CN116970611A - Nucleic acid aptamer combined with monkey pox virus surface envelope protein and application thereof - Google Patents
Nucleic acid aptamer combined with monkey pox virus surface envelope protein and application thereof Download PDFInfo
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- CN116970611A CN116970611A CN202310061388.5A CN202310061388A CN116970611A CN 116970611 A CN116970611 A CN 116970611A CN 202310061388 A CN202310061388 A CN 202310061388A CN 116970611 A CN116970611 A CN 116970611A
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a nucleic acid aptamer for combining a monkey pox virus surface envelope protein and application thereof, belonging to the technical field of biology, and comprising at least one of nucleic acid aptamers HIM-A29-1, HIM-A29-2, HIM-A29-3, HIM-A29-4, HIM-A29-5 and HIM-A29-6. The aptamer has high specificity, small molecular weight, stable chemical property, easy preservation and marking, and can be used for biochemical analysis, environmental monitoring, basic science, new drug synthesis and the like. The use of the above-mentioned aptamer includes at least one of (1) to (4): (1) use in detecting a monkey poxvirus in an environment; (2) Use in the manufacture of a medicament for the treatment of monkey pox virus pneumonia; (3) Use in the preparation of a developing agent and/or a tracer for the detection of monkey poxviruses; (4) The application of the kit in preparing a reagent and/or test paper and/or a kit for detecting the monkey poxvirus.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nucleic acid aptamer combined with a monkey pox virus surface envelope protein and application thereof.
Background
The monkey poxvirus is a DNA virus first found in monkeys in 1958 and has similar symptoms of infection to smallpox. However, hosts for monkey poxviruses are not only monkeys, but also rodents, and even other animals, including humans. Recent data show that cases of infection with the monkey pox virus have been found in up to thirty countries, and the number of deaths is gradually increasing, and the risk of global outbreaks of the monkey pox virus is rapidly increasing, so that development of an efficient diagnosis method for the monkey pox virus is urgently needed.
Aptamer refers to a single-stranded DNA or RNA molecule with high specificity and high affinity to a target, which is isolated by exponential enrichment of ligand systematic evolution (SELEX) screening. The target of the aptamer is widely composed of proteins, metal ions, small molecules, polypeptides, even whole cells and the like, and has wide prospects in the research fields of biochemical analysis, environmental monitoring, basic science, new drug synthesis and the like. After renaturation, the aptamer can be folded to form various secondary structures, such as a stem-loop structure, a hairpin structure and the like, which are complementary with part of the space structure of the target, and then is specifically combined with the target under the action of hydrophobic action, hydrogen bond, electrostatic action, van der Waals force and other intermolecular forces. Nucleic acid aptamers have many commonalities with antibodies, such as being capable of specifically recognizing and binding to a certain protein, having good affinity and higher specificity to its target, but nucleic acid aptamers are more advantageous than antibodies in many aspects, mainly represented by the following points: (1) The aptamer is easier to synthesize and chemically modify, can be synthesized manually and chemically by a solid-phase synthesis method, has small batch-to-batch difference and low synthesis cost, can modify certain chemical groups during synthesis, enhances the stability of the aptamer, and can facilitate subsequent drug coupling and the like. In contrast, the synthesis of antibodies requires preparation of antigens, selection of immunized animals, cell fusion and expansion of cultures, etc., and a series of purification steps are required in the following, the whole procedure is very complex and time-consuming, and there are some differences between different batches of synthesized antibodies; (2) The stability of the aptamer is better, the aptamer is stored only in a refrigerator with the temperature of-20 ℃, and the storage time is long. The preservation condition of the antibody is severe, and the activity of the antibody is reduced after a few months of storage, so that the use of the antibody is affected; (3) the nucleic acid aptamer has lower immunogenicity than the antibody; (4) The target range of aptamer is wider, and can be selected from various small molecules, metal ions, polypeptides or proteins, nucleic acids, cells and the like, and the targets of antibodies are mostly proteins.
