KR20220159283A - Cells sensitive to botulinum toxin via lentivirus-mediated genomic integration - Google Patents

Abstract

The present invention relates to a cell for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same.

Description

Cells sensitive to botulinum toxin via lentivirus-mediated genomic integration

The present invention relates to a cell for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same.

Botulinum toxin is a neurotoxin produced by Clostridium botulinum, a gram-positive anaerobic bacterium that grows on rotten canned food and spoiled meat. It is classified into 8 neurotoxins, 7 of which (A, B, C, D, E, F, G) can cause nerve paralysis. The size is about 150 kDa, and it is composed of a non-toxin protein complex in addition to the botulinum toxin protein, and the size of each complex is generated up to 900 kDa depending on the type of neurotoxin. Depending on the type of botulinum toxin, the type of action, target, and active period are different. Among them, botulinum toxin type A is known as one of the deadliest biological agents. This botulinum toxin blocks the signal that causes muscle spasm or contraction to cause paralysis. Since being approved by the US FDA in 1989 as a biological agent based on this function, it has been widely used for therapeutic or cosmetic purposes. . For treatment purposes, diseases such as strabismus, torticollis, blepharospasm, inability to relax, dentition, back pain, etc., and for cosmetic purposes, wrinkles, frown lines, square jaw treatment, hyperhidrosis, etc. is used for the treatment of

In order to use botulinum toxin for treatment and beauty, it requires biological verification before use, and this verification is generally confirmed through a mouse LD ₅₀ , that is, a lethality test using a mouse. Substantially, the unit on the label of the pharmaceutical preparation is the mouse LD ₅₀ unit. However, in order to provide statistically useful mouse LD ₅₀ data, it has limitations such as the large number of mice required, the high cost for the test, and the difficulty in seeing differences according to serotypes of botulinum toxin.

Therefore, in order to overcome these disadvantages, there is a need for a new simple and highly sensitive method for measuring botulinum toxin activity, which can evaluate all steps required for botulinum toxin absorption without using animals.

Domestic Patent Publication 10-2012-0134154

The present invention was made to solve the above problems in the prior art, and an object of the present invention is to provide a cell for measuring botulinum toxin activity and a method for measuring botulinum toxin activity using the same. These cells are cells overexpressing SEPTIN2 or Thioredoxin (TXN) genes, and can evaluate all of botulinum toxin binding, cellular uptake, cytoplasmic translocation and protease activity, as well as significantly improved sensitivity to botulinum toxin, resulting in low A dose of botulinum toxin can also be verified with high accuracy. In addition, the activity of the botulinum toxin pharmaceutical composition in the form of raw materials and finished drugs can also be measured.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. will be.

The present invention provides cells for measuring botulinum toxin activity overexpressing SEPTIN2 or Thioredoxin (TXN).

In one embodiment of the present invention, the cell may overexpress the gene by inserting the SEPTIN2 or TXN gene into the cell, preferably by a method such as transduction or transfection, but generally known It is not limited thereto as long as it is a method of inserting genes into existing cells. The SEPTIN2 is preferably a human SEPTIN2 gene, more preferably a gene expressing a 3,726 bp mRNA sequence of NCBI accession number NM_001008491.2, but variants thereof are also included in the scope of the present invention. Specifically, it may include a sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more to the mRNA sequence of NM_001008491.2. “Percentage of sequence homology” is determined by comparing the optimally aligned sequence to the comparison region, wherein a portion of the nucleotide sequence in the comparison region is added or added compared to the reference sequence (not including additions or deletions) to the optimal alignment of the sequences. It may contain deletions (ie gaps). And the TXN is preferably a human TXN gene, more preferably a gene expressing a 737bp mRNA sequence of NCBI accession number NM_003329.4, but variants thereof are also included in the scope of the present invention. Specifically, it may include a sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more to the mRNA sequence of NM_003329.4. “Percentage of sequence homology” is determined by comparing the optimally aligned sequence to the comparison region, wherein a portion of the nucleotide sequence in the comparison region is added or added compared to the reference sequence (not including additions or deletions) to the optimal alignment of the sequences. It may contain deletions (ie gaps).

In another embodiment of the present invention, the cells are characterized in that their susceptibility to botulinum toxin intoxication is increased compared to wild type cells due to overexpression of SEPTIN2 or TXN genes.

In another embodiment of the present invention, the cells are preferably SiMa cells, LAN-2 cells, PC12 cells, Neuro-2a cells, LA1-55n cells, N18 cells, SH-SY5Y cells, Kelly cells, NB69 cells .

In another embodiment of the present invention, the botulinum toxin may be selected from the group consisting of botulinum serotypes A, B, C, D, E, F and G.

In addition, the present invention comprises the steps of a) treating and culturing the botulinum toxin to the cells for measuring the activity of the botulinum toxin; b) lysing the cultured cells and collecting the cell lysate; and c) measuring the amount of SNAP-25 cleavage product in the cell lysate.

In one embodiment of the present invention, the amount of the SNAP-25 cleavage product is characterized by measuring using a sandwich immunoassay (ELISA), Western blot, etc., but is known as a method used to detect proteins If there is a method, it is not limited thereto.

In addition, the present invention provides a kit for measuring botulinum toxin activity, comprising the cell for measuring botulinum toxin activity as an active ingredient.

In addition, the present invention provides a use for measuring botulinum toxin activity of the cell for measuring botulinum toxin activity.

Cells for measuring botulinum toxin activity according to the present invention have significantly improved sensitivity to botulinum toxin by overexpressing SEPTIN2 or Thioredoxin, so even low doses of botulinum toxin can be verified with high accuracy, as well as botulinum toxin binding and cell absorption , it is expected to be able to replace a number of animal experiments because it can evaluate both translocation into the cytoplasm and protease activity. In addition, since the activity of not only botulinum toxin but also botulinum toxin pharmaceutical compositions in the form of raw materials and finished drugs can be measured, it is expected that the method can be easily applied to industries in various fields using botulinum toxin.

1 is a diagram briefly showing the functions of SEPTIN2 and Thioredoxin (TXN).
2 is a diagram briefly illustrating a pLenti-C-Myc-DDK-P2A-Puro vector map.
3 is a view showing the results of microscopic examination of the cultured 293FT cell line according to an embodiment of the present invention.
4 is a schematic diagram showing a method for preparing a transduced cell line using a lentivirus according to an embodiment of the present invention.
5 is a view showing a method of separating and culturing cell lines into individual colonies using a cloning cylinder according to an embodiment of the present invention.
6 is a view showing the results of Western blotting for proteins expressed in the transduced cell line according to an embodiment of the present invention.
7 is a diagram showing binding sites of primers prepared for PCR according to an embodiment of the present invention.
8 is a view showing the result of confirming the introduced gene by PCR according to an embodiment of the present invention.
9 is a schematic diagram showing the principle of sandwich ELISA.
10 is a diagram showing the result of confirming the sensitivity to botulinum toxin between a control group and a cell line transduced with the SEPTIN2 gene according to an embodiment of the present invention by comparing the amount of SNAP-25 cut.
11 is a view showing the result of confirming the sensitivity to botulinum toxin between a control group and a transduced cell line according to an embodiment of the present invention through a raw data ratio between each group.
12 is a view showing the result of confirming the sensitivity to botulinum toxin between a control group and a cell line transduced with a Thioredoxin gene according to an embodiment of the present invention by comparing the amount of SNAP-25 cut.
13 is a diagram showing the results of measuring the activity of a botulinum toxin pharmaceutical composition (raw drug product) according to an embodiment of the present invention using a cell line transduced.
14 is a diagram showing the results of measuring the activity of a botulinum toxin pharmaceutical composition (finished drug product) according to an embodiment of the present invention using a cell line transduced.
15 is a diagram briefly showing the entire development process of the present invention.

