JP2007314548A - Fragment encoding swine tsh (thyroid-stimulating hormone) receptor and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA fragment encoding a swine TSH (thyroid-stimulating hormone) receptor and uses thereof. <P>SOLUTION: The DNA fragment is a DNA having a base sequence encoding a specific amino acid sequence or another specific base sequence in which one or a plurality of bases are deleted, substituted, inserted or added in the sequences or hybridizing with the DNAs under stringent conditions and encoding a swine-derived TSH receptor. Furthermore, an expression vector is prepared by inserting any DNA fragment described above into a plasmid or a phage and a recombinant swine TSH receptor is obtained by using the expression vector, transforming a host cell and culturing the resultant transformant. A method for producing the recombinant swine TSH receptor is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a DNA fragment encoding a porcine TSH (Thyroid-stimulating hormone) receptor and its use.

  Thyroid-stimulating hormone receptor (hereinafter referred to as TSH receptor) is a receptor for thyroid-stimulating hormone (TSH) present on the thyroid cell membrane. Graves disease (Basedow disease), which is a typical disease of the thyroid gland, is a disease in which thyroid function is enhanced, and is considered to develop due to autoantibodies to the TSH receptor.

Autoantibodies against the TSH receptor include at least an antibody that binds to the TSH receptor and exhibits thyroid stimulating activity (thyroid stimulating antibody) and an antibody that binds to the TSH receptor and prevents the stimulating activity of TSH against the TSH receptor (thyroid stimulating inhibitory antibody). It is known that antibodies with two different activities exist.
The measurement of autoantibodies against the TSH receptor is used for Graves' disease diagnosis, disease grasp, etiology analysis, etc., and can be roughly classified into two according to the measurement principle.

  That is, in one method, the solubilized porcine thyroid membrane fraction is reacted with patient serum and isotope-labeled TSH, and the inhibition rate of binding of labeled TSH to the TSH receptor by the TSH receptor autoantibodies in the test serum is measured. This is a method of measuring TSH receptor autoantibody values (Non-patent Document 1).

In another method, a test serum fractionated by polyethylene glycol treatment is used as a measurement sample, and when this is reacted with porcine thyroid cells, the thyroid stimulating antibody (TSAb) in the sample is transferred to the TSH receptor on the porcine thyroid cell membrane. Since it binds and adenylate cyclase in the cell is activated to produce cAMP, it is a method of calculating TSAb activity from the ratio of the amount of cAMP produced in test serum and control serum (Non-patent Document 2).
Methods in Enzymology, 74, 405-420 (1981), Endocr. Rev., 9, 106-120, (1988) Endoclin.Vol.125, No.1, pp410- (1985)

  However, preparation of porcine-derived thyroid cells or membrane fractions used in the above method has not always been simple. For example, thyroid tissue was obtained from slaughtered pigs, and the tissue was further separated by collagenase and trypsin treatment and then stored frozen and thawed for use in the assay. In addition, the possibility of differences among lots of cells due to individual differences in pigs used for cell preparation, conditions during processing operations, storage conditions, etc. could not be denied.

  For this reason, if a recombinant TSH receptor can be used, preparation of a reagent or kit for measuring an autoantibody against the TSH receptor is simplified, and it is expected that the risk of causing a lot difference can be reduced. In particular, it is considered that the use of a porcine-derived recombinant TSH receptor is less likely to dissociate from the measured value by the conventional method than the use of a recombinant TSH receptor derived from humans or dogs. It was considered that it was preferable to use a pig-derived material.

  However, reports on TSH receptor genes such as humans and dogs and attempts to produce human TSH receptors etc. by transforming CHO cells and myeloma cells using a virus containing the genes and culturing these cells have been reported. However, regarding the TSH receptor derived from swine, not only the analysis of the gene encoding the receptor, but also the amino acid sequence has not been reported. is there.

Therefore, in order to clone a gene encoding a TSH receptor derived from porcine thyroid cells, the present inventor prepared a primer based on the thyroid TSH receptor gene sequence derived from bovine, sheep, human, mouse, and rat, As a result of screening of cDNA synthesized using the RNA of the above as a template, a gene encoding a protein having 764 amino acids and a molecular weight of 86,641 was found, and the base sequence of the gene was similar to that of a known animal-derived TSH receptor gene. I confirmed.
Further, as a result of expressing this gene and confirming its function, it was confirmed that the gene was a gene encoding porcine TSH receptor, and the obtained recombinant porcine TSH receptor was used for measurement of autoantibodies against TSH receptor. The present invention has been completed by finding out what can be done.

