KR101670188B1 - Botulinum neurotoxin type E-specific polypeptides and uses thereof - Google Patents
Botulinum neurotoxin type E-specific polypeptides and uses thereof Download PDFInfo
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- KR101670188B1 KR101670188B1 KR1020150156393A KR20150156393A KR101670188B1 KR 101670188 B1 KR101670188 B1 KR 101670188B1 KR 1020150156393 A KR1020150156393 A KR 1020150156393A KR 20150156393 A KR20150156393 A KR 20150156393A KR 101670188 B1 KR101670188 B1 KR 101670188B1
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Abstract
Description
More particularly, the present invention relates to a polypeptide specifically binding to a botulinum neurotoxin type E, a polynucleotide encoding the same, a recombinant vector, a transformant and a method for producing the same. A kit for detecting the botulinum neurotoxin E type and botulinum toxinosis diagnosis using the polypeptide, and a method of detecting and diagnosing the bot.
Botulinum toxinosis is a forcible infectious disease of the 4th group caused by botulinum neurotoxin produced by Clostridium botulinum , a gram-positive anaerobic bacterium, which causes severe diastolic nerve palsy. Botulinum neurotoxin is one of the most potent toxins produced in nature and one of the most dangerous biological terrorist weapons of Class A, along with anthrax, fest, and potholes, among the categories selected by the American Centers for Disease Control and Prevention (CDC). In particular, botulinum toxin is a fatal toxin produced by bacteria, and the toxin itself has a strong killing effect.
Botulinum neurotoxin is classified into eight serotypes (A to H) according to antigen specificity, among which A, B, and E cause diseases in the human body, and F type is rare. C and D are present in mammals other than birds and humans, and the mechanism of action and specificity of the G-type have not been clearly established (see Table 1 below). E-type Clostridium botulinum bacteria producing E-type are mainly isolated from Suseo area and are related to the occurrence of botulinum toxin by seafood or fish.
The botulinum toxin is composed of heavy chain (100 kDa), light chain (50 kDa) and hemagglutinin (Non-Patent Document 1). After the botulinum toxin enters the neuronal cytoplasm, the protein SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors), a protein associated with the neurotransmitter, is degraded to render acetylcholine non-secreted from the neuron. The muscle contraction does not occur, and the relaxation state is maintained constantly, resulting in a relaxation paralysis symptom.
Since the action of botulinum toxin is an irreversible reaction, the diagnosis of botulinum toxin should be made at the early stage of clinical manifestation before the botulinum toxin is completely bound, so that effective clinical management is possible.
The international standard test method for diagnosing botulinum toxinosis and toxin identification has been internationally accepted as a mouse test method. Currently, the laboratory diagnosis process includes sample pretreatment, mouse test, ELISA for botulinum toxin identification, . However, the mouse test has problems such as animal ethics, sensitivity, and promptness, and it is difficult to anaerobically cultivate botulinum. For botulinum neurotoxin type E, diagnosis and detection methods are relatively inferior to other serotypes A and B, which are human hosts.
Disclosure of Invention Technical Problem [8] Accordingly, the present invention provides a botulinum neurotoxin detection method and neuropathy diagnostic method which solve the animal ethics problem of the conventional botulinum neurotoxin detection and neuropathy diagnosis method, and have excellent rapidity, sensitivity and specificity.
In order to achieve the above object, the present invention provides a polypeptide specifically binding to a botulinum neurotoxin and comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 8.
According to one aspect of the present invention, there is provided a polynucleotide comprising a nucleotide sequence encoding said polypeptide.
According to one aspect of the present invention, there is provided a recombinant vector comprising the polynucleotide.
According to one aspect of the present invention, there is provided a transformant transformed with the above recombinant vector.
According to one aspect of the present invention, there is provided a kit for detecting botulinum neurotoxin and a kit for diagnosing botulinum neuropathy comprising the polypeptide described above.
