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Stilbene Estrogen Nucleic Aptamer and Its Application

A stilbene, nucleic acid aptamer technology, applied in biochemical equipment and methods, material testing products, instruments, etc., can solve the problems of time-consuming, labor-intensive, production, transportation, storage constraints, etc.

Active Publication Date: 2021-10-01
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two main types of detection methods for stilbene estrogens: the first type is the detection of physical and chemical properties, including gas chromatography or liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. Professional and technical personnel are required to operate expensive instruments and equipment, and sample pretreatment is required, and the whole process is time-consuming and labor-intensive; the second type is immunoassay, and currently the ELSA method is mainly used to detect the residual amount of stilbene estrogen in animals , but the antibodies required for immunoassays are essentially proteins, which are subject to great constraints in production, transportation, storage, etc.

Method used

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  • Stilbene Estrogen Nucleic Aptamer and Its Application
  • Stilbene Estrogen Nucleic Aptamer and Its Application
  • Stilbene Estrogen Nucleic Aptamer and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 2

[0036] Example 1 Screening of stilbene estrogen DNA aptamers

[0037] The SELEX process of this embodiment is shown in figure 1 , using streptavidin-modified magnetic beads as the stationary phase, using immobilized libraries and target elution for screening.

[0038] 1. Immobilization of nucleic acid library and streptavidin-modified magnetic beads

[0039] In the first round of screening, take the primary library lib dry powder 1OD (sequence information: 5'-ttcagcactccacgcatagc(SEQ ID NO.04)-n40-cctatgcgtgctaccgtgaa(SEQ ID NO.05)-3') synthesized from TaKaRa Company, and put it into Centrifuge at 12000rpm / min, centrifuge for 10min, then add 260μL DPBS (0.9mMCaCl 2 , 2.7mM KCl, 0.5mM MgCl 2 6H2O, 0.137M NaCl, 1.1mM KH 2 PO4, 8.1mM Na 2 HPO 4 ) dissolved. After dissolving, shake with a vortex for about 1 min, and then centrifuge at 12000 rpm / min for 10 min to prepare a 5 μM library solution. Then take 5nmol of bio-P3 primer dry powder synthesized from GenScript, centrif...

Embodiment 2

[0051] Example 2 Cloning and sequencing of nucleic acid aptamers and prediction of secondary structure of candidate aptamers

[0052] 1. Recovery of PCR products

[0053] Using non-modified upstream and downstream primers, the 14th round of screening ( figure 2 ) to amplify the secondary library obtained, followed by separation on 3% agarose gel, cutting the gel to recover the target band, and using the Axygan brand agarose gel recovery kit to recover the target product.

[0054] 2. Ligation and transformation of ligation products

[0055] 1) Add 1 μL full gold pEASY-T5 Zero vector and 4 μL aptamer PCR product into a microcentrifuge tube;

[0056] 2) Ligate at 25°C for 10 minutes (the pEASY-T5 Zero vector kit comes with a ligase);

[0057] 3) Add the ligated product to 50 μL DH5α competent cells, and place in ice for 20 minutes;

[0058] 4) After heat shock at 42°C for 30s, place on ice for 2min;

[0059] 5) Add 250 μL of LB medium (without resistance) preheated at 37°C,...

Embodiment 3

[0064] Example 3: Specific detection of nucleic acid aptamers and stilbene estrogens

[0065] 1. Detection specificity of gold nanometer colorimetric method

[0066] 1) Preparation of nano-gold: set the total volume in one tube of this example to 400 μL (total system), add 100 μL of prepared nano-gold to a 1.5mL centrifuge tube, add 200 μL ddH 2 O;

[0067] 2) Add aptamer to protect gold nanoparticles: then add aptamer Apt-7 with a final concentration of 300nM (that is, add 12 μL of 10 μM DPBS aptamer to it), take the above tube on a circular mixer at 10r / min, and incubate at room temperature for 20min ;

[0068] 3) Add target: 40 μL of 10 mM diethylstilbestrol (DES), hexestrol (HEX), dienestrol (DIES), estrone (E1) and β-estradiol were sequentially added to tubes 1-5 of the experimental group (E2) solution (the final concentration of the target solution and the analog solution is 1000 μM; add 40 μL of matrix solution to tube 0 of the control group;

[0069] 4) Take the ab...

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Abstract

The invention discloses a stilbene estrogen nucleic acid aptamer, including at least one of Apt-7, Apt-21 and Apt-31, wherein the sequence of Apt-7 is shown in SEQ ID NO.01, Apt-7 The sequence of 21 is shown in SEQ ID NO.02, and the sequence of Apt-31 is shown in SEQ ID NO.03. The nucleic acid aptamer of the present invention can recognize and bind stilbene estrogens with high specificity and high affinity, and can be applied to related methods for detecting stilbene estrogens, in terms of detection of stilbene estrogens and It is of great significance in reducing the damage of stilbene estrogens to the human body.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a stilbene estrogen nucleic acid aptamer and its application. Background technique [0002] Stilbene-like estrogens are a class of artificially synthesized non-steroidal estrogens. At present, there are mainly three substances: Diethylstilbestrol (DES), Hexestrol (HEX) and Dienestilbestrol (Dienesterol). , DIES); this type of hormone was widely used in the 1960s to treat uterine bleeding, menstrual disorders, fetal induction and animal fattening; stilbene estrogens are highly fat-soluble and can enter the human body through the food chain. Clinical studies have found that the use of this type of estrogen The second generation and even the third generation of hormones are all affected negatively. Females have vaginal malignant tumors, breast cancer, male sperm decrease, etc., and at the same time interfere with the human endocrine system. Therefore, it is highly desirable...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/74G01N33/53
CPCC12N15/115C12N2310/16G01N33/5308G01N33/74
Inventor 林俊生苏艺蒋灵丽杜烨芃郭方可
Owner HUAQIAO UNIVERSITY
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