FMT donor screening method based on PFOR enzyme activity guidance and application of FMT donor screening method
A screening method and enzyme activity technology, which is applied in the field of medical testing, can solve the problems of being unable to cultivate Clostridium difficile FMT donors and the harsh culture conditions of Clostridium difficile, so as to inhibit the growth of Clostridium difficile, and is suitable for widespread application and operation. convenient effect
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Embodiment 1
[0044] This example provides a PFOR enzyme activity-oriented screening method for FMT donors, targeting the PFOR enzyme in Clostridium difficile, and testing the inhibition rate of the fecal liquid provided by FMT donors to PFOR enzymes, screening prediction and / or evaluate the therapeutic effect of FMT on Clostridium difficile infection.
[0045] Its technical principle is as figure 1 As shown, the PFOR enzyme is involved in the pyruvate metabolism of Clostridium difficile, that is, PFOR (pyruvate ferredoxin oxidoreductase) catalyzes the oxidative decarboxylation of pyruvate to generate acetyl-CoA (acetyl-CoA) and CO 2 , Pyruvate decarboxylation reaction is the first step for Clostridium difficile to obtain energy through glycolysis, and inhibiting the activity of PFOR can effectively inhibit Clostridium difficile.
[0046] The specific steps of its screening method are as follows:
[0047] (1) cultivating Escherichia coli with PFOR enzyme plasmid;
[0048] (2) inducing E...
Embodiment 2
[0054] On the basis of the foregoing examples, the present embodiment further limits the culture method of cultivating Escherichia coli with the PFOR enzyme plasmid:
[0055] 1. Preparation of LB medium: liquid medium (broth): 5g LB powder dissolved in 500ml distilled water, solid medium (agar): 3.5gLB powder dissolved in 100mL distilled water. Place the prepared medium in a high-pressure steam sterilizer, and sterilize it under high pressure at 121°C for 15 minutes. The liquid volume in the culture bottle should not exceed 1 / 3 of the range of the bottle itself. Take the medium out of the high-temperature sterilizer at about 65°C , add ampicillin (100 μg / mL) before solidification of the solid medium and mix well, then pour the medium into petri dishes, and add about 10 mL of medium for each. Then let it cool and solidify. Add ampicillin to the liquid medium after it cools down. 2. Bacterial plate inoculation: Pick a single colony from the existing medium, streak the plate in...
Embodiment 3
[0057] In this embodiment, the E. coli with the PFOR enzyme plasmid cultivated above was induced and extracted with PFOR enzyme, specifically as follows:
[0058] 1. Take about 50mL of liquid culture medium (with ampicillin added) in a centrifuge tube, pick a single colony from the plate obtained by growing bacteria, put it in a centrifuge tube, and place it in a shaking incubator at 37°C, 220rpm, for about 200min. After shaking for about 200 minutes, add isopropyl-β-D-thiogalactoside (IPTG) for induction (final concentration of IPTG is 0.5mMol / ml), place in a shaking incubator, 25°C, 220rpm, 20h .
[0059] 2. Take 50ml of the induced bacteria solution, centrifuge at 12000g, 4°C, 5min, and remove the supernatant. Resuspend in an equal volume of pure water and centrifuge again to remove the supernatant. Then resuspend in 10mL pure water. Sonicate 10mL of bacterial solution on ice at 400W for 5s with an interval of 5s for about 10min. The obtained solution is the crude enzyme...
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