Attenuated Salmonella pullorum and application thereof
A technology for salmonella and avian salmonella, applied in the direction of bacteria, antibacterial drugs, medical preparations containing active ingredients, etc., can solve problems such as environmental and food safety impacts, and achieve good immunogenicity
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Embodiment 1
[0023] Example 1 Construction of Double Gene Deletion Attenuated Salmonella (Salmonella pullorum) SM091-DED Strain
[0024] 1. Test materials and methods
[0025] 1.1 Strains and plasmids
[0026] The Salmonella avian strain was isolated and identified by the inventor's laboratory and named SM091. Plasmid pKD46 (temperature-sensitive, containing gam, bet and exo genes regulated by the arabinose promoter, Ampr), pKD3 (containing the chloramphenicol resistance gene with FRT sites on both sides, Cmr), pCP20 (temperature-sensitive , encoding FLP recombinase, which can specifically recognize the FLP site).
[0027] 1.2 Reagents
[0028] dNTP and Taq DNA polymerase were purchased from MBI Company, plasmid extraction kit was purchased from QIAGEN Company, DNA gel recovery kit was purchased from Shanghai Huasun Company, L-arabinose was purchased from Sigma Company, and primer synthesis was completed by Nanjing Boya Company.
[0029] 1.3 Construction method of double-gene deletion ...
Embodiment 2
[0049] The preparation of embodiment 2 attenuated salmonella vaccine
[0050] 1. Test material: the attenuated Salmonella SM091-DED strain constructed in Example 1;
[0051] 2. Test method
[0052] The frozen attenuated Salmonella strains were diluted with sterilized saline, streaked on LB agar plates, and cultured overnight at 37°C. On the next day, pick a single colony and inoculate it into non-resistant LB liquid medium, and culture it at 37°C for 18-20 hours. Centrifuge at 3000rpm, wash 3 times with sterilized PBS, remove the medium components, and then count the bacteria to 10 7 CFU, divided into cryopreservation tubes, freeze-dried and stored to make attenuated Salmonella vaccine.
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