Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method

A technology of porcine pleuropneumonia and actinobacillus, which is applied in the direction of biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve the problems of destruction, poor effect of line protection of porcine pleuropneumonia, low efficiency, etc.

Active Publication Date: 2013-07-17
WUHAN KEQIAN BIOLOGY CO LTD
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Problems solved by technology

Moreover, the virulence factor produced by APP destroys the lung defense system, so that the disease is often mixed or secondary infected with Haemophilus parasuis, porcine pneumonia, porcine reproductive and respiratory syndrome, porcine pseudorabies, etc. Further increase the harm of the disease, making it one of the most serious diseases in the pig industry
Vaccine immunization is an important means of disease prevention and control. With the in-depth research on the pathogenicity and immune protection of the virulence factors of this bacteria, the country has targeted the prevention and treatment of Actinobacillus pleuropneumoniae serotypes 1, 3, There are reports of monovalent or multivalent vaccines for type 5 and type 7 infections, but due to the huge differences in the toxins and surface antigens secreted by different serotypes, the protective effect on other serotypes of porcine pleuropneumonia is often poor, and due to the geographical It is vast, and live pigs are imported from many countries. As a result, while the existing vaccines achieve the control effect on one or several serotype strains, they will also lead to an increase in the isolation rate of other serotypes, and Actinobacillus pleuropneumoniae serotype 2 It is a serotype that is widely prevalent in the world, so it is particularly necessary to screen a type 2 strain with a good antigenic effect
On the other hand, in the key production technology of bacterial vaccines in my country for a long time, the large-scale high-density culture technology of bacteria has lagged behind seriously, and there is no systematically optimized culture medium and culture for its growth characteristics for the culture of Actinobacillus pleuropneumoniae. Conditional control methods lead to high production costs and low efficiency of enterprises, resulting in relatively high product prices and affecting the immunization rate

Method used

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  • Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method
  • Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method
  • Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: strain isolation and identification

[0045] Pig lungs, throat tonsils and other tissues were aseptically taken from dying disease materials sent for inspection in pig farms, and inoculated on TSA agar medium, 10% CO 2 After culturing at 37°C for 24-36 hours, select a typical single colony for purification and culture. The purified single colony was selected and inoculated in TSB liquid culture medium, and cultivated overnight at 37°C on a shaker (220r / min).

[0046] Culture characteristics: the bacteria in 10% CO 2 , It grows well under the condition of 37 ℃, and grows vigorously on the liquid medium. On TSA (containing NAD) solid medium, in 10% CO 2 , At 37°C, after culturing for 24 hours, transparent and round colonies with a diameter of 1-2 mm are formed.

[0047] Morphological characteristics of the pathogen: Microscopic examination of the lungs and pure cultures of naturally infected pigs and experimentally infected mice showed Gram-negative, str...

Embodiment 2

[0055] Embodiment 2: culture medium optimization

[0056] Firstly, the nutrient components of Actinobacillus pleuropneumoniae serotype 2 XT high-density medium were preliminarily screened through a two-level orthogonal test (Table 1, Table 2), and the medium was established in combination with the cost of raw materials and the simplicity of operation. The component factors include: yeast powder, glucose, sodium glutamate, K 2 HPO 4 , NaH 2 PO 4 , MgSO 4 , FeSO 4 ·7H 2 O, coenzyme I, and the four component factors that have the greatest impact on the amount of live bacteria are obtained, in order: yeast powder>K 2 HPO 4 > Sodium glutamate > Glucose, then through the climbing experiment to find the most suitable concentration range of the four most influential components, and then through the central combination experiment and response surface analysis to determine the final concentration. The concentration range of the optimized medium formula obtained from the experime...

Embodiment 3

[0063] Embodiment 3: culture medium preparation

[0064] Medium components and ratio / L

[0065] Glucose 3g; Yeast powder 30g;

[0066] Sodium glutamate 3g; K 2 HPO 4 4.5g;

[0067] NaH 2 PO 4 1g; MgSO 4 0.8g

[0068] FeSO 4 ·7H 2 O 0.1g NAD 0.02g

[0069] Prepared by the following method:

[0070] A. Weigh yeast powder, glucose, sodium glutamate, K 2 HPO 4 , NaH 2 PO 4 , MgSO 4 , FeSO 4 ·7H 2 O. Coenzyme I, set the volume to 900ml, adjust the pH to 7.4, and sterilize under high-pressure steam at 115°C for 25 minutes;

[0071] B. Weigh K by volume 2 HPO 4 , NaH 2 PO 4 , dilute to 100ml, and sterilize under high-pressure steam at 121°C for 15 minutes;

[0072] C. Weigh 2gNAD, set the volume to 100ml, filter through a 0.2μm pore size filter, and take 1ml for later use;

[0073] D. Mix the above three solutions evenly to obtain the XT high-density fermentation medium of Actinobacillus pleuropneumoniae serotype 2.

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Abstract

The invention discloses an actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method, which is actinobacillus pleuropneumoniae serotype 2XT, wherein the CCTCC NO. is M2011410. The preparation method comprises the following steps: 1) medium preparation, A) weighting yeast powder, glucose, monosodium glutamate, MgSO4, FeSO4.7H2O, dissolving into pure water, regulating pH value and disinfecting; B) weighting NaH2PO4 and K2HPO4 to prepare mother liquor and disinfecting; C) weighting NAD to prepare mother liquor, filtering by a filter membrane; D) mixing the solutions obtained in the step A, the step B and the step C according to amount to obtain the medium; 2) fermentation and culture, a) activating the freeze-drying seeds by NAD-contained TSA plates until single colony is grown out; b) selecting single colony and culturing by shaking a bottle; c) transferring cultured seed liquid to the fermentation medium; d) low stirring at initial fermentation period, and low ventilating; e) raising rotating speed at logarithmic phase and ventilating, and on-line controlling pH value; f) improving dissolved oxygen, and placing into a tank and collecting bacterium. The actinobacillus pleuropneumoniae serotype 2 bacterial strain suitable for preparing inactivated vaccine has the advantages of strong toxicity, good antigen effect, low price, fast mycelium growth, highdensity and easy control.

Description

technical field [0001] The invention belongs to the technical field of animal vaccine engineering, more specifically relates to a strain of Actinobacillus pleuropneumoniae serum type 2 XT, and also relates to a preparation method of Actinobacillus pleuropneumoniae, which is suitable for high-density fermentation of porcine pleuropneumoniae The strains of Fibrobacter serotype 2 XT and other serotypes can be used to prepare antigens in large quantities for monovalent or multivalent vaccines against porcine infectious pleuropneumonia. technical background [0002] Porcine Contagious Pleuropneumonia (PCP) is an important porcine respiratory infectious disease characterized by pleuropneumonia and hemorrhagic necrotizing pneumonia caused by Actinobacillus pleuropneumoniae (APP). The pathogen Actinobacillus pleuropneumoniae is a Gram-negative pathogenic bacterium that is highly specific and parasitic in the respiratory tract of pigs. The pathogen itself has been divided into differ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/01
Inventor 徐高原王杨波周明光康超陈章表陈关平金梅林陈焕春
Owner WUHAN KEQIAN BIOLOGY CO LTD
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