CN118006616A - Nucleic acid aptamer specifically combined with porcine pseudorabies virus gE protein - Google Patents

Abstract

The invention provides a nucleic acid aptamer specifically binding to porcine pseudorabies virus gE protein, which has a nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2. the invention provides application of a nucleic acid aptamer or a derivative thereof in preparation of a product for detecting pseudorabies virus PRV. The invention screens and obtains the nucleic acid aptamer capable of combining with gE protein of PRV virus, and the provided nucleic acid aptamer has specific affinity to gE protein of PRV virus, thus providing effective products for detection and prevention of PRV virus.

Description

Nucleic acid aptamer specifically combined with porcine pseudorabies virus gE protein

Technical Field

The invention belongs to the field of molecular immunity, and particularly relates to a nucleic acid aptamer specifically binding to porcine pseudorabies virus gE protein.

Background

Porcine pseudorabies (Pseudorabies, PR), also known as Aujeszky's disease, is an acute infectious disease caused by porcine pseudorabies virus (Pseudorabies Virus, PRV). Wherein, the pig is a natural host and a reservoir of PRV virus, PRV can cause abortion, dead fetus, mummy fetus, male pig sterility, high fever and neurological symptoms of piglets, and seriously infected pigs and even die in failure. Moreover, PRV virus infection is easy to form latent infection, so that infected pigs carry toxin and expel toxin for a long time; the virus is mainly excreted from nasal secretions, saliva, milk and urine of sick pigs.

Since the first discovery of the PRV virus in the united states in 1902, porcine pseudorabies caused by the virus has been widely prevalent worldwide, with an economic loss of billions of dollars each year caused by the disease becoming one of the major infectious diseases that seriously jeopardizes the healthy development of the pig industry.

Studies have shown that pseudorabies virus (PRV) is a double stranded DNA virus of the herpesviridae family, and mature PRV virus is in the form of oval or spherical particles of 150-180 nm. PRV co-encodes more than 70 proteins, wherein the gE protein is the main virulence factor of PRV, and deletion of gE gene can significantly reduce PRV virus virulence, but the immunogenicity is hardly affected. Commercial PRV attenuated vaccines and inactivated vaccines with deletion of gE gene are widely used in various countries. After PRV vaccine immunization, antibodies against gE are not detected in animals, but gE genes exist and are expressed in all wild strains, and gE antibodies can be detected in animals infected with the wild strains. Therefore, how to detect the gE protein becomes a key for identifying wild virus infection and vaccine immunity.

Disclosure of Invention

The invention aims to provide a nucleic acid aptamer capable of specifically binding porcine pseudorabies virus gE protein, which can specifically bind PRV gE protein, so that the nucleic acid aptamer can be applied to the field of PRV detection or prevention and treatment.

One aspect of the invention provides a nucleic acid aptamer specifically binding to porcine pseudorabies virus gE protein, which has a nucleotide sequence of 5'-GGTTATTGCCGAGCGGGGGTGCAAGCGTCAGGTGTGTAAA-3' (SEQ ID NO: 1);

in another aspect, the nucleic acid aptamer has a nucleotide sequence 5'-GGGGTTGGCATTTCAGTACTTGCATGTGTGGGTTAAGTAG-3' (SEQ ID NO: 2);

in another aspect, the invention also provides a nucleic acid aptamer derivative, wherein the nucleic acid aptamer derivative is modified at the 5 'or 3' segment of the nucleic acid aptamer.

The modification is performed by using biotin, digoxin, fluorescent substances, amino groups, amino acids and derivatives thereof, vitamins, nano luminescent materials, enzymes or colloidal gold.

In a further aspect, the invention also provides the use of the aptamer or derivative thereof in the preparation of a preparation for detecting pseudorabies virus PRV.

In a further aspect, the invention also provides application of the aptamer or the derivative thereof in preparing a medicament for preventing or treating rabies virus PRV.

In another aspect, the present invention provides a reagent or kit for detecting PRV, which comprises the aptamer or the derivative thereof.

The invention screens and obtains the nucleic acid aptamer capable of combining with gE protein of PRV virus, and the provided nucleic acid aptamer has specific affinity to gE protein of PRV virus, thus providing effective products for detection and prevention of PRV virus.

