A kind of Bacillus subtilis with cephalosporin resistance and high expression sir2 protein and application thereof
A Bacillus subtilis, high expression technology, applied in the field of screening and application mechanism in the new army, can solve the problem of no related research, and achieve the effect of improving mitochondrial respiration and enhancing aerobic oxidation
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Embodiment 1
[0029] 1. Experimental methods and materials
[0030] 1.1 Preliminary screening of probiotics
[0031] Collect 1.0g of feces samples from 3 young patients in Nanyang People's Hospital, amplify and culture with LB medium, remove plasmid with ethidium bromide method, dilute with distilled water to 10-8, a total of 9 dilutions, take 1mL, and spread Put it on the LB solid medium, place it in the incubator, and cultivate it for 48 hours at 37 hours. After the culture is over, take out the plate and pick out rough and opaque surface, stained white or slightly yellow colonies, and transfer to LB liquid medium for 48 hours at 37°C, and store in a refrigerator at 4°C for later use.
[0032] 1.2 Morphological observation of strains.
[0033] Inoculate the pure culture of isolated and purified strains into the enrichment liquid medium, revive after 37 hours of cultivation for 24 hours, and perform Gram staining, observe the morphological characteristics of the cells under an optical mi...
Embodiment 2
[0093] Example 2 Bacillus subtilis BS16sir2 (BS-sir2) gene transfected cells and intracellular function research
[0094] 1. Experimental materials
[0095] 1.1 Bacteria, plasmids and cells
[0096]
[0097] 1.2 Main media and reagents
[0098] (1) Buffer A: 150 mM NaCl, 500 mM Tris-HCl, 0.1% TritoX-100, pH 8.0.
[0099] (2) Buffer B: Consists of 50 mM Tris-HCl, 0.1% TritonX-100, pH 8.0.
[0100] (3) Antibody diluent: Weigh 10 g of BSA powder and dissolve it in 200 mL of TBST buffer to form a 5% BSA solution, and store it at 4°C for later use.
[0101] (4) Cell growth medium: fetal bovine serum (final concentration: 10%) and penicillin-streptomycin solution (final concentration: 100 units / ml) were added to the culture medium, and stored at 4°C for later use.
[0102] (5) Cell maintenance solution: Fetal bovine serum and penicillin-streptomycin solution (final concentration: 100 units / mL) (final concentration: 2%) were added to the culture medium and stored at 4°C for la...
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