CN110885828A - Aptamer DT01 of diphtheria toxin and application thereof - Google Patents
Aptamer DT01 of diphtheria toxin and application thereof Download PDFInfo
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- CN110885828A CN110885828A CN201911273536.XA CN201911273536A CN110885828A CN 110885828 A CN110885828 A CN 110885828A CN 201911273536 A CN201911273536 A CN 201911273536A CN 110885828 A CN110885828 A CN 110885828A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Abstract
The invention relates to a nucleic acid aptamer DT01 of diphtheria toxin and application thereof, wherein the sequence of the nucleic acid aptamer DT01 is as follows: 5'-GTCGCATGGAAGGAGCGACGCCTTAGCCTCGCGTGTTCGCGTGCGGTGGAGCCGCGCAATCGGGACCGGCGGTCCAAGT-3' are provided. The diphtheria toxin nucleic acid aptamer DT01 can be combined with diphtheria toxin with high affinity and high specificity, and can effectively neutralize the toxic effect of diphtheria toxin as an antagonist and relieve clinical symptoms of diphtheria toxin poisoning.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-affinity aptamer DT01 specifically bound with diphtheria toxin and application thereof.
Background
Diphtheria is a potentially fatal disease caused by infection with corynebacterium diphtheriae and the potent Diphtheria Toxin (DT) secreted therefrom. Corynebacterium diphtheriae is a gram-positive bacterium, the pathogen of diphtheria. The toxic effect of diphtheria toxin is mainly to mediate adenosine diphosphate ribosylation to inhibit elongation factor 2(EF-2), and to cause tissue necrosis of toxin production sites such as respiratory tract by forming pathogenic pharyngeal false membrane and edema of local airways. Dissemination of blood-borne diphtheria toxin can also cause cranial nerve dysfunction, peripheral neuropathy and cardiotoxicity, which are responsible for death in 50-75% of diphtheria cases.
Although widespread vaccination with diphtheria toxoid vaccines has greatly reduced the incidence of diphtheria cases, the threat of diphtheria has not disappeared and diphtheria remains prevalent in some countries and regions. Periodic outbreaks of diphtheria have also recently occurred in the seas, nigeria, south africa, indonesia, laos, bangladesh, also menmen and indonesia, with fatality rates as high as 10%. International travel and susceptible population migration also exacerbate the risk of diphtheria becoming a global disease. Therefore, the national quality supervision inspection and quarantine bureau of China issues bulletins for a plurality of times to remind people of preventing the multi-national diphtheria epidemic situation from entering China. Therefore, China should not worry about the occurrence and treatment of diphtheria infection and should be fully prepared.
The most effective method for treating diphtheria is timely injection of Diphtheria Antitoxin (DAT) to neutralize diphtheria toxin and prevent further tissue damage, combined with antibiotic treatment to eliminate diphtheria bacillus and stop toxin production, which can greatly reduce the morbidity and mortality of diphtheria. DAT is currently predominantly equine diphtheria toxin antibody, and because of the risk of severe allergic reactions and the risk of developing serum disease and certain co-morbidities in humans and animals, many manufacturers have stopped production, and worldwide supply of DAT is extremely limited. Therefore, there is a need for safer and more efficient antagonists that antagonize the toxic effects of diphtheria toxin in order to address the potential risk of diphtheria onset and effectively treat diphtheria patients.
Aptamers are also known as "synthetic antibodies", "chemical antibodies", and their chemical nature is that a single-stranded oligonucleotide molecule (ssDNA or RNA) folds into a specific three-dimensional structure that binds to a target substance with high affinity and specificity. Aptamers were obtained by an in vitro screening process using Systematic evolution of ligands by exponentiation technology (SELEX). The aptamer has the characteristics of high affinity, high specificity, capability of being synthesized in vitro, capability of changing the function and the pharmacokinetic characteristic thereof through modification, no immunogenicity, economy and the like. Aptamer drugs developed based on the above advantages can specifically block biological functions of targets, for example, as neutralizing antagonists for toxins, inhibitors for cytokines, tumor therapeutic drugs for blocking transcription factors, and the like. Therefore, screening the aptamer which is combined with diphtheria toxin with high specificity and high affinity as an antagonist of the toxic effect of the aptamer has important scientific research and clinical values.
