A nucleic acid aptamer specifically recognizing sesame allergen protein Ses i 3, its preparation method and application

Abstract

The present invention relates to a nucleic acid aptamer that specifically recognizes the sesame allergen protein Ses i 3, and a screening method and application thereof. The screening method synthesizes the sesame protein Ses i 3 by means of prokaryotic expression, and screens for the nucleic acid aptamer of the sesame protein Ses i 3 through the systematic evolution of ligands by exponential enrichment (SELEX) technology based on magnetic beads. It has high affinity and high specificity and can be used for detecting sesame allergen proteins and other purposes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, it relates to a nucleic acid aptamer that specifically recognizes the sesame allergen protein Ses i3, and particularly to a method for screening nucleic acid aptamers of sesame allergen Ses i 3 based on magnetic bead-SELEX technology and its applications. Background Art

[0002] Sesame (Sesamum indicum L.) belongs to the Pedaliaceae family and Sesamum genus, and has high nutritional value, being rich in fat, protein and other nutrients. It is a common food in life. However, sesame can also cause severe allergic reactions. The main symptoms of sesame allergy include: rashes, urticaria, asthma, etc. In severe cases, it can lead to anaphylactic shock and even death. According to the recommendations put forward by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO / WHO) in 2022, sesame is listed as one of the eight new allergens.

[0003] Related research indicates that Ses i 3 can be recognized by the sera of 75% of sesame-allergic patients and is one of the main allergens of sesame. Ses i 3 is a 7S vicilin, belonging to the Cupin superfamily, consisting of 585 amino acids, lacking disulfide bonds, with a molecular weight of approximately 45 kDa. It has a protein super-secondary structure of β-barrel in its three-dimensional spatial structure and can resist thermal denaturation and enzymatic digestion. And Ses i 3 has more than 40% homology with the 7S vicilin family in pistachios, hazelnuts, peanuts, and pecans, which also makes Ses i3 in sesame have the possibility of clinical cross-allergic reactions with other nuts. At the same time, Ses i 3 has different reactions to different enzymatic digestions. It is easily digested by pepsin, while it has strong resistance to trypsin and chymotrypsin. Due to Ses i 3 having strong anti-digestion ability and thermal stability, and having a high possibility of clinical cross-allergic reactions, Ses i 3 has a strong risk of sensitization.

[0004] Aptamers are artificially synthesized single-stranded DNA or RNA segments that can specifically bind to target substances through their three-dimensional structures. They have the advantages of strong affinity, high specificity, low cost, easy synthesis, low immunogenicity, stable structure, and easy modification, and are widely used in various fields such as medicine, pharmacy, biology, and chemistry. The targets that aptamers can recognize include not only common antigens such as proteins and cells, but also small molecule substances such as toxins and metal molecules. Based on the diversity of single-stranded nucleic acid structures and conformations, aptamers generate highly specific binding forces with target molecules through various interactions such as van der Waals forces, hydrogen bond interactions, electrostatic interactions, and shape matching. Based on the stable structure and easy modification of aptamers, aptamers can be group-modified and combined with biosensors to detect target substances. In addition, due to their low immunogenicity, aptamers can also be applied in the fields of drug delivery and disease diagnosis.

[0005] At present, although the existing technology has obtained aptamers by screening from a random oligonucleotide library through the systematic evolution of ligands by exponential enrichment (SELEX) technology, there is no report on screening sesame allergen protein aptamers through SELEX technology. How to screen for Ses i 3 aptamers with high affinity and strong specificity and use them for allergen protein detection is of great significance. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention provides an aptamer that specifically recognizes the sesame allergen protein Ses i 3, and the aptamer has high affinity and high specificity.

[0007] The present invention provides a method for screening an aptamer that specifically recognizes the sesame allergen protein Ses i 3. The method screens the aptamer through the magnetic bead-SELEX systematic evolution of ligands by exponential enrichment technology. The advantage of the method is that the spherical structure of the magnetic beads is conducive to the full display of the target, and the unbound oligonucleotides are separated by magnetism, with simple operation and saving the dosage of the target molecule.

[0008] The present invention provides a kit for an aptamer that specifically recognizes the sesame allergen protein Ses i 3.

[0009] The present invention provides the application of an aptamer that specifically recognizes the sesame allergen protein Ses i 3 in the preparation of a kit for detecting the allergen protein Ses i 3.

[0010] The present invention provides the application of an aptamer that specifically recognizes the sesame allergen protein Ses i 3 in the preparation of a kit for detecting sesame allergen proteins, or the use of an aptamer that specifically recognizes the sesame allergen protein Ses i 3 for detecting sesame allergenic proteins.

