Beta-conglycinin aptamer based on automatic Capture-SELEX (systematic evolution of ligands by exponential enrichment) screening

Abstract

The invention discloses a beta-conglycinin aptamer based on automatic Capture-SELEX (systematic evolution of ligands by exponential enrichment) screening, and belongs to the technical field of food safety biology. According to the method, candidate sequences are screened out through Capture-SELEX equipment integrated with automatic library hybridization, library magnetic bead coupling, magnetic bead washing, positive screening and negative screening, and the affinity of candidate aptamers is determined through mutual verification of two affinity determination methods; and finally, verifying and predicting the binding mechanism of the candidate aptamer beta-conglycinin by combining circular dichroism and molecular docking simulation. The screened beta-conglycinin aptamer has relatively high affinity and relatively high specificity to beta-conglycinin, dissociation constants measured by an ITC method and an FP method of the aptamer are 6.59 + / -6.78 mu M and 18.24 + / -2.42 nM, and a good application prospect is provided for detection of sensitization protein in food.

Description

Technical Field

[0001] The invention relates to a beta-conglycinin aptamer based on automated Capture-SELEX screening, and belongs to the field of food safety biotechnology. Background Art

[0002] β-conglycinin is a major storage protein in soybeans, accounting for about 30% of soybean protein, and is the main food allergen in soybeans. It has strong thermal stability and resistance to digestive enzymes. Conventional thermal processing or the digestive system of the animal body cannot completely remove the potential threat posed by soybean allergenic proteins to humans and animals. Effective identification or detection technology plays a very positive role in avoiding the occurrence of soybean allergic reactions. The recognition molecules for constructing biosensors with rapid detection functions are mainly antibodies and nucleic acid aptamers. Compared with antibodies, aptamers have the advantages of convenient synthesis and modification, high stability, negligible immunogenicity, and programmable base sequence and structure. Aptamers are a class of functional single-stranded oligonucleotides that can recognize targets through geometric matching or multiple non-covalent interactions. However, at present, most of the recognition molecules for recognizing β-conglycinin are antibodies, and there are few studies on β-conglycinin aptamers. In addition, the affinity of existing β-conglycinin aptamers is measured by only one method, which lacks objectivity, and the reverse screening step is not involved in the screening process, and the specificity is weak. At the same time, there is currently no research on the binding mechanism between β-conglycinin aptamer and β-conglycinin.

[0003] At present, the main method for obtaining aptamer sequences is the systematic evolution of ligands by exponential enrichment (SELEX), which can obtain aptamers with significant affinity and specificity through iterative selection and amplification of aptamers. However, SELEX has also encountered a large number of problems in practical applications. First, due to the complexity of SELEX operation, operators need to undergo long-term professional training before screening; the screening process involves many liquid handling steps, and the nucleic acid solution frequently contacts the air, which is very easy to cause aerosol contamination; in the manual operation process, it is inevitable that poor repeatability and accuracy will be shown, and the reliability of the screening results cannot be guaranteed. As a result, the time, labor and financial resources associated with the aptamer screening process have increased significantly due to these problems.

[0004] Therefore, it is necessary to establish an efficient method for screening aptamers to screen an aptamer sequence with high affinity and high specificity in order to obtain a recognition molecule for a biosensor for detecting β-conglycinin. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a β-conglycinin aptamer based on automated Capture-SELEX screening, with the aim of constructing an automated Capture-SELEX device by combining automation technology with Capture-SELEX to screen out aptamer sequences of β-conglycinin as recognition molecules for constructing a β-conglycinin biosensor.

[0006] The first technical solution provided by the present invention is a β-conglycinin aptamer, wherein the aptamer is a polynucleotide containing a nucleotide sequence as shown in SEQ ID NO:1.

[0007] In certain embodiments, the aptamer may be linked to a functional group or molecule. Optionally, the functional group or molecule may be linked to the 5' end or the 3' end of the aptamer.

[0008] In certain embodiments, the functional group or molecule may be an isotope, a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, an enzyme marker, a magnetic substance, biotin, an affinity ligand and / or a thiol group.

