CN113980970A

CN113980970A - Aptamer specifically combined with actinia cytolysin GT-4 and application thereof

Info

Publication number: CN113980970A
Application number: CN202111142658.2A
Authority: CN
Inventors: 胡波; 于豪冰; 刘小宇; 朱玉平; 张晓娟; 焦炳华; 王梁华
Original assignee: Chinese Peoples Liberation Army Naval Characteristic Medical Center
Current assignee: Chinese Peoples Liberation Army Naval Characteristic Medical Center
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2022-01-28

Abstract

The invention relates to the technical field of biomedical engineering, in particular to a group of aptamers specifically combined with activethin GT-4 and application thereof. The aptamer obtained by screening can be used for preparing an aptamer sensor or a detection reagent, and the aptamer sensor or the detection reagent is applied to detection of actinia cytolysin GT-4 in drinking water, seawater samples and aquatic products, so that a foundation is laid for removal of actinia cytolysin GT-4 in water or aquatic products.

Description

Aptamer specifically combined with actinia cytolysin GT-4 and application thereof

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to a high-affinity aptamer specifically bound with activetolysin GT-4 and obtained by screening based on a magnetic bead SELEX technology, which can be used for rapid detection and diagnosis of activetolysin GT-4 in clinical samples and monitoring of activetolysin GT-4 in water and food.

Background

The sea anemone belongs to the phylum of echinocystis, the tentacle contains a large number of echinocytes and stinging capsules, after a human body is stabbed, the local part of the sea anemone can generate burning pain and stabbing pain within a few minutes, then blisters, bleeding or ulcers are generated, the general symptoms mainly comprise cardiovascular injury, pulmonary edema, renal dysfunction and the like, and severe people can cause organ failure and even death. The strong and extensive toxic action of actinia cytolysin is the basis of poisonous actinia sting. The inventor separates and extracts a new cytolysin Gigantoxin-4(GT-4) from the representative toxic sea anemone S.gigantea in south China sea in the early period, and deeply researches the biological function of the new cytolysin Gigantoxin-4 to find that the new cytolysin has very strong hemolytic activity and half of hemolytic concentration is about 40 ng/mL; intravenous administration to rats results in cardiovascular, pulmonary, renal, and other organ damage and failure (Hu, B., et al, Purification and characterization of gigantoxin-4, a new microorganism from the sea animal tissue culture, int J Biol Sci,2011.7(6): 729-39.).

At present, the detection methods of the anemolysin mainly comprise a biological detection method, a chromatography/mass spectrometry method, an immunological detection method and the like, but all have respective limitations. The mouse biological method has the advantages that the operation is relatively simple, the defects are that the time consumption is long, the specificity and the sensitivity can not meet the requirements, meanwhile, the animal protection regulations are violated, and the western countries do not claim the use; high Performance Liquid Chromatography (HPLC) analysis and liquid chromatography-mass spectrometry combined method (LC-MS) have the advantages of high separation efficiency, strong selectivity, high detection speed, low detection limit, repeatability and the like, but the detection technology needs standard toxin and is complex to operate, needs professional staff, is expensive in instrument and cannot perform large-scale detection; the immunological detection method has the advantages of sensitivity, good specificity, small sampling amount, capability of detecting a large number of samples and the like, but an antibody with high sensitivity and high specificity needs to be prepared, the antibody is protein and is easily influenced by temperature to cause great fluctuation of the stability of the antibody, meanwhile, the preparation of the antibody needs to be subjected to animal experiments or cell experiments, the period is relatively long, the cost is high, the titer of the antibody is probably influenced by improper labeling and modification, the detection can be interfered by cross reaction of the antibody and false positive of structural analogues, the operation is more complicated, the operation generally needs 2-4 h, and the further development and the application of the technology are limited. Therefore, it is highly desirable to establish a novel rapid and sensitive detection method.

An aptamer is a single-stranded oligonucleotide having a specific spatial structure that specifically recognizes and binds to a target molecule through intermolecular interactions, such as hydrophobic interactions, van der waals forces, and hydrogen bonding. As a novel molecular recognition element, aptamers are similar to, but superior to, antibodies. For example, aptamers are capable of recognizing a variety of targets, such as proteins, amino acids, and metal ions, even cells and viruses; the aptamer can be connected with various groups, and modification is convenient; the aptamer has stable chemical property and is not easy to denature; the aptamer can be directly chemically synthesized, and the cost is low. In recent years, with rapid progress in analytical techniques, aptamer-based bioanalytical patterns have been developed, and aptamer-based biosensors have received increasing attention. The greatest advantage of aptamer biosensors compared to antibodies is their reusability, and their small size allows efficient, high-density immobilization on biochips. The BLI biosensor is an advanced technology which relies on a biomembrane interference technology and is label-free and real-time monitoring, and the dynamic parameters of the intermolecular interaction can be measured in a high-flux manner. The detection process is simple, rapid, high-throughput, low in cost and easy to maintain, and the technology can be used as an effective substitute for analysis methods of ELISA, HPLC and SPR technologies in many fields.