As a result of intensive research on a method for measuring botulinum toxin activity, the present inventors have prepared a cell line with improved sensitivity for measuring botulinum toxin activity, and confirmed that the activity of botulinum toxin can be stably measured using the cell line of the present invention. has been completed.

As used herein, a "botulinum toxin" may be produced bacterially or by recombinant techniques, but includes any known type of botulinum toxin and any subsequently discovered type, including engineered variants or fusion proteins. Means botulinum toxin. Botulinum toxin is classified into 8 neurotoxins, and 7 types of botulinum toxin serotypes A, B, C, D, E, F and G can cause nerve paralysis. These proteins are divided into proteins that contain complexes and proteins that do not contain complexes. The pure toxin protein has a molecular weight of 150 kDa, and various proteins are produced, such as 300 kDa, 500 kDa, and 900 kDa, depending on the presence or absence of complex formation. The botulinum toxin of the present invention may alternatively be a botulinum toxin derivative, ie, a compound having botulinum toxin activity but containing one or more chemical or functional modifications relative to the native or recombinant, native botulinum toxin. For example, a botulinum toxin can be a modified neurotoxin (e.g., a neurotoxin having one or more amino acid deletions, modifications or substitutions relative to the original or recombinantly produced neurotoxin, derivative or fragment thereof). have. For example, a botulinum toxin can be a botulinum toxin that has been modified in a way that enhances its properties or reduces its undesirable side effects, but still retains the desired botulinum toxin activity. Alternatively, the botulinum toxin may be a toxin produced using recombinant or synthetic chemical techniques (e.g., a recombinant peptide, fusion protein, or hybrid neurotoxin prepared from subunits or domains of different botulinum toxin serotypes). (See, eg, US Pat. No. 6,444,209)). A botulinum toxin may also be part of an entire molecule that has been shown to have the requisite botulinum toxin activity, and in such cases, is used as such or as part of a combination or conjugate molecule, such as a fusion protein. It can be. In addition, the botulinum toxin may be in the form of a precursor of botulinum toxin, which may itself be non-toxic, such as a non-toxic zinc protease, which may become toxic upon proteolytic degradation.

In the present specification, "cell" refers to all eukaryotic cells susceptible to botulinum toxin poisoning by botulinum toxin, or all eukaryotic cells capable of absorbing botulinum toxin. Eukaryotic cells refer to cells derived from various mammals, such as, for example, mice, rats, pigs, cattle, sheep, horses, primates and humans. In the present specification, "manufactured cell line" is synonymous with an established cell line, an immortal cell line, or a transformed cell line, and means a cell selected for infinite proliferation. Transformed cell lines disclosed herein exhibit consistent sensitivity to botulinum toxin activity over multiple cell passages, and "sensitivity to botulinum toxin activity" refers to a signal detected by a non-treated control or background signal. It means the lowest botulinum toxin concentration that can be consistently measured.

In the present specification, "vector" or "plasmid" means a DNA fragment, nucleic acid molecule, etc. delivered into a cell, and the vector replicates DNA and can be independently remanufactured in a host cell. May be used interchangeably with the term "delivery vehicle". "Expression vector" means a recombinant DNA molecule comprising a coding sequence of interest and appropriate nucleic acid sequences necessary to express an operably linked coding sequence in a particular host organism. In addition, the recombinant vector or plasmid of the present invention includes a gene encoding SEPTIN2 or Thioredoxin, and thus refers to all vectors capable of overexpressing SEPTIN2 or Thioredoxin in cells.

In the present specification, “kit” refers to a device capable of measuring the activity of botulinum toxin by including the cells for measuring botulinum toxin activity of the present invention, including botulinum toxin itself, raw material for botulinum toxin, botol It can be used to measure the activity of botulinum toxin, such as finished pharmaceuticals of linum toxin, and the kit of the present invention includes a cell lysing agent for lysing cells in addition to the cells for measuring the botulinum toxin activity of the present invention, antibodies and reagents for ELISA, A manual or the like may be additionally included, but anything that can be used in the method for measuring botulinum toxin activity of the present invention may be additionally included.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1: Cultivation of cell line 293FT for lentivirus production

293FT, a cell line commonly used for lentivirus production, is derived from the 293F cell line, and is a cell line that stably expresses the SV40 large T antigen through pCMVSPORT6TAg.neo plasmid, and is a representative cell line used for lentivirus production. Since the 293FT cell line encodes the neomycin resistance gene in the plasmid, it was cultured using a medium containing geneticin, a neomycin analog, during all cell cultures except for cell thawing. More specifically, 293FT cell stock (Thermo, R700-07) was mixed with DMEM ( Gibco) medium and centrifuged at 200xg for 3 minutes to remove the supernatant and obtain the cells. The obtained cells were resuspended using a culture medium, then dispensed into a 100 mm cell culture dish and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. Afterwards, the culture medium was removed and 10% FBS (Gibco), 1X NEAA (Gibco), 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco) and 500 μg/mL geneticin selective antibiotic (Gibco) were added. After replacement with the DMEM (Gibco) medium, it was cultured. And when the cell confluency increased to 90% or more, subculture was performed again. In the subculture, the existing culture medium was first removed and 5 mL of DPBS (Gibco) was added, which is 50% of the culture volume, and washed with water. Then, DPBS was removed, 2 mL of 20% of the culture volume of TrypLE (Gibco) was added, and reacted for 2 minutes in a 37°C, 5% CO ₂ incubator. After the reaction was completed, a culture medium was additionally added, and the supernatant was removed by centrifugation at 800xg and 4° C. for 2 minutes. And after resuspending using a culture medium, the number of cells is measured using trypan blue (Gibco) and a hematocytometer, and subcultured by dispensing 2x10 ⁶ cells with 10 mL of culture medium in a 100 mm cell culture dish. And subculture was carried out in the same way as above every 3 to 4 days.