[0009] The present invention provides the design and preparation of an automated Capture-SELEX device, which mainly includes the following contents:

[0010] (1) Designed 3D drawings of the automated Capture-SELEX equipment using SolidWorks;

[0011] (2) Using CNC machine tools to cut, the ten-row metal test tube rack and equipment housing are manufactured according to the dimensions on the 3D drawing;

[0012] (3) The heating module of the equipment was built by building the circuits and installing the components of the thermistor, heating coil, solid-state relay, and heating controller;

[0013] (4) The pollution control module of the equipment was completed by installing the UV lamp, purification fan, and stretchable glass door;

[0014] (5) The magnetic bar motion module of the equipment was built by building the circuits and installing the components of the XZ-axis stepper motor, XZ-axis linear module, and motion controller;

[0015] (6) The magnetic separation module of the equipment is completed by installing components such as electromagnets, magnetic sleeves, and magnetic rods;

[0016] (7) The vibration module of the equipment is completed by building the circuits of the vibrator and vibration knob and installing the components.

[0017] (8) The prepared automated Capture-SELEX device integrates the following steps of Capture-SELEX: ① hybridization of library and complementary DNA by thermal denaturation; ② washing and activation of magnetic beads; ③ coupling of library to magnetic beads; ④ movement of magnetic beads; ⑤ positive screening; ⑥ reverse screening.

[0018] The present invention also provides a method for screening a high-affinity and high-specificity aptamer targeting β-conglycinin, which mainly comprises the following steps:

[0019] (1) Using an automated Capture-SELEX device, the library is denatured and hybridized with complementary DNA, and then the magnetic beads are moved in the grooves in sequence through a magnetic separation module to perform library coupling with magnetic beads, reverse screening, and forward screening;

[0020] (2) amplifying the single-stranded DNA bound to β-conglycinin obtained after the positive screening by PCR, and then purifying it by PCR, preparing single-stranded DNA by enzyme digestion, and obtaining a secondary library for the next round of screening by ethanol precipitation;

[0021] (3) After 14 rounds of screening, the candidate library was subjected to high-throughput sequencing, and three candidate sequences were selected based on the frequency and ΔG value of the sequences;

[0022] (4) Affinity determination of the three candidate sequences was performed using fluorescence polarization (FP) and isothermal titration calorimetry (ITC);

[0023] (5) The two aptamers with higher affinity were further tested for specificity using fluorescence polarization assay;

[0024] (6) Circular dichroism was used to verify the binding of the candidate aptamer β-5 with the best affinity and specificity to β-conglycinin;

[0025] (7) The binding mechanism of the candidate aptamer β-5 and β-conglycinin was identified through molecular docking simulation.

[0026] The second technical solution provided by the present invention is a probe, wherein the probe is a substance obtained by labeling the β-conglycinin aptamer described in the first technical solution with a labeling substance.

[0027] In certain embodiments, the label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and can be attached to a nucleic acid aptamer. Labels include, but are not limited to, dyes; radioactive labels, such as 32P; conjugated coupling groups, such as biotin; haptens, such as digoxin (DIG); chemiluminescent, phosphorescent or fluorescent moieties; fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum through fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight measurement, quantum dots, electrochemistry, X-ray diffraction or absorption, magnetism, immunoenzyme label reaction, immunoassay affinity precipitation of base filter paper, affinity chromatography, enzyme activity, microscopic projection or scanning imaging, super-resolution imaging, cell tracking, live nanoparticle tracking imaging of animals or humans, nanoflow, adjustable resistive pulse induction, fluorescence correlation spectroscopy surface plasmon resonance, fluorescence polarization, surface enhanced Raman spectroscopy, electrochemical sensing, microfluidics or microfluidics, chip analysis, proteomics, genomics, metabolomics, microbiome, RNA (mRNA, 1nRNA, SnRNA), miRNA, etc. The label can be a charged part (positive or negative charge) or can be selected as needed, and can be charge neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence). The label can also be used for targeted drug delivery.

[0028] In certain embodiments, the label is a fluorophore, a colorimetric label, a quantum dot, biotin, and other label molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cyclic olefins for click chemistry reactions, and initiator groups for polymer labeling), and can also be selected from polypeptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as peptoids), non-natural nucleic acids and their analogs (nucleotide mimics) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced emission molecules, rare earth ion ligand molecules, polymetallic oxygen clusters, etc.).

[0029] In certain embodiments, the fluorophore can be selected from fluorescein dyes, rhodamine dyes and other common dyes.