Disclosure of Invention
One of the objects of the present invention is to provide a nucleic acid aptamer DT01 for diphtheria toxin having high specificity and high affinity; the invention also aims to provide the application of the aptamer DT01 in the aspects of preparing reagents for separating and enriching diphtheria toxin in samples, preparing reagents or kits for detecting diphtheria toxin, preparing drugs for neutralizing or antagonizing diphtheria toxin, and the like.
The purpose of the invention is realized by the following technical scheme: a nucleic acid aptamer DT01 for diphtheria toxin, the sequence of which is shown below:
5'-GTCGCATGGAAGGAGCGACGCCTTAGCCTCGCGTGTTCGCGTGCGGTGGAGCCGCGCAATCGGGACCGGCGGTCCAAGT-3'(SEQ ID NO:1)。
the aptamer DT01 of diphtheria toxin is obtained by an in vitro SELEX screening technology based on aptamers, wherein carboxyl magnetic beads are used as a solid phase medium, diphtheria toxin is used as a target, and the aptamer specifically bound with diphtheria toxin is obtained by screening from a ssDNA library through the diphtheria toxin magnetic beads and is named as aptamer DT 01.
The aptamer DT01 for diphtheria toxin of the present invention may be chemically modified at its 5 'end or 3' end with a fluorophore, amino group, biotin, digoxigenin, polyethylene glycol, or the like.
The nucleic acid aptamer DT01 of diphtheria toxin has the function of antagonizing the toxicity of diphtheria toxin and can be used as a potential neutralization antagonist of diphtheria toxin. The aptamer DT01 of diphtheria toxin can be used for preparing drugs for neutralizing or antagonizing diphtheria toxin.
The application of the aptamer DT01 of diphtheria toxin in preparing a reagent for separating and enriching diphtheria toxin in a sample.
The application of the aptamer DT01 of diphtheria toxin in preparing diphtheria toxin detection reagents or kits.
The aptamer DT01 of diphtheria toxin can also be applied to targeted therapy with diphtheria toxin as an effector molecule.
Compared with the prior art, the invention has the advantages that:
1. the aptamer DT01 of the invention has the advantages of no toxicity, small molecular weight, good permeability and easy synthesis and labeling.
2. The synthesis cost of the aptamer DT01 is lower than that of antibody preparation, and the aptamer DT01 has short period and good reproducibility.
3. The aptamer DT01 of the present invention binds to diphtheria toxin with high affinity and high specificity, has a dissociation constant of 23.7pM (95% IC: 15.94-35.15pM), and does not bind to other control proteins.
4. The aptamer DT01 has wide application prospect and important scientific and social value in the fields of diagnosis and treatment of corynebacterium diphtheriae infection, targeted therapy mediated by diphtheria toxin and the like, particularly has the function of antagonizing the toxicity of diphtheria toxin, and can be used as a potential neutralization antagonist of diphtheria toxin.
Drawings
FIG. 1 is a bioinformatics mimic diagram of the secondary structure of aptamer DT 01.
FIG. 2 is a diagram showing the specificity of aptamer DT01 analyzed by fluorescence binding rate experiment. In FIG. 2, the abscissa represents the analyzed protein, and the ordinate represents the fluorescence binding rate.
FIG. 3 is a graph plotting dissociation constants of the aptamer DT01 binding to diphtheria toxin according to fluorescence binding rate experiment. Dissociation constant (Kd) was 23.7pM (95% IC: 15.94-35.15 pM). In FIG. 3, the abscissa represents the DNA concentration (pM) and the ordinate represents the fluorescence binding rate.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification:
a nucleic acid aptamer DT01 for diphtheria toxin, having the sequence:
5'-GTCGCATGGAAGGAGCGACGCCTTAGCCTCGCGTGTTCGCGTGCGGTGGAGCCGCGCAATCGGGACCGGCGGTCCAAGT-3'(SEQ ID NO:1)。
the aptamer DT01 of diphtheria toxin at 25 ℃ and 100mM Na+，1mM Mg2+Under the conditions of (a), the spatial structure thereof is as follows:
the aptamer DT01 of diphtheria toxin is chemically modified at the 5 'end or the 3' end of the aptamer DT01, including but not limited to a fluorophore, an amino group, biotin, digoxin, polyethylene glycol and the like.
