METHOD FOR THE DETECTION OF CLOSTRIDIUM NEUROTOXINS USING A NOVEL SUBSTRATE
20220390461 · 2022-12-08
Assignee
Inventors
Cpc classification
C07K14/705
CHEMISTRY; METALLURGY
C07K2319/60
CHEMISTRY; METALLURGY
G01N33/542
PHYSICS
G01N33/48714
PHYSICS
International classification
Abstract
The present invention provides a method for detecting and assaying Clostridium neurotoxins and identification of serotypes of botulinum neurotoxins in various food matrices and clinical samples. This method is also used for detection of BoNT inside the neuronal and epithelial cells. The method comprises detecting and assaying the presence of a Clostridium neurotoxin in a sample by: exposing the sample containing a Clostridium neurotoxin to a sample comprising a novel SNAMPXIN/SNAMP universal recombinant substrate fusion protein capable of producing a detectable FRET, following cleavage; detecting and assaying the presence of the Clostridium neurotoxin by measuring a change in the energy transfer or the luminescence signal; and detecting and assaying an electrophoretic mobility pattern of one or more cleaved protein bands or a degraded protein, using a high throughput automated system to identify the different serotypes of the Clostridium neurotoxin. SNAMPXIN/SNAMP is formed from parts of BoNT substrates SNAP-25 and VAMP.
Claims
1. A method for detecting and assaying the presence of a Clostridium neurotoxin in a sample comprising: a. exposing the sample containing a Clostridium neurotoxin to a sample comprising a universal recombinant substrate fusion protein capable of producing a detectable Foster resonance energy transfer, or a luminescence signal change, following cleavage of recombinant substrate fusion protein by the Clostridium neurotoxin; b. detecting and assaying the presence of the Clostridium neurotoxin by measuring a change in the Foster resonance energy transfer or the luminescence signal in the sample; and c. determining an electrophoretic mobility pattern of one or more cleaved protein bands or a degraded protein, using a high throughput automated system to identify the different serotypes of the Clostridium neurotoxin.
2. The method of claim 1, wherein the Clostridium neurotoxin is a tetanus neurotoxin or a botulinum neurotoxin, or their natural or artificial variants.
3. The method of claim 2, wherein the botulinum neurotoxin is a botulinum neurotoxin serotype A, B, C, D, E, F or G, and/or their natural and artificial variants.
4. The method of claim 3, wherein when the botulinum neurotoxin is the serotype A or C, the serotype is differentiated using the steps comprising: a. exposing the sample containing the botulinum neurotoxin A or C to a SNAMPAXIN recombinant protein that is capable of producing the detectable foster resonance energy transfer or the luminescence signal change following cleavage of the recombinant SNAMPAXIN by the botulinum neurotoxin; b. wherein the detection of the presence of the Clostridium neurotoxin by measuring a change in Foster resonance energy transfer or luminescence signal in the sample further comprises using a SNAMP substrate; and c. wherein the detection of the electrophoretic mobility pattern of the cleaved protein bands or the degraded protein, using a high throughput automated system, further comprises utilizing an artificial neural network to identify the serotype of Clostridium neurotoxin.
5. The method of claim 2, wherein the botulinum neurotoxin is in an active form.
6. The method of claim 1, wherein the universal recombinant substrate fusion protein comprises: a SNAP-25 amino acid sequence; and a VAMP protein is in between a FRET or luminescence reporter to provide SNAMP recombinant substrate fusion protein.
7. The method of claim 6, wherein the universal recombinant substrate fusion protein comprises: a. a fragment of a human SNAP-25 amino acid sequence of as set forth in SEQ ID NO: 1; b. a fragment of the human VAMP amino acid sequence of as set forth in SEQ ID NO: 4; c. optionally, comprising a human syntaxin amino acid sequence as set forth in SEQ ID NO: 5; and/or d. a Foster resonance energy transfer (FRET) or a luminescence reporter.
8. The method of claim 7, wherein the fragment of the human SNAP-25 sequence is the amino acid 141 to the amino acid 206 of SEQ ID NO: 1.
9. The method of claim 7, wherein the fragment of the human VAMP sequence is the amino acid 27 to the amino acid 94 of SEQ ID NO: 4.
10. The method of claim 7, wherein the foster resonance energy transfer (FRET) reporter is an Enhanced Green Fluorescence Protein (EGFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP) or combinations thereof.
11. The method of claim 7, wherein the universal recombinant substrate fusion protein comprises: a. the fragment of the human SNAP-25 sequence from the amino acid 141 to the amino acid 206 of SEQ ID NO: 1; b. the fragment of the human VAMP sequence of SEQ ID NO: 4 from the amino acid 27 to the amino acid 94 of SEQ ID NO: 4; and c. to create a SNAMP universal recombinant substrate fusion protein.
