RAGE antagonistic polypeptide and application thereof
An antagonistic and derivative technology, applied in the field of biotechnology and biomedicine, can solve the problems of reduced action time and easy degradation of siRNA by the body, and achieves huge social and economic benefits.
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Embodiment 1
[0038] Example 1 QPCR detection of RAGE protein expression levels in different cancer cells
[0039] Take normal breast cells MCF-10A, estrogen positive (ER+) cells MCF-7 and triple negative cells MDA-MB-231 cells.
[0040]RNA extraction: Take a 6cm petri dish as an example. When the cells grow well and the confluence reaches 80-90%, discard the cell culture medium and add 1×PBS to wash the cells to remove the remaining culture medium. Then add 300-500 μL of Trizol (the amount of Trizol is determined according to the cell density) into a 6 cm petri dish, and use a pipette to blow repeatedly until the cells are broken and there is no obvious precipitation in the lysate. Put it in a 1.5mL EP tube (note the use of RNase-free pipette tips and EP tubes, and it is best to wear a mask); place it on ice for 5 minutes, then add chloroform at a ratio of Trizol to chloroform of 5:1, Vigorously shake for 15s, chloroform can effectively separate the organic phase from the inorganic phase,...
Embodiment 2
[0050] Example 2 Screening, Synthesis and Identification of RAGE Antagonistic Polypeptides
[0051] 1. Screening of RAGE antagonistic polypeptides
[0052] (1) Establishment of 293T cell line with permanent high expression of RAGE: 293T-RAGE + / + / LRH
[0053] ①Select vigorously growing human-derived 293T cells, and the day before transfection, use 5×10 5 cells / well, seeded in a 6-well plate, cultured until the second day, the cell confluency was 60%;
[0054] ②Transfection was carried out on the second day, using one culture well of a 6-well plate as a unit, dilute 3 μg RAGE expression plasmid with 200 μL opti-MEM medium, and dilute 6 μL liposome Lipofectamine2000 with 200 μL opti-MEM medium, respectively After mixing gently, let stand at room temperature for 5 minutes;
[0055] ③ Gently mix the two tubes of dilutions, let stand at room temperature for 20 minutes, then gently add 600 μL of opti-MEM medium to the mixed dilutions;
[0056] ④ Gently rinse the cells to be tra...
Embodiment 3
[0083] The specific binding of RAGE antagonistic polypeptide to RAGE
[0084] FITC-labeled RAGE antagonistic polypeptides (Shanghai Qiangyao Biological Co., Ltd.), inoculated MDA-MB-231 cells on confocal plates, washed three times with PBS, fixed with paraformaldehyde for 15 minutes, washed three times with PBS, blocked with BSA for 1.5 hours, and incubated RAGE antibody overnight, using Alexa 594-linked anti-rabbit fluorescent secondary antibody was incubated at room temperature for 1 hour, and then incubated with FITC-labeled RAGE antagonistic polypeptide at a final concentration of 100 μmol / mL for 2 hours at room temperature, washed three times with PBST, stained with DAPI, and confocal laser scanning microscope (German Zeiss Co., Ltd. ) observation, see the results Figure 4 .
[0085] in, Figure 4 A is the fluorescent confocal microscope image of DAPI nuclear staining (shown in blue in the original fluorescent color map);
[0086] Figure 4 B after incubation of MD...
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