A cd133 antagonistic polypeptide and its derivatives and applications
A derivative and antagonistic technology, applied in the field of biotechnology and biomedicine, to achieve huge social and economic benefits, and promote the effect of apoptosis
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0047] Example 1: Panning, amplification, purification, sequencing and synthesis of CD133 antagonistic polypeptide HL12-CP5 were performed.
[0048] The purpose of this example is to screen and obtain 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 phages 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] ①Choose vigorously growing luminescent human 293T cells, and culture them at 5 × 10 cells one day before transfection. 5 Cells / well, seeded in 6-well plates, and after the second day of culture, the cell confluence was 60%;
[0052] ②Transfection was carried out on the second day. Using one culture well of a 6-well plate as a unit, 3 μg of plasmid...
Embodiment 2
[0070] Example 2 Enzyme-linked immunosorbent assay to detect the binding of phage monoclonal to CD133
[0071] First, a single clone of HL12-CP5 phage was picked, inoculated into LB medium containing ER2738, and cultured at 37°C with shaking at 230 rpm for 7 hours. Centrifuge at 5000rpm for 15min at room temperature, collect the supernatant and store at 4°C. 293T wild-type cells, 293TCD133 + / + Cells, HCT-116 cells and HT-29 cells were plated at 5 × 10 per well 3 were 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, 100 μL of phage dilution (1:100) was added to each well for 2 hours. Then wash with PBST (PBS+0.1% Tween-20) 3 times for 5 min each time to remove unbound phage. 100 μL of diluted (1:5000) peroxidase-linked anti-M13 phage antibody (purchased from GE) was added to each well, and allowed to...
Embodiment 3
[0072] Example 3 HL12-CP5 can significantly inhibit the proliferation of colorectal cancer cells HCT-116
[0073] ①The colorectal cancer cells HCT-116 were divided into 5×10 3 Cells / well were inoculated into 96-well cell culture plates with a medium volume of 200 μL per well, cultured for 24 hours, and then starved overnight;
[0074] ②Add different concentration gradients (100μM, 10μM, 1μM, 0.1μM, 0.01μM, 0.001μM) of HL12-CP5 polypeptide 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 a cell growth curve.
[0077] In the preliminary work of the inventor's laboratory, it was found that the colorectal cancer cell HCT-116 highly expressed CD133 by...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More