Mutant of thyroid stimulating hormone receptor as well as preparation method and application of mutant

Abstract

The invention relates to the field of bioengineering, in particular to a mutant of a thyroid stimulating hormone receptor as well as a preparation method and application of the mutant. The invention provides a mutant of a thyroid stimulating hormone receptor. One or more of amino acids from the 321 site to the 360 site of wild type amino acids of the thyroid stimulating hormone receptor are deleted. According to the mutant provided by the invention, the stability of protein is improved, the temperature sensitivity is improved, the functions of gradient, titer and the like are improved, and the capability of combining with an enzyme-labeled antibody and an autoantibody is not changed.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a mutant of a thyroid stimulating hormone receptor and a preparation method and application thereof. Background Art

[0002] Thyroid-stimulating hormone receptor (TSHR) exists on the plasma membrane of thyroid follicular epithelial cells and other cells. It is a seven-transmembrane glycoprotein receptor and a G protein-coupled receptor. TSHR can bind to thyroid-stimulating hormone (TSH) and is the most common antigen target of T lymphocytes and autoimmune thyroid disease (AITD) TSHR antibodies (TRAb). TSHR has a total of 764 amino acids, which are divided into the intramembrane region, the transmembrane region and the extramembrane region; the extracellular region contains 418 amino acids and is the recognition and binding site of TSH and TRAb. TSHR extracellular region (ECD): 11 cysteines, 5 pairs of disulfide bonds.

[0003] There are three main types of TSHR-related antibodies in the human body: TSAb: can activate TSH-like biological effects, causing hyperthyroidism, autoantibody epitope 6-168aa; TSBAb: after binding to TSHR, it blocks the binding of TSH to the receptor and inhibits the biological effects of TSH, causing hypothyroidism; autoantibody epitope 261-370aa; neutral TSH receptor antibody: neither activates nor blocks the effects of other ligands on TSHR.

[0004] Clinical significance of TSHR autoantibody detection: Anti-TSHR is an autoantibody of the TSH receptor on the thyroid cell membrane. It has a thyroid stimulating function similar to TSH and is not controlled by the negative feedback system. Stimulating thyroid function TRAb is one of the important causes of hyperthyroidism and recurrence of hyperthyroidism in patients with Graves' disease. Evaluation of the efficacy of medication in patients with Graves' disease: a decrease in TRAb concentration during antithyroid drug treatment may indicate remission of the disease and is an important indication for evaluating the timing of drug discontinuation and predicting recurrence. Risk assessment of thyroid dysfunction in pregnant women: the determination of serum TRAb helps guide the control of thyroid dysfunction in pregnant women during pregnancy and helps evaluate pregnancy outcomes.

[0005] TSHR is a 7-transmembrane protein. The binding of antigen and antibody depends on the conformational activity of the antigen, so it is quite challenging to successfully express an active antigen. Currently, TSHR is extracted directly from cells using detergents. The detection reagents have poor precision, especially near the low-value boundary (≤2.0IU / L), with large concentration variations of about 10% to 12%. Poor instruments can reach 16%, resulting in inconsistencies in the initial and repeated tests of samples near the boundary, resulting in positive and negative contrasts. There are two main reasons: one is that the reaction gradient in the low-value zone is small, and the other is that the luminescence value varies greatly (usually 2.5% to 5%). The reaction gradient is mainly limited by the raw material TSHR; one of the reasons for the large variation in luminescence values ​​is the temperature sensitivity of the reagent, which is mainly caused by the poor stability of TSHR. When the incubation plate changes by ±1°C, the signal value deviation is 15%-20%, and the concentration value deviation is large, with a deviation of about 50% near the boundary value, which is more obvious on instruments with poor temperature control effects; another reason may be that the crude TSHR is a crude product with low potency and a high feed ratio (1 / 25-1 / 30), which may introduce some impurities.

