CN107058329B

CN107058329B - Aptamer specifically binding to Newcastle disease virus and screening method and application thereof

Info

Publication number: CN107058329B
Application number: CN201610719675.0A
Authority: CN
Inventors: 任涛; 谭阳通; 谢鹏; 高潇祎; 王峰
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2016-08-24
Filing date: 2016-08-24
Publication date: 2020-06-19
Anticipated expiration: 2036-08-24
Also published as: CN107058329A

Abstract

The invention discloses an aptamer specifically binding to Newcastle disease virus, and a screening method and application thereof. The aptamer is a specific aptamer B53 obtained by screening by using a SELEX technology. Specifically, a random single-stranded DNA library and primers are constructed and synthesized in vitro, prokaryotic expression newcastle disease HN protein and newcastle disease virus GM strain are used as target molecules, and the single-stranded DNA library which is obtained by screening and is specifically combined with the newcastle disease virus GM strain is subjected to screening, PCR amplification, forward and reverse screening, and is subjected to clone sequencing to obtain a specific aptamer B53. The aptamer B53 has the ability of inhibiting virus replication, virus hemagglutination activity and plaque formation, can specifically recognize HN protein of Newcastle disease virus and GM strain of Newcastle disease virus, has no reactivity to avian influenza virus, infectious bursal disease virus and allantoic fluid of SPF chick embryo, and can be used for detecting Newcastle disease virus.

Description

Aptamer specifically binding to Newcastle disease virus and screening method and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to an aptamer specifically binding to Newcastle disease virus, and a screening method and application thereof.

Background

Newcastle Disease (ND) is a highly contagious, lethal and virulent infectious Disease caused by Newcastle Disease Virus (NDV), and is characterized mainly by lesions of the respiratory, digestive and central nervous systems after infection of birds. NDV belongs to the family of paramyxoviridae, is a single-stranded, nonsegmented, enveloped, negative-strand RNA virus, has a genome structure of 3 '-NP-P-M-F-HN-L-5', encodes 6 structural proteins, and currently has only one serotype. The disease is distributed worldwide, which causes great economic loss to the poultry industry. For most national poultry industry, the method not only causes huge economic loss due to the outbreak of Newcastle disease, but also has higher cost for controlling the disease. Immunization is an effective means for preventing newcastle disease, but requires repeated monitoring and is relatively expensive.

Nucleic acid aptamers, also known as aptamers or aptamers, as "chemical antibodies", are capable of specifically recognizing and binding to target molecules. In 1990, Tuerk obtained RNA capable of specifically binding to T4DNA polymerase by in vitro screening technology, and defined this technology as the exponential Enrichment of ligand phylogenetic technology (SELEX). In the same year, Ellington discovered RNA fragments that bind to small organic dyes and named aptamers (Aptamers). The aptamer is a single-stranded oligonucleotide with a specific three-dimensional conformation, the length of the oligonucleotide is generally 60-100 nt, and an oligonucleotide sequence capable of being combined with target molecules with high specificity and high affinity is obtained through multiple rounds of screening and separation from an artificially synthesized random nucleotide sequence library by an exponential enrichment ligand phylogenetic technology. Aptamers are based on their stable three-dimensional conformation and mainly comprise hairpin structures, stems, triplexes, pseudoknots and G-quartets. Through self conformation change and three-dimensional folding, a site capable of specifically binding to a target molecule is formed. Nucleic acid molecules play an important role in the biological processes of storing, transporting, processing and expressing genetic information, and aptamers broaden the conceptual understanding of nucleic acids as traditional genetic material due to the specific affinity recognition property of aptamers and target molecule molecules. The aptamer serving as a chemical antibody can be used as a function blocker to influence the processes of virus replication, translation and the like, so that the occurrence of diseases is prevented; in addition, aptamers can also replace the specific functions of antibodies for the detection of related viral diseases. In recent years, aptamers have been widely used as ideal recognition elements for detection of target molecules. The pattern of aptamer recognition of a target substance is similar to that of conventional antibody recognition, and nucleic acid aptamers have more advantages than conventional antibodies, as shown in table 1.

