Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof

A technology for porcine pleuropneumoniae and Actinobacillus, which is applied in the field of bacterial genetic engineering, can solve the problems of inability to prevent pulmonary lesions and chronic infection, and inability to provide cross-protection for heterologous serotype infection, and achieves the effect of broad market application prospects.

Inactive Publication Date: 2012-06-27
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Claims
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AI Technical Summary

Problems solved by technology

[0008] The currently used whole-bacteria inactivated vaccines and subunit vaccines can alleviate the clinical symptoms and reduce the mortality caused by homologous serotype infection pigs, but they cannot prevent lung lesions and chronic infection, and cannot provide a good response to heterologous serotype infection. Good cross-protection, while natural or experimental infection can induce protection against any heterologous serotype

Method used

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  • Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof
  • Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof
  • Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of APP serotype 7 clpP single gene deletion mutant

[0071] 1.1 Construction of recombinant suicide vector pUCΔclpP

[0072] 1.1.1 Primer design and PCR amplification of upper and lower homology arms of ClpP protease gene

[0073] Two pairs of primers were designed to amplify the upper homology arm ClpPS and the lower homology arm ClpPX of the clpP gene according to the reported APP-7 AP76 strain sequence (refer to the gene sequence of GenBank accession number CP001091.1), and the size of the amplified fragment They are 1200bp and 1249bp, respectively. EcoR I restriction site is designed at the 5' end of the upstream primer of the upper homology arm, and BamH I restriction site is designed at the 5' end of the downstream primer of the lower homology arm. The above primers were synthesized by Beijing Huada Gene Company.

[0074] The primer sequences for amplifying the upper homology arms are as follows:

[0075] clpSF: 5'-CG (EcoR I)GGGGCGTTAC...

Embodiment 2

[0092] Example 2 Construction of APP serotype 7 clpP and apxIIC double gene deletion mutants

[0093] 2.1 Construction of recombinant suicide vector pUCΔapxIIC

[0094] 2.1.1 Primer design and PCR amplification of upper and lower homology arms of apx II C gene

[0095] Two pairs of primers were designed to amplify the upper homology arm apx II CS and the lower homology arm apx II of the apx II C gene according to the reported sequence of the APP-7 AP76 strain (refer to the gene sequence of GenBank accession number CP001091.1). Cx, the amplified fragment sizes are 1412bp and 1443bp, respectively. EcoR I restriction site is designed at the 5' end of the upstream primer of the upper homology arm, and BamH I restriction site is designed at the 5' end of the downstream primer of the lower homology arm. The above primers were synthesized by Beijing Huada Gene Company.

[0096] The primer sequences for amplifying the upper homology arms are as follows:

[0097] II CSF: 5'-CG (Ec...

Embodiment 3

[0114] Example 3 Virulence identification and immune protection experiment of APPΔclpPΔapx II C mutant strain

[0115] Experimental animals: SPF grade Balb / C female mice aged 4-6 weeks, purchased from the Laboratory Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0116] 3.1 Toxicity identification

[0117] The mice were randomly divided into two groups with 10 mice in each group. The specific vaccination schedule is as follows:

[0118] The first group (test group): APPΔclpPΔapx II C prepared in Example 2 was inoculated, diluted to the concentration (CFU) listed in Table 1, and the dose of intraperitoneal inoculation of each mouse was 0.1 ml.

[0119] The second group (control group): inoculated with Actinobacillus pleuropneumoniae CVCC265, diluted to the concentration (CFU) listed in Table 1, and the dose of intraperitoneal inoculation of each mouse was 0.1 ml.

[0120] The survival of mice is shown in Table 1.

[0121] T...

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Abstract

The invention discloses a resistance marker-free porcine actinobacillus pleuropneumoniae (APP) serum type 7 double-gene defective strain, and belongs to the technical field of bacterial gene engineering. The recombinant strain APPdeltaclpPdeltaapx II C of APP is obtained by inactivating ClpP protease in the APP and a coded gene of a hemolysin activated factor Apx II C by adopting a directional homologous recombination technology, and expression of the ClpP protease and the Apx II C protein is destroyed. The obtained double-gene defective strain has lower toxicity compared with a parent strain, is safe to animals, provides an important basis for transformation of porcine contagious pleuropneumonia (PCP) vaccines and research of matched identification and diagnosis reagents, and has great significance for promoting elimination and purification of the global PCP.

Description

technical field [0001] The invention relates to a double gene deletion strain of Actinobacillus pleuropneumoniae ClpP and ApxIIC without resistance markers, a construction method and application thereof, and belongs to the technical field of bacterial genetic engineering. Background technique [0002] Porcine contagious pleuropneumonia (PCP) is a highly contagious and lethal 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 important infectious diseases that is internationally recognized as an important threat to the modern pig industry. With the large-scale and intensive development of my country's modern breeding industry, the occurrence of this disease is showing an outbreak trend, and the positive rate of some pig farms has reached more than 70%, which has seriously hindered the healthy development of my ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09A61K39/02A61P31/04C12R1/01
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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