CN110885827B - Aptamer specifically bound with vomitoxin, preparation method and application - Google Patents

Aptamer specifically bound with vomitoxin, preparation method and application Download PDF

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CN110885827B
CN110885827B CN201911170189.8A CN201911170189A CN110885827B CN 110885827 B CN110885827 B CN 110885827B CN 201911170189 A CN201911170189 A CN 201911170189A CN 110885827 B CN110885827 B CN 110885827B
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vomitoxin
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CN110885827A (en
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韩芹芹
秦鸣蔚
夏雪山
张晓萌
宋玉竹
张金阳
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Heilongjiang Hehe Medical Laboratory Co ltd
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Kunming University of Science and Technology
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Abstract

The invention belongs to the technical field of biomedicine, and discloses a nucleic acid aptamer specifically combined with vomitoxin, a preparation method and application thereof, wherein the nucleic acid aptamer is ssDNA and consists of 82 nucleotides, and the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1 is shown in the specification; its secondary structure contains prominent rings and stems, among which DONA15 gibbs free energy DG ═ 8.84, and the aptamer has a G-quadruplex structure, which is more stable. A series of property evaluations such as specificity, affinity and sensitivity of the aptamer are carried out based on an enzyme-linked oligonucleotide adsorption assay (ELONA), and the fact that DONA15 can be specifically combined with vomitoxin is shown, so that the aptamer has the characteristics of high specificity and high affinity, and can be used in subsequent rapid detection of the vomitoxin in a test strip.

