Bran coat source peroxidase anti-tumor active fragment as well as preparation method and application thereof
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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.
Active Publication Date: 2018-05-18
SHANXI UNIV
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[0003] There are no research reports on anti-tumor...
Method used
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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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Abstract
The invention provides a bran coat source peroxidase anti-tumor active fragment as well as a preparation method and application thereof. The preparation method of the fragment comprises the followingsteps: extracting mRNA of rice seedling tissues by a Trizol method, taking the mRNA as a template, performing enzymatic catalysis, and performing inverse transcription to obtain cRNA; obtaining a protein gene sequence in Genebank according to FMBP mass spectrum identification results of the rice anti-tumor active protein, respectively performing truncated screening according to different functional regions, determining a gene sequence of Ca<2+> binding sites as an anti-tumor effect structural domain, and designing a specific primer thereby; taking the rice cDNA as a template, and amplifying the FMBP-C gene sequence by a PCR (Polymerase Chain Reaction) method; respectively introducing BamH I and HindIII restriction enzyme sites at the upstream and downstream of the sequence, connecting thesites with a pMal-s vector, constructing a pMal-s-FMBP-C recombinant plasmid, transferring E.coli DH5alpha competent cells, and screening to obtain a positive transformant; performing induced expression on a target protein through IPTG (Isopropyl Thiogalactoside), performing affinity purification, desalting and concentrating, and detecting functions of resisting tumors and reversing multiple drugresistance of tumors by an MTT method. The results show that the active fragment has obvious activities of resisting tumors and reversing multiple drug resistance of the tumors.
Description
technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to an antitumor active fragment of gluten bran-derived peroxidase and its preparation method and application. Background technique [0002] Millet, scientific name: Setaria italica, also known as millet, is called millet after peeling. Botanically, it belongs to the genus Setaria of the family Poaceae. It originated in China. It is a dominant crop in the north and one of the characteristic agricultural products in Shanxi Province. Bran is a by-product of the processing of millet into millet. At present, there are not many developments and researches on bran. Bran is rich in nutrients and beneficial ingredients. Such as: triglycerides, lipoproteins, oryzanol, vitamin B, vitamin E, glucose, cellulose, and trace elements. In recent years, it has been found that there are a series of active molecules with medicinal value in millet. These active molecules have antioxidant ac...
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