CN114854760A

CN114854760A - DNA aptamer of mouse antibody and application thereof

Info

Publication number: CN114854760A
Application number: CN202210736395.6A
Authority: CN
Inventors: 李招发; 赵浩迪; 薛依萱; 徐甜甜; 折婵; 史文亮; 毛雨锐; 王年锋; 林叶子
Original assignee: Huaqiao University
Current assignee: Huaqiao University
Priority date: 2022-06-27
Filing date: 2022-06-27
Publication date: 2022-08-05

Abstract

The invention discloses a DNA aptamer of a mouse antibody and application thereof. The DNA aptamer is a mouse-derived aptamer of an antibody constant region and comprises an aptamer 2 and an aptamer 4, wherein the sequence of the aptamer 2 is shown as SEQ ID No.1, and the sequence of the aptamer 4 is shown as SEQ ID No. 2. Meanwhile, the invention prepares the Circ-Apt-2 and Circ-Apt-4 by looping the aptamer 2 and the aptamer 4. The single-chain and cyclic nucleic acid aptamer can be used for high-specificity recognition and high-affinity binding of mouse-derived antibodies, and the cyclic aptamer amplifies signals through rolling circle amplification, so that a new idea can be developed for traditional immunological detection.

Description

DNA aptamer of mouse antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a mouse antibody DNA aptamer and application thereof.

Background

The immunolabeling technology such as ELISA is the most commonly used detection method in immunological detection, but is not suitable for detecting low-content virus due to the limited detection range. Molecular biological detection such as RT-PCR can be only used in special institutions due to the need of expensive equipment, so that the detection conditions are greatly limited, and therefore, the development of a virus detection method which does not need a large-scale instrument and has the characteristics of high sensitivity, strong specificity, wide detection range, rapid detection and the like has practical significance. The continuous development of science and technology has prompted the development of DNA aptamers, and DNA aptamers and antibodies have the advantages of high affinity, high specificity, high stability and the like, so that great effects are exerted in clinical diagnosis, the binding of aptamers and target molecules is similar to the effects of antigens and antibodies, and aptamers have many characteristics obviously superior to antibodies, such as relatively simple preparation and stronger stability, and the circular aptamers can be directly used for rolling circle amplification to achieve the effect of amplifying signals, so that the development of a virus detection method based on the circular aptamers and the rolling circle amplification has good application prospects. Through the development of the last ten years, aptamer technology is beginning to be widely applied to the fields of analysis and detection, food safety, medicine and environmental detection.

Disclosure of Invention

The invention aims to provide a DNA aptamer of a mouse antibody and application thereof, in particular to a nucleic acid aptamer of a constant region of a mouse-derived antibody.

One of the technical schemes adopted by the invention for solving the technical problems is as follows:

a DNA aptamer of a mouse antibody, wherein the DNA aptamer comprises at least one of Apt-2 or Apt-4, the sequence of Apt-2 is shown as SEQ ID No.1, and the sequence of Apt-4 is shown as SEQ ID No. 2.

Preferably, the Circ-Apt-2 prepared by looping Apt-2 is also included.

Preferably, the Circ-Apt-4 prepared by looping Apt-4 is also included.

The second technical scheme adopted by the invention for solving the technical problems is as follows:

the application of the DNA aptamer of the mouse antibody in immunology.

Preferably, the DNA aptamer of the mouse antibody is used for immunological detection.

Preferably, a nanogold colorimetric detection method is used.

The sequence related by the invention is as follows:

SEQ ID No.1：

5’-GATACTGCGTGCTTGTTCCATAGGGGTGGTGTGGTGTTGTGTTCGTCGTGTTGTGGTTGGGTTGACAGTAAGTGAGAAGTTGCC-3’

SEQ ID No.2：

5’-GATACTGCGTGCTTGTTCCATACGGTAGTCCTGTTAGTGGTTGTGTTGTGCGGTGGGGGTGGTGACAGTAAGTGAGAAGTTGCC-3’

SEQ ID No.3：

5’-GATACTGCGTGCTTGTTCCATA-3’

SEQ ID No.4：

5’-TGACAGTAAGTGAGAAGTTGCC-3’

SEQ ID No.5：

5’-GGCAACTTCTCACTTACTGTCA-3’

SEQ ID No.6：

5’-GATACTGCGTGCTTGTTCCATANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGACAGTAAGTGAGAAGTTGCC-3’

the equipment, reagents, processes, parameters and the like related to the invention are conventional equipment, reagents, processes, parameters and the like except for special description, and no embodiment is needed.

