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Escherichia coli O157:H7 attenuated strain and application thereof

A technology of O157 and Escherichia coli, which is applied in the direction of bacteria, antibacterial drugs, and medical preparations containing active ingredients, etc., can solve the problems of high cost, complicated antigen preparation method, and complicated preparation method, and achieve low cost, simple method, good immunogenic effect

Active Publication Date: 2018-05-11
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there are commercial vaccines on the market, the preparation method is complicated and the cost is high
For example, the Econiche developed by Canada’s Bioniche company, the main antigenic component is the extract of Escherichia coli O157:H7-type III secreted protein (TTSP). Reduce the spread of E. coli O157:H7 in cattle to a certain extent, but the antigen preparation method is complicated and costly, and the development of a cheaper and more efficient vaccine is a research hotspot, and it is also an area of ​​concern for scholars all over the world

Method used

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  • Escherichia coli O157:H7 attenuated strain and application thereof
  • Escherichia coli O157:H7 attenuated strain and application thereof
  • Escherichia coli O157:H7 attenuated strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation and Identification of Escherichia coli O157:H7 JSC2 Strain

[0034] (1) Stool collection:

[0035] Several portions of fresh cow dung were collected from the Zhujiashan dairy farm in Nanjing, placed in sealed bags, and stored at low temperature.

[0036] (2) Bacterial enrichment culture:

[0037] Weigh 25g of each feces sample into a sterile Erlenmeyer flask, add 225mL of sterile normal saline, and put it in a 37°C incubator for 2 hours of shaking; after each sample is processed, take 10mL and add it to 90mL of mEC broth, Cultivate with shaking at ℃ for 6 hours to obtain the enrichment solution.

[0038] (3) Immunomagnetic bead enrichment:

[0039] Take out 1mL of the enrichment solution obtained in step (2) into a sterile centrifuge tube, add 20uL Dynabeads®anti-E.coli O157 (E. Rack enrichment; stand still, suck away the supernatant in the tube, wash the magnetic beads 3 times with 0.01mM, pH7.0 PBS buffer, and collect the bacterial solution. T...

Embodiment 2

[0051] Example 2 Pathogenicity test of Escherichia coli O157:H7 JSC2 strain

[0052] (1) Preparation of strains EDL933 and JSC2

[0053] Take out the cryopreservation tubes of Escherichia coli O157:H7 EDL933 strain and JSC2 strain from the cryogenic freezer, dip in the inoculation loop, streak on the SMAC plate, culture at 37°C for 12-20 hours, pick a single colony and inoculate it on mEC For the broth, after incubating at 37°C for 7-8h, centrifuge at 12000r / min for 5min, resuspend the bacteria sludge in PBS buffer (0.01mM, pH7.0), and challenge the animals for later use.

[0054] (2) Pathogenicity test in BALB / c mice

[0055] Twenty 6-week-old clean-grade BALB / c mice (provided by the Medical College of Yangzhou University) were selected and randomly divided into two groups, A and B, with 10 mice per group. Group A, oral administration of Escherichia coli O157:H7 JSC2 strain bacterial liquid, 1.0×10 10 CFU / cat; B group, the same volume of Escherichia coli O157:H7 EDL933 str...

Embodiment 3

[0065] Example 3 Escherichia coli O157: H7 JSC2 strain immune challenge protection experiment

[0066] Forty 6-week-old clean BALB / c mice were selected and randomly divided into four groups: A, B, C, and D, with 10 mice per group. Groups A and B, subcutaneous injection of JSC2 strain bacterial solution on the back (preparation method is the same as that of Example 2 Title 1), 1.0×10 6 CFU / rat; groups C and D were injected with the same volume of PBS buffer (0.01mM, pH7.0) in the back. After 28 days, groups A and C were given EDL933 strain bacterial liquid orally at the same time, 1.0×10 10 CFU / only, observe the morbidity and death of mice. 72 hours after the challenge, some of the mice in group C showed poor spirits, fluffy coat, lethargy, anorexia, preference to gather together, thin feces attached to the anus, and died one after another until the end of the monitoring period, and the mortality rate reached 50% (see Figure 4 ); while the mice in group A were in good menta...

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Abstract

The invention provides an Escherichia coli O157:H7 attenuated strain and an application thereof, and belongs to the field of biotechnology. The preservation number of the Escherichia coli O157:H7 attenuated strain JSC2 is CCTCC M 2017356. A solution of the attenuated strain is prepared, and a result of toxicity attack test of the BALB / c mice and young rabbits with the attenuated strain solution shows that the strain does not cause death in the mice and young rabbits and does not cause obvious histopathological changes. After a mouse is subcutaneously inoculated with the attenuated strain, a high-level IgG antibody can be induced, the antibody has a long duration, a good immune effect is provided for vaccinated animals, and the attenuated strain can be used for developing Escherichia coli O157:H7 vaccines and diagnostic reagents.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli O157:H7 attenuated strain and application thereof. Background technique [0002] Escherichia coli ( Escherichia coli , E. coli ) O157:H7, is a common enterohemorrhagic Escherichia coli, which is an important food-borne zoonotic pathogen. In the past 20 years, food poisoning caused by Escherichia coli O157:H7 has broken out in different scales all over the world, and its animal host range is very wide, among which ruminants have a high carrier rate. Relatively speaking, animals are more dangerous than humans as a source of infection, and they are often the source of food contamination from animals. Escherichia coli O157:H7 infection has become a global public health problem because of its outbreak tendency, strong pathogenicity and lethality, and the exacerbation of the disease by antibiotic treatment. At present, there is no ideal control strategy...

Claims

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Application Information

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IPC IPC(8): C12N1/20G01N33/569A61K39/108A61P31/04C12R1/19
CPCA61K39/0258A61K2039/522C12N1/20C12N1/205C12R2001/19G01N33/56916G01N2333/245Y02A50/30
Inventor 张雪寒张碧成俞正玉叶青孙小涵郭芸芸汪伟茅爱华周俊明何孔旺
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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