A nucleic acid aptamer capable of specifically binding to fpv- vp2 protein

Abstract

The application provides a nucleic acid aptamer capable of specifically combining with FPV-VP2 protein, and the sequence is 5'-ATCGTCTGCTCCGTCCAATA-(N)45-TTTGGTGTGAGGTCGTGC-3'; wherein the sequence of (N)45 is any one of SEQ ID NO:1-4. The nucleic acid aptamer screened by the application has excellent specificity and affinity for the FPV-VP2 protein, and provides important technical support for further research on FPV detection technology.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology, and in particular relates to a nucleic acid aptamer that can specifically bind to the FPV-VP2 protein. Background Technology

[0002] Feline panleukopenia virus (FPV) was first discovered in the 1920s. This virus is primarily transmitted orally and nasally, and infected animals can also shed the virus in their feces. FPV can infect domestic cats and wild animals such as felines, raccoons, and mustelids, with young animals being more susceptible, posing a significant threat to both wild animals and pet cats. The main clinical symptoms caused by this virus are vomiting, diarrhea, loose stools, a pronounced biphasic fever, and a significant decrease in white blood cells in cats. FPV belongs to the Parvoviridae family and the Parvovirus genus, and is a single-stranded DNA virus mainly composed of two proteins: structural proteins (VP1 and VP2) and non-structural proteins (NS1 and NS2). The VP2 protein is the most important component of the parvovirus capsid; changes in the amino acids at certain key sites in this protein can alter the antigenic characteristics and cytotropy of the parvovirus, and also change the pathogenesis of parvovirus. Virus-like particles composed of the VP2 protein can be used to prepare candidate vaccines for a variety of viruses and can effectively induce humoral and cellular immunity. They are often used as diagnostic antigens.

[0003] Nucleic acid aptamers (or simply aptamers) are single-stranded DNA or RNA fragments that can specifically bind to a variety of target substances, obtained through systematic evolution of ligands by exponential enrichments (SELEX) technology. Compared with antibodies, nucleic acid aptamers have advantages such as a wide range of target substances they can bind to (viruses, bacteria, cells, proteins, peptides, heavy metal ions, etc.), low immunogenicity, good stability, low cost, rapid and simple preparation, easy labeling, and small molecular weight. Therefore, nucleic acid aptamers have broad application prospects in biomolecular detection, disease diagnosis and treatment, and drug residue detection. Aptamers will become a powerful tool in the field of biological and chemical molecular detection and disease treatment.

[0004] Existing methods for detecting FPV suffer from various drawbacks, including high cost, low specificity and sensitivity, and difficulty in commercialization and mass production. In particular, some antibody-based detection methods are limited in clinical application due to the instability of antibodies and the complex and time-consuming preparation process. Nucleic acid aptamers are superior to antibodies in many aspects and can overcome the above-mentioned shortcomings. Summary of the Invention

[0005] The purpose of this invention is to provide a nucleic acid aptamer that can specifically bind to the FPV-VP2 protein, which can be used to detect feline panleukopenia virus (FPV), thereby overcoming the shortcomings of the prior art.

[0006] The nucleic acid aptamer that specifically binds to the FPV-VP2 protein provided by the present invention has the sequence 5′-ATCGTCTGCTCCGTCCAATA-(N)45-TTTGGTGTGAGGTCGTGC-3′; wherein the sequence of (N)45 is any one of SEQ ID NO:1-4.

[0007] Furthermore, the nucleic acid aptamer is coupled with a fluorescent group, a quenching group, biotin, nanomaterials, a thiol group, an amino group, or an enzyme;

[0008] The nucleic acid aptamer can also be conjugated with drugs used to prevent and treat feline panleukopenia virus.

[0009] Furthermore, the present invention provides the application of the aforementioned nucleic acid aptamer in the preparation of anti-FPV drugs.

[0010] Furthermore, the present invention provides a reagent containing the aforementioned nucleic acid aptamer.