[0006] In order to improve stability, improve temperature sensitivity, increase the potency, reduce the feed ratio, purify the antigen and avoid the introduction of impurities, it is necessary to develop TSHR with better performance. Summary of the invention

[0007] In view of this, the present invention provides a mutant of thyroid stimulating hormone receptor and its preparation method and application. The mutant provided by the present invention improves the stability of the protein, improves the temperature sensitivity, and improves the functions such as gradient and titer, without changing the ability to bind to enzyme-labeled antibodies and autoantibodies.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a mutant of a thyroid stimulating hormone receptor, wherein one or more of the amino acids at positions 321 to 360 of the wild-type amino acids of the thyroid stimulating hormone receptor are deleted.

[0010] In some embodiments of the present invention, the mutant deletes amino acids 321 to 360 of the wild-type amino acids of the thyroid stimulating hormone receptor.

[0011] In some embodiments of the present invention, the mutants described above have:

[0012] (1), the amino acid sequence shown in SEQ ID NO: 1; or

[0013] (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0014] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0015] In some embodiments of the present invention, the sequence of the mutant is shown in SEQ ID NO: 1:

[0016]

[0017]

[0018] In some embodiments of the present invention, the wild-type amino acid sequence of the thyroid stimulating hormone receptor in the mutant is as shown in SEQ ID NO: 3:

[0019] In some embodiments of the present invention, the N-terminus or C-terminus of the mutant further includes a tag protein; the tag protein includes: one or more of His, GST, MBP, Strep, Flag or FC.

[0020] In some embodiments of the present invention, the tag protein in the above mutant is: Flag.

[0021] The present invention also provides a nucleic acid molecule encoding the mutant.

[0022] In some embodiments of the present invention, the above-mentioned nucleic acid molecule has:

[0023] (4) the nucleotide sequence shown in SEQ ID NO: 2; or

[0024] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0025] (6) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5).

[0026] In some embodiments of the present invention, the sequence of the nucleic acid molecule is as shown in SEQ ID NO: 2:

[0027]

[0028] The present invention also provides a recombinant vector, comprising: the above nucleic acid molecule and acceptable gene elements.

[0029] The present invention also provides a host for transforming and / or transfecting the above recombinant vector.

[0030] The present invention also provides a method for preparing the mutant, and the mutant is obtained after obtaining the host, culturing, crude purification, re-dissolving, fine purification, balancing, and dissociation.

[0031] In some embodiments of the present invention, in the above-mentioned preparation method, the re-dissolution includes the step of membrane protein descaling; the membrane protein descaling adopts a detergent; the detergent includes: sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, sodium deoxycholate, sodium cholate, n-octyl-β-D-pyranoglucoside, dodecyl β-D-maltoside, TritonX-100, TritonX-114, Tween-20, CHAPS and CHAPSO. One or more.

[0032] The present invention also provides the use of the mutant, the nucleic acid molecule, the recombinant vector, the host and / or the mutant obtained by the preparation method in any of the following items:

[0033] (I), preparing a product for detecting TSHR autoantibodies; and / or

[0034] (II), detecting TSHR autoantibodies as analytes; and / or

[0035] (III) preparing products for detecting TSHR monoclonal antibodies or patient TRAbs; and / or

[0036] (IV), preparing a medicament for absorbing TRAb from the patient's circulation; and / or

[0037] (V) Preparation of drug skeletons for screening small molecule fragments to identify new small molecules.

[0038] In some embodiments of the present invention, the amino acid sequence of mutant 29 (mutant Mut-29) is based on the wild-type sequence such as SEQ ID NO: 3, with H at position 443 mutated to N.

[0039] In some embodiments of the present invention, the amino acid sequence of mutant 11 (mutant Mut-11) is based on the wild-type sequence such as SEQ ID NO: 3, with L at position 59 mutated to F.

[0040] In some embodiments of the present invention, the amino acid sequence of mutant 18 (mutant Mut-18) is based on the wild-type sequence such as SEQ ID NO: 3, with D at position 151 mutated to E.

[0041] In some embodiments of the present invention, the amino acid sequence of mutant 25 (mutant Mut-25) is based on the wild-type sequence such as SEQ ID NO: 3, with I at position 253 mutated to R.