Description

Aptamer specifically bound with vomitoxin, preparation method and application
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a nucleic acid aptamer specifically bound with vomitoxin, a preparation method and application thereof.
Background
Deoxynivalenol (DON), also known as vomitoxin (vomitoxin), is mainly one of trichothecene compounds, which are toxic metabolites produced by some species of fusarium and have similar chemical structures and biological activities. The major producer of DON is fusarium graminearum (fusarium graminearum), some other fusarium species have been reported to be produced. DON is widely existed in the world, mainly pollutes cereal crops such as wheat, barley, corn and the like, also pollutes grain products, and people and animals can generate wide toxic effects after eating the cereal crops polluted by the toxin by mistake. In addition, it often contaminates crops with other mycotoxins, such as aflatoxin, which can interact with each other after entering the body. In recent years, DON is discovered to be possibly related to human esophageal cancer and IgA nephropathy, and poses a threat to the health of human beings and animals. DON belongs to a virulent or moderate poison, and researches show that DON can accumulate to a certain extent in vivo, but does not have a special target organ and has strong cytotoxicity. After people and livestock ingest food/feed polluted by DON, acute poisoning symptoms such as anorexia, vomit, diarrhea, fever, unstable standing, slow response and the like can be caused, and the hematopoietic system is damaged to cause death in severe cases, but different animals have different degrees of sensitivity to the DON, and pigs are the most sensitive animals. Research shows that DON may have influence on immune system, obvious embryotoxicity and certain teratogenicity, and genetic toxicity, but no carcinogenic and mutagenic effect. Because of serious harm of DON, the method draws general attention of various countries. There are strict limits on the content of DON in cereals and feeds.
Aptamers (aptamers) are oligonucleotide sequences with high specificity and affinity for a target substance, which are screened from random oligonucleotide libraries based on the SELEX technique. A random oligonucleotide library with a library capacity of 1014-1015nt is artificially and chemically synthesized, the total length of the random oligonucleotide library is generally 70-100nt, and the random oligonucleotide library comprises 20-40nt random sequences in the middle. And incubating the library and a target substance for a certain time to form a nucleic acid-target compound, removing the library sequence which is not combined with the target by using a certain method, thermally dissociating the compound to obtain a sequence combined with the target, and carrying out PCR amplification by using the sequence as a template to further prepare a next-level library. The oligonucleotide sequence with high specificity and high affinity to the target, namely the aptamer, is obtained through 8-20 rounds of continuous screening. The aptamer is cloned and sequenced to obtain a corresponding nucleic acid sequence for subsequent research. Since then, aptamers have been widely used in many fields such as cell imaging, new drug development, disease treatment, and microorganism detection, but there is no method or product for directly detecting vomitoxin.
Disclosure of Invention
The invention provides a nucleic acid aptamer specifically combined with vomitoxin, a preparation method and application, and does not solve the problems in the prior art. The method is realized by the following technical scheme:
an aptamer that specifically binds to emetic toxin having the sequence set forth in SEQ ID NO: 1.
Further, the secondary structure of the nucleic acid aptamer binding specifically to emetic toxin has a protruding loop and stem, gibbs free energy DG ═ 8.84.
Further, the aptamer specifically binding to emetic toxin has a G-quadruplex structure.
Further, the Aptamer corresponding primer comprises an Aptamer Fw and an Aptamer Rv, wherein the sequence of the Aptamer Fw is shown as SEQ ID NO: 2 and SEQ ID NO: 3, the sequence of Aptamer Rv is shown as SEQ ID NO: 4 and SEQ ID NO: 5.
a method for preparing an aptamer that specifically binds to emetic toxin, the method comprising the steps of:
step one, screening, namely screening a nucleic acid aptamer population capable of being specifically combined with vomitoxin through a column capable of coupling hydroxyl by adopting SELEX technology;
step two, selecting a monoclonal, designing a primer for PCR amplification, selecting the monoclonal, utilizing a PMD 19-T carrier to connect and transform a PCR product into a competent cell, and marking the competent cell connected and transformed on a culture medium plate with ampicillin at 37 ℃ overnight;
and step three, separating, namely separating a large amount of nucleic acid by using a scribing method, and shaking bacteria to obtain the single nucleic acid required by people to obtain the aptamer DON A15.
The application of the aptamer specifically bound with the vomitoxin can be used in a kit for directly detecting the vomitoxin.
The invention has the beneficial effects that: compared with the existing vomitoxin detection technology on the market, the aptamer DON A15 screened by the SELEX technology can identify and combine the vomitoxin with high affinity and high specificity, so that the subsequent detection technology based on the aptamer can realize the direct detection of the vomitoxin in the food; the specificity, affinity and sensitivity identification of the aptamer DON A15 ensure the accuracy of the detection result of the residual vomitoxin in the food. The detection method based on the aptamer does not need complex sample pretreatment steps, reduces the loss of vomitoxin in sample liquid, and improves the reliability of detection results. Therefore, the invention fills the blank of the current method for directly detecting the vomitoxin at home and abroad.
Drawings
FIG. 1 is a block diagram of the aptamer DON A15 of the present invention;
FIG. 2 is a specific analysis of aptamer DON A15 by the ELONA method provided in the examples of the present invention;
FIG. 3 is an analysis of the affinity of the aptamer DON A15 by the ELONA method provided in the examples of the present invention;
FIG. 4 is an analysis of the sensitivity of aptamer DON A15 to vomitoxin based on the ELONA method provided in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
Example 1:
cloning, isolation and sequencing of aptamers and prediction of secondary structure of single-stranded DNA
Screening: the SELEX technology is adopted to screen a nucleic acid aptamer population capable of being specifically combined with vomitoxin through a column capable of being coupled with hydroxyl.
Picking a single clone: PCR products were ligated into competent cells using the PMD 19-T vector, and the ligated competent cells were streaked out onto ampicillin-bearing medium plates overnight at 37 ℃.
Separation: a large amount of nucleic acid is separated by a scribing method, and single nucleic acid required by people is obtained by shaking bacteria.
And (3) prediction: the temperature was set to 26 ℃ by MFOLD software, Na+At a concentration of 150mM, Mg2+Secondary structure prediction of the aptamer DON A15 single-stranded DNA molecule that specifically binds to emetic toxin was performed at a concentration of 1mM QGRS mapping. The results show that the aptamer contains prominent rings and stems, the DON A15 Gibbs free energy DG is-8.96, and the aptamer has a G-quadruplex structure and has high stability (see figure 1).
II, specificity, affinity and sensitivity to vomitoxin of aptamer DON A15
1. Aptamer DON A15 specific detection