[0011] This invention screened and obtained nucleic acid aptamers that can specifically bind to the FPV-VP2 protein, with their core sequences being SEQ ID NO: 1-4. The nucleic acid aptamers screened in this invention exhibit excellent specificity and affinity for the FPV-VP2 protein, providing important technical support for further research on FPV detection technology. Attached Figure Description

[0012] Figure 1 : Secondary structure diagram of nucleic acid aptamers, where a, b, c and d are the secondary structures of VP2-FPV-1, VP2-FPV-10, VP2-FPV-13 and VP2-FPV-46, respectively;

[0013] Figure 2 Nucleic acid aptamer specificity identification diagram;

[0014] Figure 3 Nucleic acid aptamer affinity identification diagram;

[0015] Figure 4 : Identification of the binding ability of nucleic acid aptamers to different recombinant proteins;

[0016] Figure 5 : Sandwich ELISA assay for aptamer sensitivity identification. Detailed Implementation

[0017] Unless otherwise specified, the experimental methods in the embodiments of this invention are conventional methods; unless otherwise specified, the materials used in the embodiments are conventional biochemical reagents that can be purchased from the market.

[0018] The specific sources of the experimental materials in the examples are as follows:

[0019] The protein screening kit (mainly including: carboxyl magnetic beads, real-time PCR Mix, regular PCR Mix, ePCR microdroplet generating oil, micro-nucleic acid dialysis membrane, etc.) was purchased from Onpu Topmay Biotechnology Co., Ltd.

[0020] The FPV-VP2 protein gene was synthesized by Shanghai Sangon Biotech Co., Ltd., and recombinant FPV-VP2, FPV-VP1, FPV-NS1 and FPV-NS2 proteins with His tag were obtained by prokaryotic expression at the Qingdao National Animal Health Products Engineering Center.

[0021] Mouse anti-FPV-VP2 protein monoclonal antibodies (6D5 and 10B10) were preserved by the Qingdao National Animal Health Products Engineering Center;

[0022] His protein, 1 mL Ni-NTA chromatography column was purchased from Shanghai Sangon Biotech Co., Ltd.;

[0023] Nitrocellulose membrane (NC membrane) was purchased from HRP-Streptavidin, and TMB colorimetric solution and TMB colorimetric termination solution were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0024] The ECL luminescence kit was purchased from Affinity Biosciences, and ethyl [3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich.

[0025] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0026] Example 1: Screening for specific nucleic acid aptamers for FPV-VP2 protein

[0027] 1.1 Chromatographic purification of FPV-VP2 protein

[0028] The FPV-VP2 protein was coupled to Ni-NTA media using the histidine (His) tag on the recombinant protein. The Ni-NTA media immobilized the recombinant FPV-VP2 protein can then be used for subsequent aptamer screening.

[0029] The specific steps for immobilizing FPV-VP2 protein in Ni-NTA medium are as follows: Take 1 mL of FPV-VP2 protein and thaw it at 4°C. Use a 1 mL Ni-NTA chromatography column, discard the preservation solution, and wash the Ni-NTA chromatography column with 5-10 mL of ddH2O to remove residual solution. Load the thawed recombinant FPV-VP2 protein into the chromatography column containing Ni-NTA medium. Incubate at 25°C on a shaker at 180 rpm for approximately 4 hours.

[0030] 1.2 Screening of FPV-VP2 protein aptamers

[0031] Using stringent screening conditions yields nucleic acid aptamers with high specificity and high affinity. During the screening process, the incubation time between ssDNA and recombinant protein is progressively reduced, the FPV-VP2 protein concentration is decreased, the number of washes is increased, and negative screening steps are implemented.

[0032] The SELEX scheme for screening nucleic acid aptamers in this embodiment is shown in Table 1.

[0033] Table 1: SELEX Screening Protocol for FPV-VP2 Protein Aptamers

[0034] Filtering order Recombinant protein Number of washes after incubation Negative screening 1～3 20μg 3 + 4～6 17.5μg 4 - 7～10 15μg 5 + 11～14 12.5μg 6 - 15～20 10μg 7 + 20～30 10μg 8 +

[0035] Note: (+) indicates that a negative filtering step is added at the beginning of the filtering process; (-) indicates that a negative filtering step is not added at the beginning of the filtering process.

[0036] 1.3 Negative screening of nucleic acid aptamers

[0037] Because nucleic acid aptamer libraries contain extremely abundant nucleic acid sequences, including sequences capable of binding to Ni-NTA mediators, a negative selection process is required before binding recombinant proteins to the nucleic acid aptamer library. This process involves incubating the library with a blank control Ni-NTA medium to remove some nucleic acid ligands that are affinityless for Ni-NTA mediators, thereby improving the efficiency of target molecule screening and the specificity of nucleic acid aptamers.

