A nucleic acid aptamer targeting a104r protein and application thereof in african swine fever detection

Abstract

The application belongs to the technical field of biology and particularly relates to a nucleic acid aptamer targeting African swine fever virus A104R protein and use thereof. First, the nucleic acid aptamer APT-1 capable of specifically combining with A104R protein is screened out by taking African swine fever virus A104R protein as a combination antigen, the nucleic acid aptamer is single-stranded DNA, and the nucleotide sequence is shown as SEQ ID NO. 1. Second, the nucleic acid aptamer APT-1 has good affinity and reactivity with African swine fever virus protein A104R and has no cross reaction with p30 protein, and has good specificity. The nucleic acid aptamer APT-1 is used for detecting African swine fever virus antibody, has high sensitivity and specificity, and has no cross reaction with porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine parvovirus, foot-and-mouth disease virus and swine fever virus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical or biopharmaceutical technology, specifically relating to a nucleic acid aptamer APT-1 targeting the A104R protein and its use in the detection of African swine fever virus or its antibody. Background Technology

[0002] African swine fever (ASF) is an acute, high-febrile, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) in domestic and wild pigs. This disease is extremely damaging, with a mortality rate that can reach 100%, causing enormous economic losses to my country's pig farming industry and related sectors. The World Organisation for Animal Health (WOAH) lists it as a notifiable animal disease, and it is also classified as a Class A animal disease in my country. The incubation period for ASF is generally 4–19 days. Based on its clinical manifestations, it can be classified into hyperacute, acute, subacute, chronic, and asymptomatic forms. The disease is mainly transmitted through direct contact; soft ticks can serve as reservoir hosts and transmission vectors. The clinical symptoms and pathological changes caused by ASF are related to viral virulence. Overall, they are similar to other febrile and hemorrhagic diseases in pigs, such as classical swine fever, porcine reproductive and respiratory syndrome (PRRS), and pseudorabies, making differential diagnosis difficult clinically. Laboratory testing is therefore crucial for the prevention and eradication of this disease. With the continued occurrence and spread of African swine fever worldwide, the prevalent strains present an extremely complex picture, posing new challenges to the diagnosis and control of the disease. Currently, there are no safe and effective vaccines or treatments for African swine fever. Developing new diagnostic and testing technologies and antiviral agents will provide crucial technical support for the prevention and control of this disease.

[0003] Nucleic acid aptamers are oligonucleotides obtained through SELEX screening that possess specificity and affinity for target molecules, enabling them to bind to target molecules through their three-dimensional structure. The binding properties of nucleic acid aptamers to target molecules are similar to the interaction between antibodies and antigens, hence they are also called chemical antibodies. However, nucleic acid aptamers have many advantages over traditional antibody molecules. They can recognize target molecules with high specificity and exhibit high affinity; their preparation is simple, easy to synthesize and chemically modify; they have small molecular weight, low immunogenicity, and good penetrability, making them less likely to induce immune responses when applied in vivo; and they exhibit strong stability under different temperature and pH conditions. These advantages make them show great promise for applications in biomedical fields such as disease diagnosis, targeted drug delivery, biosensing technology, antiviral and antibacterial therapies.

[0004] Current technologies for the diagnosis, antiviral treatment, and pathogenesis of African swine fever (ASF) rely on traditional antibody molecules, including polyclonal antibodies, monoclonal antibodies, and nanobodies. These methods suffer from drawbacks such as high cost, difficulty in obtaining sufficient resources, and limited commercialization and large-scale application. The A104R protein, a structural protein of the ASF virus and a DNA-binding protein, is a crucial target for ASF diagnosis and antiviral research. Therefore, screening for nucleic acid aptamers that specifically recognize and bind to the A104R protein of the ASF virus will overcome the limitations of existing antibody-based approaches in diagnosis and antiviral treatment. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a nucleic acid aptamer APT-1 targeting the A104R protein and its use in the detection of African swine fever virus or its antibodies. Specifically, it includes the following:

[0006] In a first aspect, the present invention provides a nucleic acid aptamer APT-1 that targets the A104R protein of African swine fever virus, the sequence of which is shown in SEQ ID No. 1.