TABLE 1 comparison of characteristics of aptamers and antibodies

At present, various improved SELEX screening methods emerge endlessly, and no fixed standard exists so far. The library and target molecules to be screened are mixed in a certain ratio and appropriate conditions (medium, temperature, buffer system, time, etc. for incubation) are selected, although simple, different conditions will affect the efficiency of the screening and the affinity of the ligand. How to obtain an aptamer with higher specificity and affinity, remove a non-specific sequence and a sequence with poorer specificity, and improve the screening efficiency is a problem to be solved urgently.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the technical defects of the existing protein antibody and provide the nucleic acid aptamer which has high affinity with a target molecule, high specific binding, easy obtaining, no dependence on animal, cell and internal environment in the process, short preparation period, low cost, more stability than an antibody and good repeatability and is specifically bound with Newcastle disease virus.

Another object of the present invention is to provide a method for screening the above aptamer.

It is still another object of the present invention to provide the use of the above aptamer for detecting newcastle disease virus.

The above purpose of the invention is realized by the following technical scheme:

an aptamer specifically binding to Newcastle disease virus, wherein the sequence of the aptamer is shown as SEQ ID NO. 1.

The two end positions on the nucleotide sequence of the aptamer are aminated, carboxylated, sulfhydrylated or isotopically esterified.

The nucleotide sequence of the nucleic acid aptamer is combined with a fluorescent marker, biotin modification, a nano luminescent material or an enzyme marker.

The screening method of the aptamer specifically binding to the Newcastle disease virus comprises the following steps:

s1, synthesizing a random nucleic acid DNA library and an upstream primer for PCR amplification:

the sequence of the random nucleic acid library is shown as SEQ ID NO. 1;

the sequence of the upstream primer F is shown as SEQ ID NO. 2;

the sequence of the downstream primer R is shown as SEQ ID NO. 3;

the Biotin modified upstream primer Biotin-F is shown as SEQ ID NO. 4;

s2, screening of aptamers: incubating the random nucleic acid library and the target molecules in the step S1, and separating by using a nitrocellulose filter membrane to obtain oligonucleotides combined with the target molecules, namely the screened nucleic acid aptamer library specifically combined with the Newcastle disease virus;

s3, amplifying the nucleic acid aptamer library: performing PCR amplification on the oligonucleotide obtained in S2 by using the primer synthesized in S1 to obtain double-stranded DNA;

s4, preparing a single-stranded DNA library: preparing single-stranded DNA by using the double-stranded DNA obtained in the step S3 as a template, using the upstream primer and the downstream primer and using the double-stranded DNA obtained in the step S3 as a template, amplifying by using an asymmetric PCR method, and recovering an amplified single-stranded DNA library in an ethanol precipitation mode;

s5, repeated screening: replacing the random library in step S2 with the single-stranded DNA library in step S4, and repeating the above process of steps S2-S4 at least 5 times;

s6, reverse screening: incubating the single-stranded DNA library obtained by screening in the step S5 with non-target molecules for 30min at the temperature of 15-37 ℃, and separating by using a microplate to obtain oligonucleotides which are not combined with the target molecules;

s7, taking the oligonucleotide obtained in the step S6 as a template, repeating the steps S3-S4, and preparing a secondary library for the next round of screening;

s8, circulating screening: replacing the random nucleic acid library in S2 with the secondary library in S7, and repeating the steps S2-S7-10 times to obtain a single-stranded DNA library specifically binding to the GM strain of Newcastle disease virus; and (2) amplifying to obtain Biotin modified single-stranded DNA by taking Biotin-F and R as primers and a secondary library obtained by screening in each round as a template, determining the affinity of the single-stranded DNA library and a target molecule by an ELISA method until the affinity requirement is met, and performing clone sequencing analysis on the obtained product to finally obtain the aptamer.

Preferably, the target molecule in step S2 is newcastle disease HN protein or newcastle disease virus GM, and the non-target molecule in step S6 is avian influenza virus, SPF chick embryo allantoic fluid, or bovine serum albumin.

Preferably, the incubation time in the step S2 is 15-60 min; the incubation time in step S6 was 30 min.

Preferably, in the step S3, when preparing the double-stranded DNA, the PCR process conditions are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 7min, and amplification for 9-15 cycles.

Preferably, when the single-stranded DNA is prepared by asymmetric PCR in the step S4, the content ratio of the upstream primer to the downstream primer is 50-100: 1.

Preferably, in the step S4, when preparing the single-stranded DNA, the PCR process conditions are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 30s, and finally extension at 72 ℃ for 7min, and amplification is carried out for 30-40 cycles.

In addition, the application of the aptamer in detecting Newcastle disease virus is also within the protection scope of the invention. The aptamer B53 has the capability of inhibiting virus replication, hemagglutination activity and plaque formation, can specifically recognize HN protein and Newcastle disease virus GM strain, has no reactivity to avian influenza virus, infectious bursal disease virus and SPF chick embryo allantoic fluid, and can be used for detecting Newcastle disease virus.

