RECOMBINANT POLYNUCLEOTIDE CODING FOR POLYPEPTIDE COMPRISING REPORTER MOIETY, SUBSTRATE MOIETY AND DESTABILIZING MOIETY, HOST CELL COMPRISING SAME AND USE OF SAME

Abstract

Provided are a recombinant polynucleotide encoding a polypeptide including a reporter moiety, a substrate moiety, and a destabilization moiety, a host cell including the same, and use thereof to measure the level of a protease by using the recombinant polynucleotide.

Claims

1. A recombinant polynucleotide comprising a first polynucleotide that encodes a polypeptide that includes: a reporter moiety; a destabilization moiety; and a substrate moiety operatively linking the reporter moiety at an N-terminus to the destabilization moiety at a C-terminus, wherein the substrate moiety comprises a cleavage site of protease activity.

2. The recombinant polynucleotide of claim 1, wherein the protease is a neurotoxin polypeptide, and the neurotoxin polypeptide is botulinum toxin serotype A (BoNT/A), BoNT/B, BoNT/C, BoNT/CD, BoNT/D, BoNT/DC, BoNT/E, BoNT/F, BoNT/FA, BoNT/G, or tetanus neurotoxin (TeNT).

3. The recombinant polynucleotide of claim 1, further comprising a polynucleotide encoding: a bicistronic sequence linked upstream or downstream of the first polynucleotide; and an internal standard reporter operably linked upstream or downstream of the bicistronic sequence.

4. The recombinant polynucleotide of claim 3, wherein the bicistronic sequence is a internal ribosomal entry site (IRES) sequence or a nucleotide sequence that allows ribosome to skip forming a peptide bond.

5. A host cell containing the recombinant polynucleotide of claim 1.

6. The host cell of claim 5, wherein the host cell is a cell that is capable of translocating a neurotoxin polypeptide into a cytoplasm.

7. The host cell of claim 5, wherein the host cell is NT2 cell, SiMa cell, or NG108-15 cell.

8. The host cell of claim 5, wherein the host cell comprises an exogenous polynucleotide encoding a protease, for example, a neurotoxin polypeptide having proteolytic activities.

9. A method of determining proteolytic activities of a neurotoxin polypeptide in a sample, the method comprising contacting the host cell of claim 5 with a sample suspected of containing a neurotoxin polypeptide; and measuring a signal emitted by the reporter moiety in a product obtained by the contacting.

10. The method of claim 9, wherein the recombinant polynucleotide further comprises a polynucleotide encoding a bicistronic sequence linked upstream or downstream of a first polynucleotide and an internal standard reporter operably linked upstream or downstream of the bicistronic sequence.

11. The method of claim 9, further comprising, prior to the contacting, culturing the host cell in a culture medium to differentiate into neuronal cells.

12. The method of claim 11, wherein the host cell is NT2 cell, SiMa cell, or NG108-15 cell.

13. A method of measuring an ability of a host cell in expressing or inhibiting a protease, the method including: introducing a polynucleotide encoding a protease polypeptide into the host cell of claim 5; and culturing the host cell to which the polynucleotide has been introduced and measuring signals emitted by the reporter moiety or the internal standard reporter or a product of the reporter moiety or the internal standard reporter in a culture.

14. The method of claim 13, wherein the culturing is performed in the presence of a test material.

Description

DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 shows a view illustrating the structure of a recombinant polynucleotide according to an aspect.

[0054] FIG. 2 shows the structure of a recombinant polynucleotide according to an aspect and a reaction between a polypeptide expressed thereby and a protease.

[0055] FIG. 3 shows a case where a recombinant polynucleotide according to an aspect contacts a protease in a cell.

[0056] FIG. 4 shows a view showing a case where a recombinant polynucleotide having a nucleotide sequence encoding an internal standard reporter according to an aspect is in contact with a protease in a cell.

[0057] FIG. 5 shows measurements of activities of a reporter moiety after NG108-15 cells transfected with pFB vector 1 were differentiated into neurons and then intoxicated with botulinum toxin A.

[0058] FIG. 6 shows fluorescent microscopic images of NG108-15 cells transfected with pFB vector 2 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A.