Drawings

FIG. 1, plots of ssDNA library binding rates for each round,

Fig. 2: the secondary structure diagram of the aptamer, wherein the secondary structure diagrams of PRV-gE-37, PRV-gE-6, PRV-gE-28 and PRV-gE-22 are respectively in a clockwise order,

Fig. 3: the aptamer affinity assay result in example 4 of the present invention,

Fig. 4: the invention discloses a result diagram of detecting the specificity of the aptamer,

FIG. 5 is a graph showing the result of detection of sensitivity of the aptamer of the invention.

Detailed Description

The nucleic acid aptamer, also called aptamer or aptamer, is an oligonucleotide (DNA or RNA) fragment which is screened by an in vitro screening technology and can specifically bind to target substances such as metal ions, polypeptides, proteins, viruses, cells and the like, the length of the oligonucleotide fragment is generally 60-100 nt, under proper conditions, the aptamer single-stranded DNA or RNA molecule can be adaptively folded to form structures such as hairpin, bulge loop, tetrad and the like, and sites which can specifically bind to the target molecules are formed through self conformational changes and three-dimensional folding. Compared with the traditional monoclonal antibody, the aptamer has similar affinity and specificity, and has the advantages not possessed by the antibody, such as rapid and simple preparation, easy labeling, wide target molecule range, low immunogenicity, stable chemical property, small molecular weight and the like, so that the aptamer has wide application prospect and rapid development trend in basic research, clinical diagnosis and treatment.

The present invention will be further described with reference to the following examples and drawings, but it should be noted that the scope of the present invention is not limited to the above description, and similar substitutions and modifications will be made by those skilled in the art without departing from the principles of the embodiments of the present invention. The experimental methods in this example were all carried out according to the conventional methods unless otherwise specified, and the reagents involved in the examples were all conventional reagents or were prepared according to the conventional methods unless otherwise specified.

The initial aptamer library in the following examples was synthesized by the division of biological engineering (Shanghai) and recombinant proteins PRV-gE, PRV-gB, PRV-gC and PRV-gD were obtained by the Qingdao national institute of England engineering and technology using baculovirus expression systems, and both of them had histidine (His) tags, and two mouse anti-PRV gE protein monoclonal antibodies (6G 7 and 10B 10) were screened by the Qingdao national institute of England engineering and technology.

Example 1: screening of PRV gE protein-specific nucleic acid aptamers

1. Screening of nucleic acid aptamers

In this embodiment, a microplate method is used to screen specific nucleic acid aptamers of PRV gE protein, target molecules are bound to a microplate through physical adsorption, then an aptamer library is added for incubation, unbound aptamers are discarded, and finally the aptamers bound to the target molecules are eluted, wherein the specific screening steps are as follows:

① Pretreatment of oligonucleotide library: mu.L (100. Mu.M) of the initial aptamer library was taken and mixed well in 100. Mu.L of PBS buffer. Immediately after that, the oligonucleotide was renatured by ice bath for 15 minutes to restore the three-dimensional space structure.

② Coating a target antigen: the recombinant PRV gE protein was diluted with carbonate coating buffer to coating concentration, 100. Mu.L of PRV gE protein solution was pipetted into a 96-well ELISA microplate and coated 2 wells (screening wells) in total. Meanwhile, in order to prevent the non-specific binding of the aptamer with the microplate and the BSA in the screening process, adding a reverse screening process in the screening process, coating two blank control holes (reverse screening holes) only added with coating liquid, and incubating for 1h at 37 ℃;

③ Closing: the coating solution was discarded, washed three times with PBST solution (PBS solution containing 0.05% Tween 20), and 200. Mu.L of BSA solution (PBS solution of 3% BSA) was added to the screening well and the counter-screen well, and incubated at 37℃for 2 hours;

④ Adding an aptamer library for incubation: the blocking solution was discarded and washed three times with PBST solution. The pretreated initial oligonucleotide library was added to the blocked counter-screen wells and incubated at 37℃for 1h, after which unbound ssDNA was transferred to the screening wells and incubation continued at 37℃for a progressively decreasing incubation time according to the increase in screening rounds as shown in Table 1.

