Mutants of the thyroid stimulating hormone receptor and methods of making and using the same

Abstract

The present application relates to the field of bioengineering, and particularly relates to a mutant of thyroid stimulating hormone receptor and a preparation method and application thereof. The present application provides a mutant of thyroid stimulating hormone receptor, wherein one or more of the 321st-360th amino acids of the wild-type amino acid of the thyroid stimulating hormone receptor are deleted. The mutant provided by the present application improves the stability of the protein, improves the temperature sensitivity, and improves the gradient and potency and other functions, and does not change the ability of binding with enzyme-labeled antibodies and autoantibodies.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to mutants of the thyroid-stimulating hormone receptor, their preparation methods, and applications. Background Technology

[0002] The thyroid-stimulating hormone receptor (TSHR) is located on the plasma membrane of thyroid follicular epithelial cells and many other cells. It is a seven-transmembrane glycoprotein receptor belonging to the G protein-coupled receptor family. TSHR binds to thyroid-stimulating hormone (TSH) and is the most common antigenic target for T lymphocytes and TSHR antibodies (TRAb) in autoimmune thyroid disease (AITD). TSHR consists of 764 amino acids, divided into an intramembrane region, a transmembrane region, and an extramembrane region. The extramembrane region, containing 418 amino acids, is the recognition and binding site for TSH and TRAb. The TSHR extracellular domain (ECD) contains 11 cysteine ​​residues and 5 disulfide bonds.

[0003] There are three main types of antibodies in the human body that are associated with TSHR: TSAb: can activate TSH-like biological effects, causing hyperthyroidism, with an autoantibody epitope of 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, with an autoantibody epitope of 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 thyroid cell membranes. It has a thyroid-stimulating function similar to TSH, is not controlled by a negative feedback system, and stimulates thyroid function. TRAb is one of the important causes of hyperthyroidism and hyperthyroidism relapse in patients with Graves' disease. For evaluating the efficacy of medication in Graves' disease patients, a decrease in TRAb concentration during antithyroid drug treatment may indicate disease remission and is an important indicator for evaluating the timing of drug discontinuation and predicting relapse. For risk assessment of thyroid dysfunction in pregnant women, serum TRAb measurement helps guide the control of thyroid dysfunction during pregnancy and helps assess pregnancy outcomes.

[0005] TSHR is a 7-transmembrane protein, and the binding of antigen and antibody depends on the conformational activity of the antigen, making successful expression of active antigens quite challenging. Currently, TSHR is crudely extracted directly from cells using detergents. However, the detection reagents suffer from poor precision, especially near the low-value threshold (≤2.0 IU / L), where concentration variations are significant (around 10%–12%, with some inferior instruments reaching 16%). This leads to inconsistencies between initial and repeat tests near the threshold, resulting in positive and negative results. The main reasons are twofold: firstly, the reaction gradient is small in the low-value region; secondly, the luminescence value varies considerably (typically 2.5%–5%). The reaction gradient is mainly limited by the raw material TSHR; one of the reasons for the large variation in luminescence value is the temperature sensitivity of the reagent, mainly due to the poor stability of TSHR. When the temperature plate changes by ±1℃, the signal value deviation is 15%-20%, and the concentration value deviation is larger, with a deviation of about 50% near the threshold. This is more obvious on instruments with poor temperature control. Another reason may be that the crude TSHR is crude and has low potency, and the feeding ratio is high (1 / 25 to 1 / 30), which may introduce some impurities.

[0006] To improve stability, enhance temperature sensitivity, increase potency, reduce feed ratio, purify antigens, and avoid introducing impurities, it is necessary to develop a TSHR with superior performance. Summary of the Invention

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

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The present invention provides a mutant of the thyroid-stimulating hormone receptor by deleting one or more amino acids from position 321 to position 360 of the wild-type amino acid of the thyroid-stimulating hormone receptor.