The basic principle of the ligand systematic evolution (SELEX) technology for in vitro exponential enrichment is that the interaction between an oligonucleotide library and a target molecule is utilized, a nucleic acid sequence capable of specifically binding the target molecule is screened and separated from a random single-stranded oligonucleotide library which is constructed in advance, and then the Polymerase Chain Reaction (PCR) in vitro amplification technology is utilized to enable the sequence with specificity in the library to be exponentially enriched. Therefore, screening of the aptamer with the envelope protein binding to the surface of the monkey pox virus is of great significance to diagnosis and treatment.
Disclosure of Invention
The invention aims to provide a highly specific, small-molecular-weight, stable-chemical-property and easy-to-store and label nucleic acid aptamer combined with a monkey pox virus surface envelope protein, which can be used for loading and targeting delivery of a medicament for treating monkey pox virus pneumonia, can be used as a developer or tracer of the monkey pox virus, and is used for preparing reagents and/or test paper and/or a kit for detecting monkey pox.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a nucleic acid aptamer that binds to a monkey poxvirus surface envelope protein, comprising at least one of the following (1) - (6):
(1) The aptamer HIM-A29-1 shown in SEQ ID No. 1;
(2) The aptamer HIM-A29-2 shown in SEQ ID No. 2;
(3) The aptamer HIM-A29-3 shown in SEQ ID No. 3;
(4) The aptamer HIM-A29-4 shown in SEQ ID No. 4;
(5) The aptamer HIM-A29-5 shown in SEQ ID No. 5;
(6) The aptamer HIM-A29-6 as shown in SEQ ID No. 6.
The aptamer has the advantages of high specificity, small molecular weight, stable chemical property, easy preservation and marking. The aptamer can be used for loading and targeting a drug for treating the pneumonia of the monkey pox virus, and can be used as a developer or tracer of the monkey pox virus to prepare a reagent and/or test paper and/or a kit for detecting the monkey pox.
In the aptamer, the affinity KD value of the aptamer is less than 40nM. Preferably, the aptamer has an affinity KD value of < 20nM. Further preferably, the aptamer has an affinity KD value of < 10nM.
The invention also provides a composition for screening the aptamer, which comprises,
-a solid support comprising magnetic beads and/or agarose gel beads; and
-a target molecule or a part of a target molecule bound to a solid support, the target molecule being the monkey poxvirus surface envelope protein a29 and/or DNA.
The composition can improve the screening efficiency, and can obtain the high-affinity aptamer only by 7 rounds of screening.
In the composition of the invention, the magnetic beads and the agarose gel beads can be directly, efficiently and quickly connected with target molecules.
In the composition, the magnetic beads comprise carboxyl-modified magnetic beads and/or histone-modified magnetic beads.
In the composition, the agarose gel beads comprise streptavidin-modified agarose gel beads.
The invention also discloses a preparation method of the composition, which comprises the following steps:
taking carboxyl magnetic beads, activating surface carboxyl groups, and then adding monkey pox virus surface envelope protein A29 for incubation; after incubation, sealing unreacted sites on the surface of the magnetic beads, and washing to obtain a composition; or,
taking agarose gel beads, adding the monkey pox virus surface envelope protein A29 for incubation, and washing after incubation to obtain the composition.
In this preparation method, activation of carboxyl groups was performed using N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).
The invention also provides a reaction system for screening the aptamer, which comprises,
-a single-stranded DNA library comprising at least one nucleotide chain capable of specifically recognizing a target molecule or a part of a target molecule, the target molecule being a monkey poxvirus surface envelope protein a29 and/or DNA;
-primers, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO. 8;
-the composition of claim 2;
-a buffer.
In the reaction system, the sequence of the random single-stranded DNA library is shown as SEQ ID NO. 9.
In this reaction system, the buffer includes a DPBS buffer.
The invention also provides a method for screening the aptamer, which comprises the following steps:
-synthesizing a single-stranded DNA library and primers;
-magnetic bead method and/or agarose gel bead method screening comprising formulating the reaction system of claim 3, forward and reverse screening, PCR amplification and single strand preparation.
In this method, a reaction system for screening for a nucleic acid aptamer is formulated, comprising preparing a composition for screening for a nucleic acid aptamer.
