JP2006204225A - Method for establishing cell line - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for establishing a cell line expressing the objective protein, for further detail, a method for establishing the cell line expressing a TSHR (thyroid-stimulating hormone receptor) capable of detecting a TSHR antibody with high sensitivity and to provide a system for evaluating functions of an anti-TSHR antibody. <P>SOLUTION: The method for establishing the cell line comprises a step of introducing a gene encoding the objective protein and a vector carrying a gene encoding a carrier protein into an animal cell and a step of carrying out cloning of the cell line stably expressing the objective protein with a system for magnetically separating the cell. Furthermore, the cell line stably expressing the TSHR obtained by the method is stimulated with a blood serum and an activity for elevating cAMP in the cell line is assayed to assay the activity of a stimulating type antibody and an inhibitory type antibody for the TSHR in the blood serum. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for establishing a cell line that stably expresses a target protein. More specifically, the present invention relates to a method for establishing a cell line that stably expresses a thyroid-stimulating hormone receptor (hereinafter referred to as TSHR), and a TSHR-expressing cell line established by the method.

  Transfectants (stable expression cell lines) in which the target gene is integrated into genomic DNA can be constitutively expressed, and are therefore an effective tool for protein functional analysis. However, it took a lot of time and effort to establish such stable expression cell lines.

  Basedow's disease, an organ-specific autoimmune disease, develops when autoantibodies to TSHR bind to TSHR and enhance receptor stimulating activity. Elucidation of the autoantibody production mechanism against TSHR is indispensable for analyzing the pathological condition of Graves' disease, and for this purpose, a highly sensitive detection system for the autoantibody is indispensable.

Anti-TSHR antibodies include stimulating antibodies and inhibitory antibodies (Non-patent Document 1). Stimulatory antibody against TSHR (TSAb) is a TSH receptor that binds to TSH receptor and exhibits TSH-like thyroid stimulating action (increased cAMP production in thyroid cells, increased thyroid hormone production / secretion), but suppressive antibody against TSHR (TSBAb) binds to TSH receptor but does not have thyroid stimulating activity, inhibits TSH thyroid receptor binding, and suppresses thyroid stimulating action of TSH. (Non-patent document 1).
The activity of a stimulating antibody against TSHR is generally evaluated by the cAMP-elevating activity of cells expressing TSHR. Inhibitory antibodies are also detected as the presence of antibodies that suppress cAMP-elevating activity of cells expressing TSHR due to the addition of TSH.
Yoshida T, Ichikawa Y, Ito K, Homma M :, J Biol Chem (1988), 31, pp16341-16347

  An object of the present invention is to establish a target protein-expressing cell line, specifically a TSHR-expressing cell line capable of detecting an anti-TSHR antibody with high sensitivity, and a function evaluation system for the anti-TSHR antibody.

  The present invention has established a method for establishing a cell line that stably expresses a target protein in a simple, safe and efficient manner using a magnetic cell separation system. The construction of a TSHR antibody functional evaluation system was achieved and the present invention was completed.

That is, the present invention
1. A method for establishing a cell line comprising the following steps;
1) introducing a vector carrying a gene encoding a target protein and a gene encoding a carrier protein into an animal cell;
2) a step of cloning a cell line that stably expresses the target protein using a magnetic cell separation system;
2. 2. The method according to item 1 above, wherein the carrier protein is a protein expressed on the cell membrane.
3. 3. The method according to item 1 or 2 above, wherein the carrier protein is CD4.
4). 4. The method according to any one of items 1 to 3, wherein the target protein is a thyroid stimulating hormone receptor (TSHR).
5. 5. The method according to item 4 above, wherein the TSHR gene is DNA encoding the extracellular portion of TSHR.
6). 6. The method according to 4 or 5 above, wherein the TSHR gene is derived from human or pig.
7). A cell line stably expressing the target protein obtained by the method according to any one of 1 to 6 above,
8). A method for analyzing the function of the target protein using the cell line stably expressing the target protein obtained by the method according to any one of the preceding items 1 to 6;
9. A method for detecting an antibody against a target protein in serum using a cell line stably expressing the target protein obtained by the method according to any one of items 1 to 6;
10. A method for measuring the activity of stimulatory and inhibitory antibodies against TSHR in serum, comprising the following steps;
1) Stimulating a TSHR stable expression cell line obtained by the method according to any one of 4 to 6 with the serum,
2) Measure cAMP increasing activity in the cell line,
11. The method according to 9 or 10 above, wherein the serum is derived from a mammal,
12 12. The method according to item 11 above, wherein the serum is derived from a Graves' disease patient,
13. 13. The method according to item 12 above, wherein the serum is derived from a mouse.
14 14. The method according to item 13 above, wherein the mouse is a Graves' disease model mouse;
Consists of.