All ranges recited herein include all point values within the range.

In the invention, the room temperature, namely the normal environment temperature, can be 10-30 ℃.

Compared with the background technology, the technical scheme has the following advantages:

the single-chain form and the cyclic nucleic acid aptamer form of the DNA aptamer can be used for high-specificity recognition and high-affinity binding of mouse-derived antibodies, and the signal is amplified by the cyclic aptamer through rolling circle amplification, so that a new thought can be provided for traditional immunological detection.

Drawings

FIG. 1 is a schematic flow chart of a SELEX DNA single-strand aptamer screening technique.

FIG. 2 is a schematic diagram showing the secondary structure of two DNA aptamers Apt-2 and Apt-4 in example 2 of the present invention.

FIG. 3 is a graph showing the Kd value calculation results of the aptamer Apt-2 mouse anti-rabbit in example 3 of the present invention.

FIG. 4 is a graph showing the calculation results of Kd values of mouse anti-human aptamers Apt-2 in example 3 of the present invention.

FIG. 5 is a Kd value calculation result chart of mouse anti-rabbit for aptamer Apt-4 in example 3 of the present invention.

FIG. 6 is a graph showing the calculation results of Kd values of mouse anti-human aptamers Apt-4 according to example 3 of the present invention.

FIG. 7 is a graph showing the results of the colorimetric detection of the mouse antihuman nanogold colorimetric method of Apt-2 in example 3 of the present invention.

FIG. 8 is a graph showing the results of the rabbit-resistant nanogold colorimetric detection of Apt-2 mice in example 3 of the present invention.

FIG. 9 is a graph showing the results of the colorimetric detection of the mouse antihuman nanogold colorimetric method of Apt-4 in example 3 of the present invention.

FIG. 10 is a graph showing the results of the rabbit-resistant nanogold colorimetric detection of Apt-4 mice in example 3 of the present invention.

FIG. 11 is a diagram showing the results of the specific detection of Apt-2 by the nanogold colorimetric method in example 3 of the present invention.

FIG. 12 is a diagram showing the results of specific detection of Apt-4 by nanogold colorimetry in example 3 of the invention.

FIG. 13 is a new method verification diagram for Circ-Apt-2 and Circ-Apt-4 in example 4 of the present invention, in which: virus concentration 16.1pmol/L, pore 1: Circ-Apt-2, pore channel 2: blank, channel 3: Circ-Apt-4, pore channel 4: blank.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1 SELEX-based screening of DNA aptamers to mouse antibodies

The SELEX procedure of this example is shown in fig. 1, in which a carboxyl magnetic bead is used as a stationary phase, two mouse anti-human and mouse anti-rabbit targets derived from a mouse are alternately screened, and a DNA aptamer is screened from a library by capturing, eluting and the like.

1.1 Carboxylic magnetic bead binding target

The beads were shaken up with reference to the instructions for use of the beads, 15. mu.L of the beads were placed in a 1.5mL Ep tube, and the supernatant was discarded. The beads were washed 3 times with 300. mu.L of coupling buffer. And adding 120 mu L of coupling buffer solution, 40 mu L of newly prepared EDC solution and 40 mu L of newly prepared NHS solution into the carboxyl magnetic beads, uniformly mixing by vortex, uniformly mixing and incubating for 60min on a vortex mixer at room temperature, carrying out magnetic separation and removing the supernatant. Then 100. mu.L of coupling buffer and 150. mu.L of 1mg/mL target were added to Ep tube and incubated on a disc mixer for 1h at room temperature. The supernatant was discarded, 200. mu.L of 3% BSA solution was added, and the mixture was incubated on a disc mixer at room temperature for 2 hours. Discard the supernatant, wash the beads 3 times with 1mL blocking buffer, block for 1h with 1mL blocking buffer, magnetically separate, and aspirate the supernatant.