  Accordingly, the present invention relates to a porcine TSH comprising an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence represented by SEQ ID NO: 1, wherein one or several amino acids are deleted, substituted, inserted or added. The present invention relates to a DNA fragment encoding a receptor.

  Further, the present invention has a base sequence represented by SEQ ID NO: 2 or a base sequence represented by SEQ ID NO: 2, wherein one or several bases are deleted, substituted, inserted or added, Alternatively, the present invention relates to a DNA fragment that hybridizes with these DNAs under stringent conditions and that encodes a porcine-derived TSH receptor.

  Furthermore, the present invention provides an expression vector prepared by inserting any of the above DNA fragments into a plasmid or phage, and a host cell transformed using the expression vector, and the resulting transformant is cultured. The present invention relates to a recombinant porcine TSH receptor and a method for producing the same.

The present inventor can specify for the first time a DNA fragment encoding a porcine-derived TSH receptor, and a recombinant Vita TSH receptor prepared using such a DNA fragment is useful for measuring an anti-TSH receptor antibody.
In particular, compared to the conventional method using normal porcine thyroid cells, the amount of cAMP produced is higher with the recombinant Vita TSH receptor and can be measured with higher sensitivity.

The present invention relates to a DNA fragment encoding a porcine TSH receptor consisting of the amino acid sequence represented by SEQ ID NO: 1.
The receptor consisting of the amino acid sequence represented by SEQ ID NO: 1 is not limited to the amino acid sequence represented by SEQ ID NO: 1 as long as the TSH receptor activity is maintained. An amino acid sequence in which several amino acids are deleted, substituted, modified, or added may be used.

If the DNA fragment of this invention is illustrated with a base sequence, the DNA fragment which has a base sequence shown by sequence number 2 can be mentioned.
A DNA fragment having the base sequence represented by SEQ ID NO: 2 was prepared from cDNA synthesized using porcine thyroid cell RNA as a template, as described in detail in the Examples below. It is the same as the sequence represented by nucleotide numbers 54 to 2345 in FIG. 1-2, and corresponds to the structural gene of the TSH receptor.

  In the present invention, as long as a porcine TSH receptor can be produced, a DNA fragment in which one or more bases in the base sequence represented by SEQ ID NO: 2 have been deleted, substituted, inserted or added, or those A DNA fragment that hybridizes with this DNA fragment under stringent conditions can also be used. The term “hybridization under stringent conditions” used herein means 5 × SSC (1 × SSC is 8.76 g of sodium chloride, 4.41 g of sodium citrate dissolved in 1 liter of water), 0. Using a solution containing 1% w / v N-lauroylsarcosine sodium salt, 0.02% w / v SDS, 0.5% w / v blocking reagent, a hybridization reaction was performed at 60 ° C. for about 20 hours. Sometimes it means to hybridize.

  Preparation of the DNA fragment of the present invention, that is, cloning of a target gene from cDNA synthesized using RNA extracted and purified from animal tissue as a template, preparation of an expression vector using the cloned DNA fragment, and use of an expression vector Production of recombinant TSH receptor and the like is a technique well known to engineers belonging to the field of molecular biology. Specifically, for example, “Molecular Cloning” (edited by Maniatis et al., Cold Spring Harbor Laboratories, Cold Spring Harbor , New York (1982)).

  For example, first, primers are synthesized with reference to the sequence of a similar gene that has already been cloned. Specifically, since TSH receptor genes such as cattle, sheep, and humans have already been cloned (see Biochemical and Biophysical Research Communications, 165, 1184 (1984), etc.), primers are synthesized using these sequences as they are. Alternatively, the TSH receptor genes of known animals may be compared, and a primer may be synthesized using the base sequence of the common part.

  Next, a DNA fragment containing a gene encoding a TSH receptor from a cDNA synthesized by using a synthesized primer as a probe and RNA extracted from porcine thyroid cells as a template (or a cDNA library prepared by a known method from cDNA) To clone. Examples of cloning methods include known methods such as plaque or colony hybridization methods (Molecular Cloning, Cold Spring Harbor Laboratory, (1982)) using oligonucleotides chemically synthesized based on amino acid sequences as probes. .