12. The method of claim 7, wherein the SNAMP universal recombinant substrate fusion protein comprises the amino acid sequence of SEQ ID NO: 2.
13. The method of claim 7, wherein the universal recombinant substrate fusion protein comprises: the fragment of the human SNAP-25 sequence of SEQ ID NO: 1; the fragment of the human VAMP sequence of SEQ ID NO: 4; and a foster resonance energy transfer (FRET) or luminescence reporter to provide a SNAMP universal recombinant substrate fusion protein.
14. The method of claim 1, wherein the universal recombinant substrate fusion protein comprises: the fragment of the human SNAP-25 sequence of SEQ ID NO: 1; the fragment of the human VAMP sequence of SEQ ID NO: 4; and the syntaxin protein of SEQ ID NO: 5 to provide SNAMPXIN recombinant substrate fusion protein of SEQ ID NO: 3.
15. The method of claim 7, wherein the universal recombinant substrate fusion protein comprises: the fragment of the human SNAP-25 of SEQ ID NO: 1; fragment of the human VAMP of SEQ ID NO: 4; the syntaxin protein of SEQ ID NO: 5; and, a Foster resonance energy transfer (FRET) or Luminescence reporter to provide an another recombinant universal substrate fusion protein SNAMPAXIN.
16. The method of claim 7, wherein the universal recombinant substrate fusion protein comprises human syntaxin amino acid sequence as set forth in SEQ ID NO: 5;
17. The method of claim 16, wherein the fragment of the human syntaxin sequence is the amino acid 221 to the amino acid 288 of SEQ ID NO: 5.
18. The method of claim 4, wherein the universal recombinant substrate fusion protein is expressed in E. coli using the vector of
19. The method of claim 4, wherein the universal recombinant substrate fusion protein is optionally tagged by GST or Histidine.
20. The method of claim 4, wherein the universal recombinant substrate fusion protein comprises an amino acid sequence of SEQ ID NO: 3.
21. The method of claim 1, wherein the sample is a food sample, or a blood, or a serum sample from a human or an animal.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0094]
[0095]
[0096]
[0097]
DETAILED DESCRIPTION OF THE INVENTION
[0098] The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein.
[0099] The present invention employs the use of a novel universal substrate of the present invention for the detection of Clostridium neurotoxin, especially serotypes of botulinum neurotoxin (BoNT). Clostridium botulinum produces seven serotypes of botulinum neurotoxins (A-G). The 150 kDa BoNT consists of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) linked through a di-sulfide bond. The HC plays an accessory role of binding to neuronal cells and the trafficking of the LC into the cytosol. The intracellular toxic action of the BoNT is caused by the endopeptidase activity of the LC, which is a zinc metalloprotease enzyme. A unique feature of the catalytic domain of BoNT is that while the seven BoNT serotypes contain identical Zn.sup.2+ binding motif in the active site, the substrates are either different proteins, or have different cleavage sites within the same protein. Each of the BoNT serotypes is extremely substrate selective (except for BoNT/C) with specific cleavage sites as shown in prior art Table 1. Specificity of cleavage site comes from the fact that the enzyme recognizes the tertiary structure of its substrate.
TABLE-US-00001 TABLE 1 Prior Art Table 1: Substrates and cleavage sites of different serotypes of botulinum and tetanus toxin Toxin Type Substrate Cleavage Site BoNT/A SNAP-25 Gln.sup.197-Arg.sup.198 BoNT/B VAMP Gln.sup.76-Phe.sup.77 BoNT/C SNAP-25 Arg.sup.198-Ala.sup.199 BoNT/D VAMP Lys.sup.59-Leu.sup.60 BoNT/E SNAP-25 Arg.sup.180-Ile.sup.181 BoNT/F VAMP Gln.sup.58-Lys.sup.59 BoNT/G VAMP Ala.sup.81-Ala.sup.82 TeNT VAMP Gln.sup.76-Phe.sup.77 BoNT/C Syntaxin Lys.sup.253-Ala.sup.254
[0100] Due to the requirement of the tertiary structure for substrate recognition, the use of short peptides with cleavage site does not provide the specificity and recognition required for the detection of the active toxic enzyme. Therefore, a novel substrate has been developed for the detection and identification of Clostridium toxin, especially all BoNT serotypes causing human botulism. The substrate developed is not only capable of detecting and assaying the presence of Clostridium neurotoxin; but, it also helps in detecting and assaying different serotypes of BoNT and tetanus. For this the present inventors have developed novel SNAMP (SEQ ID NO: 2) and SNAMPAXIN chimeric protein (SEQ ID NO: 3).