[0011] The present invention provides the application of a nucleic acid aptamer that specifically recognizes the sesame allergen protein Ses i 3 in the preparation of a diagnostic reagent for preventing sesame allergy, or the use of a nucleic acid aptamer that specifically recognizes the allergen protein Ses i 3 for preventing sesame allergy. Detailed Description of the Invention

[0013] To achieve this purpose, the present invention provides the following technical solutions.

[0014] In the first aspect, a nucleic acid aptamer that specifically recognizes the sesame allergen protein Ses i 3 is provided, and the nucleotide sequence of the nucleic acid aptamer comprises any one of the DNA sequences shown in SEQ ID NO.1-2:

[0015] SEQ ID No.1:

[0016] 5'-CGCGTGGGGGCACCCCCAACTGGACCTATAGCCATTCAAC-3',

[0017] SEQ ID No.2:

[0018] 5'-AGTCCCCTTCATCTTAGGGGAGGGCAGTATGTTCTTAGTT-3'.

[0019] In some embodiments, the nucleic acid aptamer has high specificity and can significantly recognize Ses i 3 in the sesame allergen protein.

[0020] In the second aspect, a screening method for a nucleic acid aptamer that specifically recognizes the sesame allergen protein Ses i 3 is provided, and the screening method comprises the following steps:

[0021] (1), Synthesize the recombinant sesame allergen protein Ses i 3;

[0022] (2), Synthesize the ssDNA library and primers;

[0023] (3), Screening of the Ses i 3 nucleic acid aptamer based on the magnetic bead-SELEX technology;

[0024] (4), Prepare the secondary single-stranded DNA library;

[0025] (5), Reverse screening;

[0026] (6), Monitoring of the screening process;

[0027] (7), Determination of the affinity of the nucleic acid aptamer.

[0028] In some embodiments, step (1) includes expressing the Ses i 3 protein through genetic recombination technology.

[0029] In some embodiments, step (1) includes the following operations: inserting the gene of Ses i 3 into the expression vector pET28a through the cloning sites BamHI (GGATCC)-XhoI (CTCGAG), and introducing it into the Rosetta strain (Rosetta(DE3) E. coli Strain). Inoculate the preserved bacteria and culture until the OD of the bacteria 600 is 0.6 - 0.8, add IPTG (isopropyl-β-D-1-thiogalactoside) to the culture and then culture to induce the expression of the fusion protein, harvest the bacteria and prepare samples for SDS-PAGE analysis.

[0030] Preferably, the final concentration of IPTG added is 1 mM;

[0031] Preferably, the culture conditions are: culture at 37 °C and 220 rpm for 3 - 5 h, preferably 4 h.

[0032] In some embodiments, the sesame protein Ses i 3 is identified, and the molecular weight is determined to be 67 kDa.

[0033] In some embodiments, the ssDNA library in step (2) is a random library with a base length of 80 nt, and the random library is:

[0034] 5’-TCGCACATTCCGCTTCTACC-(40 nt)-TTCGCACACACGGACTTACG-3’;

[0035] In some embodiments, the primer has a base length of 20 nt, specifically as follows:

[0036] 5’ primer: 5’-TCGCACATTCCGCTTCTACC,

[0037] 3’ primer: 3’-CGTAAGTCCGTGTGTGCGAA;

[0038] In some embodiments, the 5’ primer in the primer is labeled with 6-FAM (6-carboxyfluorescein), and the 3’ primer is labeled with biotin.

[0039] In some embodiments, step (3) includes immobilizing sesame protein Ses i 3 on the surface of magnetic microspheres to form a magnetic bead-protein complex; adding an oligonucleotide library to the magnetic bead-protein complex system and incubating for a certain period of time; washing away the unbound ssDNA with a buffer, and the remaining ssDNA is detached from the protein surface by heat denaturation, collected, subjected to PCR amplification, and a single-stranded secondary library is prepared by using paramagnetic microspheres labeled with streptavidin. After repeating the screening for 5-7 rounds, an enriched nucleic acid aptamer can be obtained.

[0040] Preferably, the affinity constant of the enriched nucleic acid aptamer is determined and its specificity is verified by enzyme-linked immunosorbent assay;

[0041] Optionally, the secondary structure of the nucleic acid aptamer is predicted by the mfold online website.

[0042] In some embodiments, step (3) includes the following steps:

[0043] (a) Dissolve sesame protein Ses i 3 in a buffer, add magnetic microspheres, and slowly shake to immobilize the protein on the surface of the magnetic microspheres to form a magnetic bead-protein complex.

[0044] (b) Dilute the synthesized ssDNA library with a buffer, take a certain amount of the diluted library, heat it for a certain period of time, and immediately place it on ice to restore the natural spatial structure of the ssDNA. Then add it to the magnetic bead-protein complex to obtain a mixture.