At present, many researchers have applied aptamers to the field of marine biotoxin detection, and combined with biosensor platforms (such as electrochemistry, fluorescence technology, surface plasmon resonance, and the like), developed many rapid and novel detection methods. However, there have been no reports on GT-4 aptamers.

Disclosure of Invention

The invention aims to provide a plurality of single-stranded DNA aptamers capable of carrying out high-affinity specific binding with activetin GT-4, and the affinity between the aptamers and GT-4 is tested to obtain an affinity constant (K)_D) The smallest aptamer apt-g4 a. A second object of the present invention is to provideThe application of the aptamer, such as the application of the aptamer in preparing reagent for separating and enriching anemolysin GT-4, the application in preparing reagent, kit or sensor for detecting anemolysin GT-4, the application in the rapid detection of GT-4 in a tap water sample, and the foundation for removing GT-4 in a water body or an aquatic product.

In order to achieve the purpose, the invention adopts the following main technical scheme: a single-stranded DNA aptamer (apt-g4a) capable of specifically binding to GT-4 with high affinity is obtained by screening with the magnetic bead-SELEX technology. By combining with the biosensor platform, a GT-4 aptamer sensor can be prepared and used for rapid detection of GT-4.

In a first aspect of the invention, there is provided a set of aptamers that specifically bind to actins GT-4, the nucleotide sequences of which are shown in SEQ ID Nos. 1-6 (Table 1), respectively.

TABLE 1 nucleic acid aptamer sequences

Further, the aptamer is chemically modified at the 3 'end or the 5' end by biotin, a fluorescent molecule, an isotope, electrochemistry, an enzyme, a thiol group, or the like.

In a second aspect of the invention, there is provided the use of an aptamer that specifically binds to anemolysin GT-4 as described above in the preparation of a reagent for capturing, isolating, enriching and purifying anemolysin GT-4.

In a third aspect of the invention there is provided the use of an aptamer that binds specifically to activetin GT-4 as described above in the preparation of an activetin GT-4 detection reagent, kit or sensor.

In a fourth aspect of the invention, there is provided the use of an aptamer that specifically binds to activetin GT-4 as described above for the rapid detection of activetin GT-4 in a body of water or an aquatic product. Preventing people from poisoning mainly caused by drinking polluted water and eating polluted aquatic products by mistake.

In a fifth aspect of the invention, the application of the aptamer specifically binding to actinia cytolysin GT-4 in preparing a kit for rapidly detecting actinia cytolysin GT-4 in water or aquatic products is provided.

According to a sixth aspect of the present invention, there is provided a reagent or kit for rapid detection of actinolin GT-4, said reagent or kit comprising an aptamer as defined above which specifically binds to actinolin GT-4.

The invention has the beneficial effects that:

1. the invention provides an aptamer specifically bound with actinia cytolysin GT-4 and application thereof, and experiments prove that the aptamer can be rapidly and specifically bound with actinia cytolysin GT-4, wherein the affinity between the aptamer apt-g4a and GT-4 is highest and reaches 13.7 nM. Therefore, the aptamer obtained by screening can be used for preparing an aptamer sensor or a detection reagent and is applied to the detection of GT-4 in drinking water and seawater samples. In addition, the aptamers can also lay a foundation for removing GT-4 in a water body or an aquatic product.

2. In addition, the aptamer specifically bound with actinia cytolysin GT-4 provided by the invention is used as a novel molecular recognition probe, has the advantages of low cost, stable property, convenience in modification and the like, and is suitable for large-scale application in industrial production of biological medicines.

Drawings

FIG. 1 is a 10% non-denaturing polyacrylamide gel electrophoresis result, lane 1: DL 500bp DNA marker, lane 2: initial solution control, lane 3: GT-4 protein, lane 4: magnetic bead control;

FIG. 2 is a fitted curve of aptamer sensor response values;

FIG. 3 is a specificity analysis of aptamer sensors.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1: preparation of screening libraries

Buffer A (1 XPBS pH 7.4, 1mM MgCl₂，0.05％Tween20)

Buffer B (1 XPBS pH 7.4, 1mM MgCl₂，0.05％Tween20，2mg/mL BSA)

1. The aptamer library (Biotechnology, Inc., cat # 1004728-.

2. The supernatant was carefully discarded and only about 100. mu.L of supernatant remained in the tube (when the supernatant was discarded, it was ensured that the microspheres were all retained in the tube). Add 3mL buffer B and vortex on a vortex mixer.