  The base sequence of the DNA thus obtained can be determined by known methods such as the Maxam-Gilbert method (Pro. Natl. Acad. Sci., USA 74,560 (1977)), the dideoxy method (Nucl. Acids. Res., 9,309). (1981)), or the Deaza method (Nucl. Acids. Res., 14, 1319 (1986)).

  By comparing the determined nucleotide sequence or the amino acid sequence deduced therefrom with that of a known animal-derived TSH receptor, the presence of a DNA fragment encoding a porcine-derived TSH receptor is confirmed. By repeating the above method, the base sequence of the porcine TSH receptor gene is determined.

  Since it is difficult to set PCR primers at both ends of the gene, that is, around the 5 ', N-terminal, C-terminal, or 3', it is often impossible to determine the base sequence by the above method. In such a case, use the 5 ′ RACE method and the 3 ′ RACE method (3′-Full RACE Core Set and 5′-Full RACE Core Set, etc., manufactured by Takara Shuzo Co., Ltd.) for the arrangement of these regions. Can be determined.

  The TSH receptor is prepared by inserting the cloned TSH receptor gene into a known plasmid or phage having a promoter or the like to prepare an expression vector, and cells transformed with the expression vector (CHO, COS, etc.) And a method of producing a TSH receptor by culturing the cells derived from mammalian cells or insect cells such as Sf9 and Sf21) according to a method known per se (for example, Biochemical and Biophysical Research Communications Vol.165, No.3, 1250-1255 (1989), JP 4-506675, J. Immunol. 158, 2798-2804 (1997), etc.).

  From the prepared culture, cells produced by the TSH receptor are recovered by membrane separation or centrifugation. The collected cells can be used as they are for the measurement of anti-TSH receptor antibody. In addition, the cells are disrupted by sonication, etc., separated by centrifugation, gel filtration, etc., and then heat treated, ammonium sulfate. It is also possible to isolate and purify several kinds of analysis treatment, dialysis treatment, various chromatographic treatments, etc., and use them for the measurement.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, it is clear that this invention is not limited to an Example.

Example 1: Determination of base sequence of porcine TSH receptor gene (1) Primer As shown in FIGS. 2-1 to 2-3, TSH of already cloned human, bovine, sheep, dog, mouse and rat Compare the base sequence of the receptor gene, select the region that is almost 100% conserved in 5 kinds of animals in the range of 20-30 bases, 3 kinds of 5 ′ primers, 3 kinds of 3 ′ primers, a total of 6 kinds The primers were synthesized. An odd number was used as the primer number for the 5 ′ primer, and an even number was used for the 3 ′ primer. In addition, as a corresponding location, the location of the human TSH receptor gene corresponding to each primer is represented by +1 of A of the start codon ATG.

Primer 1: 5'-GACTTCAGAGTCACCTGCAAGG -3 '
(22 bases, corresponding location +106 to +127, GC content 54.5%)
Primer 2: 5'-CAGATGCCAAACTTGCTGAG-3 '
(20 bases, corresponding location +2074 to +2093, GC content 50.0%)
Primer 3: 5'-ATGGGCTACAAGTTCCTGAG -3 '
(20 bases, corresponding location +1234 to +1253, GC content 50.0%)
Primer 4: 5'-GCCAGTCGATGGCATGGTTGT -3 '
(21 bases, corresponding location +1445 to +1465, GC content 57.1%)
Primer 5: 5'-CTGGATGCTGTTTACCTGAAC -3 '
(21 bases, corresponding locations +604 to +624, GC content 47.6%)
Primer 6: 5'-GGATTCCTCTGATTTTCTTCTG -3 '
(22 bases, corresponding location +865 to +886, GC content 40.9%)

(2) Preparation of RNA from porcine thyroid cells Porcine thyroid tissue was treated with proteases such as collagenase and trypsin and dispersed into individual cells. Using about 6 g of porcine thyroid cells as the starting material, RNA was analyzed by the AGPC method (Experimental Medicine (Yodosha) Vol.9 No.15 p99 (1991), Bio-Experiment Illustrated (Shujunsha) Volume 2 p161)). Prepared. As a result, 1.75 mg of total RNA was obtained. Furthermore, mRNA was purified from this total RNA using Oligotex-dT30 (Takara Shuzo Co., Ltd.). As a result, about 40 mg of mRNA was obtained, and the OD ₂₆₀ / OD ₂₈₀ value was 1.758.

