JP2000232880A - Production of gene recombinant human thyroid stimulating hormone receptor or its derivative using insect cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably and in large quantities obtain gene recombinant human thyroid stimulating hormone receptor(TSHR) used in the measurement or the like of an anti-TSHR autoantibody by cultivating an insect cell infected with a virus containing a gene of human TSHR or the like under a high oxygen concentration. SOLUTION: The objective gene recombinant human thyroid stimulating hormone receptor(TSHR) or its derivative useful in the measurement or the like of an anti-TSHR autoantibody for diagnosing thyroid autoimmune diseases represented by Basedow disease is obtained by cultivating an insect cell infected with a virus containing a gene coding for human TSHR or its derivative under a high oxygen concentration condition to produce insoluble human TSHR or its derivative, solubilizing the resultant insoluble human TSHR or its derivative in the presence of a recfucing agent and a protein denaturing agent and not only separating it from impurities but also decreasing the concentration of the above reducing agent and protein denaturing agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a recombinant human thyroid stimulating hormone receptor (hereinafter, a human thyroid stimulating hormone receptor is simply referred to as TSHR unless otherwise specified) using an insect cell as a host. More specifically, a method characterized by culturing insect cells under high oxygen concentration conditions, or after culturing, solubilize the culture solution in the presence of a reducing agent and a protein denaturing agent, separate from contaminants, and The present invention relates to a method characterized by lowering the concentration of a reducing agent and a protein denaturing agent.

[0002] The recombinant TSHR produced according to the present invention,
In particular, after culturing the insect cells, the recombinant TSHR produced by solubilizing the culture solution in the presence of a reducing agent and a protein denaturing agent, separating the contaminants and reducing the concentration of the reducing agent and the protein denaturing agent, Anti-TSH retaining reactivity with autoantibodies to human thyroid stimulating hormone receptor
It is useful as a reagent for measuring R autoantibodies.

[0003]

2. Description of the Related Art TSHR (Thyroid Stimu)
rating hornone receptor)
Is a thyroid stimulating hormone (Thyroid Stimu)
When TSH secreted from the pituitary gland binds to TSHR on the thyroid follicular cell membrane, the thyroid glands T3 and T4, which are regulatory hormones for metabolic function. Secrete. Details of the structure and the like of human TSHR have already been reported, and its molecular weight is about 87,000, a seven-transmembrane receptor, and the molecular weight of the extracellular domain is about 45,000.

[0004] Basedon's disease is caused by the production of thyroid hormone,
Hyperthyroidism due to increased secretion. One cause is the presence of stimulants in serum that stimulate thyroid hormone secretion. Studies have shown that serum from patients with Graves' disease has autoantibodies against thyroid components that stimulate thyroid hormone production and secretion,
It has been shown to eventually cause thyroid tissue breakdown.

[0005] As a reagent for diagnosing thyroid autoimmune diseases represented by Graves' disease and performing early treatment,
For example, reagents for measuring autoantibodies to thyroid peroxidase (TPO), thyroglobulin (Tg), TSHR and the like are known. Since anti-TSHR autoantibodies are known to be strongly involved in various diseases associated with the above-mentioned diseases, a reagent for measuring an autoantibody to TSHR is important among the above reagents.

Conventionally available reagents for measuring anti-TSHR autoantibodies are described in Smith et al. (Method in E).
nzymology, 74, 405-420, 1981
Year and Endocr. Rev .. , 9, 106-120,
1988). In these reagents, the presence of anti-TSHR autoantibodies in the patient's serum was determined by competitively binding 125 I-labeled bovine TSH and anti-TSHR autoantibodies in the patient's serum to TSHR in the porcine thyroid membrane fraction. And to measure the abundance.

[0007]

In the conventional reagents for measuring anti-TSHR autoantibodies which are commercially available, since a radioisotope of 125I is used as a labeling substance, handling of the reagents such as storage conditions and execution conditions is not sufficient. There are problems that it is troublesome and that the waste liquid after measurement must be strictly controlled. In addition, since TSHR in the porcine thyroid membrane fraction is an essential component, there is also a problem that industrial mass production is difficult. Furthermore, TSHR in the porcine thyroid membrane fraction
There is also a problem that the reactivity between the antibody and the human anti-TSHR autoantibody to be measured is not the same as the reactivity between the human TSHR and the human anti-TSHR autoantibody.