  The cell line establishment method of the present invention has the advantage of being simple, safe, and efficient, and is useful for functional analysis of various target proteins, pathological analysis and pathological evaluation of diseases related to the proteins. In addition, the TSHR-expressing cell line of the present invention can be used for pathologic analysis, pathologic evaluation of Graves 'disease, and evaluation of the model mouse by detecting anti-TSHR antibody in the serum of Graves' disease model mouse.

  Hereinafter, embodiments of the present invention will be described in more detail. The following detailed description is exemplary and illustrative only and is not intended to limit the invention in any way.

One aspect of the present invention is a method for establishing a cell line comprising the following steps;
1) introducing a vector carrying a gene encoding a target protein and a gene encoding a carrier protein into an animal cell;
2) A step of cloning a cell line stably expressing the target protein using a magnetic cell separation system. Hereinafter, each process will be described.

1) A step of introducing a gene encoding a target protein and a vector carrying a gene encoding a carrier protein into an animal cell. Step 1) includes a gene encoding a target protein and a vector encoding a carrier protein. Introducing and selecting into animal cells. Here, the carrier protein is preferably a protein expressed on a cell membrane for selection of an expression cell, and an example thereof is CD4.

  The target protein may be any useful protein or physiologically active protein, and is preferably a protein expressed on the cell membrane. Such proteins include various hormones [thyroid hormone (TSH), pituitary hormone-releasing factor, oxytocin, vasopressin, parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), growth hormone (GH), prolactin, Receptors for gastrin, secretin, cholecystokinin, insulin, glucagon, calcitonin], various lymphokines or cytokines [for example, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5 IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, tumor necrosis factor (TNF), interferon α (IFNα), Interferon β, interferon γ, interferon ω, interferon τ), hematopoietic factors (eg, erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony sting) Receptive factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), blood stem cell growth factor (SCF)] receptor, various growth factors or growth factors [eg , Vascular epidermal growth factor (VEGF), nerve growth factor (NGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factor β (TGF-β), leukocyte migration inhibitory factor (LIF) ), Ciliary neurotrophic factor (CNTF), oncostatin M (OSM)] receptor, immunoglobulin [eg, human antibody, chimeric antibody, humanized antibody or fragment thereof, Fv, scFv (single chain Fv) ), ScFv dimer] and the like.

  When the target protein is TSHR, the TSHR gene to be used may be derived from a mammal, but a human (human TSHR: NM174206), pig (porcineTSHR: NM214297), bovine (bovineTSHR: NM174206) is preferable, The thing derived from a pig is more preferable.

  The gene encoding the target protein may exist in nature, or may be a gene in which mutation such as substitution, deletion, addition or insertion is introduced into the gene. A means known per se can be used for introducing the mutation. As a preferred mutation, when the target protein is TSHR, a gene that expresses an extracellular portion of TSHR (amino acids 1 to 289 from the N terminus) or substitution of an N-type sugar chain binding site is preferable.