[0030] In certain embodiments, the fluorescein dye includes standard fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC), fluorescein amylase (FAM), tetrachlorofluorescein (TET), cy5, cy3, Quasar670, Alexa Fluor 488 / 647, and the like.

[0031] In certain embodiments, the rhodamine dye includes R101, tetraethyl rhodamine (RB200) or carboxytetramethylrhodamine (TAMRA) and the like.

[0032] In certain embodiments, the general dye is mainly selected from two categories, one is thiazole orange (T0), oxazole orange (Y0) series and dimer dyes thereof, and the other is polymethine series cyanine dyes.

[0033] In certain embodiments, the fluorophore can also be selected from the following dyes: stilbene, naphthalimide, coumarins, acridines, pyrenes, and the like.

[0034] Fluorophores are usually labeled at the 5' end of the probe sequence, but can also be placed at the 3' end by changing the modification bond (eg, -OH or -NH bond).

[0035] The third technical solution provided by the present invention is a cell containing the aptamer described in the first technical solution or the probe described in the second technical solution.

[0036] In certain embodiments, the cell may be an animal cell, a plant cell, or a microbial cell.

[0037] The fourth technical solution provided by the present invention is a biosensor containing the aptamer described in the first technical solution or the probe described in the second technical solution.

[0038] The fifth technical solution provided by the present invention is the use of the aptamer described in the first technical solution, the probe described in the second technical solution, the cell described in the third technical solution or the biosensor described in the fourth technical solution in the preparation and detection of β-conglycinin.

[0039] The sixth technical solution provided by the present invention is a reagent or a kit for detecting β-conglycinin, wherein the reagent or the kit contains the aptamer described in the first technical solution or the probe described in the second technical solution.

[0040] The seventh technical solution provided by the present invention is a method for detecting β-conglycinin, which comprises labeling a reporter group on the aptamer described in the first technical solution, allowing the aptamer labeled with the reporter group to interact with the sample to be tested, and detecting β-conglycinin by detecting the signal of the reporter group.

[0041] The eighth technical solution provided by the present invention is the application of the aptamer described in the first technical solution, the probe described in the second technical solution, the cell described in the third technical solution, the biosensor described in the fourth technical solution, the reagent or kit described in the sixth technical solution, or the method described in the seventh technical solution in the field of food, biology or medicine.

[0042] The technical effects of the present invention are as follows:

[0043] The present invention constructs an automated Capture-SELEX device for efficient screening of β-conglycinin. The device integrates functions such as constant temperature heating, vibration, magnetic bead transfer, and pollution control, and integrates all steps such as library hybridization, library coupling magnetic beads, magnetic bead washing, positive screening, and reverse screening in Capture-SELEX, effectively reducing the difficulty of the screening experiment, reducing the intervention of experimental instruments and manpower investment, and the screening experiment can be completed through simple operating instructions; secondly, before the screening experiment, sterilization can be completed by ultraviolet light irradiation. During the screening process, the stretchable glass is lowered to achieve the closure of the experimental environment. During magnetic separation, the electromagnet controls the magnetic rod to descend, and the purification fan is turned on to achieve dust-free in the area; the affinity of the candidate aptamer is mutually verified by two affinity determination methods; finally, the binding mechanism of the candidate aptamer β-conglycinin is verified and predicted by combining circular dichroism spectroscopy and molecular docking simulation. The β-conglycinin aptamer screened by the present invention has high affinity and high specificity for β-conglycinin, and its dissociation constants determined by ITC method and FP method are 6.59±6.78 μM and 18.24±2.42 nM, respectively, which provides a good application prospect for the detection of allergenic proteins in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a 3D design of the automated Capture-SELEX equipment.

[0045] Figure 2 This is a schematic diagram of the process flow of the automated Capture-SELEX equipment.

[0046] Figure 3 This is a feasibility analysis diagram of the automated Capture-SELEX equipment. (A) Electrophoresis diagram of library hybridization complementary DNA verification; (B) Optimization of the vibration frequency of library-coupled magnetic beads; (C) Comparison of magnetic bead solution before and after magnetic separation; (D) Verification of the magnetic bead recovery capability of the automated equipment; (E) Repeatability analysis of library-magnetic bead coupling using automated equipment; (F) Electrophoresis diagram of the pollution control capability of the automated equipment.