Example 2: Gene selection and coding plasmid construction for cell line production susceptible to BoNT/A poisoning

As shown in Figure 1, SEPTIN2 (SEPT2) serves to protect BoNT / A from degradation in cells, and Thioredoxin (TXN) acts to separate the disulfide bond between the heavy and light chains of BoNT / A. It is known. In order to overexpress the two proteins in cells, Origene pLenti-ORF plasmids (SEPTIN2: RC224864L3, TXN: RC208876L3) into which the genes encoding each protein were inserted were inserted into the pLenti-C-Myc-DDK-P2A-Puro vector. It was commissioned by the company. The amino acid sequence of SEQ ID NO: 8 (DNA sequence: SEQ ID NO: 9) was used to prepare SEPTIN2, and the amino acid sequence of SEQ ID NO: 10 (DNA sequence: SEQ ID NO: 11) was used to prepare Thioredoxin. The pLenti-C-Myc-DDK-P2A-Puro vector map is shown in FIG. 2 . After the tube containing the lyophilized pLenti-ORF plasmid was centrifuged at 5000xg for 3 minutes, 100 μL of distilled water was added, pipetted, and stored in a -20°C freezer until use.

To construct a strain transformed with the pLenti-ORF plasmid, the plasmid was thawed at room temperature, and 100 μL of competent cells (RBC bioscience) was thawed at 4°C. In addition, 2 μL of the thawed plasmid was treated with water-soluble cells, reacted at 4 ° C for 10 minutes, subjected to heat shock at 37 ° C for 1 minute, and then added with 700 μL of LB broth and stirred in a 37 ° C shaking incubator for 15 minutes. cultured for a while. After the incubation was complete, 20 μL of the culture solution was added to an LB agar plate to which 34 μg/mL of chloramphenicol was added and spread evenly with a spreader. The agar plate treated with bacteria was incubated for 16 hours in a 37 ℃ incubator. And each colony generated on the agar plate was inoculated into 1.5 mL of LB broth to which chloramphenicol was added, and cultured with shaking in a 37°C incubator for 16 hours. After the incubation was complete, the culture medium was centrifuged at 13,000 rpm for 1 minute to remove the supernatant, and the remaining pellet was used for plasmid purification using the DNA-spin Plasmid DNA Purification Kit (iNtRON) according to the provided protocol. In addition, the nucleotide sequence of the purified plasmid was analyzed by requesting Cosmogenetech. Primer sequences used for sequencing are shown in Table 1. As a result of sequencing, it was confirmed that SEPTIN2 exactly matches NCBI's accession number NM_001008491.2 and TXN exactly matches NM_003329.4. Through this, it was confirmed that SEPTIN2 or TXN, that is, a plasmid in which the target gene was normally inserted was constructed. did

Name Primer sequence (5' -> 3') sequence number SEPTIN2 V2 AGAGCTCGTTTAGTGAA One SEPTIN2-600F GGATGAAATTGAAGAACATA 2 TXN V2 AGAGCTCGTTTAGTGAA 3

In order to mass-produce the pLenti-ORF plasmid, 500 μL of the strain culture medium transformed with the plasmid was inoculated into 100 mL of LB broth supplemented with 34 μg/mL of chloramphenicol, followed by shaking culture at 37° C. for 16 hours. Then, the culture solution was transferred to a 500 mL Centrifuge bottle (Nalgene), centrifuged at 6000xg, 4° C. for 15 minutes, and the supernatant was removed. Then, the plasmid was purified from the pellet using the HiSpeed Plasmid Midi Kit (Qiagen). The purified plasmid was quantified using a Life Science UV/Vis Spectrophotometer DU 730 (Beckman Coulter). The results are shown in Table 2.

Gene A260 A280 260/280 ratio DNA concentration (ng/μL) TXN 1.119 0.595 1.880 559 SEPTIN2 1.059 0.562 1.884 529

Example 3: Production of lentivirus through transfection in 293FT cell line

293FT cell line, which is a cell line with high lentivirus production efficiency, was subcultured in the same manner as in Example 1, and 2.5x10 ⁶ cells were dispensed into a 100 mm cell culture dish. And it was confirmed under a microscope that cell saturation reached 40-50%. The results are shown in Figure 3. For transfection, 1.5 mL of opti-MEM (Gibco) and 5 μg of pLenti-ORF plasmid were added to a 1.5 mL tube and mixed. Then, 6 μg of 0.5 μg/μL packaging plasmid (Origene) contained in distilled water was added and mixed, and then 33 μL of TurboFectin (Origene) was additionally added and mixed. The mixed solution was reacted at room temperature for 15 minutes, then treated in a cell culture dish and cultured for 2 days. In addition, the supernatant of the medium containing the virus was collected and stored at 4 ° C., and a new medium was added and cultured for another 1 day. Then, the medium supernatant was collected again, mixed with the medium recovered the day before, filtered using a 0.45 μm pore filter (Sartorius), and stored at 4° C. until use.

Example 4: Construction of transduced cell lines using lentivirus

4x10 ⁶ SiMa (DSMZ, ACC164) cell lines subcultured in the same manner as in Example 1 were cultured in RPMI1640 (Gibco) supplemented with 10% FBS, 2 mM L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). It was dispensed into a 60 mm cell culture dish to which 4 mL of medium was added and cultured for 16 hours. And after removing the culture medium, 1 mL of the lentivirus solution obtained in the same manner as in Example 3 and 3 mL of new culture medium were mixed and added, and polybrene (Sigma-Aldrich) was added so that the final concentration was 8 μg / mL added and incubated. The next day, after removing the culture medium and adding a new medium, the cells were cultured for another 1 day, and replaced with a new culture medium supplemented with 1 μg/mL of puromycin (Sigma-Aldrich). Thereafter, only transduced puromycin drug-resistant cells were selected while replacing the culture medium containing puromycin at intervals of 3 to 4 days. A method for preparing a cell line transduced using lentivirus is briefly shown in FIG. 4 .

And, in order to separate the cultured cell lines into individual colonies, as shown in FIG. 5, spaces were physically partitioned in the culture dish using a cloning cylinder (Sigma-Aldrich). More specifically, after removing the medium used for culture and washing the cells by treating them with 3 mL of DPBS, cloning cylinders were attached to each colony, and only DPBS in the cylinders was removed separately. Then, 50 μL of trypLE was treated in the cloning cylinder and reacted for 3 minutes in a 37°C, 5% CO ₂ incubator. Thereafter, the trypLE and the cell mixture were mixed by pipetting, and then each was dispensed into a 96-well plate containing 150 μL of culture medium supplemented with 1 μg/mL of puromycin. In addition, the cell lines were named in the order of distribution. Thereafter, the cells were scaled up and cultured according to the growth of the cells.