Preferably, a method of preparing a composition for screening for a nucleic acid aptamer comprises the steps of:
taking carboxyl magnetic beads, activating surface carboxyl groups, and then adding monkey pox virus surface envelope protein A29 for incubation; after incubation, sealing unreacted sites on the surface of the magnetic beads, and washing to obtain a composition; or,
taking agarose gel beads, adding the monkey pox virus surface envelope protein A29 for incubation, and washing after incubation to obtain the composition.
More preferably, the activation of the carboxyl group is performed using N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).
In this method, the positive screening includes performing the positive screening using the composition described above.
In this method, counter-screening comprises counter-screening using His magnetic beads and/or His sepharose beads.
In this method, the number of screening rounds by the magnetic bead method and/or the agarose gel bead method is at least 7.
The aptamer screened by the method has the advantages of high specificity, small molecular weight, stable chemical property, easy preservation and marking, and the affinity KD can reach below 10nM.
The invention also provides the use of the aptamer, which comprises at least one of (1) - (4):
(1) Use in detecting a monkey poxvirus in an environment;
(2) Use in the manufacture of a medicament for the treatment of monkey pox virus pneumonia;
(3) Use in the preparation of a developing agent and/or a tracer for the detection of monkey poxviruses;
(4) The application of the kit in preparing a reagent and/or test paper and/or a kit for detecting the monkey poxvirus.
The invention also provides a reagent for detecting the monkey poxvirus, which comprises the nucleic acid aptamer marked by the fluorescent dye.
In this reagent, the fluorescent dye-labeled aptamer is a FAM fluorescent dye-labeled aptamer.
The invention also provides a test paper for detecting monkey pox virus, which comprises,
-a nitrocellulose membrane comprising a detection line comprising antibodies to the monkey poxvirus a29 protein and a quality control line comprising antibodies to streptavidin;
-a binding pad comprising the above-mentioned nucleic acid aptamer;
-a sample pad; and
-absorbent paper.
The invention also provides application of the reagent or the test paper in detecting the monkey pox virus in the environment.
The invention also provides a method for detecting the monkey pox virus in the environment, which comprises the following steps:
sampling a region to be detected in the environment;
mixing and incubating the sample with the reagent;
after incubation of the mixture, the fluorescence intensity in the mixture was measured, and the result was determined.
The invention also provides a method for detecting the monkey pox virus in the environment, which comprises the following steps:
sampling a region to be detected in the environment;
mixing a sample with the aptamer to obtain a mixture;
detecting whether the mixture contains a monkey pox virus-aptamer complex.
In this method, the mixture is tested for the presence of a monkey pox virus-aptamer complex using the test strip described above.
The invention has the following beneficial effects: the invention uses the SELEX technology to screen out the aptamer capable of specifically combining with the monkey pox virus surface envelope protein A29, and has high affinity and KD value of below 10nM. In addition, the single-stranded DNA library designed by the invention can improve the screening efficiency, and only 7 rounds of screening are needed to obtain the high-affinity aptamer. The aptamer has the characteristics of small molecular weight, better stability, easy modification, no immunogenicity, short preparation period and the like, and can be used for biochemical analysis, environmental monitoring, basic science, new drug synthesis and the like. Therefore, the invention provides a highly specific, small-molecular-weight, stable-chemical-property and easy-to-store and label nucleic acid aptamer combined with the surface envelope protein of the monkey pox virus, which can be used for loading and targeting delivery of a medicament for treating the pneumonia of the monkey pox virus, can be used as a developer or tracer of the monkey pox virus, and is used for preparing reagents and/or test paper and/or a kit for detecting the monkey pox.
Drawings
FIG. 1 shows the binding of the library obtained by the different rounds of screening in example 1 to the target protein;
FIG. 2 is affinity detection data for aptamer HIM-A29-1;
FIG. 3 is affinity detection data for aptamer HIM-A29-2;
FIG. 4 is affinity detection data for aptamer HIM-A29-3;
FIG. 5 is affinity detection data for aptamer HIM-A29-4;
FIG. 6 is affinity detection data for aptamer HIM-A29-5;
FIG. 7 is affinity detection data for aptamer HIM-A29-6;
FIG. 8 shows the results of detection of the A29 protein of the monkey poxvirus.