[0042] In some embodiments of the present invention, the amino acid sequence of mutant 32 (mutant Mut-32) is obtained by mutating the M at position 463 to V based on the wild-type sequence such as SEQ ID NO: 3.

[0043] In some embodiments of the present invention, the amino acid sequence of mutant 38 (mutant Mut-38) is based on the wild-type sequence such as SEQ ID NO: 3, with V at position 595 mutated to I.

[0044] In some embodiments of the present invention, the method for detecting TSHR antibodies: using a competitive method, in human serum, TRAb competes with TSHR antibodies for the antigen TSHR is the most important method for measuring and diagnosing Graves' disease and other thyroid diseases, and the present invention also adopts this method.

[0045] In some embodiments of the present invention, the strategy for improving TSHR stability is: previous patents use one or more amino acid mutations in the 1-260 amino acid fragment, while the present invention deletes one or more or the entire amino acid fragment between 316-366 on the basis of the whole protein, and uses tag purification to obtain a high-purity and high-stability membrane protein. The protein does not change the properties of antibody binding, and does not affect the judgment and measurement of Graves' disease and other thyroid diseases.

[0046] The present invention provides a mutant of a thyroid stimulating hormone receptor, wherein one or more of the amino acids at positions 321 to 360 of the wild-type amino acids of the thyroid stimulating hormone receptor are deleted.

[0047] The present invention uses appropriate purification tags and one or more mutants to purify a new TSHR with high titer, high gradient and good temperature sensitivity, which better meets the performance requirements of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0049] Figure 1 Shown is a schematic diagram of TSHR transmembrane;

[0050] Figure 2 Shown is the SDS-PAGE picture after purification; the target band is around 65KD;

[0051] Figure 3 The TSHR Western Blot antibody is a tag antibody. It can be seen from the figure that under electrophoresis conditions, TSHR has monomer, dimer and polymer forms.

[0052] Figure 4 The 7 peaks of HPLC are shown from right to left; among them: Peak 1: 1KD, Peak 2: 17KD, Peak 3: 45KD, Peak 4: 150KD, Peak 5: 300KD, Peak 6: 670KD, Peak 7: 1340KD; From the figure, TSHRMut-6 is a high polymer form with a molecular weight of more than 1340KD;

[0053] Figure 5 Shows the clinical relevance of TSHR and the correlation between Mut-29 and Roche 2 =0.9344, Mut-6 and Roche R 2 =0.9719 (the medium-high value is closer to the Roche value), the correlation R between Mut-6 and Mut-29 2 =0.9461; the coincidence rate between Mut-6 and Roche was slightly higher than that between Mut-29 and Roche (96.72% VS 95.62%. DETAILED DESCRIPTION

[0054] The invention discloses a mutant of a thyroid stimulating hormone receptor and a preparation method and application thereof.

[0055] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0056] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0057] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0058] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0059] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0060] The present invention deletes amino acids in TSHR by mutation technology on the basis of adding purification tags to plasmid pCMV2, obtains a mutant (such as SEQ ID No: 1) in which one or more or the entire amino acid fragments between 316-366 are deleted, transiently transfected into 293 cells, fermented and cultured for 6 days, centrifuged to remove the supernatant, and retains the precipitate. After detergent treatment for 1 hour, centrifuged, the precipitate is discarded and the supernatant is retained, and then purified by purification tags, all purification operations are performed on ice, and finally a purified membrane protein is obtained.

[0061] The protein has improved stability, improved temperature sensitivity, and improved functions such as gradient and titer without changing the ability to bind to enzyme-labeled antibodies and autoantibodies.

[0062] In Example 1, Comparative Example 1, Comparative Example 2, Verification Example 1 and Verification Example 2 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0063] The present invention will be further described below in conjunction with embodiments:

[0064] Example 1

[0065] 1. Construction of mutants: The mutants of the present invention were synthesized by the nucleic acid synthesis department of our company and the enzyme chain was cut into plasmid pCMV2.