Example 5: Identification of Transduced Cell Lines

5.1. Verification of the expression of the introduced protein

In order to confirm the transduced cell line prepared in the same manner as in Example 4, the primarily expressed protein was confirmed using Western blotting. In more detail, the medium of the 100 mm cell culture dish cultured by scale-up was removed, and washed with 5 mL of 4° C. DPBS. After removing DPBS, cells were treated with 200 μL of RIPA lysis buffer (iNtRON) supplemented with cOmplete™ and EDTA-free Protease Inhibitor Cocktail (Roche). After transferring the cell lysate to a 1.5 mL tube using a cell lifter (SPL), it was reacted at 4°C for 20 minutes without movement. After centrifugation at 17,000 rpm and 4°C for 30 minutes, the supernatant was transferred to a new 1.5 mL tube. The amount of protein in the supernatant was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher), sampled with 4X Laemmli Sample Buffer (Bio-Rad), and then heated at 100 ° C for 10 minutes to prepare a sample for Western blotting. . And Western blotting was performed using a 15% polyacrylamide gel. Proteins in the sample were separated according to size through electrophoresis, and transferred to Immobilon-P PVDF Membrane (Merck) under conditions of 100 V for 1 hour. The protein transfer pattern was confirmed by staining the membrane with Ponceau S (Sigma-Aldrich). Soak Membrane in 0.1% polysorbate 20 in PBS (PBST), wash with water 4 times for 5 minutes each using a Digital Orbital Shaker (DAIHAN Scientific) to remove Ponceau S, add blocking buffer (5% BSA PBST), and incubate at room temperature for 1 hour Blocking was performed using a Digital Orbital Shaker. When the target gene in the pLenti-C-Myc-DDK-P2A-Puro vector, that is, the inserted gene is expressed, Myc and DDK tags are expressed at the C-terminus of the expressed protein. Therefore, the membrane after blocking was treated with a primary antibody (Myc-tag, Cell Signaling Technology, 2278S, 1:1000 v/v in 2% BSA PBST) that specifically binds to the Myc tag, and then subjected to a Digital Orbital Shaker at 4°C. was reacted for 16 hours. After the reaction was completed, the membrane was soaked in PBST and washed 4 times for 5 minutes each using a Digital Orbital Shaker, and then treated with secondary antibodies (Anti-Rabbit HRP, abcam, ab6721, 1:10000 v/v in PBST) at room temperature. reacted for 1 hour. Thereafter, the membrane was immersed in PBST, washed four times for 5 minutes each using a Digital Orbital Shaker, treated with Pierce ECL Western Blotting Substrate (ThermoFisher), and then protein was detected with ImageQuant LAS 500 (Cytiva). The results are shown in FIG. 6 .

As shown in FIG. 6, it was confirmed that Thioredoxin protein or Septin-2 protein was expressed in the transduced SiMa cell line. However, unidentified bands other than Septin-2 were detected in the SEPTIN2-1 cell line.

5.2. Verification of gene introduction

In order to confirm the introduction of the target gene in the transduced cell line prepared in the same manner as in Example 4, genomic DNA was isolated from the cell line and then the transgene portion was amplified using PCR. In order to distinguish the transduced gene from the endogenous gene, primers for PCR were designed using the 3'-terminal sequence of the transduced gene only, and then commissioned to CosmoGenetech to manufacture. did Primer sequences used for PCR are shown in Table 3. As shown in Figure 7, forward primers for amplification of the TXN or SEPTIN2 genes were designed to bind to the 5'-end sequence of each gene, and the reverse primer common to both genes was DDK- It was designed to bind to the tag sequence. In addition, primers (GH20 and GH21) capable of amplifying the β-globin gene were used as positive controls.

Purpose Name Primer sequence (5' -> 3') sequence number PCR TXN forward CTTTTCAGGAAGCCTTGG 4 SEPTIN2 forward GTCTAAGCAACAGCCAAC 5 DDK reverse (common) CTTATCGTCGTCATCCTTG 6 Positive control GH20 GAAGAGCCAAGGACAGGTAC 7 GH21 GGAAAATAGACCAATAGGCAG 8

For PCR, the transduced SiMa-TXN-3, SiMa-SEPTIN2-1, SiMa-SEPTIN2-2, and SiMa-SEPTIN2-3 cell lines were each 1 μg/mL of puromycin added to the culture medium containing 100 ml. Divided into mm cell culture dishes, and when the cell confluency reached about 80%, the genomic DNA of each cell line was extracted using the Wizard® Genomic DNA Purification Kit (Promega) according to the protocol provided, and the extracted genomic DNA was It was quantified using Life Science UV/Vis Spectrophotometer DU 730 (Beckman Coulter). The results are shown in Table 4.

Cell line 260/280 ratio DNA concentration (ng/uL) SiMa-TXN-3 1.937 1072.0 SiMa-SEPTIN2-1 1.830 887.5 SiMa-SEPTIN2-2 1.939 947.0 SiMa-SEPTIN2-3 1.916 1053.0

In addition, 50 ng of genomic DNA, 25 μL of 2X Platinum SuperFi PCR Master Mix, and 2.5 μL each of 10 μM forward and reverse primers were mixed, and finally, distilled water was added to 50 μL to prepare a PCR sample. PCR was performed. . PCR conditions are shown in Table 5.

Cycle Procedure Temperature (℃) Time One Denaturation 95 30 seconds 30 Denaturation 95 30 seconds Annealing 50 30 seconds Elongation 72 1 min 30 sec One Elongation 72 5min One Storage 4 Overnight

After PCR was completed, electrophoresis was performed using a 1.5% (w/v) DNA agarose gel mixed with 0.01% (v/v) EcoDye™ Nucleic Acid Staining Solution (Biofact) and the electrophoresis kit Mupid-One (Advance). moved Electrophoresis was performed at 100 V for 35 minutes, and imaging was performed using a Gel Documentation System LSG 1000 (iNtRON). The results are shown in FIG. 8 .

As shown in FIG. 8 , it was confirmed that the beta globin gene corresponding to the positive control and the TXN gene including the transduced DDK-tag were normally amplified in the SiMa-TXN-3 cell line. In addition, it was confirmed that all of the SEPTIN2 genes including the transduced DDK-tag were amplified in the cell lines of SiMa-SEPTIN2-1, SiMa-SEPTIN2-2, and SiMa-SEPTIN2-3. Through the above results, it was confirmed that the transduced cell line overexpressing the transduced gene was normally produced.

Example 6: Verification of BoNT/A biological activity through sandwich ELISA

6.1. Preparation of cell lysate of SiMa-SEPTIN2-3 cells treated with BoNT/A

In order to confirm the biological activity verification system of the cell-based botulinum toxin, a cell lysate of cells treated with the botulinum toxin was first prepared. In order to treat botulinum toxin, as a control, the SiMa cell line was cultured in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin at 1.2 X 10 ⁵ cells/100 μL/ At the concentration of the well, the experimental group SiMa-SEPTIN2-3 cell line was placed in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 μg/mL puromycin. Each was dispensed at a concentration of 1.2 X 10 ⁵ cells/100 μL/well. After each cell line was seeded and cultured for 2 days, the medium was removed and 1X B-27™ Plus Supplement (Gibco), 1X N-2 Supplement (Gibco), 2 mM L-glutamine and 25 μg/mL Trisialoganglioside GT _1B ( Matreya) was added thereto and treated with 100 μL of differentiation medium and cultured for 2 days to induce differentiation. After 2 days of differentiation, the medium was removed, and the BoNT/A complex was serially diluted 1.35 times in RPMI1640 supplemented with 1X B-27™ Plus Supplement, 1X N-2 Supplement, and 0.25% Human Serum Albumin (Green Cross). and finally added to each well to make 100 μL. After treatment with botulinum toxin and incubation for 4 days, the medium was removed and lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl ₂ and 1% Triton X-100) supplemented with protease inhibitor cocktail 110 μL was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.