Table 1: PRV gE protein nucleic acid aptamer screening protocol table

⑤ Eluting: the oligonucleotide library was discarded and washed four times with 300. Mu. LPBST solution. 100. Mu.L of the nucleic acid eluent was added to the microwells and eluted at 80℃for 10min.

⑥ Extracting nucleic acid aptamer: collecting the eluent in ELISA microwells, extracting the aptamer in the eluent by phenol extraction, and phenol: chloroform: the ratio of isoamyl alcohol was 25:24:1, and the aptamer candidates were recovered using Bioteke short fragment DNA recovery kit.

2. Preparation of ssDNA secondary library

And (3) performing conventional PCR by using the eluted and recovered nucleic acid aptamer as a template, amplifying to obtain double chains, and performing asymmetric PCR by using the double chains as the template, namely performing PCR amplification by adding unequal amounts of upstream and downstream primers, wherein when one end primer is consumed in the amplification process, single-chain DNA (ssDNA) is amplified under the guidance of the remaining other end primer. The ssDNA obtained was used as a secondary library for the next round of screening. High purity dsDNA and ssDNA were prepared by preferred PCR conditions, upstream primer F:

5′-ATGGACCTGACGACGCAGCTC-3'，

The downstream primer R:5'-ACGTGAACCGACCGTGTCCAG-3', the preferred PCR annealing temperature is 65 ℃, the number of reaction cycles is 15, the ratio F of the upstream primer to the downstream primer of the asymmetric PCR is R=100:1, and the number of reaction cycles is 35. The PCR reaction system is shown in Table 2.

Table 2: DNA library amplification System Table

Reagent(s)	Conventional PCR volume (μL)	Asymmetric PCR volume (μL)
			2×Premix Taq	25	25
Upstream primer	1	1
			Downstream primer	1	0.01
cDNA	3	5
			DdH2O	Supplement and fill to 50	Supplement and fill to 50

The 50 μl system configuration was completed, and the conventional PCR reaction procedure was: 95 ℃ for 5min;95 ℃,30s,65 ℃,30s,72 ℃,30s,15 cycles; 72 ℃ for 5min; the reaction was terminated at 4 ℃. The asymmetric PCR reaction procedure is 95 ℃ for 5min;95 ℃,30s,65 ℃,30s,72 ℃,30s,35 cycles; 72 ℃ for 5min; the reaction was terminated at 4 ℃.

In order to obtain a purer secondary library, the obtained product is subjected to agarose gel electrophoresis after the asymmetric PCR amplification is finished, and is recovered by a Bioteke short-fragment DNA gel recovery kit, so that ssDNA is obtained, and the ssDNA can be used in the next screening.

The above procedure was repeated for a total of 12 rounds of screening. The aptamer screening conditions generally range from wide pine to stringent, and the early screening conditions are too stringent to easily cause loss of part of the aptamer sequence, and stringent screening conditions are suitably used in the later stages of screening in order to obtain a high-specificity and high-affinity aptamer. In the screening process, the PRV gE target protein concentration was gradually reduced, the washing times were appropriately increased, and the incubation time of ssDNA and PRV gE protein was gradually shortened, and the overall protocol for screening PRV gE protein nucleic acid aptamers was as shown in Table 1.

2. Each round of ssDNA library binding rate determination

Taking ssDNA library after each round of screening to carry out binding rate measurement, and detecting the screening effect of each round through the binding rate condition, wherein the specific operation steps are as follows:

① The total amount of eluted ssDNA was calculated by measuring the content of eluted ssDNA after each round of screening by Nano Drop2000, and multiplying the total volume of elution.

② The total amount of ssDNA dosed for each round of screening was calculated according to the method of step ①.

③ The total amount of ssDNA obtained by elution was divided by the total amount of ssDNA added for each round, and the ssDNA binding rate for each round of screening was calculated, and the results are shown in FIG. 1. With the change of screening conditions and the increase of the number of screening rounds, the binding rate of the screened ssDNA library is continuously increased, and the increase after 10 rounds of screening is gradually flattened, so that the sequence enrichment degree is not greatly increased with the increase of the number of screening rounds.

Example 2: sequencing of PRV gE protein nucleic acid aptamer

1. Ligation transformation:

after the 12 th round of screening was completed, the resulting library was amplified into double-stranded DNA by PCR, agarose gel electrophoresis and cut gel recovery, and the resulting library was recovered and cloned into pMD19-T Vector. A connection system was prepared as shown in Table 3, and the mixture was homogenized and connected at 16℃for 4 hours.