[0038] The steps for negative filtering are as follows:

[0039] ① cDNA single-stranded library preparation: Mix 25 μL of aptamer library (150 μM) with 200 μL of coupling buffer. Heat at 98 °C for 10 min and then cool at 4 °C for later use.

[0040] ② Washing of Ni-NTA media: Using a pre-assembled Ni-NTA media chromatography column, discard the storage solution. Wash the Ni-NTA media with 5-10 volumes of ddH2O to remove residual solution.

[0041] ③ Column loading of single-stranded libraries: Load the aptamer library onto the Ni-NTA medium, adding 2 mL of coupling buffer and stirring with a glass rod until the Ni-NTA medium is completely suspended. Shake at 120 rpm for 2 hours at room temperature.

[0042] ④ Collection of starting library for screening: Collect the flow-through from the chromatography column, and wash the Ni-NTA medium with 2 mL of coupling buffer as described in Table 1.1, continuously collecting the flow-through. This flow-through is then used as the starting library for screening.

[0043] 1.4 Forward screening of nucleic acid aptamers

[0044] Because of the His tag on the FPV-VP2 protein, the recombinant protein can be coupled to Ni-NTA medium. The recombinant FPV-VP2 protein is immobilized in Ni-NTA medium, thus facilitating subsequent aptamer screening.

[0045] The forward screening steps for nucleic acid aptamers are as follows:

[0046] ① Take the assembled chromatography column and set it aside. Tighten the bottom outlet of the chromatography column, load the recombinant protein-Ni-NTA complex into the chromatography column, open the outlet, and wash the chromatography column with coupling buffer as per Table 1.1. Discard the buffer.

[0047] ② For the first round of screening, 25 μL (150 μM) of the primary nucleic acid aptamer library (synthesized by Shanghai Bioengineering Co., Ltd.) was directly incubated with Ni-NTA medium. In other screening steps, the prepared secondary nucleic acid aptamer library and conjugate were mixed thoroughly and incubated at room temperature for 1 hour.

[0048] ③ Removal of non-specific nucleic acid aptamers: Wash the chromatography column with coupling buffer as required in Table 1.1 to remove non-specifically bound single-stranded libraries.

[0049] ④ Construction of secondary nucleic acid aptamer library: Elute the single-stranded DNA-FPV-VP2 protein complex using 3-4 column volumes of elution buffer (50mM EDTA, 10mM PBS solution), and simultaneously recover the ssDNA from the collected solution:

[0050] a. Add all the collected solution to a final volume of 20% isopropanol to increase the recovery rate of ssDNA. After mixing, load the liquid onto the adsorption column in multiple portions, centrifuge at 12000 rpm for 1 min, and then discard the waste liquid.

[0051] b. To effectively remove impurities such as proteins from the sample, add 700 μL of 80% anhydrous ethanol to the collection tube for rinsing, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat the washing once.

[0052] c. Centrifuge at 12000 rpm for 2 minutes to remove residual liquid. Place at room temperature for 10-15 minutes to air dry.

[0053] d. Collect ss DNA. Transfer the adsorption column to a clean EP tube, add 30–80 μL of preheated sterile ddH2O, incubate at room temperature for 10 min, then centrifuge at 12,000 rpm for 2 min to collect the single-stranded library of nucleic acid aptamers. (The filtrate can be added back to the adsorption column and centrifuged again to improve the recovery rate of ss DNA.)

[0054] ⑤ PCR amplification was performed to obtain a secondary selection library. The recovered solution was labeled, and PCR was performed using the collected ss DNA as a template to prepare the secondary selection library. The PCR reaction system is shown below, with the upstream primer being 5'-CCTATGCGTGCTACCGTGAA-3' and the downstream primer being 5'-TTCAGCACTCCACGCATAGC-3'.