6.2. EC using sandwich ELISA ₅₀ Confirm

Botulinum toxin serotype A (BoNT/A) acts on the presynapse of the neuromuscular junction and causes the cleavage of Synaptosomal-Associated Protein, 25kDa (SNAP-25) molecule bound to the presynaptic cell membrane. Since it is known that this principle is applied, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) test method using each antibody capable of recognizing cleaved SNAP-25 and intact SNAP-25 is designed did The principle is briefly shown in FIG. 9 .

Mouse Synaptosomal Protein 25kDa (SNAP-25, aa 183-197) cleaved Monoclonal Antibody (Mybiosource, MBS312597), a capture antibody that specifically binds to cleaved SNAP-25, was mixed with 0.1 M sodium carbonate coating buffer (coating After diluting to a concentration of 0.2% (v/v) using buffer, pH 9.6), 100 μL was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and moved at 4°C for 16 hours. reacted without Thereafter, antibodies that did not bind to the wells were removed, and washed three times using 200 μL of 0.1% polysorbate 20 in PBS (PBST). Then, 200 μL of blocking buffer (5% BSA PBST) was applied to each well, and placed on a shaker at room temperature at 75 rpm for 1 hour. After 1 hour, the blocking buffer was removed, washed three times with 200 μL of PBST, and 90 μL of the cell lysate obtained in the same manner as in Example 6.1 was added and reacted for 2 hours on a shaker at room temperature at 75 rpm. . Then, the cell lysate was removed and washed three times with 200 μL of PBST. Then, after diluting the detection antibody, Anti-SNAP-25 antibody (Sigma-Aldrich, S9684) to a concentration of 0.1% (v/v) using a blocking buffer, 100 μL was treated in each well, It was reacted for 1 hour in a shaker at 75 rpm at room temperature. Thereafter, all unbound antibodies were removed by washing three times with 200 μL of PBST, and the secondary antibody, Anti-Rabbit HRP (abcam, ab6721) was diluted to 0.01% (v/v) using a blocking buffer, , 100 μL of the diluted antibody solution was treated in each well and reacted for 1 hour on a shaker at 75 rpm at room temperature. And after washing three times with 200 μL of PBST, the TMB solution of the TMB Peroxidase EIA Substrate Kit (Bio-Rad) and the hydrogen peroxide solution were mixed at a ratio of 9: 1, and 50 μL was applied to each well, and then incubated at 37 ° C in an incubator. Reacted for 30 minutes. Then, after treating each well with 50 μL of 2 N sulfuric acid, the absorbance (OD450) in the 450 nm wavelength band was measured using a SpectraMax Plus 384 Microplate Reader (Molecular devices). For the obtained raw data, a dose-response curve (four parameters) was created using GraphPad Prism Version 7.00 (GraphPad Software, Inc.), and the EC ₅₀ value was calculated based on the result of the company's mouse titer test (unit). Calculated. Afterwards, all experiments were repeated at least three times, and the results were expressed as mean values ± standard deviations. The results are shown in FIG. 10 .

As shown in FIG. 10, the EC ₅₀ of the control SiMa cell line was 7.35 U/mL (0.49 pM), and the EC ₅₀ of the SEPTIN2-3 transduced cell line was 5.38 U/mL (0.36 pM). Through this, it was confirmed that sensitivity can be improved by overexpressing SEPTIN2.

In addition, in order to compare the difference in sensitivity between cell lines in more detail, the OD _{450 nm} value of each group of SiMa cell line was divided by the OD _{450 nm} value of each group of SEPTIN2-3 cell line, and the degree of response of the cell line to the same botulinum toxin treatment was compared. The results are shown in FIG. 11 .

As shown in FIG. 11, the OD450 _SEPTIN2 /OD450 _WT value without toxin treatment was 1, whereas when toxin was treated, the value rose to about 1.7, confirming the difference in sensitivity to toxin between cell lines. . Through this, it was confirmed that the transduced cell line overexpressing the SEPTIN2 gene had increased sensitivity to botulinum toxin compared to the control group (wild type).

6.3. Preparation of cell lysate of SiMa-Thioredoxin cells treated with BoNT/A

In order to confirm the biological activity verification system of the cell-based botulinum toxin, a cell lysate of cells treated with the botulinum toxin was first prepared. To treat botulinum toxin, as a control, the SiMa cell line was cultured in 8 X 10 ⁴ cells/100 μL/well in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin. At the concentration of the well, SiMa-TXN3 cell line as an experimental group was plated 8 X in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 μg/mL puromycin. Each was dispensed at a concentration of 10 ⁴ cells/100 μL/well. After each cell line was seeded and cultured for 2 days, the medium was removed and 1X B-27™ Plus Supplement (Gibco), 1X N-2 Supplement (Gibco), 2 mM L-glutamine and 25 μg/mL Trisialoganglioside GT _1B ( Matreya) was added thereto and treated with 100 μL of differentiation medium and cultured for 2 days to induce differentiation. After 2 days of differentiation, the medium was removed, and the BoNT/A complex was prepared in RPMI1640 at concentrations of 0, 1, 5, 10, 50, 100, 500, 1000, and 2000 (pM), and finally 100 μL was added to each well. . After treatment with botulinum toxin and incubation for 4 days, the medium was removed and lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl ₂ and 1% Triton X-100) supplemented with protease inhibitor cocktail 110 μL was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.

6.4. EC using sandwich ELISA ₅₀ Confirm

In the same manner as in Example 6.2, the sensitivity of botulinum toxin to the transduced cell line overexpressing the TXN gene was confirmed using the cell lysate of Example 6.3. The results are shown in FIG. 12 .

As shown in FIG. 12, it was confirmed that the transduced cell line overexpressing the Thioredoxin gene had increased sensitivity to botulinum toxin compared to the control group (wild type).

Example 7: Verification of Biological Activity of BoNT/A Pharmaceutical Composition (Raw Material) by Sandwich ELISA

In order to confirm whether the cell-based botulinum toxin biological activity verification system can be applied to the botulinum toxin pharmaceutical composition (drug substance), sandwich ELISA was performed using the SiMa-SEPTIN2-3 cell line. More specifically, the SiMa-SEPTIN2-3 cell line was cultured in 1.2 X 10 ⁵ in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 μg/mL puromycin. It was dispensed at a concentration of cells/100 μL/well. After culturing for 2 days, the medium was removed and treated with 100 μL of differentiation medium supplemented with 1X B-27™ Plus Supplement, 1X N-2 Supplement, 2 mM L-glutamine and 25 μg/mL Trisialoganglioside GT _1B . and cultured for 2 days to induce differentiation. After 2 days of differentiation, the medium was removed, and the BoNT/A complex (botol, a raw material from which other substances such as preservation solution were removed from ATGC-100) was added to RPMI1640 to which 1X B-27™ Plus Supplement and 1X N-2 Supplement were added. linum toxin) was serially diluted 1.55 times (serial dilution), and the cells were treated by concentration. After treatment with botulinum toxin and incubation for 4 days, the medium was removed and lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl ₂ and 1% Triton X-100) supplemented with protease inhibitor cocktail 110 μL was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.