According to one aspect of the present invention, the polypeptide included in the botulinum neurotoxin detection kit and the botulinum neuropathy diagnosis kit includes a chromogenic enzyme, a radioactive isotope, a chromopore, a biotin, a fluorescent substance, a fluorescer ). ≪ / RTI >
According to one aspect of the present invention, there is provided a chip for detecting botulinum neurotoxin comprising the polypeptide described above and for diagnosing botulinum neuropathy.
According to one aspect of the present invention, the substrate on which the polypeptide is immobilized is a solid substrate and is characterized in that it is at least one selected from the group consisting of polymer, glass, gold, paper and biological film.
According to one aspect of the present invention, there is provided a method for detecting botulinum neurotoxin and a method for diagnosing botulinum neuropathy comprising the step of reacting a sample with the above-mentioned polypeptide.
According to one aspect of the present invention, the sample is any one or more selected from a tissue, a cell, a whole blood, a plasma, a serum, a blood, a saliva, a sputum, a lymphatic fluid, a cerebrospinal fluid, an intracellular fluid and a urine sample.
Provided is a method for diagnosing botulinum neurotoxin and toxin, which is excellent in the rapidity, sensitivity, and specificity, by solving the animal ethics problem of the detection and diagnosis method which is used in the past, using the polypeptide specific to the botulinum neurotoxin according to the present invention can do. This may be helpful in the direction of treatment of botulinum toxinosis and may be used as a prognostic tool for the diagnosis of botulinum toxinosis. Furthermore, it can be applied not only to botulinum neurotoxin E type but also to other serotypes of botulinum neurotoxin and various toxins causing diseases in humans and animals, and it can be utilized as a diagnostic sensor technology capable of detecting various toxins through technical application .
Fig. 1 schematically shows a phage display process performed on botulinum toxin type E; Fig.
FIG. 2 shows the result of PCR amplification of a random sequence of single-stranded DNA of a phage to confirm a random sequence of the selected phage, followed by purifying only the band of the predicted product size (184 bp) and confirming by gel electrophoresis. Lane M means a 100 bp DNA ladder and lanes 1 to 20 are amplified random sequences of each plaque.
FIG. 3 is an example of a detection chip using a botulinum toxin type E specific binding peptide as a functional material.
In order to accomplish the above object, the present invention provides a polypeptide specifically binding to a botulinum neurotoxin type E, comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 8, a polynucleotide encoding the polypeptide, a recombinant vector , Transformants, compositions for the detection and diagnosis of botulinum neurotoxin, and methods of detecting and diagnosing botulinum neurotoxin. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.
The present invention provides a polypeptide which specifically binds to Botulinum neurotoxin and which comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-8. The present inventors carried out a phage display technique on the botulinum neurotoxin E type to isolate and identify polypeptides specifically binding thereto, thereby completing the present invention.
The polypeptides (or peptides) of the present invention include all kinds of peptides, proteins, peptide mimics, compounds and biologics, and specifically refers to targeting botulinum toxinosis by binding specifically to botulinum neurotoxin type E. Preferably, it may be a low molecular peptide consisting of 11 or 12 amino acids. Low molecular weight peptides are small in size and stabilized three-dimensionally. They are easy to pass through the mucosa and can recognize molecular targets even in deep tissues. These low molecular peptides have the advantage of being able to diagnose target toxins or diseases early because the stability can be ensured through local injection and the immunoreactivity can be minimized. The low molecular weight peptide according to the present invention is relatively simple in mass production and has a low toxicity. In addition, the low molecular peptide according to the present invention has a high binding strength to a target substance rather than an antibody, and does not undergo denaturation during thermal / chemical treatment.