[0059] FIG. 7 shows measurements of activities of a reporter moiety of NG108-15 cells transfected with pFB vector 3 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A.

[0060] FIG. 8 shows measurements of activities of a reporter moiety of SiMa cells transfected with pFB vector 3 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A.

[0061] FIG. 9 shows measurements of activities of a reporter moiety of NT2 cells transfected with pFB vector 3 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A.

[0062] FIG. 10 shows measurements of activities of a reporter moiety and an internal standard reporter protein each expressed by NG108-15 cells transfected with pFB vector 6 after the cells were differentiated into neurons and then transient-transfected with pCDNA4_BLC vector expressing a light chain of botulinum toxin B.

[0063] FIG. 11 shows measurements of signals emitted by a reporter moiety protein expressed by NT2 cells transfected with pFB vector 2 after the cells are established as a stabilized monoclonal-derived cell line, differentiated into neurons, and then, intoxicated with different concentrations of BoNT/A.

[0064] FIGS. 12 to 14 show measurements of signals emitted by a reporter moiety protein or an internal standard reporter each expressed by NT2 cells transfected with pFB vector 2 after the cells are established as a stabilized monoclonal-derived cell line, differentiated into neurons, and then, intoxicated with different concentrations of BoNT/A.

MODE OF THE INVENTION

[0065] Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1

[0066] Preparation of recombinant polynucleotide encoding a reporter moiety-substrate moiety-destabilization moiety (hereinafter also referred to as R-S-D) polypeptide and cells containing the recombinant polynucleotide

(1) Preparation of Recombinant Polynucleotide Encoding a Reporter Moiety-Substrate Moiety-Destabilization Moiety (Hereinafter Also Referred to As R-S-D) Polypeptide

[0067] Two recombinant polynucleotide having different structures were prepared. One is to encode a polypeptide having an R-S-D structure and the other is to encode a polypeptide having an I-B-R-S-D structure. Here, I indicates a nucleotide sequence encoding an internal standard reporter, and B indicates a bicistronic sequence. The recombinant polynucleotide is in the form of a plasmid vector.

[0068] A nucleotide sequence encoding a luminescence or fluorescence protein was used as the internal standard reporter or the reporter moiety. Examples of the sequence encoding the luminescence protein are a nucleotide sequence (for example, SEQ ID NO: 1) encoding a firefly luciferase (FLuc) protein (for example, SEQ ID NO: 9) and a nucleotide sequence (for example, SEQ ID NO: 2) encoding a nano-luciferase (NLuc) protein (SEQ ID NO: 10), and examples of the sequence encoding the fluorescence protein are a nucleotide sequence (SEQ ID NO: 3) encoding an enhanced green fluorescence protein (eGFP) (SEQ ID NO: 11) or a nucleotide sequence (SEQ ID NO: 4) encoding an mCherry protein (SEQ ID NO: 12).

[0069] The bicistronic sequence used herein is a nucleotide sequence (P2A) (SEQ ID NO: 5) encoding a self-cleaving peptide derived from porcine teschovirus-1.

[0070] The substrate moiety of a proteolytic enzyme used herein is, in the case of botulinum toxin type A, a nucleotide sequence (SEQ ID NO: 6) encoding an amino acid sequence (SEQ ID NO: 14) from 145 to 207 of the total of 207 amino acid sequence as a C-terminal region of human SNAP25, or, in the case of botulinum toxin type B, a nucleotide sequence (SEQ ID NO: 17) encoding human VAMP2 (SEQ ID NO: 18).

[0071] The destabilization moiety used herein is a nucleotide sequence (SEQ ID NO: 8) encoding the PEST sequence (SEQ ID NO: 16) and a nucleotide sequence (SEQ ID NO: 7) encoding CLI sequence (SEQ ID NO: 15).