Mouse Synaptosomal Protein 25kDa (SNAP-25, aa 183-197) cleaved Monoclonal Antibody (Mybiosource), a capture antibody that specifically binds to truncated SNAP-25, was added to 0.1 M sodium carbonate coating buffer , pH 9.6) after dilution to a concentration of 0.8% (v/v), 100 μL was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and left at 4°C for 16 hours without movement. reacted Thereafter, antibodies that did not bind to the wells were removed, and washed three times using 200 μL of 0.1% polysorbate 20 in PBS (PBST). Then, 200 μL of blocking buffer (5% BSA PBST) was applied to each well, and placed on a shaker at room temperature at 75 rpm for 2 hours. After 2 hours, the blocking buffer was removed, 90 μL of the obtained cell lysate was added, and the mixture was treated for 2 hours in a shaker at 75 rpm at room temperature. Then, the cell lysate was removed and washed three times with 200 μL of PBST. Then, after diluting the detection antibody, Anti-SNAP-25 antibody (Sigma-Aldrich, S9684) to a concentration of 0.1% (v/v) using a blocking buffer, 100 μL was treated in each well, It was reacted for 1 hour in a shaker at 75 rpm at room temperature. Thereafter, all unbound antibodies were removed by washing three times with 200 μL of PBST, and the secondary antibody, Anti-Rabbit HRP (abcam, ab6721) was diluted to 0.01% (v/v) using a blocking buffer, , 100 μL of the diluted antibody solution was treated in each well and treated for 1 hour on a shaker at 75 rpm at room temperature. And after washing three times with 200 μL of PBST, 100 μL of TMB (ThermoFisher, 34028) solution was applied to each well and reacted at room temperature for 15 minutes. Then, after treating each well with 100 μL of 2 N sulfuric acid, the absorbance (OD450) in the 450 nm wavelength band was measured using a SpectraMax Plus 384 Microplate Reader (Molecular devices). For the obtained raw data, a dose-response curve (four parameters) was created using GraphPad Prism Version 7.00 (GraphPad Software, Inc.), and the EC ₅₀ value was calculated based on the company's mouse titer test result (unit). Calculated. The results are shown in FIG. 13 .

As shown in FIG. 13, it was confirmed that the amount of SNAP-25 cleavage increased in a botulinum toxin dose-dependent manner. And it was confirmed that the EC ₅₀ value was 16.98 U/mL.

Example 8: Verification of biological activity of BoNT/A pharmaceutical composition (drug product) through sandwich ELISA

In order to confirm whether the cell-based botulinum toxin biological activity verification system can be applied to another botulinum toxin pharmaceutical composition (finished drug product), sandwich ELISA was performed using the SiMa-SEPTIN2-3 cell line. More specifically, the SiMa-SEPTIN2-3 cell line was cultured in 1.2 X 10 ⁵ in a 96-well plate containing RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and 1 μg/mL puromycin. It was dispensed at a concentration of cells/100 μL/well. After culturing for 2 days, the medium was removed and treated with 100 μL of differentiation medium supplemented with 1X B-27™ Plus Supplement, 1X N-2 Supplement, 2 mM L-glutamine and 25 μg/mL Trisialoganglioside GT _1B . and cultured for 2 days to induce differentiation. After 2 days of differentiation, the medium was removed, and the BoNT/A finished product (ATGC-100) was serially diluted 1.4 times in RPMI1640 to which 1X B-27™ Plus Supplement and 1X N-2 Supplement had been added. It was treated by concentration. After treatment with botulinum toxin and incubation for 4 days, the medium was removed and lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl ₂ and 1% Triton X-100) supplemented with protease inhibitor cocktail 110 μL was applied to each well to lyse the cells. Then, the cell lysate was transferred to a 1.5 mL tube, and the supernatant was obtained by centrifugation at 17,000 rpm and 4° C. for 5 minutes to prepare a cell lysate for sandwich ELISA.

Mouse Synaptosomal Protein 25kDa (SNAP-25, aa 183-197) cleaved Monoclonal Antibody (Mybiosource), a capture antibody that specifically binds to truncated SNAP-25, was added to 0.1 M sodium carbonate coating buffer , pH 9.6) after dilution to a concentration of 0.8% (v/v), 100 μL was applied to each well of a Clear Flat-Bottom Immuno Nonsterile 96-Well Plate (ThermoFisher) and left at 4°C for 16 hours without movement. reacted Thereafter, antibodies that did not bind to the wells were removed, and washed three times using 200 μL of 0.1% polysorbate 20 in PBS (PBST). Then, 200 μL of blocking buffer (5% BSA PBST) was applied to each well, and placed on a shaker at room temperature at 75 rpm for 2 hours. After 2 hours, the blocking buffer was removed, 90 μL of the obtained cell lysate was added, and the mixture was treated for 2 hours in a shaker at 75 rpm at room temperature. Then, the cell lysate was removed and washed three times with 200 μL of PBST. Then, after diluting the detection antibody, Anti-SNAP-25 antibody (Sigma-Aldrich, S9684) to a concentration of 0.1% (v/v) using a blocking buffer, 100 μL was treated in each well, It was reacted for 1 hour in a shaker at 75 rpm at room temperature. Thereafter, all unbound antibodies were removed by washing three times with 200 μL of PBST, and the secondary antibody, Anti-Rabbit HRP (abcam, ab6721) was diluted to 0.01% (v/v) using a blocking buffer, , 100 μL of the diluted antibody solution was treated in each well and treated for 1 hour on a shaker at 75 rpm at room temperature. And after washing three times with 200 μL of PBST, 100 μL of TMB (ThermoFisher, 34028) solution was applied to each well, followed by reaction at room temperature for 15 minutes. Then, after treating each well with 100 μL of 2 N sulfuric acid, the absorbance (OD450) in the 450 nm wavelength band was measured using a SpectraMax Plus 384 Microplate Reader (Molecular devices). For the obtained raw data, a dose-response curve (four parameters) was created using GraphPad Prism Version 7.00 (GraphPad Software, Inc.), and the EC ₅₀ value was calculated based on the result of the company's mouse titer test (unit). Calculated. The results are shown in FIG. 14 .

As shown in FIG. 14, it was confirmed that the amount of SNAP-25 cleavage increased in a botulinum toxin dose-dependent manner. And it was confirmed that the EC ₅₀ value was 5.42 U/mL.