[0045] (c) Place the mixture at room temperature and slowly shake, perform magnetic separation, discard the supernatant to remove the unbound DNA, add a certain amount of PBS solution to resuspend, heat for a certain period of time to separate the bound DNA, perform magnetic separation, and collect the supernatant.

[0046] Preferably, the buffer in step (a) is carbonate buffer (CBS);

[0047] Preferably, the buffer in step (b) is phosphate buffer (PBS); the dilution concentration is 1 μM;

[0048] Preferably, the ice bath time in step (b) is 8-12 min, more preferably 9, 10, 11 min, and most preferably 10 min.

[0049] Preferably, the slow shaking conditions in steps (b) and (c) are: the shaking time is 1-3 h, and the shaking temperature is room temperature; more preferably, the shaking time is 2 h;

[0050] Preferably, the heating conditions in steps (b) and (c) are: heating at 95 °C for 5 min.

[0051] In some embodiments, step (3) further includes activating the magnetic microspheres, which includes the following steps: Take Affimag FEO magnetic microspheres (purchased from Tianjin Baisile Chromatography Technology Development Center, product number: 3632) in a centrifuge tube, place it on a magnetic rack for magnetic separation, remove the supernatant, wash it with PBS solution 1-3 times, add EDC (1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution, and slowly shake at room temperature to activate the carboxyl groups on the surface of the magnetic microspheres. Subsequently, perform magnetic separation, remove the supernatant, and wash it with PBS solution 1-3 times to obtain activated magnetic microspheres.

[0052] In some embodiments, step (4) includes: using the supernatant collected in the previous step as a DNA template for PCR amplification, and preparing the secondary single-stranded DNA library required for screening Ses i 3 aptamers through paramagnetic microspheres labeled with streptavidin.

[0053] In some embodiments, step (4) includes the following steps:

[0054] (d), Using the supernatant collected in the previous step as a DNA template for PCR amplification to obtain a PCR product;

[0055] (e), Add streptavidin-labeled paramagnetic microspheres to the PCR product, then slowly shake. After the PCR product is fixed on the surface of the microspheres, wash off the unbound PCR product with a buffer, resuspend the remaining part in a buffer, heat it, unwind the DNA double strand, perform magnetic separation, and collect the supernatant, which is the prepared secondary library.

[0056] Preferably, the shaking condition in step (e) is slow shaking at 37°C for 1-2 h, more preferably shaking for 1 h;

[0057] Preferably, the buffer in step (e) is PBS; the number of washing times is 1-3 times;

[0058] Preferably, the heating condition in step (e) is heating at 95°C for 5 min.

[0059] In some embodiments, steps (3)-(4) are repeatedly screened 5-7 times, preferably 6 times;

[0060] In some embodiments, the reverse screening in step (5) is: start adding reverse screening from the first repeated screening, and use bovine serum albumin (BSA) as the reverse screening target; the purpose is to increase the specificity of the screening, exclude the DNA that binds to the reverse screening target (BSA), and then incubate the remaining DNA with the positive screening target (sesame protein Ses i 3).

[0061] The reverse screening step is as follows: Couple BSA with magnetic microspheres, and the operation is the same as in step (3). First, add the secondary library prepared in step (4) to the complex system of BSA and magnetic microspheres, slowly shake at room temperature for 30 min, take the supernatant, and use the DNA supernatant containing unbound DNA as the screening library to repeat step (3).

[0062] In some embodiments, the reverse screening

[0063] In some embodiments, the steps of step (6) include: Measure the fluorescence value of the supernatant (magnetic microsphere - sesame protein Ses i 3 - DNA system) screened in step (4) with a fluorescence spectrometer until the fluorescence value tends to be stable and no longer rises, and then terminate the screening.

[0064] In some embodiments, the principle of step (6) is that since the secondary library prepared in step (4) is labeled with carboxyfluorescein, after binding to the target protein, the formed magnetic microsphere - sesame protein Ses i3 - DNA system is fluorescently labeled, and the fluorescence value can be measured with a fluorescence spectrometer. By continuously repeating steps (3) - (4), the DNA that can bind to the target in the secondary single - stranded DNA library is continuously enriched until the fluorescence intensity tends to be stable and no longer rises, and at this time, it is used as a sign to judge the termination of the screening process.

[0065] In some embodiments, the fluorescence measurement conditions in step (6) are: excitation wavelength is 492 nm, and emission wavelength is 518 nm.

[0066] In some embodiments, the determination of the affinity of the aptamer in step (7) is to measure the affinity constant of the Ses i 3 aptamer by enzyme - linked immunosorbent assay.

