A recombinant thyroid-stimulating hormone receptor protein, its preparation method and application

Abstract

This invention provides a recombinant thyroid-stimulating hormone receptor (TSHR) protein, its preparation method, and its applications. The recombinant TSHR protein comprises an extracellular TSHR protein segment and an intracellular TSHR protein segment, with the C-terminus of the extracellular TSHR protein segment linked to the N-terminus of the intracellular TSHR protein segment. The protein achieves extracellular soluble expression, high purity, high activity, and good stability.

Description

Technical Field

[0001] This invention relates to a recombinant thyroid-stimulating hormone receptor protein, its preparation method, and its application, belonging to the technical field of thyroid-stimulating hormone receptor protein preparation. Background Technology

[0002] The thyroid-stimulating hormone receptor (TSHR) is a G protein-coupled receptor located on the membrane of thyroid follicular cells. Under normal physiological conditions, TSHR regulates the growth, differentiation, and function of thyroid follicular cells by binding to thyroid-stimulating hormone (TSH) through the adenylate cyclase (AC-cAMP) pathway and the phospholipase C-diacylglycerol and phosphoinositol pathway. TSHR not only mediates the physiological functions of TSH but is also an important autoantigen. Its corresponding autoantibody, thyroid-stimulating hormone receptor antibody (TSHRAb, TRAb), plays a crucial role in the development of autoimmune thyroid disease (AITD). TRAb mediates the occurrence and development of AITD and is considered an important indicator for the detection of AITD patients.

[0003] Currently, there are two main types of methods for detecting TRAb: competitive assays and double-antigen sandwich assays. Competitive assays primarily utilize the patient's own TRAb to competitively bind to TSHR (transferase-stimulating hormone) with labeled recombinant human autostimulatory monoclonal M22 to quantify TRAb. This method offers higher sensitivity and better detection stability compared to the traditional method of TRAb competing with TSH for TSHR binding, and is currently the dominant TRAb detection method. Double-antigen sandwich assays use two or more TSHR antigens sandwiched together to detect TRAb. This type of method is not yet widely used clinically. Regardless of the method used, TRAb quantification requires the binding of TRAb with TSHR. Therefore, as a key raw material for TRAb detection, the performance of TSHR, both clinically and industrially, is crucial.

[0004] Since the value of TRAb in AITD was discovered, researchers and technicians in this field have never stopped studying methods for preparing TSHR protein. Currently, the main methods for preparing TSHR protein are natural extraction and genetic engineering techniques.

[0005] Natural extraction involves extracting TSHR from other species (mainly pigs) to replace human TSHR. However, because TSHR itself is present in very low concentrations on thyroid follicular cells, and because TSHR is a seven-membrane perforating protein with low solubility (requiring the addition of high concentrations of surfactants), coupled with the inherent instability of TSHR, the difficulty of natural extraction is significantly increased. To date, the purity and content of the extracted natural antigens are relatively low, the process is relatively complex and cumbersome, and there are significant batch-to-batch variations. The high concentration of surfactants in the extracts also makes the antigens unusable directly. These factors limit the development of subsequent research.

[0006] Since Nagayama successfully cloned human TSHR cDNA in 1989, many researchers have attempted various methods of recombinant TSHR expression using different expression systems. Loosfelt and Harfst et al. attempted to express the full-length and extracellular segments of TSHR using *E. coli*, but failed to achieve soluble expression; the protein existed as inclusion bodies, requiring urea denaturation and electroelution to obtain high-purity protein. However, these purification methods damaged the protein's structure, preventing it from binding to TSH and TRAb and thus hindering its application in subsequent research. In 1992, Harfst, in 1993, Huang, and in 1995, Fowler et al. attempted to express the full-length TSHR using a rod-insect expression system. Although high levels of mRNA were detected, TSHR expression was unsuccessful; these proteins were either insoluble in water, denatured, or lacked glycosylation. In 1995, Chazenbalk and Rapoport achieved extracellular segment expression by introducing an early promoter, but the expression level remained low. For a long time, researchers believed that the rod system was not suitable for the recombination expression of TSHR. Wedlock et al.'s attempts to recombinantly express the full-length and extracellular segments of TSHR in a yeast expression system by introducing the leader sequence STE2 and α factor all ended in failure.

[0007] To date, many researchers have considered the expression of TSHR in mammalian cells as the most effective method for recombinant expression of full-length TSHR. However, despite this, the expression level is still very low, and extracellular secretion expression has not been achieved. Protein expression is intracellular, requiring multiple steps such as cell collection, cell washing, cell disruption, and centrifugation of the disruption products to collect the supernatant in order to further purify and obtain recombinant TSHR protein. Given the instability of TSHR itself, each additional step increases the uncertainty of the purified product and affects the stability of the product's application effect.