Through the above results, as shown in FIG. 15, by using a cell line overexpressing SEPTIN2 or TXN gene using the lentivirus of the present invention, the potency of not only botulinum toxin but also botulinum toxin pharmaceutical compositions, that is, raw materials and finished drugs It was confirmed that can be easily measured on a cell basis and the sensitivity can be significantly improved. Therefore, it is expected that the cell line of the present invention can be applied to various industrial fields using botulinum toxin.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> ATGC CO., LTD. <120> Cells sensitive to botulinum toxin via lentivirus-mediated genomic integration <130> MP21-050P1 <150> KR 10-2021-0066040 <151> 2021-05-24 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> SEPTIN2 V2 <400> 1 agagctcgtt tagtgaa 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > SEPTIN2-600F <400> 2 ggatgaaatt gaagaacata 20 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TXN V2 <400> 3 agagctcgtt tagtgaa 17 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TXN forward <400> 4 cttttcagga agccttgg 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SEPTIN2 forward <400> 5 gtctaagcaa cagccaac 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DDK reverse <400> 6 cttatcgtcg tcatccttg 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GH20 <400> 7 gaagagccaa ggacaggtac 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GH21 <400> 8 ggaaaataga ccaataggca g 21 <210> 9 <211> 361 <212> PRT <213> Homo sapiens <400> 9 Met Ser Lys Gln Gln Pro Thr Gln Phe Ile Asn Pro Glu Thr Pro Gly 1 5 10 15 Tyr Val Gly Phe Ala Asn Leu Pro Asn Gln Val His Arg Lys Ser Val 20 25 30 Lys Lys Gly Phe Glu Phe Thr Leu Met Val Val Gly Glu Ser Gly Leu 35 40 45 Gly Lys Ser Thr Leu Ile Asn Ser Leu Phe Leu Thr Asp Leu Tyr Pro 50 55 60 Glu Arg Val Ile Pro Gly Ala Ala Glu Lys Ile Glu Arg Thr Val Gln 65 70 75 80 Ile Glu Ala Ser Thr Val Glu Ile Glu Glu Arg Gly Val Lys Leu Arg 85 90 95 Leu Thr Val Val Asp Thr Pro Gly Tyr Gly Asp Ala Ile Asn Cys Arg 100 105 110 Asp Cys Phe Lys Thr Ile Ile Ser Tyr Ile Asp Glu Gln Phe Glu Arg 115 120 125 Tyr Leu His Asp Glu Ser Gly Leu Asn Arg Arg His Ile Ile Asp Asn 130 135 140 Arg Val His Cys Cys Phe Tyr Phe Ile Ser Pro Phe Gly His Gly Leu 145 150 155 160 Lys Pro Leu Asp Val Ala Phe Met Lys Ala Ile His Asn Lys Val Asn 165 170 175 Ile Val Pro Val Ile Ala Lys Ala Asp Thr Leu Thr Leu Lys Glu Arg 180 185 190 Glu Arg Leu Lys Lys Arg Ile Leu Asp Glu Ile Glu Glu His Asn Ile 195 200 205 Lys Ile Tyr His Leu Pro Asp Ala Glu Ser Asp Glu Asp Glu Asp Phe 210 215 220 Lys Glu Gln Thr Arg Leu Leu Lys Ala Ser Ile Pro Phe Ser Val Val 225 230 235 240 Gly Ser Asn Gln Leu Ile Glu Ala Lys Gly Lys Lys Val Arg Gly Arg 245 250 255 Leu Tyr Pro Trp Gly Val Val Glu Val Glu Asn Pro Glu His Asn Asp 260 265 270 Phe Leu Lys Leu Arg Thr Met Leu Ile Thr His Met Gln Asp Leu Gln 275 280 285 Glu Val Thr Gln Asp Leu His Tyr Glu Asn Phe Arg Ser Glu Arg Leu 290 295 300 Lys Arg Gly Gly Arg Lys Val Glu Asn Glu Asp Met Asn Lys Asp Gln 305 310 315 320 Ile Leu Leu Glu Lys Glu Ala Glu Leu Arg Arg Met Gln Glu Met Ile 325 330 335 Ala Arg Met Gln Ala Gln Met Gln Met Gln Met Gln Gly Gly Asp Gly 340 345 350 Asp Gly Gly Ala Leu Gly His Val 355 360 <210> 10 <211> 8138 <212> DNA <213> Artificial Sequence <220> <223> SEPTIN2 expression construct sequence <400> 10 gtcgacggat cgggatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca 240 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420 ccattga cgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc 840 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 900 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 960 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1020 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1080 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1140 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1200 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 1260 attaaaacat atagtatgggcaagcaggga gctagaacga ttcgcagtta atcctggcct 1320 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1380 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1440 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1500 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1560 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1620 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1680 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1740 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1800 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1860 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1920 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1980 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2040 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2100 aagaatgaac aagaattatt ggaatt agat aaatgggcaa gtttgtggaa ttggtttaac 2160 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580 cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940 ctggcattat gcccagtaca tgaccttacg g gactttcct acttggcagt acatctacgt 3000 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240 tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt 3300 accgaggaga tctgccgccg cgatcgccat gtctaagcaa cagccaactc agtttataaa 3360 tccagaaaca cctggctatg ttggatttgc aaacctcccc aatcaagttc accgaaaatc 3420 agtgaaaaaa ggttttgagt tcacactgat ggtggtcggt gaatcaggtc taggaaaatc 3480 gactctcata aacagcctat tcctaactga tctgtaccca gaaagagtca tacctggagc 3540 agcagaaaaa attgaaagaa ctgtccagat tgaggcttca actgttgaaa ttgaagagcg 3600 aggggtcaag ctacgcctga cagtggtaga tacccctggc tatggtgacg ctatcaactg 3660 cagagattgt tttaagacaa ttatctccta tattgatgag caatttgaga ggtacctgca 3720 tgacgagagc ggcttgaaca ggcggcacat cattgataat agggtgcatt gttgctttta 3780 ctttatttca ccttttggac atggacttaa gccctta gat gtggcgttta tgaaggcaat 3840 acacaacaag gtgaatattg tgcctgtcat tgcaaaagct gacactctca ccctgaagga 3900 acgggagcgg ctgaagaaaa ggattctgga tgaaattgaa gaacataaca tcaaaatcta 3960 tcacttacct gatgcagaat cagatgaaga tgaagatttt aaagagcaga ctagacttct 4020 caaggctagc atcccattct ctgtggttgg atccaatcag ttgattgaag ccaaaggaaa 4080 gaaggtcaga ggccgcctct acccctgggg tgttgtggaa gtggagaacc cagagcacaa 4140 tgactttctg aagctgagaa ccatgctcat cacccacatg caggatctcc aggaggtgac 4200 ccaggacctt cattatgaaa acttccgttc tgagagactc aagagaggcg gcaggaaagt 4260 ggagaatgag gacatgaata aagaccagat cttgctggaa aaagaagctg agctccgccg 4320 catgcaagag atgattgcaa ggatgcaggc gcagatgcag atgcagatgc agggcgggga 4380 tggcgatggc ggggctctcg ggcaccacgt gacgcgtacg cggccgctcg agcagaaact 4440 catctcagaa gaggatctgg cagcaaatga tatcctggat tacaaggatg acgacgataa 4500 ggtttgggta ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga 4560 ggagaaccct ggacctatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga 4620 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc ga ctaccccg ccacgcgcca 4680 caccgtcgat ccggatcgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac 4740 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt 4800 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat 4860 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc 4920 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca 4980 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg 5040 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg 5100 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg 5160 caagcccggt gcctgatgta cacccaaacg gccggccgcg gtctgtacaa gtaggattcg 5220 tcgagggacc taataacttc gtatagcata cattatacga agttatacat gtttaagggt 5280 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca 5340 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat 5400 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct 5460 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccg tt gtcaggcaac 5520 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca 5580 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca 5640 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg 5700 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga 5760 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt 5820 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga 5880 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta 5940 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg 6000 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga 6060 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 6120 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca 6180 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg 6240 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat 6300 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tga cccggag 6360 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag 6420 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 6480 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 6540 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 6600 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6660 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6720 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6780 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6840 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6900 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6960 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7020 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7080 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 7140 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaaca a 7200 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7260 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7320 tcacgttaag ggattttggt catgattacg ccccgccctg ccactcatcg cagtactgtt 7380 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa 7440 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg 7500 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg 7560 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt 7620 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt 7680 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt 7740 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag 7800 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct 7860 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag 7920 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg 7980 tatatccagt gatttttttc tccatactct tcctttttca atattattga agcatttatc 8040 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 8100 gggttccgcg cacatttccc cgaaaagtgc cacctgac 8138 <210> 11 <211> 105 <212> PRT <213> Homo sapiens <400> 11 1 5 10 15 Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 20 25 30 Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys 35 40 45 Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp 50 55 60 Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe 65 70 75 80 Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys 85 90 95 Leu Glu Ala Thr Ile Asn Glu Leu Val 100 105 <210> 12 <211> 7370 <212> DNA <213> Artificial Sequence <220> <223> Thioredoxin expression construct sequence <400> 12 gtcgacggat cgggatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca 240 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc 840 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 900 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 960 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1020 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1080 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1140 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1200 gagaattaga tcgcgatggg aaaa aattcg gttaaggcca gggggaaaga aaaaatataa 1260 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 1320 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1380 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1440 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1500 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1560 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1620 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1680 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1740 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1800 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1860 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1920 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1980 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2040 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2100 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2160 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580 cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880 tcaagt gtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3000 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240 tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt 3300 accgaggaga tctgccgccg cgatcgccat ggtgaagcag atcgagagca agactgcttt 3360 tcaggaagcc ttggacgctg caggtgataa acttgtagta gttgacttct cagccacgtg 3420 gtgtgggcct tgcaaaatga tcaagccttt ctttcattcc ctctctgaaa agtattccaa 3480 cgtgatattc cttgaagtag atgtggatga ctgtcaggat gttgcttcag agtgtgaagt 3540 caaatgcatg ccaacattcc agttttttaa gaagggacaa aaggtgggtg aattttctgg 3600 agccaataag gaaaagcttg aagccaccat taatgaatta gtcacgcgta cgcggccgct 3660 cgagcagaaa ctcatctcag aagaggatct ggcagcaaat gatatcctgg attacaagga 3720 tgacgacgat a aggtttggg taggaagcgg agctactaac ttcagcctgc tgaagcaggc 3780 tggagacgtg gaggagaacc ctggacctat gaccgagtac aagcccacgg tgcgcctcgc 3840 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 3900 cgccacgcgc cacaccgtcg atccggatcg ccacatcgag cgggtcaccg agctgcaaga 3960 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc 4020 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 4080 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg 4140 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 4200 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 4260 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta 4320 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 4380 gtgcatgacc cgcaagcccg gtgcctgatg tacacccaaa cggccggccg cggtctgtac 4440 aagtaggatt cgtcgaggga cctaataact tcgtatagca tacattatac gaagttatac 4500 atgtttaagg gttccggttc cactaggtac aattcgatat caagcttatc gataatcaac 4560 ctctggatta caaaatt tgt gaaagattga ctggtattct taactatgtt gctcctttta 4620 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 4680 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 4740 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 4800 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 4860 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 4920 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 4980 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 5040 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 5100 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc 5160 gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct 5220 gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag 5280 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa 5340 aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg 5400 atctaccaca cacaaggcta ct tccctgat tggcagaact acacaccagg gccagggatc 5460 agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta 5520 gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg 5580 gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac 5640 atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc 5700 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 5760 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 5820 cccttttagt cagtgtggaa aatctctagc agcatgtgag caaaaggcca gcaaaaggcc 5880 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5940 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 6000 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 6060 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 6120 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 6180 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 6240 cacgacttat cgccactggc agcagcca ct ggtaacagga ttagcagagc gaggtatgta 6300 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 6360 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 6420 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 6480 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 6540 tggaacgaaa actcacgtta agggattttg gtcatgatta cgccccgccc tgccactcat 6600 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat 6660 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca 6720 tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga 6780 aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat 6840 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga 6900 aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg 6960 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga 7020 actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt 7080 gcttattttt ctttacggtc tttaaaaagg ccg taatatc cagctgaacg gtctggttat 7140 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata 7200 tatcaacggt ggtatatcca gtgatttttt tctccatact cttccttttt caatattatt 7260 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 7320ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 7370

