Nucleic acid aptamer targeting cell surface antigen mfi2 protein and its application
A nucleic acid aptamer and surface antigen technology, which can be used in medical preparations, anti-tumor drugs, and pharmaceutical formulations with non-active ingredients, which can solve problems such as high price and life-threatening
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Embodiment 1
[0013] Example 1: Affinity of aptamer MS2-1 to MFI2+ / +Panc-1 cells.
[0014] 293T and Panc-1 cells were washed twice with binding buffer, digested with EDTA and prepared into single cell suspension. After counting, add 500,000 / group to a centrifuge tube and resuspend in binding buffer. Add 250nM DNA to each tube and make up to 100μl with binding buffer. Incubate at 37°C in the dark for 30 min, centrifuge to remove the supernatant, and wash twice with binding buffer. Add 500μl binding buffer, mix well, and detect by flow cytometry.
[0015] The affinity curve was detected and drawn by flow cytometry, and the calculated Kd value was 39nM, indicating that the nucleic acid aptamer MS2-1 has a good affinity with Panc-1 cells, see figure 1 .
Embodiment 2
[0016] Example 2: Endocytosis of aptamer MS2-1 in MFI2 positive cells and MFI2 negative cells.
[0017] The small discs seeded with 293T and Panc-1 cells in advance were clamped out from the 6-well plate, fixed with 4% paraformaldehyde for 30 min, and washed twice with binding buffer. Configure 250nM MS2 aptamer solution with binding buffer, add 50ul dropwise to each small disc, and incubate at 4°C and 37°C in the dark for 0.5h, 1h, and 3h, respectively. Wash 3 times with binding buffer, add DAPI to mount.
[0018] The results showed that the nucleic acid aptamer MS2-1 could bind to the membrane surface of MFI2+ / +Panc-1 cells after incubation for 3 hours at 4°C, but could not be endocytosed by the cells. At 37°C, the nucleic acid aptamer MS2-1 began to be endocytosed by Panc-1 cells after 30 min of incubation, while MFI2- / -293T cells could not be endocytosed after 3 h of incubation, indicating that the nucleic acid aptamer MS2-1 can specifically Sexually binds and is endocyt...
Embodiment 3
[0019] Example 3: The coupling of aptamer MS2-1 and doxorubicin specifically inhibits the proliferation of MFI2-positive pancreatic cancer cells.
[0020] NB4 cells and Panc-1 cells were planted in a 96-well plate, with 10,000 cells per well, and 10ul of different concentrations of aptamer MS2 and doxorubicin-coupled drug MS2-1-Dox or doxorubicin single drug was added, After incubation for 72 hours, 20 ul of MTT with a concentration of 5 mg / ml was added, 100 ul of triple solution was added after 4 hours, and the absorbance was detected at a wavelength of 595 nm after incubation for 16 hours.
[0021] The results showed that MS2-1-Dox, the conjugated drug of nucleic acid aptamer MS2-1 and doxorubicin, could specifically inhibit the proliferation of MFI2+ / +Panc-1 cells, while for MFI2- / - NB4 cells, the conjugated drug could not inhibit its proliferation, but could significantly reduce the toxicity of doxorubicin, indicating that the conjugated drug MS2-1-Dox can specifically de...
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