Escherichia coli O157:H7 attenuated strain and application thereof

[0033] Example 1 Isolation and identification of Escherichia coli O157:H7 JSC2 strain

[0034] (1) Stool collection:

[0035] Several fresh cow dung was collected from Zhujiashan Dairy Farm in Nanjing City, placed in airtight bags, and stored at low temperature.

[0036] (2) Enrichment culture:

[0037] Weigh 25 g of each fecal sample into a sterile conical flask, add 225 mL of sterile normal saline, and place it in a 37°C incubator for 2 h; Shake culture at ℃ for 6h to obtain enrichment solution.

[0038] (3) Immunomagnetic bead enrichment:

[0039] Take 1mL of the enrichment solution obtained in step (2) into a sterile centrifuge tube, add 20uL Dynabeads® anti-E. rack for enrichment; stand still, aspirate the supernatant from the tube, wash the magnetic beads three times with 0.01 mM, pH 7.0 PBS buffer, and collect the bacterial liquid. The washing method is as follows: resuspend the magnetic beads with PBS buffer, place them in a magnetic stand, let them stand, and aspira

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

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

[0053] Take out the cryopreservation tube of E. coli O157:H7 EDL933 strain and JSC2 strain from the cryogenic freezer, dip the inoculation loop, streak it on the SMAC plate, and cultivate it at 37°C for 12-20 h, pick a single colony and inoculate it on mEC The broth was incubated at 37°C for 7-8 h, centrifuged at 12000 r / min for 5 min, and the bacterial slurry was resuspended in PBS buffer (0.01 mM, pH 7.0), and the animals were challenged for use.

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

[0055] Twenty 6-week-old clean-grade BALB / c mice (provided by Yangzhou University School of Medicine) 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, 1.0×10 10 CFU / only; group B, oral administration of the same volume of E. coli O157:H7 EDL933

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

[0066] Forty 6-week-old clean-grade BALB / c mice were selected and randomly divided into four groups: A, B, C, and D, 10 mice / group. Groups A and B, the back subcutaneous injection of JSC2 strain bacterial solution (the preparation method is the same as that of Example 2 title 1), 1.0 × 10 6 CFU / only; Groups C and D, the same volume of PBS buffer (0.01mM, pH 7.0) was injected back. After 28 days, groups A and C were given oral administration of EDL933 strain at the same time, 1.0×10 10 CFU / mouse, observe the morbidity and death of mice. 72 hours after the challenge, some mice in group C showed poor spirit, fluffy coat, lethargy, anorexia, like to get 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 mental state and did no