Salmonella choleraesuis double-gene-deletion strain free of resistance marker and application thereof

A technology of Salmonella and resistance markers, applied in the field of genetic engineering of animal bacteria, can solve the problems of lytic death and inability to form cell walls, etc.

Inactive Publication Date: 2012-10-17
HENAN UNIV OF SCI & TECH
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AI Technical Summary

Problems solved by technology

The aspartate β-galactose dehydrogenase encoded by the asd gene is an enzyme necessary for the synthesis of diaminopimelic acid (DAP), and at the same time, DAP is the main chemical component of the cell wall of Gram-negative bacteria, the peptidoglycan tetrapeptide side chain A component of the asd gene, therefore, the mutant strains of the asd gene cannot form a complete cell wall in the absence of exogenous DAP, and eventually die by lysis

Method used

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  • Salmonella choleraesuis double-gene-deletion strain free of resistance marker and application thereof
  • Salmonella choleraesuis double-gene-deletion strain free of resistance marker and application thereof
  • Salmonella choleraesuis double-gene-deletion strain free of resistance marker and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of Salmonella choleraesuis C78-2crp asd gene deletion strain C7822

[0032] 1. ASD-related primer design

[0033] Reference literature (Xu Yindi, et al. Construction and identification of a balanced lethal vector system for the ΔcrpΔasd deletion strain of Salmonella choleraesuis C500 strain. Acta Biological Engineering, 2006, 22(3): 366-372) and http: / / www.ncbi.nlm.nih.gov / The registered asd gene sequence of Salmonella typhimurium LT2 strain (GenBank No: AE006468.1) designed 7 asd-related primers (see figure 1 , Table 1), used for the construction and identification of Salmonella choleraesuis C78-2 (commercial strains purchased from the National Veterinary Microorganism Collection Center of China Veterinary Drug Administration) and double gene deletion strain ΔcrpΔasdC78-2. All primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0034] Table 1PCR primers

[0035]

[0036] 2. Cloning of the upstream and downstream fragments asd1 (up...

Embodiment 2

[0043] Example 2 Bacteriological characteristics of Salmonella choleraesuis ΔcrpΔasd gene deletion strain C7822

[0044] 1. Phenotype identification of gene deletion strain C7822

[0045] The serotype of the mutant strain C7822 was identified according to the instruction manual of Salmonella single-factor serum (purchased from Zhejiang Ningbo Tianrun Biopharmaceutical Co., Ltd.). The standard strain C78-2 and the strain C7822 (experiment number: ΔcrpΔasdC78-2) were streaked and inoculated on the corresponding MacConkey agar plates with and without 1% maltose, and then transferred to glucose, maltose, lactose, sucrose, arabinose, xylose , mannitol and other carbon sources and biochemical identification tubes such as H2S (purchased from Hangzhou Tianhe Microbial Reagent Co., Ltd., and when C7822 was inoculated, an appropriate amount of DAP was added to the biochemical tube) to study its biochemical characteristics. The results showed that the C7822 serotype was consistent with ...

Embodiment 3

[0053] Example 3 Safety Evaluation of Salmonella choleraesuis Gene C7822

[0054] The plasmid PYA3493 containing the asd gene was electrotransformed into C7822 (experiment number: ΔcrpΔasdC78-2(PYA3493)), which was used to determine the safety evaluation of C7822.

[0055] 1. Toxicity determination of gene deletion strain C7822 of the present invention

[0056] To test the toxicity of the constructed gene deletion strain C7822 (experiment number: ΔcrpΔasd C78-2) to BALB / c mice, 40 BALB / c mice were equally divided into 2 large groups, and each large group was divided into 4 groups . The mice in the first group were orally infected with ΔcrpΔasd C78-2 (PYA3493), and each dose in each group was 4×10 5 CFU, 2×10 6 CFU, 1×10 7 CFU and 5×10 7 CFU; The second largest group was orally infected with the parental bacteria C78-2, and each dose of each group was 1.6×10 3 CFU, 8.0×10 3 CFU, 4.0×10 4 CFU and 2.0×10 5 CFU. Record the death situation and calculate the median lethal ...

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Abstract

The invention relates to a Salmonella choleraesuis double-gene-deletion strain free of a resistance marker, Salmonella choleraesuis C7822 (with an accession number of CCTCC NO: M2011402 ). The strain is obtained by carrying out deletion of the asd gene, one of the important nutrition and metabolism genes of Salmonella choleraesuis, on Salmonella choleraesuis C7821 (with an accession number of CCTCC NO: M2010102 ) which has undergone deletion of the crp virulence gene. The gene-deletion strain C7822 loses the capability of synthesizing diaminopimelic acid (DAP), so the strain cannot survive in a DAP negative environment. After plasmids containing the asd gene are transformed into C7822, the asd gene in the plasmids can form a complementary gene with C7822, which enables C7822 to regain the capability of surviving in a DAP negative environment. The double-gene-deletion strain C7822 obtained in the invention has distinct genetic background, strong security and no resistance marker and can stably carry and express exogenous antigens; the double-gene-deletion strain is a good carrying vector and expression strain for exogenous antigens and has a wide application prospect.

Description

technical field [0001] The invention relates to a Salmonella choleraesuis double-gene deletion mutant bacterium without a resistance marker, and also relates to the application of the Salmonella choleraesuis double-gene deletion bacterium, which belongs to the technical field of animal bacteria genetic engineering. Background technique [0002] Salmonella Choleraesuis (Salmonella Choleraesuis) is one of the main pathogens that cause paratyphoid fever in piglets, often causing a large number of weaned piglets to suffer from the disease, which has brought huge losses to the pig industry. Certain strains of the bacterium can also infect humans through the food chain, causing salmonellosis characterized by diarrhea and sepsis, which is also of great significance in medicine and public health. Vaccine immunization is considered to be an effective means to prevent and control the disease (MOHAMED I.HUSSEINY, MICHAEL H.2008.Construction of highly attenuated S almonella enteric a se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/74C12R1/42
Inventor 张春杰张明亮程相朝赵战勤李正王慧郁川
Owner HENAN UNIV OF SCI & TECH
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