  Desirably, the means for introducing the gene into the animal cell is preferably an integration method such as the liposome method, the calcium phosphate method, or the electroporation method. However, a transient expression method such as a liposome method, a calcium phosphate method, an electroporation method, a viral vector method, an atelocollagen method, or the like may be used. The vector to be used is not particularly limited, and applies to all recombinant vectors capable of introducing an inserted DNA fragment by a usual recombination experiment, such as a known plasmid, phage, cosmid, BAC, YAC, recombinant virus, transposon, etc. be able to.

  The vector can be constructed together with a promoter and an enhancer which are naturally suitable for a combination known per se. For example, a commercially available protein expression vector in which a promoter suitable for the host is inserted can be used.

  Further, it is desirable that a ribosome binding site (IRES) sequence is located between the target protein of the vector and the carrier protein. By simultaneous translation from the 5 ′ end of the transcript and the IRES sequence by the IRES sequence, both the target protein and carrier protein therapy are synthesized from a single transcript in one cell. This increases the probability that both proteins are expressed in the cell.

  Further, the vector preferably contains DNA encoding an antibiotic resistance selection marker. The selection marker allows cloning with antibiotics. Examples of the antibiotic include neomycin, pleomycin, hygromycin, puromycin and the like. Specific examples of vectors used in the present invention include pC1-IRES-Vector (Novagen, discontinued).

  The animal cell into which the gene is introduced is not particularly limited, but is preferably established as a cultured cell, and preferred examples include CHO-K1 cell, COS cell, HEK293 cell and the like.

2) A step of cloning a cell line stably expressing the target protein using a magnetic cell separation system.
Step 2) is a step of cloning cells expressing the target protein using a magnetic cell separation system using an antibody against the carrier protein introduced into the cell in step 1). Specifically, the cells into which the vector has been introduced are magnetically labeled in step 1) using an antibody against a carrier protein to which magnetic microbeads are bound. Thereafter, the cells are passed through a separation column provided with a magnet, and then cells retained in the separation column are eluted to remove non-target protein-expressing cells and obtain target protein-expressing cells. Commercially available kits can be widely used for the magnetic cell separation system, and a specific example is the MACS system (Daiichi Chemical Co., Ltd.).

  In addition, after selecting a cell expressing the marker using the antibiotic resistance selection marker, a magnetic cell separation system is implemented using the cell, thereby increasing the cloning efficiency. Specifically, the antibiotic is added to a cell culture medium, and the cells expressing the marker are selected by culturing the cells.

  Furthermore, a cell line expressing a target protein can be cloned from cells eluted from a separation column in a magnetic cell separation system using a known cell line cloning method such as a limiting dilution method or a pickup method. The limiting dilution method is a method of growing in a culture vessel (for example, a 96-well plate) at a concentration of 1 cell or less. The pick-up method is a method in which a colony derived from a rare cell is isolated and separated by a cylindrical cylinder (cloning cylinder).

  The target gene introduction efficiency in the target protein-expressing cell line can be evaluated by known means such as an antibody against a carrier and a flow cytometer using an antibody against the target protein.

  Another aspect of the present invention is a cell line stably expressing the target protein obtained by the above method. The cell line stably expressing the target protein can be used for functional analysis of the target protein. Therefore, another aspect of the present invention is a method for analyzing the function of a target protein using a stable expression strain of the target protein obtained by the above method.

  Yet another embodiment of the present invention is a method for detecting an antibody against a target protein in serum using a cell line stably expressing the target protein obtained by the above method. As a specific example, the activity of stimulatory and inhibitory antibodies against TSHR in serum can be measured using a TSHR stably expressing cell line.

  Accordingly, one aspect of the present invention is a method for measuring the activity of stimulatory and inhibitory antibodies against TSHR in serum, comprising the following steps: 1) A TSHR stably expressing cell line obtained by the above method is used in serum Stimulate 2) Measure cAMP elevating activity in the cell line. The cAMP elevating activity can be measured using a commercially available kit. For example, kit “Yamasa” (Yamasa Soy Sauce) can be used.