The polypeptide of the present invention can be easily produced by chemical synthesis known in the art (Non-Patent Document 2). Representative methods include liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemical methods (Non-Patent Documents 3 and 4), but are not limited thereto. First, a DNA sequence encoding the peptide is prepared according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using automated DNA synthesizers (such as those sold by Biosearch or Applied Biosystems). The constructed DNA sequence is operatively linked to the DNA sequence and contains one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence , And the host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformant is cultured under appropriate medium and conditions to express the DNA sequence, and the substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using methods known in the art (e.g., chromatography). By "substantially pure peptide" herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. The genetic engineering method for synthesizing the polypeptide of the present invention can refer to various methods known to those skilled in the art (see Non-Patent Documents 4 to 8)
According to still another aspect of the present invention, there is provided a polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1, a recombinant vector comprising the polynucleotide, and a transformant transformed with the polynucleotide. The polynucleotides and recombinant vectors can be obtained as described above.
As used herein, the term " polypeptide " (or peptide, protein) is interpreted to include an amino acid sequence that exhibits substantial identity to the amino acid sequence of interest. The above substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence to the greatest correspondence and analyzing the aligned sequence using algorithms commonly used in the art to obtain a homology of at least 60% , More preferably at least 80% homology, and most preferably at least 90% homology.
For example, the polypeptide may comprise at least about 60%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4% , 99.5% or more, or 99.9% or more of the amino acid sequence, and specifically binds to the botulinum neurotoxin and detects and detects botulinum toxin. In general, the higher the percent identity, the more preferable.
In addition, the polypeptide having the above-mentioned identity includes a polypeptide which specifically binds to a botulinum neurotoxin, including an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and / or added in the polypeptide of the specific amino acid sequence described . In general, the smaller the number of elimination, substitution, insertion, and / or addition is, the more preferable.
As used herein, the term " polynucleotide " (or nucleotide, nucleic acid) encompasses both DNA (gDNA and cDNA) and RNA molecules. Nucleotides, which are basic constituent units in nucleic acid molecules, include not only natural nucleotides, Also included are analogs in which the sugar or base moieties are modified.
The polynucleotide of the present invention is not limited to a nucleic acid molecule encoding the above-described specific amino acid sequence (polypeptide), and may be a polynucleotide having an amino acid sequence exhibiting substantial identity to a specific amino acid sequence as described above or a poly Is interpreted to include nucleic acid molecules encoding the peptides. The above substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence to the greatest correspondence and analyzing the aligned sequence using algorithms commonly used in the art to obtain a homology of at least 60% , More preferably at least 80% homology, and most preferably at least 90% homology.
The polypeptide having the corresponding function includes, for example, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added. Such a polypeptide comprises a polypeptide which is composed of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and / or added as described above and which can specifically bind to a botulinum neurotoxin (particularly, type E) And it is preferable that the number of disappearance, substitution, insertion, and / or addition of amino acid residues is small. In addition, the polypeptide includes an amino acid sequence having an identity of about 60% or more with the specific amino acid sequence described above and having a function of detecting botulinum neurotoxin and diagnosing botulinum toxin. The higher the identity, the better Do.
The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. The vector of the present invention may be a conventional cloning vector or an expression vector, and the expression vector may contain an expression regulatory sequence such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer Signal sequence or leader sequence, and can be produced variously according to the purpose. The vector also includes a selection marker for selecting a host cell containing the vector and, if the vector is a replicable vector, a replication origin.
The transformant of the present invention may be one which has been transformed by said vector. Preferably, the transformant is produced by transforming a vector comprising a polynucleotide encoding the polypeptide of the present invention with a method known in the art, such as, but not limited to, transient transfection, microinjection, Transfection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextranmediated transfection, polybrene-mediated transfection, transfection, electroporation, gene gun, and other known methods for introducing a nucleic acid into a cell. The transformant may be Escherichia coli ), Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Other nasal pillow kusu (Staphylococcus), may be an Agrobacterium tumefaciens Tome Pacific Enschede (Agrobacterium tumefaciens), but is not limited thereto.