(3) PCR
RNA LA PCR ^™ Kit (AMV) ver. 1.1 (Takara Shuzo Co., Ltd.) was used to synthesize cDNA using total RNA prepared from porcine thyroid cells as a template. The reverse transcription reaction primer was Oligo dT-Adaptor primer (attached to the kit), the enzyme was Avian Myeloblastosis Virus-derived reverse transcriptase, and the reaction was carried out at 55 ° C. for 30 minutes.

  Next, PCR was performed using the synthesized cDNA as a template and the above primers as a pair of No. 1 and No. 6, No. 3 and No. 2, and No. 5 and No. 4. Amplification of the gene by PCR was performed by repeating the steps of heat denaturation (94 ° C., 30 seconds), annealing (55 ° C., 1 minute), and extension reaction (72 ° C., 1 minute) 50 times.

  When calculated from the base sequence of the human TSH receptor gene, a fragment with a size of 781 bases for primer 1 and primer 6, 864 bases for primer 3 and primer 2, and 862 bases for primer 5 and primer 4 is amplified. It is expected that When each of the three DNA fragments amplified by PCR was subjected to agarose electrophoresis, a DNA fragment having a size consistent with that expected was detected. Each DNA fragment was separated by agarose electrophoresis, and the portion containing the fragment was recovered and purified using DNACELL (Daiichi Chemical). The base sequence of the purified DNA fragment was determined, the corresponding amino acid sequence was deduced, and compared with base sequences and amino acid sequences derived from other animals, it was shown that they were very similar. Therefore, based on the obtained base sequence, the following primers were further synthesized and PCR was performed in the same manner as described above to determine the base sequence of almost the entire region of the TSH receptor structural gene.

Primer 9 5'-CACTGACTTCATGTGCATGGC-3 '
(21 bases, corresponding location +1893 to +1913, 52.4%)
Primer 10 5'-GGACTGTGATTCCAGCTGCTG-3 '
(21 bases, corresponding location +268 to +288, 57.1%)
Primer 11 5'-GACATCAATCCCTGCGAATGC-3 '
(21 bases, corresponding locations +492 to +512, 52.4%)
Primer 13 5'-GTGAATGCTGTAAATGGTCCC-3 '
(21 bases, corresponding location +943 to +963, 47.6%)
Primer 15 5'-ACGCCTACGCCATCATGGCTG-3 '
(21 bases, corresponding location +1610 to +1630, 61.9%)
Primer 16 5'-AACAGCATCCAGCTTTGTCCC-3 '
(21 bases, corresponding location +595 to +615, 52.4%)
Primer 18 5'-GTCGTAATGGCTGTCAAAGGC-3 '
(21 bases, corresponding location +1138 to +1158, 52.4%)
Primer 20 5'-AGAGGAGTCTCAGTGTCCATG-3 '
(21 bases, corresponding location + 1313 to + 1733, 52.4%)

(4) Determination of base sequence of C-terminal and 3 ′ peripheral region The 5 ′ and 3 ′ peripheral regions on both sides of the structural gene were different from the regions encoding amino acids among the animals compared. Since the base sequence of the obtained structural gene was relatively close to that of cattle and sheep compared to humans, mice, and rats, the primers corresponding to the 3 ′ peripheral region were based on the base sequences of cattle or sheep. Set up.

Primer 12 5'-CAGCCTAAGTCCTTGTACCACTTA-3 '
(24 bases, GC content 45.8%)
Primer 14 5'-GTGTCGATGGTTGGAATGATGCTC-3 '
(24 bases, GC content 50.0%)
When PCR was performed by combining the two 3 ′ primers with the primer 9 and the primer 15, amplification of a DNA fragment having the expected size when the primer 14 was used was observed. When the nucleotide sequence of the DNA fragment obtained using primer 9 and primer 14 was determined, it was found that there was a site that coincided with the nucleotide sequence of the C-terminal region of the obtained porcine TSH receptor gene and was derived from the gene. Indicated. This revealed the C-terminal region and the downstream base sequence.

(5) Determination of the base sequence of the N-terminal region and the 5 ′ peripheral region In the same manner as when the base sequence of the 3 ′ peripheral region was determined, three types of 5 ′ peripheral region were determined with reference to the base sequence derived from bovine or sheep. A 5 ′ primer was set.

