Nucleic acid aptamer specifically combined with African swine fever virus A104R protein and application of nucleic acid aptamer

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid aptamer specifically combined with African swine fever virus A104R protein and application of the nucleic acid aptamer. According to the present invention, the exponential enrichment ligand systematic evolution technology (SELEX) is adopted, the African swine fever virus A104R protein is adopted as the target, 6 nucleic acid aptamers capable of combining the A104R protein are screened from a random nucleic acid library, and the sequences of the nucleic acid aptamers are represented by SEQ ID No.1-6; the nucleic acid aptamer has good affinity and reactivity with African swine fever virus protein A104R, has no cross reaction with p30 protein, has good specificity, and can be used for detection and analysis of African swine fever virus or A104R protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine or biotechnology, and specifically relates to a nucleic acid aptamer specifically binding to the A104R protein of African swine fever virus and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by infection with African swine fever virus (ASFV). There are 24 genotypes of ASFV, which can be divided into high-virulence strains, medium-virulence strains and low-virulence strains according to the difference in virus virulence; the mortality rate of pigs caused by infection with high-virulence strains is usually as high as 100%, which seriously threatens the pig farming industry in my country and the world. The clinical symptoms caused by ASFV infection are diverse and similar to some other diseases of pigs, making it difficult to make a differential diagnosis clinically. At present, there is no safe and effective vaccine or treatment for ASFV, and the prevention and control of the disease mainly depends on rapid and accurate laboratory testing and strict purification measures.

[0003] Nucleic acid aptamers are functional oligonucleotides with high specificity and affinity for target molecules obtained through SELEX technology screening. Most of them are between 15 and 60 bases in length. Because their binding ability with target substances is comparable to that of antibodies, they are also called "chemical antibodies". Nucleic acid aptamers have the advantages of low immunogenicity, simple preparation, good penetration and easy modification. They have broad application prospects in biomedical fields such as target molecule detection, disease diagnosis, imaging, drug delivery and targeted therapy.

[0004] A104R protein is a structural protein of African swine fever virus, present in the nucleoid of the virus, a DNA binding protein, involved in viral transcription, auxiliary and genome packaging, and an important target protein for diagnosis and antiviral research. Therefore, screening nucleic acid aptamers that can specifically recognize and bind to the A104R protein of African swine fever virus is of great value and significance for the diagnosis, treatment and analysis of the pathogenic mechanism of African swine fever. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a nucleic acid aptamer that specifically binds to the A104R protein of African swine fever virus and its application. Specifically, it includes the following contents:

[0006] In a first aspect, the present invention provides a nucleic acid aptamer targeting the A104R protein of African swine fever virus, the sequence of the nucleic acid aptamer being shown in any one of SEQ ID No. 1-6.

[0007] Preferably, the sequence of the nucleic acid aptamer 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 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 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 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 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 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 the tenth aspect, the present invention provides an African swine fever virus detection reagent, which comprises: (i) the nucleic acid aptamer described in the first aspect above, or the nucleic acid aptamer conjugate described in the fifth aspect above; 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 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 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 targeting the African swine fever virus A104R protein, which can specifically recognize and bind to the African swine fever virus A104R protein.

[0029] (2) The nucleic acid aptamer 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 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

[0031] Figure 1 The results of Western blot identification of A104R recombinant protein, where M is protein marker, 1 is negative control, and 2 is purified A104R protein;

[0032] Figure 2 The SDS-PAGE was used to identify the coupling effect of A104R protein and Ni-NTA magnetic beads, where M is the molecular weight standard, 1 is the Mag-A104R magnetic bead complex, and 2 is the magnetic separation supernatant.

[0033] Figure 3 The results of PCR amplification of ssDNA after the tenth round of SELEX screening, where M is the molecular weight standard and 1-6 are amplified products;

[0034] Figure 4 ELISA was used to identify the reactivity of nucleic acid aptamers with African swine fever virus protein A104R. DETAILED DESCRIPTION

[0035] After extensive and in-depth research, namely a large amount of screening, the inventors of the present application obtained a nucleic acid aptamer that can bind to the A104R protein of the African swine fever virus. 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, and can be used for the detection and analysis of the African swine fever virus or the A104R protein, as well as 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 nucleic acid aptamers that can recognize the A104R protein of the African swine fever virus. Then, nucleic acid aptamers with high affinity and specificity for the A104R protein of the African swine fever virus are identified by methods such as ELISA.

[0037] Among them, the nucleic acid aptamers that bind to the A104R protein of African swine fever virus are represented by APT-2, APT-4, APT-5, APT-6, APT-7 and APT-8 (the oligonucleotide sequences are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively).