[0055] Table 2: Single-stranded DNA library amplification system

[0056] reagents Volume (μL) Premix Taq 10 Upstream primer (100m M) 1 Downstream primer (100 mM) 1 cDNA 3 <![CDATA[DdH2O]]> 5 Total volume 20

[0057] After preparing the 20 μL system, the PCR reaction program was as follows: 94℃, 5 min; 94℃, 30 s, 55℃, 30 s, 72℃, 20 s, 35 cycles; 72℃, 5 min; 4℃ to terminate the reaction.

[0058] The PCR-amplified library was recovered using agarose gel electrophoresis to purify the library fragments. The recovered solution was heated in a water bath at 95°C for 10 min, and then completely cooled at 4°C. This yielded ssDNA, which was used for the next stage of library screening.

[0059] ⑥ Repeat the above steps for a total of 30 rounds of screening. Add the Ni-NTA of the conjugated FPV-VP2 protein to the chromatography column. First, wash the conjugation complex multiple times with buffer, then add 10 μL of the secondary library prepared in the previous round for binding. Thereafter, add a negative screening step every 3 rounds until the 20th round. By reducing the concentration of the recombinant protein and increasing the number of elutions, highly specific and high-affinity nucleic acid aptamers can be obtained. Detailed screening system is shown in Table 1.

[0060] ⑦ After completing 30 rounds of SELEX screening, the obtained PCR amplification products were subjected to agarose gel electrophoresis and recovered. The recovered ssDNA was then used for subsequent cloning and sequencing.

[0061] Example 2: Sequencing of FPV-VP2 protein aptamers

[0062] The preparation steps for E. coli TOP10 competent cells are as follows:

[0063] ① TOP10 bacterial cell activation: Streak frozen Escherichia coli TOP10 strain onto LB agar plates and incubate overnight at 37°C.

[0064] ②TOP10 expansion culture: Select healthy TOP10 single colonies and incubate them in 5 mL of LB liquid medium at 37°C with shaking until OD. 600 The inoculum concentration is approximately 0.5. Inoculate the bacterial culture into 500 mL of LB liquid medium at a 1:100 ratio and incubate until the OD value reaches [value missing]. 600 It is around 0.6.

[0065] ③ TOP10 bacterial cell collection: Transfer the bacterial solution to a sterile EP tube and centrifuge at 4℃ and 5000rpm for 10min.

[0066] ④ Discard the culture medium, invert the EP tube to remove any remaining culture medium.

[0067] ⑤ Resuspend the bacterial cells: Add pre-cooled 0.1M CaCl2 solution.

[0068] ⑥Recover the bacterial cells: Centrifuge at 4000 rpm for 10 min at 4℃, then invert the EP tube to remove the culture medium.

[0069] ⑦ Preparation of competent cells: Resuspend the cells in pre-cooled 0.1M CaCl2 solution, and mix with glycerol solution. Aliquot the cells into centrifuge tubes and store at -80℃ for later use.

[0070] Table 3: Construction System of pMD-18T Nucleic Acid Aptamer Library Vector

[0071] reagents volume cDNA recovered from gel 1μg pMD-18T 3μL Solution I 1μL <![CDATA[ddH2O]]> Bring the volume to 10 μL

[0072] The steps of cloning and sequencing are as follows:

[0073] Prepare the 10 μL system described above and incubate overnight at 16°C. After nucleic acid electrophoresis, transform the pMD-18T-aptamer library vector into the host TOP10.

[0074] Vector transformation: ① Take competent TOP10 cells and thaw them on ice. ② Add the ligated mixture to 50 μL of competent TOP10 cells and incubate on ice for 30 min. ③ Incubate at 42℃ for 90 s, then incubate on ice for another 10 min. ④ Centrifuge at 12000 rpm for 1 min and discard the supernatant. Add 1 mL of fresh LB medium and gently pipette the cells. ⑤ Incubate at 37℃ on a shaker at 180 rpm for 1 h. ⑥ Centrifuge at 8000 rpm for 1 min and discard 800 μL of supernatant. ⑦ Resuspend the cells in 200 μL of medium and spread them on LB agar plates containing ampicillin. Incubate the spread plates at 37℃ overnight, inverted.

[0075] Bacterial sequencing: Single colonies were picked and incubated in 1 mL of LB broth containing ampicillin at 37°C and 180 rpm for 2 h. Simultaneously, bacterial PCR amplification was performed, and PCR-positive single colonies were selected for sequencing. The samples were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results were compared and analyzed.