[0008] In recent years, attempts have been reported to transform CHO cells and myeloma cells with a virus containing the human TSHR gene and culture these cells to produce human TSHR industrially. However, it has not been reported that a large amount of human TSHR has been produced so far, which is sufficient for a measurement reagent using a radioisotope capable of high-sensitivity detection as a labeling substance as described above. However, there is a problem that the quantity is insufficient to use, for example, an enzyme or the like as a labeling substance in order to solve the problem contained in the measurement reagent. On the other hand, attempts have been made to express human TSHR using Escherichia coli as a host, but human THSR produced by Escherichia coli has poor reactivity and is difficult to use as a reagent for measuring anti-TSHR autoantibodies. There are issues.

In general, in order to use a low-sensitivity but easily handled enzyme or the like as a labeling substance instead of a radioisotope as a labeling substance, and to obtain the same sensitivity as when a radioisotope is used as a labeling substance, It is said that at least 1 mg of TSHR having high reactivity with anti-TSHR autoantibodies must be able to be produced.

[0010] As a solution to the above-mentioned problems, there has been reported that TSHR containing TSHR having reactivity with autoantibodies is expressed in insect cells (GS See).
tharamaiah et al. Immunol. Fifteen
8, 2798-2804, 1997). However, it is difficult to set conditions for TSHR expression by insect cells, and there is a problem that the TSHR expression level in each culture is unstable. Further, there is a problem that the expressed TSHR is insoluble and it is difficult to carry out a purification operation as it is. Furthermore, insoluble TSHR has a problem that most of it has no reactivity with anti-TSHR autoantibodies and cannot be used as a reagent for measuring anti-TSHR autoantibodies.

Accordingly, a first object of the present invention is to provide a method for producing TSHR suitable for stably expressing a large amount of TSHR using insect cells. A second object of the present invention is to provide a method for producing TSHR, which is suitable for obtaining TSHR expressed by insect cells in a state excellent in reactivity with anti-TSHR autoantibodies.

[0012]

Means for Solving the Problems A first invention made to achieve the first object is an insect cell infected with a virus containing a gene encoding a human thyroid stimulating hormone receptor or a derivative thereof. Is cultivated under high oxygen concentration conditions. A method for producing a human thyroid stimulating hormone receptor or a derivative thereof. In order to achieve the second object, a second invention is directed to an insoluble insoluble cell obtained by culturing insect cells infected with a virus containing a gene encoding a human thyroid stimulating hormone receptor or a derivative thereof. A human thyroid stimulating hormone receptor or a derivative thereof is solubilized in the presence of a reducing agent and a protein denaturing agent, separated from contaminants, and reduced in concentration of the reducing agent and the protein denaturing agent. This is a method for producing a hormone receptor. Hereinafter, the first and second inventions will be described in detail.

[0013] Insect cells can be obtained by using TS
A gene encoding HR (hereinafter simply referred to as TSHR gene) is introduced. The production method of the present invention can be used not only for production of TSHR but also for production of TSHR of other mammals. That is, in the following description, when a human mammalian TSHR gene is used, human TSHR can be produced, and when another mammal's TSH gene is used, the other mammalian TSH gene can be used. It becomes possible to manufacture TSHR. Hereinafter, the present invention will be described with reference to an example in which human TSHR is produced.

The TSHR gene introduced into insect cells is T
In addition to those encoding the full-length SHR, those encoding a TSHR derivative in which a mutation is introduced into a part thereof may be used. In addition to the one that encodes the full length TSHR,
It may encode a part of the derivative.
Codes for some of the derivatives include, for example, T
A gene encoding the extramembrane region of SHR can be exemplified. Such a gene may be a known DNA sequence (eg, B
BRC, 165, p. 1184, 1984) and the like, or can be obtained from human cells using a DNA sequence synthesized based on the sequence.

The virus containing the TSHR gene can be prepared according to a conventionally known method (for example, Am
annual of methods for Bacu
lowvirus Vectors and Insec
t Cell Culture Producers.
Bulletin, no. 1555, Texas ag
ricultural experiment sta
Tion, College Station, Texa
s, 1987).

