Listeria ivanovii balanced lethal system and construction method and application thereof
A Listeria, balanced lethal technology, applied in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of unreported, pathogenic mechanism, lack of data on in vivo and in vitro biological characteristics research, etc. problem, to achieve the effect of stable genetic composition and good growth in vitro
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Embodiment 1
[0036] Example 1. Basic method for constructing dal and dat gene targeting plasmids
[0037] 1.1 Preparation of linear fragments of targeting plasmid vectors
[0038] 1.1.1 Plasmid extraction
[0039] The plasmid pCW107 was extracted according to the instructions of the OMEGA plasmid extraction kit (Wang C, Zhang F, YangJ, Khanniche A, Shen H. Expression of Porcine Respiratory and Reproductive Syndrome Virus membrane-associated proteins in Listeria ivanovii via a genomesite-specific integration and expression system. J Mol Microbiol Biotechnol, 2014,24(3):191-195.), eluted with 35μL Elution Buffer.
[0040] 1.1.2 Digestion
[0041] Plasmid pCW107 was digested with restriction endonucleases XbaI and SpeI, and the digestion system was carried out according to the instructions of the reagents. After enzyme digestion at 37°C for 1 hour, add 0.5 μL alkaline phosphatase (CIAP), and dephosphorylate in 37°C water bath for 30 minutes.
[0042] 1.1.3 Glue recovery
[0043] The dige...
Embodiment 2
[0065] Embodiment 2. Knockout of Listeria ovis dal, dat gene
[0066] 2.1 Plasmid extraction
[0067] Extract the plasmids pCW107-LI dal and pCW107-LI dat, respectively, with 35 μL sterile ddH 2 O elutes.
[0068] 2.2 Preparation of Competent Cells
[0069] Pick freshly cultured LIΔactAplcB-lacZ (LIΔ for short) on BHI plates (Wang C, Zhang F, Yang J, Khanniche A, Shen H. Expression of Porcine Respiratory and Reproductive Syndrome Virus membrane-associated proteins in Listeria ivanovii via a genomesite-specific integration and expression system. J Mol Microbiol Biotechnol, 2014, 24 (3): 191-195.) Inoculate the colonies in 15 mL of BHI broth containing 0.5 mol / L sucrose, and culture overnight at 180 rpm at 37°C; Inoculate in 250mL BHI broth containing 0.5mol / L sucrose, incubate at 37°C at 180rpm, adjust to zero with BHI broth containing 0.5mol / L sucrose, and measure A 600 value; when A 600 When the value reaches 0.4, add penicillin G (final concentration is 12.5 μg / mL), mix...
Embodiment 3
[0087] Example 3. Construction of an anaplerotic strain of Listeria ovis
[0088] 3.1 Remove Ery R Gene complementation plasmid construction
[0089] 3.1.1 Plasmid extraction
[0090] The plasmid pCW-gfp was extracted according to the instructions of the OMEGA plasmid extraction kit (Zhang X, Su L, Huang H, Jiang M, Liu S, Li Y, Liu T, Zhou Y, Tang T, Mahdy SE, Wang C. Coating with chitooligosaccharides enhances the Cytokine induction of Listeria ivanovii-based vaccine strain. J Pharm Sci, 2019, 108(9): 2926-2933.), eluted with 35 μL ElutionBuffer, and its concentration and purity were detected by Nanodrop.
[0091] 3.1.2 Removal of Ery by enzyme digestion R Gene
[0092] The plasmid pCW-gfp was digested with restriction endonucleases Sal Ⅰ and Spe Ⅰ. After enzyme digestion at 37°C for 1 hour, add 0.5 μL CIAP, and dephosphorylate in 37°C water bath for 30 minutes.
[0093] 3.1.3 Glue recovery
[0094] The enzyme digestion mixture was electrophoresed, and the gel contain...
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