[0076] After 30 rounds of screening, the obtained 30th round library was cloned and sequenced to obtain the full-length nucleic acid sequence in the library.

[0077] In this invention, 48 positive monoclonal clones were selected for sequencing analysis. A large number of repetitive sequences were found among the 48 sequences, and these repetitive sequences were enriched during the SELEX screening process. Cloning and sequencing yielded four non-repetitive nucleic acid aptamer sequences (Table 4), among which VP2-FPV-1 and VP2-FPV-10 sequences appeared most frequently.

[0078] Table 4: Information on first-generation sequencing of nucleic acid aptamers

[0079]

[0080] Example 3: Prediction of the secondary structure of nucleic acid aptamers

[0081] Bioinformatics analysis was performed using Clustal X software. Sequence alignment analysis was conducted on the sequences obtained in Table 4. The secondary structures of the screened nucleic acid aptamers were predicted and analyzed using online software (http: / / unafold.rna.albany.edu / ?q=mfold, conditions set to 25℃, sodium ion concentration 145mM, magnesium ion concentration 1mM). The secondary structures of the four most frequently occurring nucleic acid aptamers were predicted sequentially using the online Mfold software. The results are shown below. Figure 1 As shown in the figure, the prediction results indicate that nucleic acid aptamers possess common stem-loop structures, frequently found in tRNAs as recognition structures. This suggests that stem-loop structures in the secondary structure of nucleic acid aptamers are the primary form in which they recognize target molecules.

[0082] A comparison of the Gibbs free energies of the four aptamer nucleotides revealed that aptamer VP2-FPV-10 had the lowest Gibbs free energy, indicating greater stability compared to the other aptamers. Observation of the secondary structure prediction diagrams for these four aptamers showed that VP2-FPV-1, VP2-FPV-10, and VP2-FPV-13 possessed similar structures, potentially recognizing the same recognition site on the target molecule. In contrast, VP2-FPV-46 exhibited significantly different secondary structures from other nucleic acid aptamers, suggesting it might recognize a different site on the target molecule.

[0083] Example 4: Specific screening of FPV-VP2 protein aptamers

[0084] The experimental steps for specific identification of FPV-VP2 protein aptamers are as follows:

[0085] ① Antigen coating: Coat FPV-VP2 protein, dissolve recombinant protein 0-25nM in 50mM carbonate coating buffer, add 100μL per well to a 96-well microplate, and incubate overnight at 4℃.

[0086] ② Blocking: On the second day, discard the coating solution, wash three times with PBST on a horizontal shaker, add 150 μL to each well, and block with 1% BSA for 1 h.

[0087] ③ Incubate with biotin-labeled nucleic acid aptamers: Discard the blocking solution, wash 3 times with PBST on a horizontal shaker, add 100 μL of PBS solution containing 20 nM aptamers; incubate at 37°C for 2 h.

[0088] ④ Incubation of streptavidin-labeled horseradish peroxide: Wash three times with PBST, add 100 μL of SM-HRP secondary antibody diluted 1:500 to 1:1000 to each well, and incubate at 37°C and 100 rpm for 1 h on a constant temperature shaker. After incubation, add 200 μL of PBST washing buffer to each well and shake at 37°C on a constant temperature shaker. Wash a total of 5 times.

[0089] ⑤ Color development: Add 100 μL of TMB substrate and develop color for 20 min in the dark. Then add 2 M H2SO4 to terminate the reaction and measure the absorbance at 450 nm. To reduce experimental error, three replicates were set up for each sample.

[0090] ELISA was used to measure the absorbance of different FPV-VP2 protein concentrations and different concentrations of aptamers VP2-FPV-1, VP2-FPV-10, VP2-FPV-13, and VP2-FPV-46 at a wavelength of 450 nm. The results are as follows: Figure 2As shown, the absorbance of VP2-FPV-13 and VP2-FPV-46 with the target molecule was significantly higher than that of aptamers VP2-FPV-1 and VP2-FPV-10, indicating that these two aptamers have a high affinity for the recombinant FPV-VP2 protein, and the affinity of VP2-FPV-46 is higher than that of VP2-FPV-13.