Standing in water bath at 3.95 deg.C for 5 min. Naturally cool to room temperature (requiring more than 30 min) in order to allow the oligonucleotide to "anneal" to its lowest energy-consuming conformation, forming a stable tertiary structure.

4. 7mL of buffer A was added. Then vortexed on a vortex mixer. Centrifuge at 3000g for 10min, carefully discard the supernatant and add fresh buffer A to a total volume of 1.8 mL. Finally, the microspheres containing the oligonucleotide library were quantitatively transferred to a 2mL centrifuge tube.

Example 2: screening of GT-4 aptamers

The screening process mainly comprises four steps, namely negative screening, coupling, specific screening and amplification, and comprises the following specific processes:

1. negative selection

1) M-280 streptavidin magnetic beads (Invitrogen, cat. 11205D) were mixed well on a vortex apparatus and pipetted 250. mu.L into a 1.5mL tube. Placing on a magnetic frame, standing for 1min, and removing supernatant with a precision pipettor. Add 500. mu.L buffer A, spin wash the beads, discard the supernatant, repeat the wash 2 times.

2) The beads were resuspended in 50. mu.L of buffer A and then added to the entire aptamer library all over before and incubated for 1 hour at room temperature with rotation. The magnetic particles and any bound aptamers were removed with a magnetic separator and the non-adsorbed library was transferred to a 15mL centrifuge tube. The wash transfer was repeated until all the non-adsorbed libraries were transferred. Discard the magnetic beads and all the adsorbed adapter strip libraries.

3) The obtained aptamer library was washed 3 times with 10mL of buffer a. The washed aptamer library was finally resuspended in buffer A and then transferred to a 2mL tube to a final solution volume of 1.8mL for GT-4 screening, designated library A.

Coupling of GT-4 to magnetic beads

1) The magnetic beads (10mg/mL) were spun down and 50. mu.L (0.5mg) was pipetted into a 1.5mL tube. A1.5 mL tube containing 50. mu.L of the spun-up magnetic beads was placed on a magnetic stand and allowed to stand for 1min, and the supernatant was discarded. Add 250 u L buffer B, spin washing magnetic beads 3 times.

2) 100 μ L of buffer B was added at room temperature, followed by 10-15 μ g GT-4, and incubated for 30 minutes at room temperature. Place on magnetic rack, discard buffer, and wash 3 times with 200. mu.L buffer B. Finally, 100. mu.L of buffer A was added to obtain a conjugate.

3. Specific screening

1) The library A and the conjugate are mixed at room temperature, are incubated for 90min in a rotating mode at room temperature, and are adsorbed to the library which is non-covalently coupled to the magnetic beads for 1-2min by using a magnetic frame. The supernatant and unbound aptamer library were discarded to obtain library B. The library B was washed with buffer A, magnetic beads were adsorbed on a magnetic rack, and the supernatant was discarded until the wash became clear and free of unbound aptamer library. The final library B was resuspended in 50. mu.L of buffer B.

2) To the above 50. mu.L of library B, 50. mu.L of 1N NaOH was added. Incubate at 65 ℃ for 30 min. Then add 2M Tris-Cl 40. mu.L to neutralize NaOH. Adsorbing the magnetic beads by using a magnetic frame, transferring the supernatant into a DNA adsorption column, centrifuging for 2min at 1500g, and collecting about 140-.

3) Library C was divided into 3 tubes of 45. mu.L each. No.1 is an initial solution control tube, No. 3 is a magnetic bead negative control tube, 105 mu L of buffer solution A is added into each tube of No.1 and No. 3, and the total volume reaches 150 mu L. The final reaction volume was 150. mu.L of labeled GT-4 added to tube 2 at a GT-4 concentration of 100nM (see Table 2 below for details). Library C and GT-4 were vortex incubated for 1 hour at room temperature.

TABLE 2

4) 60 mu.L of the magnetic beads are taken, washed 2 times by buffer B, finally the magnetic beads are resuspended by 30 mu.L of buffer B, 10 mu.L of each magnetic bead is added into a No. 2-3 tube, and vortex incubation is carried out for 30min at room temperature. The magnetic beads were magnetically separated using a magnetic stand and washed 3 times with 150. mu.L of buffer B per tube. Finally, 100. mu.L of buffer B was added to each of the 2-3 tubes to obtain magnetic beads. mu.L of each was used for PCR experiments.