[0066] Plasmid extraction: The plasmid is transformed into Top10, screened on an antibiotic plate, and verified by enzyme digestion and PCR. The verified clones are picked, amplified and cultured, and the plasmid is extracted using an endotoxin-free kit. The concentration, size and purity of the plasmid are measured, and the qualified ones are verified, such as: diluting the concentration to 400-500ng / μL with high-pressure purified water, and the A260 / A280 ratio should be between 1.8 and 2.0, and then entering the transfection.

[0067] 2. Plasmid transfection: After 3-4 passages, take samples aseptically for cell counting on the day of cell transfection. The cell density should reach (3.0-5.0)×10 6cells / mL, with a viability of ≥95%. Prepare A and B solutions using PEI. Add the plasmid to tube A and shake gently to mix. Add 2mg / mL PEI to tube B and shake gently to mix. Incubate A and B solutions at room temperature for 5 minutes, then use a pipette to add B solution to A solution to prepare AB mixed solution. Shake gently to mix. Incubate at room temperature for 10 minutes. Slowly add the AB mixed solution to a 2L shake flask, gently shake while adding the sample to mix it evenly at 37°C and 5% CO. 2 , 130rpm (amplitude 25mm) in a shaker. Samples were taken on the 6th or 7th day after transfection to count the cells. The cell suspension was centrifuged at 8000rpm×20min. After centrifugation, the supernatant was discarded and the precipitated cells were retained.

[0068] 3. Protein crude purity:

[0069] Calculate the volume of buffer used in the entire purification process, in which the cells are resuspended at 5×10 8 Resuspend the cell pellet with 60 mL of buffer; after disruption, re-dissolve at 5 × 10 8 Resuspend the cell pellet with 5 mL of buffer.

[0070] The buffer used for purification was 10 mM Tris-HCl + 50 mM NaCl pH 7.5 (Roche protease inhibitors must be added immediately, one tablet of preparation per 200 mL of buffer). 8 Resuspend the cell pellet with 60mL buffer, calculate the volume of solution, add the appropriate volume of protease inhibitor (for example, 400mL buffer, need to add 2 tablets of protease inhibitor), stir and mix well, and place on ice. Use a 5mL pipette tip to resuspend the cells, and pour the cell suspension into a suitable beaker, and stir in an ice bath for 20min. High-pressure homogenizer breaks the cells, and the high-pressure homogenized sample is placed in a 500mL high-speed centrifuge cup, and centrifuged after balancing. Use a Thermo high-speed refrigerated centrifuge and A27-6*500 rotor, and the centrifugation conditions are: 4℃, 12000g, 30min. Collect the precipitate after centrifugation, pour out the supernatant, and turn the centrifuge cup upside down on the clean toilet paper laid in advance for 30s to allow the liquid to drain completely, and then place the centrifuge cup with the precipitate on ice.

[0071] 4. Redissolve the cell pellet and add 5×10 8 Resuspend the cell pellet with 5 mL of buffer prepared with 1% Triton X-100 in advance, and take an appropriate volume of the prepared buffer (e.g. 5 × 10 8For cells, 5 mL of 1% detergent TritonX-100 buffer is required. Resuspend the precipitate with a 5 mL pipette, transfer to a 50 mL high-speed centrifuge tube and mix thoroughly with a mixer. Place in a 4°C environment and let stand for 60 minutes. Centrifuge after balancing the balance, using a Thermo centrifuge and A27-8*50 rotor, centrifugation conditions: 4°C, 47000g, 60 minutes. After centrifugation, the supernatant is determined by A280 for total protein concentration (using the 1Abs=1mg / mL method of A280 detection of Thermo NanoDr op2000 micro-volume UV spectrophotometer to determine the total protein concentration: the calibration solution is 10mM Tris-HCl+50mM NaCl pH7.5).

