CN117487812A - Nucleic acid aptamer specifically binding aflatoxin B1 small molecule and application thereof - Google Patents
Nucleic acid aptamer specifically binding aflatoxin B1 small molecule and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention discloses a nucleic acid aptamer specifically combined with aflatoxin B1 small molecules and application thereof, and the nucleic acid aptamer comprises a nucleotide sequence shown as SEQ ID No. 1; alternatively, a nucleotide sequence having high homology with the nucleotide sequence of SEQ ID No.1 and capable of specifically binding to the aflatoxin B1 small molecule; or a nucleotide sequence which is derived from the nucleotide sequence shown in SEQ ID No.1 and can specifically bind to the aflatoxin B1 small molecule. The aptamer has the advantages of high specificity, high stability, convenient synthesis, low molecular weight and easy modification, can replace complex mass spectrographs and antibodies, and provides the aptamer capable of being specifically combined with the aflatoxin B1 to detect the aflatoxin B1, so that a novel method is provided for detecting the aflatoxin B1.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer specifically combined with aflatoxin B1 small molecules and application thereof.
Background
Aflatoxin (Aflatoxin) is a series of difuranyl compounds produced by certain strains such as Aspergillus flavus and Aspergillus parasiticus. It includes about 20 toxins, of which Aflatoxin B1 (Aflatoxin B1) is the most toxic of all aflatoxins. Aflatoxin B1 has very strong oncogenic, mutagenic and teratogenic properties and is also very stable when heated. The probability of occurrence of the peanut, corn, grain and oil in food and feed in hot and humid areas is highest, peanut, corn, grain and oil are easily polluted by aflatoxin B1, and food polluted by aflatoxin B1 can enter the body through a food chain and accumulate, so that liver chronic damage and even death are caused.
Aflatoxin B1 has been listed by the international cancer research institute as a class I carcinogen, and the contents of aflatoxin B1 in various foods and agricultural products are well defined by the world health organization and the various national health organizations, and detection of aflatoxin B1 is an important barrier for human life safety. Current methods of detection for aflatoxin B1 are varied, traditional instrumentation methods such as High Performance Liquid Chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), but these methods require highly trained personnel to perform the operations, and are costly and time consuming. While various products based on ELISA kits and related immunosensors are commercially available. However, the production cost, batch stability, cross-reaction sensitivity, time consumption, false positive or false negative results, and matrix interference effects of antibodies limit their widespread use.
In recent years, aptamer (aptamer) has become a new type of biosensing element for detecting analytes from ions to cells. Nucleic acid aptamer refers to DNA or RNA molecules obtained by screening and separating by an exponential enrichment ligand system evolution (SELEX) technology, and can be combined with other targets such as proteins, metal ions, small molecules, polypeptides and even whole cells with high affinity and specificity. Compared with the traditional method and the antibody, the aptamer has the advantages of low cost, good stability, simple operation, suitability for various analysis methods and the like. Based on the above, the invention develops a nucleic acid aptamer capable of specifically binding with aflatoxin B1 to detect the aflatoxin B1, which provides a new method for detecting the aflatoxin B1.
Disclosure of Invention
In order to solve the problems, the invention provides a nucleic acid aptamer specifically binding to aflatoxin B1 small molecules, which firstly obtains the aflatoxin B1 nucleic acid aptamer with high affinity, high specificity, low molecular weight and easy modification, and replaces complex mass spectrographs and antibodies.
The invention also provides application of the nucleic acid aptamer, and a method for detecting the content of aflatoxin B1 by utilizing aptamer fluorescence competition.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a nucleic acid aptamer specifically combined with aflatoxin B1 small molecules, which comprises a nucleotide sequence shown in SEQ ID No. 1; alternatively, a nucleotide sequence having high homology with the nucleotide sequence of SEQ ID No.1 and capable of specifically binding to the aflatoxin B1 small molecule; or a nucleotide sequence which is derived from the nucleotide sequence shown in SEQ ID No.1 and can specifically bind to the aflatoxin B1 small molecule.
As a preferred embodiment of the present invention, the high homology means at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology with the nucleotide sequence shown in SEQ ID No. 1.
As a preferred embodiment of the invention, the nucleic acid aptamer comprises a nucleotide sequence complementary to the nucleotide sequence and maintains affinity.
As a preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid aptamer comprises a base modification and maintains affinity.