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

[0011] In some embodiments of the present invention, the above-mentioned mutant has:

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

[0013] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has 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 above-mentioned 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 above-mentioned mutant is shown in SEQ ID NO:3:

[0019] In some embodiments of the present invention, the N-terminus or C-terminus of the above 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 above-mentioned mutant.

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

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

[0024] (5) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (4), and which has 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 above-mentioned nucleic acid molecule is shown in SEQ ID NO:2:

[0027]

[0028] The present invention also provides a recombinant vector comprising: the above-described nucleic acid molecule and an acceptable gene element.

[0029] The present invention also provides a host, transformation and / or transfection of the above-mentioned recombinant vector.

[0030] The present invention also provides a method for preparing the above-mentioned mutant, wherein the mutant is obtained after obtaining the above-mentioned host, culture, crude purification, reconstitution, refined purification, equilibration, and dissociation.

[0031] In some embodiments of the present invention, the resolution in the above preparation method includes a membrane protein descaling step; the membrane protein descaling uses a detergent; the detergent includes one or more of the following: sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, sodium dodecyltrimethylammonium bromide, sodium deoxycholate, sodium cholate, n-octyl-β-D-glucopyranoside, dodecyl β-D-maltodextrin, Triton X-100, Triton X-114, Tween-20, CHAPS, and CHAPSO.

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

[0033] (I) Products for preparing autoantibodies against TSHR analytes; and / or

[0034] (II) Detection of TSHR autoantibodies; and / or

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

[0036] (IV) Preparation of the drug for the absorption of the patient's TRAb in the circulation; and / or

[0037] (V) Preparation and screening of small molecule fragments to identify novel small molecule drug skeletons.

[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 the 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 the 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 the 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 based on the wild-type sequence such as SEQ ID NO:3, with the M at position 463 mutated to V.

[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 the V at position 595 mutated to I.

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

[0045] In some embodiments of this invention, the strategy for improving TSHR stability is as follows: Previous patents used one or more amino acid mutations in a 1-260 amino acid fragment. This invention, however, involves deleting one or more or the entire amino acid fragment between 316 and 366 from the whole protein and then using tag purification to obtain a high-purity, highly stable membrane protein. This does not alter the protein's antibody binding properties and does not affect the diagnosis and measurement of Graves' disease and other thyroid diseases.

[0046] The present invention provides a mutant of the thyroid-stimulating hormone receptor by deleting one or more amino acids from position 321 to position 360 of the wild-type amino acid of the thyroid-stimulating hormone receptor.

[0047] This invention employs a suitable purification tag and one or more mutants to purify new TSHR reagents with high titers, high gradients, and good temperature sensitivity, thus better meeting the performance requirements of the reagent kit. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0049] Figure 1 Schematic diagram of TSHR transmembrane;

[0050] Figure 2 The SDS-PAGE image after purification shows the target band at approximately 65 kDa.

[0051] Figure 3The TSHR Western Blot antibody is a tag antibody; as shown in the figure, under electrophoretic conditions, TSHR exists in monomeric, dimeric, and polymeric forms.

[0052] Figure 4 The HPLC results show the sizes of the seven peaks from right to left: peak 1: 1 KD, peak 2: 17 KD, peak 3: 45 KD, peak 4: 150 KD, peak 5: 300 KD, peak 6: 670 KD, and peak 7: 1340 KD. The graph indicates that TSHRMut-6 is a polymer with a molecular weight exceeding 1340 KD.

[0053] Figure 5 Clinical relevance of TSHR, Mut-29 relevance to Roche R 2 =0.9344, Mut-6 and Roche R 2 =0.9719 (the mid-to-high value is closer to the Roche value), correlation R between Mut-6 and Mut-29 2 =0.9461; Compliance rate, the overall compliance rate of Mut-6 with Roche is slightly higher than that of Mut-29 with Roche (96.72% vs 95.62%). Detailed Implementation

[0054] This invention discloses mutants of the thyroid-stimulating hormone receptor, their preparation methods, and applications.