[0091] Example 5: Detection of FPV-VP2 protein aptamer affinity

[0092] The binding of target molecules to nucleic acid aptamers can be considered a chemical dynamic equilibrium. Nucleic acid aptamers and target molecules continuously bind and dissociate until the rates of binding and dissociation reach equilibrium. The amounts of nucleic acid aptamers, target molecules, and the complex remain in balance. The affinity of nucleic acid aptamers can be characterized using the dissociation constant. Based on the ELONA method, a quantitative amount of target molecules is immobilized in enzyme-labeled wells and incubated with nucleic acid aptamers. Measuring the dissociation constant of the binding between the nucleic acid aptamer and the target molecule can be used to characterize the affinity of the nucleic acid aptamer. The experimental steps are as follows:

[0093] ① Coating: Dissolve the recombinant protein in 50mM carbonate coating buffer to a final concentration of 10μg / mL, add 100μL of protein solution to a 96-well microplate, and incubate overnight at 4℃.

[0094] ② Blocking: On the second day, discard the coating solution, wash three times with PBST on a horizontal shaker, add 150 μL to each well, and block with 1% BSA for 1 h.

[0095] ③ Incubate with biotin-labeled nucleic acid aptamers: Discard the blocking solution, wash 3 times with PBST on a horizontal shaker, add 100 μL of PBS solution containing 1, 5, 10, 15, and 20 nM aptamers; incubate at 37°C for 2 h.

[0096] ④ Incubation of streptavidin-labeled horseradish peroxide: Wash three times with PBST, add 100 μL of SM-HRP secondary antibody diluted 1:500 to 1:1000 to each well, and incubate at 37°C and 100 rpm for 1 h on a constant temperature shaker. After incubation, add 200 μL of PBST washing buffer to each well and shake at 37°C on a constant temperature shaker. Repeat the washing process three times.

[0097] ⑤ Color development: Add 100 μL of TMB substrate and develop color in the dark for 20 min, then add 2 M H2SO4 to terminate the reaction. Measure the absorbance at 450 nm. To reduce experimental error, set up 3 replicates for each sample.

[0098] KD values ​​were measured using the ELONA method. With a fixed concentration of recombinant FPV-VP2 protein, absorbance was measured at 450 nm for different concentrations of aptamers VP2-FPV-1, VP2-FPV-10, VP2-FPV-13, and VP2-FPV-46. OD values ​​were plotted on the x-axis as aptamer concentration. 450 A nonlinear fitting curve was established using the ordinate as the vertical axis, and the KD value was calculated. The results are as follows: Figure 3 As shown, the dissociation constants (KD) between VP2-FPV-13 and VP2-FPV-46 and the target molecule are both in the nanomolar range. The dissociation constant of VP2-FPV-13 is 5.455 nM, and the dissociation constant of VP2-FPV-46 is 3.456 nM. This indicates that these two aptamers have a high affinity for the recombinant FPV-VP2 protein, and the affinity of VP2-FPV-46 is higher than that of VP2-FPV-13.

[0099] Example 6: Gel retardation verification of the specific binding of nucleic acid aptamers to recombinant proteins

[0100] Electrophoretic mobility shift assay (EMSA), also known as gel retardation assay, is a commonly used affinity electrophoresis technique used to study protein-DNA or RNA interactions. To verify whether an interaction exists between the recombinant protein and the nucleic acid aptamer, this invention utilizes EMSA technology to verify the interaction between the nucleic acid aptamer and the recombinant FPV-VP2 protein.

[0101] 20 μg of recombinant FPV-VP2 protein was incubated with 20 μM nucleic acid aptamers VP2-FPV-13 and VP2-FPV-46 in 20 mM phosphate buffer (pH 7.0) for 3 h. A 5% TAE-PAGE gel was prepared as shown in Table 5, and after solidification, electrophoresis was performed in 1×TAE solution. The samples incubated with the nucleic acid aptamers and recombinant FPV-VP2 protein were mixed with the non-denaturing loading buffer and loaded onto the gel. Electrophoresis was performed at 118 V for 30 min. After electrophoresis, the gel was stained with SYBR Green I for 30 min, and the results were observed using a gel imaging system.

