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Insulin aptamer INS5 and preparation method thereof

A nucleic acid aptamer and insulin technology, which is applied in the field of protein detection, can solve the problems of lack of research on efficient and specific recognition of insulin, and no report on the preparation method of insulin screening. Effect

Inactive Publication Date: 2017-05-10
LIUZHOU LIJIE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is still a lack of efficient and specific recognition of insulin based on aptamers, and the aptamers for insulin and their screening and preparation methods have not been reported yet.

Method used

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  • Insulin aptamer INS5 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Implementation 1 Preparation of the nucleic acid aptamer INS5 specifically binding to insulin

[0029] Construction of random sequence oligonucleotide library: artificially synthesized single-stranded DNA sequence, the construction capacity of the library is 1×10 5 The single-stranded DNA random sequence oligonucleotide library, the DNA sequence included in the single-stranded DNA random oligonucleotide library is:

[0030] 5'-GGATCCACCAGCGTCATCAGCA-N 25~40 -AGATAGTAAGTGCAATCTGGC-3'

[0031] The DNA sequence of the single-stranded DNA random oligonucleotide library includes an intermediate random sequence N 25~60 and fixed sequences at both ends, the middle random sequence N 25~40 It is a random sequence of 30-40 bases, and the fixed sequences at both ends are: 5'-GGATCCACCAGCGTCATCAGCA, 3'-CGGTCTAACGTGAATGATAGA, and the fixed sequences at both ends are PCR amplification primer binding regions.

[0032] 1) Add 0.5ml (1x10 9 Microparticles) Invitrogen’s micro-beads...

Embodiment 2

[0040] Example 2 Detecting the Binding Ability of Nucleic Aptamer INS5 and Insulin by Flow Cytometry

[0041] 1) Synthesize an insulin aptamer labeled with a fluorescent group FAM at the 5' end.

[0042] 2) Use 0 nml / L, 5 nml / L, 10 nml / L, 20 nml / L, 50 nml / L, 100 nml / L, 200 nml / L concentration gradient of FAM-labeled aptamer-linked insulin (INS) The micro-magnetic beads were used to determine the dissociation constant (kd) of the insulin aptamer. Add 150 nmol / L insulin-linked micro-magnetic beads to the aptamers at various concentrations above diluted with 200 μL of binding buffer, and incubate at 37°C for 30 min. After washing the beads with binding buffer, resuspend in 250 µL of binding buffer. Oligonucleotide fragments of random sequences and micro-magnetic beads connected with insulin-like (IN) were set as controls. Insulinoids are synthetic insulin analogs (insulin detemir).

[0043] 3) Use BD's flow cytometer to measure the fluorescence of the microbeads, and then use...

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Abstract

The invention discloses an insulin aptamer, which has high specificity and high affinity, and preparation method thereof. A nucleotide sequence of the insulin aptamer is TAGGCCTCGG ACGTTATATA CACGATCTCT ACGACCGTTC TGGCTGCGTT TCGGACTGT, and is named INS5. The preparation method includes the steps of: constructing a random sequence library, screening a target substance-nucleic acid compound, separating a target oligonucleotides aptamer, performing PCR amplification on the target oligonucleotides aptamer, cyclically screening a highly-specific aptamer, and modifying the insulin aptamer. The aptamer has no toxicity and is low in molecular weight, is easy to synthesize and mark, can only specifically the insulin and cannot recognize and be matched with other atypical-para-insulin or analogues thereof, and can be used as a molecular beacon for detection of content of insulin.

Description

technical field [0001] The invention belongs to the technical field of protein detection and relates to a nucleic acid, in particular to an insulin nucleic acid aptamer with high specificity and high affinity and a preparation method thereof. Background technique [0002] Insulin is a protein hormone secreted by the islet β cells in the pancreas when stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, and glucagon. Insulin is the only hormone in the body that lowers blood sugar, and at the same time promotes the synthesis of glycogen, fat, and protein. Exogenous insulin is mainly used in the treatment of diabetes. [0003] The biosynthesis rate of insulin is affected by the concentration of plasma glucose. When the concentration of blood glucose rises, the content of proinsulin in β cells increases and the synthesis of insulin accelerates. Insulin is synthesized in the beta cells of the pancreas. Insulin has a molecular weight of 5...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/74G01N33/53
CPCC12N15/115C12N2310/16G01N33/53G01N33/74G01N2333/62C12N2330/31
Inventor 陈东
Owner LIUZHOU LIJIE TECH
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