Detailed Description
The present invention will be further described in detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1:
screening method of aptamer combined with monkey pox virus surface envelope protein
Random single stranded DNA libraries and primers shown in the following sequences were synthesized:
1. random single-stranded DNA library:
SEQ ID NO.9：5’-TCCAGCACTCCACGCATAAC（N36）GTTATGCGTGCTACCGTGAA-3’；
wherein "N36" represents a sequence of 36 arbitrary nucleotide bases joined together. The library was synthesized by the division of biological engineering (Shanghai).
The primer information is shown as SEQ ID NO.7 and SEQ ID NO.8, and is synthesized by Jin Weizhi Biotechnology Co.
SEQ ID NO.7：TCCAGCACTCCACGCATAAC；
SEQ ID NO.8：GTTATGCGTGCTACCGTGAA。
The primers were ddH 2 O was dissolved to prepare a 50. Mu.M stock solution, which was stored at-40℃until use.
2. Magnetic bead method screening
The magnetic bead method is adopted for screening, 7 rounds of screening are combined, and the specific screening method is as follows:
1. carboxyl magnetic bead immobilized monkey pox virus surface envelope protein A29
200 mu L of carboxyl magnetic beads are taken, washed with 200 mu L of ultrapure water for 4 times, and the magnetic beads are fished by a magnet, and the supernatant is removed. Respectively taking 100 mu L of prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), mixing in equal volume, adding into magnetic beads, incubating at room temperature for 20min to activate carboxyl groups on the surfaces of the magnetic beads, and then washing with a DPBS buffer solution for 1 time for later use;
taking 80 mu L of monkey pox virus surface envelope protein A29 (purchased from Yiqiao Shenzhou, 40891-V08E, concentration of 0.5 mg/mL), adding 260 mu L of 10mM sodium acetate solution with pH of 4.5, mixing well, and adding into the activated magnetic beads; placing the mixture on a vertical mixer for incubation for 2 hours at room temperature, wherein the envelope protein A29 on the surface of the monkey pox virus is coupled to the surface of the magnetic beads through the amino groups on the surface of the protein;
after the coupling, the coupling tube was placed on a magnetic rack, the supernatant was removed, the solution was washed 1 time with DPBS buffer, 200. Mu.L of 1M ethanolamine pH 8.5 was added to the beads, and incubated on a vertical mixer for 15 minutes at room temperature, blocking unreacted activation sites on the bead surface. Then, the mixture was placed on a magnetic rack, the blocking solution was aspirated, washed 3 times with 200. Mu.L of DPBS buffer, and finally dissolved in 200. Mu.L of DPBS buffer.
2. Reverse screening and screening
Preparation of reverse sieve magnetic beads: the His small peptide is coupled with magnetic beads, the His small peptide is synthesized by Hangzhou Dangang biotechnology limited company and is 9 continuous histidines, and the step of coupling the His small peptide is the same as the step of coupling the monkey pox virus surface envelope protein A29. His small peptide concentration is 20mg/mL, with pH 4.0 10mM NaAC solution dilution, specifically, 4 u L His small peptide, adding 396 u L pH 3.6 10mM NaAC solution mixing, adding 500 u L ethanol amine to close, finally dissolved in 500 u L DPBS, the other steps the same.
Library solubilization and variational treatment: taking a 1OD random single-stranded nucleotide library, centrifuging at 13000rpm for 2min, adding PBS buffer for dissolution, putting into a metal bath at 95 ℃ for denaturation for 10min, then putting into ice for cooling for 5min, and standing at room temperature for 15min to form a stable conformation.
Incubation of library with magnetic beads: taking a certain amount of His magnetic beads, washing the His magnetic beads with PBS buffer solution, adding the treated library into the His magnetic beads, adding a certain amount of herring sperm DNA, BSA and sodium chloride solution, uniformly mixing, and then incubating for a period of time on a vertical mixer at room temperature; placing the mixture on a magnetic frame, collecting supernatant, and carrying out positive screening on the supernatant and monkey pox virus surface envelope protein A29 magnetic beads; in the first round, only the positive screening is carried out, from the second round, his magnetic beads are used for carrying out the reverse screening before the positive screening taking the surface envelope protein A29 of the monkey pox virus as a target is carried out, 2 mu L of His protein of 20mg/mL and the surface envelope protein A29 magnetic beads of the monkey pox virus are added into the supernatant obtained by the reverse screening as a single-stranded nucleotide library for carrying out the positive screening; adding 100 mu L of magnetic beads after forward screening and reverse screeningddH 2 O, a metal bath at 95 ℃ for 10min, and cooling on ice for a period of time, and then magnetically sucking the supernatant as an solution; to further increase the screening pressure, virus lysate and human serum were added starting from the fifth round.
