Inonotus sp. CM01 and application thereof
A technology of microporous bacteria and preparations, applied in the field of microorganisms
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Embodiment 1
[0042] The fruiting bodies of this strain are collected from Yarlung Zangbo Nature Reserve in Xinjiang. The surface of the fruiting bodies is disinfected with 75% alcohol, the fruiting bodies are cut from the middle part of the fruiting bodies with a sterile scalpel, and the fruiting bodies are excavated and cut with a sterile inoculation hook. The tissue in the middle of the surface is placed in the middle of the culture dish containing PDA medium. Place the petri dish in a constant temperature incubator at 26°C until the hyphae germinate. Then repeated transfers were carried out in PDA medium to obtain a purified mycelial culture of the strain.
Embodiment 2
[0043] The identification of embodiment 2 CM01 bacterial strains
[0044] Morphological identification: the fruiting body of this strain is annual, sessile, woody structure, cap semicircular, surface yellow, with plush structure, with inconspicuous concentric circles, blunt edges, the lower surface is light yellow, with obvious Porous fungal tubes, basidiospores are oval, 8.0x5.7μm in size, yellowish brown, thick-walled. The mycelium is yellow on the PDA medium, and can form sclerotium on the PDA medium. The mycelium structure has branches but no septum, and the shape conforms to the characteristics of the photozoa. See figure 1 .
[0045] Molecular identification: the genome of mycelia was extracted using the fungal genome kit. The extracted genome was used to amplify the ITS sequence. The primers are ITS1 (5'-TCCGTAGGTGGTGAACCTGCGG-3'), ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), the 30.0μL reaction system is 10×PCR Buffer 5.0μL, dNTP 3.0μL, DNA template 2.0μL, forward and revers...
Embodiment 3
[0049] Example 3 Antitumor activity of CM01 bacterial strain
[0050] The mycelium was inoculated onto the PDA liquid shake flask culture medium by the punching method (10 mm aperture), and 4 mycelium blocks were inoculated per 100 mL of liquid culture medium. Cultivate on a shaker at 26°C and 130r / min for 10 days, filter and remove the mycelium to obtain the fermentation supernatant, and dry the fermentation supernatant by a rotary evaporator until the sample is viscous (moisture content below 10%) (40°C) , reconstituted with sterile water to 50mg / mL is the fermentation supernatant sample, treated the PDA medium in the same way as a negative control, inoculated tumor cells Hela, B16, MCF-7 and normal cells HVUEC in a 96-well plate , the seeding volume per well was 1×10 3 cells, with a volume of 100 μL, placed in 5% CO 2 In the incubator, cultured at 37°C for 24 hours, added 10 μL of fermentation supernatant sample and negative control sample respectively, and continued to c...
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