The invention firstly synthesizes ssDNA library with the total length of 81nt in vitro, wherein two ends are fixed sequences for PCR amplification, and the middle is a random sequence of 40 nt. And analyzing the optimization result of the PCR condition by utilizing PAGE and denaturing PAGE, and further determining the annealing temperature, the cycle number and the asymmetric primer content of the PCR. Taking prokaryotic expression newcastle disease HN protein and newcastle disease virus GM strain as target molecules, screening by a nitrocellulose membrane filtration method and a reverse screening by a microplate method, and obtaining an aptamer specifically binding the HN protein and the newcastle disease virus GM strain through 10 rounds of repeated screening. And (3) carrying out primary structure analysis on the sequencing result by using DNAMAN software, and predicting the secondary structure by using MFold. The affinity and specificity of the aptamer were analyzed by ELISA and Dot blot assays, and aptamer biological activity was analyzed by hemagglutination inhibition assay and plaque inhibition assay. The result shows that the aptamer specifically binding to the Newcastle disease virus has the characteristics of high affinity, high specific binding, easy obtaining, independent animal, cell and internal environment in the process, short preparation period, low cost, more stability than an antibody and good repeatability and can be used for detecting the Newcastle disease virus.

Various improved SELEX screening methods currently mix the library and target molecule to be screened in a certain ratio and select appropriate conditions (medium, temperature, buffer system, time, etc. for incubation) that, although simple, affect the efficiency of the screening and the affinity of the ligand. The invention makes the following improvements on the basis of the cellulose nitrate membrane filtering method: the SELEX method of combining nitrocellulose filter membrane with a microporous plate is adopted, and different incubation media are alternately used, so that the aptamer with higher specificity and affinity can be obtained. Gradually adjusting the screening conditions, such as reducing the input amount of the target molecules and the secondary library, shortening the incubation time and the like, so as to obtain the oligonucleotide with stronger bonding force with the target molecules, which is more stable than an antibody and has good repeatability, and can effectively reduce the batch difference in the actual production. Proper reverse screening step is favorable for eliminating non-specific sequence and sequence with poor specificity, and the screening efficiency is improved.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention adopts a SELEX screening method combining a nitrocellulose filter membrane and a microporous plate, and obtains the aptamer B53 of the Newcastle disease virus with higher specificity and affinity by screening through alternately using different incubation media. The aptamer B53 has the capacity of inhibiting virus replication, hemagglutination inhibition activity and plaque formation, and can reduce 44.2% of GM strain virus plaques; can specifically recognize HN protein of Newcastle disease and GM strain of Newcastle disease virus, has no reactivity to avian influenza virus, infectious bursal disease virus and allantoic fluid of SPF chick embryo, and can be used for detecting Newcastle disease virus.

2. The invention is evolved through an in vitro screening process, and the process is independent of animals, cells and internal environment; the preparation period is short, the cost is low and the preparation method is easy to obtain; the screening conditions are gradually optimized by reducing the input amount of the target molecules and the secondary library, shortening the incubation time and the like, so as to obtain the oligonucleotide with stronger binding force with the target molecules. Compared with an antibody, the antibody is more stable and has good repeatability, and the difference among batches in actual production can be effectively reduced; meanwhile, the addition of a reverse screening process is beneficial to removing non-specific sequences and sequences with poor specificity, and the screening efficiency is improved. The aptamer which is obtained by screening and specifically binds to the Newcastle disease virus can provide a new idea for the detection and treatment of the Newcastle disease virus.

Drawings

FIG. 1 shows the primary and secondary structures of aptamer B53 obtained by SELEX screening, wherein A represents the primary structure of the aptamer and B represents the secondary structure of the aptamer.

FIG. 2 shows the affinity assay results of ssDNA libraries obtained from each round of screening.

FIG. 3 is the result of ELISA test of aptamer B53 obtained by SELEX screening, wherein A represents the affinity of the aptamer to different strains F48E9, GM and La Sota of Newcastle disease virus; b shows the affinity of the aptamers to different viruses.

FIG. 4 shows the Dot-blot detection result of aptamer B53 obtained by SELEX screening. Graph A shows the results of aptamers against different strains of Newcastle disease virus F48E9, GM and La Sota; b shows the results of aptamers against different viruses.

FIG. 5 shows the Dot-blot analysis results of aptamer B53 obtained by SELEX screening.