As insect cells, normal insect cells can be used. Among them, sf9 and s which are easy to obtain
In addition to f21 cells (manufactured by INVITROGEN), silkworm larvae may be directly infected with a virus described below. These insect cells can be cultured in a usual medium. For example, a commercially available medium (Gracco, Grac)
e's Insect Cell Medium, IP
L41, Sf-900IISFM or TC-100I
nsec Medium).
As a procedure for culturing insect cells, a culture flask may be used for a small scale, and a spinner flask or a culture apparatus for culturing a suspension cell may be used for a larger scale.

The introduction of the TSHR gene into insect cells may be carried out by previously introducing the TSHR gene into a virus capable of infecting insect cells and using this virus. For example, insect cells are cultured in advance to an appropriate cell density,
A method of infecting the cells by adding a virus solution after collecting the cells by centrifugation or the like, or a method of directly adding a virus to a cell suspension of insect cells can be exemplified. The amount of virus to be added was O. I (Multiplicity Of)
Infection) is preferably about 0.1 to 10, but there is no particular problem even below this.

There is no particular limitation on the culture time of the insect cells infected with the virus. For example, the cells expressing TSHR may be recovered about 2 to 10 days after the infection.

The first invention of the present invention is to cultivate insect cells infected with this virus under high oxygen concentration conditions. According to the findings of the present inventors, in order to stably express TSHR in large amounts using insect cells, the oxygen concentration in the culture solution was increased from 2 ppm to the saturated oxygen concentration (7. 86 ppm), and more preferably, the pressure is controlled so as to be higher than the saturated oxygen concentration. In order to achieve this object, it is possible to exemplify a method in which air having a high oxygen concentration is always supplied into the container while holding the culture solution in the closed container. More specifically, 1
For example, air to which about 0 to 30% oxygen has been added is ventilated to the same extent as the volume of the closed container per minute. Further, in order to facilitate the control, in the present invention, a dissolved oxygen sensor for measuring the oxygen concentration in the culture solution and a supply means for supplying high-oxygen-concentrated air with oxygen added to the inside of the closed vessel are provided. It is particularly preferable to use a culture vessel equipped with

When insect cells infected with a virus having the TSHR gene are cultured, insoluble TSHR, which is an expression product of the gene, is accumulated in the insect cells. Therefore,
Even if the insect cells are crushed, the produced TSHR can be produced as a purified TSHR by using a technique often used in the separation and purification of proteins, such as ion exchange, gel filtration, or affinity chromatography. I can't. Accordingly, a second invention is to provide a method for culturing insect cells infected with TSHR or a virus containing the above-mentioned TSHR derivative gene to express the gene, and to obtain soluble and anti-TSHR from the insect cells in which the insoluble TSHR as an expression product is accumulated. It is obtained in a state excellent in reactivity with autoantibodies.

The TSHR gene, insect cell or virus used in the second invention is the same as described above. Insect cell culture can be performed according to a conventional culture method, but it is particularly preferable to culture the cells under high oxygen concentration conditions using the first invention.

After the culture, the insect cells that have accumulated the insoluble TSHR are disrupted. The disruption operation is not particularly limited as long as the operation does not damage the accumulated TSHR. For example, ultrasonic treatment and gentle homogenization can be exemplified.
The cell lysate is subsequently solubilized in the presence of a reducing agent and a protein denaturant. Reducing agents and protein denaturants are insoluble TS
The HR may be contacted at once, or may be contacted sequentially. These reagents may be added at the time of the cell crushing treatment or may be added after the cell crushing treatment.

Examples of the reducing agent used in the second invention include ordinary reducing agents such as mercaptoethanol and dithiothreitol. The reducing agent may be used in an amount sufficient to reduce the expressed TSHR in consideration of the amount of the expressed TSHR and the reducing power of the reducing agent.
The use of about 0% can be exemplified. Examples of the protein denaturing agent include, for example, urea, guanidine hydrochloride, an alkaline substance and the like, and particularly, an ionic surfactant such as SDS (sodium dodecyl sulfate) is preferable. The protein denaturant also denatures the expressed TSHR in consideration of the amount of the expressed TSHR and the protein denaturing power of the denaturant to lose its higher-order structure (incorrect higher-order structure of the insolubilized TSHR). It is sufficient to use a sufficient amount for
In the case of DS, it can be exemplified to use about 0.1 to 2% as a standard.