[0047] Figure 4 It is the specificity analysis diagram of candidate aptamers β-1 and β-5.

[0048] Figure 5 This is an analysis diagram of the binding mechanism between β-5 and β-conglycinin; (A) circular dichroism analysis; (B) molecular docking simulation. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0050] The raw materials used in the examples are:

[0051] β-conglycinin and glycinin were extracted from low-temperature defatted soybean meal (Henan Kunhua) by isoelectric precipitation and purified by AKTA avant (Superdex G200).

[0052] Trypsin inhibitor, lectin, ovalbumin (OVA), bovine serum albumin (BSA), acrylamide / bisacrylamide 30% solution, Tris (2-carboxyethyl) phosphine, and N,N,N',N'-tetramethyleneethylenediamine were purchased from Sigma-Aldrich.

[0053] Gel red nucleic acid stain was purchased from Botium.

[0054] The aptamer random library was purchased from Takara Biotechnology (Dalian) Co., Ltd.

[0055] Dynabead M-270 streptavidin magnetic beads used for SELEX were from Thermo Fisher Scientific.

[0056] Lambda exonuclease and 1× Lambda exonuclease reaction buffer were purchased from New England Biolabs.

[0057] Primers, all PCR reagents and other compounds used for electrophoresis were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0058] Other conventional reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0059] Example 1 Screening of β-conglycinin

[0060] 1. Construction of the automated Capture-SELEX platform

[0061] The automation equipment was designed using SolidWorks modeling software. From the appearance, Figure 1As shown in the figure, the device consists of a central control platform and a screening reaction chamber. The central control platform includes: a temperature controller, a motion module controller, a lighting / UV switch, a vibration switch, a heating switch, a magnetic bar lifting switch, and a vibration frequency adjustment knob. The screening reaction chamber includes: X-axis and Z-axis stepping modules; a ten-row aluminum alloy test tube rack that can load 1.5mL centrifuge tubes; a constant temperature heating module; a vibration module that can vibrate the test tube rack with variable frequency; a lighting lamp and a UV lamp; a magnetic bar system with an exhaust fan installed above the magnetic bar; and a plexiglass baffle. The models of the components of the equipment are shown in Table 1.

[0062] Table 1 Equipment parts model

[0063]

[0064] From the functional point of view, the automation equipment is divided into 6 modules, such as Figure 1 As shown, they are:

[0065] (1) The row test tube rack is located at the bottom center of the screening reaction chamber. The row test tube rack is made of aluminum alloy blocks by CNC machine cutting. There are 10 grooves on the test tube rack, numbered 1-10 from left to right. Each groove has a diameter of 11mm and a depth of 30mm, which can accommodate a 1.5mL centrifuge tube. In order to facilitate the observation of the internal state of the centrifuge tube, a gap is cut in each groove. Among them, groove No. 5 is separated from the grooves on both sides by cutting to reduce the heat transfer generated by the groove during the heating process.

[0066] (2) Heating module, located in groove No. 5 of the row test tube rack. The module consists of a heating rod, a temperature detector, a solid-state relay, and a temperature controller. The bottom of groove No. 5 in the test tube rack is filled with a heating rod, and a thermistor for temperature measurement is attached to the wall of the groove. These two components are connected with the solid-state relay and the temperature controller on the central control platform to form a constant temperature heater, which can continuously heat the groove at a constant temperature with a temperature deviation of ±1°C.

[0067] (3) The magnetic rod motion module is located directly above the row of test tube racks. The motion controller can be operated to control the stepper module to drive the magnetic rod separation system to smoothly move up, down, left, and right between the test tube groove No. 1 and the test tube groove No. 10. The module consists of a controller, two stepper motors, a 300mm X-axis linear motion guide, and a 100mm Z-axis linear motion guide. By installing the lines in an orderly manner, the function of carrying the magnetic rod movement can be played. In addition, the controller can set the magnetic rod to run in a single step, that is, to move from one position to another at a specific speed.