[0102] Table 5: Formulation of 5% TAE-PAGE Gel

[0103] reagents volume 10×TAE solution 2.0mL 40% Acrylamide 2.5mL <![CDATA[ddH20]]> 15.5mL APS 0.05g TEMED 12μL

[0104] Using BSA as a blank control, the specificity of two nucleic acid aptamers, VP2-FPV-13 and VP2-FPV-46, was specifically identified using the ELONA method. Negative controls included recombinant FPV-VP1, FPV-NS1, and FPV-NS2, all of which were proteins expressed in prokaryotes in the laboratory and all carried a His tag. All four nucleic acid aptamers obtained from sequencing were tested. Results are as follows... Figure 4 As shown, BSA, FPV-VP1, FPV-NS1, and FPV-NS2 showed no specific recognition. Since FPV-VP1, FPV-NS1, and FPV-NS2 all contain a His tag, this indicates that the nucleic acid aptamers do not specifically recognize the His tag. The nucleic acid aptamers VP2-FPV-13 and VP2-FPV-46 specifically recognize the recombinant FPV-VP2 protein.

[0105] Example 7: Sandwich ELISA Method for Determining FPV-VP2 Protein Based on Nucleic Acid Aptamers

[0106] (1) The concentration of the purified mouse anti-FPV-VP2 protein monoclonal antibody (10B10) was determined by NanoDrop2000, diluted to 4 μg / mL and coated into 96-well plates, 100 μL per well, incubated at 37℃ for 1 h, and washed 3 times with PBST for 3 min each time.

[0107] (2) Discard the washing solution and add 100 μL of blocking solution to each well, and incubate at 37°C for 1 h;

[0108] (3) FPV-VP2 protein was diluted with diluent to 10 μg / mL, 7.5 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, 0.5 μg / mL and 0.25 μg / mL and added to the sealed wells, 100 μL per well. PBS control wells were also set up. The mixture was incubated at 37℃ for 1 h and washed 3 times with PBST for 3 min each time.

[0109] (4) Dilute the biotin-labeled aptamer VP2-FPV-46 to 100 nM, denature at 95℃ for 5-8 min, and then quickly incubate on ice for 15 min. Add the treated aptamer to the test wells, 100 μL per well. Add mouse anti-FPV-VP2 monoclonal antibody (6D5) diluted 1:400 to the positive control wells. Feline calicivirus (FCV), feline herpesvirus (FHV), canine parvovirus (CPV) and canine distemper virus (CDV) are used as negative controls. Cell supernatant and PBS are used as blank controls. Incubate at 37℃ for 1 h, and wash 3 times with PBST for 3 min each time.

[0110] (5) Add HRP-labeled streptavidin to the biotin-labeled aptamer test wells and add HRP-labeled goat anti-mouse IgG to the control wells incubated with mouse anti-FPV-VP2 monoclonal (6D5). All were diluted 1:1000 times, 100 μL per well, and incubated at 37°C for 30 min. Wash three times with PBST for 3 min each time.

[0111] Add 100 μL of TMB two-component colorimetric solution to each well, incubate in the dark for 15 min, and then measure the OD. 450 The result is as follows Figure 5 As shown, a positive result is defined as an OD value ≥ negative control OD value × 2.1. Therefore, the minimum detectable amount of FPV-VP2 protein by aptamer VP2-FPV-46 is 0.25 μg / mL, which is better than the minimum detectable amount of mouse anti-FPV-VP2 monoclonal antibody (10B10) which is 0.5 μg / mL.

[0112] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention. sequence list <110> Qingdao National Engineering Research Center for Animal Health Co., Ltd. Anhui Gongjieshan Agricultural Technology Co., Ltd. <120> A nucleic acid aptamer that specifically binds to the FPV-VP2 protein <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <400> 1 gagtaatctg cctcaatgat taagtatgta cacaccccag ctcgg 45 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <400> 2 ccggtcgaat acgtggtaga gcatgtaaaa tcaccagcca gctcg 45 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 ccaagactgt taagttccta aagtcaggta caagccatgc acgac 45 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <400> 4 cgatcgaact gtaacgggtc agaggaaagt acacggcaag cacga 45

Claims

1. A nucleic acid aptamer, characterized in that, The sequence of the nucleic acid aptamer is shown in SEQ ID NO:3 or SEQ ID NO:

4.

2. The use of the nucleic acid aptamer according to claim 1 in the preparation of a reagent for detecting feline panleukopenia virus.

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