[0008] In summary, current methods for preparing TSHR proteins suffer from several drawbacks, including low expression levels, low purity, low yield, complex and cumbersome extraction processes, poor stability, large batch-to-batch variability in extracted products, high concentrations of surfactants in the extracted products, and inability to express them in an extracellular soluble manner. Consequently, these methods cannot be directly used for TRAb detection. Summary of the Invention

[0009] This invention provides a recombinant thyroid-stimulating hormone receptor protein, its preparation method, and its application, which can effectively solve the above-mentioned problems.

[0010] This invention is implemented as follows:

[0011] A recombinant thyroid-stimulating hormone receptor protein includes an extracellular TSHR protein segment and an intracellular TSHR protein segment, wherein the C-terminus of the extracellular TSHR protein segment is attached to the N-terminus of the intracellular TSHR protein segment.

[0012] As a further improvement, the amino acid sequence of the extracellular segment of the TSHR protein is shown in SEQ ID NO: 12, and the amino acid sequence of the intracellular segment of the TSHR protein is shown in SEQ ID NO: 13.

[0013] As a further improvement, its N-terminus is also linked to an exogenous secretory signal peptide.

[0014] As a further improvement, the C-terminus of the intracellular segment of the TSHR protein is also linked to a purified label sequence.

[0015] A method for preparing the above-mentioned recombinant thyroid-stimulating hormone receptor protein includes the following steps:

[0016] S1, construct a recombinant plasmid, the recombinant plasmid comprising an extracellular segment gene fragment of TSHR protein and an intracellular segment gene fragment of TSHR protein; the C-terminus of the extracellular segment of TSHR protein is connected to the N-terminus of the intracellular segment of TSHR protein;

[0017] S2, the recombinant plasmid is transferred into a eukaryotic expression system for the expression and identification of the target protein;

[0018] S3, Purify the target protein.

[0019] As a further improvement, the method for constructing the recombinant plasmid involves first using overlap PCR to connect the extracellular and intracellular gene fragments of the TSHR protein into the target gene fragment, and then using the Gibson cloning method to construct the target gene fragment into the plasmid vector.

[0020] As a further improvement, the method for constructing the recombinant plasmid involves first chemically synthesizing a target gene fragment including an extracellular segment gene fragment of the TSHR protein and an intracellular segment gene fragment of the TSHR protein, and then constructing the target gene fragment into a plasmid vector.

[0021] As a further improvement, the eukaryotic expression system is selected from the rod-insect expression system, the CHO cell expression system, the 293 cell expression system, or the yeast expression system.

[0022] Application of the above-mentioned recombinant thyroid-stimulating hormone receptor protein in the detection of thyroid-stimulating hormone receptor antibodies.

[0023] A thyroid-stimulating hormone receptor antibody detection kit, comprising the aforementioned recombinant thyroid-stimulating hormone receptor protein.

[0024] The beneficial effects of this invention are:

[0025] This invention expresses the extracellular and intracellular segments of TSHR in tandem, enabling the intracellular protein to provide structural support for the extracellular protein, thereby increasing the stability of the extracellular protein and improving its activity. This method can be applied to the development of TRAb detection reagents. At the same time, removing the transmembrane segment increases the probability of extracellular soluble expression, simplifying the purification process. The protein preparation method is simple and the protein yield is high. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is the T1, T2, and T3 fragment recovery and identification spectrum provided in Embodiment 1 of the present invention. Lane 1: T1 fragment, Lane 2: T2 fragment, Lane 3: T3 fragment, Lane 4: Nucleic acid marker.

[0028] Figure 2 This is the T4 fragment recovery and identification chromatogram provided in Embodiment 1 of the present invention. Lane 1: T4 fragment; Lane 2: Nucleic acid marker.

[0029] Figure 3 This is the T5 fragment recovery and identification chromatogram provided in Embodiment 1 of the present invention. Lane 1: T5 fragment; Lane 2: Nucleic acid marker.

[0030] Figure 4 This is a morphological diagram of H5 cells before infection with baculovirus, provided in Embodiment 1 of the present invention.

[0031] Figure 5 This is a cell morphology diagram of H5 cells after infection with baculovirus, provided in Embodiment 1 of the present invention.

[0032] Figure 6 This is the SDS-PAGE and corresponding WB results of the target protein provided in Example 1 of this invention. Lane 1: Expression supernatant; Lane 2: Purified target protein; Lane 3: Unrelated protein fused with human FC tag; Lane 4: Protein marker.

[0033] Figure 7 This is a prototype reagent quantitative curve provided in Example 2 of the present invention.