The aptamer DT01 of diphtheria toxin is obtained by carrying out chemical modification including but not limited to fluorescent group, amino group, biotin, digoxin, polyethylene glycol and the like on the product obtained by carrying out truncation, elongation or partial base replacement on the aptamer DT 01.
The aptamer DT01 of diphtheria toxin is obtained by an in vitro SELEX screening technology based on aptamers, carboxyl magnetic beads are used as a solid phase medium, diphtheria toxin is used as a target, and the aptamer specifically bound with diphtheria toxin is screened from a ssDNA library through the diphtheria toxin magnetic beads.
The screening method of the aptamer DT01 of diphtheria toxin comprises the following steps:
(1) preparation of screening library: a random ssDNA library was prepared as shown by the following sequence:
5’-GTCGCATGGAAGGAGCGACGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGGGACCGGCGGTCCAAGT-3’；
(2) coupling diphtheria toxin with carboxyl magnetic beads to prepare diphtheria toxin magnetic beads;
(3) subjecting the ssDNA library to a thermal activation treatment;
(4) incubating the ssDNA library obtained in the step (3) with the diphtheria toxin magnetic beads obtained in the step (2);
(5) magnetically separating the diphtheria toxin magnetic beads after the step (4), and washing away ssDNA which is not combined, weakly combined and non-specifically combined on the surfaces of the diphtheria toxin magnetic beads; heating the diphtheria toxin magnetic beads, and collecting ssDNA specifically combined with the diphtheria toxin magnetic beads, namely a ssDNA enrichment library;
(6) and (3) PCR amplification: and (3) carrying out PCR amplification on the ssDNA enrichment library obtained in the step (5), wherein primers used for the PCR amplification are as follows:
primer DTup: 5 '-FAM-GTCGCATGGAAGGAGCGACG-3'
Primer DTdown: 5 '-Biotin-ACTTGGACCGCCGGTCCCG-3';
(7) purification of PCR products: purifying the PCR product by using a small fragment DNA purification kit; incubating the purified dsDNA with streptavidin magnetic beads, washing the streptavidin magnetic beads combined with the dsDNA, melting the dsDNA, separating by using a magnetic frame, and collecting supernatant; precipitating the supernatant by ethanol to obtain a secondary ssDNA library for the next round of screening;
(8) and (3) circulating screening: and (3) taking the FAM-labeled secondary ssDNA library obtained in the step (7) as a secondary library for the next round of screening, and repeating the screening processes of the steps (3) to (7).
The first embodiment is as follows: screening for aptamer DT01
The screening method of the aptamer DT01 of diphtheria toxin comprises the following steps:
(1) preparation of screening library: designing a random ssDNA library, the sequence of which is: 5 '-GTCGCATGGAAGGAGCGACGNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNCGGGACCGGCGGTCCAAGT-3', which includes a fixed sequence region at both ends (20 nucleotides at the 5 'end and 19 nucleotides at the 3' end) and a random sequence region in the middle (40 random sequence nucleotides), and was synthesized by Competition Biotechnology, bioengineering, Inc.
(2) Diphtheria toxin coupling with carboxyl magnetic beads: the diphtheria toxin is derived from Corynebacterium diphtheriae NCTC10648 strain with a purity of > 98%, available from The Native Antigen Company, United kingdom, and The carboxylated magnetic beads and coupling reagents thereof are available from Bangs Laboratories, Inc., USA. Diphtheria toxin coupling to magnetic beads procedures refer to the manufacturer's instructions. Measuring the change of protein concentration in diphtheria toxin solution before and after coupling by BCA method, and calculating the coupling efficiency of the magnetic beads to be 83%; the magnetic beads of diphtheria toxin were dispersed in PBS buffer and stored at 4 ℃.
(3) A random library of 1nmol ssDNA was dissolved in 500. mu.L selection buffer (50mM Tris-HCl, 100mM NaCl, 1mM MgCl)25mM KCl, pH 7.4) and then heat activated. The thermal activation treatment method comprises the following steps: after denaturation at 95 ℃ for 5min, the mixture was immediately placed in an ice-water bath for 10min, followed by room temperature for 10 min.