The method is improved on the basis of the traditional ELISA method, the selected aptamer is used for replacing an antibody, and a biotin-avidin amplification system is used for detecting a sample to be detected.
(1) Coating of toxins
0.05M pH 9.6 carbonate buffer was mixed with vomitoxin at a volume ratio of 1:1 to give a final vomitoxin concentration of 50. mu.g/mL. Adding 100 mu L of PBST cleaning solution into each hole, adding 200 mu L of PBST cleaning solution into each hole, shaking and washing for 3 times on a horizontal constant temperature shaking bed, and patting dry on clean absorbent paper after washing for 2 minutes each time. A blank control and a negative control are set simultaneously (the blank control is skim milk, the negative control is non-target substances including AFB1, AFG1, ZEN, OTA, and the concentration of the positive group is kept consistent with that of the positive group, and the method is the same as the method).
(2) And sealing the container
And (3) adding 100 mu L of 5% skim milk into each well of the enzyme label plate coated with vomitoxin, sealing the wells with adhesive sticker, incubating the wells for 2 hours at 37 ℃ on a 100rpm oscillator, discarding the liquid in the wells after sealing, and repeating the washing step in the step (1).
(3) Adding aptamer with biotin label for incubation
The aptamer DON A15 capable of binding to vomitoxin is obtained by screening and sent to Shanghai bio-organisms for synthesis, and the DNA is labeled with Biotin (Biotin) DON A15. When the aptamer is used, a short-time centrifugation is firstly carried out, so that the aptamer which is marked with biotin is totally gathered at the bottom of a test tube. According to the instructions, the biotin-labeled aptamer was dissolved in sterile water sufficiently to have a concentration of 10-4The storage solution of M, in order to avoid repeated freezing and thawing, can be divided into small portions.
The aptamer DON A15 labeled with biotin was diluted with 1 XPBS to a working concentration of 400nM, and then 100. mu.L of the aptamer was added to each well, sealed with a pressure-sensitive adhesive or a sealing film, incubated at 37 ℃ for 2 hours on a 100rpm shaker, and after incubation, the well liquid was discarded, and the washing step in (1) was repeated.
(4) Incubation with enzyme conjugate
mu.L of streptavidin-labeled horseradish peroxidase conjugate was added to each well, followed by sealing with a pressure-sensitive adhesive, followed by incubation at 37 ℃ for 1 hour on a 100rpm shaker, after which the well liquid was discarded, and the washing step in (1) was repeated.
(5) And color development
100. mu.L of a TMB solution was added to each well, followed by development in the dark at 37 ℃ for 20 minutes.
(6) And terminate
Finally, 50. mu.L of stop solution (2M sulfuric acid) was added to each well, and the absorbance at 450nm of each well was measured by a microplate reader within 10 minutes from the termination of the reaction to obtain OD450 nm.
The results show that aptamer DON a15 is capable of specific binding to emetic toxin (see figure 2).
2. Affinity K of aptamer DON A15dValue calculation
(1) 0.05M pH 9.6 carbonate buffer and vomitoxin are mixed evenly according to the volume ratio of 1:1, and the final concentration of the vomitoxin is 50 mu g/mL. Adding 100 mu L of washing solution into each hole, sealing with adhesive, incubating for 2 hours on an oscillator with the temperature of 37 ℃ and the rpm of 150, discarding liquid in each hole after incubation, adding 200 mu L of washing solution into each hole, and performing oscillation washing on a horizontal shaking table for 3 times, 2 minutes each time, and drying on clean absorbent paper each time;
(2) adding 100 mu L of 5% skim milk into each hole in an enzyme label plate coated with vomitoxin, sealing with adhesive sticker, incubating for 2 hours on an oscillator at 37 ℃ and 100rpm, discarding liquid in the holes after sealing, and repeating the washing step in step 1;
(3) diluting the biotin-labeled aptamer with 1 XPBS to 1nM, 5nM, 10nM, 20nM, 40nM, 80nM, 100nM, 200nM and 400nM, adding 100 muL of the biotin-labeled aptamer into each well, sealing with a self-adhesive sticker or a sealing film, incubating for 2 hours at 37 ℃ on a 100rpm oscillator, discarding the liquid in the well after incubation, and repeating the washing step in step 1;
(4) adding 100 mu L of horseradish peroxidase conjugate into each hole, sealing with adhesive sticker, incubating for 1 hour on a vibrator with the speed of 150rpm at the temperature of 37 ℃, discarding liquid in the holes, and repeating the washing step in step 1;
(5) adding 100 mu L of TMB color developing agent into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
(6) adding 50 mu L of stop solution (2M sulfuric acid), and measuring the absorbance value OD450nm at 450nm of each hole by using a microplate reader within 10 minutes after the reaction is stopped; the results show that K of the aptamer DON A15d28.77 ± 7.585nM (see fig. 3).
3. Detection of sensitivity of aptamer DON A15 to vomitoxin
(1) And 0.05M of carbonate buffer solution with pH of 9.6 and the vomitoxin are uniformly mixed according to the volume ratio of 1:1, so that the vomitoxin forms different concentration gradients. Adding 100 mu L of washing solution into each hole, sealing with adhesive, incubating for 2 hours on an oscillator with the temperature of 37 ℃ and the rpm of 150, discarding liquid in each hole after incubation, adding 200 mu L of washing solution into each hole, and performing oscillation washing on a horizontal shaking table for 3 times, 2 minutes each time, and drying on clean absorbent paper each time;
(2) adding 100 mu L of 5% skim milk into each hole in an enzyme label plate coated with vomitoxin, sealing with adhesive sticker, incubating for 2 hours on an oscillator at 37 ℃ and 100rpm, discarding liquid in the holes after sealing, and repeating the washing step in step 1;
(3) diluting the biotin-labeled aptamer to 400nM with 1 XPBS, adding 100 muL of the biotin-labeled aptamer into each well, sealing the wells with adhesive sticker or sealing film, incubating the wells for 2 hours at 37 ℃ on a 100rpm oscillator, discarding the liquid in the wells after the incubation is finished, and repeating the washing step in step 1;
(4) adding 100 mu L of horseradish peroxidase conjugate into each hole, sealing with adhesive sticker, incubating for 1 hour on a vibrator with the speed of 150rpm at the temperature of 37 ℃, discarding liquid in the holes, and repeating the washing step in step 1;
(5) adding 100 mu L of TMB color developing agent into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
(6) adding 50 mu L of stop solution (2M sulfuric acid), and measuring the absorbance value OD450nm at 450nm of each hole by using a microplate reader within 10 minutes after the reaction is stopped;
the results showed that the lowest concentration of vomitoxin detected by aptamer DON A15 was 7.81. mu.g/mL (see FIG. 4).
And (4) conclusion: the aptamer DON A15 is successfully screened in the SELEX technology, and successfully shows better specificity, sensitivity and affinity, and plays an important role in detecting vomitoxin in subsequent actual samples.
Sequence listing
<110> university of Kunming science
<120> nucleic acid aptamer specifically bound with vomitoxin, preparation method and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> DNA
<213> Artificial sequence (Artificial)
<400> 1
gacatattca gtctgacagc gcgctcacaa taaaaggtct tggatgaggg aaagcatccc 60
ggatggacga atatcgtcta gc 82
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial)
<400> 2
gacatattca gtctgacagc 20
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
cgctgtcaga ctgaatatgt c 21
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 4
gctagacgat attcgtccat c 21
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 5
gatggacgaa tatcgtctag c 21