[0055] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0057] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0058] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0059] Furthermore, 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 precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0060] This invention utilizes a purification tag on plasmid pCMV2 to delete amino acids from TSHR via mutation technology, resulting in a mutant (e.g., SEQ ID No:1) with one or more or the entire amino acid fragment deleted between 316-366. This mutant is transiently transfected into 293 cells, fermented for 6 days, centrifuged to remove the supernatant, and the precipitate is retained. After treatment with a detergent for 1 hour, centrifuged again, the precipitate is discarded, and the supernatant is retained. The supernatant is then purified using the purification tag. All purification operations are performed on ice, ultimately yielding the purified membrane protein.

[0061] This protein improves stability and temperature sensitivity, while also enhancing gradient and titer functions, without altering its ability to bind to enzyme-labeled antibodies and autoantibodies.

[0062] In Examples 1, 1, 2, 1 and 2 of this invention, the raw materials and reagents used were all commercially available.

[0063] The present invention will be further illustrated below with reference to the embodiments:

[0064] Example 1

[0065] 1. Construction of mutants: The mutants of this invention are synthesized by our company's nucleic acid synthesis department and the enzyme chains are digested into plasmid pCMV2.

[0066] Plasmid extraction: The plasmid is transformed into Top10, screened on antibiotic plates, and verified by enzyme digestion and PCR. Verified clones are picked, amplified, and plasmids are extracted using an endotoxin-free kit. The plasmid concentration, size, and purity are measured. For verification (e.g., diluting with autoclaved aerated water to 400–500 ng / μL, with an A260 / A280 ratio between 1.8 and 2.0), the plasmid is then introduced for transfection.

[0067] 2. Plasmid transfection: After 3-4 passages, aseptically sample cells on the day of transfection for cell counting, aiming for a cell density of (3.0-5.0) × 10⁻⁶. 6Cells / mL, viability ≥95%, prepared solutions A and B using PEI. Plasmid was added to tube A and gently shaken to mix. 2 mg / mL PEI was added to tube B and gently shaken to mix. Solutions A and B were incubated separately at room temperature for 5 min. Then, solution B was added to solution A using a pipette to prepare the AB mixture. The mixture was gently shaken to mix and incubated at room temperature for 10 min. The AB mixture was slowly added to a 2 L shake flask while gently shaking to ensure thorough mixing. The cells were cultured at 37°C, 5% CO2, 130 rpm (25 mm amplitude) on a shaker. Samples were taken on day 6 or 7 post-transfection for cell counting. The cell suspension was centrifuged at 8000 rpm for 20 min. The supernatant was discarded after centrifugation, and the cell pellet was retained.

[0068] 3. Crude protein purity:

[0069] Calculate the total volume of buffer solution used in the entire purification process, including cell resuspending at 5 × 10⁻⁶. 8 The cell pellet was resuspended in 60 mL of buffer solution; after lysis, it was reconstituted at 5 × 10⁻⁶ ppm. 8 The cell pellet was resuspended in 5 mL of buffer solution.

[0070] The purification buffer used was 10 mM Tris-HCl + 50 mM NaCl, pH 7.5 (Rochete protease inhibitor needs to be added fresh; add one tablet of the preparation per 200 mL of buffer). Purification was performed at a rate of 5 × 10⁻⁶. 8 The cell pellet was resuspended in 60 mL of buffer solution. The calculated solution volume was adjusted, and an appropriate volume of protease inhibitor was added (e.g., 2 tablets of protease inhibitor for 400 mL buffer solution). The mixture was stirred and placed on ice. The cells were resuspended using a 5 mL pipette tip, and the cell suspension was poured into a suitable beaker and stirred on ice for 20 min. The cells were then homogenized using an autoclave. The homogenized sample was transferred to a 500 mL high-speed centrifuge cup, balanced, and centrifuged using a Thermo high-speed refrigerated centrifuge with an A27-6*500 rotor. Centrifugation conditions: 4℃, 12000 g, 30 min. After centrifugation, the pellet was collected. The supernatant was discarded, and the centrifuge cup was inverted on a clean sheet of paper for 30 seconds to allow the liquid to drain completely. The centrifuge cup containing the pellet was then placed on ice.