1.2 screening of DNA aptamers from nucleic acid libraries

Taking primary library lib dry powder 1OD (shown as SEQ ID No.6 as sequence information, namely, 5 '-GATACTGCGTGCTTGTTCCATA (SEQ ID No.3) -N40-TGACAGTAAGTGAGAAGTTGCC (SEQ ID No.4) -3') synthesized by TaKaRa company, putting the powder into a centrifuge at 8000rpm/min, centrifuging for 5min, and adding ribozyme-free water into a fume hood to dissolve the powder to 100 mu M.

mu.L of the library and 50. mu.L of binding buffer were mixed well, and then subjected to metal bath at 95 ℃ for 10min, 4 ℃ for 15min, and left at room temperature for 30 min. (first round 12. mu.L primary library + 188. mu.L binding buffer), the library was added to magnetic beads coupled to the target, the mixer incubated for 2h at room temperature with mixing, the supernatant was discarded, the beads were washed 4 times with 300. mu.L washing buffer, and the supernatant was aspirated. Add 200. mu.L of Elution buffer, heat-elute at 95 ℃ for 15min, centrifuge the magnetic separation, collect the supernatant, and use it as a PCR recovery library.

1.3 screening of target aptamers

The PCR reaction system is (the total volume of the system is 50 mu L): ddH ₂ O29.9. mu.L, 10 XPCR Buffer 4. mu.L, 5. mu.M forward primer 0.8. mu.L, 5. mu.M reverse primer 0.8. mu.L, dNTPs 0.32. mu.L, and the recovered library as a template 5. mu.L, r-Taq 0.24. mu.L. PCR reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; 10min at 72 ℃; storing at 4 ℃.

Example 2 cloning of aptamers, sequencing, prediction of candidate aptamer Secondary Structure and cyclization of candidate aptamers to prepare cycloaptamers

2.1 recovery of PCR products

The upstream primer was used as 5'-GATACTGCGTGCTTGTTCCATA-3' (SEQ ID No.3), and the downstream primer was used as: 5'-GGCAACTTCTCACTTACTGTCA-3' (SEQ ID No.5), amplifying the secondary library obtained by the 10 th round screening, cutting gel and recovering a target strip, and recovering a target product by using a gel recovery kit.

2.2 cloning and colony PCR identification

(1) Before the experiment, the superclean bench is irradiated by an ultraviolet lamp for 0.5h, and air is blown for 0.5 h.

(2) Adding 1 mu L T5 cloning vector and 4 mu L aptamer into an EP tube, recovering and purifying the product, uniformly mixing by blowing and sucking, centrifuging, connecting for 15min at 25 ℃ by a PCR instrument, and placing on ice after the reaction is finished.

(3) Trans1-T1 competent cells were thawed on ice for 5 min.

(4) After thawing, 50. mu.L of Trans1-T1 competent cells and 5. mu.L of ligation product were added to the EP tube, mixed by pipetting, and ice-cooled for 0.5 h.

(5) The ice bath is finished, and the competent cells are heat-shocked in a water bath at 42 ℃ for 90s and immediately ice-bathed for 120 s.

(6) After the ice bath is finished, 500 mu L of LB liquid culture medium is added, the mixture is evenly mixed by blowing and sucking, and the mixture is cultured for 60min by a constant temperature shaking table at 37 ℃ and 220 rpm.

(7) Centrifuging at 2000Xg for 2min, discarding 400 μ L of supernatant, and mixing with bacterial solution.

(8) And (3) sterilizing the coater in advance in the ultra-clean workbench, uniformly dripping the uniformly mixed bacteria liquid into a culture medium containing ampicillin, and uniformly coating.

(9) And (4) after the incubator is placed for 60min at 37 ℃, the incubator is placed upside down for culture for 12-17 h.

(10) Ampicillin was added to the liquid culture to a final concentration of 0.01%, and the mixture was mixed well and 4mL was taken in a shake tube.

