CD133 antagonistic polypeptide as well as derivative and application thereof
A derivative and antagonistic technology, applied in the field of biotechnology and biomedicine, to achieve the effect of promoting apoptosis and huge social and economic benefits
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Embodiment 1
[0047] Example 1: Panning, amplification, purification, sequencing and synthesis of CD133 antagonistic polypeptide HL12-CP5.
[0048] This example is mainly for the purpose of screening positive phages that specifically bind to CD133, and then by amplifying and purifying the positive phages, extracting phage single-stranded DNA (ssDNA) for sequencing, analyzing and comparing the obtained sequences, and finally synthesizing high-purity The antagonistic polypeptide HL12-CP5.
[0049] details as follows:
[0050] 1. Establishment of 293T cell line with permanent high expression of CD133: 293T-CD133 + / +
[0051] ①Select vigorously growing luminescent human 293T cells, and the day before transfection, use 5×10 5 cells / well, inoculated in a 6-well plate, cultured until the second day, the cell fusion degree was 60%;
[0052] ② Transfect on the second day, take one culture well of a 6-well plate as a unit, dilute 3 μg of plasmid with 200 μL of opti-MEM medium, and dilute 6 μL of ...
Embodiment 2
[0070] Example 2 Enzyme-linked immunosorbent assay detection of phage monoclonal binding to CD133
[0071] Firstly, a single clone of HL12-CP5 phage was picked, inoculated in LB medium containing ER2738, and cultured with shaking at 230 rpm for 7 hours at 37°C. Centrifuge at room temperature for 15 minutes at 5000 rpm, collect the supernatant and store at 4°C. 293T wild-type cells, 293TCD133 + / + cells, HCT-116 cells and HT-29 cells at 5×10 per well 3 seeded in 96-well plates and cultured for 24 hours. The next day, the cell culture medium was discarded, and blocking solution (DMEM+5%BSA) was added to each well to block for 2 hours. After discarding the blocking solution, add 100 μL of phage dilution solution (1:100) to each well for 2 hours. Then wash with PBST (PBS+0.1% Tween-20) 3 times, each time for 5min to remove unbound phage. Add 100 μL diluted (1:5000) peroxidase-linked anti-M13 phage antibody (purchased from GE) to each well, and let it react for 1 hour at room t...
Embodiment 3
[0072] Example 3 HL12-CP5 can significantly inhibit the proliferation of colorectal cancer cell HCT-116
[0073] ①The colorectal cancer cells HCT-116 were treated with 5×10 3 Each cell / well was inoculated in a 96-well cell culture plate with a medium volume of 200 μL in each well, cultured for 24 hours, and then starved overnight;
[0074] ② Add HL12-CP5 polypeptides with different concentration gradients (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM) and culture for 24 hours, 48 hours, and 72 hours, respectively;
[0075] ③ Add 20 μL of MTT working solution to each well, and continue to culture in a carbon dioxide incubator for 4 hours;
[0076] ④ Discard the supernatant in the culture plate, add 150 μL DMSO (dimethyl sulfoxide), shake for 10 minutes, select a wavelength of 490 nm on a microplate reader for detection, and draw the growth curve of the cells.
[0077] In the previous work of the inventor's laboratory, it was found by q-PCR test that the colorectal cancer ...
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