[0072] The recombinant polynucleotide was constructed as follows:

[0073] First, a nucleotide sequence (SEQ ID NO: 1) (hereinafter referred to as FLuc sequence) encoding firefly luciferase (FLuc)(SEQ ID NO: 9) was obtained from pGL4.31 vector (Promega), a nucleotide sequence (SEQ ID NO: 2) (hereinafter referred to as NLuc sequence) encoding nano-luciferase (NLuc) (SEQ ID NO: 10) was obtained from pNL1.1 vector (Promega), a nucleotide sequence(SEQ ID NO: 3)(hereinafter referred to as eGFP sequence) encoding enhanced green fluorescence protein (eGFP) (SEQ ID NO: 11) was obtained from pEGFP-C1 vector (clontech), and a nucleotide sequence (SEQ ID NO: 4)(hereinafter referred to as mCherry sequence) encoding mCherry (SEQ ID NO: 12) was obtained from pmCherry-C1 vector. A nucleotide sequence (SEQ ID NO: 6) encoding an amino acid sequence from 145 to 207 of the total of 207 amino acid sequence as a C-terminal region of human SNAP25 (hereinafter referred to as SNAP25 sequence), and a nucleotide sequence encoding human VAMP2 (hereinafter referred to as VAMP2 sequence) were synthesized by RT-PCR from mRNA of human-derived cell lines. A sequence encoding CL1-PEST polypeptide in which CL1 is fused with PEST (hereinafter referred to as CL1-PEST sequence) and a nucleotide sequence encoding a self-cleaving peptide P2A derived from porcine teschovirus-1 (hereinafter referred to as P2A sequence) were obtained by gene synthesis.

[0074] Next, recombinant polynucleotides having the structure of R-S-D or I-B-R-S-D were constructed by gene cloning using restriction enzyme using a combination of the sequences, and ligated to the BarnHI/XhoI restriction site of the pCDNA4 vector (Invitrogen) or pFB vector (Agilent) to construct recombinant vectors. For example, a vector (hereinafter referred to as pCDNA4 vector 1) in which a recombinant polynucleotide consisting of NLuc sequence, SNAP25 sequence, and CLI-PEST sequence is introduced to BamhI/XhoI enzyme site of pCDNA4 vector or a vector (hereinafter referred to as pFB vector 1) in which a recombinant polynucleotide consisting of NLuc sequence, SNAP25 sequence, and CL1-PEST sequence is introduced to BamHI/XhoI enzyme site of pFB vector were constructed. For example, a vector (hereinafter referred to as pCDNA4 vector 2) in which a recombinant polynucleotide consisting of mCherry sequence, SNAP25 sequence, and CL1-PEST sequence is introduced to BamH/IXhoI enzyme site of pCDNA4 vector or a vector (hereinafter referred to as pFB vector 2) in which a recombinant polynucleotide consisting of mCherry sequence, SNAP25 sequence, and CL1-PEST sequence is introduced to BamHI/XhoI enzyme site of pFB vector were constructed. For example, a vector (hereinafter referred to as pCDNA4 vector 3) in which a recombinant polynucleotide consisting of FLuc sequence, P2a sequence, NLuc sequence, SNAP25 sequence, and CL1-PEST sequence is introduced to BamHI/XhoI enzyme site of pCDNA4 vector or a vector (hereinafter referred to as pFB vector 3) in which a recombinant polynucleotide consisting of FLuc sequence, P2a sequence, NLuc sequence, SNAP25 sequence, and CL1-PEST sequence is introduced to BamHI/XhoI enzyme site of pFB vector were constructed. A pFB vector (hereinafter referred to as pFB vector 4) in which a recombinant polynucleotide consisting of FLuc sequence, P2a sequence, NLuc sequence, VAMP2 sequence, and CLI-PEST sequence is introduced to BamH/XhoI enzyme site of pFB vector, was constructed.

[0075] Here, the pCDNA4 vector is a plasmid-derived vector and used to transiently express the recombinant polynucleotides in cells. The pFB vector used herein was a vector derived from moloney murine leukemia virus (MMLV), and used to construct a cell line modified by transfection, that is, a cell line in which the recombinant polynucleotides were introduced into a chromosome.

[0076] The structures of the constructed plasmid vectors and the viral vectors were confirmed by sequencing, and the plasmid vector DNA was purified and isolated to the level for cell culture.