  Serum may be derived from mammals such as humans and mice. As an example, by stimulating a TSHR stably expressing cell line with the serum of a Graves 'disease patient and measuring cAMP-elevating activity, the autoantibodies in the serum can be detected and the pathological condition of Graves' disease can be evaluated. Moreover, TSHR stable expression strains can also be used to establish Graves' disease model mice. Specifically, in the production of a Graves 'disease model mouse, the mouse serum was stimulated with a stable expression strain of TSHR, and cAMP-elevating activity was measured to detect autoantibodies in the serum, and the Graves' disease model mouse produced was evaluated. can do.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to the following Example.

Example 1: Establishment of TSHR-expressing cell line <Materials and methods>
1) Preparation of TSHR expression vector
PC1-IRES-ΔCD4 (FIG. 1) was used as a TSHR expression vector. The full-length human TSHR gene (human TSHR: AY429111) and the extracellular region were incorporated into this vector at the EcoRI site to prepare pC1-TSHR-IRES-ΔCD4. The base sequence at the junction of the insert was confirmed by DNA sequencing. As shown in FIG. 2, by using pC1-TSHR-IRES-ΔCD4, it is possible to express TSHR simultaneously in all cells that theoretically express CD4.

2) Cell culture Chinese hamster ovary cell-derived CHO-K1 cells were cultured at 37 ° C. in the presence of 5% CO ₂ using F-12 (HAM) medium containing 10% FBS.

3) Gene transfer method
Introduce 0.3 to 0.5 μg of pC1-TSHR-IRES-ΔCD4 into 6 × 10 ⁵ cells / 9.6 cm ² dish of CHO-K1 cells using FuGENE 63 μL / well (Roche-Diagnostic)) did.

4) Concentration of ΔCD4-expressing cells As shown in FIG. 3, the cells were concentrated using a magnetic cell separation system (MACS) and an anti-CD4 antibody bound with magnetic microbeads. Twenty-four hours after gene transfer, ΔCD4-positive cells were magnetically labeled by reacting the antibody against CD4 with the gene-transferred cells. These cells were passed through a separation column installed in a magnet, and then ΔCD4-positive cells retained in the separation column were eluted. The interval between MACS treatments was about one week, and during that time G418 was added to the medium at a concentration of 1 mg / ml to remove TSHR non-expressing cells. G418 is a selection reagent in genetic engineering experiments. It is an aminoglycoside antibiotic and is inactivated by the aminoglycoside 3′-phosphotransesterase encoded by the neo gene of transposon Tn5. In general, in order to select only the transformed cells by isolating the cells in which the target gene transfected with the neo gene is constitutively expressed using this, Used in addition to.

5) Cloning of TSHR stable expression cell line A TSHR stable expression cell line was isolated by a limiting dilution method. Cloning was carried out by seeding the cells obtained in 4) in a 96-well plate at a concentration of 0.3 cells / well.

6) Confirmation of TSHR gene transfer efficiency
Using a FACS caliber (manufactured by Becton Dickinson), the efficiency of TSHR gene transfer in TSHR-transfected cells was confirmed. TSHR-introduced cells were analyzed using PE (phycoerythrin) -labeled anti-CD4 antibody, monoclonal anti-TSHR antibody (2C11) and FITC (Fluoresceinisothiocyanate) -labeled anti-mouse Igs antibody. 2C11 is an antibody whose epitope is the amino acid sequence of positions 354 to 359, which is the extracellular region of TSHR.

7) cAMP measurement
After collecting TSHR-expressing cells with PBS containing 1 mM EDTA (pH 8.0) and 0.3% BSA, Tyrode-Hepes buffer (14 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl ₂ , 12 mM NaHCO ₃ , 5.6 mM D-Glucose , 0.49 mM MgCl ₂ , 0.37 mM NaH ₂ PO ₄ , 25 mM Hepes, pH 7.4), suspended in 2 × 10 ⁴ cells / 100 μl / tube, and added with various concentrations of TSH in the presence of 0.5 mM IBMX (isobutylmethylxanthine) 37 Incubated for 30 minutes at ° C. A final concentration of 0.1 M hydrochloric acid was added, and the amount of cAMP in the cell extract after boiling was measured using a cyclic AMP kit “Yamasa” (Yamasa Soy Sauce).

