Nucleic acid aptamer specifically binding to african swine fever virus a104r protein and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a nucleic acid aptamer specifically combined with African swine fever virus A104R protein and application thereof. The application uses the system evolution of ligand by exponential enrichment (SELEX) to screen 6 nucleic acid aptamers capable of being combined with A104R protein from a random nucleic acid library by taking African swine fever virus A104R protein as a target, and the sequences of the nucleic acid aptamers are shown as SEQ ID No. 1-6. The nucleic acid aptamer has good affinity and reactivity with African swine fever virus protein A104R, and 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] This invention belongs to the field of biomedicine or biotechnology, specifically relating to a nucleic acid aptamer that specifically binds to the A104R protein of African swine fever virus and its application. Background Technology

[0002] African swine fever (ASF) is an acute, febrile, and highly contagious disease of pigs caused by the African swine fever virus (ASFV). ASFV has 24 genotypes, which can be classified into highly virulent, moderately virulent, and low-virulence strains based on viral virulence. Infection with highly virulent strains typically results in a 100% mortality rate in pigs, seriously threatening the pig industry in my country and globally. The clinical symptoms caused by ASFV infection are diverse and similar to some other swine diseases, making differential diagnosis difficult clinically. Currently, there are no safe and effective vaccines or treatments for ASF; prevention and control mainly rely on rapid and accurate laboratory testing and strict eradication measures.

[0003] Nucleic acid aptamers are functional oligonucleotides with high specificity and affinity for target molecules, obtained through SELEX screening. They are mostly 15-60 bases in length and, due to their binding affinity to target substances comparable to antibodies, are also known as "chemical antibodies." Nucleic acid aptamers possess advantages such as low immunogenicity, simple preparation, good penetration, and ease of modification, and have broad application prospects in biomedical fields such as target molecule detection, disease diagnosis, imaging, drug delivery, and targeted therapy.

[0004] The A104R protein is a structural protein of African swine fever virus (ASFV), located in the viral nucleus. It is a DNA-binding protein involved in viral transcription, helper genome packaging, and is also an important target protein for diagnostic and antiviral research. Therefore, screening nucleic acid aptamers that can specifically recognize and bind to the A104R protein of ASFV is of great value and significance for research on the diagnosis, treatment, and pathogenesis of ASFV. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a nucleic acid aptamer that specifically binds to the African swine fever virus A104R protein and its applications. Specifically, it includes the following:

[0006] In a first aspect, the present invention provides a nucleic acid aptamer targeting the A104R protein of African swine fever virus, wherein the sequence of the nucleic acid aptamer is 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 includes at least one of phosphorylation, methylation, amination, thiolation, fluorination, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopization.

[0009] Secondly, the present invention provides the application of the nucleic acid aptamer 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 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 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 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, luminescent nanomaterials, 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 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 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 the eleventh aspect, the present invention provides a reagent for detecting African swine fever virus A104R protein, the reagent containing 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 generating the nucleic acid aptamer 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 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 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 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

[0031] Figure 1 The results of Western blot identification of recombinant A104R protein are shown in the figure, where M is the protein marker, 1 is the negative control, and 2 is the purified A104R protein.

[0032] Figure 2 The coupling effect of A104R protein with Ni-NTA magnetic beads was identified by SDS-PAGE, 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 screening using SELEX technology are shown. M represents the molecular weight standard, and 1-6 represent the amplification products.

[0034] Figure 4 To identify the reactivity of nucleic acid aptamers with African swine fever virus protein A104R using ELISA. Detailed Implementation

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

[0036] Specifically, this invention utilizes SELEX technology to screen random oligonucleotide libraries, thereby obtaining nucleic acid aptamers capable of recognizing the African swine fever virus A104R protein. Then, nucleic acid aptamers with high affinity and specificity for the African swine fever virus A104R protein are identified using methods such as ELISA.

[0037] The nucleic acid aptamers that bind to the African swine fever virus A104R protein are represented as 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: Induction and purification of recombinant African swine fever virus A104R protein

[0039] The recombinant plasmid pET-30a-A104R, which was correctly identified by sequencing, was transformed into BL21(DE3) competent cells. The bacterial culture was inoculated 1:100 into liquid LB medium (Kan+) and incubated at 37°C with shaking at 200 rpm. OD 600 When the protein concentration reaches 0.6–0.8, 0.5 mmol / L IPTG is added, and expression is induced at 37°C for 8 hours. The induced bacterial culture is centrifuged at 10,000 rpm for 10 minutes, the supernatant is discarded, and the precipitate is vortexed with PBS and washed three times. The washed precipitate is sonicated, centrifuged at 10,000 rpm for 30 minutes, and the precipitate and supernatant are collected separately. A104R protein is purified using nickel column affinity chromatography. The sonicated supernatant is added to a treated nickel column, and the column is mixed at room temperature for 4 hours. The bound liquid is collected. Impurities are eluted with different concentrations of imidazole buffer, and finally, the target protein is eluted with 300 mmol / L imidazole buffer to obtain the purified A104R protein.

[0040] The purified A104R protein was quantified and analyzed using Western blotting. The identification results are as follows: Figure 1 As shown.

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

[0042] The recombinant ASFV A104R protein with a His tag 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 specific procedure was as follows: 1 mL of ASFV A104R protein was taken and allowed to thaw completely at 4°C. The Ni-NTA magnetic beads were thoroughly mixed, and 1 mL of the bead suspension was placed in a centrifuge tube. The centrifuge tube was placed on a magnetic separator, and after the solution became clear, the supernatant was discarded. The solution was repeatedly washed with deionized water to remove any residual preservation solution. The thawed A104R recombinant protein was transferred to magnetic beads containing binding buffer and incubated overnight at 4°C with gentle shaking at 200 rpm. The magnetically conjugated A104R protein (Mag-A104R) was analyzed using SDS-PAGE to verify successful conjugation.

[0043] The results are as follows Figure 2 As shown, the A104R recombinant protein was successfully coupled to 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 is 5'-ATCCAG AGT GAC GCA GCA-(N)40-TGG ACA CGG TGG CTT AGT-3', where (N)40 represents 40 bases randomly composed of A, T, G, and C. The library was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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'), and the amplification results were analyzed by agarose gel electrophoresis. Figure 3 As shown, the amplified products were purified by gel extraction and sent to Sangon Biotech (Shanghai) Co., Ltd. for high-throughput sequencing.

[0052] Analyzing the sequencing results, sequences with high enrichment frequencies were selected for synthesis. The ssDNA sequences that can specifically recognize and bind to the African swine fever virus A104R protein were identified by ELISA. These are the selected nucleic acid aptamers, and the 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.

[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 to prepare a coupling compound of HRP-Streptavidin-Bio-APT.

[0055] 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 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. 100 μL / well of the prepared HRP-Streptavidin-Bio-APT 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 determined. Meanwhile, 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 this application all exhibit good reactivity with the African swine fever virus protein A104R, while showing no cross-reactivity or weak reactivity with the p30 protein.

[0057] The embodiments described above only illustrate the basic principles, main features, and preferred embodiments of the present invention. Any improvements made to the technical solutions of the present invention without departing from the scope of the present invention should be protected within the scope 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 of SEQ ID No. 1-6.

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 according to claim 1; the substance for labeling, detection, and diagnosis is attached to the 5' end and / or 3' end of the nucleic acid aptamer; the substance for labeling, detection, and diagnosis is selected from at least one of the following: fluorescent markers, radioactive substances, biotin, digoxigenin, and nanoluminescent materials.

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 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.

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