(2) Production of Cells Expressing R-S-D or I-B-R-S-D Polypeptide

[0077] In order to construct a cell line that stably and constantly expressing the vectors constructed in (1), transduction with MMLV-derived viral vector, that is, pFB vector, was performed. The transfection with MMLV-derived viral vector is a widely practiced method in the art, and the reference (Felts Ka et al., Mol Biotechnol. 2002 Sep. 22(1):25-32.). Briefly, HEK-293T cells (ATCC-CRL-3216), a cell for packaging, were seeded in 10 ml of DMEM medium containing 10% fetal bovine serum (FBS) at the population of 1?10.sup.7 cells in a 55 cm.sup.2 culture dish, and cultured for 24 hours in a 37? C. and 5% CO.sub.2 incubator, and then, the recombinant polynucleotide pFB vector according to the present invention, that is, pFB vector 1, 2, 3, or 4, and pCMV-gagpol (Cell Biolabs, inc.) and pCMV-VSV-G (Cell Biolabs, inc.) were mixed at a ratio of 3:2:1. At this time, pFB vector was used in an amount of 7.5 ?g. The mixtures of this vector was used for transfection with Lipofectamine? 3000 (Invitrogen) and the transduction method was performed according to the manufacturer's instructions. Cells were cultured under the same conditions for 48 hours after transduction. Thus, an infectious packaged pFB vector 1,2,3 or 4-containing virus was produced from the HEK 293T cells.

[0078] Then, the infectious packaged pFB vector 1,2,3 or 4-containing virus was transfected into a target cell to produce a cell line that enables stable, constant expression. The target cell used herein was NG108-15 cell(ATCC-HB-12317), SiMa cell(DSMZ: ACC-164), and NT2 cell(ATCC-CRL-1973). NG108-15 cells are hybrid cells of neuroblastoma of rat and glioma of rat, and a cell line that has been identified as susceptible to botulinum toxin type A through differentiation into neurons (Whitemarsh RC et al., Biochem Biophys Res Commun. 2012 Oct. 19;427(2):426-30). SiMa cells are a cell line that is derived from human neuroblastoma and have been identified as susceptible to botulinum toxin type A through differentiation into neurons (Fernandez-Salas E et al., PLoS One. 2012;7(11):e49516). NT2 cells are an embryonal carcinoma cell line derived from human testicles, have multiple differentiation potential, and may undergo differentiation into neurons under specific conditions, and when differentiated into neurons, the NT2 cells were found to be susceptible to botulinum toxin type A (Tegenge et al., Cell Mol Neurobiol. 2012 Aug;32(6):1021-9).

[0079] The specific procedure of a cell line that expresses a target polypeptide stably is as follows.

[0080] First, a target cell was seeded in a 24-well plate containing 0.5 ml of DMEM medium containing 10% FBS at the population of about 1?10.sup.5 cells per well one day before infection, and then, cultured in a 5% CO.sub.2 incubator at a temperature of 37? C. for one day. The culture supernatant of HEK-293T cells collected therefrom was filtered using a 0.45-?m syringe filter to obtain a viral solution in which cells and cell debris were removed. The viral solution was added to wells in which the target cell had been cultured and from which the culture medium had been removed, with a concentration of 1 ml/well (24-well), and the cells were cultured under the same conditions for 6 hours to induce infection. Polybrene was added at a level of 8 mg/ml to increase the efficiency of infection into a target cell. After 6 hours, the medium was replaced with DMEM medium containing 10% FBS as a medium for normal cell culture, and the cells were cultured under the same conditions for 2 days.

[0081] Next, the infected target cell in the 24-well was cultured for 2 days, and then, transferred to a 55 cm.sup.2 culture dish, and cultured after 500 to 1000 ?g/ml of geneticin (Gibco), a selective antibiotic of the pFB vector, was added thereto. The appropriate dose of antibiotic was determined based on the titration of the target cell and geneticin. When NT2 cells were the target cell, they were cultured in a culture dish containing 10 ml of cell culture medium in the presence of 800 ?g/ml of geneticin for 5 days.

[0082] Next, to form a single cell clone, the target cell was transferred to a 96-well well at the concentration of 2 cells/well. In each well of a 96-well plate containing 100 ?l of the same medium used in the culturing in 55 cm.sup.2 culture dish, colonies formed with single cell clones while being cultured under the same conditions for 4 weeks were selected by a cell culture microscope, and the selected colonies were subjected to expansion culture.