[0068] (4) Magnetic separation module, fixed on the Z-axis linear motion guide of the magnetic rod motion module. The module consists of an impact electromagnet, a magnetic rod, a magnetic block and a magnetic rod sleeve. When the impact electromagnet is powered on, it can push the magnetic rod to the bottom of the rod sleeve. When the magnetic rod moves into the magnetic bead solution, the magnetic beads can be magnetically attracted. On the contrary, when the power is off, the impact electromagnet can pull the magnetic rod back to the top of the rod sleeve. Then the rod sleeve moves up on the Z-axis motion guide and away from the groove, and the magnetic beads gradually fall off the rod sleeve. Since the magnetic rod needs to move to the bottom of the test tube during the magnetic attraction process, the cross-sectional area of ​​the magnetic rod we selected is 4 mm. Since the size of the magnetic rod is small, the accompanying magnetic force is weak. To ensure that the magnetic rod absorbs all the magnetic beads in the test tube, an N52 strong magnet is added above the magnetic rod to increase the magnetic strength of the magnetic rod.

[0069] (5) Vibration module, the vibrator is located at the bottom of the row test tube rack. The vibration frequency of the vibrator can be adjusted to 1-10 gears through the controller. Through this vibration function, the reagents in different test tubes are fully mixed. In addition, the vibration amplitude required for different reactions is also different. This purpose can be achieved by adjusting the vibration knob. In addition, low-frequency vibration will also make the magnetic beads fall off the magnetic rod sleeve more completely.

[0070] (6) Pollution control module. According to the design concept of the clean bench, an ultraviolet sterilization lamp is installed on the top of the screening reaction chamber, a small fan is installed next to the electromagnet of the magnetic separation module, and a plexiglass sliding door is installed from the top to the bottom. Before the equipment is working, the working area can be sterilized by ultraviolet irradiation; when working, a closed space is formed by closing the plexiglass sliding door. When the magnetic rod is displaced by electromagnetic attraction of magnetic beads, the small centrifugal fan will automatically open, and the airflow has a uniform cross-sectional wind speed, which can remove the original air in the working area and take away dust particles and biological particles to form a sterile and highly clean working environment.

[0071] 2. Methods and steps for screening β-conglycinin based on automated Capture-SELEX

[0072] The design principle of this automated equipment is based on the Capture-SELEX technology. Every operational detail in the Capture-SELEX screening process is automatically mapped one by one. By operating the buttons and knobs of the equipment, each step of Capture-SELEX can be completed step by step.

[0073] The schematic diagram of this technology is shown in Figure 2As shown, from the groove No. 3 to the groove No. 10 of the ten-row test tube rack, centrifuge tubes containing 200 μL (1 mg / mL) of magnetic bead solution, 300 μL of PBS buffer (10 mM, pH = 7.4), 300 μL of single-stranded DNA (ssDNA) library and biotin-modified complementary DNA (library is 0.1 nmol, complementary DNA is 0.15 nmol), 300 μL of binding buffer, 300 μL of reverse screening target solution (soybean globulin) (50 μg / mL), 300 μL of binding buffer, 300 μL of target solution (β-conglycinin) (50 μg / mL), and 300 μL of PBS buffer are placed in sequence. First, the ssDNA library and the biotin-modified complementary DNA are heated to 95°C in the groove No. 5 of the heating module, and are heat-denatured at a constant temperature for 10 minutes. After cooling, the two complementarily hybridize to form a ssDNA-cDNA complex. Then, the magnetic beads were moved from groove 3 to groove 4 under the combined action of the magnetic rod motion module and the magnetic separation module. After vibration washing, they were transferred to groove 5 containing the library-cDNA complex solution, and the library was coupled to the magnetic beads with streptavidin-biotin by vibration incubation for 2 hours. Thereafter, the magnetic beads with the library were washed in groove 6 to remove the library with poor surface binding. Then, the magnetic beads were successively incubated in grooves 7, 8, and 9 for 2 hours to complete the steps of reverse screening, removal of nonspecific ssDNA, washing, and positive screening. Finally, the magnetic beads were moved from groove 9 to groove 10 to obtain the ssDNA specifically bound to β-conglycinin in groove 9.