In order to easily identify, detect and quantify whether the polypeptide of the present invention binds to botulinum neurotoxin type E, the peptide of the present invention can be provided in a labeled state. That is, they may be provided by linking (e.g., covalently binding or bridging) to a detectable label. The detectable label is a chromogenic enzyme (e.g., peroxidase (peroxidase), alkaline phosphatase (alkaline phosphatase)), radioactive isotopes (for example: 124 I, 125 I, 111 In, 99 mTc, 32 P, 35 S), Such as chromophore, biotin, luminescent material or fluorescent material such as FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, Phycoerythrin, quantum dots, GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein), DsRed (Discosoma sp. Red fluorescent protein), CFP Fluorescent Protein), CGFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), Cy3, Cy5 and Cy7.5). Similarly, the detectable label may be an antibody epitope, a substrate, a cofactor, an inhibitor or an affinity ligand. Such labeling may be performed during the synthesis of the peptide of the present invention, or may be performed in addition to the peptide already synthesized. Fluorescence-mediated tomography (FMT) can be used to detect a fluorescent substance as a detectable marker. For example, the peptide of the present invention labeled with a fluorescent substance can be circulated into the blood and the fluorescence by the peptide can be observed by fluorescence tomography. If fluorescence is observed, it is diagnosed as botulinum toxinosis.
In addition, the present invention provides a kit for detecting botulinum neurotoxin and botulinum toxin comprising the above-mentioned polypeptide. More specifically, a polypeptide comprising the amino acid sequence selected from SEQ ID NOS: 1-8.
As used herein, the term diagnosis is used to identify the presence or characteristic of a pathological condition, to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder Determining the prognosis (e.g., identifying the disease state, determining the stage of the disease or determining the responsiveness of the disease to treatment), or providing information about the therapeutic efficacy of an object that is afflicted with a particular disease or disorder And monitoring the status of the object. For the purpose of the present invention, the presence of botulinum neurotoxin is identified and diagnosed and observed for botulinum toxinosis.
The peptides included in the diagnostic kit of the present invention may have the amino acid sequences shown in SEQ ID NOS: 1 to 8, and may be those obtained by chemical or genetic engineering methods as described above. Since the peptides of the present invention are usefully targeted to the botulinum neurotoxin type E, they can be usefully used for the diagnosis of botulinum toxinosis. In addition to the peptide of the present invention, the diagnostic composition may further contain a buffer or reaction solution which stably maintains the peptide structure or physiological activity. In addition, in order to maintain stability, Lt; RTI ID = 0.0 > C, < / RTI >
The polypeptide may be labeled with any one or more selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, biotin, luminescent materials, and fluorescers as described above, . ≪ / RTI > When the peptide of the present invention is provided without labeling, the kit for the diagnosis of arteriosclerosis of the present invention further comprises a component for searching whether the peptide of the present invention binds to a botulinum neurotoxin in vitro or in vivo . Said component may be a known compound for the labeling of the peptides of the invention or may be an antibody against the peptide of the invention or a secondary antibody thereto and a reagent for detection thereof for searching through an antigen-antibody reaction. At this time, the label of the peptide of the present invention and related compounds are as described above.
3 is an example of a detection chip using a botulinum toxin type E specific binding peptide. The botulinum neurotoxin E-type specific binding peptide is immobilized on the detection chip. When a mixture of botulinum neurotoxin type E and another substance is treated, the botulinum neurotoxin type E is specifically bound and the remaining non-binding substances are released. Subsequent treatment of the fluorescent substance-labeled peptide results in a fluorescence response when bound to the peptide-botulinum neurotoxin E-type complex immobilized on the chip. Detection of the fluorescent signal can detect botulinum neurotoxin E type.
The kit of the present invention may further comprise a tube, a well plate, an instructional material describing a method of use and the like to be used for mixing the components as necessary. Experimental procedures, reagents and reaction conditions which can be used in the above methods can be those conventionally known in the art, which is obvious to a person skilled in the art.