[0071] 4. Redissolve the cell pellet at a concentration of 5 × 10⁻⁶. 8 The cell pellet was resuspended in 5 mL of pre-prepared 1% Triton X-100 detergent buffer. An appropriate volume of freshly prepared buffer (e.g., 5 × 10⁻⁶) was then added. 8For the cells, use 5 mL of 1% Triton X-100 detergent buffer. Resuspend the pellet in a 5 mL pipette and transfer to a 50 mL high-speed centrifuge tube. Mix thoroughly with a mixer. Incubate at 4°C for 60 min. After balancing, centrifuge using a Thermo centrifuge with an A27-8*50 rotor at 4°C, 47000 g, for 60 min. Determine the total protein concentration of the supernatant using the A280 assay (1 Abs = 1 mg / mL method using the A280 assay on a Thermo NanoDrop 2000 micro UV spectrophotometer: calibration solution was 10 mM Tris-HCl + 50 mM NaCl, pH 7.5).

[0072] Protein purification (Flag label):

[0073] 5. Equilibration: Place the magnetic bead rack in an ice box, remove the magnetic plate, and add 500 μL of 10 mM Tris-HCl + 50 mM NaCl + 0.05% Triton X100 + mixed protease inhibitor cocktail tablets (pH 7.5) to each centrifuge tube. Gently mix with a pipette, then insert the magnetic plate. Once the magnetic beads have adhered to the magnetic plate, gently aspirate the supernatant from the edge of the centrifuge tube away from the magnetic beads and transfer it to the waste container. Repeat the above steps three times. Prepare the magnetic bead rack in advance and place it in an ice box. Insert 1.5 mL centrifuge tubes into the magnetic bead rack, mix the magnetic bead solution, and add 20 μL of the solution to each centrifuge tube sequentially.

[0074] Add 1 mL of sample to each tube, place it on a rotary culture apparatus, rotate at 25–30 rpm, and incubate overnight at 4°C.

[0075] 6. Peptide dissociation: The overnight incubated samples were sequentially and evenly added to centrifuge tubes on a magnetic bead plate. The samples were reequilibrated three times using the equilibration buffer following step 5. The magnetic plate was then removed, and 25 μL of peptide elution buffer was added to each centrifuge tube (a total of 150 μL for 6 tubes). The tubes were then incubated at 4°C for 30 min on a rotary incubator. After incubation, the magnetic plate was reinserted. Once all the magnetic beads had adhered to the plate, the supernatant was aspirated using a pipette, filtered through a 0.22 μm filter, and the concentration was measured. The samples were then stored at -80°C.

[0076] Comparative Example 1 (Strep Tag)

[0077] Following steps 1-4 of Example 1, after adding the Strep purification tag to plasmid pCMV2, the following steps were taken:

[0078] Equilibration: Add 20 mL of equilibration buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) to the chromatography column and wash the column. Loading: Load the crude pure solution onto the column. Washing: Add 20 mL of equilibration buffer to the chromatography column and wash the column to remove non-specifically bound proteins. Elution: Add 5 mL of washing buffer (containing 2.5 mM dethiobiotin equilibration buffer) to the chromatography column and elute the target protein. Collect the eluent in a centrifuge tube, concentrate to at least 0.5 mg / mL using an ultrafiltration tube, filter through a 0.22 μm filter membrane, measure the concentration, and store at -80°C.