FIG. 6 shows the results of hemagglutination inhibition assay of aptamer B53 obtained by SELEX screening.

FIG. 7 shows the results of plaque inhibition assay for aptamer B53 obtained by SELEX screening.

Detailed Description

The following description and specific examples, taken in conjunction with the accompanying drawings, further illustrate the present invention and should not be construed as limiting the invention. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Example 1 in vitro screening of Newcastle disease Virus aptamers

S1, constructing and synthesizing a random nucleic acid library with the length of 81nt, wherein the sequence is shown as SEQ ID NO.1, and 5'-CCGGAATTCCTAATACGACTC- (N) 40-TATTGAAAACGCGGCCGCGG-3'; wherein, the two ends are respectively fixed sequences of 20 and 21 bases, and the middle 40 bases are random sequences.

The sequences of the upstream primer F and the downstream primer R synthesized for PCR amplification are as follows:

an upstream primer F: 5'-CCGGAATTCCTAATACGACTC-3', as shown in SEQ ID NO. 2;

a downstream primer R: 5'-CCGCGGCCGCGTTTTCAATA-3', as shown in SEQ ID NO. 3;

biotin modified upstream primer Biotin-F: 5'-Biotin-CCGGAATTCCTAATACGACTC-3' as shown in SEQ ID NO. 4.

S2. incubation and isolation of the ssDNA library with HN protein, 35.5. mu.g of the ssDNA library was added to 100. mu.L of binding buffer (50mM Tris-HCl, 25mM NaCl, 5mM MgCl2, 10mM DTT (dithiothreitol), pH 7.5), water bath at 95 ℃ for 10min, followed by rapid ice-cooling for 10 min. To reduce non-specific ssDNA binding to the membrane, the membrane was soaked with binding buffer prior to screening, the library was filtered 3 times on nitrocellulose membrane in a Pop-top filter, and the filtered ssDNA was used for the next screening.

S3, nitrocellulose membrane screening: loading the nitrocellulose membrane into a Pop-top filter, ensuring tight binding of the nitrocellulose membrane to the inner wall of the syringe, wetting the membrane with a binding buffer, adding the ssDNA library filtered in the previous step into the HN protein solution, and incubating at room temperature for 90min (the amount of ssDNA library per screening cycle and the amount of HN protein decrease with increasing number of screening cycles). And the nucleic acid and protein incubated solution was filtered through a filter and then washed with a binding buffer to wash away oligonucleotides that were not bound to the protein.

S4, reverse screening: and adding a reverse screening link during the 5 th screening, namely coating irrelevant target molecules (avian influenza virus, SPF chick embryo allantoic fluid and bovine serum albumin) in an ELISA plate and sealing the ELISA plate by using a BSA solution, firstly adding ssDNA and target molecule incubation solution into the ELISA plate, incubating at room temperature for 30min, then sucking the solution out, adding the solution into HN protein solution, and incubating at room temperature. The filter is used for filtration, and then the binding buffer is used for washing away the oligonucleotides which are not bound with the protein.

S5, recovering ssDNA by using an ethanol coprecipitator, amplifying a target band through PCR, and preparing the ssDNA through asymmetric PCR. The ssDNA obtained was used as a secondary library for the next round of screening.

S6, determining the affinity of the ssDNA secondary library obtained in each round, wherein the basic principle is that the eluted specific ssDNA sequence is amplified into double chains by a PCR method, the double chains are used as templates after agarose gel electrophoresis identification and recovery, a large amount of amplification is carried out by an asymmetric PCR method by using BioF and R with biotin labels as primers, and the synthesized secondary library is recovered by an ethanol precipitation method and is measured for later use. The affinity of the library to the target molecule was determined by ELISA using ssDNA labeled with biotin as the primary antibody and streptavidin labeled with HRP as the secondary antibody. The method comprises the following specific steps:

(1) coating antigen: coating a microplate with the HN protein of the newcastle disease virus, adding the same amount of coating solution into a blank control well, and standing overnight at 4 ℃;

(2) and (3) sealing: the liquid in the plate was discarded and rinsed with PBST. Adding 1% BSA blocking solution, and blocking in a 37 ℃ thermostat for 2 h;

(3) add Biotin-ssDNA library: the liquid in the plate was discarded and rinsed with PBST. Putting the processed sub-libraries into a micro-porous plate, wherein the input amount of each hole is 500pmoL, and incubating for 2h in a constant temperature box at 37 ℃;