[0083] In this process, a pre-selection process may be performed according to the structure of a produced recombinant polynucleotide. That is, when an internal standard reporter protein is a fluorescence protein, the pre-selection process may be performed by using a fluorescence microscopy, or when an internal standard reporter protein is a luminescence protein, the pre-selection process may be performed by luminescent analysis.

[0084] To pre-select a recombinant polynucleotide in which the internal standard reporter protein is not present or to pre-select a monoclonal cell line having an excellent sensitivity from among monoclonal cell lines in which an internal standard reporter protein is present, proteasome inhibitor MG132 (Sigma) was added at a concentration of 10 ?M medium, or when the recombinant polynucleotide includes SNAP25 sequence, light chain DNA of botulinum toxin type A was used with Lipofectamine? 3000 (Invitrogen), and when the recombinant polynucleotide includes VAMP2 sequence, light chain DNA of botulinum toxin type B was used with Lipofectamine? 3000 (Invitrogen), and the transduction was performed according to the manufacturer instructions. At this time, BoNT/A LC and BoNT B LC were introduced into the BamHI/XhoI site of pCDA4. In detail, regarding only to cells in which a monoclonal colony was identified while culturing in a 96-well for about 3 to 4 weeks, the same number of cells were seeded seperately in two 96-well plates: one plate for a screening experiment, and the other plate for maintenance culture.

[0085] Monoclone-derived cell lines obtained through these screening procedures were subjected to expansion culture, and stored frozen. The lyophilization was carried out by freezing the general mammalian cells. Briefly, 5?10 to 1?10.sup.7 cells were diluted in 1 ml of a culture medium containing 5% or 10% DMSO and the diluted cells were placed in a freezing vial. The temperature was lowered to ?80? C. and then stored in the gas phase of a liquefied nitrogen tank.

[0086] As a result, cell line NG108-15 cells, SiMa cells, and NT2 cells, which stably, constantly express the pFB vector 1, 2, 3, 4, or 6 containing virus, were established.

(3) Differentiation of Established NG108-15 cells, SiMa cells, and NT2 cells into Neurons and Toxin Intoxication

[0087] NG108-15 cells, SiMa cells, and NT2 cells are neuroblastoma cell lines or embryonal carcinoma cell line, which are known to be susceptible to botulinum toxin when differentiated into neurons. Therefore, these cells were differentiated into neurons according to the following procedure.

[0088] NG108-15 cells were maintenance-cultured in DMEM medium supplemented with 10 (v/v) % FBS in a 5% CO.sub.2 incubator at a temperature of 37? C. For neuronal differentiation, cells were seeded at a concentration of 2?10.sup.4 cells/well in each well of a 96-well matrigel (BD science)-coated plate for cell culture, and incubated in a neurobasal medium (Gibco) supplemented with 50 ?M of retinoic acid and 25 ?M of purmophamine for about 5 days. The neurobasal medium contained B27 (Gibco), Glutamax (Gibco), and non-essential amino acid (Gibco) as supplements in an amount of 1? each.

[0089] SiMa cells were maintenance-cultured in DMEM medium supplemented with 10 (v/v) % FBS in a 5% CO.sub.2 incubator at a temperature of 37? C. For neuronal differentiation, cells were seeded at a concentration of 5?10.sup.4 cells/well in each well of a 96-well matrigel (BD science)-coated plate for cell culture, and incubated in a serum-free MEM medium (Welgene) supplemented with 50 ?M of retinoic acid for about 5 days. The MEM medium contained B27 (Gibco), N2 (Gibco), Glutamax (Gibco), HEPES (Gibco) and non-essential amino acid (Gibco) as supplements in amounts of 1? each.

[0090] NT2 cells (also referred to as NTteraA2) were maintenance-cultured in a-MEM medium (Welgene) supplemented with 10 (v/v) % FBS in a 5% CO2 incubator at a temperature of 37? C. For neuronal differentiation, the cells were seeded at a concentration of 1?10.sup.5 cells/ml in each well of a Petri dish and cultured in differentiation medium supplemented with 50 ?M retinoic acid for 1 week under the same conditions while the medium was exchanged every 2 to 3 days. For the differentiation medium, DMEM/F12 (Welgene) medium supplemented with 10 (v/v)% FBS was used.