Primer 7 5'-ATCGCCGAGCACGCAGAGGTAG-3 '
(22 bases, GC content 63.6%)
Primer 17 5'-ATGAGAGGGAGGCGATC-3 '
(17 bases, GC content 58.8%)
Primer 19 5'-GGGCCCGGAGGACGATG-3 '
(17 bases, GC content 76.5%)

PCR was performed using cDNA as a template and the above primer as a 5 'primer, and primers 10 and 16 as the 3' primer. However, no clearly amplified DNA fragment was detected, so the cDNA PCR Library Kit and cDNA A cDNA PCR Library was prepared using a Synthesis kit (both manufactured by Takara Shuzo Co., Ltd.).
PCR was performed using the prepared porcine thyroid cell-derived cDNA PCR Library as a template and a CA primer as a 5 ′ primer (attached to the cDNA PCR Library Kit) and a primer 20 as a 3 ′ primer. The PCR reaction solution was subjected to agarose electrophoresis, and a DNA fragment having about 1500 bases or more was recovered and purified from the gel.

  PCR was carried out using the prepared DNA as a template and further using the CA primer and primer 16, and the obtained DNA fragment was used as a template and PCR was carried out using the CA primer and primer 10. Amplification of the DNA fragment was observed. When the DNA fragment was recovered and purified and the nucleotide sequence was determined, the nucleotide sequence upstream of the previously known primer 10 was detected, indicating that this fragment was derived from the TSH receptor gene. As a result, the base sequence from the N-terminal region of the TSH receptor gene and 53 bases upstream from the ATG start codon was clarified.

Example 2 Cloning of Porcine TSH Receptor Gene Based on the nucleotide sequence revealed above, primer 21 which is a 5 ′ primer corresponding to the 5 ′ peripheral region and 3 ′ primer corresponding to the 3 ′ peripheral region are used. A primer 22 was designed and synthesized.

Primer 21 5'-GCTGAAGATGAAGAGATAGCC-3 '
(21 bases, corresponding location -18 to -38, GC content 47.6%)
Primer 22 5'-CTAAGACACCAGCCTAAGTCC-3 '
(21 bases, corresponding locations +2312 to +2332, GC content 52.4%)

  Using mRNA prepared from porcine thyroid cells as a template, cDNA was synthesized using Oligo dT-adapter primer (attached to cDNA PCR Library Kit) and Avian Myeloblastosis Virus-derived reverse transcriptase. Furthermore, PCR reaction was performed using the primer 21 and the primer 22 using this cDNA as a template. The PCR reaction was performed by repeating the steps of heat denaturation (98 ° C., 10 seconds), annealing (55 ° C., 30 seconds), and extension reaction (72 ° C., 2 minutes) 25 times. The amplified TSH receptor gene (cDNA) was separated by agarose electrophoresis, recovered and purified.

  The purified TSH receptor gene fragment was cloned into a plasmid using a perfecty brand cloning kit (manufactured by Novagen, sold by Takara Shuzo Co., Ltd.). The base sequence of the obtained TSH receptor gene was identical to the base sequence determined above, and no mutation occurred.

Example 3: Preparation of recombinant porcine TSH receptor (1) Incorporation of cloned porcine TSH receptor gene (cDNA) into pCMV-Script Cloned TSH receptor cDNA derived from porcine thyroid tissue was used as a template, and is shown below. PCR was performed according to a conventional method using primer 23 (TSR37) and primer 24 (TSR22).
Primer 23 5'-GCC CGG CCA CCA TGG GTC TGA CGC CC-3 '
Primer 24 5'-CTA AGA CAC CAG CCT AAG TCC-3 '

  The amplified PCR fragment was named TSHR37 / 22. TSHR37 / 22 was subjected to agarose electrophoresis to separate and excise the DNA fragment, and then recovered and purified by DNACELL. The obtained TSHR37 / 22 was cloned into pCMV-Script using pCMV-Script PCR Cloning kit (Stratagene), and the resulting plasmid was named pCMV: pTSHR. The cloning method was performed according to the instructions attached to the kit.