[0074] The collected ssDNA was used as a PCR amplification template for PCR amplification. The total volume of the system was 50 μL, including 0.5 μL FAM-labeled forward primer (10 μmol / L), 0.5 μL phosphate-labeled reverse primer (10 μmol / L), 1 μL dNTPmix (5 mmol / L), 5 μL 10× PCR amplification buffer, 41 μL sterile ultrapure water, 2 μL template and 0.50 μL Taq DNA polymerase (5 U / μL). The amplification conditions were: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, and multiple cycles followed by extension for 2 min. The obtained amplified product was stored at 4°C. The product was subjected to 10% polyacrylamide gel electrophoresis to determine the optimal number of PCR cycles, and the PCR product was purified using a PCR product purification kit. In order to prepare the next round of ssDNA library, the phosphorylated antisense strand of the PCR product was specifically digested with λ exonuclease. The digestion time is that the double PCR product is mixed with 2μL λ exonuclease (5U / μL) and 35μL 10×λ exonuclease buffer (670mM Glycine-KOH, 25mM MgCl2) and incubated at 37°C. The digestion time is determined by 10% denaturing polyacrylamide gel electrophoresis. Once complete digestion to ssDNA is confirmed, the digestion mixture is heated to 75°C for 10 minutes to inactivate the enzyme. Finally, the ssDNA is purified by phenol-chloroform extraction and redissolved in binding buffer as a sublibrary for the next round of screening.

[0075] 3. Feasibility Verification of Automated Capture-SELEX

[0076] (1) Verification of hybridization between ssDNA library and cDNA: This screening method is to fix the library on streptavidin magnetic beads by complementary hybridization between the designed biotinylated cDNA and the ssDNA library. According to the screening experiment, the library and cDNA are mixed and placed in the heating groove of the automated equipment for hybridization. The electrophoresis experiment shows that Figure 3 A. After the heat denaturation hybridization step, the cDNA is almost consumed. After the original ssDNA library is hybridized, the structure changes to a certain extent, and the band shifts upward. The ssDNA library and cDNA are successfully hybridized.

[0077] (2) Verification of library coupling to magnetic beads: Use 5 vibration gears, 1, 3, 5, 7, and 9, to incubate the system to promote the coupling of the library to the magnetic beads. Coupling rate = (fluorescence value of the library solution before fixation - fluorescence value of the library solution after fixation) / fluorescence value of the library solution before fixation × 100%. Figure 3 B, when the vibration frequency is 5, the maximum coupling rate of the library is shown. Therefore, the vibration frequency of 5 is selected as the optimal coupling condition.

[0078] (3) Verification of library transfer: The movement of the library between the grooves is driven by the magnetic beads. The recovery rate of the magnetic beads is calculated by weighing the mass of the magnetic beads before and after the automated screening experiment to verify the transfer effect of the library. According to the screening experiment, the magnetic bead solution before and after the experiment is dried and weighed. Figure 3 CD, through 6 parallel experiments, the recovery rate of magnetic beads ranged from 93.75% to 95.45%, with a coefficient of variation of <1%. The overall average recovery rate of magnetic beads was 94.73%.

[0079] (4) Verification of the reproducibility of the screening experiment: The reproducibility of the screening experiment under the action of the automated equipment was verified by conducting experiments on the magnetic bead-coupled library under the same conditions. Figure 3 E, after 5 experiments, the library coupling range is 70.20% to 73.98%, and the coefficient of variation is <2%. This automated system greatly eliminates potential errors and variations in human operations, thereby ensuring that the execution method and conditions of each experiment are consistent, further enhancing the repeatability of the experiment.

[0080] (5) Verification of contamination control: Three screening experiments were conducted, and the ssDNA of the screening products was collected and verified by 10% non-denaturing polyacrylamide gel electrophoresis. Figure 3 F, the electrophoresis lane where the product is located after screening using automated equipment has no stray bands. Comparing with the initial library in lane 4, it can be determined that the lane where the product solution is located is the ssDNA of the shedding library.

[0081] 4. Analysis of aptamer binding characteristics

[0082] The final round of screening products were sequenced using high-throughput sequencing. The top two sequences in frequency and the sequence with the largest Gibbs free energy change were selected based on the sequence frequency and the Gibbs free energy of the secondary structure. A total of three candidate aptamers (Table 2) were used for affinity experiments.

