Bran coat source peroxidase anti-tumor active fragment as well as preparation method and application thereof
An anti-tumor activity and peroxidase technology is applied in the fields of biotechnology and medicine to achieve the effect of reversing the multidrug resistance of tumors, low cost and simple and easy method.
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Embodiment 1
[0025] Example 1: Cloning and sequence analysis of recombinant gluten bran-derived peroxidase gene, the steps are as follows:
[0026] (1) Extraction of total RNA from millet seedling tissue (both using professional tips and EP tubes). Take 0.3g of millet seedlings, add liquid nitrogen to grind into fine powder, add 5ml RNA iso plus (trizol), and distribute in imported EP tubes (store at -80°C if RNA is not extracted immediately); add 200μl chloroform, shake vigorously 15s, stand still for 5min; centrifuge at 13000rpm, 4°C for 15min. Separate the layers (colorless aqueous phase, phenol-chloroform phase, light red phase) and draw the supernatant (500-600 μl) and store it in a sterile EP tube; add an equal volume of isopropanol, invert the centrifuge tube up and down, and mix well , in an environment of 15-30°C, let stand for 10min; centrifuge at 13000rpm, 4°C for 10min, a white precipitate can be seen; pour off the supernatant carefully, and slowly add 1ml of 75% ethanol along...
Embodiment 2
[0042] Example 2: Functional verification of gluten bran peroxidase active fragment (Re-FMBP-C)
[0043] ① Functional verification of Re-FMBP-C anti-tumor function
[0044] HCT-116, Hela, and DLD1 in the logarithmic growth phase were divided into 6×10 3 per well into a 96-well culture plate. 37°C with 5% CO 2 After incubating in the incubator for 24 hours, Re-FMBP-C was added at concentrations of 0.025, 0.05, and 0.1 μg / μl, respectively, and five replicate wells were set, and pMal-s tagged protein (MBP) was used as a control. HCT-8 / MDR was operated in the same way as above by adding Re-FMBP-C at a concentration of 0.025, 0.05, and 0.1 μg / μl and incubating for 48 hours, then discarding the culture medium in the well, washing it twice with PBS, and adding new culture medium to each well. Add 20 μl of MTT solution (5 mg / ml), continue to incubate for 4 h, and terminate the culture. Carefully aspirate and discard the culture supernatant in the wells, add 150 μl DMSO to each wel...
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