PCR and electrophoretic analysis

PCR amplification was performed on each of the above reaction tubes using 100. mu.L of 1 XPCR buffer (2.5mM MgCl. for each PCR reaction tube)₂0.2mM dNTP, 0.4. mu.M forward primer, 0.4. mu.M reverse primer and 1U Taq enzyme) under the following amplification conditions: pre-denaturation at 94 deg.C for 1min under circulation conditions of 94 deg.C, 30s, 58 deg.C, 30s and 72 deg.C for 1min, and finally extension at 72 deg.C for 3min, and setting 25 cycles. The PCR product was subjected to 10% native polyacrylamide electrophoresis.

Table 3 information and affinity values of the selected representative sequences

Example 3: preparation and evaluation of GT-4 aptamer sensor

The biomembrane interference technology is a label-free technology and can provide real-time and high-flux biomolecular interaction information. As a novel label-free technology, the technology of biological membrane interference has occupied an important position in the research of the interaction between biological molecules. As the thickness and mass density of the surface biological membrane of the SSA (super streptavidin) chip are larger, a larger number of aptamer molecules can be assembled on a spatial structure, so that the interaction with GT-4 can cause larger change of signals, and therefore, the SSA chip with a significant response value is selected to be used for preparing a GT-4 aptamer sensor.

The 5' end of the aptamer is modified by biotin, and the aptamer is fixed on the surface of a Super Streptavidin (SSA) sensor through the interaction of biotin and streptavidin. Prior to fixation, the aptamers were first renatured (95 ℃ water bath for 10 minutes, ice bath quenched for 5 minutes, and left at room temperature for 10 minutes) to help refold the aptamers into a stable spatial structure. Buffer, biotin-labeled aptamer solution, buffer, and GT-4 solution were sequentially added to different columns of a 96-well plate at a loading volume of 200 μ L prior to detection with the biofilm interferometer OctetRED 96. The programming of the Octet RED 96 system is set as: (1) sensor equilibration (2 minutes); (2) aptamer coupling (3 min); (3) sensor rebalancing (2 min); (4) GT-4 binding (5 min); (5) GT-4 was dissociated (5 min). All steps were performed at room temperature. And after the detection is finished, deducting the response value of the control group sensor from the response value of the experimental group sensor by using Octet data analysis software CFR Part 11 Version 6.x to obtain a corrected actual response value. In addition, response data were fitted using a 1:1 binding mode to obtain binding-dissociation curves of aptamers to GT-4 and various kinetic parameters.

First, the performance parameters of the aptamer sensor are measured. The response of the aptamer sensors to interaction with GT-4 (10-2000 nmol/mL) at different concentrations was determined separately (FIG. 2). The results show that the response value of the BLI aptamer sensor continuously increases along with the increase of GT-4 concentration, the response value of the sensor reaches a platform at 250s, the response value at the moment is recorded, and a corresponding curve is obtained through fitting. In addition, in the range of GT-4 concentration from 20 nM to 800nM, the aptamer sensor showed good linear detection (FIG. 3), with a limit of detection (LOD) of 0.43 nM.

By utilizing the aptamer sensor, a cross-reactivity experiment is further carried out on the aptamer and toxins such as BTX, NOD-R, OA, STX, GYM and the like, and the result is shown in figure 3, the response value of the sensor caused by GT-4 is 0.48 which is obviously higher than the response values generated by other toxins, and the aptamer sensor has great practical application value in the aspect of GT-4 toxin detection.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

SEQUENCE LISTING

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Claims

1. The nucleotide sequences of the aptamers which are specifically combined with the actinolysin GT-4 are respectively shown as SEQ ID No.1-SEQ ID No. 6.

2. The aptamer according to claim 1, wherein biotin, a fluorescent molecule, an isotope, electrochemistry, an enzyme, or a thiol modification is performed at the 3 'end or the 5' end thereof.

3. Use of an aptamer according to claim 1 or 2 that specifically binds to activetine GT-4 for the preparation of a reagent for capturing, isolating, enriching and purifying activetine GT-4.

4. Use of an aptamer according to claim 1 or 2 that specifically binds to activetine GT-4 for the preparation of an activetine GT-4 detection reagent, kit or sensor.

5. Use of an aptamer according to claim 1 or 2 that specifically binds to activetin GT-4 for the rapid detection of activetin GT-4 in an aqueous body or an aquatic product.

6. Use of an aptamer according to claim 1 or 2 that specifically binds to actinia cytolysin GT-4 in the preparation of a kit for the rapid detection of actinia cytolysin GT-4 in an aqueous body or an aquatic product.

7. A reagent or kit for rapid detection of activetin GT-4 comprising an aptamer according to claim 1 or 2 that specifically binds to activetin GT-4.