(4) And (3) incubating the ssDNA library obtained in the step (3) with the diphtheria toxin magnetic beads (with diphtheria toxin loading of 20ng) and yeast tRNA (molar weight 5 times of that of the ssDNA library) obtained in the step (2) at room temperature for 1 h.
(5) Magnetically separating the diphtheria toxin magnetic beads after the step (4), and washing ssDNA (single stranded deoxyribonucleic acid) which is not bound, weakly bound and non-specifically bound on the surfaces of the diphtheria toxin magnetic beads by using a selection buffer solution containing 0.2% BSA (bovine serum albumin); the diphtheria toxin magnetic beads were then washed with 200. mu.LddH2And (4) resuspending, placing in a magnetic frame for 1-2min after a hot water bath at 100 ℃ is carried out for 5min, and collecting supernatant to obtain ssDNA (single-stranded deoxyribonucleic acid) specifically bound with the diphtheria toxin magnetic beads, namely a ssDNA enrichment library.
(6) And (3) PCR amplification: adding the ssDNA enrichment library obtained in the step (5) into 1mL of PCRmix; after vortex oscillation and uniform mixing, subpackaging according to 50 mu L of each tube for PCR amplification, wherein the amplification conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30S, annealing at 63 ℃ for 30S, and extension at 72 ℃ for 30S, 15-25 cycles.
Wherein 1mL of PCRmix contains: 10 XPCR buffer 100. mu.L; pfu enzyme 3. mu.L; dNTP 20 u L; primer DTup: 5 '-FAM-GTCGCATGGAAGGAGCGACG-3' and primer DTdown: 3. mu.L of each of 5 '-Biotin-ACTTGGACCGCCGGTCCCG-3'; the primer DTup and the primer DTdown are synthesized by the company of biological engineering, Inc.
(7) Purification of PCR products: PCR products labeled with biotin and a fluorophore FAM at both ends, respectively, were purified using a small fragment purification kit (said small fragment purification kit is available from Biotechnology, Ltd.), the purified dsDNA was incubated with streptavidin magnetic beads (available from Invitrogen-Dynal) at 37 ℃ for 20min, the dsDNA-bound streptavidin magnetic beads were washed three times with a washing buffer (5mM Tris-HCl, pH 7.5, 1M NaCl, 500. mu.M EDTA), and then incubated with 50. mu.L NaOH solution (0.1M) at 37 ℃ for 30min to melt the dsDNA; the supernatant was collected by magnetic frame separation and ethanol precipitated to obtain the FAM-labeled secondary ssDNA library and dissolved in selection buffer as the secondary library for the next round of screening.
(8) The screening process was performed for 12 rounds. From the second round, the secondary libraries were used in an amount of 30pmol each.
Example two: acquisition and analysis of aptamer DT01 sequence:
(1) after 12 rounds of screening, the enriched ssDNA library was collected and submitted to the Beijing Xinnuo Bai Shi medical laboratory Ltd to analyze the sequence of the library by high throughput sequencing technology, the analysis process was: amplifying the enrichment library by PCR, and adding a sequencing joint and an Index part; selecting a purified library by gel electrophoresis; using Nanodrop one to measure the concentration and purity of DNA for quality control analysis; using illumina novaseqTM6000 platform, using single chain library as template to do bridge PCR amplification, sequence primer annealing, synthesizing and sequencing; and comparing and enriching the sequencing result.
(2) According to the enrichment degree of the aptamer in the library, ssDNA with high enrichment degree is selected as a candidate aptamer, wherein the aptamer DT01 accounts for 12.4% of the enrichment library, and the sequence of the aptamer is shown as SEQ ID NO. 1.
(3) Analysis at 25 ℃ with UNAFold network platform 100mM Na+，1mM Mg2+Under the conditions of (1), the secondary structure of the aptamer DT01 sequence. The schematic diagram of the secondary structure of the sequence of the aptamer DT01 is analyzed and shown in FIG. 1.
Example three: specific analysis of aptamer DT 01:
(1) FAM-labeled aptamer DT01 was chemically synthesized in vitro and dissolved in selection buffer.