[0079] Comparative Example 2 (His Tag)

[0080] Following steps 1-4 of Example 1, after adding the His purification tag to plasmid pCMV2, the following steps were taken:

[0081] Equilibration: Add 20 mL of 20 mM PBS pH 7.4 equilibration buffer to the chromatography column and wash the column. Loading: Load the crude purified solution (dialyzed overnight) onto the column three times to ensure thorough binding of the His-recombinant protein in the sample to the protein purification packing material. Washing: Add 20 mL of 20 mM PBS pH 7.4 and 20 mL of 1.5 M NaCl washing buffer to the chromatography column to wash the column and remove non-specifically bound proteins. Elution: Add 5 mL of 300 mM imidazole PBS elution buffer to the chromatography column to elute the target protein. Collect the eluent in a centrifuge tube, concentrate it to at least 0.5 mg / mL using an ultrafiltration tube, filter through a 0.22 μm filter membrane, measure the concentration, and store at -80℃. The experimental results are shown in Table 1.

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

[0083]

[0084]

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

[0086] Verification Example 1 Performance Evaluation

[0087] ANTI-FITC antibody (Thermo Fisher Scientific, 701078) is coated with magnetic beads. The target site is an intracellular amino acid, which is used as the capture antibody. The capture antibody is conjugated with FITC. The enzyme-labeled antibody is an HRP-labeled enzyme antibody. The first-generation crude purified TSHR and the second-generation purified mutant Mut-6 are used as antigens. Serum is 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: Mut-6 mutant

[0090] Control group: Mut-29 mutant (selected from mutants with poor efficacy)

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

[0092] Experimental results:

[0093] Table 2

[0094] TRAb1 TRAb2 Mutant strain 6-1 / 200 Mutant strain 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 mutant protein's titer, gradient, temperature sensitivity, repeatability, and intermediate precision have all been improved.

[0100] Clinical relevance:

[0101] Depend on Figure 5 It can be seen that Mut-29 is related to Roche's R... 2 =0.9344, Mut-6 and Roche R 2 =0.9719 (higher values ​​are closer to Roche values) 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; the comparison of mutant 6 with the preferred mutants 11, 18, 25, 32 and 38 in terms of gradient and temperature sensitivity concluded that mutant 6 was superior to the other mutants in terms of overall performance in 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 description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant, characterized in that, Its sequence is as shown in SEQ ID NO:1, which is an amino acid sequence.

2. A fusion protein, characterized in that, It consists of the mutant as described in claim 1 and a tag protein; the tag protein is located at the N-terminus or C-terminus of the mutant.

3. A nucleic acid molecule encoding the mutant as described in claim 1.

4. The nucleic acid molecule as described in claim 3, characterized in that, Its sequence is as shown in SEQ ID NO:2, which is a nucleotide sequence.

5. A recombinant vector, characterized in that, include: The nucleic acid molecule as described in claim 3 or 4, and the acceptable gene element.

6. The host, characterized in that, Transformation and / or transfection with the recombinant vector as described in claim 5.

7. The method for preparing the mutant as described in claim 1, characterized in that, After obtaining the host, culture, crude purity, reconstitution, refined purity, equilibration, and dissociation as described in claim 6, the mutant is obtained.

8. The preparation method according to claim 7, characterized in that, The resolution includes a membrane protein descaling step; the membrane protein descaling uses a detergent; the detergent includes one or more of the following: sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, sodium dodecyltrimethylammonium bromide, sodium deoxycholate, sodium cholate, n-octyl-β-D-glucopyranoside, dodecyl β-D-maltodextrin, Triton X-100, Triton X-114, Tween-20, CHAPS, and CHAPSO.

9. The use of the mutant as described in claim 1, the fusion protein as described in claim 2, the nucleic acid molecule as described in claim 3 or 4, the recombinant vector as described in claim 5, the host as described in claim 6, and / or the mutant obtained by the preparation method as described in claim 7 or 8 in any of the following: ( ), reagents or kits for preparing autoantibodies against TSHR analytes; and / or ( ), and prepare reagents or kits for detecting TSHR monoclonal antibodies or patient TRAb.

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