[0067] In some embodiments, the determination of the affinity of the aptamer includes the following steps:

[0068] (h), Take the product in step (4) of the last round of screening for PCR amplification. The amplification conditions are: pre - denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 62 °C for 20 s, extension at 72 °C for 20 s, cycle 12 times, 72 °C for 5 min, and store at 4 °C. Sequence the amplified product, synthesize the five sequences with the highest abundance in the sequencing results, which are the candidate aptamers, and modify the 5' end with biotin;

[0069] (i), Fix sesame protein Ses i 3 on the enzyme - linked immunosorbent assay plate;

[0070] (j), Gradient - dilute the candidate aptamer modified with biotin as the primary antibody, add it to the enzyme - linked immunosorbent assay plate, incubate for a certain time, add streptavidin labeled with horseradish peroxidase as the secondary antibody, and incubate at room temperature;

[0071] (k), Use an enhanced chemiluminescence reagent (ECL) as the color development solution for color development, terminate the reaction, and detect the absorbance (OD 450 ) at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader;

[0072] Preferably, in step (j), the aptamer treatment is as follows: Dilute the candidate aptamer to different concentrations with PBS solution, add 100 μL to each well, and incubate; the dilution concentrations are 50 nM, 100 nM, 150 nM, 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM; the incubation is at 37 °C for 1 h.

[0073] Optionally, (m), Calculate the aptamer affinity constant by non-linear fitting.

[0074] In some embodiments, the five sequences screened in step (h) are as follows:

[0075] SEQ ID No.1:

[0076] 5'-CGCGTGGGGGCACCCCCAACTGGACCTATAGCCATTCAAC-3',

[0077] SEQ ID No.2:

[0078] 5'-AGTCCCCTTCATCTTAGGGGAGGGCAGTATGTTCTTAGTT-3',

[0079] SEQ ID No.3:

[0080] 5'-TCTGAAGTACGAGCACCTGACGGCATCGTAAATCTTCCCG-3',

[0081] SEQ ID No.4:

[0082] 5'-TACCGTGCGTTGTTCACATGGGCGGATGTCTTGGGTGTCC-3',

[0083] SEQ ID No.5:

[0084] 5'-CCGGGGCATCAAGTGTCTCGTTATAAATCAGTTGCTGGAG-3';

[0085] In some embodiments, the non-linear fitting formula in step (m) is:

[0086] Y = Bmax*X / (Kd + X),

[0087] Where Y represents absorbance, X is the aptamer concentration, Bmax is the maximum binding concentration of the aptamer, and Kd is the aptamer affinity constant. The affinity constant of the aptamer is calculated by performing a non-linear fitting formula on the experimental results.

[0088] In some embodiments, in step (7), through the determination of the affinity of the nucleic acid aptamer, SEQ ID NO.1 and SEQ ID NO.2 are determined to be nucleic acid aptamers having an affinity for the protein Ses i 3.

[0089] In some embodiments, in step (7), the treatment method of immobilizing the protein on the ELISA plate is as follows: Dilute the protein with PBS solution, coat 100 μL per well on the ELISA plate, incubate, wash with PBS solution 1 - 3 times (preferably 3 times), and add 1% gelatin solution to block at room temperature for 1 - 2 hours (preferably 1.5 h);

[0090] The dilution concentration of streptavidin in PBS is: 1:200; the incubation time at room temperature is 30 min;

[0091] The reaction terminator is 0.2 M H2SO4 solution.

[0092] In some embodiments, the specificity of the nucleic acid aptamer is verified, that is, the specificity of the Ses i 3 nucleic acid aptamer is verified by an enzyme-linked immunosorbent assay.

[0093] In some embodiments, the verification of the specificity of the nucleic acid aptamer includes the following steps: Immobilize sesame protein Sesi 3 and other total proteins to be tested on the ELISA plate; Gradient dilute the nucleic acid aptamers SEQ ID NO.1 and SEQ ID NO.2 modified with biotin as the primary antibody, add them to the ELISA plate, incubate for a certain time, add streptavidin labeled with horseradish peroxidase as the secondary antibody, and incubate at room temperature; Use an enhanced chemiluminescence reagent (ECL) as the chromogenic solution for color development, terminate the reaction, and measure the OD 450 value. Perform a significance analysis on the OD 450 value. Judgment criterion: Compare the OD 450 values of different proteins with the OD 450 value of Ses i 3. If p < 0.05, it is considered significantly different; if p < 0.01, it is considered extremely significantly different and has specificity, otherwise it is considered non-specific.