(11) The single colony successfully transformed is carefully picked by the gun head and directly injected into LB liquid culture solution containing ampicillin.

(12) The cells were incubated overnight at 37 ℃ with constant temperature shaking at 220 rpm.

(13) After the culture, the bacterial solution became turbid, and 1. mu.L of the turbid bacterial solution was used for colony PCR.

(14) And (3) carrying out non-denaturing gel electrophoresis on the colony PCR product, and picking 200-300 bp of bacterial liquid to be sent to a biological company for sequencing.

(15) The sequences were subjected to homology analysis using DNAMAN software.

Two different candidate aptamer sequences, named aptamer 2(Apt-2) and aptamer 4(Apt-4), were obtained by primary screening:

Apt-2(SEQ ID No.1)：

Apt-4(SEQ ID No.2)：

5’-GATACTGCGTGCTTGTTCCATACGGTAGTCCTGTTAGTGGTTGTGTTGTGCGGTGGGGGTGGTGACAGTAAGTGAGAAGTTGCC-3’。

2.3Apt-2 and Apt-4 Secondary Structure prediction

Secondary Structure prediction was performed using RNA Structure software. The result shows that the secondary structures Apt-2 and Apt-4 are stem-loop structures and have higher stability, and the secondary structures are shown in figure 2.

2.4 looping Apt-2 and Apt-4 to prepare Circ-Apt-2 and Circ-Apt-4

mu.L of 10. mu.M Apt-2(SEQ ID No.1), Apt-4(SEQ ID No.2) padlock circular sequence, 1. mu.L of 50. mu.M complementary sequence, 12. mu.L of nuclease-free water and 2. mu.L of 10 XT 4 buffer are respectively added into an EP tube, mixed uniformly, denatured at 95 ℃ for 5 minutes by a PCR instrument, reacted at 25 ℃ for 20 minutes, and 2. mu. L T4 DNA ligase is added after the reaction is finished. Mixing, placing in PCR instrument, performing enzyme-linked reaction at 25 deg.C for 2 hr, and reacting at 70 deg.C for 12 min. After the reaction, 1.6 mu L of 20000U/mL Exonuclease I Exonuclease and 0.8 mu L of 100000U/mL Exonuclease III Exonuclease are added, and after the enzyme digestion reaction at 37 ℃ for 2 hours, the inactivation at 80 ℃ is carried out for 22 minutes.

Example 3 detection of affinity and specificity of DNA aptamers to influenza A H1N1 Virus

3.1 detection of Apt-2 and Apt-4 Kd values by ELISA

(1)10 x ELISA coating solution, with Milli-Q pure water dilution to 1 x, the target with 1 x ELISA coating solution dilution, after mixing, in 96 hole plate each well with 100 u L diluted target solution, and placed at 4 degrees C overnight, light-proof.

(2) The coating solution was discarded, 300. mu.L of 3% BSA was added to each well, and the wells were blocked on a shaker at low speed for 60min at room temperature.

(3) After the blocking, the blocking solution was aspirated, 200. mu.L of washing solution was added to each well, and the mixture was rinsed on a shaker at room temperature for 5min each time for 3 times.

(4) The biotin-modified aptamers were diluted with PBS to different concentrations and treated with a metal bath at 95 ℃ for 10min, 4 ℃ for 15min, and room temperature for 25 min.

(5) And (3) adding 100 mu L of biotin modified aptamer with different concentrations into the wells according to a concentration gradient, and incubating for 60min at room temperature on a shaking table.

(6) After incubation, the liquid was discarded, and washing solution was added to each well at room temperature of 200. mu.L, and washed on a shaker at room temperature for 5min 4 times.

(7) HRP was calculated as 1: 2000, diluted with PBS, mixed well and 100. mu.L was added to the wells and incubated on a shaker at room temperature for 40 min.

(8) After incubation, the wells were discarded, 200. mu.L of washing solution was added to each well, and the wells were rinsed on a shaker at room temperature for 5min each time for 3 times.