(4) adding a secondary antibody: the liquid in the plate was discarded and rinsed with PBST. Adding HRP-labeled streptavidin diluted at a ratio of 1:5000 as a second antibody into a microplate, incubating for 1h at a constant temperature of 37 ℃ in a 100 mu L/hole manner;

(5) color reading value: the liquid in the plate was discarded and rinsed with PBST. After adding 100. mu.L/well TMB for color development, the same amount of stop buffer was added and OD was read₄₅₀The value is obtained. As a result, as shown in FIG. 2, it was observed that the positive ratio increased and the OD was increased with the increase in the number of screening rounds₄₅₀The increase is gradual, the increase is not obvious after the 9 th round, and the increase is not obviously different from the 10 th round, which indicates that the increase of the number of screening rounds can not improve the enrichment degree of the sequence any more, and the screening process is finished.

S7, cloning, identifying and sequencing the secondary library screened in the last round to obtain the aptamer sequence.

Example 2 enzyme-linked immunosorbent assay (ELISA)

The aptamer obtained by screening is sent to the company of Biotechnology engineering (Shanghai) GmbH for synthesis, biotin modification is carried out, and the specificity is determined by ELISA method. Different samples were selected with a coating of 2. mu.L/well and a blank control was set up. Coating overnight at 4 deg.C, sealing with 1% BSA at 37 deg.C, adding different biotin-labeled aptamers into the wells, incubating, adding horse radish peroxidase-labeled avidin, incubating, adding substrate, developing, stopping developing, and measuring OD with enzyme-labeling instrument₄₅₀The value is obtained.

FIG. 3 is an ELISA test of aptamer B53 obtained by SELEX screening, and A shows the affinity of the aptamer to different strains of Newcastle disease virus F48E9, GM and La Sota. As shown in fig. 3, the results indicate that aptamers B53 all had affinity for newcastle disease virus F48E9, GM, and La Sota strains, with highest affinity for GM; the B-plot shows the affinity of the aptamers to different viruses, and the results show that aptamers B53 all showed specific affinity to NDV GM strain and were non-reactive to AIV, IBDV and SPF chick embryos.

EXAMPLE 3 Dot hybridization assay (Dot-blot)

Dropping 2 μ L HN protein (1mg/mL), GM strain purified virus, AIV, IBV, SPF chick embryo allantoic fluid into NC membrane, and simultaneously setting up a blank control group without dropwise adding solution; after the NC membrane is dried, adding 3% BSA blocking solution, blocking for 2 hours at 37 ℃ by a shaking table, and washing the membrane by PBST solution; adding the biotin-labeled ssDNA with the concentration of 5 pmol/. mu.L to an NC membrane, incubating for 2h at 37 ℃ in a shaking table, and washing the membrane with a PBST solution; adding the NC membrane into a Streptavidin-HRP solution diluted by 1:5000, incubating for 1h at a constant temperature of 37 ℃ by using a shaking table, and washing the membrane for 3 times with a PBST solution, wherein each time lasts for 3 min; stopping the color development after 10min of DAB display, taking out the membrane and observing the color development condition of the membrane.

The result of aptamer specificity analysis is shown in fig. 4, fig. 4A shows the result of aptamer on different strains of newcastle disease virus, F48E9, GM and La Sota, and B53 can specifically bind to different strains of newcastle disease virus to generate spots with different gray scales. The GM strain has the strongest gray scale, which indicates that the affinity of the aptamer B53 to GM is stronger than that of F48E9 and La Sota; FIG. 4B shows the results for different viruses with aptamers, B53 was able to specifically bind HN protein and the GM strain of Newcastle disease virus, spots appeared with different degrees of lightness and darkness, and spots appeared lighter at NDV than at HN and no spots appeared at allantoic fluid of AIV, IBV and SPF chick embryos. The aptamer B53 has higher specificity. The results are consistent with the results of the ELISA. The affinity analysis results of the aptamer for newcastle disease virus and HN protein are shown in FIG. 5, wherein the left graph shows the identification results of the aptamer for HN protein with different concentrations, and the right graph shows the identification results of the aptamer for NDV with different concentrations. As the HN protein and GM concentrations decreased, the DAB-developed spots became darker, and when the HN protein concentration was less than 0.25mg/mL and the GM was less than 16HAU, no spots appeared after DAB development.