[0091] The cultured cells form spheres, and after 1 week of culturing, the spheres were collected and transferred to a regular cell culture plate having the same area. The cells were cultured, as adherent cells, on the differentiation medium supplemented with 50 ?M of retinoic acid for 1 week while the medium was exchanged every 2 to 3 days. Subsequently, the adhered cells were deaggregated and detached by using trypsin, and the number of cells was counted.

[0092] 1?10.sup.7 cells were transferred to a 175T flask (Nunc) and cultured in the differentiation medium supplemented with a mitotic inhibitor for 10 days under the same conditions while the medium was exchanged every 2 to 3 days. The mitotic inhibition used herein was 1 ?M AraC, 10 ?M Uridine, and 10 ?M Floxuridine. Cells differentiated into neurons were detached by using trypsin, and the obtained cells were stored frozen. A medium for freezing was used as the medium for the storing frozen, and the freezing was performed by cell freezing method. The differentiated neurons were seeded at the population of 1?10.sup.5 cells per well in a 96-well matrigel-coated plate for cell-culture and cultured for 10 days or more in the differentiation medium, and the medium was changed every 2 to 3 days.

(4) Intoxicating of Differentiated Neurons with Botulinum Toxin

[0093] Intoxicating of differentiated neurons with botulinum toxin type A was performed as follows. For intoxicating with a purified toxin protein, the purified toxin protein was diluted to an appropriate concentration in the differentiation medium and then exchanged with the culture medium of the neuron cultured in a 5% CO.sub.2 incubator at a temperature of 37? C. Thereafter, the cells were cultured under the same conditions for 24 hours to induce toxin-intoxication. Then, the medium was replaced with the differentiation medium and the cells were cultured for 72 hours under the same conditions.

[0094] The differentiation medium was added to a commercially available botulinum toxin drug vial (Meditoxin injectable, Neuronox) to suspend lyophilized toxin protein and excipient, and then, the differentiation medium was exchanged with the culture medium of the cells being maintainance-cultured in a 5% CO.sub.2 incubator at a temperature of 37? C. The toxin potency test on final product vials was performed by using a toxin placebo vial to make the amount of excipient treated the same. The toxin placebo vial was prepared by removing only the toxin protein from the final product toxin vial.

[0095] That is, the same amount of the final product toxin vial and the toxin placebo vial were suspended in the same amount, and then, the total amount of the intoxicating medium was treated in the same manner according to each treatment concentration.

(5) Quantitative Analysis of Reporter Moiety or Internal Standard Reporter

[0096] Quantitative analysis of the reporter moiety or internal standard reporter protein expressed from the recombinant polynucleotide was performed as follows.

[0097] Regarding the recombinant polynucleotide according to the present invention, when a luminescence protein was used as a reporter moiety or an internal standard reporter protein, the luminescence protein was quantitatively analyzed by a luminescence assay. When the reporter moiety is NLuc, the Nano-luciferase assay kit (Promega) was used.

[0098] When the reporter moiety was NLuc and the internal standard reporter protein was FLuc, the One-glo dual nano-luciferase assay kit (Promega) was used and the test was performed according to the supplier's manual. The luminescence was measured by using SpectraMax (Molecular Device Inc). Normalization was performed by using a resultant value obtained by dividing the measured NLuc value by the internal standard reporter protein FLuc value.

[0099] Regarding the recombinant polynucleotide according to the present invention, when a fluorescence protein was used as a reporter moiety or an internal standard reporter protein, the fluorescence values were quantitatively analyzed by a fluorescence meter. When the reporter moiety is mCherry and the internal standard reporter protein is eGFP, mCherry was excited at a wavelength of 610 nm and emitted light at a wavelength of 507 nm was measured by using SpectraMax (Molecular Device), a fluorescence meter. For eGFP, eGFP was excited at the wavelength of 488 nm and emitted light at a wavelength of 507 nm was measured. Normalization was performed by using a resultant value obtained by dividing the measured mCherry emission value by the internal standard reporter protein eGFP emission value. In addition, fluorescence values of GFP and RFP may be measured by using an Incucyte device (Essen Bioscience Inc.) to perform normalization. eGFP belongs to a GFP family and mCherry belongs to an RFP family.