[0094] In some embodiments, in the specificity verification of the nucleic acid aptamer, the treatment method of immobilizing the protein on the enzyme-linked immunosorbent assay (ELISA) plate is as follows: Dilute the protein with PBS solution, coat 100 μL per well on the ELISA plate, and incubate. Wash with PBS solution 1 - 3 times (preferably 3 times). Add 1% gelatin solution to block at room temperature for 1 - 2 hours (preferably 1.5 h);

[0095] The dilution concentration of streptavidin in PBS is 1:200; the incubation time at room temperature is 30 min;

[0096] The reaction terminator is 0.2 M H2SO4 solution.

[0097] In some embodiments, it further includes the prediction of the secondary structure of the nucleic acid aptamer;

[0098] Preferably, the secondary structure of the nucleic acid aptamer is predicted through the mfold online website (http: / / www.mfold.org).

[0099] In a third aspect, a kit for specifically recognizing the allergen protein Ses i 3 with a nucleic acid aptamer is provided.

[0100] In some embodiments, the kit contains any one of the DNA sequences shown in SEQ ID NO.1 - 2, and an instruction manual. The kit includes, but is not limited to, developing ELISA kits, chemiluminescence detection kits, tissue rapid detection kits, etc.

[0101] In a fourth aspect, the application of the nucleic acid aptamer specifically recognizing the allergen protein Ses i 3 in the preparation of a kit for detecting the allergen protein Ses i 3 is provided.

[0102] In some embodiments, the kit containing at least one DNA sequence shown in SEQ ID NO.1 - 2 is used to detect the sesame allergen protein Ses i 3.

[0103] In a fifth aspect, the application of the nucleic acid aptamer specifically recognizing the sesame allergen protein Ses i 3 in the preparation of a kit for detecting the sesame allergen protein, or the use of the nucleic acid aptamer specifically recognizing the allergen protein Ses i 3 for detecting the sesame allergen protein is provided.

[0104] In a sixth aspect, the application of the nucleic acid aptamer specifically recognizing the sesame allergen protein Ses i 3 in the preparation of a diagnostic reagent for preventing sesame allergy, or the use of the nucleic acid aptamer specifically recognizing the sesame allergen protein Ses i 3 for preventing sesame allergy is provided.

[0105] Beneficial effects

[0106] The present invention screens and obtains nucleic acid aptamers of sesame allergen protein Ses i 3 through the magnetic bead-SELEX technique. With multiple rounds of screening and the increase of selection pressure, aptamers with high affinity are enriched. After sequencing, nucleic acid aptamer sequences with high affinity and high specificity for the target protein can be obtained. It can be used for detecting sesame allergen protein Ses i 3 and for preventing sesame allergy, with fast detection speed, high detection accuracy and specificity.

[0107] The screening method of the present invention screens out oligonucleotides that can bind to the target from a random library. The magnetic bead-SELEX technique immobilizes the target on magnetic microbeads through covalent or non-covalent binding. Subsequently, a random library is added. Part of the oligonucleotides in the library bind to and are immobilized on the target, and the unbound ones are eluted. Then, the bound oligonucleotides are separated for PCR amplification and the next round of screening. The advantage of the method of the present invention is that the spherical structure of the magnetic beads is conducive to the full display of the target, and the unbound oligonucleotides are separated by magnetism, with simple operation and saving the dosage of the target molecule.

[0108] The screening method of the present invention uses BSA as the counter-screening target, increasing the specificity of screening. The screened aptamers have strong specificity and an affinity reaching the nM level.

[0109] The present invention can achieve the screening of nucleic acid aptamers of sesame allergen protein Ses i 3. This method immobilizes the protein on the surface of magnetic microbeads, saving costs and having no impact on the protein structure, increasing the specificity of the screened nucleic acid aptamers, and being able to accurately and specifically recognize sesame protein Ses i 3.

[0110] The present invention also synthesizes immunologically active sesame protein Ses i 3 by prokaryotic expression and characterizes its molecular weight by SDS-PAGE. Description of the Drawings

[0111] Figure 1 is the SDS-PAGE characterization of Ses i 3;

[0112] Figure 2 is the result of the affinity determination of Ses i 3 nucleic acid aptamers. (a) is the result of the affinity determination of SEQ ID NO.1, and (b) is the result of the affinity determination of SEQ ID NO.2;

[0113] Figure 3 is the result of the specificity analysis of Ses i 3 nucleic acid aptamers. (a) is the specificity result of SEQ ID NO.1, and (b) is the specificity result of SEQ ID NO.2;

[0114] Figure 4It is the prediction result of the secondary structure of the Ses i 3 nucleic acid aptamer. Detailed implementation mode

[0115] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0116] Example 1 Screening experiment of nucleic acid aptamer specifically recognizing sesame allergen protein Ses i 3

[0117] (1) Synthesize recombinant sesame allergen protein Ses i 3:

[0118] Prokaryotic expression of sesame protein Ses i 3: Obtain the amino acid sequence of Ses i 3 from the UniProt server, insert the gene of Ses i 3 into the pET28a vector through the BamH1 and XhoI1 restriction sites, and introduce it into the Rosetta strain (Rosetta E. coli Strain). Select positive clones and expand the culture at 37 °C until the OD 600 value is 0.6 - 0.8, then add 1 mmol / L IPTG to induce protein expression, centrifuge to collect the strain, resuspend the cells and perform ultrasonic disruption. After high-speed centrifugation, take the supernatant for Ni column affinity chromatography purification, and finally obtain sesame protein Ses i 3.