After the reducing agent and the protein denaturing agent are brought into contact with TSHR as described above, TSH is reduced and denatured.
R is separated from various contaminants derived from insect cells, such as crushed membrane fragments of insect cells. Known techniques such as centrifugation and gel filtration can be used for the separation. By reducing the concentration of the reducing agent and the protein denaturing agent added to the fraction containing the separated TSHR, the recombination of SS bonds cleaved by the reducing agent and the correct higher-order structure of the lost higher-order structure are reduced. It folds into a structure.

The operation of lowering the concentration of the reducing agent and the protein denaturing agent may be, for example, lowering the concentration to which the concentration of the reducing agent or the protein denaturing agent in the buffer or the like used in the above-mentioned separation operation is added, or after the separation operation. For example, the fraction containing TSHR is subjected to dialysis to reduce the concentration of the reducing agent and the protein denaturing agent, or to add a reagent that weakens each action of the reducing agent and the protein denaturing agent.

In the second invention, mercaptoethanol and SDS are used as a reducing agent and a protein denaturing agent,
Separation was performed by SDS gel electrophoresis (SDS-PA) using a buffer solution free from mercaptoethanol and SDS.
GE) is particularly preferred. In such an embodiment, the concentration of the reducing agent and the protein denaturing agent in the fraction (gel) containing TSHR after the separation is reduced, and only the cutout is carried out.
This is because it becomes possible to produce TSHR having reactivity with SHR autoantibodies. As an apparatus used here, a commercially available SDS-PAGE apparatus (manufactured by Bio-Rad Inc., Model 491 Prepcel, etc.) can be exemplified.

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples are one embodiment of the present invention and do not limit the present invention.

Example 1 Acquisition of Human TSHR Gene The following series of genetic recombination operations such as extraction of m-RNA, cleavage of DNA with restriction enzymes, etc. were performed by the method of Maniatis et al. (Molecular cloning, Cold S.
spring Harbor Laboratory, 19
1982) and the method of Harwin et al. (DNA Cloni)
ng, a Practical Approach, v
ol. 1, p49, Oxford 1985). Screening of a lambda gt11 cDNA library with an antibody was performed using a commercially available vector (Pharmacia, trade name lambda).
Directional Cloning Vect
or).

First, human thyroid cells (thyroidectomy tissue)
M-RNA was extracted from, and a cDNA library was prepared using lambda gt11 (Clontech).
Next, a human-derived monoclonal antibody to TSHR (TRMo, JBC 263, p168341,
1988) were used to extract about 500,000 clones.

When the extracted clones were analyzed, Ra
report et al. (BBRC 165, p118).
4, 1989) was confirmed to have the same sequence as the human TSHR gene.

Example 2 Construction of Human TSHR Extracellular Domain Virus Using the human TSHR gene extracted in Example 1 as a material,
A vector for expressing the extracellular region of TSHR was constructed.

First, cDN encoding the entire length of TSHR
A is a commercially available vector (pUC118, manufactured by Takara Shuzo Co., Ltd.)
After cloning into the EcoRV site downstream of the signal peptide encoded by the TSHR gene,
In order to introduce a stop codon at the end of the extracellular region encoded by the SHR gene and an EcoRI site immediately downstream of the stop codon, oligomers for mutagenesis of SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized, and commercially available reagents ( Amersham
Company name, product name in vitro mutagenes
is system).

After the introduction of the oligomer, the vector EcoRV
And EcoRI to obtain a fragment of about 1.2 Kbp. The extracted fragment was used as a commercially available expression vector for insect cells (pharm GP, trade name pAcGP67).
B) and transfer T in insect cells according to the usual method.
A virus for expressing the R gene was obtained.

Example 3 Expression of TSHR in Culture Plate Insect cells (sf21 cells) were cultured in 50 ml of a culture solution (manufactured by GIBCO, trade name: sf900II-SFM).
Was cultured in a culture flask at 27 ° C. After the insect cells were cultured until they became almost monolayer, 2 ml of the virus solution prepared in Example 2 was added, the mixture was left at 27 ° C. for 1 hour, and the culture solution was removed together with the virus. Thereafter, the insect cells infected with the virus were transferred into 5 ml of a fresh culture solution and cultured at 27 ° C. for 3 days. During culturing, air, 50 ml / min.
Oxygen gas was blown at a rate of ml / min.

