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Fusion protein aptamer screening method and kit

A nucleic acid aptamer and fusion protein technology, applied in the field of genetic engineering, can solve the problems of high screening cost, complicated operation, single target, etc., and achieve the effect of improving high efficiency, small adsorption effect, and good biocompatibility

Inactive Publication Date: 2019-02-12
廖世奇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these screening methods have certain limitations, such as high screening cost, long cycle, complex operation, single target, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. The method for screening the expression of Strep-TagII in Escherichia coli cells using the fusion protein nucleic acid aptamer screening kit

[0051] (1) Centrifuge 100 µL of Escherichia coli fusion protein expressing bacteria solution and 100 µL of non-expressing protein bacterial solution at 4°C, 12,000 r / min for 30 minutes, discard the supernatant, and incubate with 100 µL agar magnetic beads at 37±0.3°C for 30 minutes, wash Wash once with buffer solution, add protein blocking solution and seal at 37±0.3°C for 1 hour, remove the supernatant, wash once with washing buffer solution, and magnetically separate to obtain positive sieve tubes (magnetic bead E. coli fusion expression protein bacterial solution) And anti-sieve tube (magnetic beads E. coli non-expressing protein bacteria liquid).

[0052] (2) Forward binding: In the first round of screening, the random oligonucleotide library (ssDNA library) was dissolved in binding buffer, heated at 95°C for 5 min...

Embodiment 2

[0058] Example 2, the method of screening the expression of GST by Pichia pastoris using the fusion protein nucleic acid aptamer screening kit

[0059] (1) Centrifuge 100 µL Pichia fusion protein expressing bacteria solution and 100 µL non-expressing protein bacterial solution at 4°C, 12,000 r / min for 30 minutes, discard the supernatant, and incubate with 100 µL agar beads at 37±0.3°C for 30 minutes, Wash once with washing buffer, add protein blocking solution and seal at 37±0.3°C for 1 hour, remove the supernatant, wash once with washing buffer, and magnetically separate to obtain positive screen tubes (Pichia magnetic beads fusion expressing proteobacteria) liquid) and anti-sieve tube (Pichia magnetic beads non-expressing protein bacteria liquid).

[0060] (2) Forward binding: In the first round of screening, the random oligonucleotide library (ssDNA library) was dissolved in binding buffer, heated at 95°C for 5 minutes and cooled rapidly in ice water for 10 minutes, then ad...

Embodiment 3

[0066] Example 3, the method for screening the expression of His-tag by baculovirus using fusion protein nucleic acid aptamer screening kit

[0067] (1) Centrifuge 100 µL of baculovirus fusion protein-expressing bacterial solution and 100 µL of non-expressing protein bacterial solution at 4°C, 12,000 r / min for 30 minutes, discard the supernatant, and incubate with 100 µL of agar beads at 37±0.3°C for 30 minutes. Wash once with washing buffer, add protein blocking solution and seal at 37±0.3°C for 1 hour, remove the supernatant, wash once with washing buffer, and magnetically separate to obtain positive screen tubes (magnetic bead baculovirus fusion expressing proteobacteria) solution) and anti-sieve tube (magnetic bead baculovirus non-expressing protein bacteria solution).

[0068] (2) Forward binding: In the first round of screening, the random oligonucleotide library (ssDNA library) was dissolved in binding buffer, heated at 95°C for 5 minutes and cooled rapidly in ice water...

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Abstract

The invention provides a fusion protein aptamer screening method and kit in gene engineering, and belongs to the field of molecular biology. The fusion protein aptamer screening method utilizes a two-way heat cycling subtraction systematic evolution of ligands by exponential enrichment (SELEX), adopts fusion proteins in the gene engineering as a screening target, and epoxy (or carboxyl, NHS magnetic beads and streptomycin) agar magnetic beads as a vector, couples a fusion tag protein antibody (or biotinylated aptamer) onto the magnetic beads to form capturing magnetic beads so as to capture the fusion tag protein expressed in the gene engineering, and utilizes a random oligonucleotide library with large capacity to be combined with magnetic bead-tag protein antibody-tag protein-target protein to form a complex, thereby screening out the aptamer specifically combined with the target protein. The fusion protein aptamer screening method has the advantages of high sensitivity, high specificity, rapidness, high efficiency and the like, and has important significance in detection and medicine research for the secretion or intracellular expression gene engineering fusion protein of bacteria, yeast, insects and mammalian cells.

Description

technical field [0001] The present invention relates to a method for screening fusion protein nucleic acid aptamers in genetic engineering, in particular to a fusion protein nucleic acid aptamer in genetic engineering that is established by organically combining nano magnetic bead materials, SELEX screening technology and high-throughput nucleic acid sequencing The screening method, and the present invention also relates to a test kit based on the screening method, which belongs to the technical field of genetic engineering. Background technique [0002] SELEX stands for Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This technology uses a large-capacity random oligonucleotide library to interact with target proteins, and screens out nucleic acid adapters that specifically bind to the target. Ligand. The method has the advantages of high sensitivity, strong specificity, rapidity and high efficiency. Since Tuerk first used this technology to screen speci...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6869G01N33/53
CPCC12N15/1058C12N2320/13C12Q1/6869G01N33/53C12Q2535/122
Inventor 廖世奇田彩平袁红霞魏政丽
Owner 廖世奇
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