[0072] Protein purification (Flag tag):

[0073] 5. Balance: Place the magnetic bead rack in an ice box, remove the magnetic plate, add 500μL 10mM Tris-HCl + 50mM NaCl + 0.05% TritonX100 + mixed protease inhibitor cocktail tablets pH7.5 to each centrifuge tube, blow gently with a gun to evenly mix, then insert the magnetic plate, and when the magnetic beads are adsorbed on the magnetic plate, use a gun to gently suck up the clean supernatant from the side of the centrifuge tube away from the magnetic beads and put it into the waste liquid bucket. Repeat the above steps and balance three times in total. Prepare the magnetic bead rack in advance and place it in an ice box. Insert a 1.5mL centrifuge tube into the magnetic bead rack, mix the magnetic bead solution, and suck up 20μL of the magnetic bead solution into each centrifuge tube in turn.

[0074] Add 1 mL of sample to each tube, place on a rotating incubator at 25-30 rpm, and incubate overnight in a 4°C refrigerator.

[0075] 6. Peptide dissociation: The samples incubated overnight were evenly added to the centrifuge tubes on the magnetic bead plate in turn, and the balance solution was used to balance three times according to step 5. The magnetic plate was removed, and 25 μL of peptide elution solution was added to each centrifuge tube (a total of 150 μL elution 6 tubes), and the plate was placed on a rotating incubator and incubated in a refrigerator at 4 degrees for 30 minutes. After the incubation, the magnetic plate was inserted, and when the magnetic beads were all adsorbed to the magnetic plate, the supernatant was sucked with a gun, filtered with a 0.22 μm filter membrane, and the concentration was measured and stored at -80°C.

[0076] Comparative Example 1 (Strep tag)

[0077] According to the experiment of steps 1-4 in Example 1, the plasmid pCMV2 is added with the Strep purification tag, and then the steps are as follows:

[0078] Balance: Add 20mL of balance buffer (100mM Tris-HCl, 150mMNaCl, 1mM EDTA, pH 8.0) to the column to wash the column. Loading: Load the crude pure liquid onto the column. Washing: Add 20ml of balance buffer to the column to wash the column and remove non-specifically bound impurities. Elution: Add 5mL of washing solution (containing 2.5mM desthiobiotin balance buffer) to the column to elute the target protein. Collect the outflowing target protein with a centrifuge tube, concentrate it to at least 0.5mg / mL with an ultrafiltration tube, filter it with a 0.22μm filter membrane, measure the concentration, and store it at -80℃.

[0079] Comparative Example 2 (His tag)

[0080] According to the experiment of steps 1-4 in Example 1, the plasmid pCMV2 is added with a his purification tag, and then the steps are as follows:

[0081] Balance: Add 20mL 20mM PBS pH7.4 balance buffer to the chromatography column to wash the column. Loading: The crude pure solution dialyzed overnight was repeatedly loaded on the column 3 times to allow the His-recombinant protein in the sample to fully bind to the protein purification filler. Washing: Add 20mL 20mMPBS pH7.4 and 20mL 1.5M NaCL washing solution to the chromatography column to wash the column respectively to remove non-specifically bound impurities. Elution: Add 5mL 300mM imidazole PBS eluent to the chromatography column to elute the target protein. Use a centrifuge tube to collect the outflowing target protein, concentrate it to at least 0.5mg / mL with an ultrafiltration tube, filter it with a 0.22μm filter membrane, measure the concentration, and store it at -80℃. The experimental results are shown in Table 1.

[0082] Table 1 Comparison of purification performance of different tags (temperature sensitive)

[0083]

[0084]

[0085] As shown in Table 1, the temperature sensitivity of TSHR purified using the Flag tag is better than that using the His and Strep tags.

[0086] Verification Example 1 Performance Evaluation

[0087] ANTI-FITC antibody (Thermo Fisher, 701078) was coated on magnetic beads, and the antibody whose target site was intracellular amino acids was used as the capture antibody. The capture antibody was coupled with FITC, and the enzyme-labeled antibody was HRP-labeled enzyme-labeled antibody. The first-generation crudely purified TSHR and the second-generation purified mutant Mut-6 were used as antigens, and serum was used as the sample to compare the performance differences between the two, and to detect the mutant protein titer, gradient, temperature sensitivity, repeatability, intermediate precision and other properties.