qPCR monitors the progress of the reaction:
using nucleic acid molecules in the cells as templates, and performing amplification by qPCR; the method comprises the following steps: 2. Mu.L of the positive and negative screen solutions were added to 18. Mu.L of qPCR mix, respectively, and qPCR amplified for 30 cycles, and the data were analyzed.
3. PCR amplification in large quantities
The amplification cycle number is determined according to qPCR data, and the rest positive screening is added into PCR mix to amplify the corresponding cycle number.
4. Preparation of single strands
Since the double-stranded DNA obtained after PCR amplification has biotin modified on one strand, the double-stranded DNA is captured by the action of streptavidin and biotin using agarose gel beads labeled with streptavidin in this example, and then the target single-stranded DNA is dissociated by the denaturation of NaOH, and the specific experimental procedure is as follows:
1) Incubation with agarose gel beads: 200. Mu.L of the gun head with the filter element was placed in a 1.5mL EP tube, 60. Mu.L of streptavidin-coated agarose gel beads were added, the mixture was washed twice with DPBS, 100. Mu.L of each time was used, the double-stranded DNA product obtained by PCR 3 amplification was added to the gun head with agarose gel beads after washing, 100. Mu.L of each time was used, a 200. Mu.L gun was used to hit the liquid into a waste liquid cup, and the mixture was washed twice with DPBS after the addition of the product.
2) 100. Mu.L of 40mM sodium hydroxide solution was added and collected in a new EP tube, neutralized by adding 4. Mu.L of 1M HCl solution, and then by adding 104. Mu.L of 2 XPBS solution to obtain the library for the next round of screening.
3) Concentration measurement: the final yield was obtained by measuring the a260 value by NanoDrop times the OD value, the ssDNA concentration, and the volume.
3. Repeated screening
The magnetic bead method is used for repeatedly screening 7 rounds, each operation uses the secondary library obtained in the previous operation as an initial nucleic acid library, after the library is subjected to renaturation treatment, herring sperm DNA and BSA with corresponding volumes are added, the concentration of sodium chloride is regulated, and then the herring sperm DNA and BSA are incubated with magnetic beads coupled with proteins; detecting the change of the recognition capability of the DNA single-stranded library on the surface envelope protein A29 of the monkey pox virus by using a flow cytometry after screening, and when the recognition capability of the DNA single-stranded library on the surface envelope protein A29 of the monkey pox virus meets the requirement, namely the binding capability of the screened DNA single-stranded library and target protein is higher than that of the library (figure 1) which is screened and initially put into, wherein R1, R2, R3, R4, R5, R6 and R7 respectively represent the libraries obtained by screening of round 1, round 2, round 3, round 4, round 5, round 6 and round 7, the library obtained by round 7 can be seen to have much higher affinity than the library obtained by screening of round 7 with the first round and the target, the sequencing requirement is met, and the obtained library is subjected to high-throughput sequencing analysis.
4. Analysis and identification
After high-throughput sequencing analysis is carried out on the obtained enriched library product, a plurality of sequences are selected to be synthesized by general biology, and the affinity is detected;
in the subsequent detection, it was confirmed that 4-6 sequences had strong binding ability, and after 3 sequences were truncated, the nucleic acid aptamers shown in SEQ ID NO. 1-6 were respectively designated HIM-A29-1, HIM-A29-2, HIM-A29-3, HIM-A29-4, HIM-A29-5, HIM-A29-6, as shown in Table 1.