(6) Results

[0100] FIG. 5 shows measurements of activities of a reporter moiety after NG108-15 cells transfected with pFB vector 1 were differentiated into neurons and then intoxicated with botulinum toxin A. Referring to FIG. 5, production of NG108-15 cells transfected with pFB vector 1, differentiation into neurons, intoxication with botulinum toxin A, and measurement of reporter moiety activity are the same as described in (3), (4), and (5). Referring to FIG. 5, to measure the activity of nano-luciferase, luminescence of the cells that had been cultured for being intoxicated for the total of 72 hours were measured by using the Nano-luciferase kit. As shown in FIG. 5, the measured activity of the nano-luciferase, which was evaluated by relative luminescence unit, was significantly increased when the cells were intoxicated with 10 nM botulinum toxin A compared with when not intoxicated therewith.

[0101] FIG. 6 shows fluorescent microscopic images of NG108-15 cells transfected with pFB vector 2 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A. Referring to FIGS. 6, the production of NG108-15 cells transfected with pFB vector 2, differentiation into neurons, and intoxication with botulinum toxin A are the same as described in (3) and (4). FIG. 6 shows fluorescence images of cells, cultured for the total of 72 hours for intoxication, obtained by using an Olympus FSX-100 device, which is a fuorescence microscope.

[0102] Referring to FIG. 6, Control is of a control group with 0 nM of a botulinum toxin A, and BoNT/A 1 nM is of a test group with 1 nM of a botulinum toxin A. As shown in FIG. 6, the test group showed a significant increase in the number of cells with red fluorescence.

[0103] FIGS. 5 and 6 show that when the cleavage site of SNAP25 is not cleaved by botulinum toxin A, the polypeptides of the NLuc-SNPA25 substrate moiety-CL1-PEST structure are easily degraded and the relative luminescent unit is low, and when the cleavage site of SNAP25 is cleaved by botulinum toxin A, the polypeptides of the NLuc-SNPA25 substrate moiety-CL1-PEST structure is stabilized from degradation, and the relative luminescent unit is high.

[0104] FIG. 7 shows fluorescent microscopic images of NG108-15 cells transfected with pFB vector 3 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A. Referring to FIG. 7, production of NG108-15 cells transfected with pFB vector 3, differentiation into neurons, intoxication with botulinum toxin A, and measurement of reporter moiety activity are the same as described in (3), (4), and (5). Referring to FIG. 7, the activities of FLuc and NLuc were measured by measuring the luminescence of the cells cultured for intoxication for the total of 72 hours by using a One-glo nano dual luciferase kit.

[0105] As shown in FIG. 7, the relative activity of NLuc with respect to FLuc, which was evaluated by a relative luminescence unit, was significantly increased when the cells were intoxicated with 10 nM botulinum toxin A compared to when not intoxicated therewith. Referring to FIG. 6, BoNT/A is of a control group with 0 nM of a botulinum toxin A, and BoNT/A 1 nM is of a test group with 1 nM of a botulinum toxin A. As shown in FIG. 7, the measured activity of NLuc, which was evaluated by relative luminescence, was significantly increased when the cells were intoxicated with 10 nM botulinum toxin A compared with when not intoxicated therewith.

[0106] FIG. 8 shows fluorescent microscopic images of SiMa cells transfected with pFB vector 3 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A.

[0107] FIG. 9 shows fluorescent microscopic images of NT2 cells transfected with pFB vector 3 after the cells were differentiated into neurons and then intoxicated with botulinum toxin A.

[0108] FIGS. 8 and 9 show results obtained in the same manner as used to obtain results shown in FIG. 7, except that SiMa cells and NT2 cells were used instead of NG108-15 cells. As shown in FIGS. 8 and 9, the measured activity of NLuc, which was evaluated by relative luminescence, was significantly increased when the cells were intoxicated with 10 nM botulinum toxin A compared with when not intoxicated therewith.