(9) And (3) sucking 100 mu L of TMB color development liquid by a pipette gun, adding the TMB color development liquid into a 96-well plate, developing for 10min, sucking 100 mu L of 1mol/L HCl by the pipette gun after the development is finished, and adding the HCl into each well to terminate the reaction.

(10) Measuring absorbance value by a microplate reader at the wavelength of 450nm and 650nm, and storing the result for analysis.

The horizontal axis is the target concentration and the vertical axis is (F0-F)/F0, statistical affinity data are calculated, and Kd values are obtained by using a fitted curve in graphpad software.

The Kd value for each aptamer was calculated according to equation (1).

Wherein y represents saturation, namely the mass fraction of aptamer-bound target protein to total target protein; x represents the concentration of the aptamer added, nmol/L; bmax is the maximum number of aptamers bound to the target protein; kd represents the dissociation constant of both, nmol/L.

As shown in fig. 3, fig. 4, fig. 5 and fig. 6, the color of the solution changes from light yellow to dark yellow with increasing aptamer concentration, which indicates that the binding force is increasing. Saturation is reached when the absorbance no longer increases, indicating that the binding of the aptamer to the target is saturated at this point. Nonlinear fitting using GraphPad Prism6 gave Kd values. Aptamer 2 Kd values for mouse anti-rabbit and mouse anti-human are (4.67012 + -1.02294) nmol/L and (5.7693 + -0.98448) nmol/L, respectively; aptamer 4 Kd values for mouse anti-rabbit and mouse anti-human are (1.89114 + -1.16338) nmol/L and (5.83748 + -0.53644) nmol/L, respectively.

3.2 detection of DNA aptamers Apt-2 and Apt-4 and detection sensitivity and specificity of target protein by nanogold colorimetric method

3.2.1 preparation of gold nanoparticles

(1) Sterilizing a 100mL round bottom flask and a 100mL brown bottle autoclave, and drying for later use.

(2) Soaking the round-bottom flask and the brown bottle in aqua regia for 5 h. Pouring out the aqua regia after soaking, rinsing with Milli-Q pure water for 6 times, and drying for later use.

(3) A round bottom flask was charged with 98mL of water and 2mL of a 1% prepared chloroauric acid solution.

(4) And (3) placing a magnetic stirrer into the round-bottomed flask, fixing the round-bottomed flask by using an iron support, placing the fixed round-bottomed flask into an oil bath pan, and sealing the bottle mouth by using a sealing film.

(5) The magnetic stirrer was turned on and the oil bath temperature was set to 140 ℃, heating the solution.

(6) After the solution boiled, 7mL of 1% trisodium citrate solution was added to the round bottom flask and the solution was observed for color change.

(7) The color in the round bottom flask will change from colorless to dark blue, the dark blue solution gradually changes to reddish brown with the change of heating time, and the reddish brown solution slowly changes to wine red with the change of heating time.

(8) And after the solution turns to wine red, continuing to heat for 15min, and finishing the preparation of the nano gold.

(9) Closing the instrument, removing the fixing device, taking down the round-bottom flask, wrapping the round-bottom flask with tin foil paper, and naturally cooling the round-bottom flask to room temperature in a dark place.

(10) The nanogold solution cooled to room temperature was transferred to a brown bottle and stored at 4 ℃ in the dark.

3.2.2Apt-2 and Apt-4 and mouse anti-human and mouse anti-rabbit sensitivity detection

(1) The target was diluted to different concentrations with a gradient of Milli-Q pure water and mixed well, with diluted target concentrations of 10, 20, 30, 40, 50. mu.g/mL, respectively.

(2) Adding 100 mu L of nano gold into the enzyme label strip hole, adding 45 mu L of aptamer with the concentration of 500nmol/L, uniformly mixing by blowing and sucking, and incubating for 30min at room temperature on a shaking table.

(3) After the incubation is finished, 0, 10, 20, 30, 40 and 50 mu g/mL target is added into each hole in sequence and is evenly mixed by blowing and sucking, and the mixture is incubated for 20min at room temperature on a shaking table.

(4) After the incubation is finished, 20 mu L of 1mol/L NaCl is added into each hole respectively, and the mixture is fully blown, sucked and mixed evenly.