According to another aspect of the present invention, there is provided a chip for detecting a botulinum neurotoxin (particularly, type E) and a botulinum toxinase including a polypeptide. In order to provide the kit and the chip, the polypeptide of the present invention may be immobilized on one or more solid substrates selected from the group consisting of polymers, glass, gold, paper and biological membranes. More specifically, there may be mentioned, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethylcellulose, Polyvinylidene-ether-maleic acid copolymer, amino-acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or Magnetic particles and the like can be used. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The substrate may have any possible shape, such as spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip). When the polypeptide of the present invention is implemented in a chip, one or more polypeptides can be used as a chip in which the polypeptide is arranged at a predetermined position on a solid substrate and immobilized at a high density. The presence or absence of the neurotoxin in the sample can be confirmed by treating the chip with the target sample to determine whether the botulinum neurotoxin (in particular, E type) in the sample and the polypeptide form a complex.
According to another aspect of the present invention, there is provided a method for detecting botulinum neurotoxin and a method for diagnosing botulinum toxin comprising the step of reacting a sample with the above-mentioned polypeptide. More preferably, the polypeptide has the above-mentioned detection label and can bind to the botulinum neurotoxin type E, and the binding of the neurotoxin and the peptide can be detected through a conventional detection method for the detection label.
A sample of the present invention refers to any substance presumably containing or containing a botulinum neurotoxin. The sample may be natural or synthetic and may be obtained by any means known to one of ordinary skill in the art. The sample may be a sample of an in vitro cell culture component such as a tissue, a cell, a whole blood, a plasma, a serum, a blood, a saliva, a sputum, a lymphatic fluid, a cerebrospinal fluid, an intracellular fluid or a urine sample, Recombinant cells, and the like. On the other hand, it may be obtained not only from a sample obtained from a human body, but also from other animals, for example, fish, seafood, or foods using the same.
Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.
1. botulinum neurotoxin type E and phage display
Peptides
Selection of library
1.1. Botulinum neurotoxin E type selection
Botulinum neurotoxin type E consists of heavy chain and light chain and is linked by a disulfide bond. Among the two chains, the light chain is a zinc metalloprotase activity and is a region showing substantial toxicity in the cell. In the present invention, the botulinum neurotoxin E used for screening peptides is C. botulinum BoNT-E Light Chain (R & D Systems), which is a light chain region that is substantially toxic, and has six Histidine There is a His tag.
1.2. Phage Display Peptides library( Phage display peptide library )
The phage display peptide library used in the present invention is " Phage Display Libraries-12 (New England BioLab.) &Quot;, which comprises 12 After artificially sequencing the peptide to express a random amino acid sequence, Escherichia It is composed of recombinant bacteriophages expressing different peptides by infection with E. coli .
2. Specific binding
Peptides
By phage display technique for screening
Panning
process
In the present invention, a phage display technique was used for screening botulinum neurotoxin E-type specific binding peptides, and this was proceeded easily on an eppendorf tube (E-tube). The phage display technique consists of the following steps.
(1) Binding of phage-peptide library to botulinum neurotoxin type E
(2) Separation of phage-botulinum neurotoxin complex using Ni-NTA agarose beads
(3) Cleaning process
(4) Phage collection process
(5) Phage titration course
(6) Phage amplification process
The above procedure is referred to as a panning process. After repeated rounds several times, the base sequence of the phage DNA was analyzed to identify peptides that specifically bind to botulinum neurotoxin type E (see FIG. 1).
2.1 Phage - Peptides Binding process between the library and botulinum neurotoxin type E
The amount of phage injected per round and the concentration of botulinum neurotoxin type E reacting with the phage were kept constant. In this experiment, the initial amount of phage was fixed at 1.95 × 10 11 PFU. The concentration of the botulinum neurotoxin E-type to be reacted with the phage library was fixed at 0.5 nM and the final volume was adjusted to 200 μL using a 1 × PBST (0.1% [v / v] Tween-20) buffer. The phage and botulinum neurotoxin E were present in an Eppendorf tube and reacted for 30 minutes at room temperature to proceed with panning.