[0007] Preferably, the sequence of the nucleic acid aptamer APT-1 is chemically modified.

[0008] Preferably, the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, fluorination, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopization.

[0009] In a second aspect, the present invention provides the application of the nucleic acid aptamer APT-1 described in the first aspect above in the analysis, purification, imaging, or concentration detection of African swine fever virus or African swine fever virus A104R protein for non-detection purposes.

[0010] Thirdly, the present invention provides the use of the nucleic acid aptamer APT-1 described in the first aspect above in the preparation of reagents or kits for detecting African swine fever virus or antibodies.

[0011] Fourthly, the present invention provides the use of the nucleic acid aptamer APT-1 described in the first aspect above in the preparation of a drug for the prevention or treatment of African swine fever virus infection.

[0012] Fifthly, the present invention provides a nucleic acid aptamer conjugate, wherein the nucleic acid aptamer conjugate is a substance for labeling, detection, diagnosis or treatment attached to the sequence of the nucleic acid aptamer APT-1 described in the first aspect above.

[0013] Preferably, the substance used for labeling, detection, diagnosis, or treatment includes at least one of the following: fluorescent markers, radioactive substances, therapeutic substances, biotin, digoxigenin, nanoluminescent materials, peptides, or siRNA.

[0014] Preferably, the substance for labeling, detection, diagnosis or treatment is attached to the 5' end and / or 3' end of the nucleic acid aptamer APT-1.

[0015] Preferably, the substance used for labeling, detection, diagnosis, or treatment is selected from at least one of polypeptides, antigens, antibodies, detection markers, gold nanoparticles, luminescent nanomaterials, therapeutic drugs, and enzymes.

[0016] Preferably, the substance used for labeling, detection, diagnosis or treatment is streptavidin or biotin.

[0017] In a sixth aspect, the present invention provides the application of the nucleic acid aptamer conjugate described in the fifth aspect above in the analysis, purification, imaging, or concentration detection of African swine fever virus or African swine fever virus A104R protein for non-detection purposes.

[0018] In a seventh aspect, the present invention provides the use of the nucleic acid aptamer conjugate described in the fifth aspect above in the preparation of reagents or kits for detecting African swine fever virus or antibodies.

[0019] Eighthly, the present invention provides the use of the nucleic acid aptamer conjugate described in the fifth aspect above in the preparation of a medicament for the prevention or treatment of African swine fever virus infection.

[0020] In a ninth aspect, the present invention provides a pharmaceutical composition comprising: (i) the nucleic acid aptamer APT-1 described in the first aspect above, or the nucleic acid aptamer conjugate described in the fifth aspect above; and (ii) a pharmaceutically acceptable carrier.

[0021] In a tenth aspect, the present invention provides an African swine fever virus detection reagent, the detection reagent comprising: (i) the nucleic acid aptamer APT-1 described in the first aspect above, or the nucleic acid aptamer conjugate described in the fifth aspect above; and (ii) a detection-acceptable carrier.

[0022] In one aspect, the present invention provides a reagent for detecting African swine fever virus A104R protein, the reagent containing the nucleic acid aptamer APT-1 described in the first aspect, or the nucleic acid aptamer conjugate described in the fifth aspect, or the detection reagent described in the tenth aspect.

[0023] In a twelfth aspect, the present invention provides a method for generating the nucleic acid aptamer APT-1 described in the first aspect above, the method comprising the following steps:

[0024] (1) Design and synthesis of random initial oligonucleotide libraries;

[0025] (2) Using SELEX technology, ssDNA sequences that bind to African swine fever virus A104R protein were forward screened from the synthesized random oligonucleotide library, and African swine fever virus p30 protein was used as the reverse screening protein. After ten rounds of screening, ssDNA sequences with high affinity and specificity to A104R protein were obtained.