Example 4 hemagglutination inhibition assay

The aptamer is diluted to 50 pmol/mu L by using a binding buffer solution, heated in a water bath at 95 ℃ for 10min, immediately subjected to ice bath for 10min, and incubated with the treated aptamer and the Newcastle disease virus GM strain for 30min at room temperature. Firstly, taking a 96-hole micro-reaction plate, and adding 25 mu L PBS into each hole of 1-12 holes by using a micropipette; then 25 mul of the aptamer and virus mixed solution is sucked and added into the 1 st hole, and the mixture is blown and beaten evenly; then sucking 25 microliter of mixed solution from the 1 st hole, adding the mixed solution into the 2 nd hole, sucking 25 microliter of mixed solution after mixing, adding the mixed solution into the 3 rd hole, sequentially carrying out multiple dilution to the 11 th hole, and finally sucking 25 microliter of mixed solution from the 11 th hole and discarding the mixed solution, wherein the 12 th hole is a PBS blank control group; and finally, adding 25 mu L of chicken red blood cell suspension with the volume fraction of 1% into each hole, and standing at room temperature for 15min to observe the result. And simultaneously setting a virus control group, namely, using an equivalent amount of binding buffer to replace an aptamer to incubate with the Newcastle disease virus at room temperature.

As shown in FIG. 6, the selected dominant aptamers were subjected to hemagglutination inhibition assay with GM strain virus, and a virus control group was set. The results showed that the aptamers all had varying degrees of hemagglutination inhibition activity against the GM strain virus, with the hemagglutination titer of the virus control group being 11log2, the hemagglutination titer of the a 1-treated group being 9log2, the hemagglutination titer of the A3-treated group being 7log2, the hemagglutination titer of the a 20-treated group being 10log2, and the hemagglutination titers of the B7, B53 and B60-treated groups being 8log 2.

EXAMPLE 5 plaque inhibition assay

Spreading the secondary CEF on 6-well cell plate, sucking out culture medium after the cell grows to a monolayer, and culturing in each wellAdding 450 μ L of Newcastle disease virus GM strain virus diluent (diluted 10 times by DMEM medium and diluted 10 times)^-2、10^-3、10^-4、10^-5、10^-6) And an unvaccinated blank group was set, and 450. mu.L of DMEM was added. Incubate at 37 ℃ for 1h to aspirate the virus fluid and wash the cells 1 time with PBS. Covering each hole with 2mL of solid culture medium (DMEM/2% FBS + 2% low melting point agarose solution with the volume ratio of 1:1, culturing at 37 ℃ for 3d, adding 1mL of 0.1% neutral red solution into each hole, staining for 20min, removing the staining solution, culturing at 37 ℃ in a dark place for 1h, observing and counting the plaques by naked eyes, taking the virus dilution capable of clearly identifying the single plaques as the optimal dilution concentration, diluting the aptamer into 100 pmol/mu L by using a binding buffer solution, heating in a water bath at 95 ℃ for 10min, immediately carrying out ice bath for 10min, incubating the treated aptamer and the Newcastle disease virus GM strain at room temperature for 30min, diluting to the optimal dilution concentration by using DMEM, culturing at 37 ℃ for 1h, sucking out the virus solution, washing the cells for 1 time by PBS, covering each hole with 2mL of solid culture medium (DMEM/2% FBS + 2% low melting point agarose solution with the volume ratio of 1:1, culturing at 37 ℃ for 3d, adding 1mL of 0.1% neutral red solution into each well, staining for 20min, removing the staining solution, culturing at 37 deg.C in dark for 1-5h, and counting the plaques by visual observation, and setting an equal volume of binding buffer solution and a virus control group incubated with Newcastle disease virus GM at room temperature for 30 min.

As shown in fig. 7, the value 1 represents a blank set, no plaque; the number 2 indicates the number of plaques in the virus control group as 52; the number 3 indicates the aptamer B53 treatment group with a plaque number of 29. The result shows that the aptamer can reduce 44.2% of GM strain virus plaques and has strong virus plaque inhibition capacity.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations will be apparent to persons skilled in the art upon consideration of the foregoing description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An aptamer specifically binding to Newcastle disease virus, wherein the sequence of the aptamer is shown as SEQ ID No. 1.

2. The aptamer according to claim 1, wherein both end positions on the nucleotide sequence of the aptamer are aminated, carboxylated, thiolated, or isotopically substituted.

3. The aptamer according to claim 1, wherein the aptamer has a nucleotide sequence that is bound with a fluorescent label, biotin modification, a nano-luminescent material or an enzyme label.

4. Use of the nucleic acid aptamer according to any one of claims 1 to 3 in the preparation of a newcastle disease virus detection kit.