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A balanced lethal system, construction method and application of Listeria ovis

A balanced lethal technology for Listeria, applied in chemical instruments and methods, microbial-based methods, biochemical equipment and methods, etc., can solve the lack of reports, pathogenic mechanisms, and lack of data on biological characteristics in vivo and in vitro, etc. problem, to achieve stable gene composition and good growth in vitro

Active Publication Date: 2021-06-22
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the gene composition of LI is similar to that of LM, the data on the functions of LI genes, pathogenic mechanisms, and biological characteristics in vivo and in vitro are still scarce.
In particular, no studies have yet clarified the functions of the LI dal and LI dat genes, and it has not been reported whether LI will have similar phenomena to LM when the dal and / or dat genes are deleted, and the hemolytic ability of bacteria after the deletion of the above genes has not been reported. , changes in cell invasion and proliferation

Method used

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  • A balanced lethal system, construction method and application of Listeria ovis
  • A balanced lethal system, construction method and application of Listeria ovis
  • A balanced lethal system, construction method and application of Listeria ovis

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a balanced lethal system, construction method and application of Listeria ovis. On the basis of actA and plcB double-gene attenuated LI strain, four missing genes (actA, plcB, dal, dat) attenuated nutrient-deficient strain (LI△actAplcB△dal△dat), the attenuated nutrient-deficient strain cannot grow in a nutrient environment without adding D-alanine, and then the dal gene complementation plasmid is constructed and introduced into this strain, The LI balanced lethal system, that is, the replenishing bacteria (LI△actAplcB△dal△dat:dal) was obtained. The LI equilibrium lethal system obtained by the present invention is genetically stable. The complementing plasmid contained in the bacterial cells of the LI balanced lethal system obtained by the present invention can be used as a carrier for inserting exogenous antigen genes, so that the system can stably carry and express exogenous genes, and then utilize the antigen presenting ability of LI itself Relevant biological characteristics can be used as a carrier for preparing vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a balanced lethal system for Listeria ovis, a construction method and an application thereof. Background technique [0002] Listeria ivanovii (LI) belongs to the genus Listeria, can enter antigen-presenting cells such as macrophages, and proliferate in cells, but it rarely causes human diseases and is safe. Immunization of mice with recombinant bacteria obtained by inserting Mycobacterium tuberculosis antigen into LI genome can induce antigen-specific CD8 mainly secreting IFN-γ + T cell immune response confirmed that LI can be used as a vaccine vector. The construction method of the LI vector vaccine reported now is to directly insert the exogenous antigen gene into the LI genome or introduce the plasmid expressing the exogenous antigen into LI. Weaker; the latter method requires antibiotics to select plasmids, and the genetic stability of plasmids in bacterial cells also requires an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/90C12R1/01
CPCC07K14/195C12N15/74C12N15/902
Inventor 汪川周玉真刘思静张云雯
Owner SICHUAN UNIV
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