(2) Referring to step (2) of example one, BSA (available from Sigma), pertussis toxin (PTX, available from The natural Antigen Company, uk), and staphylococcus aureus enterotoxin B (SEB, available from ToxinTechnology, usa) were coupled with carboxyl magnetic beads to prepare BSA magnetic beads, PTX magnetic beads, and SEB magnetic beads, respectively.
(3) And (3) mixing 200 mu L of the aptamer DT01 solution obtained in the step (1) with the BSA magnetic beads, PTX magnetic beads, SEB magnetic beads and diphtheria toxin magnetic beads obtained in the step (2), and incubating at room temperature for 1h in a cassette, wherein blank magnetic beads are used as blank controls.
(4) The magnetic beads obtained in step (3) were washed 3 times with 0.1% PBST, and the aptamers bound to the magnetic beads were eluted by boiling 200. mu.L of a selection buffer at 100 ℃ for 5 min.
(5) The fluorescence intensities of the initial solution and the eluate were measured by a fluorescence quantitative analyzer, and the fluorescence binding ratio (initial fluorescence intensity-eluted fluorescence intensity)/initial fluorescence intensity × 100% was calculated, and the binding ratio of the aptamer DT01 to the target molecule was preliminarily represented by the calculated value.
As shown in figure 2, the binding rate of the aptamer DT01 to diphtheria toxin is significantly higher than that of BSA, PTX and SEB, which indicates that the aptamer DT01 has better specificity to diphtheria toxin.
Example four: affinity analysis of aptamer DT01
(1) And mixing FAM labeled aptamer DT01 solutions with different concentrations with diphtheria toxin magnetic beads respectively, and incubating the mixture in a cassette at room temperature for 1 h.
(2) Referring to the step (4) and the step (5) in the third example, the fluorescence binding rates of the aptamer DT01 solutions with different concentrations and the diphtheria toxin magnetic beads were obtained and calculated.
(3) The calculated value of the fluorescence binding rate was used to plot a saturation binding curve of aptamer DT01 bound to diphtheria toxin, and the dissociation constant of aptamer DT01 bound to diphtheria toxin was calculated by nonlinear regression analysis.
As shown in FIG. 3, the present inventors obtained a saturation binding curve of aptamer DT01, and calculated that the dissociation constant of aptamer DT01 was 23.7pM (95% IC: 15.94-35.15pM), indicating that aptamer DT01 binds diphtheria toxin strongly with a dissociation constant on the picomolar scale.
Example five: research on antagonism of diphtheria toxin toxicity effect of aptamer DT01
(1) Determination of diphtheria toxin test dose: the determination of the diphtheria toxin test dose is carried out according to the standard method recommended by the national institutes of health, and the main method is as follows: guinea pigs weighing around 250g (purchased from the 900 th hospital animal center) were purchased, and after being adapted to the test environment for 1d, and grouped according to diphtheria toxin doses, 5 per group, 1IU of Diphtheria Antitoxin (DAT) was mixed with different doses of Diphtheria Toxin (DT) to prepare a mixture (designated DAT-DT), and incubated at room temperature for at least 1 h. DAT-DT was injected subcutaneously at 0d per animal in a volume of 3.0 mL. The physical condition and mortality of the guinea pigs were observed, and the diphtheria toxin dose at which all the guinea pigs died within 96 hours was used as the test dose. The lowest lethal dose ≈ 4fL (4.0156fL) was observed as the test dose.
(2) The research method for antagonizing diphtheria toxin toxicity by aptamer DT01 comprises the following steps:
A. preparation of pegylated aptamer DT 01: synthesizing a nucleic acid aptamer DT01 with amino modification at the 5 'end and dT modification at the 3' end by Compton engineering bioengineering, Inc.; then polyethylene glycol (PEG) of 40-kDa is used for carrying out pegylation treatment on the modified aptamer DT01 to obtain a pegylated aptamer DT01(PEG-DT01), the pegylation process is finished by Beijing KeKai science and technology GmbH, and the PEG-DT01 is subjected to HPLC homozygosis for later use.