<Result>
When the pC1-IRES-ΔCD4-TSHR vector was transfected into CHO-K1 cells, and after 24 hours, the gene introduction efficiency was examined with a flow cytometer, it was 6.0%. Therefore, we tried to concentrate the transgenic cells using MACS system. As a result, as shown in FIG. 4, the rate of ΔCD4-positive cells after MACS treatment was 27.8% for the first time, 30.7% for the second time, and 42.8% for the third time. Finally, ΔCD4-positive cells could be concentrated to 67.3% in the fourth round.

  Thereafter, cloning was performed from the concentrated cells, and finally 34 clones were obtained. As a result of analysis using a flow cytometer, 32 CD4-positive cells and 2 negative cells were found. These clones were classified into 6 groups based on ΔCD4 expression patterns, ie, flow cytometer histograms. As a result of analyzing 6 representative clones using 2C11, there were only 2 types of TSHR positive clones. Among these two clones, clone 12 with high TSHR expression was selected and named CHO-TSHR-AHK12 (hereinafter referred to as AHK12) and used for cAMP measurement.

As shown in FIG. 5, the production of cAMP in AHK12 increased in a TSH concentration-dependent manner, and showed an increase of 80 times or more in the 10 mU / ml added group compared to the unstimulated group. The EC50 of TSH was about 0.1 mU / ml.
When this AHK12 was added with 1 mg / ml of G418 and further cultured for 2 months and then analyzed again with a flow cytometer, no decrease in TSHR expression in AHK12 was observed as shown in FIG. In addition, all the clones that recloned AHK12 showed the same TSHR expression pattern as the parent strain AHK12, suggesting that AHK12 is a completely stable TSHR-expressing cell line.

Example 2: Production of Graves' disease model mouse <Material and method>
1) Preparation of TSHR expression vector
PC1-IRES-ΔCD4 (FIG. 1) and pBacMam2 (FIG. 7) were used as TSHR expression vectors. In Graves' disease, the extracellular region up to the first transmembrane domain of TSHR is considered to be important for early antigen recognition and binding to anti-TSHR antibodies. Therefore, a total of four types of expression vectors were prepared in which TSHR-wildtype (TSHR-WT) and the extracellular region TSHR-289His (amino acids 1 to 289 from the N terminus) were incorporated into each of the two types of vectors. Furthermore, a vector in which LacZ was incorporated into pBacMam2 was prepared in order to perform a basic study of the in vivo EP method.

2) Basic study of in vivo EP method Using CUY21 EDIT (Neppagene) as an electroporator, basic study of in vivo EP method was conducted. After diluting Nembutal 10 times with saline, a dose of 50 mg / kg was administered intraperitoneally to 5-week-old female ICR mice. Under anesthesia, the skin of the thigh was cut about 0.5 cm, and a parallel needle fixed electrode was inserted into the muscle. PBacMam2-LacZ was injected into the thigh muscle between the electrodes at 50 μg / site (final 0.9% NaCl). After injection of the expression vector, 6 pulses were given under the conditions of 50 V and loading time of 50 msec. This operation was performed on the left and right thighs, and the skin was sutured after the operation.
The group subjected to DNA vaccination was used as a control for in vivo EP method. For DNA vaccination, one week before vaccination, 50 μl of 10 μM cardiotoxin is injected into the planned expression vector administration site (cardiotoxin pretreatment), and then the phosphate buffered saline-dissolved plasmid vector DNA is injected intramuscularly. did.
In order to examine the range of transgene expression, muscle tissues 3 and 7 days after introduction were collected. In addition, in order to examine temporal changes in gene expression, muscle tissues were collected 1, 3, 5, 7, 10, 14, 21 days after introduction.