After the culture, the insect cells are collected, and TE buffer (10 mM Tris-HCl buffer (pH 8.
0) 1 mM EDTA) was added and stirred well,
Centrifuged. Insect cells collected by centrifugation
Sample buffer for DS-PAGE (1M Tris-
HCl (pH 6.8) 1.25 ml, 10% SDS4m
1, glycerol 2 ml, 2-mercaptoethanol 1
5 ml of a mixture of 10 ml by mixing 1.35 ml of distilled water, 0.4 ml of saturated bromophenol blue).
In addition, a commercially available ultrasonic generator (SON manufactured by BRANSON, SON
Sonication using IFIER 250)
(control 5.0 for 30 seconds)
The mixture was heated at 0 ° C. for 5 minutes, and this was used as a sample for SDS-PAGE on a 10% gel. SDS-PAG
E is a buffer for separation (Tris 3 g, glycine 14.
A solution in which 26 g and 5 ml of 20% SDS were dissolved (diluted) in 1 liter) was used at a constant power of 12 W.

The gel after separation by SDS-PAGE was C
FIG. 1 shows the results of staining with BB (Coomassie brilliant blue). In FIG. 1, the column 1 shows the results for insect cells infected with the virus, and the column 2 shows the results when the insect cells not infected with the virus were subjected to the same operation for comparison. It is.

For comparison, the same operation was performed on insect cells cultured in a state where the flask was filled with nitrogen.
SDS-PAGE was performed, but TSHR could not be confirmed.

From the above results, it can be seen that a large amount of TSHR can be stably produced by culturing insect cells under high-concentration oxygen conditions.

Example 4 TSHR in Spinner Flask
Insect cells (sf21 cells) were cultured for 500 m using a culture solution (GIBCO, trade name: sf900II-SFM).
The culture was carried out at 27 ° C. in a 1 l spinner flask. When the cell density of the insect cells reached 1 × 10 ⁶ cells, the cells were collected, 50 ml of the virus solution prepared in Example 3 was added, the mixture was left at 27 ° C. for 1 hour, and the culture solution was removed together with the virus. The virus-infected insect cells were then transferred to 200 ml of fresh culture in a spinner flask and cultured at 27 ° C. for 3 days. At the time of culture, air at a rate of 300 ml / min.
Oxygen gas was blown at a rate of 0 ml / min.

The cultured insect cells were recovered, and
The same operation as described above, that is, a TE buffer solution was added, the mixture was sufficiently stirred, centrifuged, and SDS-PAGE was added to the collected insect cells
5 ml of a sample buffer solution was added, and the mixture was dissolved by sonication, heated at 100 ° C. for 5 minutes, and subjected to SDS-PAGE using a separation buffer as a sample. Separation was performed with a constant power of 12 W using a 10% gel.

The gel after separation by SDS-PAGE was C
The results of staining with BB are shown in FIG.

For comparison, TE cells were added to the cultured insect cells.
After adding a buffer solution, thoroughly stirring and centrifuging, sonication was performed in the same manner as described above in a TE buffer solution, and TS filtration was performed by gel filtration.
An attempt was made to purify the HR, but it could not be purified because TSHR was in an insolubilized state.

Example 5 Production of Purified TSHR The same culture operation as in Example 4 was performed to obtain 1.6 × 10 ⁸ insect cells. These cells were subjected to sonication and the like in the same manner as in Example 4, and then commercially available SDS-PA for fractionation.
GE (Bio-Rad model 491 Prep cell)
Was used to obtain the protein contained in each fraction. For the protein contained in each fraction, the same SDS-PAG as in Example 4 was used.
The samples were subjected to E and analyzed by Southern blotting. FIG. 3 shows the results of SDS-PAGE and Western blotting.

As described above, the fraction containing the target TSHR (the fraction provided in column Nos. 15 to 17 in FIG. 3) was identified, and the fraction was identified using an ultrafiltration membrane (Centriprep 30 manufactured by Amicon). From the results of measuring the amount of TSHR by comparison with a marker after concentration, in this example,
It was found that a large amount of purified TSHR of 1.75 mg could be produced from 1.6 × 10 ⁸ insect cells.