[0034] Figure 8 This is a correlation diagram between the prototype reagent constructed using this recombinant protein and commercial reagents provided in Example 2 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0037] This invention provides a recombinant thyroid-stimulating hormone receptor (TSHR) protein, comprising an extracellular TSHR protein segment and an intracellular TSHR protein segment, wherein the C-terminus of the extracellular TSHR protein segment is attached to the N-terminus of the intracellular TSHR protein segment. The TSHR protein can be from humans; or from animals closely related to humans such as pigs, cattle, or chimpanzees; or from the above sources with individual amino acid mutations, but these individual amino acid mutations do not affect active, soluble expression.

[0038] Preferably, the amino acid sequence of the extracellular segment of the TSHR protein is shown in SEQ ID NO: 12; and the amino acid sequence of the intracellular segment of the TSHR protein is shown in SEQ ID NO: 13. More preferably, the extracellular segment of the TSHR protein is the amino acid sequence corresponding to the 64-1248 bp fragment of the full-length human TSHR DNA; and the intracellular segment of the TSHR protein is the amino acid sequence corresponding to the 2035-2292 bp fragment of the full-length human TSHR DNA.

[0039] The N-terminus of the recombinant thyroid-stimulating hormone receptor protein is also linked to an exogenous secretory signal peptide, which facilitates soluble protein expression. If the selected expression vector contains its own secretory signal peptide, the N-terminus of the recombinant thyroid-stimulating hormone receptor protein may not be linked to an exogenous secretory signal peptide.

[0040] Preferably, the C-terminus of the intracellular segment of the TSHR protein is further linked to a purification label sequence, which is selected from human recombinant IgG1 FC, 6×His, and GST label sequences, but is not limited to these. On the one hand, this increases the possibility of soluble expression, and on the other hand, the affinity chromatography tag can effectively separate the target fragment from the expression supernatant, thereby improving the purity of the protein.

[0041] A method for preparing the above-mentioned recombinant thyroid-stimulating hormone receptor protein includes the following steps:

[0042] S1. Construct a recombinant plasmid comprising an extracellular segment gene fragment and an intracellular segment gene fragment of the TSHR protein; the C-terminus of the extracellular segment of the TSHR protein is linked to the N-terminus of the intracellular segment. Preferably, the recombinant plasmid further comprises a purification marker sequence gene fragment, which is linked to the C-terminus of the intracellular segment of the TSHR protein. This method tandemly expresses the extracellular and intracellular segments of TSHR, achieving structural support from the intracellular segment to the extracellular segment, thereby increasing the stability of the extracellular segment and improving protein activity. Simultaneously, removing the transmembrane segment increases the probability of soluble expression, simplifying the purification process. This removed transmembrane segment is a 7-transmembrane protein, including the intramembrane portion and the extramembrane segment in the middle of the transmembrane segment.

[0043] S2, the recombinant plasmid is transferred into a eukaryotic expression system for the expression and identification of the target protein.

[0044] S3, Purify the target protein. The purification method is a conventional protein purification technique, which will not be described in detail here.

[0045] The method for constructing the recombinant plasmid involves first ligating the extracellular and intracellular gene fragments of the TSHR protein into a target gene fragment using overlap PCR, and then constructing the target gene fragment into a plasmid vector using the Gibson cloning method. Preferably, the target gene fragment further includes a purified marker sequence gene fragment.

[0046] Alternatively, the method for constructing the recombinant plasmid involves first chemically synthesizing a target gene fragment comprising both the extracellular and intracellular segments of the TSHR protein, and then constructing the target gene fragment into a plasmid vector. Preferably, the target gene fragment further includes a purified marker sequence gene fragment. The chemical synthesis can be performed in various ways, such as synthesizing each fragment individually, combining multiple fragments in pairs, or directly synthesizing the sequence into the expression vector in one step.

[0047] As a further improvement, the eukaryotic expression system is selected from, but is not limited to, a rod-insect expression system, a CHO cell expression system, a 293 cell expression system, or a yeast expression system. More preferably, the eukaryotic expression system is a rod-insect expression system, under which the expressed protein exhibits the highest stability and activity.

[0048] Application of the above-mentioned recombinant thyroid-stimulating hormone receptor protein in the detection of thyroid-stimulating hormone receptor antibodies.

[0049] A thyroid-stimulating hormone receptor antibody detection kit, comprising the aforementioned recombinant thyroid-stimulating hormone receptor protein.

