Method for screening drugs for targeting synthesis and assembly of bacillus tubercle cell wall core
A technology of Mycobacterium tuberculosis and cell wall, which is applied in the field of screening drugs targeting Mycobacterium tuberculosis, and can solve the problem of lack of screening drugs targeting the whole cell wall of Mycobacterium tuberculosis
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Embodiment 1
[0071] The determination of embodiment 1 inoculum size
[0072] OD 600 The value can reach 6.6-7.5, diluted to OD 600 for 3. The growth rate and final concentration of the bacteria under different culture times (8h, 10h, 12h, 14h, 24h) with different proportions of inoculum (5%, 1%, 0.5%, 0.3% and 0 / 1%) , measured by OD, the growth curve after inoculation is as follows figure 1 . Select several inoculum concentrations with small differences in absorption values before culturing, and use 1 μg / ml ethambutol to evaluate them. When the inoculum amount is 0.3%, the inhibition rate difference is the most obvious, which is 56.5±5.71, while at 0.5% It was 37.4±4.22 when the inoculation amount was 1%, and 17.2±1.37 when the inoculation amount was 1%, and the inhibition of ethambutol on the bacteria in BHI medium was the most significant when the inoculation amount was 0.3%.
Embodiment 2
[0073] The determination of embodiment 2 positive control drug concentration
[0074] In BHI and BHIS medium, measure different concentration ethambutol (1ng, 10ng, 100ng, 1 μgl, 10 μg, 100 μgl, 1mg / ml) to the growth inhibitory rate of wild-type Corynebacterium glutamicum, determine the difference between the two The maximum ethambutol concentration was the concentration of the positive control drug in the screening model.
[0075] Determination of the concentration of ethambutol, the concentration of 1 μg / ml has a significant difference in the inhibition rate of wild-type Corynebacterium glutamicum in the two media, and when the concentration of 10 μg / ml is used, in the BHIS medium, sorbitol The growth of Corynebacterium glutamicum was not significantly restored. Therefore, 1 μg / ml was chosen as the measurement standard for the construction and application of drug screening models based on 96-well plates.
Embodiment 3
[0076] The feasibility verification of embodiment 3 model
[0077] Using the conditions determined above, the different effects of antibiotics with different mechanisms of action on the test strain wild-type Corynebacterium glutamicum on the two culture media were determined. For the 96-well plate method, add the known antibiotic to be tested to each well with a final concentration of 1 μg / ml. For the disk method, 50 μl of the compound at 100 μg / ml was applied to a disk with a diameter of 7 mm.
[0078] The antibiotic of table 2 different mechanism of action is to the inhibition situation of wild-type Corynebacterium glutamicum on different mediums
[0079]
[0080] a. Use concentration is 50ug / ml; b. Use concentration is 500ug / ml
[0081] In the 96-well plate-based screen, the difference in activity was represented by the difference in inhibition rate. It can be seen from Table 2 that among the drugs known to act on the cell wall of Mycobacterium tuberculosis, ethambuto...
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