An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom
A technology of Pseudomonas aeruginosa infection and Pseudomonas aeruginosa, which is applied in the direction of medical raw materials derived from viruses/phages, viruses/phages, antibacterial drugs, etc., and can solve problems such as the feasibility of hindering the treatment of phage preparations.
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Embodiment 1
[0033] Take 1L of sewage from the sewage treatment center of Tongji Hospital, add 58g of NaCl, and centrifuge at 10,000×g for 10min. Take the supernatant, add PEG-8000 to a final concentration of 10% (w / v), put it in a refrigerator at 4°C overnight, and centrifuge at 10,000×g for 20min at 4°C. Resuspend the pellet with 5ml SM Buffer. Extract with an equal volume of chloroform once. Mix 300 μl of treated sewage with 200 μl of overnight cultured host bacteria, incubate at 37°C for 20 minutes, mix with 3ml of melted top layer agar at 50°C, spread the plate, cool and invert at 37°C for overnight culture. Remove the largest plaque with a tip, dissolve it in 2ml LB culture solution, add imipenem-resistant Pseudomonas aeruginosa φA392 cultivated overnight to the culture solution at a ratio of 1:100, shake at 250 rpm at 37°C 4.5 to 5 hours. Centrifuge at 12,000 rpm for 5 minutes, and take 80% of the supernatant and store it at 4°C. Take this supernatant and co-culture with φA392 b...
Embodiment 2
[0035] The overnight cultured host bacteria φA392 was diluted 1:100, and cultured to the early logarithmic growth phase. Add 10 μl of the concentrated phage in Example 1, continue to incubate for 5 hours, add 1 / 10 volume of chloroform, continue shaking for 10 minutes, then add DNase I and RNase A to a final concentration of 1 μg / ml [3] , placed at room temperature for 30 minutes, and centrifuged to remove bacterial debris. Add 1 / 6 volume of PEG / NaCl, overnight at 4°C. The next day, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 1ml SM Buffer. Add 1 / 6 volume of PEG / NaCl to precipitate again, incubate on ice for 1 hour, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 200 μl SM Buffer to obtain amplified phage, which can reach the desired titer after repeated amplification.
Embodiment 3
[0037] Use 30 strains of diluted imipenem-resistant Pseudomonas aeruginosa to make a uniform lawn on LB medium. Divide 16-20 squares on the back of the medium and mark them, then drop the phage φA392 liquid into the corresponding squares, and after the droplets are dry, place them upside down at 37°C and incubate for 12-16h, and observe the results ( Figure 4 ). It can be seen that φA392 forms plaques on 22 strains, as follows: IMR-Pa 91, 15, 9915, 9472, 9986, 9747, 9484, 8095, 8145, 505, 9431, 7827, 1014, 1092, A392, A399 , 1423, 1505, 1612, 1182, 1578, 2024.
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