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Nucleic acid aptamer of protein, derivative of nucleic acid aptamer and application thereof

A nucleic acid aptamer and derivative technology, applied in the fields of biomedicine and analytical chemistry, to achieve the effects of good stability, high sensitivity, easy synthesis and transformation

Active Publication Date: 2021-10-08
HUNAN SAIAOWEI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on nucleic acid aptamers of NTRK1 protein at home and abroad

Method used

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  • Nucleic acid aptamer of protein, derivative of nucleic acid aptamer and application thereof
  • Nucleic acid aptamer of protein, derivative of nucleic acid aptamer and application thereof
  • Nucleic acid aptamer of protein, derivative of nucleic acid aptamer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Screening of NTRK1-specific nucleic acid aptamers

[0042] 1. Library reverse screening

[0043] 1.1 Take 25ug of His protein and incubate with 50ul of carboxyl magnetic beads activated by 0.4M EDC and 0.1M NHS at room temperature for 60min to couple to carboxyl magnetic beads; add 100ul of 1M ethanolamine and incubate at room temperature for 10min to quench unreacted Activate the carboxylic acid group, adsorb the magnetic beads on the magnetic frame, remove the supernatant, wash with an appropriate amount of DPBS 3 times, and set aside.

[0044] 1.2 Dissolve 1OD (about 1.4nmol) of the initial library in 280ul DPBS, heat at 95°C for 10min, cool in ice water for 5min, then place at room temperature for 5min, add His protein-coupled magnetic beads prepared in 1.1, and incubate at room temperature for 1h , the magnetic frame adsorbs the magnetic beads, and the collected supernatant is recorded as NTRK1-pool-1.

[0045] 1.3 Wash the magnetic beads 4 times with ...

Embodiment 2

[0064] Example 2: Dot Blot detection of Apt-TRK-1 and Apt-TRK-2 binding to NTRK1 protein

[0065] The dot blot experiment was performed using a Smart Blotter vacuum transfer instrument (Wealtec, Smart Blotter SB-10). The filter paper and nitrocellulose membrane (NC membrane) were pre-cut, and the NC membrane was soaked with transfer buffer and placed on On the filter paper, put the NC membrane and filter paper on the support plate, and make sure there are no air bubbles on it, gently close the top cover, insert and close the flip stoppers on both sides at the same time, and then assemble the Smart Blotter system to the aspirator , spot 1ug NTRK1 protein in batches into the sample wells, use the same amount of BSA as the control protein, turn on the aspirator to pump air, wait for all the protein solution to pass through the membrane, turn off the aspirator after 5min, take out the membrane, and use 3 %BSA plus 0.1% Tween-20 was blocked at room temperature for 1 h, washed 3 tim...

Embodiment 3

[0066] Example 3: Determination of Apt-TRK-1 and Apt-TRK-2 and NTRK1 Protein Affinity

[0067] In order to detect the affinity between the screened nucleic acid aptamers and NTRK1 protein, we used the enzyme-linked nucleic acid aptamer adsorption method (ELASA) and the magnetic bead fluorescent quantitative PCR method to determine the level and dissociation constant K D .

[0068] 1. ELASA determination of the binding affinity of Apt-TRK-1 and Apt-TRK-2

[0069] In a 96-well ELISA plate, add 100ng NTRK1 protein to each well, coat with gentle rotation overnight at 4°C, discard the supernatant, wash 3 times with PBST, block with 3% BSA + 100ug / mL salmon sperm DNA for 2 hours, and aspirate Blocking solution, washed 3 times with PBST, added biotin-labeled Apt-NTRK1-1 and Apt-NTRK1-1 and Apt-NTRK1-1 and Apt- NTRK1-2, repeat 3 times in each well, add PBS as blank control at the same time, incubate at room temperature for 1 hour, aspirate the supernatant, wash 4 times with PBST, ad...

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Abstract

The invention provides a nucleic acid aptamer of a protein, which is a nucleic acid aptamer specifically combined with an NTRK1 protein. Preferably, the sequence of the nucleic acid aptamer comprises at least one of SEQ ID No. 1 and SEQ ID No. 2. The nucleic acid aptamer or its truncated nucleic acid aptamer and derivative of the nucleic acid aptamer provided by the invention can be highly specifically combined with NTRK1 protein, which is a novel targeting small molecule, and has the advantages of small molecular weight, high sensitivity, good stability, easy synthesis and modification and the like compared with an antibody. According to the invention, a first nucleic acid aptamer capable of being combined with the NTRK1 protein is screened out through a magnetic bead SELEX technology, and a novel potential targeting drug is provided for treatment of human chronic pain.

Description

technical field [0001] The invention relates to the fields of biomedicine and analytical chemistry, in particular to a protein nucleic acid aptamer, its derivatives and applications thereof. Background technique [0002] Chronic pain refers to pain that lasts for more than one month. Some people compare chronic pain to an immortal cancer. At present, there are at least 100 million chronic pain patients in China. The NTRK1 gene belongs to the nerve growth factor receptor family and encodes a single transmembrane receptor tyrosine kinase protein TrkA. Nerve growth factor (NGF) is its main high-affinity ligand. TrkA dimerizes after binding to dimeric NGF ligands, autophosphorylates and activates downstream signaling, thereby regulating cell differentiation and survival. TrkA is expressed in various tissues and organs, such as peripheral nervous system, central nervous system, digestive tract, adrenal cortex, etc. Nociceptor sensitization induced by NGF-TrkA pathway signaling ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10A61K31/7105A61P25/04G01N33/68
CPCC12N15/115C12N15/1048G01N33/68A61K31/7105A61P25/04C12N2310/16G01N2333/71
Inventor 蔡娜李柱
Owner HUNAN SAIAOWEI BIOTECH CO LTD
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