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Group of aptamers for specific recognition of beta-bungatotoxin and use thereof

A technology of bungarotoxin and nucleic acid aptamer, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., and can solve problems such as poor stability, insufficient adsorption of enzyme-labeled plates, and weak affinity , to achieve the effects of small molecular weight, easy chemical modification and labeling, and short cycle time

Inactive Publication Date: 2014-05-28
中国人民解放军成都军区疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with polyclonal antibody, the affinity of monoclonal antibody is weaker, the adsorption to microtiter plate is not good enough, and the stability is also poor in the operation of enzyme or biotin labeling

Method used

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  • Group of aptamers for specific recognition of beta-bungatotoxin and use thereof
  • Group of aptamers for specific recognition of beta-bungatotoxin and use thereof
  • Group of aptamers for specific recognition of beta-bungatotoxin and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of nucleic acid aptamers specifically binding to β-bungarotoxin

[0033]1. Construct a random single-stranded DNA library and primers: design a random single-stranded DNA library with a fixed sequence of 20 bases at both ends and a random sequence of 40 bases in the middle, namely: 5′-AGC AGC ACA GAG GTC AGA TG -N40-CCT ATG CGT GCT ACC GTG AA-3'. Where N represents any one of bases A, G, C, and T, and N40 represents a random fragment length of 40 bases. Primer 1: 5'-fluorescein-AGC AGC ACA GAG GTC AGA TG-3', Primer 2: 5'-TTC ACG GTA GCA CGC ATA GG-phosphate group-3'. The library and primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. Binding buffer (20mM HEPES, pH7.4; 150mM NaCl; 5mM KCl; 2mM MgCl 2 ;2mM CaCl 2 ), primers use ddH 2 O was prepared into a 100 μM stock solution and stored at -20°C for later use.

[0034] 2. SELEX screening: Prepare β-bungarotoxin with carbonate buffer (CBS, pH 9.6) to a concentration of 10 μg / mL, t...

Embodiment 2

[0037] Example 2 Determination of the binding force of each round of nucleic acid aptamer library and β-bungarotoxin by digesting fiber membrane method

[0038] Nitrocellulose membranes (HAWP02500nitrocellulose filters, Millipore, 0.22μm) were soaked in 0.5M KOH solution for 30min, then soaked with ddH 2 O was fully washed, then placed in the binding buffer, shaken for 40min, placed in a new binding buffer, and stored at 4°C. Each round of fluorescein-labeled ssDNA library was prepared to a concentration of 1 μM, 10 μL was added to 1 mL of binding buffer, placed at 90 ° C for 10 min, then placed on ice for 10 min, and at room temperature for 10 min. Add 4 μL of 1 mg / mL β-bungarotoxin, incubate at room temperature for 1 h, and control without adding β-bungarotoxin, then filter the membrane and wash the filter membrane with 1 mL of binding buffer. The filtrates were combined, and the fluorescence intensity of the filtrate was measured with a Perkin Elmer LS55 fluorescence spect...

Embodiment 3

[0039] Example 3 Determination of the dissociation constant Kd value of the nucleic acid aptamer by the fluorescence anisotropy method

[0040] Fluorescence-labeled aptamers βB-1, βB-19, βB-32, and βB-20 were artificially synthesized, and 4 μL of aptamers (1 μM) were added to 2 mL of binding buffer to measure the fluorescence anisotropy value. Then titrate with different concentrations of β-bungarotoxin, and measure the fluorescence anisotropy value at each concentration. Determination parameters are the same as in Example 2. Use the concentration of β-bungarotoxin as the abscissa, and the increment of the fluorescence anisotropy value as the ordinate, and use SigmaPlot10.0 software to do nonlinear regression to analyze the dissociation constant (Kd) value of the aptamer.

[0041] The Kd values ​​of the aptamers βB-1 and βB-20 were both lower than 100nM, 65.9nM and 83.8nM respectively, and the measurement results were as follows image 3 shown. The Kd value of βB-19 is 540 ...

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Abstract

The invention provides a group of aptamers for specific recognition of beta-bungatotoxin and use thereof. The aptamers provided by the invention are any single-chain deoxyribonucleic acid (DNA) sequence shown in SEQ ID NO.1-4 in a sequence table. The aptamers and the beta-bungatotoxin have high affinity and specificity, and have broad application prospects in detection of the beta-bungatotoxin and diagnosis of bungarus multicintus bite.

Description

technical field [0001] The present invention relates to the technical field of biological toxins, specifically referring to the preparation of a group of nucleic acid aptamers with high specificity binding to β-bungarotoxin by using SELEX technology (exponentially enriched ligand system evolution technology) in molecular biology technology , to provide a scientific basis and a theoretical basis for the nucleic acid aptamer in the detection of β-bungarotoxin and the diagnosis of Bungare bites. Background technique [0002] β-bungaretoxin is derived from the venom of Bungarus multicinctus, and is a presynaptic polypeptide neurotoxin that exhibits Ca 2+ Dependent phospholipase A2 (Phospholipase A 2 , PLA 2 ) activity, which is the most important toxic component of Bungara venom, LD50 (i.p. mouse) is 0.019μg / g. The molecular weight (Mr) is 20500, which is composed of two chains, A chain and B chain, and connected by a pair of interchain disulfide bonds. The A chain with a la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68C12Q1/44G01N21/64
Inventor 叶锋平郑颖范泉水王熙米其利郭平余静王杰张志晓冯子良
Owner 中国人民解放军成都军区疾病预防控制中心
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