Example 6 Reactivity of TSHR with Anti-Human TSHR Antibody The reactivity of the purified TSHR produced in Example 5 with an anti-human TSHR antibody was investigated.

Various amounts of purified TSHR were added to a 96-well microtiter plate (EIA / RI, manufactured by Coaster).
A plate), and then diluted buffer (50 mM)
Tris-HCl (pH 8.1), 1 mM MgC
l ₂ , 50 mM NaCl, 0.05% Tween2
0, 0.02% (W / V) NaN ₃ , 0.5% (W / V)
V) Bovine serum albumin) was added and allowed to stand overnight to perform blocking (prevention of non-specific binding) to prepare a plate.

The biotinylated anti-human TSHR antibody was diluted with a dilution buffer, added to each well in a volume of 100 μl to a final concentration of 10 μg / ml, reacted, and then streptavidin diluted 500-fold with a dilution buffer. 100 μl of an enzyme (alkaline phosphatase; ALP) conjugated with ALP was further added, and an ALP substrate (manufactured by Sigma,
SIGMA FAST pHPP tablets were prepared according to the attached protocol, added in 100 μl portions to develop color, and the absorbance at 405 nm was measured.

FIG. 4 shows the results. According to FIG. 4, the TSHR produced in Example 5 has reactivity with the anti-human TSHR antibody, and the TSHR produced in Example 5
When measuring anti-human TSHR antibody using
It turns out that it is appropriate to use about ng / well.

From the above results, the separation operation was carried out in the presence of a reducing agent and a protein denaturing agent, and then the concentration of the reducing agent and the protein denaturing agent were reduced, whereby the insoluble TSHR expressed in the insect cells was converted to anti-TSHR. It can be seen that it can be obtained as a soluble TSHR having reactivity with an antibody (anti-TSHR autoantibody).

The following procedure confirmed that the biotinylated anti-human TSHR antibody used in this example did not affect the binding to the antigen human TSHR by biotinylation.

First, a plate was prepared in the same manner as described above, except that TSHR was adsorbed to the wells at 300 ng / well. To each well, a biotinylated anti-human TSHR antibody diluted with a dilution buffer, and various amounts of the unbiotinylated antibody diluted with a dilution buffer were added and reacted, and the absorbance was measured in the same manner as described above. It was measured. The results are as shown in FIG. 5. Since the binding of the biotinylated antibody to TSHR was inhibited by the addition of the non-biotinylated antibody and the absorbance decreased, the biotinylated treatment was performed using anti-human TS.
It was confirmed that the HR antibody did not affect the binding to TSHR.

Example 7 Preparation of reagent for measuring anti-TSHR autoantibody 1 A plate was prepared in the same manner as in Example 6 except that purified TSHR was added to a 96-well titer plate at 300 ng / well. 25 wells in the plate
Alternatively, 50 μl of a human serum obtained from a healthy person or a patient with Graves' disease was added (25 μl of a serum-added well was further added with 25 μl of a dilution buffer), and reacted at 37 ° C. for 1.5 hours.

50 μl of a solution prepared by diluting the biotinylated anti-human TSHR antibody with a dilution buffer to 50 μM was added to each well, and after one and a half hours of reaction, the well was washed with the dilution buffer. After washing, ALP conjugated with streptavidin, diluted 500-fold with dilution buffer, was prepared in Example 6.
In the same amount as in Example 6, and the color was developed.

FIG. 6 shows the results. In FIG. 6, the 2SD value of the average value when serum from a healthy person was used (serum was 25 μm) was used.
l (mean; 0.334, SD; 0.03
From 3, the average-2SD = 0.267), when 50 μl was used (mean value; 0.285, SD; 0.024)
When the average -2SD = 0.237)) was used as the cutoff value, the rate at which the measurement result of the serum obtained from a patient with Graves' disease was judged to be positive (positive rate) was as follows when 25 μl of serum was used. 44.4%, 6 when using 50 μl of serum
5.1% compared to using 25 μl of serum.
It can be seen that the positive rate is higher when 0 μl of serum is used.

Example 8 Preparation of Reagent for Measurement of Anti-TSHR Autoantibody 2 A plate was prepared in the same manner as in Example 6 except that purified TSHR was added to a 96-well titer plate at 300 ng / well. 50 wells in the plate
μl of human serum obtained from a healthy person or a patient with Graves' disease was added, and reacted at 37 ° C. for 1.5 hours.