2.2 Phage-botulinum neurotoxin complex separation process
The His tag is attached to the N-terminus of botulinum neurotoxin E, the target protein. Utilizing the strong tagging of the His tag with nickel, only the phage-botulinum neurotoxin complex specifically bound to the botulinum neurotoxin E form by Ni-NTA agarose beads was isolated.
The Ni-NTA agarose beads used in the separation procedure were washed with 1X PBST (0.1% [v / v] Tween-20) buffer. After that, Blocking buffer (0.1 M NaHCO 3 (pH 8.6), 5 mg / ml BSA) was added, and the reaction was allowed to proceed for 1 hour by shaking at 50 rpm to proceed blocking of the beads. The blocked beads were washed five times with 1X PBST (0.1% [v / v] Tween-20). The phage library reaction solution bound to botulinum neurotoxin type E was added to washed Ni-NTA agarose beads and allowed to react at room temperature for 15 minutes.
2.3 Cleaning process
After the separation process, 1 ml of 1X PBST (0.1% [v / v] Tween-20) buffer was added to the suspension to remove the phage that did not bind to the target protein, and the mixture was centrifuged (13,000 rpm, , 10 minutes). The above washing process was performed 10 times in total.
2.4 Retrieval of Phases
After the washing process, an acidic solution of pH 2.2 was treated and eluted for 10 minutes in order to elute the phage binding to the botulinum neurotoxin type E to separate the phage and the botulinum neurotoxin E type. After centrifugation (13,000 rpm, 10 minutes, 4 ℃), only the supernatant was recovered and the digestion was obtained. 150 .mu.l of a basic solution of pH 9.0 was treated to neutralize the lowered pH in the elution step, finally recovering the botulinum neurotoxin E-type specific binding protein.
2.5 phage amplification process
To introduce the same amount of phage into the next round, the eluted phage was introduced into host E. coli ER 2738 to amplify the botulinum neurotoxin E type specific binding phage. Initial log phase E. coli To the ER2738 culture medium, 500 쨉 l of a phage eluent was added, followed by shaking culture for 4 hours and 30 minutes (37 캜, 200 rpm). E. coli infected with phage The supernatant was collected by centrifugation (4000 rpm, 30 minutes, 4 ° C) of the ER2738 culture. 20% PEG / 2.5M NaCl solution was added to the recovered supernatant, and the supernatant was allowed to stand overnight at 4 ° C to precipitate the phage. Thereafter, washing with 1X PBS buffer (pH 7.4) was repeated to remove remaining cells. Thereafter, 200 μl of 1 × PBS buffer (pH 7.4) was suspended and the amplified fragments were recovered. However, the phage library that carried out the third round was not amplified.
2.6 Sound selection ( Negative selection ) To remove the nonspecific phage
In addition to botulinum neurotoxin type E, which is a target protein, negative selection was performed between 2 rounds and 3 rounds to remove phage binding to Eppendorf tubes. In the absence of botulinum neurotoxin E, the binding process with the phage library proceeded. During the separation process with Ni-NTA agarose beads, non-pellet-binding supernatant was removed to remove nonspecific binding phage to botulinum neurotoxin type E, But only the combined wave was recovered. After the phage screening, the phage obtained were not subjected to proper titration, but immediately followed by another round with the botulinum neurotoxin E type added thereto.
3. Repetitive
Panning
Fuzzy titration for course progression
3.1. Fuzzy titration Phage titering ) process
The phage obtained through one round of panning and the phage amplified by infecting the host were measured by PFU through the titration process. E. coli 5 ml of the ER2738 culture medium was cultured to the mid-log phase (OD 600 ? 0.5) and 200 占 퐇 was dispensed into Eppendorf tubes. The obtained phages were prepared by dilution stepwise, and 200 μl of
Recovery rate increased from 7.21 × 10 -6 to 1.03 × 10 -4 , especially after negative selection, the amount of phage was increased. This means that the specific binding phage is gradually enriched in the botulinum neurotoxin E type.
