Nucleic acid aptamer targeting A104R protein and application of nucleic acid aptamer in African swine fever detection

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid aptamer targeting African swine fever virus A104R protein and application of the nucleic acid aptamer. Firstly, African swine fever virus A104R protein is taken as a binding antigen, a nucleic acid aptamer APT-1 capable of being specifically bound with the A104R protein is obtained through screening, the nucleic acid aptamer is single-stranded DNA, and the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO.1; secondly, the nucleic acid aptamer APT-1 disclosed by the invention has relatively good affinity and reactivity with African swine fever virus protein A104R, does not have cross reaction with p30 protein, and has good specificity; the nucleic acid aptamer APT-1 is used for detecting the 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, swine fever virus and the like.

Description

Technical Field

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

[0002] African swine fever (ASF) is an acute, high fever, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and wild boars. The disease is serious and has a mortality rate of up to 100%, causing huge economic losses to my country's pig farming and related industries. The World Organization for Animal Health (WOAH) lists it as a reportable animal infectious disease, and it is also a Class I animal epidemic disease stipulated by my country. The incubation period of African swine fever is generally 4 to 19 days. According to its clinical manifestations, it can be divided into hyperacute, acute, subacute, chronic, and asymptomatic types. The disease is mainly transmitted through direct contact, and soft ticks can serve as reservoir hosts and transmission vectors of the disease. The clinical symptoms and pathological changes caused by African swine fever are related to the virulence of the virus. In general, it is similar to other febrile and hemorrhagic diseases of pigs, such as classical swine fever, porcine reproductive and respiratory syndrome, and pseudorabies. It is difficult to make a differential diagnosis clinically, and laboratory testing is particularly important for the prevention, control, and purification of the disease. As African swine fever continues to occur and spread around the world, the epidemic strains have become extremely complex, which has also brought new challenges to the diagnosis and prevention of the disease. At present, there is no safe and effective vaccine or treatment for African swine fever. The development of new diagnostic testing technologies and antiviral preparations will provide important technical support for the prevention and control of the disease.

[0003] Aptamers are oligonucleotides that have specificity and affinity for target molecules obtained through SELEX technology screening, and can bind to target molecules through their three-dimensional structures. The binding properties of aptamers to target molecules are similar to the interaction between antibodies and antigens, so they are also called chemical antibodies, but aptamers have many advantages over traditional antibody molecules. Aptamers can recognize target molecules with high specificity and show high affinity; they are simple to prepare, easy to synthesize and chemically modified; they have small molecular weight, low immunogenicity, good penetration, and are not easy to cause immune responses when used in vivo; they show strong stability under different environmental conditions such as temperature and pH. The above advantages make it show great application prospects in biomedical fields such as disease diagnosis, targeted drug delivery, biosensor technology, antiviral and antibacterial.

[0004] The existing technical methods for the diagnosis, antiviral and pathogenic mechanism of African swine fever are all based on traditional antibody molecules, including polyclonal antibodies, monoclonal antibodies and nano antibodies, which have the disadvantages of high cost, difficulty in obtaining, difficult commercialization and batch application. The A104R protein is one of the structural proteins of the African swine fever virus, a DNA binding protein, and an important target for the diagnosis and antiviral research of African swine fever. Therefore, screening and obtaining nucleic acid aptamers that can specifically recognize and bind to the A104R protein of the African swine fever virus will overcome the limitations of existing traditional antibodies in diagnosis and antiviral aspects. Summary of the invention

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

[0006] In a first aspect, the present invention provides a nucleic acid aptamer APT-1 targeting the A104R protein of African swine fever virus, and the sequence of the nucleic acid aptamer APT-1 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 comprises at least one of phosphorylation, methylation, amination, sulfhydrylation, fluorination, substitution of oxygen with sulfur, substitution of oxygen with selenium or isotopization.

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

[0010] In a third aspect, the present invention provides the use of the nucleic acid aptamer APT-1 described in the first aspect above in the preparation of a reagent or kit for detecting African swine fever virus or antibodies.

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

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

[0013] Preferably, the substance for labeling, detecting, diagnosing or treating comprises at least one of a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxin, a nanoluminescent material, a peptide or siRNA.

[0014] Preferably, the substance for labeling, detection, diagnosis or treatment is connected 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, nanoluminescent materials, 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 use of the nucleic acid aptamer conjugate described in the fifth aspect in the analysis, purification, imaging or concentration detection for non-detection purposes of African swine fever virus or African swine fever virus A104R protein.

[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 a reagent or kit for detecting African swine fever virus or antibodies.

[0019] In an eighth aspect, the present invention provides the use of the nucleic acid aptamer conjugate described in the fifth aspect above in the preparation of a drug for preventing or treating 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, or the nucleic acid aptamer conjugate described in the fifth aspect; and (ii) a pharmaceutically acceptable carrier.