Claims (8)

Cells for measuring botulinum toxin activity overexpressing SEPTIN2 or Thioredoxin (TXN). According to claim 1,
The cell is characterized in that the SEPTIN2 or TXN gene is transduced and overexpressed, a cell for measuring botulinum toxin activity. According to claim 1,
Cells for measuring botulinum toxin activity, characterized in that the cells have increased susceptibility to botulinum toxin intoxication. According to claim 1,
The cells are SiMa cells, LAN-2 cells, PC12 cells, Neuro-2a cells, LA1-55n cells, N18 cells, SH-SY5Y cells, Kelly cells, NB69 cells, N1E-115 cells, BE(2)-M17 cells A cell for measuring botulinum toxin activity, characterized in that it is any one selected from the group consisting of, and SK-N-BE (2) cells. According to claim 1,
The cell for measuring botulinum toxin activity, characterized in that the botulinum toxin is any one selected from the group consisting of botulinum serotypes A, B, C, D, E, F and G. a) treating the cells for measuring botulinum toxin activity according to any one of claims 1 to 5 with botulinum toxin and culturing the cells;
b) lysing the cultured cells and collecting the cell lysate; and
c) a method for measuring botulinum toxin activity, comprising measuring the amount of SNAP-25 cleavage product in the cell lysate. According to claim 6,
Characterized in that the amount of the SNAP-25 cleavage product is measured using a sandwich immunoassay (ELISA) or Western blot, a method for measuring botulinum toxin activity. A kit for measuring botulinum toxin activity, comprising the cell for measuring botulinum toxin activity according to any one of claims 1 to 5 as an active ingredient.

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