TABLE 1 sequence of nucleic acid aptamers
| Sequence number | Sequence name | Nucleic acid sequences | Number of bases | SEQ ID No. |
| 1 | HIM-A29-1 | AGCACTCCACGCATAACGGGGTCTGGTGGTCTGGCTGGTTTGGGGGTTTGGTCGTTATGCGTGCTACCGT | 70 | 1 |
| 2 | HIM-A29-2 | AGCACTCCACGCATAACGGAGCCGGGGGGCCTTGGGGGATGCAGCTTCGAAGTGTTATGCGTGCTACCGT | 70 | 2 |
| 3 | HIM-A29-3 | AGCACTCCACGCATAACGGGGGAGGAGGAGGTTCACTTTGGGCGGGTGGGAATGTTATGCGTGCTACCGT | 70 | 3 |
| 4 | HIM-A29-4 | AGCACTCCACGCATAACGGGGGAGGAGGAGGATGTTATTGGGTGGGCGGGTTTGTTATGCGTGCTACCGT | 70 | 4 |
| 5 | HIM-A29-5 | AGCACTCCACGCATAACCGGTGGGCGGGGGATGTGGGAAGGTATCGGGGTTGCGTTATGCGTGCTACCGT | 70 | 5 |
| 6 | HIM-A29-6 | CACGCATAACGTCTTGCGGGGCGGCGGGTTGAGAGGATGTCGGGTGGTTATGCGTG | 56 | 6 |
Example 2
Screening method of aptamer combined with monkey pox virus surface envelope protein
1. Synthesis of random single stranded DNA library and primers shown in the following sequences
As in example 1.
2. Magnetic bead method screening
The magnetic bead method is adopted for screening, 7 rounds of screening are combined, and the specific screening method is as follows:
1. agarose gel beads immobilized monkey pox virus surface envelope protein A29
200 mu L of agarose gel beads are taken, washed 4 times by 200 mu L of ultrapure water, and the supernatant is removed;
taking 80. Mu.L of monkey pox virus surface envelope protein A29 (purchased from Yiqiao Shenzhou, 40891-V08E, concentration 0.5 mg/mL), adding 260. Mu.L of 10mM sodium acetate solution, pH4.5, mixing well, adding to the agarose gel beads; placing the mixture on a vertical mixer for incubation for 2 hours at room temperature;
after incubation, the supernatant was aspirated, washed 3 times with DPBS buffer and finally dissolved in 200 μl DPBS buffer.
The rest of the procedure is the same as in example 1.
3. Repeated screening
The procedure is as in example 1.
4. Analysis and identification
The procedure was as in example 1, and nucleic acid aptamers HIM-A29-1, HIM-A29-2, HIM-A29-3, HIM-A29-4, HIM-A29-5, HIM-A29-6 were also obtained by screening.
Example 3
Preparation method of test paper for detecting monkey pox virus
1. Aptamer coupled latex particles
Diluting Streptavidin (SA) -modified latex particles (mass fraction 1%, provided by Hangzhou Yoghurt) with ultrapure water to a mass fraction of 0.5%; then adding 4 times excess biotin modified aptamer into SA modified latex particles with the mass fraction of 0.5% for crosslinking, wherein the crosslinking condition is that the mixture is vibrated at room temperature for 1 h;
removing the aptamer sequence which is not bound to the surface of the latex particles in the supernatant by a centrifugal method, wherein the centrifugal condition is 4 ℃,10000 rpm and 10 min; then washing the crosslinked product twice by using ultrapure water through a centrifugal method, and finally, fixing the volume by using 50 mu L of dispensing liquid for standby;
latex particle-aptamer conjugates (illustrated as HIM-A29-6) were prepared according to the procedure described above.
2. Pretreatment of
The following treatment fluid is prepared according to the formula:
sample pad treatment liquid: 1M Tris-HCl buffer, 1% PVP,1% PEG,5% BSA, pH 9.0;
bonding pad treatment liquid: 0.2M Tris-HCl buffer, 5% BSA,1% PVP,2% PEG,20% sucrose, pH 8.0;
dialysate: 0.008 mol/L NaCl, pH 7.0.