[0038] Example 1 Induced expression and purification of African swine fever virus A104R recombinant protein

[0039] The recombinant plasmid pET-30a-A104R identified by sequencing was transformed into BL21 (DE3) competent cells, and the bacterial solution was inoculated into liquid LB medium (Kan+) at a ratio of 1:100, and the culture was shaken at 200 r / min at 37°C. The OD 600 When the protein level reaches 0.6-0.8, add 0.5mmol / L IPTG and induce expression at 37℃ for 8 hours. Centrifuge the induced bacterial solution at 10000rmp for 10 minutes, discard the supernatant, vortex the precipitate with PBS and wash it 3 times. Ultrasonicate the washed precipitate, centrifuge it at 10000rmp for 30 minutes, and collect the precipitate and supernatant respectively. Purify the A104R protein using nickel column affinity chromatography. Add the supernatant solution after ultrasonication to the treated nickel column, place it on a mixer and mix it at room temperature for 4 hours, and collect the combined liquid. Elute the impurities with different concentrations of imidazole buffer in turn, and finally elute the target protein with 300mmol / L imidazole buffer, which is the purified A104R protein.

[0040] The purified A104R protein was quantified and analyzed by Western Blot. The identification results were as follows: Figure 1 shown.

[0041] Example 2: Coupling of African swine fever virus A104R protein with Ni-NTA magnetic beads

[0042] The His-tagged African swine fever virus A104R recombinant protein was coupled to Ni-NTA magnetic beads (Shanghai Biotech Co., Ltd.), and the A104R target protein was fixed by magnetic beads to facilitate the subsequent screening of nucleic acid aptamers. The specific operation is as follows: Take 1mL of ASFV A104R protein and place it at 4°C to fully melt it for use. Mix the Ni-NTA magnetic beads thoroughly, take 1ml of the magnetic bead suspension and place it in a centrifuge tube, place the centrifuge tube on a magnetic separator, wait until the solution becomes clear, discard the supernatant, add deionized water and wash repeatedly to remove the residual storage solution. Transfer the melted A104R recombinant protein to the magnetic beads containing Binding buffer, and shake slowly at 200rpm overnight at 4°C. The magnetic bead-coupled A104R protein (Mag-A104R) was analyzed by SDS-PAGE to check whether the coupling was successful.

[0043] The results are as follows Figure 2 As shown, the A104R recombinant protein was successfully coupled to the Ni-NTA magnetic beads.

[0044] Example 3 Screening of nucleic acid aptamers for A104R protein based on SELEX technology

[0045] Initial oligonucleotide library: The initial library of nucleic acid aptamers was designed and synthesized. The oligonucleotide sequence was 5'-ATCCAG AGT GAC GCA GCA-(N)40-TGG ACA CGG TGG CTT AGT-3', (N)40 represents 40 bases randomly composed of A, T, G, and C. It was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] Enriched library sequencing: 10 rounds of screening were repeated using the magnetic bead SELEX method, and the single-stranded secondary library obtained in the previous round was used as the starting library in each round of screening. 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 amplification results were analyzed by agarose gel electrophoresis. Figure 3 As shown, the amplified product was purified by gel recovery and then sent to Sangon Biotechnology (Shanghai) Co., Ltd. for high-throughput sequencing.

[0052] The sequencing results were analyzed, and sequences with higher enrichment frequencies were selected for synthesis. The ssDNA sequences that could specifically recognize and bind to the A104R protein of African swine fever virus were obtained by ELISA identification. These were the nucleic acid aptamers obtained by screening, and their sequences were shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.

[0053] Example 4 Identification of the reactivity of nucleic acid aptamers with African swine fever virus A104R protein

[0054] A biotin-labeled nucleic acid aptamer (Bio-APT) was synthesized and co-incubated with HRP-labeled streptavidin (HRP-Streptavidin) to prepare a HRP-Streptavidin-Bio-APT coupling substance.

[0055] 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, washed once, and patted dry; the prepared HRP-Streptavidin-Bio-APT 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; 2 mol / L H2SO4 solution was added 100 μL / well 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 (NC).

[0056] The results are as follows Figure 4As shown, the nucleic acid aptamers APT-2, APT-4, APT-5, APT-6, APT-7 and APT-8 described in the present application all have good reactivity with the African swine fever virus protein A104R, but have no cross-reaction or weak reactivity with the p30 protein.

[0057] The above-described embodiments only describe the basic principles, main features and preferred embodiments of the present invention. Without departing from the scope of the present invention, various improvements to the technical solutions of the present invention should fall within the scope of protection of the claims of the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to the A104R protein of African swine fever virus, characterized in that: The sequence of the nucleic acid aptamer is shown in any one of SEQ ID No. 1-6.

2. The nucleic acid aptamer according to claim 1, characterized in that The sequence of the nucleic acid aptamer is chemically modified.

3. The nucleic acid aptamer 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 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.

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

10. Use of the nucleic acid aptamer 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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