Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof
A technology of porcine pleuropneumonia and actinobacillus, applied in the field of bacterial genetic engineering, can solve the problems of being unable to prevent lung lesions and chronic infections, and the inability to provide cross-protection for infection of heterologous serotypes, and achieve the effect of broad market application prospects
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Embodiment 1
[0070] Example 1 Construction of APP Serum Type 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 the upper and lower homology arms of the ClpP protease gene
[0073] 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.
[0074] The primer sequences for amplifying the upper homology arm are as follows:
[0075] cl...
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 the upper and lower homology arms of the apx II C gene
[0095] 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 apx II CS and the lower homology arm apx II of the apx II C gene respectively Cx, the sizes of the amplified fragments 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 a 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 arm are as follows:
[...
Embodiment 3
[0114] Example 3 Toxicity identification and immune protection experiment of APPΔclpPΔapx II C mutant strain
[0115] 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.
[0116] 3.1 Virulence identification
[0117] The mice were randomly divided into two groups, 10 in each group. The specific vaccination plan is as follows:
[0118] The first group (test group): inoculated with APPΔclpPΔapx II C 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.
[0119] 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.
[0120] The survival of the mice is shown in Table 1.
[0121] Table 1
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