[0083] Table 2 Candidate sequences of β-conglycinin aptamers determined by high-throughput analysis

[0084]

[0085]

[0086] (1) Isothermal titration calorimetry (ITC) to verify affinity: Fill the cell with 300 μL of β-conglycinin (6 μM) and load 38 μL of aptamer (100 μM) into the syringe. Each ITC experiment uses a syringe speed of 750 rpm, with an initial purge injection of 0.4 μL, followed by 18 consecutive injections of 2 μL (180 s intervals) to ensure mixing after each injection. Fit the corrected binding isotherm in the MicroCalPEAQ-ITC analysis software, establish a single-site binding model, and obtain the binding affinity and thermodynamic parameters of the binding process.

[0087] (2) Verify affinity using fluorescence polarization (FP): Add 5'-FAM-modified aptamers to a series of target solution concentrations and incubate for 2 hours. The final concentration of the aptamer is 5 nM, and the final concentration of the target is 10, 20, 40, 60, 100, 150, and 200 nM, and incubate at 37°C in the dark for 2 hours. After incubation, the fluorescence polarization value is read by the Synergy H1 multi-mode microplate reader. Binding buffer is added to the system instead of the target as a blank control, and the reaction conditions are the same as above. In the specific analysis, other proteins (soybean globulin, trypsin inhibitor, lectin, bovine serum albumin, chicken serum albumin) are used instead of β-conglycinin for incubation.

[0088] Table 3 ITC and FP methods were used to characterize the affinity of the screened sequences for β-conglycinin

[0089]

[0090] The results are shown in Table 3. Both β-1 and β-5 sequences have good affinity for β-conglycinin. Next, glycinin, trypsin inhibitor, lectin, bovine serum albumin and chicken serum albumin were used to perform specific analysis of these two sequences, using the same method as the fluorescence polarization method mentioned above. Figure 4 As shown, β-1 has no cross-reaction to trypsin inhibitor, lectin, bovine serum albumin and chicken serum albumin, but has a high cross-reaction rate to soybean globulin (close to 40%). In contrast, β-5 has no obvious cross-reaction to the above-mentioned interferents (cross-reaction rates are all less than 20%), so β-5 was selected as the high-affinity and high-specificity aptamer sequence for β-conglycinin.

[0091] Example 2 Analysis of aptamer binding mechanism

[0092] This example uses circular dichroism (CD) and molecular docking to analyze the binding mechanism of the β-5 sequence screened in Example 1, as follows:

[0093] (1) Analysis of binding conformation by circular dichroism (CD): Circular dichroism was used to analyze the structure of the aptamer. Before detection, deoxygenation was performed with dry purified nitrogen (99.99%), and a nitrogen environment was maintained during the experiment. The background signal of 5 μM β-conglycinin in the binding buffer or binding buffer was first measured and subtracted from the CD spectrum. A quartz colorimetric tube with a 1 cm path and a bandwidth of 1 nm was used, and scanned 3 times at room temperature. The wavelength before and after the aptamer binding was measured at 320 to 220 nm.

[0094] (2) Molecular docking simulation analysis of binding sites: The resolution of The β-conglycinin structure was obtained by removing water molecules for the docking process. In order to construct the three-dimensional structure of the aptamer, the Vienna number was first formed through the Mfold Online Web server (http: / / unafold.rna.albany.edu / ?q=mfold), and then the corresponding three-dimensional structure with the best predicted energy was constructed using the RNAcomposer online tool. The final three-dimensional conformation was obtained by mutating the U base to the T base. The docking experiment was performed using the H-DOCK program. The docking was performed using the HDOCK program, a professional protein-protein and protein-DNA / RNA docking program. Subsequently, the docking score was calculated based on the ITScorePR iterative scoring function, and the structure with the best docking score was selected as the standard result for subsequent interaction analysis.

[0095] like Figure 5 As shown in A, β-5 has a characteristic positive peak at 276nm and a characteristic negative peak at 249nm, which are the characteristic peaks of the typical B-type DNA structure. The positive peak at 276nm is mainly formed by the superposition of bases in the complementary region, and the negative peak at 249nm is mainly formed by the right-handed helical structure. This shows that β-5 forms a hairpin structure in the solution. When β-conglycinin binds to β-5, the CD signal at 276nm increases, which may be due to the binding of the aptamer to β-conglycinin, which enhances the base stacking in the hairpin and makes the aptamer structure compact. These characteristics are also consistent with Capture-SELEX based on structure switchability.