50 μl of various concentrations of the biotinylated anti-human TSHR antibody diluted with the dilution buffer was added to each well, and after one and a half hours of reaction, the wells were washed with the dilution buffer. After washing, ALP which was diluted 500-fold with a dilution buffer and bound to streptavidin was added, and PNPP which is a substrate of ALP was further added to develop color.

FIG. 5 shows the results. From FIG. 5, 25 μM
When using a biotinylated anti-human TSHR antibody of
It can be seen that the difference between the results obtained when serum from a healthy person and serum from a patient with Graves' disease were the largest.

Example 9 Preparation of Reagent for Measurement of Anti-TSHR Autoantibody 3 Based on the results of the above Example, a suitable measurement system for measuring anti-human TSHR autoantibody using human TSHR produced according to the present invention was prepared. It was constructed.

First, 30 wells were placed in a 96-well titer plate.
A plate was prepared in the same manner as in Example 6, except that purified TSHR was added so as to be 0 ng / well. To the wells of the plate, 50 μl of human serum obtained from a healthy person or a patient with Graves' disease was added, and reacted at 37 ° C. for 1.5 hours.

The biotinylated anti-human TSHR antibody was diluted with a dilution buffer to obtain a final concentration of 25 μM.
Each μl was added to each well and allowed to react for 1.5 hours, and then the wells were washed with a dilution buffer. After washing, 5 times with dilution buffer
ALP conjugated with streptavidin, diluted 1:00
Was added, and PNPP which is a substrate of ALP was further added to develop color.

FIG. 8 shows the results. From FIG. 8, it can be seen that the 2SD value of the average value when using healthy human serum (average value; 1.18)
2, SD; average from 0.099-2 SD = 0.985)
It can be understood that by setting the cutoff value to, the measurement result of the serum obtained from a patient with Graves' disease can be determined to be positive with a high probability of 85.4%.

[0062]

According to the present invention, human TSHR and the like can be stably and mass-produced using insect cells (first invention), and excellent reactivity with anti-TSHR antibodies can be obtained using insect cells. It is possible to produce purified human TSHR or the like (second invention). The first invention and the second invention achieve different effects, and can be used separately. However, if they are applied simultaneously, human TSHR and the like having reactivity with anti-human TSHR autoantibodies can be stably obtained. And it becomes easy to mass-produce.

As described above, if human TSHR and the like having reactivity with human anti-TSHR autoantibody and the like can be produced stably and in large quantities, TSHR in the porcine thyroid membrane fraction can be produced.
-TSHR using human TSHR produced by Escherichia coli and Escherichia coli
It is possible to provide a reagent superior to a reagent for measuring an autoantibody. Further, since a large amount of TSHR can be stably produced, it is also possible to provide a reagent for measuring an anti-TSHR autoantibody which does not use a radioisotope as a labeling substance. This reagent has effects such as easier handling compared to a reagent using a radioisotope as a labeling substance.

As described above, by using the present invention to stably produce large quantities of TSHR having excellent reactivity with anti-TSHR autoantibodies, finally, the porcine thyroid membrane fraction THR and human TSH produced by Escherichia coli
It is possible to provide a reagent that is more reliable than the reagent using R and that is easy to handle.

The present invention, which enables mass production of human TSHR, also facilitates the production of polyclonal and monoclonal anti-human TSHR antibodies that require large amounts of human TSHR.

[0066]

[Sequence List] SEQUENCE LISTING <110> Tosoh Corporation <120> Method for producing recombinant human thyroid stimulating hormone receptor or its derivative using insect cells <130> PA210-9731 <160> 2 <210> 1 <211 > 34 <212> DNA <213> Artificial Sequence <220> <223> Mutagenesis oligomer <400> 1 caattctcag gaattcttag tagcccatta tgtc 34 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Oligomers for mutagenesis <400> 2 gacgaacacc ccatgatatc cgcccaggtc cctg 34

[Brief description of the drawings]

FIG. 1 is a photograph (enlarged photograph) showing the result of SDS-PAGE in Example 3. The column 1 in the figure shows the results for insect cells infected with the virus,
The second column shows the results for insect cells not infected with the virus, which were performed for comparison. Arrows in the figure indicate the produced TSHR staining bands estimated from the molecular weight.