[0119] Identification of sesame protein Ses i 3: Characterize the purified Ses i 3 by SDS-PAGE (see Figure 1 ) to determine that its molecular weight is 67 kDa.

[0120] (2) Synthesize ssDNA library and primers

[0121] According to the conventional methods in the art, synthesize a random ssDNA library with a base length of 80 nt and two primers with a length of 20 nt:

[0122] Random ssDNA library: 5’-TCGCACATTCCGCTTCTACC-(40 nt)-TTCGCACACACGGACTTACG-3’

[0123] 5’ primer: 5’-TCGCACATTCCGCTTCTACC

[0124] 3’ primer: 3’-CGTAAGTCCGTGTGTGCGAA

[0125] Among them, the 5’ primer is labeled with 6-FAM (6-carboxyfluorescein), and the 3’ primer is labeled with biotin.

[0126] (3) Screening of Ses i 3 nucleic acid aptamer of sesame protein based on magnetic bead-SELEX technology

[0127] Activation of magnetic microspheres: Take 50 μL of Affimag FEO magnetic microspheres in a 1.5 mL centrifuge tube, place it on a magnetic rack for magnetic separation, discard the supernatant, wash it three times with PBS solution, add 500 μL of 50 mg / mL EDC and 500 μL of 50 mg / mL NHS solution, and slowly shake it at room temperature for 30 min to activate the carboxyl groups on the surface of the magnetic microspheres. Subsequently, perform magnetic separation, discard the supernatant, and wash it three times with PBS solution to obtain activated magnetic microspheres.

[0128] Screening: Resuspend the activated magnetic microspheres with 500 μL of CBS solution, add 6 μg of sesame protein Ses i 3, vortex and mix well, slowly shake it at room temperature for 2 h, and adsorb the protein based on the principle that the amino group of the protein forms a peptide bond with the carboxyl group on the surface of the magnetic microspheres to fix the protein on the surface of the magnetic microspheres, obtaining a magnetic bead-protein complex. Subsequently, place the magnetic bead-protein complex on a magnetic rack for magnetic separation, discard the supernatant, and wash it three times with PBS solution. Dilute the random ssDNA library with PBS to 1 μM, take 200 μL of the diluted library, heat it at 95 °C for 5 min, and immediately ice-bath it for 10 min to restore the natural spatial structure of the ssDNA. Add it to the magnetic bead-protein complex and slowly shake it at room temperature for 30 min. Perform magnetic separation, discard the supernatant, remove the unbound DNA, add 200 μL of PBS solution to resuspend it, heat it at 95 °C for 5 min to separate the bound DNA, perform magnetic separation, and collect the supernatant.

[0129] (4) Preparation of secondary single-stranded DNA library

[0130] Use the supernatant collected in step (3) as a DNA template for PCR amplification. The amplification conditions are shown in Table 1 to obtain a PCR product.

[0131] Take 200 μL of streptavidin-coated paramagnetic microspheres in a 1.5 mL centrifuge tube, perform magnetic separation, discard the supernatant, wash it three times with PBS solution, add 500 μL of the PCR product, and slowly shake it at 37 °C for 1 h. Since the two primers are respectively labeled with carboxyfluorescein and biotin, one strand of the PCR product is labeled with carboxyfluorescein and the other is labeled with biotin. The biotin binds to streptavidin to fix the PCR product on the surface of the paramagnetic microspheres. Subsequently, perform magnetic separation and discard the supernatant, wash it three times with PBS solution to wash away the unbound PCR product, add 200 μL of PBS solution to resuspend it, quickly shake and heat it at 95 °C for 5 min, the double-stranded PCR product unwinds, and the single strand without biotin label but with carboxyfluorescein label dissociates into the supernatant. Perform magnetic separation and collect the supernatant to obtain the secondary single-stranded DNA library.

[0132] Repeated screening is carried out to prepare a secondary single-stranded DNA library for the next round of screening.

