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Porcine actinobacillus pleuropeumoniae ClpP protease gene-deleted strain containing no resistance marker, construction method thereof, and application thereof

A technology of porcine pleuropneumoniae and Actinobacillus, which is applied in the field of bacterial genetic engineering, can solve problems such as affecting the formation of biofilm, decreased viability, weakened virulence, etc., and achieves the effect of broad market application prospects.

Inactive Publication Date: 2012-05-09
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Studies have found that the insertion mutation or deletion of this gene usually reduces the ability of bacteria to withstand adverse factors, the growth of bacteria is destroyed, the viability decreases, and the formation of biofilm is affected, resulting in weakened virulence

Method used

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  • Porcine actinobacillus pleuropeumoniae ClpP protease gene-deleted strain containing no resistance marker, construction method thereof, and application thereof
  • Porcine actinobacillus pleuropeumoniae ClpP protease gene-deleted strain containing no resistance marker, construction method thereof, and application thereof
  • Porcine actinobacillus pleuropeumoniae ClpP protease gene-deleted strain containing no resistance marker, construction method thereof, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of recombinant suicide vector pUCΔclpP

[0044] 1.1 Primer design and PCR amplification of the upper and lower homology arms of the ClpP protease gene

[0045] According to the reported sequence of APP-7 AP76 strain (refer to the gene sequence of GenBank accession number CP001091.1), two pairs of primers were designed to amplify the upper homology arm ClpPS and the lower homology arm ClpPX of the clpP gene respectively, and the size of the amplified fragment was For 1200bp and 1249bp respectively, an EcoR I restriction site was designed at the 5' end of the upstream primer of the upper homology arm, and a BamH I restriction site was designed at the 5' end of the downstream primer of the lower homology arm. The above primers were synthesized by Beijing Huada Gene Company. The flow chart of the construction of Actinobacillus pleuropneumoniae recombinant suicide plasmid pUCΔclpP is as follows figure 1 shown.

[0046] The primer sequences for ampl...

Embodiment 2

[0057] Example 2 Construction of APP Serum Type 7 clpP Gene Deletion Mutant

[0058] The constructed recombinant suicide plasmid pUCΔclpP was electrotransformed into APP serum type 7 CVCC265, and the positive colonies of the single exchange strain were screened on a 10ug / mL Amp resistance plate, and primers were designed inside the upper and lower homology arms for PCR identification. The wild strain could An 858bp fragment can be amplified, a 367bp fragment can be amplified by the deletion strain, and a 858bp and 367bp fragment can be amplified by the single exchange strain at the same time. The results of PCR identification of the single exchange strain were as follows: Figure 5 shown. The above primers were synthesized by Beijing Huada Gene Company.

[0059] The primer sequences are as follows:

[0060] clpJDF: 5'-CGTGGTGTCGCTTGAAACTC-3'

[0061] clpJDR: 5'-AATTAGACCGTATTCCATCGC-3'

[0062] After the positive single colony of the single exchange strain was cultured an...

Embodiment 3

[0064] Example 3 Toxicity identification and immune protection experiment of APPΔclpP mutant strain

[0065] Experimental animals: SPF Balb / C female mice aged 4-6 weeks were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0066] 3.1 Virulence identification

[0067] The mice were randomly divided into two groups, 10 in each group. The specific vaccination plan is as follows:

[0068] The first group (test group): inoculated with APPΔclpP prepared in Example 2, diluted to the concentration (CFU) listed in Table 1, and each mouse was inoculated intraperitoneally with a dose of 0.1 ml.

[0069] The second group (control group): inoculated with Actinobacillus pleuropneumoniae CVCC265, diluted to the concentration (CFU) listed in Table 1, and each mouse was inoculated with 0.1 ml of intraperitoneal dose.

[0070] The survival of the mice is shown in Table 1.

[0071] Table 1

[0072]

[0073]...

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Abstract

The invention discloses a porcine actinobacillus pleuropeumoniae ClpP protease gene-deleted strain containing no resistance marker, and a construction method thereof. The invention belongs to the technical field of bacteria genetic engineering. According to the invention, a recombinant strain APPdeltaclpP of actinobacillus pleuropeumoniae is a strain obtained through coding gene deactivation upon ClpP protease in actinobacillus pleuropeumoniae with an oriented homologous recombination technology, and the expression of the ClpP protease is damaged. With the technology provided by the invention, the virulence of the mutant strain is lower than a parent strain, and the mutant strain is safe to animals. Therefore, an important basis is provided for the modification upon porcine contagious pleuropneumonia vaccines and the development of corresponding differential diagnosis reagents. The strain and the method provided by the invention have important significances in promoting the eradication and purification of porcine contagious pleuropneumonia all around the world.

Description

technical field [0001] The invention relates to a ClpP protease gene deletion strain of Actinobacillus pleuropneumoniae without resistance marker, its construction method and application, and belongs to the technical field of bacterial genetic engineering. Background technique [0002] Porcine contagious pleuropneumonia (PCP) is a highly contagious and fatal respiratory infectious disease caused by Actinobacillus pleuropneumoniae (APP). At present, the disease has been widely prevalent in countries all over the world, causing huge economic losses, and has become one of the internationally recognized important infectious diseases that endanger the modern pig industry. With the large-scale and intensive development of modern breeding industry in my country, the occurrence of this disease is in an explosive trend, and the positive rate of some pig farms has reached more than 70%, which seriously hinders the healthy development of pig farming in my country. [0003] According t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/74A61K39/02A61P31/04C12R1/01
Inventor 谢芳王春来张艳禾李建军朱晓凯李艳玲
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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