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Shigella flexneri phage strain and application thereof

A technology of Shigella flexneri and bacteriophage, applied in the field of bioengineering, can solve problems such as the evolution of bacteria and achieve the effect of preventing pollution

Active Publication Date: 2009-09-02
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a new method capable of specifically cleaving Shigella flexneri for the current prevention and treatment of Shigella flexneri (Shigella flexneri). Bacteriophage of Shigella and its Application

Method used

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  • Shigella flexneri phage strain and application thereof
  • Shigella flexneri phage strain and application thereof
  • Shigella flexneri phage strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, the separation and preparation of phage

[0024] The sample of the present invention is collected from the sewage of the cleaning site of the pig breeding factory of Jiangsu Academy of Agricultural Sciences, filtered through double-layer filter paper, 3000 × g.min -1 Centrifuge for 20 min, and then filter the supernatant with a 0.22 μm filter membrane.

[0025] Take 50ml of filtered supernatant, add 2ml of overnight culture of phage host bacteria, and then add CaCl 2 After mixing the mother solution (sterilized by a 0.22 μm filter membrane) to a final concentration of 1 mM, add 20 ml of LB medium, incubate at room temperature for 1 h, and then place it at 37 ° C for 12 to 16 h. The next day, take the above-mentioned culture and wash it at 14000×g.min -1 Centrifuge for 30min, take the supernatant; 14000×g.min -1 Centrifuge again for 20 min, take the supernatant and add 0.1% chloroform to form a phage stock solution.

[0026] Divide the ordinary nutrient...

Embodiment 2

[0028] Embodiment 2, purification culture and amplification of bacteriophage

[0029] Pick a single phage plaque with the same shape and size on the double-layer plate forming the phage plaque, puncture the target phage plaque with an inoculation needle, insert the inoculation needle into 3-5ml LB medium after puncture, and repeat 3-5 times After that, add 0.1ml of phage host bacterial solution, mix well, act at room temperature for 15min, incubate at 37°C for 10-14h, 14000×g.min -1 , 4°C, centrifuge for 20min, take the supernatant, add 0.1ml chloroform at the same time, react at room temperature for 20min, sterile filter (0.22μm) the supernatant;

[0030] Take 2ml of freshly cultured host bacteria, centrifuge, resuspend in 0.4ml LB medium, add 0.1ml phage (the ratio of single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Add maltose (0.2%), MgSO 4 (10mM), incubate at 37°C for 20min to make the phage particles adsorb to the host bacteria; add 100ml LB...

Embodiment 3

[0033] Embodiment 3, the influence of temperature and pH on the stability of bacteriophage Sh72

[0034] Take 0.1ml 1×10 respectively 8 Pfu / ml of purified phage was reacted in a water bath at 40°C to 90°C for 30min and 1h, and the titer was measured after the sample was cooled in an ice bath; peptone water and 2×10 9Pfu / ml purified phages were mixed in equal amounts, and the potency was measured after 2 hours in a water bath at 37°C.

[0035] The results show that if figure 2 As shown, after the phage was treated at 40°C and 50°C for 30 minutes, its activity did not change significantly; at 50°C for 1 hour, the activity decreased; after the action at 70°C, the titer decreased to less than 50% of the initial titer; at 90°C , almost inactivated.

[0036] The results after the phage were treated with different pH image 3 , the titers of phages were all at 10 after pH5.0~9.0 9 Above, its activity has no obvious change; when the pH is 4.0 and 10.0, its titer drops to 5×10 8...

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Abstract

The invention relates to a phage strain and application thereof. The preserving number of the phage strain is CCTCC NO: M209029; and the strain has strong cracking function for Shigella flexneri. After the phage strain is purified, amplified and cultured, the phage strain can be used for inhibiting the Shigella flexneri. The phage strain has the following advantages: because the Shigella flexneri with drug resistance can be killed by adopting the specificity of the phage strain to crack the Shigella flexneri, simultaneously the evolution of the Shigella flexneri and the generation of drug-resistant strains can be prevented; because the phage strain has an aqueous phase, flushing liquor or leacheate for sterilizing environments or apparatuses can be easily prepared by the prior method; and because the phage strain can specifically crack the Shigella flexneri and has no toxic and side effects, the phage strain can be used as a food additive to prevent the Shigella flexneri from polluting food, and used as a medicament for treating the diseases caused by the Shigella flexneri.

Description

technical field [0001] The invention relates to a phage strain and its application, especially a phage capable of specifically lysing Shigella flexneri and its application. Belongs to the field of bioengineering. Background technique [0002] Shigella is one of the main pathogens of gastrointestinal disease infection. Shigella is divided into 4 species and at least 47 serotypes, among which Shigella flexneri is the dominant bacterium of dysentery in developing countries Every year, about 164 million people worldwide get sick, and the death toll is as high as 1.106 million, and 69% of them are children under 5 years old. Studies have shown that children's diarrhea caused by Shigella flexneri is mainly due to eating food contaminated by Shigella flexneri. At present, bacterial contamination is a serious problem in food safety and food production industry, and the death toll of food poisoning caused by bacterial contamination is still on the rise. Facing the emergence of inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01N63/00A01P1/00A23L3/3571A61K35/76A61P1/12A61P31/04C12R1/92
CPCY02A50/30
Inventor 张辉王冉魏瑞成陈明
Owner JIANGSU ACAD OF AGRI SCI
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