[0026] (3) The ssDNA sequences obtained in the tenth round of screening were amplified by PCR, and the PCR products were purified and recovered and then subjected to high-throughput sequencing to synthesize ssDNA sequences with high enrichment frequency. Biotin labeling was performed, and the nucleic acid aptamer APT-1 targeting the A104R protein of African swine fever virus was identified by ELISA.

[0027] The beneficial effects of this invention are:

[0028] (1) The present invention provides a nucleic acid aptamer APT-1 that targets the A104R protein of African swine fever virus, wherein the nucleic acid aptamer can specifically recognize and bind to the A104R protein of African swine fever virus;

[0029] (2) The nucleic acid aptamer APT-1 described in this invention has good affinity and reactivity with African swine fever virus protein A104R, and does not cross-react with p30 protein, thus exhibiting good specificity;

[0030] (3) The nucleic acid aptamer APT-1 described in this invention has no cross-reactivity with porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine parvovirus, foot-and-mouth disease virus and classical swine fever virus, and can be used for the specific detection of African swine fever virus antibodies.

[0031] (4) The nucleic acid aptamer APT-1 described in this invention can be used for the detection and analysis of African swine fever virus or A104R protein. Compared with traditional antibodies, nucleic acid aptamers have advantages such as high affinity, high specificity, simple preparation, small molecular weight, stable chemical properties, large-scale synthesis, and easy storage and modification. Attached Figure Description

[0032] Figure 1 The results of ELISA identification of the reactivity of nucleic acid aptamer APT-1 with African swine fever virus A104R protein in Example 2;

[0033] Figure 2The figures show the ROC curves and interactive scatter plots of positive and negative sera for detecting African swine fever virus based on nucleic acid aptamer APT-1 in Example 3; where A is the ROC curve; B is the interactive scatter plot; P represents antibody-positive serum; N represents antibody-positive serum;

[0034] Figure 3 This is the specificity analysis result of detecting African swine fever virus antibodies based on nucleic acid aptamer APT-1 in Example 4. Detailed Implementation

[0035] Through extensive and in-depth research and screening, the inventors of this application have identified a nucleic acid aptamer, APT-1, that targets the African swine fever virus protein A104R. Experimental results show that the nucleic acid aptamer described in this invention can specifically recognize and bind to the African swine fever virus protein A104R, and can be used for the diagnosis, prevention, or treatment of African swine fever.

[0036] Specifically, this invention utilizes SELEX technology to screen random oligonucleotide libraries, thereby obtaining the nucleic acid aptamer APT-1, which can recognize the African swine fever virus A104R protein. Then, methods such as ELISA were used to identify that this nucleic acid aptamer has high affinity and specificity for the African swine fever virus A104R protein.

[0037] The nucleic acid aptamer targeting the A104R protein of African swine fever virus is referred to as APT-1 (the oligonucleotide sequence is shown in SEQ ID No. 1).

[0038] Unless otherwise specified, the experimental reagents described in the following examples are all common commercially available reagents; the operations in the experiments are all conventional operating methods known in the art unless otherwise specified.

[0039] Example 1: Screening of nucleic acid aptamers for A104R protein using SELEX technology

[0040] A104R protein conjugation with Ni-NTA magnetic beads: The His-tagged African swine fever virus A104R recombinant protein was conjugated with Ni-NTA magnetic beads (Sangon Biotech (Shanghai) Co., Ltd.) to immobilize the A104R target protein for subsequent nucleic acid aptamer screening. The A104R recombinant protein was transferred to Ni-NTA magnetic beads containing binding buffer, and incubated overnight at 4°C with gentle shaking at 200 rpm. After magnetic separation, the supernatant was discarded, and the magnetic beads were washed three times with PBS solution to obtain the A104R protein-magnetic bead conjugate (Mag-A104R).