4. Identification of random peptide sequences
4.1 Phage amplification process for single strand DNA extraction
Prepare IPTG, X-gal containing LB plate cultured for titration of 3 round phage eluent. The number of blue plaques formed on the LB plate was 100 or less, and the plates were set within 3 days after the incubation. In addition, 300 ml of the E. coli ER2738 seed culture was inoculated into 30 ml of the LB liquid medium, and each ml was dispensed into a test tube. Twenty plaques on the plate were inoculated into each test tube and incubated for 4 hours and 30 minutes with shaking (37 ° C, 200 rpm). After culturing, centrifugation (13,000 rpm, 30 seconds) was repeatedly carried out to recover only the cell-free supernatant and to obtain a purging eluent.
4.2 Single strand DNA extraction of botulinum neurotoxin E-type specific binding phage
200 쨉 l of a 20% PEG / 2.5M NaCl solution was added to 500 쨉 l of the phage eluent which had been amplified, and the phage was precipitated by incubating at room temperature for 20 minutes. The supernatant was then removed by repeated centrifugation (13,000 rpm, 4 캜, 10 min). 100 쨉 l of Iodide buffer was added to remove the surface proteins of the phage. After that, 250 쨉 l of pure ethanol was added, followed by incubation at room temperature for 20 minutes. The supernatant was removed by centrifugation (13,000 rpm, 4 ° C, 10 minutes), and the phage pellet was washed with 70% ethanol. Single-stranded DNA was extracted from the washed pellet by suspending 30 占 퐇 of 1 占 TE buffer in an aliquot.
4.3 Random Peptide Sequencing and Sequencing of the Phage Library
In the single stranded DNA of the extracted phage, the random peptide sequence was amplified by PCR. The PCR reaction composition was 1 μl of template DNA, 5 μl of 10 × PCR buffer, 4 μl of each 2.5 mM dNTP mixture, 2 μl of 10 pmol forward primer, 2 pmol of 10 pmol reverse primer, 0.3 占 퐇 (1 unit / 占 퐇) of Ex Taq polymerase (Takara, Japan) and 35.7 占 퐇 of distilled water. The primers used consisted of the following sequences (Table 3).
(Forward primer)
(Reverse primer)
The PCR reaction conditions were denatured at 95 ° C for 4 minutes, followed by 25 cycles of reaction at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by extension at 72 ° C for 7 minutes Respectively. PCR amplification products were identified by agarose gel electrophoresis and only the band of the predicted product size (184 bp) was cut, gel-purified and confirmed by agarose gel-electrophoresis. After the PCR reaction, 4 μl was taken and 2% agarose gel electrophoresis was performed to confirm whether the band appeared at 184 bp. The random peptide sequence obtained by PCR was recovered using 50 쨉 l of distilled water (see Fig. 2) after cutting only the 184 bp band using Gel purification kit (Nucleogen, Korea).
The amplified random peptide sequence was inserted into a T-vector, and then E. coli DH5a. ≪ / RTI > A random peptide sequence was identified by extracting and sequencing the plasmid in which the random peptide sequence was inserted. A total of 8 peptide sequences were obtained (Table 4).
The obtained peptide can be used as a functional material for detecting botulinum neurotoxin E type which causes serious relaxation paralysis in humans, and can be fixed on a chip, and a kit using the same can be produced (refer to FIG. 3).
