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A kind of preservation method and special protective agent of Salmonella choleraesuis vaccine strain

A technology of Salmonella cholera and Salmonella, which is applied in the field of preparation of auxiliary preparations for vaccine products, can solve the problems of increased intracellular solute concentration, increased solute damage, excessive dehydration of cells, etc., and achieves good storage stability, easy operation, and high survival rate high effect

Active Publication Date: 2020-11-06
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the cooling rate is too slow, the cells will be excessively dehydrated, the intracellular solute concentration will increase, and the solute damage will be increased (Fields et al 1997)

Method used

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  • A kind of preservation method and special protective agent of Salmonella choleraesuis vaccine strain
  • A kind of preservation method and special protective agent of Salmonella choleraesuis vaccine strain
  • A kind of preservation method and special protective agent of Salmonella choleraesuis vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Screening of protective agents for vaccine bacteria under liquid nitrogen ultra-low temperature freezing conditions

[0019] 1.1 Fermentation and concentration of bacterial liquid

[0020] Take the bacterium liquid of the Salmonella choleraesuis vaccinia strain that has been correctly identified by enzyme digestion (the S. choleraesuis vaccinia strain is C500 / pGS / 2SS) and inoculate it in 5ml of LB liquid LB medium according to the volume ratio of 1:100 , placed on an air bath constant temperature shaker at 37°C at 200r / min for 14h, then inoculated the cultured bacterial solution into 350ml LB liquid LB medium at a ratio of 1:100 by volume, at 37°C, 200r / min Shake culture for 15 hours, centrifuge at 4000r / min for 10 minutes, discard the supernatant, and use sterile phosphate buffered saline (abbreviated as PBS, i.e. phosphate buffered saline, pH7.2, concentration 0.01M: weigh disodium hydrogen phosphate (Na 2 HPO 4 12H 2 (O) 2.90g, potassium dihydrogen phos...

Embodiment 2

[0050] Example 2: Compatibility optimization of protective agent for vaccine bacteria C500 / pGS / 2SS cryopreserved in liquid nitrogen

[0051] 2.1 Orthogonal design

[0052] Table 6 L9(3 4 ) Orthogonal design table

[0053]

[0054] Table 7 Liquid nitrogen ultra-low temperature freezing multi-factor optimization test arrangement

[0055]

[0056] Mix the adjusted bacterial solution and the above protective agent formula in equal volumes according to the volume ratio of 1:1 to make the final concentration as shown in Table 7, and then distribute them in cryopreservation tubes. Each group has three replicates and placed in - Place it in a refrigerator at 80°C for 12 hours, then put it into liquid nitrogen for storage for a week, then immediately place the liquid nitrogen-preserved Salmonella choleraesuis vaccine strain in a 37°C water bath for 30 minutes, and then detect the survival rate of the bacteria to screen out the protective bacteria. The most effective protectant...

Embodiment 3

[0065] Embodiment 3: Detection of storage stability of Salmonella choleraesuis vaccine strain C500 / pGS / 2SS

[0066] 3.1 Experimental design

[0067] Under the best protective agent formula screened out, the bacterial liquid of Salmonella choleraesuis vaccine strain C500 / pGS / 2SS with different concentrations was tested, and its storage stability was tested for 1 week and 4 weeks, and in At the end of the experiment, the plasmid stability was checked.

[0068] 3.2 Plasmid mini-extraction and enzyme digestion identification

[0069] On the ultra-clean workbench, use a pipette to take 1ml of the bacterial solution (vaccine bacteria C500 / pGS / 2SS), and follow the instructions of the plasmid mini-extraction kit produced by Tiangen Biochemical (Beijing) Technology Co., Ltd. to extract the plasmid. The specific operation steps are as follows:

[0070] (1) Take 1.5 mL of bacterial liquid in a 2 mL clean centrifuge tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and...

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Abstract

The invention belongs to the technical field of preparation of animal vaccine auxiliary preparations and relates to a method for preserving a salmonella choleraesuis vaccine strain and a special protective agent thereof. The method for preserving the salmonella choleraesuis vaccine strain deposited under liquid nitrogen conditions is obtained by screening and characterized in that a protective agent is added to a salmonella choleraesuis vaccine solution during the preservation of the salmonella choleraesuis vaccine strain. The protective agent is prepared from the following components in massratio: 2.5% of sucrose, 7% of glucan, 6% of dimethylacetamide, and 0.025% of methionine. The special protective agent is prepared from the following components in mass ratio: 2.5% of sucrose, 7% of glucan, 6% of dimethylacetamide, and 0.025% of methionine. The special protective agent has the effects of permeability, semi-permeability, non-permeability and anti-oxidation. A survival rate of different concentrations of vaccine bacteria at different preservation times under a selected protective agent formula has no significant difference, and the survival rate maintains 70% or higher.

Description

technical field [0001] The invention belongs to the technical field of preparation of auxiliary preparations of vaccine products, and in particular relates to a preservation method of Salmonella choleraesuis vaccine bacteria and a special protective agent thereof. Background technique [0002] The preliminary work of the present invention is to prepare a vaccine bacterium called C500 / pGS / 2SS (patent application number: 2008101979818, strain preservation number: CCTCC NO: M208194), which is an attenuated strain that lacks asd gene and crp gene Salmonella, which contains non-resistance screening hepatitis B surface antigen S gene and double-copy somatostatin (SS) gene plasmid, the main function is to promote animal growth, which is important for shortening the slaughter time of fattening pigs, improving feed utilization and reducing production costs significance. [0003] The preliminary research of the present invention shows that the C500 / pGS / 2SS vaccine strain has achieved...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/04C12N1/20C12R1/42
CPCC12N1/04C12N1/20
Inventor 杨利国梁爱心于垚垚刘文浩牛凯峰王力军刘爽王亚平姜婷婷乔同
Owner HUAZHONG AGRI UNIV
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