(5) Incubating for 5min at room temperature on a shaking bed, and measuring the absorbance at 650nm and 520nm by using an enzyme-labeling instrument after the incubation is finished.

(6) Storing the experimental results, calculating A ₆₅₀ /A ₅₂₀ And analyzing the result.

Measuring the absorption spectrum of 400-800 nm by using a multifunctional microplate reader, and calculating A ₆₅₀ /A ₅₂₀ With target concentration as abscissa, A ₆₅₀ /A ₅₂₀ As ordinate, make scatter diagram, and find target concentration and A ₆₅₀ /A ₅₂₀ As well as their range, as shown in fig. 7, 8, 9 and 10.

3.2.3 detection of specificity of Apt-2 and Apt-4 with targets

(1) BSA, influenza B virus was diluted with Milli-Q purified water to the same concentration as the target.

(3) After the incubation is finished, water, BSA and a target are sequentially added into each hole, the mixture is evenly blown and sucked, and the mixture is incubated on a shaking table for 20min at room temperature.

The upper graph in fig. 11 shows the change of color of the nanogold added with different targets by Apt-2, and it can be seen that the nanogold added with the mouse anti-human antibody and the mouse anti-rabbit antibody is purple, and wine red when other targets or blank control is added. In FIG. 11, the lower panel is Δ A for the target and other negative controls ₆₅₀ /A ₅₂₀ In comparison, independent sample t test is carried out on the samples in the sps, and delta A is carried out between an experimental group and a control group ₆₅₀ /A ₅₂₀ The aptamer Apt-2 is obviously different from other interference targets, so that the aptamer Apt-2 can be specifically combined with the targets without being combined with the other interference targets. The upper graph in fig. 12 shows the change of color of the aptamer Apt-4 added with different targets, and it can be seen that the color of the nanogold added with mouse anti-human and mouse anti-rabbit is purple, and the color of the nanogold added with other targets or blank control is wine red. In FIG. 12, the lower panel is Δ A for the target and other negative controls ₆₅₀ /A ₅₂₀ In comparison, independent sample t test is carried out on the samples in the sps, and delta A is carried out between an experimental group and a control group ₆₅₀ /A ₅₂₀ The significant difference is that the aptamer Apt-4 can not bind to other interference targets, and can specifically bind to the constant region of the mouse-derived antibody.

Example 4 immunological detection method verification of DNA aptamers Circ-Apt-2 and Circ-Apt-4 instead of enzyme-labeled secondary antibody

(1) The beads were vortexed and pipetted 1. mu.L into a 1.5mL EP tube and placed on a magnetic rack, and the supernatant was detached and aspirated.

(2) Add 150. mu.L of coupling buffer, vortex on the vortex apparatus for 12s, magnetically separate and aspirate the supernatant.

(3) And (3) repeating the step (2) twice.

(4) And adding 100 mu L of coupling buffer solution, 30 mu L of newly prepared EDC solution and 30 mu L of newly prepared NHS solution into the carboxyl magnetic beads, and uniformly mixing by vortex. (Note: this step is to be carried out rapidly, since the intermediates of the amine reaction are unstable)

(5) Incubate on the homogenizer for 25min at room temperature, magnetically separate and aspirate the supernatant.

(6) The beads were washed with 150. mu.L of coupling buffer, vortexed for 12s, magnetically separated and the supernatant aspirated.

(7) EP tube 1 was charged with 100. mu.L of 3% BSA; 100 μ L of virus was added to EP tube 2 and incubated for 1h on a disc mixer at room temperature.

(8) And finishing the incubation. Add 1000. mu.L of quench buffer to the EP tube, mix by inversion for 12s, magnetically separate and aspirate the supernatant.

(9) Add 1000. mu.L of quench buffer to the EP tube, mix and incubate in a disc mixer for 1h at room temperature, magnetically separate and aspirate the supernatant.

(10) Add 1000. mu.L of quench buffer to the EP tube, mix by inversion for 12s, magnetically separate and aspirate the supernatant.