(2) Transfection of pCMV: pTSHR into CHO-K1 cells and isolation of G-418 resistant strain Using lipofectamine (GIBCO BRL), pCMV: pTSHR DNA was transferred to CHO-K1 cells (Dainippon Pharmaceutical). Introduced. The transfection method was performed according to the instructions accompanying the lipofectamine.
After the transfection operation, the treated cells were cultured in Ham'sF-12 medium (GIBCO BRL) for 48 hours. After CHO cells were detached from the plate by trypsin treatment, one-tenth amount of cells were subcultured in a medium to which G-418 (gentamicin derivative, 160 mg / L) was newly added.

  The medium was changed every 2 to 3 days, the dead cells were removed, and the culture of the cells that grew in the presence of G-418 was continued. When cells grew in a medium containing G-418 to some extent, CHO cells transfected by trypsin treatment were detached and the number of cells was counted. Dilution was performed stepwise in a medium supplemented with G-418 according to the cell concentration, and each diluted solution was cultured in a 96-well plate. Furthermore, it observed with the microscope 2-3 days later, and the thing which has only one colony in one well was looked for, and about 11 strains were isolated. At the stage where these 11 isolates were cultured and grown so as to cover the bottom of the well to some extent, they were peeled off by trypsin treatment and continued to be transferred to a plate having a diameter of 100 mm.

(3) Search for CHO cells (transfectants) introduced with pCMV: pTSHR Each of the 11 G-418-resistant CHO cell lines isolated was 25,000 cells / well in a 22 mm diameter well. And cultured. After culturing for 3 days, the medium was removed, and 0.5 ml of a binding buffer having the following composition (hereinafter simply referred to as a binding buffer) was added to each well. Further, 5 μl of I ^125- labeled bovine-derived TSH (hereinafter referred to as I ¹²⁵ -bTSH) was added and incubated at 37 ° C. for 2 hours.

After the incubation, the medium was removed, and the medium was further washed once with 0.5 ml of ice-cold binding buffer. After washing, 0.5 ml of Lysis buffer (0.1N NaOH, 1% SDS, hereinafter simply referred to as Lysis buffer) was added to each well to solubilize cells adhering to the bottom surface. The solubilized cells were collected in a test tube, and further washed with 0.5 ml of an ice-cooled binding buffer. The solubilized cell solution and this washing solution were combined and the amount of I ¹²⁵ -bTSH bound to the TSH receptor was measured with a γ-counter. As a control, 4 cell lines transfected with pCMV-Script were also cultured and examined in the same manner. The results are shown in Table 1 below.

  As is apparent from Table 1, since the binding amount (cpm) of the four strains transfected with pCMV-Script cDNA is considered to be a basal level, pCMV: pTSHR trans showing a clearly higher binding amount than this. Fectant No. 2, no. 3, no. 4, no. 5, no. 6, no. 7, no. 8, no. 9 and no. It was suggested that a total of 9 strains of 11 expressed the TSH receptor. Among these, No. showing higher binding. 3, no. 5, no. 6 and no. 8 strains were selected for further study.

  In the table, Porcine represents pCMV: pTSHR, and Script represents a CHO cell line transfected with pCMV-Script.

Binding buffer composition (g / L)
KCl 0.40
KH ₂ PO ₄ 0.06
NaHCO ₃ 0.35
Na ₂ HPO ₄ 0.048
D-Glucose 1.00
Sucrose 95.84
Bovine Serum Albumin (BSA) 2.5

(4) Confirmation of CHO / pCMV: pTSHR transfectant pCMV: pTSHR transfectant No. 3, no. 5, no. 6, no. 8 total 4 strains (hereinafter referred to as P3, P5, P6, and P8, respectively) and pCMV-Script transfectant no. 1 and 2 strains (hereinafter, referred to as C1, C2) was used to examined the I ¹²⁵ -bTSH binding inhibitory effect by binding and not modified with an isotope bTSH (cold bTSH) added with I ¹²⁵ -bTSH .

That is, each transfectant was planted to a plate having a diameter of 100 mm so as to be 5 × 10 ⁵ cells / plate. The cells were cultured in a CO ₂ incubator at 37 ° C. for 3 days. After culturing, the medium was removed and replaced with 4 ml of binding buffer kept at 37 ° C. To this, 40 μl of I ¹²⁵ -bTSH was added and incubated at 37 ° C. for 2 hours to examine binding. Furthermore, the inhibition of binding of I ¹²⁵ -bTSH by addition of 20 μl coldTSH (50 mIU / ml) was also examined. Each test was performed in duplicate. Also. For the control C1 and C2 strains, only binding was examined.