[0088] Gradient comparison experiment:

[0089] Experimental group: mutant Mut-6

[0090] Control group: mutant Mut-29 (a mutant with poor selection effect)

[0091] Experimental steps: The experimental steps for gradient comparison, the culture and purification methods of mutant Mut-6 and mutant Mut-29 are the same as those in Example 1 above.

[0092] Experimental results:

[0093] Table 2

[0094] TRAb1 TRAb2 Mutant 6-1 / 200 Mutant 29-1 / 200 p6 22,765,000 50,842,795 p6 22,784,180 50,495,172 p27 8,595,891 20,537,492 p27 8,399,546 50,503,990 p14 4,018,444 10,300,474 p14 4,005,859 9,992,425 p33 285,658 779,832 p33 269,743 779,035 p6 22,774,590 50,669,074 p27 8,497,719 20,520,741 p14 4,012,152 10,146,450 p33 277,701 779,434 2.68 2.47 2.12 2.02 14.45 13.02 82.01 65.01

[0095] As shown in Table 2, mutant Mut-6 is slightly better than mutant Mut-29 in terms of gradient.

[0096] Table 3

[0097]

[0098]

[0099] As shown in Table 3, the performance of the mutant protein, such as titer, gradient, temperature sensitivity, repeatability, and intermediate precision, has been improved.

[0100] Clinical relevance:

[0101] Depend on Figure 5 It can be seen that Mut-29 is correlated with Roche 2 =0.9344, Mut-6 and Roche R 2 =0.9719 (middle-high value is closer to Roche value) Correlation R between Mut-6 and Mut-29 2 =0.9461.

[0102] Verification Example 2

[0103] The experimental steps were the same as steps 1 to 5 in Example 1; mutant 6 was compared with preferred mutants 11, 18, 25, 32, and 38 in terms of gradient and temperature sensitivity. It was concluded that mutant 6 was superior to other mutants in terms of these two aspects.

[0104] Table 4

[0105]

[0106]

[0107]

[0108] Table 5

[0109]

[0110]

[0111]

[0112] Table 6

[0113]

[0114]

[0115] Table 7

[0116]

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of a thyroid stimulating hormone receptor, characterized in that One or more of the amino acids at positions 321 to 360 of the wild-type amino acid sequence of the thyroid stimulating hormone receptor are deleted.

2. The mutant according to claim 1, characterized in that It has: (1), the amino acid sequence shown in SEQ ID NO: 1; or (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

3. The mutant according to claim 1 or 2, characterized in that The N-terminus or C-terminus of the mutant further comprises a tag protein; the tag protein comprises: one or more of GST, MBP, Flag or FC.

4. A nucleic acid molecule encoding the mutant according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that It has: (4) the nucleotide sequence shown in SEQ ID NO: 2; or (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or (6) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5).

6. A recombinant vector, characterized in that include: The nucleic acid molecule according to claim 4 or 5 and an acceptable genetic element.

7. A host, characterized in that Transform and / or transfect the recombinant vector as claimed in claim 6.

8. The method for preparing a mutant according to any one of claims 1 to 3, characterized in that: The mutant is obtained after obtaining the host as claimed in claim 7, culturing, crude purification, re-dissolving, fine purification, balancing and dissociation.

9. The preparation method according to claim 8, characterized in that: The re-dissolution comprises a membrane protein descaling step; the membrane protein descaling adopts a detergent; the detergent comprises one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, sodium deoxycholate, sodium cholate, n-octyl-β-D-pyranoglucoside, dodecylβ-D-maltoside, TritonX-100, TritonX-114, Tween-20, CHAPS and CHAPSO.

10. Use of the mutant according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the recombinant vector according to claim 6, the host according to claim 7 and / or the mutant obtained by the preparation method according to claim 8 or 9 in any of the following items; (I), preparing a product for detecting TSHR autoantibodies; and / or (II), detecting TSHR autoantibodies as analytes; and / or (III) preparing products for detecting TSHR monoclonal antibodies or patient TRAbs; and / or (IV), preparing a medicament for absorbing TRAb from the patient's circulation; and / or (V) Preparation and screening of small molecule fragments to identify new small molecule drug skeletons.

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