B. Animal model experiment of aptamer DT 01: guinea pigs weighing around 250g (purchased from the 900 th hospital animal center) were purchased and, after being adapted to the test environment for 1d, divided into 5 per group according to the dose groups of diphtheria antitoxin and aptamer, different doses of pegylated aptamer DT01(PEG-DT01), Diphtheria Antitoxin (DAT) were mixed with 4fL Diphtheria Toxin (DT) to prepare mixtures (designated PEG-DT01-DT and DAT-DT, respectively) and incubated at room temperature for at least 1 h. PEG-DT01-DT and DAT-DT were injected subcutaneously in a volume of 3.0mL per animal at 0d, respectively. Guinea pigs were observed for physical condition and mortality, with the observation time extended from the standard 96h to 30d to allow greater discrimination of dose effects and to assess signs of toxin-induced end organ damage at 30d post toxin exposure. Animals were observed daily for signs of diphtheria toxin intoxication and weekly body weights were measured as a measure of overall health. Establishing a digital scoring system of clinical symptoms: asymptomatic is 0; sloppy fur 1; lethargy is 2; dehydration 3; dragging one or two rear legs to 4; moribund ═ 5; death was 6. Animals that were either dying or had a 20% reduction in body weight from baseline were euthanized.
(3) The results of the study of the antagonism of diphtheria toxin by aptamer DT 01: as shown in table 1, in the animal model receiving DAT-DT injection, all guinea pigs receiving DAT dose ≦ 1.25IU died, and the lower DAT dose, the earlier the guinea pig died; all guinea pigs receiving doses of DAT ≧ 1.75IU survived. Survival in guinea pigs receiving doses of DAT ranging from 1.5IU to 1.6IU was variable. In animal models receiving PEG-DT01-DT injection, all guinea pigs receiving PEG-DT01 dose of 16 μ g or less died, and the lower the dose, the earlier the guinea pig died; all guinea pigs receiving PEG-DT01 dose ≧ 48 μ g survived. Survival rates in guinea pigs receiving doses of PEG-DT01 of 16-48 μ g were variable, suggesting that the aptamer DT01 acts similarly to diphtheria antitoxin. In addition, clinical symptoms of tested guinea pigs are observed, and the clinical performance scores of all guinea pigs are less than or equal to 3 when the dosage of PEG-DT01 is more than or equal to 64 mu g, which indicates that aptamer DT01 can be used as an antagonist to effectively neutralize the toxic effect of diphtheria toxin and relieve the clinical symptoms of diphtheria toxin poisoning.
TABLE 1 protective Effect of aptamer DT01 and diphtheria antitoxin on lethal guinea pig model of leukotoxin
The foregoing is only a preferred embodiment of the present invention, and it should be noted that a person skilled in the art may make several changes, improvements and modifications without departing from the spirit of the present invention, and these changes, improvements and modifications should be construed as the protection scope of the present invention.
Sequence listing
<110> winter medical technology Limited of Baoai in Changle region, Fuzhou city
<120> aptamer DT01 of diphtheria toxin and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>79
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>1
gtcgcatgga aggagcgacg ccttagcctc gcgtgttcgc gtgcggtgga gccgcgcaat 60
cgggaccggc ggtccaagt 79
<210>2
<211>20
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>2
<210>3
<211>19
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>3
acttggaccg ccggtcccg 19
Claims (5)
1. An aptamer DT01 to diphtheria toxin, characterized by:
the sequence is shown as SEQ ID NO:1 is shown in the specification; and 100mM Na at 25 deg.C+，1mM Mg2+Under the condition of (2), the spatial structure is as follows:
2. the aptamer DT01 for diphtheria toxin according to claim 1, wherein: and carrying out chemical modification on the 5 'end or the 3' end of the aptamer DT01 by using a fluorescent group, an amino group, biotin, digoxin and polyethylene glycol.
3. Use of the aptamer DT01 for diphtheria toxin according to claim 1 in the preparation of an agent for the isolation and enrichment of diphtheria toxin in a sample.
4. Use of the aptamer DT01 for diphtheria toxin according to claim 1 in the preparation of a diphtheria toxin detection reagent or kit.
5. Use of the aptamer DT01 for diphtheria toxin according to claim 1 in the preparation of a medicament for neutralising or antagonizing diphtheria toxin.
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