After the incubation, the medium was removed and washed once with 2 ml of ice-cold binding buffer. 2 ml of lysis buffer was added thereto to solubilize and recover the cells adhering to the bottom of the plate. Further, it was washed with 1 ml of ice-cold binding buffer. These cell lysates and washings were combined, and the amount of I ¹²⁵ was measured with a γ-counter. The results are shown in Table 2 below. In Table 2, (-) indicates no addition of cold bTSH, and (+) indicates addition. Therefore, (−) represents the ability to bind to I ¹²⁵ -bTSH, and (+) represents the inhibitory effect due to the addition of cold TSH.
From the results of Table 2, the transfectants P3, P5, P6, and P8 have significantly higher binding ability to I ¹²⁵ -bTSH than the control, and the binding is inhibited by the addition of bTSH. It was revealed that the transfected porcine TSHR gene was expressed.

(5) Examination of binding ability with ligand (I ¹²⁵ -bTSH) P6 and C1 strains were each inoculated and cultured in a well having a diameter of 22 mm at 25,000 cells / well. After culturing for 3 days, the medium was removed, and 0.5 ml of binding buffer was added to each well. Further, I ¹²⁵ -bTSH was added in 5 steps (2, 4, 6, 10 and 20 μl) separately and incubated at 37 ° C. for 2 hours.

After the incubation, the medium was removed, and the medium was further washed once with 0.5 ml of ice-cold binding buffer. After washing, 0.5 ml of lysis buffer was added to each well to solubilize cells adhering to the bottom surface. The solubilized cells were collected in a test tube, and further washed with 0.5 ml of an ice-cooled binding buffer. The solubilized cell solution and this washing solution were combined and the amount of I ¹²⁵ -bTSH bound to the TSH receptor was measured with a γ-counter. The results are shown in Table 3. Each test was performed in duplicate.

As is apparent from Table 3, the amount of P6 bound increased in proportion to the amount of added I ¹²⁵ -bTSH up to 10 μl (= about 3000 cpm), and the amount of added ligand was much different between about 3000 and 6000. From this, it can be seen that the bond has reached saturation. On the other hand, the binding amount of C1 is almost constant irrespective of the amount of added ligand, suggesting nonspecific binding.

(6) Examination of Ligand (I ¹²⁵ -bTSH) Binding Inhibitory Effect by Addition of Cold bTSH P6 and C1 strains were each inoculated and cultured in a 22 mm diameter well at 25,000 cells / well. After culturing for 3 days, the medium was removed, and 0.5 ml of binding buffer was added to each well. To this, 10 μl of I ¹²⁵ -bTSH amount was added, and the added cold bTSH amount was added in 7 steps and incubated at 37 ° C. for 2 hours. The amount of cold bTSH added is 0, 5, 15, 50, 150, 500 and 1500 μIU per well, and the final concentrations are 0, 0.01, 0.03, 0.1, 0.3, 1 0.0 and 3.0 mIU / ml.

After the incubation, the medium was removed, and the medium was further washed once with 0.5 ml of ice-cold binding buffer. After washing, 0.5 ml of lysis buffer was added to each well to solubilize cells adhering to the bottom surface. The solubilized cells were collected in a test tube, and further washed with 0.5 ml of an ice-cooled binding buffer. The solubilized cell solution and this washing solution were combined and the amount of I ¹²⁵ -bTSH bound to the TSH receptor was measured with a γ-counter. The results are shown in Table 4. Each test was examined in duplicate.

As is clear from Table 4, the amount of ligand binding of control C1 is constant without being affected by the amount of cold bTSH added, whereas P6 has a relatively low amount of ligand bound as the concentration of cold bTSH added increases. 100% binding is inhibited at a concentration of 1 mIU / ml. Therefore, it was shown that the binding of P6 to I ¹²⁵ -bTSH is specific binding via the pTSH receptor.

(7) Examination of cAMP production ability by stimulation with bTSH and TSAb The in vitro diagnostic pharmaceutical TSAb kit “Yamasa” (manufactured by Yamasa Soy Sauce) (hereinafter simply referred to as “kit”) was used for measurement. The measurement method was performed according to the package insert attached to the kit. That is, P6 and C1 strains were each planted and cultured in a well of 11 mm in diameter so as to be 6,000 / well. When the cells were almost full after culturing for 6 days, the medium was removed and the cells were further washed with 0.5 ml of cell washing solution attached to the kit. After adding 50 μl of cell washing solution to the well, 200 μl of cell activity determination solution (bTSH 100 mIU / ml) attached to the kit or an IgG fraction sample prepared from human serum containing TSAb was added and stirred.

