Nucleic acid aptamer wh6 specifically binding to annexin a2 and its application
An annexin and nucleic acid aptamer technology, applied in the field of aptamers, can solve the problems of no ANXA2 reporting or publication
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Embodiment 1
[0023] A nucleotide library sequence with a length of 80 nt was synthesized (Shanghai Sangon Bioengineering (Shanghai) Co., Ltd.), the specific sequence is as follows: 5'-ACCGAC CGT GCT GGA CTC A (N)42 A CTA TGA GCGAGC CTG GCG-3 ', both ends are fixed sequences, 42N in the middle of this library represents 42 random bases, which can guarantee a library capacity of about 10 14 , a sufficiently large library capacity can form different three-dimensional spatial structures, thereby ensuring the existence of sequences with spatial structures that bind to the target, and will be screened out in the subsequent screening process. The sequence of the library is the P80 ss library. The random oligomeric ssDNA library was then amplified with the following primers:
[0024] P80-SF: 5'-FAM-ACC GACCGT GCT GGA CTC A-3' (SEQ ID NO. 1)
[0025] P80-AP: 5'-biotin-CGC CAG GCT CGC TCA TAG T-3' (SEQ ID NO.2)
[0026] Random ssDNA library (5000 pmol in the first round of screening, equivalent to...
Embodiment 2
[0033] The total protein of multiple myeloma ANBL-6 and NCI-H929 cells was extracted using RIPA protein lysate (P0013B, Beyotime Institute of Biotechnology), and the protein concentration was determined with a BCA kit (purchased from Promega Company), electrophoresis, and membrane transfer. Incubate with ANXA2 antibody (sc86235, Santa Cruz Biological Company, dilution ratio 1:1000) and GAPDH antibody (dilution ratio 1:5000) respectively, add the corresponding HRP-labeled secondary antibody, incubate for 2 h, wash the membrane with PBST, and pass through ECL The reaction solution is developed. The expression of ANXA2 protein in different multiple myeloma cell lines was detected.
[0034] see results figure 2 , found that the expression of ANXA2 was higher in multiple myeloma ANBL-6 cells, but lower in NCI-H929 cells.
Embodiment 3
[0036] Multiple myeloma ANBL-6 cells and NCI-H929 cells were cultured to the logarithmic growth phase, and counted to adjust the cells to 1×10 6 cells / mL; take 0.3 mL cells each, and wash twice with Wash Buffer; dilute FITC fluorescent group-modified wh6 and DNA-lib to 250 nM with Binding Buffer; incubate with cells in ice-water mixture for 50 min, wash with BindingBuffer was washed twice, each time for 2 min; the cells were resuspended to 400 μL with PBS, detected by flow cytometry and the results were analyzed.
[0037] see results image 3 It was shown that the wh6 nucleic acid aptamer had significant binding to multiple myeloma ANBL-6 cells, but had no obvious binding to multiple myeloma NCI-H929 cells.
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