[0133] Table 1 PCR amplification conditions

[0134]

[0135] (5) Reverse screening

[0136] Start reverse screening from the first repeated screening. Couple BSA with magnetic beads (the operation is the same as in step (3)). First, add the prepared secondary single-stranded DNA library to the complex system of BSA and magnetic beads, slowly shake at room temperature for 30 min, take the supernatant, and use the unbound DNA as the screening library to repeat step (3).

[0137] (6) Screening process monitoring

[0138] Measure the fluorescence value of the supernatant (magnetic bead - sesame protein Ses i 3 - DNA system) obtained by screening in step (4) using a fluorescence spectrometer. After 7 rounds of screening in steps (3) - (5), the fluorescence value of the magnetic bead - sesame protein Ses i 3 - DNA system tends to be stable and no longer increases, then terminate the screening.

[0139] The fluorescence measurement conditions are: excitation wavelength is 492 nm, and emission wavelength is 518 nm.

[0140] (7) Aptamer affinity determination

[0141] Take the product in step (4) of the last screening for PCR amplification (the amplification conditions are shown in Table 1), sequence the amplified product, synthesize the five sequences with the highest abundance in the sequencing results (i.e., SEQ ID No.1 - 5), which are the candidate aptamers, and modify the 5' end with biotin;

[0142] SEQ ID No.1:

[0143] 5'-CGCGTGGGGGCACCCCCAACTGGACCTATAGCCATTCAAC-3',

[0144] SEQ ID No.2:

[0145] 5'-AGTCCCCTTCATCTTAGGGGAGGGCAGTATGTTCTTAGTT-3',

[0146] SEQ ID No.3:

[0147] 5'-TCTGAAGTACGAGCACCTGACGGCATCGTAAATCTTCCCG-3',

[0148] SEQ ID No. 4:

[0149] 5'-TACCGTGCGTTGTTCACATGGGCGGATGTCTTGGGTGTCC-3',

[0150] SEQ ID No. 5:

[0151] 5'-CCGGGGCATCAAGTGTCTCGTTATAAATCAGTTGCTGGAG-3';

[0152] The affinity was determined by enzyme-linked immunosorbent assay (ELISA): Ses i 3 of sesame protein was diluted to 6 μg / ml with PBS solution, and 100 μL per well was coated on the enzyme-linked immunosorbent assay plate and incubated at 37 °C for 1.5 h. It was washed three times with PBS solution, and 1% gelatin solution was added for blocking at room temperature for 1.5 hours. The biotinylated candidate aptamers were diluted to different concentrations (50 nM, 100 nM, 150 nM, 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM) with PBS solution, and 100 μL was added to each well and incubated at 37 °C for 1 h. It was washed three times with PBS solution, and 50 μL of streptavidin labeled with horseradish peroxidase (1:200, diluted with PBS) was added to each well and incubated at room temperature for 30 min. It was washed three times with PBS solution, 100 μL of ECL chromogenic solution was added to each well and incubated in the dark at 37 °C for 15 min, and then 50 μL of 0.2 M H2SO4 solution was added to each well to terminate the reaction, and the absorbance (OD 450 ) was detected with an enzyme-linked immunosorbent assay reader. The absorbance results are shown in Table 2-3 below.

[0153] Table 2 Absorbance (OD 450 ) results of the affinity of nucleic acid aptamer SEQ ID NO.1

[0154]

[0155] Note: ABC are three groups of parallel experiments.

[0156] Table 3 Absorbance (OD 450 ) results of the affinity of nucleic acid aptamer SEQ ID NO.2

[0157]

[0158] Note: ABC are three groups of parallel experiments.

[0159] Table 4 Absorbance (OD 450 ) results of the affinity of nucleic acid aptamer SEQ ID NO.3

[0160]

[0161] Note: ABC are three groups of parallel experiments.

[0162] Table 5 Affinity absorbance (OD 450 ) results of nucleic acid aptamer SEQ ID NO.4

[0163]

[0164] Note: ABC are three groups of parallel experiments.

[0165] Table 6 Affinity absorbance (OD 450 ) results of nucleic acid aptamer SEQ ID NO.5

[0166]

[0167] Note: ABC are three groups of parallel experiments.

[0168] As described in Tables 2-6, for SEQ ID NO.1-2, as the aptamer concentration increases, the absorbance gradually increases and stabilizes after 600 nM, indicating that the aptamer has a certain affinity for the protein. Nonlinear fitting can be used to obtain the affinity constant. For SEQ ID NO.3-5, as the aptamer concentration increases, the absorbance does not show a trend of changing with the concentration increase, and the affinity constant cannot be obtained by nonlinear fitting.

[0169] Use Graphpad Prism software to perform nonlinear fitting on the experimental results. Calculate the affinity constant of the aptamer through the formula Y = B max *X / (Kd + X), where Y represents the absorbance, X is the aptamer concentration, and B max is the maximum binding concentration of the aptamer, and Kd is the aptamer affinity constant.