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

[0022] In the eleventh aspect, the present invention provides a reagent for detecting the A104R protein of the African swine fever virus, wherein the reagent contains 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 producing the nucleic acid aptamer APT-1 described in the first aspect, the method comprising the following steps:

[0024] (1) Designing and synthesizing a random initial oligonucleotide library;

[0025] (2) Using the SELEX technique, ssDNA sequences that bind to the A104R protein of African swine fever virus were positively screened from a synthetic random oligonucleotide library, and the p30 protein of African swine fever virus was used as a counter-screening protein. After ten rounds of screening, ssDNA sequences with high affinity and specificity for the A104R protein were obtained;

[0026] (3) The ssDNA sequences obtained from the tenth round of screening were amplified in large quantities by PCR, and the PCR products were purified and recovered for high-throughput sequencing. The ssDNA sequences with higher enrichment frequencies were synthesized and biotin-labeled, and the nucleic acid aptamer APT-1 targeting the A104R protein of African swine fever virus was identified by the ELISA method.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention provides a nucleic acid aptamer APT-1 targeting 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 the present invention has good affinity and reactivity with the African swine fever virus protein A104R, has no cross-reaction with the p30 protein, and has good specificity;

[0030] (3) The nucleic acid aptamer APT-1 described in the present invention has no cross-reaction with porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, 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 the present 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 the advantages of high affinity, high specificity, simple preparation, small molecular weight, stable chemical properties, large-scale synthesis, and easy storage and modification. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2The ROC curve and interactive dot plot of the positive and negative serum of African swine fever virus detected by nucleic acid aptamer APT-1 in Example 3; wherein A is the ROC curve; B is the interactive dot plot; P represents antibody-positive serum; N represents antibody-positive serum;

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

[0035] After extensive and in-depth research, i.e., a large amount of screening, the inventors of the present application identified a nucleic acid aptamer APT-1 that targets the African swine fever virus protein A104R. The experimental results show that the nucleic acid aptamer of the present 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, the present invention uses SELEX technology to screen a random oligonucleotide library to obtain a nucleic acid aptamer APT-1 that can recognize the A104R protein of the African swine fever virus. Then, ELISA and other methods are used to identify that the nucleic acid aptamer has a high affinity and specificity with the A104R protein of the African swine fever virus.

[0037] Among them, the nucleic acid aptamer targeting the A104R protein of African swine fever virus is represented by APT-1 (the oligonucleotide sequence is shown in SEQ ID No. 1).

[0038] The experimental reagents described in the following examples are all common commercially available reagents unless otherwise specified; 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 based on SELEX technology

[0040] A104R protein was coupled with Ni-NTA magnetic beads: The African swine fever virus A104R recombinant protein with a His tag was coupled with Ni-NTA magnetic beads (Shanghai Sangon Biotech Co., Ltd.), and the A104R target protein was fixed with magnetic beads to facilitate the subsequent screening of nucleic acid aptamers. The A104R recombinant protein was transferred to the Ni-NTA magnetic beads containing the Binding buffer, and the mixture was shaken slowly at 200 rpm overnight at 4°C, magnetically separated, the supernatant was discarded, and the magnetic beads were washed three times with PBS solution to obtain the A104R protein and magnetic bead conjugate (Mag-A104R).

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

[0042] Preparation of ssDNA single-stranded library: Take an appropriate amount of nucleic acid aptamer initial library and dissolve it in binding buffer. After heating at 95℃ for 10 minutes, take it out and put it in ice bath for 15 minutes, and then put it at room temperature for 15 minutes to equilibrate and set aside.

[0043] Forward screening: Take 50 μL of magnetic beads coupled to A104R protein (Mag-A104R), add the prepared ssDNA single-stranded library, slowly mix, incubate at 4°C overnight, saturate the binding of ssDNA, wash the magnetic beads 3 times with washing buffer, 1 mL each time. After the last wash, try to dry the liquid in the tube, add 50 μL of deionized water, heat at 95°C for 10 minutes, and remove the supernatant by magnetic separation for the preparation of the secondary library.

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

[0045] Preparation of single-stranded secondary library: Take the prepared PCR product, add Lambda exonuclease for digestion, prepare ssDNA, and digest according to the following reaction system and conditions: digestion system: dsDNA 5μg; Reaction buffer (10×) 5μL; Lambda Exonuclease 1μL (5units); ddH2O up to 50μL; digestion conditions: 37℃30min; 75℃10min; 4℃10min. Purify and recover the digestion product to prepare a single-stranded secondary library for the next round of screening.

[0046] Reverse screening: African swine fever virus p30 protein was coupled to Ni-NTA magnetic beads, and the operation steps were as described above. Take 50 μL of magnetic bead-coupled p30 protein (Mag-p30), add the prepared single-chain secondary library, slowly mix, incubate at 37°C for 45 minutes, and remove the supernatant by magnetic separation for use in forward screening.

[0047] Enriched library sequencing: 10 rounds of screening were repeated using the magnetic bead SELEX method, and each round of screening used 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, and PCR amplification was performed using primers without phosphorylation modification (5'-ACTAAGCCACCGTGTCCA-3' and 5'-ATCCAGAGTGACGCAGCA-3'). The amplified product was purified by gel recovery and sent to Sangon Biotech (Shanghai) Co., Ltd. for high-throughput sequencing.