As a preferred embodiment of the present invention, the base modification is a thio modification, a phospho modification, a methylation modification, an amination modification, a sulfhydrylation modification, a selenium-substituted oxygen modification or a linking isotope modification.
It will be appreciated by those skilled in the art that, as an improvement over the above-described techniques, a modification may be made at a position on the nucleotide sequence of the above-described aptamer, e.g., phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or ligation isotopicization, etc., provided that the aptamer sequence thus modified has desirable properties, e.g., may have an affinity for binding the aflatoxin B1 small molecule equal to or greater than the parent aptamer sequence prior to modification, or, although the affinity is not significantly improved, has greater stability.
As a preferred embodiment of the invention, the nucleotide sequence of the nucleic acid aptamer comprises a tag and maintains affinity.
As a preferred embodiment of the present invention, the label is a fluorescent label, a radioactive label, a therapeutic label, a biotin label, a digoxigenin label, a nano luminescent material label, a small peptide label, an siRNA label or an enzyme label.
It will be appreciated by those skilled in the art that, as an improvement to the above-described technical scheme, a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano-luminescent material, a small peptide, siRNA or an enzyme label, etc. may be attached to the nucleotide sequence of the above-described nucleic acid aptamer, provided that the nucleic acid aptamer sequence thus modified has desirable properties, for example, may have an affinity for binding to the aflatoxin B1 small molecule equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or may have higher stability although the affinity is not significantly improved.
The invention also provides application of the nucleic acid aptamer specifically combined with the aflatoxin B1 small molecule in detecting the content of the aflatoxin B1.
As a preferable scheme of the invention, the content of aflatoxin B1 small molecules is detected by adopting a fluorescence competition method.
Compared with the prior art, the invention has the following beneficial effects:
1) Compared with an antibody, the nucleic acid aptamer has the advantages of small molecular weight, better stability, easy transformation and modification, no immunogenicity, short preparation period, capability of avoiding a series of processes of animal immunization, feeding, protein extraction, purification and the like by artificial synthesis and the like, so that the nucleic acid aptamer is a very ideal molecular probe. Nucleic acid aptamers directed against small molecules of aflatoxin B1 have not been published and applied by humans, and therefore there is a need in the art for nucleic acid aptamers having high binding affinity against small molecules of aflatoxin B1.
2) Nucleic acid aptamers are more stable than antibodies, and have little batch-to-batch variation due to chemical synthesis; and the aptamer is successfully used for detecting aflatoxin B1 small molecules.
3) The aptamer obtained by the invention has the advantages of high specificity, high stability, convenient synthesis, low molecular weight and easy modification, and can replace complex mass spectrographs and antibodies.
4) The invention also provides a nucleic acid aptamer capable of specifically binding to aflatoxin B1 to detect the aflatoxin B1, which provides a novel method for detecting the aflatoxin B1.
Drawings
FIG. 1 is a schematic diagram of the fluorescence competition method for detecting aflatoxin B1 small molecules.
FIG. 2 is a schematic diagram of the experimental detection of AFB1-05 aptamer with aflatoxin B1 small molecule in example 1 iTC.
FIG. 3 is an absorption spectrum of an aptamer solution before and after immobilization in example 2.
FIG. 4 is a fluorescence spectrum of aflatoxin B1 at different concentrations of example 2.
FIG. 5 is a graph of fluorescence intensity as a function of aflatoxin B1 concentration for example 2.
FIG. 6 is a graph of fluorescence intensity as a function of aflatoxin B1 concentration for example 2.
Detailed Description
In order to facilitate understanding of the technical means, the creation characteristics, the achievement of the objects and the effects achieved by the present invention, the present invention is further described below with reference to specific examples, but the following examples are only preferred examples of the present invention, not all of which are described in detail below. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1
Isothermal calorimetric titration (iTC) verifies the affinity of the AFB1-05 aptamer to the aflatoxin B1 small molecule, comprising the steps of:
1. aflatoxin B1 (available from Shanghai Biotechnology Co., ltd., cat# A606874) was dissolved in dimethyl sulfoxide, diluted to 1mM (1% dimethyl sulfoxide) with DPBS buffer, AFB1-05 monoclonal (SEQ ID No.1: TCCTCGGTACACACGGTCCAGGCTGGTCTCCCTCCC) was synthesized by DPBS, dissolved to 5. Mu.M from general Biotechnology Co., ltd., anhui) and 1% dimethyl sulfoxide was contained in the solution, and finally a DPBS buffer containing 1% dimethyl sulfoxide was prepared.
