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.
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Examples
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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Application Information
- IPC
- C12N1/20; C12N15/09; A61K39/02; A61P31/04; C12R1/01
- Inventors
- 谢芳; 王春来