Preparation of SNAMP
[0101] The present invention comprises a designed, cloned, expressed, and purified chimeric protein herein referred to as “SNAMP” of SEQ ID NO: 2; and comprising: SNAP25 (141-206)-VAMP (27-94)), which has cleavage sites for all the serotypes of BoNT and tetanus neurotoxin. This chimeric protein is tagged with GST for purification. Another embodiment of this invention is to clone the SNAMP gene in the His(6x) containing pBR322 vector containing tac promoter to obtain a His-tagged SNAMP, using standard cloning protocols that are readily known to those skilled in the art and widely carried out in laboratories (Li and Singh, 1999). Using a pattern recognition method of cleavage products of SNARE, chimeric protein detection of Clostridium toxin and serotype of BoNT was achieved. The design of this plasmid is shown in
[0102] In the preferred embodiment, the cell paste (from 1 litre culture) was suspended in about 15 ml, 1×PBS pH 7.4 (called basic buffer), added with protease inhibitors. After sonication, the cell lysate was centrifuged at 12,000 rpm for about 1 hour by using Thermo Scientific Sorvall Legend RT Centrifuge and FIBERLite F15-8x50C rotor, then the supernatant is poured into a clean tube. The extract is thus obtained and is loaded to the pre-equilibrated GST or Ni-NTA column.
[0103] After loading to the GST or Ni-NTA column, 1×PBS or 10 mM imidazole was added to the basic buffer as a washing step, respectively. Then protein binding to the column was eluted by either by 20 mM glutathione (in case of GST tag) or 200 mM imidazole (in case of His-tag). Next, the pool was combined, which had less contaminated bands; and the pool was concentrated by using Centriprep-30. After concentration, the protein concentration was measured by UV. Purification results of SNAMP obtained in this embodiment are shown in
Cleavage Assay
[0104] As mentioned supra, BoNT serotypes cleave their unique substrates at different sites, thus generating a different pattern of cleaved products on a monitoring platform. In a set of experiments to demonstrate endopeptidase activity of three different serotypes and forms of BoNT and TeNT, 50 nM of the enzyme was incubated with 5 μM of SNAMP substrate in a reaction buffer (1×PBS, pH 7.4) containing 2 mM DTT. The reaction was allowed for 1 hr at 37° C. and was stopped with the addition of 0.8×SDS-PAGE sample buffer. The reaction was monitored on a SDS-PAGE gel (
Cellular Assay System
[0105] To build a universal BoNT sensor that can detect the cleavage of all the serotypes of botulinum toxin inside a sensitive neuronal cell, the plasmids with AcGFP-SNAMP-DsRed or variant constructs are transfected into primary rat spinal cord neuron cells (RSpN). Reasons for selecting the primary cell, RSpN, are as follows: a) it is a highly sensitive cell for toxin detection (BoNT/A sensitivity (EC50) is 0.3-1.0 unit (Pellett, 2013); b) it has significant expression of all the receptors required for BoNT uptake; and c) it is a perfect system to develop rapid detection of SNARE cleavage.
[0106] To culture RSpN cells, a 24 well plate is first coated with Neuron coating solution I (Cell Application Inc., CA). Thawed RSpN cells (Cell Application Inc., CA) are transferred into a 50 ml tube and Rat Neuron Plating medium (Cell Application Inc., CA) is added. After gentle mixing, the cell suspension in the 50 ml tube is pipetted and aliquoted into each well of the pre-coated 24-well plate. Cells are grown in a 37° C., 5% CO.sub.2 humidified incubator. After 24 hours (h), media is changed to Rat Neuron Culture medium (Cell Application Inc., CA). Cells are maintained by changing half of the Rat Neuron Culture medium every three day. After maturation, cells are transfected with plasmids. For transfection, the NeuroPORTER (Amsbio, CA) transfection reagent is used and transfection of the construct is performed according to the manufacturer's protocol. After two hours of incubation (with the mixture of transfection reagents and plasmid), one additional volume of fresh culture medium containing a 2× concentration of B27 onto the cells is added. This assay can readily be developed by an artisan in 6-well, 12-well or 96-well formats, too, depending on the requirement.
[0107] Measurements of energy transfer are used for measuring the intracellular enzymatic activity. The FRET mechanism is by the fluorescence emission spectrum of the energy donor chromophore overlaps the absorption spectrum of the energy acceptor chromophore, and distance between donor and acceptor should be within 10-100 Å. In one of the constructs, GFP-SNAMP-RFP, GFP and RFP are the donor and acceptor, respectively, with a Foster distance of the pair as 57 Å. The GFP-RFP FRET pair is successfully used (Basavanna et al., 2013) for the full-length SNAP-25 with 206 residues, and also with the SNAMP substrate of 134 residues. The endpoint of the FRET analysis obtains significant FRET frequency (I.sub.FRET/I.sub.D should be greater than I.sub.FRET/I.sub.A, where I, D and A is intensity, donor and acceptor, respectively) or fluorescence emission ratio from both reporters after incubation with different concentrations of BoNT toxins for 24 and 48 h. This helps in reducing the time by 50-75% compared to MBA.