3) Confirmation of β-gal expression in muscle tissue
The thigh muscle at the muscle injection site of the TSHR expression vector was excised, and β-gal expression in the muscle tissue was confirmed. A slice of muscular tissue and a cryosection (LEICA, Jung Frigocutt 2800E) frozen section cut to 10 μm were fixed with 10% formalin solution, and then a substrate solution containing X-gal was added dropwise to develop a blue color. Was observed.

4) Measurement of β-gal activity in muscle tissue
Β-gal activity was measured at the intramuscular injection site of TSHR expression vector. Remove the thigh muscle from the expression vector intramuscular injection site, and buffer A (15 mM Tris-HCl (pH 8.0), 60 mM KCl, 15 mM EDTA, 500 mM DTT, 0.4 mM p-ABSF) 3 times the wet weight ) And homogenized on ice. 15 cycles of ultrasonic grinding [Duty50, Output 2] were performed on ice, and the supernatant obtained by centrifugation at 14000 rpm, 4 ° C. for 10 minutes was collected. The absorbance of o-nitrophenol produced by adding ONPG (o-nitrophenyl-β-D-galactoside) as a substrate and incubating at 37 ° C. for 3 hours was measured at a wavelength of 415 nm. After further protein quantification, the activity value (unit) / mg protein was converted.

5) Production of Graves' disease model mouse
The same procedure as 2 was performed using 5-week-old female BALB / c mice. In the first onset experiment, pC1-IRES-ΔCD4 was used as an expression vector. As a control, three expression vectors incorporating pC1-IRES-ΔCD4, TSHR-WT, and TSHR-289His were introduced into the muscle at 50 μg / site using the in vivo EP method. This operation was performed 3 times every 3 weeks.
Moreover, in order to measure TSAb activity, the serum of the 7th day was extract | collected after the 2nd and 3rd EP, respectively. The number of cases was 25 for each of pC1-IRES-ΔCD4-TSHR-WT and TSHR-289His, 15 controls, and 9 sham.
In the second onset experiment, pBacMam2 was used as an expression vector, and the conditions such as the procedure, administration schedule, number of cases, etc. were the same as in the first onset experiment.

<Result>
As shown in the left of FIG. 8, expression of β-gal along muscle fibers was observed in the group into which pBacMam-LacZ was introduced by the in vivo EP method. Further, as shown in the right side of FIG. 8, when observed from the cross section of the muscle fiber, the activity of β-gal was observed concentrated on a part of the region subjected to in vivo EP treatment.

  As shown in FIG. 9, in the in vivo EP treated group, the enzyme activity of β-gal showed a maximum value on the 7th day and almost disappeared on the 14th day. In the DNA vaccination treatment group, the maximum value was observed after 1 day, and it decreased to a value almost the same as that of the control group on the 3rd day. Compared to the group that had undergone DNA vaccination after cardiotoxin pretreatment, up to 60-fold β-gal activity was shown after 7 days in the in vivo EP treated group.

Example 3: Measurement of TSAb in Grapes' model disease mouse serum using TSHR-expressing cell line <Material and method>
Using the TSHR-expressing cell line established in Example 1, TSAb in the Graves' disease model mouse serum prepared in Example 2 was measured. Blood collected from the mouse prepared in Example 1 was centrifuged at 12000 rpm at 4 ° C. for 10 minutes to collect serum. TSAb activity was evaluated using the human TSHR expression strain CHO-K1-TSHR-AHK12 established in Example 1. After stimulating CHO-K1-TSHR-AHK12 with 5% mouse serum, the cAMP increasing activity of TSAb was measured in the same manner as 7) of Example 1 using cAMP kit “Yamasa”. Further, the thyroid gland was removed from the mouse, the thyroid gland was observed for morphological changes, and the thyroid tissue was observed by HE staining.

