Screening method for protein nucleic acid aptamer

A nucleic acid aptamer and screening method technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low aptamer enrichment efficiency, aptamer affinity and specificity reduction, and achieve Good enrichment, high affinity, and improved screening efficiency

Inactive Publication Date: 2019-01-11
HUAIHAI INST OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods require chemical modification of the aptamer screening library, such as binding streptavidin-labeled magnetic beads or agarose microspheres. Chemical modification may change the inherent structure of the target protein, making the screened aptamer The enrichment efficiency is low, and the affinity and specificity of the obtained aptamers are reduced

Method used

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  • Screening method for protein nucleic acid aptamer
  • Screening method for protein nucleic acid aptamer
  • Screening method for protein nucleic acid aptamer

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: A screening method for protein nucleic acid aptamers: embed the protein on the surface of a polystyrene 96-well plate or other utensils that absorb protein; add trypsin or protease to digest and degrade the protein, and make the protein and oligonucleotide The acid-binding complex is detached from the surface of the container, the protease is inactivated by heating and the ssDNA is released; the ssDNA that specifically binds to the protein is enriched, and aptamers with high affinity are screened.

Embodiment 2

[0036]Example 2: A screening method for protein nucleic acid aptamers: the specific steps are as follows: protein molecules are coated in a 96-well polystyrene plate, blocked with BSA or FBS, and then the oligonucleotide sequence ssDNA is added to the Incubate in the plate wells coated with protein for a certain period of time, remove the unbound oligonucleotide sequences with buffer, digest the protein-oligonucleotide complexes in the 96-well polystyrene plate with trypsin diluent, and then Boil the entire solution system in boiling water until the protein is denatured; centrifuge at 12,000g for 20 minutes to remove the precipitate to remove protein residues; use PCR to enrich the secondary library, use asymmetric PCR and denaturing polyacrylamide gel cutting to prepare and purify the oligo Nucleotide single strand; enriched nucleic acid aptamers can be obtained after cycling the above steps 7-10 times.

Embodiment 3

[0037] Embodiment 3, the screening method experiment of protein nucleic acid aptamer:

[0038] In this example, the surface recombinant antigen HP-Ag of Helicobacter pylori, natural carcinoembryonic antigen CEA, and tumor markers CA125 and CA199 were used as targets, and the following screening methods were used to screen nucleic acid aptamers. The specific steps are as follows:

[0039] 1 Synthesize 80nt random single-stranded DNA library and primers;

[0040] Random library: 5'-AAC TAC GCT CAG ACA GAC AC-40N-CTG TGA TGC GAC TAT CTG AG -3';

[0041] 5' Primer: 5'-AACTACGCTCAGACAGACAC-3'

[0042] 3' Primer: 5'-CTCAGATAGTCGCATCACAG-3'

[0043] 2 Screening: Dilute the four proteins with protein coating solution and coat them in 96-well polystyrene plates, shake slowly at 37°C for 2 hours, remove the solution and wash with PBS, add an equal volume of 1mg / mL bovine serum albumin, After shaking slowly at 37°C for 1 h, the protein solution was discarded and the 96-well polystyren...

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Abstract

The invention relates to a screening method for protein nucleic acid aptamer. The screening method comprises the following steps: embedding protein on the surface of a polystyrene 96 pored plate or other vessel for absorbing protein; adding trypsin or protease for digesting and degrading protein, removing the compound of protein and oligonucleotide from the container surface, heating for inactivating protease and releasing ssDNA; enriching the ssDNA specifically combined with the protein, thereby acquiring high-affinity aptamer. According to the screening method disclosed by the invention, screening library and PCR primer need not be modified, cost is saved while a target structure is not changed, so that the specificity of the screened aptamer is promoted; the ssDNA specifically combinedwith the protein is enriched, so that the screening efficiency is increased and the high-affinity aptamer is screened; the screening method is suitable for the screening of protein-targeted nucleic acid aptamer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer screening method, which is applicable to the screening of nucleic acid aptamers targeting proteins. Background technique [0002] Aptamers are a class of functionalized oligonucleotide sequences that can form specific three-dimensional structures, which can bind to proteins, peptides, toxins, metal ions, and cells with high affinity and specificity. Nucleic acid aptamers are called "chemical antibodies". Compared with traditional antibodies, their small molecular weight, strong specificity, and high stability make them potential ligands. Especially for proteins that are difficult to prepare antibodies, the screening of aptamers makes it possible to detect these proteins with high sensitivity. Nucleic acid aptamers are also widely used in the research of many fields such as biosensors, biopharmaceuticals and molecular recognition probes. A variety of protein-t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811
CPCC12Q1/6811C12Q2525/205C12Q2531/113C12Q2531/107
Inventor 王淑军吕明生闫婉丽房耀维刘姝陈丽谷利德
Owner HUAIHAI INST OF TECH
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