[0041] Initial oligonucleotide library: An oligonucleotide library with fixed sequences at both ends and random sequences in the middle (5'-ATC CAG AGT GAC GCA GCA-(random sequence)-TGG ACA CGG TGG CTT AGT-3') was designed. The initial oligonucleotide random library was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0042] Preparation of ssDNA single-stranded library: Dissolve an appropriate amount of the initial nucleic acid aptamer library in binding buffer, heat at 95°C for 10 minutes, immediately remove and incubate on ice for 15 minutes, then let it stand at room temperature for 15 minutes to equilibrate, and set aside for use.

[0043] Forward screening: Take 50 μL of magnetic beads conjugated with A104R protein (Mag-A104R), add it to the prepared ssDNA single-stranded library, mix slowly, and incubate overnight at 4°C to saturate the binding of ssDNA. Wash the magnetic beads 3 times with washing buffer, 1 mL each time. After the last wash, aspirate as much liquid as possible from the tube, add 50 μL of deionized water, heat at 95°C for 10 minutes, and then magnetically separate the supernatant for use in preparing the secondary library.

[0044] PCR amplification: Using the product selected by forward selection as a template, PCR amplification was performed using primers modified with 5' phosphorylation (5'P-ACTAAGCCACCGTGTCCA-3' and 5'-ATCCAGAGTGACGCAGCA-3') to obtain a double-stranded DNA with 5' phosphorylation modification. An appropriate number of PCR amplification cycles was selected, and the amplified product was purified and recovered.

[0045] Preparation of single-stranded secondary libraries: The prepared PCR products were digested with Lambda exonuclease to prepare ssDNA. The digestion was performed under the following reaction system and conditions: dsDNA 5 μg; Reaction buffer (10×) 5 μL; Lambda Exonuclease 1 μL (5 units); ddH2O up to 50 μL; Digestion conditions: 37℃ for 30 min; 75℃ for 10 min; 4℃ for 10 min. The digested products were purified and recovered to prepare single-stranded secondary libraries for the next round of screening.

[0046] Reverse screening: African swine fever virus p30 protein was conjugated with Ni-NTA magnetic beads, following the steps described above. 50 μL of the magnetically conjugated p30 protein (Mag-p30) was added to the prepared single-stranded secondary library, slowly mixed, and incubated at 37°C for 45 minutes. The supernatant was then magnetically separated for use in forward screening.

[0047] Enriched library sequencing: Ten rounds of screening were performed using the SELEX method with magnetic beads, with each round using the single-stranded secondary library obtained in the previous round as the starting library. The ssDNA obtained after the 10th round of screening was used as a template for PCR amplification. PCR amplification was performed using unphosphorylated primers (5'-ACTAAGCCACCGTGTCCA-3' and 5'-ATCCAGAGTGACGCAGCA-3'). The amplification products were purified by gel extraction and sent to Sangon Biotech (Shanghai) Co., Ltd. for high-throughput sequencing.

[0048] Analyzing the sequencing results, sequences with high enrichment frequencies were selected for synthesis. An ssDNA sequence that can specifically recognize and bind to the African swine fever virus A104R protein was obtained by ELISA, which is the nucleic acid aptamer APT-1, and the sequence is shown in SEQ ID No. 1.

[0049] Example 2: Identification of the reactivity of nucleic acid aptamer APT-1 with African swine fever virus protein A104R

[0050] A biotin-labeled nucleic acid aptamer, APT-1 (Bio-APT-1), was synthesized and co-incubated with HRP-labeled streptavidin to prepare a coupling compound of HRP-Streptavidin-Bio-APT-1.

[0051] African swine fever virus A104R protein was diluted to 5 μg / mL using carbonate buffer, and 100 μL / well was added to each well of an ELISA plate for overnight coating at 4°C. The plates were washed with 300 μL / well of PBST solution three times, and then blotted dry. 100 μL / well of PBST solution containing 3% BSA was added to each well, and the plates were blocked at 37°C for 1 hour. The plates were washed with 300 μL / well of PBST solution once, and then blotted dry. 100 μL / well of the prepared HRP-Streptavidin-Bio-APT-1 solution was diluted 1:20 and incubated at 37°C for 45 minutes. After repeating the washing steps, 100 μL / well of TMB substrate solution was added, and the plates were incubated at 37°C for 9 minutes. The reaction was terminated by adding 100 μL / well of 2 mol / L H2SO4 solution. The OD was measured using an ELISA reader. 450 The value was used as a control, with African swine fever virus p30 protein serving as the reference.