2. Experiments were performed using a Malvern model iTC 200 instrument.
After the start-up self-checking of the instrument is finished, the automatic cleaning of the titration needle and the titration tank is selected, ultrapure water is used for titration after the cleaning is finished, and the stability of the instrument and the cleanliness of the titration needle and the titration tank are checked. Then, sample injection was started, and 280. Mu.L of DPBS buffer containing 1% dimethyl sulfoxide was titrated with 60. Mu.L of aflatoxin B1 solution to obtain control data. The titration needle and titration cell were again cleaned, the instrument was sampled, and 280. Mu.L of AFB1-05 monoclonal solution containing 1% dimethyl sulfoxide was titrated with 60. Mu.L of aflatoxin B1 solution to obtain experimental data. The titration mode of experiment setting is that the first drop is injected with 0.4 mu L, the later 19 drops are injected with 3 mu L each time, the time length of each injection is 20s, and the injection interval time is 150s. After the experiment is finished, the titration needle and the titration cell are cleaned, and the machine is shut down.
As shown in fig. 2, the control group data is subtracted from the experimental group data, a titration curve of aflatoxin B1 and AFB1-05 monoclonal is drawn, and the area of each exothermic peak is automatically integrated by software to obtain the data of a combination isothermal curve, a combination ratio, a combination constant, enthalpy change, entropy change, gibbs free energy and the like. As can be seen from the figure, the binding constant of aflatoxin B1 small molecule and AFB1-05 monoclonal is 3.21×10 5 M -1 The dissociation constant was 3.11. Mu.M, indicating that both have higher affinity.
Example 2
Detection of aflatoxin B1 small molecule by fluorescence competition method
As shown in FIG. 1, the invention synthesizes a 3' -end fluorescent group (FAM) modified AFB1-05-F monoclonal and a 5' -end Biotin (Biotin) and 3' -end quenching group (BHQ) modified B-MT-Q monoclonal, and partial sequences of the two monoclonal can be in base complementary pairing. By utilizing the binding property of streptavidin and biotin, double-stranded DNA formed by AFB1-05-F and B-MT-Q can be fixed on M-270 streptavidin magnetic beads, and fluorescent groups and quenching groups are too close to each other, so that fluorescence quenching can be caused. The detection target aflatoxin B1 small molecule is then added through a blocking and washing step. Aflatoxin B1 competes with B-MT-Q and tends to combine with the aptamer AFB1-05-F, so that the structure of AFB1-05-F is changed, double chains are opened, at this time, the complex of aflatoxin B1 and AFB1-05-F is free in solution, and finally, supernatant is obtained through centrifugal magnetic attraction and fluorescence is measured, so that the detection of small molecules of aflatoxin B1 can be realized.
The detection method comprises the following steps:
1) 5'-Biotin (Biotin) and 3' -quencher (BHQ) modified B-MT-Q monoclonal dry powder (SEQ ID No.2:5' -Biotin-AAAAACTACGTGGGA-BHQ-3', synthesized from general biosystems (Anhui Co., ltd.), and 3' -end fluorescent group (FAM) -modified AFB1-05-F monoclonal (SEQ ID No.3:5'-TCCTCGGTACACACGGTCCAGGCTGGTCTCCCTCCCACGTAG-FAM-3', which was synthesized from general biosystems (Anhui Co., ltd.) dry powder, was centrifuged at 12000rpm for 10min, and then 100. Mu.M was obtained with DPBS, shaken and stored at 4℃until needed.
2) The dissolved AFB1-05-F and B-MT-Q were mixed and added to a PCR tube in a concentration ratio of 1:2 to slowly renature to form a double-stranded structure (provided that: keeping the temperature at 95 ℃ for 10min, slowly cooling to 60 ℃ for 1min, then slowly cooling to 25 ℃ for 10min, keeping the cooling rate at 0.1 ℃/s, and storing the mixed solution of AFB1-05-F and B-MT-Q at 4 ℃ for later use after the completion of the cooling. Subsequently, 5. Mu.L of the DNA mixture was subjected to ultraviolet concentration detection, and ultraviolet detection A260 data was recorded as A1.
