inactivated vaccines

[0101] Two weeks after the first and second immunizations, blood was collected from the tail vein of the mice, and the serum was separated and tested by ELISA. The specific operation method of ELISA is as follows:

[0102] 1) Antigen coating: PMF0 and RPmF2019s whole bacterial proteins were diluted to a working concentration of 1 μg / 100 μL using a carbonate buffer solution at pH 9.6, and 100 μL / well were added to the ELISA plate for coating at 4°C overnight.

[0103] 2) The liquid in the plate was discarded, and the plate was washed three times with 0.05% PBST buffer. 200 μL / well of blocking solution was added, and the plate was blocked at 37° C. for 1 h. After the blocking was completed, the plate was washed three times.

[0104] 3) Serum was used as the primary antibody, diluted in proportion, and 100 μL / well was added to the ELISA plate, incubated at 37°C for 1 hour, and washed three times after incubation.

[0105] 4) Dilute goat anti-mouse IgG according to the instructions, add 100 μL / well to the ELISA plate, incubate at 37°C for 1 hour, and wash.

[0106] 5) Add 100 μL of TMB colorimetric solution to each well and let it stand at room temperature in the dark for 10 minutes.

[0107] 6) Add 100 μL 1 mol / L sulfuric acid to each well to terminate the reaction and measure the OD using a microplate reader. 450 Absorbance, 5 wells for each sample, repeated three times.

[0108] Testing has shown that PMF0 can stimulate mice to produce an antibody titer of 1:12800, and RPmF2019s can stimulate mice to produce an antibody titer of 1:25600, while providing mice with protection rates of 70% and 90% respectively, indicating that RPmF2019s has better immunogenicity than PMF0 and has better candidate advantages as a vaccine strain than PMF0.

[0109] (4) Determination of immune protection rate

[0110] In the previous toxicity test, each mouse was injected with 2.42×10 13 No mortality was observed in the experimental group with CFU RPmF2019s, indicating that RPmF2019s may cause latent infection or be non-lethal, so PMF0 was used to replace RPmF2019s for infection. PMF0 grown in BHI liquid medium to the logarithmic phase was plated and counted, and 7.5×10 6 Two weeks after the second challenge with CFU, the survival of mice was observed for one week and the survival curve was drawn. Fig.13 As shown, immunization with inactivated vaccines made with PMF0 and RPmF2019s can provide 70% and 90% protection rates for mice, respectively.

[0111] 10. Whole genome sequence analysis of PMF0 and RPmF2019s

[0112] The whole genome colinearity of the two bacterial strains was compared using mauve (20150226), and the missing parts were identified using nBLAST in NCBI.

[0113] The results are shown in Table 5. The sequencing showed that the PMF0 genome size was 2,491,596 bp, the CG content was 40.27%, and it contained 2395 open reading frames (ORFs) with an average length of 921.86 bp. The ORF sequence accounted for 88.61% of the total genome length; it contained 59 tRNAs, 19 rRNAs (including 7 5S rRNAs, 6 16S rRNAs, and 6 23S rRNAs), 28 ncRNAs; and it contained 5 CRISPRs sequences. The total genome size of RPmF2019s is 2,487,092bp, with a CG content of 40%. It contains 2,396 open reading frames with an average length of 919.99bp. The ORF sequence accounts for 88.63% of the total genome length; it contains 60 tRNAs, 19 rRNAs (including 7 5S rRNAs, 6 16S rRNAs, and 6 23S rRNAs), and 28 ncRNAs; it contains 5 CRISPRs sequences.

[0114] Table 5 Basic information of PMF0 and RPmF2019s genome sequences

[0115]

[0116] The whole genome sequences of the two strains were analyzed for colinearity, and it was found that RPmF2019s had two mutation deletions compared with PMF0. The nucleotide sequences of the deleted segments were identified by BLAST, and it was found that one of the deleted sequences was a non-coding region deletion, and the other deleted region included all segments of phyA and phyB and part of fcbE, totaling 4577 bp (see Fig.14 ).

[0117] qRT-PCR identification of the missing gene: RNA of PMF0 and RPmF2019s was extracted and transcribed into cDNA, and then identified by real-time fluorescence quantitative PCR. The results are as follows: Fig.15 , it was found that there was no RNA transcript of phyA, phyB and fcbE in RPmF2019s.

[0118] 11. Application of indirect ELISA method for detection of Pm strain antibodies

[0119] PMF0 and RPmF2019s were used as coating sources, and three copies of mouse antisera against PMF0 and RPmF2019s (tested after dilution at 1:1000) prepared in the protective test of the inactivated vaccines of PMF0 and RPmF2019s were tested respectively. One replicate was made for each copy, and specific detection was performed using the positive sera of mice with Escherichia coli and Salmonella and negative sera stored in the laboratory.

[0120] Table 6 Indirect ELISA test results of different strains as coating sources

[0121]

[0122]

[0123] The results are shown in Table 6. With PMF0 and RPmF2019s as coating sources, the test results of anti-Escherichia coli mouse positive serum and anti-Salmonella mouse positive serum were all negative, and the results of mouse anti-RPmF2019s serum and mouse anti-PMF0 serum were all positive. The OD values ​​of mouse anti-RPmF2019s serum detected by ELISA with PMF0 as coating source and RPmF2019s as coating source were higher than the OD values ​​of the corresponding mouse anti-PMF0 serum, indicating that RPmF2019s has a better immune effect. The OD values ​​of mouse anti-RPmF2019s serum and mouse anti-PMF0 serum detected with RPmF2019s as coating source were higher than the detection values ​​with PMF0 as coating source, indicating that RPmF2019s is more advantageous as a coating source.

Claims

1. A Pasteurella multocida capsule mutant strain RPmF2019s, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242234.

2. Use of the capsule mutant strain RPmF2019s described in claim 1 as a coating antigen for ELISA detection of Pasteurella multocida antibodies.

3. Use of the capsule mutant strain RPmF2019s according to claim 1 in the preparation of Pasteurella multocida vaccine.

4. A kit, characterized in that: Containing the capsule mutant strain RPmF2019s according to claim 1.

5. A Pasteurella multocida vaccine, characterized in that: Contains the inactivated capsule mutant RPmF2019s according to claim 1.

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