Table 3: construction System Table of pMD 19-T-nucleic acid aptamer library vector

Composition of the components	Volume of
		Round 12 dsDNA	0.3pmoL
pMD19-T Vector	1μL
		Solution I	1μL
ddH2O	Supplement to 10 mu L

Adding 5 μL of the ligation product into 50 μL of competent DH5 alpha, ice-bathing for 30min, transferring into a water bath at 42 ℃ for heat shock for 90s, ice-bathing for 2min, adding 900 μL of LB without antibiotics, placing into a constant temperature shaking table at 37 ℃, culturing for 30min at 200r/min, centrifuging for 5min at 6000r/min, discarding 800 μL of supernatant, and coating 100 μL of resuspension thalli on an LB solid culture plate containing ampicillin, and placing into an incubator at 37 ℃ for inversion culture for 12h.

2. Culturing, identifying and sequencing transformant enrichment:

And (3) selecting monoclonal colonies for PCR identification, selecting positive clones for amplification culture, extracting plasmids according to a description of a small extraction kit of the root plasmids, randomly selecting 42 positive clones, and sequencing by a division company of biological engineering (Shanghai). 42 positive monoclonals are picked for sequencing, 28 positive monoclonals are successfully sequenced, DNAMAN 8 is utilized for comparing and analyzing the sequencing success results, 4 nucleic acid aptamer sequences with higher occurrence frequency are obtained, the fact that the repeated sequences are enriched to the greatest extent in the screening process is shown, the occurrence frequency of PRV-gE-37, PRV-gE-6, PRV-gE-22 and PRV-gE-28 is highest, the repeated occurrence frequency is 16, 9, 8 and 6 times respectively, and the statistical results are shown in table 4.

Table 4: aptamer sequencing results table

The aptamer is capable of folding to form a unique spatial structure, thereby specifically recognizing the target molecule. The secondary structure of the four selected nucleic acid aptamers was predicted using online nucleic acid structure prediction software Mfold (http:// unafild. Rna. Albany. Edu/. The secondary structure predictions show that the nucleic acid aptamers PRV-gE-37, PRV-gE-6, PRV-gE-22 and PRV-gE-28 all have stable neck-loop or hairpin structures, comparing the Gibbs free energies of the four aptamer nucleotides, with the Gibbs free energy of the aptamer PRV-gE-37 being lowest and more stable than the other aptamers.

Example 3: PRV gE protein aptamer affinity detection

Binding of the aptamer to the target molecule is a dynamic equilibrium process, and the strength of affinity between the aptamer and the target molecule is often characterized by a dissociation constant, KD, value. The specific experimental procedure of the present embodiment is as follows, based on the ELONA method, coating a target molecule with a fixed concentration in an elisa plate, adding a nucleic acid aptamer for incubation, and evaluating the affinity of the nucleic acid aptamer according to its dissociation constant with the target protein:

① Coating a target antigen: diluting the recombinant PRV gE protein with carbonate coating buffer solution to a concentration of 10 mug/mL, sucking 100 mu L of PRV gE protein solution, adding the solution into a 96-well ELISA microplate, and incubating for 1h at 37 ℃;

② Closing: the coating was discarded, washed three times with PBST solution (PBS solution containing 0.05% Tween 20), 200. Mu.L BSA solution (PBS solution of 3% BSA) was added to each well, and incubated at 37℃for 2h;

③ Adding a nucleic acid aptamer: the blocking solution was discarded and washed three times with 200. Mu.L of PBST solution, 100. Mu.L of 1nM, 5nM, 10nM, 15nM, 20nM biotin-labeled aptamer solution was added and incubated at 37℃for 1h.

④ Adding streptavidin-labeled horseradish peroxidase: the incubated aptamer solution was discarded, washed three times with PBST, 100. Mu.L of 1:1000 diluted SA-HRP secondary antibody was added to the wells and incubated for 1h in a constant temperature shaker at 37 ℃.

⑤ Color development: the SA-HRP solution was discarded, the well plate was washed four times with PBST, the washing solution remaining in the well was carefully photographed, 100. Mu. LTMB was aspirated and added to the microwells, and developed for 10min at 37℃in the absence of light.