[0096] In order to further study the binding mechanism of the aptamer and identify possible binding sites, the three-dimensional conformation of the aptamer was constructed, and the aptamer-protein complex was studied by docking. The conformation with the lowest binding energy was used to predict the binding site. Figure 5As shown in Figure B, the hydrogen bond between aptamer β-5 and β-conglycinin plays a major role in the formation of the complex, and multiple hydrogen bond sites can be clearly seen in the complex (see Table 4 for specific sites). This result proves that aptamer β-5 can bind to β-conglycinin.

[0097] Table 4 Hydrogen bonds and hydrogen bond distances between aptamer β-5 and β-conglycinin

[0098]

[0099]

[0100] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A β-conglycinin aptamer, characterized in that: The aptamer is a polynucleotide containing a nucleotide sequence as shown in SEQ ID NO:

1.

2. The aptamer according to claim 1, characterized in that The 5' end or 3' end of the aptamer is connected with a functional group or molecule, and the functional group or molecule is an isotope, a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, an enzyme marker, a magnetic substance, biotin, an affinity ligand and / or a thiol group.

3. An automated Capture-SELEX device for screening the aptamer according to claim 1 or 2, characterized in that: The automated Capture-SELEX device is built by the following steps: (1) Designed 3D drawings of the automated Capture-SELEX equipment using SolidWorks; (2) Using CNC machine tools to cut, the ten-row metal test tube rack and equipment housing are manufactured according to the dimensions on the 3D drawing; (3) The heating module of the equipment was built by building the circuits and installing the components of the thermistor, heating coil, solid-state relay, and heating controller; (4) The pollution control module of the equipment was completed by installing the UV lamp, purification fan, and stretchable glass door; (5) The magnetic bar motion module of the equipment was built by building the circuits and installing the components of the XZ-axis stepper motor, XZ-axis linear module, and motion controller; (6) The magnetic separation module of the equipment is completed by installing components such as electromagnets, magnetic sleeves, and magnetic rods; (7) The vibration module of the equipment is completed by building the circuits of the vibrator and vibration knob and installing the components; The automated Capture-SELEX device integrates the following screening steps: ① hybridization of library and complementary DNA by thermal denaturation; ② washing and activation of magnetic beads; ③ coupling of library with magnetic beads; ④ movement of magnetic beads; ⑤ positive screening; ⑥ reverse screening.

4. A method for screening aptamers for β-conglycinin, characterized in that: The main steps include: (1) using the automated Capture-SELEX device described in claim 3 to denature and hybridize the library with complementary DNA, and then moving the magnetic beads in the grooves in sequence through the magnetic separation module to perform library coupling with magnetic beads, reverse screening, and forward screening; (2) amplifying the single-stranded DNA bound to β-conglycinin obtained after the positive screening by PCR, and then purifying it by PCR, preparing single-stranded DNA by enzyme digestion, and obtaining a secondary library for the next round of screening by ethanol precipitation; (3) After multiple rounds of screening, the candidate library obtained is subjected to high-throughput sequencing, and candidate sequences are selected based on the sequence frequency and ΔG value; (4) using fluorescence polarization measurement and isothermal titration calorimetry to measure the affinity of candidate sequences and screen out aptamers with stronger affinity; (5) Using fluorescence polarization measurement method, further specificity measurement is performed on the aptamer with stronger affinity in step (4), and the aptamer with the best affinity and specificity is screened out.

5. A probe, characterized in that: The probe is a substance obtained by labeling the β-conglycinin aptamer according to claim 1 or 2 with a labeling substance.

6. A cell containing the aptamer according to claim 1 or 2 or the probe according to claim 5.

7. A biosensor comprising the aptamer according to claim 1 or 2 or the probe according to claim 5.

8. Use of the aptamer according to claim 1 or 2, the probe according to claim 5, the cell according to claim 6 or the biosensor according to claim 7 in the preparation of a biosensor for detecting β-conglycinin.

9. A reagent or kit for detecting β-conglycinin, characterized in that: The reagent or kit contains the aptamer according to claim 1 or 2 or the probe according to claim 5.

10. A method for detecting β-conglycinin, characterized in that: The method comprises labeling a reporter group on the aptamer of claim 1 or 2, allowing the aptamer labeled with the reporter group to interact with a sample to be tested, and detecting β-conglycinin by detecting a signal of the reporter group.

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