Method for screening aptamer via high performance liquid chromatography, aptamer and application
A high-performance liquid chromatography and nucleic acid aptamer technology, which can be used in pharmaceutical formulations, medical preparations with inactive ingredients, and medical preparations containing active ingredients, etc., and can solve the problems of difficulty in automation, high cost, and low efficiency. , to achieve the effect of low instrument requirements, low cost and strong universality
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Embodiment 1
[0049] A method for HPLC-assisted screening of nucleic acid aptamers, mainly using a microfluidic chip for screening, and the specific screening process includes the following steps:
[0050] (a) Optimizing the nucleic acid library: the structure of the DNA library is divided into five parts: in the middle is a fixed sequence of 26 bases, which is used for complementary hybridization with the fixed probe (BDNA); on both sides of the fixed sequence are two DNA arms, Both include 18-base random sequences and 19-base sequences necessary for PCR amplification. The immobilization probe is a DNA strand complementary to the immobilization sequence of the library labeled with biotin at one end, and is used for immobilization of the library.
[0051] Random library RS36 has the nucleotide sequence described in SEQ ID NO.1, specifically:
[0052] 5'-CCGCTTCGCCGTCTCCTAC-NNNNNNNNNNNNNNNNNN-AAAAGTGGGTAGGGCGGGTTGGAAAA-NNNNNNNNNNNNNNNNNNNN-CGCTCGTCACCCCTTCTCCT-3' (Note: N stands for any bas...
Embodiment 2
[0071] A nucleic acid aptamer 1 of docetaxel screened by the method of Example 1, the nucleotide sequence of the nucleic acid aptamer 1 has the nucleotide sequence described in SEQ ID NO.2, specifically 5'- TTGTTTCTCTGTCGATTA-3'.
Embodiment 3
[0073] A nucleic acid aptamer 2 of docetaxel screened by the method of Example 1, the nucleotide sequence of the nucleic acid aptamer 2 has the nucleotide sequence described in SEQ ID NO.3, specifically 5'- ATTAGCTGTCTCTTTGTT-3'.
[0074] The nucleotide sequences of the nucleic acid aptamers in the above-mentioned Examples 2 and 3 are selected from naturally occurring or artificially synthesized sequences, or the same sequences from any other sources.
[0075] Examples 2 and 3 are only preferred nucleic acid aptamers screened by the present invention. According to the screening method of the present invention, nucleic acid aptamers with the following nucleotide sequences can also be screened:
[0076] (1) The homology with the nucleotide sequence of the nucleic acid aptamer listed in Example 2 or 3 is more than 60% (for example, the above-mentioned nucleic acid aptamer sequence can be deleted or partially complementary nucleotides can be added);
[0077] (2) the sequence that...
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