Pasteurella multocida acellular antigen, preparation method and applications thereof

[0033] Example 1: Preparation of Pasteurella multocida cell-free antigen

[0034] Pasteurella multocida C51-2 and C51-17 strains from rabbits (both purchased from the China Veterinary Drug Administration, and the strain preservation numbers are CVCC499 and CVCC1753 respectively) and Pasteurella multocida C48-2 and 1502 strains from chickens were taken. strains (both purchased from the China Veterinary Drug Control Institute, and the strain preservation numbers are CVCC44802 and CVCC2082) freeze-dried strains were dissolved in 1-2ml sterile saline and inoculated in 2-20% (V/V) serum-modified Martin After recovery of the agar plate, take the colonies and inoculate them in 2-20% (V/V) serum-modified Martin broth, place at 37°C, shake at 200 rpm for 12 hours, add 0.15% formaldehyde solution to the bacterial solution, and inactivate at 37°C for 24 hours. Centrifuge at 10000rpm for 20min, and the supernatant is the cell-free antigen of Pasteurella multocida.

[0035] Among them: the modified Martin plate containing 2-20% (V/V) serum was prepared according to the following steps: Weigh 42g of modified Martin agar (purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd.), add 800-980ml of distilled water, shake well After mixing, heat until fully dissolved, sterilize with high pressure steam at 115°C for 30 minutes, and lower the temperature to about 60°C, add 20-200ml of newborn bovine serum (Sijiqing, purchased from Zhejiang Tianhang Biotechnology Co., Ltd.), shake well and pour plate.

[0036] The modified Martin broth containing 2-20% (V/V) serum is prepared according to the following steps: Weigh 40g of the modified Martin medium (purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd.), add 800-980ml of distilled water, and shake well Then heat until fully dissolved, sterilize with high-pressure steam at 115°C for 30 minutes, add 20-200ml of newborn bovine serum after cooling, mix well and serve.

[0037] Example 2: Detection of the main components of the cell-free antigen of Pasteurella multocida

[0038] 1 Determination of cell-free antigen protein content

[0039] The protein content of each cell-free antigen was directly measured using a nucleic acid protein analyzer (BioPhotometer plus, Eppendorf, Germany), and the results are shown in Table 1.

[0040] Table 1: Determination results of protein content in cell-free antigen of Pasteurella multocida

[0041] Strain number

[0042] 2 Determination of polysaccharide content of cell-free antigen (using phenol-sulfuric acid colorimetric method)

[0043] Using anhydrous glucose (sigma) as a reference substance, the regression equation y=1.3914x+0.0086 (R 2 =0.997), each cell-free antigen sample was diluted 20 times with sterile normal saline, and its absorbance at 490nm wavelength was measured by phenol-sulfuric acid colorimetry, and the polysaccharide content in the cell-free antigen sample was calculated by substituting it into the regression equation. The results are shown in Table 2.

[0044] Table 2: Determination of polysaccharide content in cell-free antigens

[0045]

[0046] Example 3: Establishment and application of Pasteurella multocida indirect hemagglutination test method

[0047] 1 Preparation of sensitized sheep red blood cells

[0048] 1.1 Collection and washing of sheep red blood cells

[0049] Fresh anticoagulated sheep blood was stored at 4°C and centrifuged at 2000rpm for 10min, and the supernatant and white blood cells were discarded. Wash with PBS (0.15mol/L, pH7.2) for 3-5 times, make 5% erythrocyte suspension by volume, and store at 4°C for later use.

[0050] 1.2 Formylation and tanninization of red blood cells

[0051] Mix glutaraldehyde with water to make a 2.5% glutaraldehyde solution by volume, and store it at 4°C for later use.

[0052] Place the 5% erythrocyte suspension pre-cooled at 4°C in a magnetic stirrer, stir at a low speed, and add the glutaraldehyde solution pre-cooled at 4°C dropwise at a volume ratio of 5:1. Then the erythrocyte suspension was hydroformylated in a constant temperature shaker at 30°C and 150rpm for 5h. Wash 3 times with PBS (0.15mol/L, pH 7.2), make 5% aldehydized erythrocyte suspension, and store at 4°C.