[0048] The sequencing results were analyzed, and sequences with higher enrichment frequencies were selected for synthesis. After ELISA identification, a ssDNA sequence that can specifically recognize and bind to the A104R protein of African swine fever virus was obtained, which is the nucleic acid aptamer APT-1, and the sequence is shown in SEQID No.1.

[0049] Example 2 Identification of the reactivity between nucleic acid aptamer APT-1 and 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 (HRP-Streptavidin) to prepare a coupling product of HRP-Streptavidin-Bio-APT-1.

[0051] The African swine fever virus A104R protein was diluted to 5 μg / mL using carbonate buffer, and 100 μL / well was added to the ELISA plate for overnight coating at 4°C; the ELISA plate was washed with PBST solution, 300 μL / well each time, repeated three times, and patted dry; 100 μL / well of PBST solution containing 3% BSA was added to the ELISA plate, and blocked at 37°C for 1 hour; the ELISA plate was washed with PBST solution, 300 μL / well each time, washed once, and patted dry; the prepared HRP-Streptavidin-Bio-APT-1 was diluted 1:20, 100 μL / well was added to the ELISA plate, and incubated at 37°C for 45 minutes; after repeated washing steps, TMB substrate solution was added, 100 μL / well, and color was developed at 37°C for 9 minutes; 100 μL / well of 2 mol / L H2SO4 solution was added to terminate the reaction; OD was measured using an ELISA reader 450 At the same time, the African swine fever virus p30 protein was used as a control.

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

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

[0054] The A104R protein of African swine fever virus was diluted to 0.5 μg / mL using carbonate buffer, and 100 μL / well was added to the ELISA plate and coated at 4°C overnight; the ELISA plate was washed with PBST solution, 300 μL / well each time, repeated three times, and patted dry; 100 μL / well of PBST solution containing 3% BSA was added to the ELISA plate, and blocked at 37°C for 1 hour; the ELISA plate was washed with PBST solution, 300 μL / well, washed once, and patted dry; 50 μL of negative and positive serum of African swine fever virus were mixed with 50 μL of serum diluent, respectively, and then added Add the plate to the ELISA plate and incubate at 37°C for 15 minutes; wash the plate with PBST solution, 300 μL / well each time, repeat three times, and pat dry; add 20 nm HRP-Streptavidin-Bio-APT-1 to the plate and incubate at 37°C for 45 minutes; wash the plate with PBST solution, 300 μL / well each time, repeat three times, and pat dry; add TMB substrate solution, 100 μL / well, and color at 37°C for 9 minutes; add 2 mol / L H2SO4 solution, 100 μL / well to terminate the reaction; use an ELISA reader to measure OD 450 value.

[0055] Through ROC curve and interactive dot plot analysis, it was determined that when the PI was 38.5%, the sensitivity and specificity of detecting positive and negative serum of African swine fever virus reached 95.40% and 95.60% respectively. Figure 2 shown.

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

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

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

[0059] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope 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. The nucleic acid aptamer APT-1 according to claim 1, characterized in that: The sequence of the nucleic acid aptamer APT-1 is chemically modified.

3. The nucleic acid aptamer APT-1 according to claim 2, characterized in that: The chemical modification comprises at least one of phosphorylation, methylation, amination, sulfhydrylation, fluorination, substitution of oxygen with sulfur, substitution of oxygen with selenium or isotopization.

4. A nucleic acid aptamer conjugate, characterized in that: The nucleic acid aptamer conjugate is a substance used for labeling, detection, diagnosis or treatment connected to the sequence of the nucleic acid aptamer APT-1 described in claim 1.

5. The nucleic acid aptamer conjugate according to claim 4, characterized in that: The substance used for labeling, detection, diagnosis or treatment includes: at least one of a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxin, a nanoluminescent material, a peptide or siRNA.

6. The nucleic acid aptamer conjugate according to claim 5, characterized in that: The substance for labeling, detection, diagnosis or treatment is connected to the 5' end and / or 3' end of the nucleic acid aptamer APT-1.

7. The nucleic acid aptamer conjugate according to claim 6, characterized in that: The substance used for labeling, detection, diagnosis or treatment is selected from at least one of polypeptides, antigens, antibodies, detection markers, gold nanoparticles, nanoluminescent materials, therapeutic drugs and enzymes.

8. Use of the nucleic acid aptamer APT-1 as described in any one of claims 1 to 3, or the nucleic acid aptamer conjugate as described in any one of claims 4 to 7 in the analysis, purification, imaging or concentration detection for non-detection purposes of African swine fever virus or African swine fever virus A104R protein.

9. Use of the nucleic acid aptamer APT-1 as described in any one of claims 1 to 3, or the nucleic acid aptamer conjugate as described in any one of claims 4 to 7 in the preparation of a reagent or kit for detecting African swine fever virus or antibodies.

10. Use of the nucleic acid aptamer APT-1 according to any one of claims 1 to 3, or the nucleic acid aptamer conjugate according to any one of claims 4 to 7 in the preparation of a drug for preventing or treating African swine fever virus infection.

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