⑥ Terminating the color development: the color development was stopped by adding 50. Mu.L of stop solution 2M H ₂SO₄ solution per well. The absorbance at 450nm was measured.

KD values were calculated based on the ELONA method, wherein the coated recombinant PRV gE protein was fixed, and the aptamers PRV-gE-37, PRV-gE-6, PRV-gE-22 and PRV-gE-28 were set to different concentration gradients. The result is shown in FIG. 3 below, calculated as KD, with the concentration of aptamer on the abscissa and the measured absorbance OD ₄₅₀ on the ordinate, where the dissociation constant KD between PRV-gE-37 and the target molecule is highest, PRV-gE-6 times, where the dissociation equilibrium constant KD between PRV-g-37 and the target molecule is 5.427nM; the dissociation equilibrium constant KD between PRV-gE-6 and the target molecule is 3.854nM. The results show that PRV-gE-37 and PRV-gE-6 in the four aptamers have higher affinity with the recombinant PRV gE protein.

Example 4: specific detection of PRV gE protein nucleic acid aptamer

According to the result of the affinity assay in example 3 above, PRV-gE-37 and PRV-gE-6 among the four nucleic acid aptamers showed strong affinity to PRV gE protein, so that evaluation of both aptamers was continued. In order to verify whether the aptamer has specificity to recombinant PRV gE protein, the ELONA method was used to identify the specificity of two nucleic acid aptamers PRV-gE-37 and PRV-gE-6. The negative control proteins PRV-gB, PRV-gC and PRV-gD used in the experiments are expressed by the laboratory and are all provided with his labels. Two nucleic acid-adapted PRV-gE-37 and PRV-gE-6 with better affinity are selected, PRV-gE, PRV-gB, PRV-gC and PRV-gD are coated on a micro-porous plate at 4 ℃ overnight, 3% BSA is blocked, a BSA blank is set, a biotin-labeled aptamer (5 pmol/. Mu.L) is used as a primary antibody, HRP-labeled streptavidin (1:5 000 dilution) is used as a secondary antibody, TMB is developed and terminated, and an OD450 value is measured by an enzyme-labeling instrument.

The specific detection of the nucleic acid aptamers PRV-gE-37 and PRV-gE-6 was performed using the ELONA method. His-tagged recombinant PRV-gB, PRV-gC and PRV-gD proteins were used as negative controls and BSA as blank controls. As shown in FIG. 4, the detection results show that BSA, PRV-gB, PRV-gC and PRV-gD have no specific recognition with the aptamer, and the PRV-gB, PRV-gC and PRV-gD all contain His tags, which indicates that the aptamer has no specific recognition with respect to the His tags, and the aptamer PRV-gE-37 and PRV-gE-6 can specifically recognize recombinant PRV-gE proteins.

Example 5: gel blocking to verify binding of aptamer to recombinant protein

Gel Retardation (Gel recovery), also known as electrophoretic mobility assay (Electrophoretic Mobility SHIFT ASSAY, EMSA), is a commonly used technique of affinity electrophoresis for studying protein interactions with DNA or RNA. Experiments typically radiolabeled DNA fragments are incubated with specific proteins, and the products are then electrophoretically analyzed in a non-denaturing gel. The mobility of the protein-DNA complex is reduced compared to the free DNA, so that a "blocking" of the electrophoretic band of the protein-DNA complex is observed.

To further investigate whether the aptamer interacted with recombinant PRV gE protein, the binding of the aptamer to PRV gE protein was investigated using gel blocking experiments. Mu.g of recombinant PRV-gE was mixed with 20. Mu.M aptamer PRVgE-37, PRVgE-6, respectively, and incubated for 3h to prepare 5% TAE-PAGE gels (gel formulations including 10 XTAE solution 2mL,40% acrylamide 2.5mL,APS 0.05g,TEMED 12. Mu.L, ddH ₂ 0.5 mL). And (3) uniformly mixing the non-denatured loading buffer with the incubated compound, loading the mixture into a sample, and carrying out electrophoresis for 35min at 120V. After electrophoresis, the gel was stained with SYBR Green I for 30min, and observed in a gel imaging system, a significant mobility change was observed in the gel electrophoresis, indicating that the oligonucleotide ssDNA forms a stable complex with PRV-gE-37.

