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Pasteurella multocida cell-free antigen, preparation method and application thereof

A Pasteurella, multi-kill technology, applied in the field of veterinary biological products, can solve the problems of cumbersome operation steps, difficult to achieve large-scale production, etc., to reduce side reactions, easy to scale production, good specificity and sensitivity Effect

Active Publication Date: 2017-05-17
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both can obtain outer membrane proteins with good immunogenicity, but the operation steps are cumbersome and difficult to achieve large-scale production

Method used

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  • Pasteurella multocida cell-free antigen, preparation method and application thereof
  • Pasteurella multocida cell-free antigen, preparation method and application thereof
  • Pasteurella multocida cell-free antigen, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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: th...

Embodiment 2

[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 Protein content in cell-free antigen (mg / ml) C51-2 10.2 C51-17 10.6 C48-2 8.8 1502 9.5

[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 ph...

Embodiment 3

[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...

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Abstract

The present invention relates to a cell-free antigen of Pasteurella multocida, wherein said antigen is derived from the supernatant of an inactivated Pasteurella multocida culture, said supernatant containing polysaccharides and proteins. The invention also provides an indirect hemagglutination test method for Pasteurella multocida established by using the antigen, which has good specificity and sensitivity; the invention also provides a vaccine containing the antigen. The Pasteurella multocida cell-free antigen vaccine of the invention has the same immune efficacy as the whole bacteria vaccine, and has the advantages of not affecting the growth of animals and having little local side effects at the injection site of the vaccine.

Description

technical field [0001] The invention relates to the field of veterinary biological products, in particular to a cell-free antigen of Pasteurella multocida. Background technique [0002] Pasteurell multoeida (Pm) can cause pasteurellosis in a variety of animals, such as fowl cholera in poultry, swine pneumonia, hemorrhagic sepsis in cattle, rabbits and other animals. This type of disease is divided into three categories: acute, subacute and chronic. The first two types have rapid onset and high mortality. The third type is likely to cause animal growth retardation, reduced feed conversion rate, and secondary infection of other bacteria or viruses, which in turn cause huge damage. Economic losses. Effective vaccination is an effective way to prevent and control this type of disease. Pasteurella inactivated vaccines and attenuated vaccines for various animals have made great contributions to the prevention and control of this disease. However, most of the vaccines currently u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/285A61K39/102A61P31/04
CPCA61K39/102G01N33/56911G01N33/86
Inventor 田克恭张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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