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Porcine actinobacillus pleuropneumoniae attenuated strain and porcine pleuropneumonia-preventing product prepared from same

A technology of porcine pleuropneumonia and actinobacillus, which is applied in the field of microbiology and immunology, can solve the problems that attenuated live vaccines have not yet appeared, and achieve the effect of low toxicity, high biological safety and reduced toxicity

Inactive Publication Date: 2015-06-24
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the development of APP live attenuated vaccine is still in the laboratory development stage, and commercialized live attenuated vaccine has not yet appeared

Method used

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  • Porcine actinobacillus pleuropneumoniae attenuated strain and porcine pleuropneumonia-preventing product prepared from same
  • Porcine actinobacillus pleuropneumoniae attenuated strain and porcine pleuropneumonia-preventing product prepared from same
  • Porcine actinobacillus pleuropneumoniae attenuated strain and porcine pleuropneumonia-preventing product prepared from same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Obtainment of APP serotype 7 attenuated double mutant strain

[0059] 1. Bacterial culture

[0060] The freeze-dried APP strains of different serotypes were taken, streaked and cultured on TSA plates supplemented with 5% newborn calf serum and 0.005% NAD, and cultured in a 37°C incubator for about 16-20 hours. A single colony was picked from the culture plate and inoculated into 5 mL of TSB medium supplemented with 0.005% NAD, and cultured in a shaker at 37°C for about 12-16 hours.

[0061] 2. APP genomic DNA extraction

[0062] Take 1mL of APP culture medium cultured in TSB medium and add it to a 1.5mL centrifuge tube. After centrifugation at 12,000g for 30 seconds, discard the culture solution, wash with 1mL TE buffer once, and extract with the bacterial whole genome extraction kit according to the kit instructions. Extract the genomic DNA, and finally dissolve the genomic DNA in 100 μL TE buffer and freeze for later use.

[0063] 3. Gene fragment amplification, cl...

Embodiment 2

[0087] Example 2. Detection of biological characteristics of APP mutant strains

[0088] The two mutant strains of APP7, GS7C and GS7CA, were screened and compared and analyzed on the basis of their biological characteristics and their differences with the maternal strains.

[0089] 1. Proliferation ability

[0090] Single colonies of mutant strains and maternal strains were selected to inoculate TSB+NAD culture medium and cultured overnight at 37℃; then 5mL TSB+NAD culture medium was inoculated at a ratio of 1:1000, and cultured at 37℃ for 12h with shaking every 1h Sampling and measuring the OD600 value of the bacteria, drawing the growth curve of the bacteria; at the same time, counting the viable bacteria of the maternal strains to obtain the relationship between the OD600 of the bacteria and the cell CFU. Comparing the difference in the proliferation ability of the mutant strain and the parent strain through the bacterial growth curve.

[0091] The result is image 3 As shown, th...

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Abstract

The invention discloses a porcine actinobacillus pleuropneumoniae attenuated strain and a porcine pleuropneumonia-preventing product prepared from the same, and relates to the fields of microorganisms and immunology. According to the porcine actinobacillus pleuropneumoniae attenuated strain and the porcine pleuropneumonia-preventing product prepared from the same disclosed by the invention, on the basis of a single mutant strain GS7C, a ureC gene segment in a genome is further deleted, and an apxIII-N gene segment with immunogenicity is inserted, and then the double mutant strains (GS7CA) of apxIIC- / apxIA+ and ureC- / apxIII+ which successfully express ApxIII-N proteins are screened by virtue of a sacB negative gene screening system, and the collection number is CGMCC NO. 10016. Experimental data indicates that, the hemolytic activity and urease activity of the double mutant strains are completely lost, the toxicity is greatly reduced, and stable inheritance can be realized; the attenuated live vaccine prepared by the porcine actinobacillus pleuropneumoniae attenuated strain disclosed by the invention has low toxicity, cross protection activity, high bio-safety and stable quality.

Description

Technical field [0001] The invention relates to the field of microbes and immunology, in particular to an attenuated strain of Actinobacillus pleuropneumoniae and a product prepared for preventing porcine pleuropneumonia Background technique [0002] Porcine infectious pleuropneumonia is a respiratory disease of pigs caused by Actinobacillus pleuropneumoniae (APP). The main clinical features of this disease are hemorrhagic and necrotizing pneumonia and fibrinous pleurisy. It is generally believed that this bacterium enters the pig body through the respiratory tract, and then directly enters the alveoli through the trachea and bronchi. In addition to host and environmental factors, various virulence factors are the main factors in the pathogenicity of this bacteria, including capsules, endotoxins, exotoxins, adhesin, transferrin, outer membrane proteins and secreted enzymes. [0003] Actinobacillus pleuropneumoniae is the main pathogen causing porcine pleuropneumonia. It is a Gram-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04A61P11/00C12R1/01
Inventor 徐福洲崔国林郭芳芳杨兵周宏专苏霞陈小玲王金洛刘爵
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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