[0053] Mix tannic acid and water at a ratio of 1:20000 (g/ml) to make a tannic acid solution, which is prepared and used immediately.

[0054] Take 5% formaldehyde erythrocyte suspension and mix it with an equal volume of freshly prepared tannic acid solution (1:20000), put it in a water bath at 37°C for 30 minutes, wash it with PBS (0.15mol/L, pH6.4) for 3 times, and restore to The original volume was made into 5% tanned sheep red blood cell solution, and stored at 4°C for later use.

[0055] 1.3 Sensitization of sheep red blood cells

[0056] Mix 5% tanned sheep red blood cells with an equal volume of 10-fold diluted Pasteurella multocida cell-free antigen solution, sensitize in a water bath at 37°C for 30 minutes, shake gently during this period, centrifuge at 3000rpm for 3 minutes, discard the supernatant, and use After washing with PBS (0.15mol/L, pH7.2) for 3 times, a 1% sensitized red blood cell suspension was prepared with PBS (0.15mol/L, pH7.2).

[0057] 2 Preparation of Pasteurella multocida positive serum and negative serum

[0058] Rabbit Pasteurella multocida C51-2 strain, C51-17 strain, chicken Pasteurella multocida C48-2 strain, 1502 strain, Bordetella bronchiseptica BS039 strain and Escherichia coli CMCC44149 strain were respectively Inoculate in TSA solid medium, culture at 37°C for 18 hours, take a single colony and inoculate them in 5ml TSB liquid medium respectively, culture at 37°C and shake at 180rpm for 14h, take 0.1ml and inoculate in 100ml TSB liquid medium for expansion culture, 37°C , after shaking at 200rpm for 10 hours, take a sample to count the viable bacteria, add 0.15% formaldehyde solution to inactivate the bacteria solution for 24 hours, concentrate the inactivated bacteria solution to 10 billion CFU/ml according to the results of the viable bacteria count, and add 20% aluminum hydroxide glue respectively , after mixing, they are Pasteurella multocida Pm serum (C51-2 strain) antigen, Pasteurella multocida Pm serum (C51-17 strain) antigen, Pasteurella multocida Pm serum (C48 -2 strains) antigen, Pasteurella multocida Pm serum (1502 strain) antigen, Bordetella bronchiseptica Bb serum (BS039 strain) antigen, Escherichia coli E.coli serum (CMCC44149 strain) Use antigen. Among them, the first four antigens are antigens for Pasteurella multocida positive sera, and the latter two are antigens for Pasteurella multocida negative sera.

[0059] Take 14 experimental rabbits and divide them into 7 groups at random, 2 rabbits per group. The 6 groups were inoculated with the above-mentioned antigens for Pasteurella multocida positive and negative serum respectively, that is, strain C51-2, strain C51-17, and strain C48. -2 strains, 1502 strains, BS039 strains and CMCC44149 strain antigens, the other group was inoculated with sterile saline, 2ml/cause, subcutaneously injected on the back; 14 days later, the same dose was boosted once, and each immune group was collected 21 days after the booster immunization. Rabbit serum, respectively Pm serum (C51-2 strain), Pm serum (C51-17 strain), Pm serum (C48-2 strain), Pm serum (1502 strain), Bb serum (BS039 strain), E.coli Serum (CMCC44149 strain) and negative serum.