[0052] The results are as follows Figure 1 As shown, the nucleic acid aptamer APT-1 described in this application has good reactivity with the African swine fever virus protein A104R, but no cross-reactivity with the p30 protein.

[0053] Example 3: Detection of African swine fever virus antibodies based on nucleic acid aptamer APT-1

[0054] African swine fever virus A104R protein was diluted to 0.5 μg / mL using carbonate buffer, and 100 μL / well was added to each well of an ELISA plate for overnight coating at 4°C. The plates were then washed with 300 μL / well of PBST solution three times, and the plates were blotted dry. 100 μL / well of PBST solution containing 3% BSA was added to each well, and the plates were blocked at 37°C for 1 hour. The plates were then washed once with 300 μL / well of PBST solution, and the plates were blotted dry. 50 μL of African swine fever virus negative and positive sera were mixed with 50 μL of serum diluent, respectively, and then added to the plate. Add HRP-Streptavidin-Bio-APT-1 to the microplate and incubate at 37°C for 15 minutes. Wash the microplate with PBST solution, 300 μL / well each time, repeating three times, and blot dry. Add 20 nm HRP-Streptavidin-Bio-APT-1 to the microplate and incubate at 37°C for 45 minutes. Wash the microplate with PBST solution, 300 μL / well each time, repeating three times, and blot dry. Add 100 μL / well of TMB substrate solution and incubate at 37°C for 9 minutes. Stop the reaction by adding 100 μL / well of 2 mol / L H2SO4 solution. Measure the OD using a microplate reader. 450 value.

[0055] ROC curve and interactive scatter plot analysis determined that when the PI was 38.5%, the sensitivity and specificity for detecting African swine fever virus in positive and negative sera reached 95.40% and 95.60%, respectively. The results are as follows: Figure 2 As shown.

[0056] Example 4: Specificity analysis of African swine fever virus antibody detection based on nucleic acid aptamer APT-1

[0057] The specificity of the detection of African swine fever virus antibodies based on nucleic acid aptamer APT-1 was analyzed, and positive sera for porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine parvovirus, foot-and-mouth disease virus and classical swine fever virus were detected according to the method in Example 2 above.

[0058] The results are as follows Figure 3 As shown, the nucleic acid aptamer APT-1 described in this application showed no cross-reactivity with positive sera from porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine parvovirus, foot-and-mouth disease virus, and classical swine fever virus. This indicates that the nucleic acid aptamer APT-1 described in this application can be used to specifically detect African swine fever virus antibodies in serum samples.

[0059] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer APT-1 targeting the A104R protein of African swine fever virus, characterized in that, The sequence of the nucleic acid aptamer APT-1 is shown in SEQ ID No.

1.

2. A nucleic acid aptamer conjugate, characterized in that, The nucleic acid aptamer conjugate is a substance for labeling, detection, and diagnosis attached to the sequence of the nucleic acid aptamer APT-1 as described in claim 1; the substance for labeling, detection, and diagnosis is selected from at least one of fluorescent markers, radioactive substances, biotin, digoxigenin, and nanoluminescent materials; the substance for labeling, detection, and diagnosis is attached to the 5' end and / or 3' end of the nucleic acid aptamer APT-1.

3. The nucleic acid aptamer conjugate as described in claim 2, characterized in that, The substances used for labeling, detection, and diagnosis are selected from nanoluminescent materials.

4. The use of the nucleic acid aptamer APT-1 as described in claim 1, or the nucleic acid aptamer conjugate as described in any one of claims 2-3, in the preparation of reagents or kits for detecting African swine fever virus or antibodies.

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