[0108] For faster detection of the toxins, the cultures are incubated with stimulation buffer; 10 mM HEPES (pH 7.4) containing 56 mM KCl, 82 mM NaCl, 2 mM CaCl.sub.2, and 1 mM MgCl.sub.2 is adjusted to 325 mOsm with sucrose for 4 min at 37° C., along with BoNT. BoNT-containing buffer is then removed by aspiration and cells rinsed with toxin free minimal essential medium (MEM). The culture is incubated at 37° C. in MEM for an additional few hours (6-12 h, depending on the optimized toxin concentration) and used for further experimentation.
[0109] The GFP/RFP construct design is based on the following two considerations:
a) long excitation wavelength of GFP (484 nm), which aid in preventing phototoxicity; and b) brightness of GFP/RFP signal, which provides higher signal to noise ratio. The RFP photo may get bleached easily, which can reduce sensitivity or give a false reading. This is avoided by either having a shorter exposure time at a higher intensity of excitation light, or having a less intense excitation light with a longer exposure.
[0110] SNAMPAXIN is purified using similar a protocol as SNAMP. The protein sequence of SNAMPAXIN is listed in SEQ ID NO: 3.
[0111] SNAP25 is the substrate for BoNT/A and BoNT/C toxin. But the cleavage site is just one amino acid apart. So, by using SNAMPAXIN, the artisan can distinguish between these two toxins in the sample. Whereas SYMPAXIN has both SNAP25 and Syntaxin. In SDS-PAGE we will have one extra band at ˜12 kDa for the cleavage product by BoNT/C.
Example for Detection of Toxin in a Food Sample
[0112] Food samples are processed to obtain a clear aqueous solution to be tested for toxin detection. SNAMP, or its derivatives, is prepared in a reaction mixture of about 500 ul at a concentration of (1-2 uM). 50-100 ul of test aqueous solution is mixed with the SNAMP solution and incubated for 30-120 min at 37° C. The reaction is stopped with SDS-PAGE sample buffer. The sample is boiled for 1 min before loading on to a polyacrylamide gel for electrophoresis. Electrophoretic protein bands are examined after staining with Coomassie blue for detection of toxin and recognition of serotype.
Advantages of the Invention
[0113] Present invention gives a novel SNAMP and SNAMPAXIN universal substrate. The substrate can be cleaved by all the known substrate of BoNT (A-G). The present invention also provides a novel process for the preparation of the universal substrate for in vitro and intracellular assay. The present invention also provides a method for detection of BoNT in clinical and food matrices. The present invention uses cell system containing the new SNAMP or SNAMPAXIN substrate for the assay of different serotypes of BoNT to replace MBA.
Conclusion
[0114] Accordingly, the preceding exemplifications merely illustrate the principles of the various embodiments. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the embodiments and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the various embodiments, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.
[0115] The transitional term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
[0116] Or, the technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.
[0117] The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
[0118] The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 5%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3).
[0119] As used herein, the term “substantially” refers to approximately the same shape as stated.
[0120] While several embodiments of the disclosure have been described, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Therefore, the above description should not be construed as limiting, but merely as exemplifications of embodiments.
[0121] Trademarks: the product names used in this document are for identification purposes only; and are the property of their respective owners.
LIST OF REFERENCES CITED
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[0124] Basavanna, U., Muruvanda, T., Brown, E. W., and Sharma, S. K. (2013). Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E. International Journal of Microbiology, Article ID 593219, 1-7.
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[0127] Li, L., and Singh, B. R. (1999). In vitro translation of type A Clostridium botulinum neurotoxin heavy chain and analysis of its binding to rat synaptosomes. Journal of Protein Chemistry, 18, 89-95.
[0128] Pellett S. (2013). Progress in cell-based assays for botulinum neurotoxin detection. Current topics in microbiology and immunology, 364, 257-285.
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[0130] Straughan D (2006) Progress in applying the Three Rs to the potency testing of botulinum toxin type A. Altern Lab Anim 34: 305-313.
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[0132] Whitemarsh, R. C., Strathman, M. J., Chase, L. G., et al., (2012) Novel Application of Human Neurons Derived from Induced Pluripotent Stem Cells for Highly Sensitive botulinum Neurotoxin Detection, Toxicological Sciences, 126 (2), 426-435.
[0133] Whitemarsh, R. C., Tepp, W. H., Bradshaw, M., et al. (2013). Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro. Infection and immunity, 81(10), 3894-3902.
OTHER REFERENCES CITED
[0134] US patent application 20140235490 by City of Hope that was published on Aug. 21, 2014.
[0135] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the scope of the appended claims.