3) 50. Mu.L of 10mg/mL M-270 streptavidin magnetic beads (available from Semer Feishr technologies Co., ltd., cat. No. 65305) were washed three times with 500. Mu.L of DPBS buffer. 1mL of the DNA mixture was added to the beads, mixed well, incubated at room temperature for 60min on a rotary shaker, all the beads were adsorbed by a strong magnet after the completion, and the supernatant was removed and retained. The ultraviolet A260 value A2 was detected by pipetting the micro supernatant, the concentration of DNA not immobilized on the magnetic beads in the supernatant was judged by the value of A2/A1 to judge the immobilization efficiency of DNA to magnetic beads, and as shown in FIG. 3, A2 in the supernatant was greatly reduced compared with the value A260 value A1 before the immobilization, and the immobilization efficiency was calculated to be 75%. Then, the cells were washed three times with 400. Mu.L of DPBS buffer, and blocked by adding 100. Mu.M Biotin solution for 2 hours.
4) After the end of the blocking, the cells were washed three times with 500. Mu.L of DPBS buffer and the volume was set to 500. Mu.L with DPBS. 7 separate tubes were prepared, 50. Mu.L of the blocked beads were added to each tube, 100. Mu.L of DPBS buffer diluted aflatoxin B1 sample solution (0 ng/mL, 0.18ng/mL, 0.75ng/mL, 1.36ng/mL, 2ng/mL, 8ng/mL and 20ng/mL in this order) was added after removing the supernatant by magnetic attraction, shaking, and incubation was performed for 60min at room temperature on a rotary shaker.
5) After incubation, each sample was centrifuged at 5000rpm for 2min, and all beads were attached with a strong magnet and the supernatant was transferred to a new centrifuge tube. Fluorescence intensity of the fluorescent group in each sample supernatant was measured using a fluorescence spectrophotometer (Hitachi, model F-2700) and the data was recorded.
The experimental results are shown in FIG. 4, and it can be seen that the fluorescence intensity gradually increases with the increase of the aflatoxin B1 concentration. From FIGS. 5 and 6, it can be seen that at 520nm, aflatoxin B1 concentration appears to be positively correlated with fluorescence intensity and has a better discrimination. The concentration is in the range of 0.18ng/mL-2ng/mL, and the linear relation between the concentration and the fluorescence intensity is good, which shows that the AFB1-05 monoclonal has good affinity with aflatoxin B1. Therefore, the aptamer can replace the traditional mass spectrum instrument detection and replace antibodies, thereby providing good thought and method for developing a new detection method.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (9)
1. A nucleic acid aptamer specifically binding to a small aflatoxin B1 molecule, comprising a nucleotide sequence shown in SEQ ID No. 1; alternatively, a nucleotide sequence having high homology with the nucleotide sequence of SEQ ID No.1 and capable of specifically binding to the aflatoxin B1 small molecule; or a nucleotide sequence which is derived from the nucleotide sequence shown in SEQ ID No.1 and can specifically bind to the aflatoxin B1 small molecule.
2. The aptamer of claim 1, wherein the aptamer has a high homology of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% with the nucleotide sequence shown in SEQ ID No. 1.
3. A nucleic acid aptamer that specifically binds to the small molecule aflatoxin B1 according to claim 1 or 2 wherein the nucleic acid aptamer comprises a nucleotide sequence that is complementary to the nucleotide sequence and retains affinity.
4. A nucleic acid aptamer that specifically binds to the small molecule aflatoxin B1 according to claim 1 or 2 wherein the nucleotide sequence of the nucleic acid aptamer comprises a base modification and retains affinity.
5. The aptamer of claim 4, wherein the base modification is a thio modification, a phospho modification, a methylation modification, an amination modification, a sulfhydryl modification, a selenium-substituted oxy modification or a ligation isotope modification.
6. A nucleic acid aptamer that specifically binds to the small molecule aflatoxin B1 according to claim 1 or 2 wherein the nucleotide sequence of the nucleic acid aptamer comprises a tag and retains affinity.
7. The aptamer of claim 6, wherein the label is a fluorescent label, a radioactive label, a therapeutic label, a biotin label, a digoxigenin label, a nano luminescent material label, a small peptide label, an siRNA label, or an enzyme label.
8. The use of the aptamer specifically binding to the small aflatoxin B1 molecule according to any one of claims 1 to 7, characterized in that the use of the aptamer specifically binding to the small aflatoxin B1 molecule for detecting the content of aflatoxin B1.
9. The use of the aptamer specifically binding to the small aflatoxin B1 molecule according to claim 8, wherein the content of the small aflatoxin B1 molecule is detected by fluorescence competition method.
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