[0109] FIG. 10 shows measurements of signals emitted by a reporter moiety protein and an internal standard reporter protein expressed after light chain of BoNT/B (pCDNA4-BLC) was arbitrarily expressed in NG108-15 cells that were induced to be transfected by using pFB vector 4 to stably express a polypeptide. Here, regarding the pCDNA4-BLC vector, Lipofectamine 3000 (Invitrogen) was used and transduction was performed according to the manufacturer's instructions. Cells were cultured under the same conditions for 48 hours after the transduction. Accordingly, the pCDNA4-BLC vector is transiently expressed in NG108-15 cells. In FIG. 10, the vertical axis indicates values obtained by normalizing NLuc values with respect to Flue.

[0110] As shown in FIG. 10, the activity of NLuc measured with respect to FLuc, which was evaluated by relative luminescence, was significantly increased when DNA of pCDNA4-BLC encoding the light chain of 10 ?g botulinum toxin B was introduced compared to when not introduced.

[0111] FIG. 11 shows measurements of signals emitted by a reporter moiety protein after cell line NT2 cells transducted with pFB vector 3 and stabilized were intoxicated with a different concentration of BoNT/A. In FIG. 11, BoNT/A API represents an active pharmaceutical ingredient used to manufacture a product containing BoNT/A. Herein, the active pharmaceutical ingredient is used as BoNT/A. BoNT/A API was supplied by Meditox Co., Ltd. pFB vector 3 was transduced by using Lipofectamine? 3000 (Invitrogen) according to the manufacturer's instructions. Cells were cultured under the same conditions for 48 hours after the transduction. Accordingly, pFB vector 3 is transiently expressed in NT2 cells. In FIG. 11, RLU represents a relative luciferase unit. As shown in FIG. 11, the activity of NLuc measured with respect to the FLuc, which was evaluated by relative luminescence unit, is dependent on the concentration of botulinum toxin A at the concentration of 1 pM to 10 nM. This indicates that by measuring RLU, the level of the botulinum toxin in a sample, for example, the level of type A, may be measured.

[0112] FIGS. 12 to 14 show measurements of signals emitted by a reporter moiety and an internal standard reporter protein expressed in NT2 cells that were transfected with pFB vector 3 and stabilized after the cells were intoxicated with different concentrations of BoNT/A-containing final products. Results shown in FIGS. 12 to 14 were obtained in the same manner as described in connection with FIG. 11, except that as BoNT/A, a final product containing BoNT/A was used instead of a active pharmaceutical ingredient used to produce a product containing BoNT/A. Compared to BoNT/A API, the product further contains ingredients such as excipients. FIGS. 12, 13, and 14 show FLuc values, NLuc values, and NLuc/FLuc values, respectively.

[0113] As shown in FIG. 14, the activity of NLuc with respect to FLuc, which was evaluated by relative luminescence unit, was dependent on the concentration of botulinum toxin A at a concentration of 1 U to 10 U. This indicates that by measuring RLU, the level of the botulinum toxin in a sample, for example, the level of type A, may be measured.

INDUSTRIAL APPLICABILITY

[0114] The recombinant polynucleotide according to the first aspect may be used to determine protease activities of a neurotoxin polypeptide in a host cell or sample containing the recombinant polynucleotide.

[0115] The host cell containing the recombinant polynucleotide according to the second aspect may be efficiently used in determining protease activities of a neurotoxin polypeptide in a sample.

[0116] The kit for determining proteolytic activities of a neurotoxin polypeptide according to the third and fourth aspects may be used to determine proteolytic activities of a neurotoxin polypeptide.

[0117] According to the method of determining characteristics of a neurotoxin polypeptide in a sample according to the fifth and seventh aspects, characteristics of a neurotoxin polypeptide may be efficiently determined.

[0118] According to the method of determining characteristics of a neurotoxin polypeptide in a sample according to the sixth and eighth aspects, protease activities of a neurotoxin polypeptide in a sample may be efficiently determined.

[0119] According to the method of measuring the ability of a host cell to express or inhibit a protease according to the ninth aspect, the ability of a host cell to express or inhibit a protease may be efficiently measured or it may be determined whether a test material controls protease activities.