Recombinant salmonella choleraesuis expressing mycoplasma hyopneumoniae p46 protein, preparation method and application thereof
A technology of mycoplasma hyopneumoniae and salmonella, which is applied in the field of genetic engineering of animal bacteria, can solve problems such as biological safety issues, and achieve good biological safety and good immune protection effects
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Embodiment 1
[0069] Cloning of the gene fragment of embodiment 1 Mycoplasma hyopneumoniae p46 protein
[0070] For the cloning method of the p46 protein gene fragment of Mycoplasma hyopneumoniae, see figure 2 shown.
[0071] 1. The primers required for preparing the p46 gene fragment capable of prokaryotic expression are shown in Table 1.
[0072] Table 1 prepares the primers required for the p46 gene fragment capable of prokaryotic expression
[0073]
[0074] Note in Table 1: The underlined part of the primer is the enzyme cutting site.
[0075] 2. Preparation of prokaryotic expressed p46 gene fragments
[0076] Genomic DNA extracted from the live vaccine strain Mhp168 of Mycoplasma hyopneumoniae (purchased from Nanjing Tianbang Biotechnology Co., Ltd. Beijing) Co., Ltd. Bacterial Genomic DNA Extraction Kit Instructions Operation) as a template, utilize the primer p46(BamH I) / p46(HindIII) PCR amplification p46 gene fragment as shown in Table 1, the amplification system is as foll...
Embodiment 2
[0079] Construction and identification of embodiment 2 recombinant plasmid pYA-46
[0080] For the construction method of recombinant plasmid pYA-46, see Figure 8 shown.
[0081] 1. The primers required to construct the recombinant plasmid pYA-46 are shown in Table 2.
[0082] Table 2 Primers required for recombinant plasmid pYA-46
[0083]
[0084] Note in Table 2: The underlined part of the primer is the restriction site.
[0085] 2. Construction and identification of recombinant plasmid pYA-46
[0086] Depend on Figure 8 As shown, the plasmid pGEX-KG-46 capable of prokaryotic expression of the Mycoplasma hyopneumoniae p46 gene after the site-directed mutation is used as a template, and the primers p46(SalI) / p46(HindIII) shown in Table 2 are used for PCR amplification and recovery. The product and the shuttle plasmid pYA3493 (gifted by Dr. Roy Curtiss III, University of Washington, USA) were digested with Sal I and HindIII respectively, ligated after recovery, and ...
Embodiment 3
[0087] Example 3 Construction and identification of Salmonella choleraesuis C500 (pYA-46) expressing Cpro-46 fusion protein
[0088] The construction method of Salmonella choleraesuis C500 (pYA-46) is as follows: Figure 8 shown. The recombinant plasmid pYA-46 identified above was electrotransformed (parameters: voltage 2.0KV, time 4ms, capacitance 25μF and pulse resistance 200Ω) to C500 competent cells of the asd gene deletion strain (see literature: Xu Yindi et al., Salmonella choleraesuis C500 Construction and identification of a balanced lethal vector system for strain ΔcrpΔasd deletion strain. Acta Biological Engineering, 2006, 5(3): 366-371. Attached is a commitment certificate for distributing attenuated Salmonella choleraesuis vaccine strain C500ΔcrpΔasd deletion strain to the public). The construction process of Salmonella choleraesuis C500 (pYA-46) is as follows: Figure 8 shown. Pick a single bacterium colony on the DAP negative plate and cultivate it, carry out ...
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