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> Industry Academic Cooperation Foundation of Chungbuk National University <120> Botulinum neurotoxin type E specific polypeptides and uses the <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides_1 <400> 1 Ser Ala Leu Lys Gly Leu Phe Pro Ala Asp His His 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides_2 <400> 2 Thr Tyr Thr His Ser Ser Ser Gln His Tyr Gly 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides_3 <400> 3 Ala Asp Trp Tyr His Trp Arg Ser Ser Ser Ser Ser 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides_4 <400> 4 Gly Gln Ser Glu His His Met Arg Val Ala Ser Phe 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides_5 <400> 5 Ser Ser Asp His Arg Thr His Val Thr His Ser Arg 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides-6 <400> 6 Gly Leu Glu Ala Ser Arg His Pro His Gly Ser Trp 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides-7 <400> 7 Gly Leu His Thr Ser Ala Thr Asn Leu Tyr Leu His 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Botulinum neurotoxin type E specific polypeptides_8 <400> 8 Ser Thr Pro Gly Cys Cys Ala His Asp His Phe Arg 1 5 10
Claims (10)
Wherein the polypeptide is labeled with any one or more selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, biotin, luminescent materials, and fluorescers, and the detection of botulinum neurotoxin E and botulinum toxin Kit for the diagnosis of toxicosis.
Wherein the substrate on which the polypeptide is immobilized is at least one selected from the group consisting of polymer, glass, gold, paper and biological membrane as a solid substrate, and a chip for diagnosing botulinum toxin E detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018124436A1 (en) * | 2016-12-29 | 2018-07-05 | 한국표준과학연구원 | Sensor for detection of botulinum toxin using spun carbon nanotube sheet |
| WO2020232407A1 (en) * | 2019-05-16 | 2020-11-19 | Trustees Of Tufts College | Peptide antibiotics and methods of use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080068069A (en) | 2005-10-28 | 2008-07-22 | 토소겐 게엠베하 | Botulinum neurotoxin a protein receptor and uses thereof |
| WO2010014854A2 (en) * | 2008-07-31 | 2010-02-04 | The Regents Of The University Of California | Antibodies that neutralize botulinum neurotoxins |
| KR20140071238A (en) | 2012-12-03 | 2014-06-11 | 에스알씨, 인코퍼레이트 | Real-time assay for the detection of botulinum toxin |
| KR20150098394A (en) | 2014-02-20 | 2015-08-28 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Primers set for simultaneously detecting the neurotoxins of Clostridium botulinum and multiplex PCR kits using the same |
-
2015
- 2015-11-09 KR KR1020150156393A patent/KR101670188B1/en active IP Right Grant
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080068069A (en) | 2005-10-28 | 2008-07-22 | 토소겐 게엠베하 | Botulinum neurotoxin a protein receptor and uses thereof |
| WO2010014854A2 (en) * | 2008-07-31 | 2010-02-04 | The Regents Of The University Of California | Antibodies that neutralize botulinum neurotoxins |
| KR20140071238A (en) | 2012-12-03 | 2014-06-11 | 에스알씨, 인코퍼레이트 | Real-time assay for the detection of botulinum toxin |
| KR20150098394A (en) | 2014-02-20 | 2015-08-28 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Primers set for simultaneously detecting the neurotoxins of Clostridium botulinum and multiplex PCR kits using the same |
Non-Patent Citations (9)
| Title |
|---|
| 1. Gu S et al., Science, 335(6071), pp.997-981(2012). |
| 2. Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983. |
| 3. Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997. |
| 4. A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989. |
| 5. Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982. |
| 6. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second(1998) and Third(2000) Edition. |
| 7. Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif,1991. |
| 8. Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990. |
| Zou Q. et al, Analytical Biochemistry 494:pp.4-9 (2015.10.30.).* * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018124436A1 (en) * | 2016-12-29 | 2018-07-05 | 한국표준과학연구원 | Sensor for detection of botulinum toxin using spun carbon nanotube sheet |
| KR20180077803A (en) * | 2016-12-29 | 2018-07-09 | 한국표준과학연구원 | Sensor for detecting botulinum neurotoxin using spinning carbon-nanotube |
| KR101903255B1 (en) * | 2016-12-29 | 2018-10-02 | 한국표준과학연구원 | Sensor for detecting botulinum neurotoxin using spinning carbon-nanotube |
| WO2020232407A1 (en) * | 2019-05-16 | 2020-11-19 | Trustees Of Tufts College | Peptide antibiotics and methods of use thereof |
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