(11) 100 μ L of primary antibody from mouse, which binds the virus, was added to the EP tube and incubated with a disc mixer at room temperature for 1 h.

(12) After incubation, the supernatant was aspirated and washed 4 times with 300 μ L PBS for 3min each.

(13) Diluting the loop aptamer 100 times, denaturing at 95 deg.C for 10min in a metal bath, cooling at 4 deg.C for 15min, and standing at room temperature for 25 min.

(14) Adding the denatured cycloaptamer, uniformly mixing and incubating for 40min at room temperature by a disc mixer, magnetically separating and sucking off the supernatant.

(15) Add 300 u L PBS washing 6 times, each time 3 min.

(16) After the washing, 3. mu.L of Milli-Q pure water was added and mixed by pipetting.

(17) To the EP tube was added 12.5. mu.L of ribozyme-free water, 4. mu.L of dNTPs, 3. mu.L of cycloaptamer, 1.5. mu.L of MgSO 2 ₄ 2.5 mu L of 10xBst Buffer, 0.5 mu L of Bst DNA polymerase, 0.5 mu L of upstream primer and 0.5 mu L of downstream primer are mixed evenly by vortex, and the mixture is subjected to RCA reaction and is put into a PCR instrument for 30min of rolling circle amplification at 65 ℃ and for 15min at 80 ℃. After the reaction, 2. mu.L of RCA sample, 10. mu.L of Milli-Q pure water and 2. mu.L of 6 × Loading buffer were taken, mixed well, and the results of the experiment were analyzed with non-denaturing gel. As shown in fig. 13, lanes 1 and 3 are a loop aptamer 2 and a loop aptamer 4,

lanes

2 and 4 are controls, and it can be seen from the electrophoresis result that both the loop aptamer 2 and the loop aptamer 4 have a step-shaped band, which proves that the method is effective, and proves that the loop aptamer can replace an enzyme-labeled secondary antibody to perform signal amplification to indirectly detect viruses, and that whether the circular aptamer can be bound to a magnetic bead is detected by an electrophoresis result diagram, whether a circular mouse-derived antibody exists on the magnetic bead is detected by the circular aptamer, and the existence of the viruses can be indirectly detected by detecting the antibody. In the experimental process, the virus is combined on the magnetic beads, the corresponding primary antibody is combined on the virus, and the loop aptamer is combined with the primary antibody to carry out RCA (Rolling circle amplification), so that the effect of indirectly detecting the virus can be achieved.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Sequence listing

<110> university of Chinese

DNA aptamer of <120> mouse antibody and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 84

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gatactgcgt gcttgttcca taggggtggt gtggtgttgt gttcgtcgtg ttgtggttgg 60

gttgacagta agtgagaagt tgcc 84

<210> 2

<211> 84

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gatactgcgt gcttgttcca tacggtagtc ctgttagtgg ttgtgttgtg cggtgggggt 60

ggtgacagta agtgagaagt tgcc 84

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gatactgcgt gcttgttcca ta 22

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgacagtaag tgagaagttg cc 22

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggcaacttct cacttactgt ca 22

<210> 6

<211> 84

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatactgcgt gcttgttcca tannnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

nntgacagta agtgagaagt tgcc 84

Claims

1. A DNA aptamer for a mouse antibody, comprising: the DNA aptamer comprises at least one of Apt-2 or Apt-4, wherein the sequence of Apt-2 is shown as SEQ ID No.1, and the sequence of Apt-4 is shown as SEQ ID No. 2.

2. The DNA aptamer of a mouse antibody according to claim 1, characterized in that: also comprises a Circ-Apt-2 prepared by looping the Apt-2.

3. The DNA aptamer of a mouse antibody according to claim 1, characterized in that: also comprises a Circ-Apt-4 prepared by looping Apt-4.

4. Use of a DNA aptamer of a mouse antibody according to any one of claims 1 to 3 in immunology.

5. Use according to claim 4, characterized in that: the DNA aptamers of the mouse antibody are used for immunological detection.

6. Use according to claim 4, characterized in that: and (3) adopting a nano-gold colorimetric detection method.