FIG. 2 is a photograph (enlarged photograph) showing the result of SDS-PAGE in Example 4. The arrow in the figure indicates the produced TSHR stained band estimated from the molecular weight.

FIG. 3 is a photograph showing the results of SDS-PAGE and Western blotting in Example 5.
Each column in the figure shows the eluted fraction of preparative SDS-PAGE. The column described before purification is for preparative SDS.
-Cell lysate before purification by PAGE
It is a column which shows the result subjected to S-PAGE.

FIG. 4 shows TSHR and anti-human T in Example 6.
It is a figure which shows the result of the reactivity measurement with an SHR antibody. A high signal was obtained when reacted with anti-human TSHR antibody, up to the time when 300 ng of TSHR was adsorbed per well. In the figure, the vertical axis represents the absorbance (ABS 405 nm), and the horizontal axis represents the amount (ng) of purified TSHR adsorbed to the wells.

FIG. 5 is a diagram showing that the biotinylated anti-TSHR antibody used in Example 6 did not affect the binding to TSHR by the biotinylation treatment. In the figure, the vertical axis represents the absorbance (ABS 405 nm), and the horizontal axis represents the final concentration (μm) of the co-existing non-biotinylated anti-TSHR antibody.
g / ml). The lower the measured absorbance, the greater the inhibition by untreated anti-TSHR antibody.

FIG. 6 is a diagram showing the results in Example 7, where the left side shows the case where 25 μl of serum was used and the right side shows the case where 50 μl of serum was used. The dashed line in each figure indicates the 2SD value of the average value in the case of using healthy human serum in each case, and the numerical value in each figure indicates the value obtained from a patient with Graves' disease when the 2SD value was used as a cutoff value. The rate at which the obtained serum can be determined to be positive (positive rate) is shown. In addition, the solid line in each figure shows the average value of the results of the serum of a healthy individual. In the left figure (when 25 μl of serum was used), 28 out of 63 serums (44.
4%) is below the 2SD value and is determined to be positive,
In the right figure (when 50 μl of serum is used), 65.1% is determined to be positive, and it can be seen that the positive rate is higher when 50 μl of serum is used than when 25 μl of serum is used.

FIG. 7 is a view showing a result in Example 8. In the figure, the vertical axis represents the absorbance (ABS 405 nm), and the horizontal axis represents the final concentration (μM) of the biotinylated anti-human TSHR antibody used for the measurement. In the figure, the closed circles indicate the results for the sera of healthy individuals, and the open circles indicate the results for the sera of patients with Graves' disease. It can be seen that when 25 μM biotinylated anti-human TSHR antibody was used, the difference between the results obtained when serum from a healthy subject and serum from a patient with Graves' disease were the largest.

FIG. 8 is a diagram showing the results of Example 9. The dashed line in each figure shows the 2SD value of the mean value when using healthy human serum, and the value in each figure is the measurement of serum obtained from a patient with Graves' disease when the 2SD value is used as a cutoff value. The rate at which the result can be determined as positive (positive rate) is shown. In addition, the solid line in each figure shows the average value of the results of the serum of a healthy individual. As described above, the anti-human TSHR using the purified TSHR produced according to the present invention, which was revealed in each example,
A particularly preferred assay system for measuring autoantibodies (300
ng of a purified TSHR-adsorbed well, a measurement system using 50 μl of serum, and a final concentration of 25 μM of a biotinylated anti-human TSHR antibody), 41 out of 48 serums from patients with Graves' disease fell below the 2SD value, and 85 .4
It can be seen that a high positive rate of% can be achieved.

Claims (2)

[Claims] An insect cell infected with a virus containing a gene encoding a human thyroid stimulating hormone receptor or a derivative thereof is cultured under high oxygen concentration conditions. A method for producing the derivative. 2. An insoluble human thyroid stimulating hormone receptor or a derivative thereof obtained by culturing insect cells infected with a virus containing a gene encoding a human thyroid stimulating hormone receptor or a derivative thereof is reduced with a reducing agent and a protein denatured protein. A method for producing a human thyroid stimulating hormone receptor, comprising solubilizing in the presence of an agent, separating from contaminants, and reducing the concentrations of the reducing agent and the protein denaturing agent.