<Result>
With respect to cAMP-elevating activity, the average value of the control group + 3SD was used as a reference, and a sample exceeding this was determined as TSAb positive. As a result, in the group in which pC1-IRES-ΔCD4-TSHR-WT was introduced by in vivo EP method, 0/25 TSAb-positive individuals after the second EP, 2/25 TSAb-positive individuals after the third EP, and the fourth EP There were 0/25 post-TSAb positive individuals. In TSHR-289His, as shown in FIG. 10, 10/24 were TSAb positive individuals after the second EP, 18/23 were TSAb positive after the third EP, and 11/23 were TSAb positive after the fourth EP. Met. Similarly, as shown in FIG. 10, in the pBacMam2 vector, TSHR-WT was 11/25 after the third EP and 19/25 in TSHR-289His. Moreover, the TSAb activity after 2 times of EP of pBacMam2-TSHR-289His showed a value 15 times the average value of the control at the maximum. Further, as shown in FIGS. 11 and 12, thyroid enlargement was observed histologically in the TSAb positive solid.

  The cell line establishment method of the present invention is simple, safe, and efficient, and is useful for functional analysis of various target proteins, pathological analysis and pathological evaluation of diseases associated with the proteins. In addition, the TSHR-expressing cell line of the present invention can be used for pathological analysis, pathological evaluation of Graves 'disease patients, and evaluation of model mice by detecting anti-TSHR antibodies in the serum of Graves' disease model mice. Furthermore, the target protein purified from the expressed cells can be used as an antigen or the like in another autoantibody measurement system.

2 shows the structure of pC1-IRES-ΔCD4, which is a TSHR expression vector. Example 1 By using pC1-TSHR-IRES-ΔCD4, it is possible to express TSHR simultaneously in all cells that theoretically express CD4. Example 1 The cells were concentrated using a magnetic cell separation system (MACS) and an anti-CD4 antibody bound with magnetic microbeads. Example 1 The result of having concentrated ΔCD4 positive cells by MACS is shown. Example 1 The dose-response curve of TSH in CHO-TSHR-AHK-12 is shown. Example 1 Stable expression in CHO-TSHR-AHK-12 is shown. Example 1 The structure of pBacMam2, a TSHR expression vector, is shown. (Example 2) In the group into which pBacMam2 was introduced by the in vivo EP method, the expression of β-gal along the muscle fiber (left) and the activity of β-gal in the cross section of the muscle fiber (right) are shown. (Example 2) The change of the enzyme activity of (beta) -gal by in vivo (in vivo) EP treatment is shown. (Example 2) Induction of TSAb in pC1 and pBacMam2 vectors is shown. Example 3 The thyroid gland changes in TSAb positive individuals. Example 3 A thyroid tissue image (HE staining) is shown. Example 3

Claims (14)

A method for establishing a cell line comprising the following steps;
1) introducing a vector carrying a gene encoding a target protein and a gene encoding a carrier protein into an animal cell;
2) A step of cloning a cell line stably expressing the target protein using a magnetic cell separation system. The method according to claim 1, wherein the carrier protein is a protein expressed on a cell membrane. The method according to claim 1 or 2, wherein the carrier protein is CD4. The method according to any one of claims 1 to 3, wherein the target protein is a thyroid stimulating hormone receptor (TSHR). The method according to claim 4, wherein the TSHR gene is DNA encoding an extracellular portion of TSHR. The method according to claim 4 or 5, wherein the TSHR gene is derived from human or pig. A cell line stably expressing the target protein obtained by the method according to any one of claims 1 to 6. A method for analyzing the function of a target protein using a cell line stably expressing the target protein obtained by the method according to any one of claims 1 to 6. A method for detecting an antibody against a target protein in serum using a cell line stably expressing the target protein obtained by the method according to any one of claims 1 to 6. A method for measuring the activity of stimulatory and inhibitory antibodies against TSHR in serum, comprising the following steps;
1) A TSHR stable expression cell line obtained by the method according to any one of claims 4 to 6 is stimulated with the serum.
2) The cAMP increasing activity in the cell line is measured. The method according to claim 9 or 10, wherein the serum is derived from a mammal. The method according to claim 11, wherein the serum is derived from a Graves' disease patient. The method according to claim 12, wherein the serum is derived from a mouse. The method according to claim 13, wherein the mouse is a Graves' disease model mouse.