[0060] 3 Establishment of Indirect Hemagglutination Test Method for Pasteurella multocida

[0061] Pasteurella multocida C51-2 and C48-2 were inoculated in serum-containing medium respectively, and cultured by shaking at 37°C and 200rpm for 12h, adding 0.15% formaldehyde solution to the bacterial liquid, inactivated at 37°C for 24h, and centrifuged Afterwards, the supernatants are the cell-free antigens of Pasteurella multocida C51-2 and C48-2 respectively. After diluting the antigens by 10 times, the sheep erythrocytes are sensitized according to steps 1.1, 1.2, and 1.3, and the sensitized sheep erythrocytes are taken 25 μL were mixed with 25 μL PBS (0.15mol/L, pH7.2), 25 μL Pm (C51-2 and C48-2) serum, 25 μL Bb serum (BS039), 25 μL E.coli serum (CMCC44149) and 25 μL negative serum at 96 The wells were mixed on a V-type hemagglutination plate, and placed at 37°C for 30 minutes. It was found that the sheep red blood cells sensitized by the C51-2 cell-free antigen only had an agglutination reaction with the Pm (C51-2) serum, and the rest of the wells settled into a small circle. point; C48-2 cell-free antigen-sensitized sheep red blood cells only had agglutination reaction with Pm (C48-2) serum, and the rest of the wells were settled into a small dot, indicating that the prepared sensitized sheep red blood cells can be used to detect serum Antibodies specific for Pasteurella multocida.

[0062] 4 Pasteurella multocida indirect hemagglutination test method for detecting antibodies to Pasteurella multocida in serum

[0063] 4.1 Indirect hemagglutination assay (IHA) method

[0064] Take 25 μl of PBS (0.15mol/L, pH7.2) containing 1% bovine serum albumin and put it in the well of a 96-well V-type hemagglutination plate, and take Pm serum (C51-2), Pm serum (C51-17), Pm serum 25 μl each of serum (C48-2), Pm serum (1502), Bb serum (BS039), E.coli serum (CMCC44149) and negative serum were placed in the first wells of the first to seventh rows of the two reaction plates, and the ratio Dilute to the 11th well, the 12th well is used as a blank control, and then add 25 μl of 1% sensitized red blood cell suspension to each well. Vibrate lightly on a micro-shaker for 1 min to make it fully mixed, incubate at 37°C for 30 min or at room temperature for 60 min and observe the results. Use "-", "++", "+++", "#" to indicate the agglutination intensity of red blood cells.

[0065] 4.2 Judgment criteria

[0066] "#" indicates that the agglutinated red blood cells cover the bottom of the well evenly in the form of a thin film. "+++" indicates that the agglutinated red blood cells cover the bottom of the well, but a small amount of red blood cells settle into small dots in the center. "++" indicates that the red blood cells have settled in the center of the bottom of the well, and there are still scattered agglutinated red blood cells around. "-" indicates that all red blood cells have settled in the center of the bottom of the well, and there are no scattered red blood cells around. The hemagglutination titer of the serum is the highest dilution factor of the serum where "#" appears.

[0067] 4.3 Results

[0068] The detection results of the indirect hemagglutination test method: using the indirect hemagglutination test method for Pasteurella multocida of the present invention, each serum sample to be tested was subjected to an indirect hemagglutination test, and the results showed that: 30 minutes at 37°C or 60 minutes at room temperature Afterwards, the erythrocytes in the blank control wells were completely settled into a small dot, with 100% agglutination as the judgment end point, and the IHA titer of each Pm serum measured by the C51-2 cell-free antigen-sensitized sheep erythrocytes was 1:2 1 ~1:2 6 , the highest IHA titer of Pm serum (C51-2) was 1:2 6 , Bb serum (BS039), E.coli serum (CMCC44149) and negative serum were all negative; the IHA titer of each Pm serum measured on C48-2 cell-free antigen-sensitized sheep red blood cells was 1:2 2 ~1:2 7 , the highest IHA titer of Pm serum (C48-2) was 1:2 7 , Bb serum (BS039), E.coli serum (CMCC44149) and negative serum were all negative (see Table 3 for details). The Pasteurella indirect hemagglutination test method can be used to detect whether the serum contains Pasteurella multocida antibodies, and it has good stability. The IHA titer between homologous strain antigen antibodies is higher, and heterologous strain antigen antibodies The IHA titers among them are lower, indicating that the antigen provided by the present invention has a certain crossness among different bacterial strains, but the sensitivity among homologous bacterial strains is higher.

[0069] Table 3: Test results of indirect hemagglutination test method

[0070]