Nucleic acid aptamer specifically binding to metastatic gastric cancer cell, and application thereof
A nucleic acid aptamer, combined with metastasis technology, applied in tumor/cancer cells, animal cells, vertebrate cells, etc., can solve the death of cancer patients, and there is no nucleic acid aptamer that can distinguish metastatic gastric cancer cells from non-metastatic gastric cancer cells. Ligands, incurable metastatic cancer, etc.
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Embodiment 1
[0032] Screening of nucleic acid aptamers specifically binding to metastatic gastric cancer cells
[0033] 1. Synthesize a random single-stranded DNA library and primers shown in the following sequences:
[0034] Random ssDNA library:
[0035] 5'-TTCAGCACTCCACGCATAGC-40N-CCTATGCGTGCTACCGTGAA-3',
[0036] Among them, "40N" represents a sequence formed by connecting 40 arbitrary nucleotide bases, and the library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;
[0037] 5' end primer: 5'-FAM-TTCAGCACTCCACGCATAGC,
[0038] 3' end primer: 5'-(20A)-Spacer 18-TTCACGGTAGCACGCATAGG,
[0039] Among them, "20A" represents a polyA tail composed of 20 adenosine (A), "Spacer 18" represents an 18-atom hexaethylene glycol interarm, and the structural formulas of the three "Spacer 18" are shown in the following formulas I-III , the "Spacer 18" structural formula used in the above 3' end primer is shown in formula I,
[0040]
[0041] The above primers were synthesized by ...
Embodiment 2
[0060] The binding ability and binding position of nucleic acid aptamer LW-25 and metastatic gastric cancer cell HGC-27 were detected by flow cytometry:
[0061] 1. Wash the HGC-27 cells in the logarithmic growth phase twice with PBS, then digest them with enzyme-free digestion solution (APPLYGEN, Beijing) for 5 minutes, break up, centrifuge at 2000rpm, remove the supernatant, and wash with 2ml PBS for 2 Incubate with 400nM FAM-labeled aptamer LW-25 and a random single-stranded DNA library at 4°C for 1 hour;
[0062] 2. At the same time, take the HGC-27 cells in the logarithmic growth phase and wash them twice with PBS, then treat them with 0.25% trypsin digestion solution at 37°C for 5 minutes, then add serum to stop the digestion, break up the cells, and centrifuge at 2000rpm to remove The supernatant was washed twice by centrifugation with 2ml PBS and then incubated with 400nM FAM-labeled nucleic acid aptamer LW-25 at 4°C for 1 hour;
[0063] 3. The cells incubated in step...
Embodiment 3
[0065] Flow cytometry detection of binding specificity of nucleic acid aptamer LW-25 and metastatic gastric cancer cell HGC-27:
[0066] 1. Wash HGC-27 cells, AGS cells, A549 cells, Hep3B cells, sw480 cells, MCF-7 cells, HepG2 cells, and HEK-293 cells in logarithmic growth phase twice with PBS, and then digest them with enzyme-free (APPLYGEN, Beijing) was digested and dispersed, centrifuged at 2000rpm to remove the supernatant, washed twice with 2ml PBS, and then mixed with 400nM FAM-labeled nucleic acid aptamer LW-25 and random single-stranded DNA library at 4°C Incubate for 1 hour;
[0067] 2. The cells incubated in step 1 were washed twice with washing buffer, each time 400 μl, and finally 200 μl of washing buffer was added to resuspend the cells for flow cytometry detection. Test results such as Figure 4 as shown, Figure 4 It is the detection result of binding specificity between nucleic acid aptamer LW-25 and metastatic gastric cancer cell HGC-27; Figure 4 The resu...
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