[0170] After calculation, the Kd value of SEQ ID NO.1 is: 141.9 nM, and the Kd value of SEQ ID NO.2 is: 367 nM (see Figure 2 ).

[0171] Through the affinity determination of nucleic acid aptamers, SEQ ID NO.1 and SEQ ID NO.2 were determined to be nucleic acid aptamers with affinity for sesame protein Ses i 3.

[0172] Example 2 Verification of the specificity of nucleic acid aptamers

[0173] Verify the specificity of the Ses i 3 nucleic acid aptamer by enzyme-linked immunosorbent assay

[0174] Dilute sesame protein Ses i 3, as well as the total proteins of peanut, hazelnut, Brazil nut, cashew nut, almond, and walnut to 6 μg / ml with PBS solution, and coat 100 μL per well on the ELISA plate. Incubate at 37 °C for 1.5 h, and wash three times with PBS solution. Add 1% gelatin solution and block at room temperature for 1.5 hours. Wash three times with PBS solution. Dilute the biotin-labeled aptamers SEQ ID NO.1 and SEQ ID NO.2 to 200 nM with PBS, add 100 μL per well, and incubate at 37 °C for 1 h. Wash three times with PBS solution. Add 50 μL of streptavidin labeled with horseradish peroxidase (1:200, diluted with PBS) to each well, and incubate at room temperature for 30 min. Wash three times with PBS solution. Add 100 μL of ECL chromogenic solution to each well and incubate at 37 °C in the dark for 15 min. Subsequently, add 50 μL of 0.2 M H2SO4 solution to each well to terminate the reaction, and measure the absorbance (OD 450 ) at 450 nm using an ELISA reader. The absorbance results are shown in the following table.

[0175] Table 7 Specific absorbance (OD 450 ) results of aptamer SEQ ID NO.1

[0176] Sesi3 Almond Peanut Hazelnut Walnut Brazil nut Cashew nut A 2.08 1.796 1.437 0.981 1.151 1.877 1.159 B 2.055 1.793 1.571 0.988 1.11 1.766 1.092 C 2.234 1.79 1.603 1.05 1.124 1.961 1.157

[0177] Note: ABC are three groups of parallel experiments.

[0178] Table 8 Specific absorbance (OD 450 ) results of aptamer SEQ ID NO.2

[0179] Sesi3 Almond Peanut Hazelnut Walnut Brazil nut Cashew nut A 1.938 1.604 1.101 0.966 1.145 1.497 0.828 B 1.856 1.483 1.226 0.993 1.173 1.489 0.785 C 2.04 1.635 1.229 1.034 1.221 1.434 0.865

[0180] Note: ABC are three groups of parallel experiments

[0181] After performing a significance analysis on the experimental results (see Figure 3 , where the abscissa 1-7 represents in sequence: sesame protein Ses i 3, almond, peanut, hazelnut, walnut, Brazil nut, cashew nut), the aptamers of the present invention have high specificity.

[0182] Example 3 Prediction of the secondary structure of aptamers

[0183] Predict the secondary structure of the aptamers through the mfold online website (http: / / www.mfold.org) (see Figure 4 ).

[0184] Example 4 Preparation of aptamers into a kit

[0185] According to the kit preparation method, the sequence of SEQ ID NO.1 or 2 is prepared into a test agent, and an instruction manual is added to prepare a detection kit. The kit includes, but is not limited to, enzyme-linked immunosorbent assay kits, chemiluminescence detection kits, tissue rapid detection kits, etc.

Claims

1. A nucleic acid aptamer that specifically recognizes the allergen protein Ses i 3, characterized in that: The nucleotide sequence of the nucleic acid aptamer is any one of the DNA sequences shown in SEQ ID NO.1-2: SEQ ID No. 1: 5'-TACCGTGCGTTGTTCACATGGGCGGATGTCTTGGGTGTCC-3', SEQ ID No. 2: 5'-AGTCCCCTTCATCTTAGGGGAGGGCAGTATGTTCTTAGTT-3'.

2. A kit for nucleic acid aptamers that specifically recognizes the allergen protein Ses i 3 as described in claim 1.

3. The nucleic acid aptamer that specifically recognizes the allergen protein Ses i 3 as claimed in claim 1 is used in the preparation of a kit for detecting sesame allergen protein.

4. The use as claimed in claim 3, wherein the sesame allergen protein is protein Ses i 3.

5. The nucleic acid aptamer that specifically recognizes the allergen protein Ses i 3 as claimed in claim 1 is used in the preparation of a diagnostic reagent for preventing sesame allergy.

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