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Resistance marker-free attenuated live vaccine against porcine contagious pleuropneumonia (PCP) and application thereof

A technology of porcine pleuropneumonia and resistance markers, applied in the direction of antibacterial drugs, bacterial antigen components, microbial-based methods, etc., can solve the problem of inability to prevent lung lesions and chronic infections, and the inability to provide cross-protection for heterologous serotype infections, etc. Problems, to achieve the effect of broad market application prospects

Inactive Publication Date: 2014-02-05
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The currently used whole-bacteria inactivated vaccines and subunit vaccines can alleviate the clinical symptoms and mortality caused by homologous serotype infection, but they cannot prevent lung lesions and chronic infection, and cannot provide good treatment for heterologous serotype infection. cross-protection, while natural or experimental infection can induce protection against any heterologous serotype

Method used

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  • Resistance marker-free attenuated live vaccine against porcine contagious pleuropneumonia (PCP) and application thereof
  • Resistance marker-free attenuated live vaccine against porcine contagious pleuropneumonia (PCP) and application thereof
  • Resistance marker-free attenuated live vaccine against porcine contagious pleuropneumonia (PCP) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Construction of APP Serum Type 7 clpP Single Gene Deletion Mutant

[0078] 1.1 Construction of recombinant suicide vector pUCΔclpP

[0079] The flow chart of the construction of Actinobacillus pleuropneumoniae recombinant suicide plasmid pUCΔclpP is as follows figure 1 shown.

[0080] 1.1.1 Primer design and PCR amplification of the upper and lower homology arms of the ClpP protease gene

[0081] According to the reported sequence of APP7 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 sizes of the amplified fragments were respectively 1200bp and 1249bp, the PCR amplification results of the upper homology arm clpPS and the lower homology arm clpPX are as follows Figure 4 shown. An EcoRI restriction site was designed at the 5' end of the upstream primer of the upper homology arm,...

Embodiment 2

[0100] Example 2 Construction of APP serotype 7 clpP and apxⅡC double gene deletion mutants

[0101] 2.1 Construction of recombinant suicide vector pUCΔapxⅡC

[0102] The flow chart of the construction of Actinobacillus pleuropneumoniae recombinant suicide plasmid pUCΔapxⅡC is as follows figure 2 shown.

[0103] 2.1.1 Primer design and PCR amplification of the upper and lower homology arms of the apxⅡC gene

[0104] According to the reported sequence of APP7 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 apxⅡCS and the lower homology arm apxⅡCX of the apxⅡC gene respectively, and the sizes of the amplified fragments were respectively 1412bp and 1443bp, the PCR amplification results of the upper homology arm apxⅡCS and the lower homology arm apxⅡCX are as follows Figure 10 shown. An EcoR Ⅰ restriction site was designed at the 5' end of the upstream primer of the upper ho...

Embodiment 3

[0123] Example 3 Construction of APP serotype 7 gene deletion mutants of clpP, apxⅡC and fur

[0124] 3.1 Construction of recombinant suicide vector pUCΔfur

[0125] The flow chart of the construction of Actinobacillus pleuropneumoniae recombinant suicide plasmid pUCΔfur is as follows image 3 shown.

[0126] 3.1.1 Primer design and PCR amplification of upper and lower homology arms of fur gene

[0127] According to the reported sequence of APP7 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 furS and the lower homology arm furX of the fur gene respectively, and the sizes of the amplified fragments were respectively 1412bp and 1443bp, the PCR amplification results of the upper homology arm furS and the lower homology arm furX are as follows Figure 16 shown. An EcoR Ⅰ restriction site was designed at the 5' end of the upstream primer of the upper homology arm, and a BamH Ⅰ...

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Abstract

The invention discloses a resistance marker-free attenuated live vaccine against porcine contagious pleuropneumonia (PCP) and application thereof and further discloses a resistance marker-free actinobacillus pleuropneumoniae (APP) serum 7 type three gene deletion strain used for preparing the vaccine, belonging to the field of bacterial gene engineering technology. The resistance marker-free APP serum 7 type three gene deletion strain APP-Delta clpP-Delta apxIIC-Delta fur is a strain obtained by inactivating coding genes of protease ClpP, an ApxII toxin activator ApxIIC and an iron absorption regulatory protein Fur of APP through oriented homologous recombination and destroys expression of ClpP, ApxIIC and the Fur protein. The three gene deletion strain obtained in the invention has smaller virulence, decreased by more than 100 times, compared with that of a parent strain, and is safe to animals; A pig immunized by using the strain is well protected from attacks by different serum type virulent strains of APP, and all the protection rates are greater than 80%; and the three gene deletion strain can be used as the attenuated live vaccine for immunoprophylaxis of PCP.

Description

technical field [0001] The invention relates to a porcine infectious pleuropneumonia attenuated live vaccine without resistance markers and its application, in particular to an Actinobacillus pleuropneumoniae gene deletion strain comprising ClpP protease, ApxⅡC and Fur without resistance markers The prepared attenuated live porcine infectious pleuropneumonia vaccine and its application in preventing and treating porcine infectious pleuropneumonia belong 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 mode...

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 王春来谢芳李刚张艳禾
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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