Pretreatment is carried out after preparation:
sample pad treatment: soaking glass fiber in sample pad treatment liquid, standing for 30 min, taking out, draining, and naturally drying for later use;
and (3) bond pad treatment: soaking polyester fiber in the bonding pad treatment liquid, standing for 30 min, taking out, draining off water, and naturally drying for later use;
antibody dialysis: the antibody is diluted and put into a dialysis bag, and then is put into dialysis liquid and triple distilled water in turn for dialysis 12 h.
3. Spray-coated latex particles
The latex particle-aptamer conjugate prepared in this example was sprayed onto the pretreated conjugate pad by a laminator, the mass fraction of the latex particle-aptamer conjugate was 0.5%, and the sprayed amount was 6. Mu.L/cm.
4. Scribing film
The film was drawn using a film drawing machine, and antibodies used for the detection line (T line) were: a29 protein antibody M006 at a concentration of 2 mg/mL;
the antibodies used for the quality control line (C line) were: the concentration of the anti-streptavidin antibody was 1 mg/mL.
5. Combination of two or more kinds of materials
And sequentially assembling the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper on the PVC plate to obtain the test strip.
Test example 1
Aptamer affinity detection
Surface Plasmon Resonance (SPR) was used to detect affinity and specificity of the simian poxvirus surface envelope protein a29 aptamer to the simian poxvirus surface envelope protein a 29.
1. The general biosynthetic nucleic acid aptamers HIM-A29-1, HIM-A29-2, HIM-A29-3, HIM-A29-4, HIM-A29-5, HIM-A29-6 were each diluted to 200nM with DPBS buffer.
2. Channel 2 coupling the monkey poxvirus surface envelope protein a29 to the CM5 chip surface: then mixing equal volumes of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and injecting 50. Mu.L of activated chip at a flow rate of 5. Mu.L/min; diluting the monkey pox virus surface envelope protein A29 with 10mM sodium acetate with pH4.5 to a final concentration of 50 mug/mL, and then injecting the sample with a sample volume of 50 mug, a flow rate of 5 mug/min, wherein the coupling amount of the monkey pox virus surface envelope protein A29 is 2000Ru; after the sample injection is completed, the chip is sealed by ethanolamine, the flow rate is 5 mu L/min, and the sample injection is 50 mu L; channel 1 was treated as described above, except that the step of coupling the protein was not performed, and the step of activation and blocking was exactly the same as the control channel.
3. And (3) detection: kinetic detection parameters were set using a surface plasmon resonance (GE Healthcare, model: biacore T200), and the 6 aptamer samples diluted in step 1 were sequentially passed through 1, 2, 3, 4, 5, 6 channels, each aptamer being programmed as follows: and (3) injecting 30 mu L/min×2min, dissociating 30 mu L/min×3min, regenerating 1M NaCl 30 mu L/min×0.5min, and sequentially injecting diluted nucleic acid aptamers.
The affinity assay data are shown in FIGS. 2-7, each curve being a curve after channel 2 is subtracted by channel 1, illustrating the binding capacity of the corresponding aptamer to the target protein N protein. These data demonstrate that the nucleic acid aptamers HIM-A29-1, HIM-A29-2, HIM-A29-3, HIM-A29-4, HIM-A29-5, HIM-A29-6 all detected strong binding to the monkey pox virus surface envelope protein A29 using SPR, and KD values are shown in Table 2, respectively.
TABLE 2 affinity of Simian poxvirus surface envelope protein A29 to aptamer
| Name of the name | Affinity KD (nM) |
| SEQ ID NO:1（HIM-A29-1） | 10.8 |
| SEQ ID NO:2（HIM-A29-2） | 35.4 |
| SEQ ID NO:3（HIM-A29-3） | 12.4 |
| SEQ ID NO:4（HIM-A29-4） | 18.6 |
| SEQ ID NO:5（HIM-A29-5） | 8.49 |
| SEQ ID NO:6（HIM-A29-6） | 5.63 |
As shown in Table 2, the aptamer screened by the invention has good affinity with the surface envelope protein A29 of the monkey pox virus, which can reach below 10nM, thus indicating that the aptamer can accurately and efficiently recognize and bind the monkey pox virus.
