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A nucleic acid aptamer specifically binding lipocalin-1 and its application

A technology of lipocalin and nucleic acid aptamer, which is applied in the field of ophthalmology, can solve the problems that are difficult to meet the early clinical diagnosis, and achieve the effect of high affinity, low preparation cost and good stability

Active Publication Date: 2021-12-10
EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these diagnostic methods have their own advantages, they often require combined diagnosis through multiple techniques, and are more inclined to the diagnosis of DR and the assessment of the disease, which is difficult to meet the needs of early clinical diagnosis.

Method used

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  • A nucleic acid aptamer specifically binding lipocalin-1 and its application
  • A nucleic acid aptamer specifically binding lipocalin-1 and its application
  • A nucleic acid aptamer specifically binding lipocalin-1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Immobilization of LCN 1 on the surface of magnetic beads

[0034] The LCN 1 protein is mainly immobilized on the surface of magnetic beads through the His tag at the C-terminus. The specific process is as follows: (a) Take 20 μL of Dynabeads TM His-Tag Isolation&Pulldown magnetic beads with 200μL selection buffer (1mM MgCl 2 in1xPBS, pH 7.2) and washed several times; (b) Dissolve 5 μg of LCN 1 protein in 200 μL of screening buffer, and add it to the 20 μL of the washed magnetic beads, and incubate at room temperature for 30 minutes; (c) incubate After finishing, rinse with screening buffer 3 times, resuspend in 20 μL of screening buffer, and store at 4°C.

Embodiment 2

[0035] Construction of embodiment 2.ssDNA library and its primers

[0036] Random ssDNA library: 5′-AGCAGCACAGAGGTCAGATG-N 40 -CCTATGCGTGCTACCGTGAA-3' (SEQID No.9), wherein, N represents any one of the bases A, T, C, G, N 40 Represents a random sequence of 40 nucleotides in length. Upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3' (SEQ ID No.10); Downstream primer: 5'-TTCACGGTAGCACGCATAGG-3' (SEQ ID No.11); Modified downstream primer: 5'-poly(dA20)-Spacer18 -TTCA CGGTAGCACGCATAGG-3' (SEQ ID No. 12).

Embodiment 3

[0037] Example 3. Screening of LCN 1 nucleic acid adaptation

[0038] Such as figure 1As shown, in order to obtain high-affinity nucleic acid aptamers that specifically bind to LCN 1, a total of 8 rounds of screening were performed. The screening protocol is shown in Table 2. The specific screening process is as follows: (a) Take 20 μL LCN 1 magnetic beads, add blocking buffer (screening buffer containing 0.1 mg / ml yeast tRNA, 1 mg / mL BSA) and incubate at room temperature for 15 min Afterwards, rinse twice with screening buffer; (b) Put a certain amount of ssDNA library in a water bath at 95°C for 10 minutes, quench in an ice bath for 5 minutes, and place it at room temperature for 10 minutes, then add it to the blocked magnetic beads, and incubate with rotation at room temperature; (c) After incubation, rinse the magnetic beads with 200 μL of screening buffer several times, magnetically separate, remove unbound ssDNA, add 100 μL of sterile water, and bathe at 95°C for 10 min...

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Abstract

The invention relates to the field of ophthalmology, in particular to a nucleic acid aptamer specifically binding to lipocalin-1 (LCN 1) and its application. The present invention obtains a set of high-affinity nucleic acid aptamers specifically binding to LCN 1 through magnetic bead SELEX technology screening, and further improves the nucleic acid aptamers by truncation and construction of highly stable mini-hairpin structures and other optimization strategies. performance. The nucleic acid aptamer of the present invention has broad application prospects, and can be used for the capture of LCN 1 in clinical samples, the detection of LCN 1 in vivo and in vitro, the development of LCN 1 therapeutic drugs in eye diseases, and the construction of targeted drug delivery systems. 1 The development of relevant detection technology, the development of clinical diagnostic methods and the research and development of nucleic acid drugs provide effective tools.

Description

technical field [0001] The invention relates to the field of ophthalmology, in particular to a nucleic acid aptamer specifically binding to lipocalin-1 and its application. Background technique [0002] Diabetic retinopathy (Diabetic Retinopathy, DR) is one of the most serious microvascular complications of diabetes, and has become the first blinding eye disease among working-age people in the world. Due to the complex pathogenesis of DR, there is no effective cure for it in clinical practice so far. Only through early diagnosis and intervention can the risk of blindness be effectively reduced. Currently, the commonly used clinical diagnostic methods for DR mainly include fundus examination, fundus angiography, optical coherence tomography, and electrophysiology. Although these diagnostic methods have their own advantages, they often require combined diagnosis through multiple techniques, and they are more inclined to the diagnosis of DR and the assessment of the disease, w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C07K14/47C07K1/14G01N33/68G01N33/53A61K47/54A61P3/10A61P9/10
CPCC12N15/115C07K14/47G01N33/6893G01N33/5308A61K47/549A61P3/10A61P9/10C12N2310/16G01N2800/164G01N2800/042G01N2333/47
Inventor 高顺祥吴继红张圣海孙兴怀
Owner EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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