Example 6: establishment of PRV-gE protein sandwich ELISA method based on aptamer determination

① Coating: diluting a mouse anti-PRV gE protein monoclonal antibody (6G 7) to a concentration of 5 mug/mL by using a carbonate coating solution, sucking 100 mug of the solution, adding the solution into a 96-well ELISA microplate, and incubating for 1h at 37 ℃;

② Closing: the coating was discarded, washed three times with PBST solution (PBS solution containing 0.05% Tween 20), 200. Mu.L BSA solution (PBS solution of 3% BSA) was added to each well, and incubated at 37℃for 2h;

③ PRV gE protein addition: the blocking solution was discarded and washed three times with 200. Mu.L of PBST solution, PRV gE protein was diluted with PBS to 0.25. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 7.5. Mu.g/mL, 10. Mu.g/mL, 100. Mu.L was added per well, control wells were set up with PBS, and incubated for 1h at 37 ℃.

④ Adding a nucleic acid aptamer: biotin-labeled aptamer PRV-gE-37 (100 nM) was pretreated, denatured at 95℃for 10min, and then rapidly placed on ice for 15min. 100 μl of the pretreated aptamer was added to the experimental wells, a 1:400-fold dilution of murine anti-PRV gE monoclonal antibody (10B 10), PEDV, CSFV, PRRSV and PCV2 as negative controls, cell supernatants and PBS as blank controls were added to the positive control wells, incubated for 1h at 37deg.C, and PBST washed 3 times;

⑤ 100. Mu.L of 1:6000 diluted SA-HRP was added to the assay wells incubated with biotin-labeled aptamer, 1:1000-fold diluted HRP-labeled goat anti-mouse IgG antibody was added to control wells incubated with mouse anti-PRV gE monoclonal antibody (10B 10), 100. Mu.L of each well was incubated for 1h at 37℃and PBST was washed 3 times;

⑥ Color development: the incubated solution was discarded, the well plate was washed four times with PBST, the washing solution remaining in the well was carefully photographed, 100. Mu. LTMB of the color development solution was aspirated and added to the microwells, and developed at 37℃for 10min in the dark.

⑦ Terminating the color development: the color development was stopped by adding 50. Mu.L of stop solution (2M H ₂SO₄) per well. As a result of measuring the absorbance OD ₄₅₀ at 450nm, the lowest detection amount of the aptamer PRV-gE-37 against the PRV-gE protein was 0.3. Mu.g/mL, and the lowest detection amount of the aptamer PRV-gE-37 was 0.5. Mu.g/mL, which is superior to the murine anti-PRV-gE monoclonal antibody (10B 10).

It will be appreciated by persons skilled in the art that the above-described embodiments are merely illustrative of the preferred embodiments of the present invention and not intended to limit the scope of the invention, and that various changes and modifications may be made to the technical solution of the present invention without departing from the spirit and scope of the invention, which is defined in the claims.

Claims (8)

1. A nucleic acid aptamer, wherein the nucleotide sequence of the nucleic acid aptamer is SEQ ID NO:1.

2. The nucleic acid aptamer of claim 1, wherein the nucleotide sequence of the nucleic acid aptamer is SEQ ID NO:2.

3. A nucleic acid aptamer derivative, wherein the nucleic acid aptamer derivative is modified at the 5 'or 3' segment of the nucleic acid aptamer of claim 1 or 2.

4. The aptamer derivative according to claim 3, wherein the modification is performed using biotin, digoxin, a fluorescent substance, an amino group, an amino acid and a derivative thereof, a vitamin, a nano-luminescent material, an enzyme, or colloidal gold.

5. Use of a nucleic acid aptamer according to claim 1 or 2 or a derivative thereof for the preparation of a preparation for the detection of pseudorabies virus PRV.

6. The use of claim 5, wherein the article is a test kit.

7. Use of a nucleic acid aptamer or a derivative thereof according to claim 1 or 2 for the preparation of a medicament for the prophylaxis or treatment of rabies virus PRV.

8. A reagent or kit for detecting pseudorabies virus PRV, comprising the aptamer or derivative thereof according to claim 1 or 2.