Test example 2
Detection of monkey poxvirus A29 protein
The situation that the strip appears on the test strip for detecting the monkey pox virus is observed by dripping buffer solutions containing the monkey pox virus A29 protein with different concentrations:
the monkey pox virus is prepared into standard solution with the concentration of 1 mug/mL, 500ng/mL, 100ng/mL, 50ng/mL and 10ng/mL by using a virus diluent, the standard solution is sucked by a rubber head dropper, 3 drops of the standard solution are dropped on a sample pad, and after the dropping, the standard solution is stood for 15 minutes, and then the result is judged:
positive (+): two bands appear, a detection line and a quality control line, which indicate that the monkey pox virus or the monkey pox virus exists in the sample;
negative (-). Only one band appears on the quality control line, and no band appears on the detection line, which indicates that the sample has no monkey pox virus A29 protein;
invalidation: the quality control line does not appear as a red band, possibly due to incorrect operation or reagent failure, should be retested.
The detection results are shown in FIG. 8. As can be seen from FIG. 8, the detection effect is good, which shows that the nucleic acid aptamer screened by the invention can effectively detect the monkey pox virus in the environment.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art, and are not described herein.
While the foregoing embodiments have been described in detail in connection with the embodiments of the invention, it should be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like made within the principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A nucleic acid aptamer that binds to a monkey poxvirus surface envelope protein, comprising at least one of the following (1) - (6):
(1) The aptamer HIM-A29-1 shown in SEQ ID No. 1;
(2) The aptamer HIM-A29-2 shown in SEQ ID No. 2;
(3) The aptamer HIM-A29-3 shown in SEQ ID No. 3;
(4) The aptamer HIM-A29-4 shown in SEQ ID No. 4;
(5) The aptamer HIM-A29-5 shown in SEQ ID No. 5;
(6) The aptamer HIM-A29-6 as shown in SEQ ID No. 6.
2. A composition for screening for the aptamer of claim 1, comprising,
-a solid support comprising magnetic beads and/or agarose gel beads; and
-a target molecule or a part of a target molecule bound to the solid support, said target molecule being monkey poxvirus surface envelope protein a29 and/or DNA.
3. A reaction system for screening the aptamer of claim 1, comprising,
-a single-stranded DNA library comprising at least one nucleotide chain capable of specifically recognizing the target molecule or a part of a target molecule, which target molecule is monkey pox virus surface envelope protein a29 and/or DNA;
-a primer having the sequence shown in SEQ ID No.7 and SEQ ID No. 8;
-the composition of claim 2;
-a buffer.
4. A method of screening for the aptamer of claim 1, comprising the steps of:
-synthesizing a single-stranded DNA library and primers;
-magnetic bead method and/or agarose gel bead method screening comprising formulating the reaction system of claim 3, forward and reverse screening, PCR amplification and single strand preparation.
5. The use of the aptamer of claim 1, comprising at least one of (1) - (4):
(1) Use in detecting a monkey poxvirus in an environment;
(2) Use in the manufacture of a medicament for the treatment of monkey pox virus pneumonia;
(3) Use in the preparation of a developing agent and/or a tracer for the detection of monkey poxviruses;
(4) The application of the kit in preparing a reagent and/or test paper and/or a kit for detecting the monkey poxvirus.
6. A reagent for detecting a monkey poxvirus comprising the nucleic acid aptamer of claim 1 labeled with a fluorescent dye.
7. A test paper for detecting monkey pox virus comprises,
-a nitrocellulose membrane comprising a detection line comprising antibodies to the monkey poxvirus a29 protein and a quality control line comprising antibodies to streptavidin;
-a binding pad comprising the nucleic acid aptamer of claim 1;
-a sample pad; and
-absorbent paper.
8. Use of the reagent of claim 6 or the test strip of claim 7 for detecting monkey poxvirus in an environment.
9. A method of detecting a monkey poxvirus in an environment comprising:
sampling a region to be detected in the environment;
incubating the sample with the reagent of claim 6;
after incubation of the mixture, the fluorescence intensity in the mixture was measured, and the result was determined.
10. A method of detecting a monkey poxvirus in an environment comprising:
sampling a region to be detected in the environment;
mixing the sample with the aptamer of claim 1 to obtain a mixture;
detecting whether the mixture contains a monkey pox virus-aptamer complex.
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