After 4 hours incubation at 37 ° C., 10 μl of stop solution was added. 25 μl of this reaction solution was transferred to a glass tube, and 100 μl of I ¹²⁵ -cAMP solution and 100 μl of cAMP antibody solution were added and stirred well. After incubation at 4 ° C. overnight, 500 μl of cAMP second antibody solution was added and stirred. After incubation at 4 ° C. for 30 minutes, the mixture was centrifuged under conditions of 1860-1970 g, 4 ° C., 15 minutes. After removing the supernatant with an aspirator, I ¹²⁵ of the precipitate was measured with a γ-counter. The TOTAL and NSB samples were prepared and the cAMP standard curve was prepared according to the package insert. The measurement results are shown in Table 5.

As is clear from Table 5, the value of bTSH or IgG fraction did not change in the control C1, indicating that it did not react with the stimulating substance. On the other hand, since P6 produces considerably high cAMP in response to bTSH and TSAb, the CHO / pTSHR (P6) cell of the present invention specifically binds to TSH, and binds to TSH and is stimulated by TSAb. As a result of cAMP production, it was revealed that the receptor protein was expressed in a functional form in CHO cells transfected with pTSHR cDNA.
Furthermore, the amount of cAMP produced is higher with the use of the recombinant porcine TSH receptor than when normal porcine thyroid cells are used, and measurement can be performed with higher sensitivity.

It shows the nucleotide sequence of a DNA fragment containing a porcine TSH receptor structural gene (2292 bp, encoding a polypeptide having a molecular weight of 86,641 consisting of 764 amino acids). In the figure, Met represents the translation start codon of the porcine TSH receptor structural gene, and stop represents its stop codon. It shows the nucleotide sequence of a DNA fragment containing a porcine TSH receptor structural gene (2292 bp, encoding a polypeptide having a molecular weight of 86,641 consisting of 764 amino acids). In the figure, Met represents the translation start codon of the porcine TSH receptor structural gene, and stop represents its stop codon. The amino acid sequence of the pig-derived TSH receptor of the present invention is shown in comparison with the amino acid sequences of human, bovine, sheep, mouse, rat, and dog-derived TSH receptors. In the figure, porcine is a pig, human is a human, Bos taurus is a cow, Ovis aries is a sheep, Mus musculus is a mouse, Rattus is a rat, and Canine is an amino acid sequence of a TSH receptor derived from a dog. Moreover, the alphabet in a figure is the one-letter code of an amino acid, and shows the following amino acids. The amino acid sequence of the pig-derived TSH receptor of the present invention is shown in comparison with the amino acid sequences of human, bovine, sheep, mouse, rat, and dog-derived TSH receptors. In the figure, porcine is a pig, human is a human, Bos taurus is a cow, Ovis aries is a sheep, Mus musculus is a mouse, Rattus is a rat, and Canine is an amino acid sequence of a TSH receptor derived from a dog. Moreover, the alphabet in a figure is the one-letter code of an amino acid, and shows the following amino acids. The amino acid sequence of the pig-derived TSH receptor of the present invention is shown in comparison with the amino acid sequences of human, bovine, sheep, mouse, rat, and dog-derived TSH receptors. In the figure, porcine is a pig, human is a human, Bos taurus is a cow, Ovis aries is a sheep, Mus musculus is a mouse, Rattus is a rat, and Canine is an amino acid sequence of a TSH receptor derived from a dog. Moreover, the alphabet in a figure is the one-letter code of an amino acid, and shows the following amino acids. A: Alanine, R: Arginine, N: Asparagine, D: Aspartic acid, C: Cysteine, Q: Glutamine, E: Glutamic acid, G: Glycine, H: Histidine, I: Isoleucine, L: Leucine, K: Lysine, M : Methionine, F: phenylalanine, P: proline, S: serine, T: threonine, W: tryptophan, Y: tyrosine, V: valine

Claims (1)

  A recombinant porcine TSH receptor having an amino acid sequence represented by SEQ ID NO: 1 prepared using a DNA fragment encoding a porcine TSH (Thyroid-stimulating hormone) receptor having a base sequence represented by SEQ ID NO: 2.