Claims (5)

1. An aptamer that specifically binds to emetic toxin having the sequence set forth in SEQ ID NO: 1.
2. The aptamer specific for binding to emetic toxin according to claim 1, wherein the secondary structure of said aptamer specific for binding to emetic toxin has overhanging loops and stems with gibbs free energy DG-8.84.
3. The aptamer specific for binding to emetic toxin according to claim 1, wherein the aptamer specific for binding to emetic toxin has a G-quadruplex structure.
4. The Aptamer specific binding for emetic toxin of claim 1, wherein the Aptamer-corresponding primer comprises AptamerFw and Aptamer Rv, wherein the AptamerFw has the sequence of SEQ ID NO: 2 and SEQ ID NO: 3, the sequence of Aptamer Rv is shown as SEQ ID NO: 4 and SEQ ID NO: 5.
5. use of the aptamer specifically binding to emetic toxin according to claim 1 in the preparation of a kit for detecting emetic toxin.
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CN111500586B (en) * 2020-05-21 2022-11-04 昆明理工大学 Aptamer specifically combined with rabies virus L protein capping region and application thereof
CN113295858A (en) * 2021-06-04 2021-08-24 南京师范大学 Method for detecting vomitoxin by using GelRed-based non-labeled aptamer sensor and application
CN113774063B (en) * 2021-08-30 2023-05-23 北京市农林科学院 Vomitoxin specific nucleic acid aptamer and application thereof

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CN110066804A (en) * 2019-05-06 2019-07-30 昆明理工大学 A kind of aptamer with borax specific binding

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CN110066804A (en) * 2019-05-06 2019-07-30 昆明理工大学 A kind of aptamer with borax specific binding

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基于无模板随机合成鸟嘌呤四聚体的荧光"开启"生物传感器用于粮食中呕吐毒素的快速检测;韩逸陶等;《食品科学》;20190531;第41卷(第14期);第300-306页 *

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