[0050] The reagents and instruments used in the embodiments of this invention are as follows:

[0051] PCR reagents (including phusion polymerase, buffer, and dNTPs) were purchased from TransGen Biotech Ltd.; T4 ligase, DNA Marker, protein Marker, restriction endonucleases such as BamHI, and alkaline phosphatase substrate (NBT / BCIP) were purchased from Thermo Fisher Scientific; Gibson assembly solution was prepared by the applicant; agarose was purchased from Biowest; Lipofectamine™ 2000 was purchased from Thermo Fisher; the primary antibody used for Western blotting was produced by the applicant, and the secondary antibody was purchased from KPL; DNA purification and recovery kits and plasmid extraction kits were purchased from TIANGEN; ampicillin and other reagents were purchased from BBI; linearized Baculovirus DNA and ESF921 cell culture medium were purchased from Expression Systems; SIMHF cell culture medium was purchased from Sino Biological Inc.; fetal bovine serum was purchased from PAN Biotech; Ni medium was purchased from GE Healthcare; and trypan blue was purchased from Sigma. Stimulating TRAb antibody was purchased from RSR; magnetic microparticles were purchased from Life Sciences AS; and acrid esters were from Suzhou Zhongcheng Chemical Co., Ltd.

[0052] The following equipment was purchased: a standard plate PCR instrument from Biometra; a horizontal electrophoresis apparatus (RDY-SP1Z) from Shanghai Shanfu; a constant temperature incubator from Shanghai Zhicheng; a constant temperature shaker (ZWYR-D2401) from Shanghai Zhicheng; a clean bench (BSC-1000IIA2) from Shanghai Boxun Industrial Co., Ltd.; a centrifuge from Thermo Scientific; an inverted microscope from Guangzhou Yuexian; a vertical electrophoresis apparatus from Bio Rad; a transfer apparatus from Bio Rad; a gel imaging system (805) from Shanghai Shanfu; a cell counter from ALIT; and an MK10 dry thermostat from Hangzhou Aosheng. A fully automated chemiluminescence analyzer (Caris200) was purchased from Xiamen Youmaike Medical Instruments Co., Ltd.

[0053] The cell lines, bacterial strains, and plasmids used in the embodiments of this invention are as follows:

[0054] The full-length human TSHR (hTSHR, human thyroid-stimulating hormone receptor) gene and PCR primers were synthesized by General Biotech; plasmid pAcGP67-B was a commercial vector; SF9 and H5 cells were commercial cells; and Trans5α competent cells were purchased from Kangti Life Insurance Co., Ltd.

[0055] Example 1: Preparation method of recombinant thyroid-stimulating hormone receptor protein

[0056] Cloning design

[0057] Using Primer 5.0, primers were designed to amplify the 64-1248 bp (extracellular segment) and 2035-2292 bp (intracellular segment) of hTSHR based on the cDNA sequence of human TSHR (hTSHR) (e.g., SEQ ID NO: 1) and the cDNA sequence of human antibody IgG1 (e.g., SEQ ID NO: 2). The upstream primer sequence PF1 is shown in SEQ ID NO: 3; the upstream primer sequence PF2 is shown in SEQ ID NO: 4; the upstream primer sequence PF3 is shown in SEQ ID NO: 5; the downstream primer sequence PR1 is shown in SEQ ID NO: 6; the downstream primer sequence PR2 is shown in SEQ ID NO: 7; and the downstream primer sequence PR3 is shown in SEQ ID NO: 8. BamHI and NotI were used as restriction enzyme sites. All the above-mentioned sequences and primers were synthesized by General Biotech.

[0058] The optimized nucleotide sequence of hTSHR from codons 64-1248bp is shown in SEQ ID NO: 9; the optimized nucleotide sequence of hTSHR from codons 2035-2292bp is shown in SEQ ID NO: 10.

[0059] The amino acid sequence of human TSHR (hTSHR) is shown in SEQ ID NO: 11.

[0060] The amino acid sequence of the extracellular segment of hTSHR is shown in SEQ ID NO: 12; the amino acid sequence of the intracellular segment of hTSHR is shown in SEQ ID NO: 13.

[0061] Construction of recombinant plasmids:

[0062] 1. The pAcGP67-B vector (which contains a secretory signal peptide and does not require additional ligation) was double-digested with QuickCut BamHI and QuickCut NotI. After incubating at 37°C for an appropriate time, the digested vector was subjected to 1% agarose gel electrophoresis and purified using a gel extraction kit to obtain the linear vector.

[0063] 2. Using hTSHR DNA as a template, the hTSHR DNA sequence from positions 64 to 1248 was amplified using primers PF1 and PR1 (T1); the hTSHR DNA sequence from positions 2035 to 2292 was amplified using primers PF2 and PR2 (T2); and the human antibody IgG1 DNA sequence from the constant region of the human antibody was amplified using primers PF3 and PR3 (T3). The T1 (positions 64-1248), T2 (positions 2035-2292), and T3 (human antibody constant region sequence) DNA fragments amplified by PCR were subjected to 1% agarose gel electrophoresis, purified and identified using a gel extraction kit. The identification results are as follows: Figure 1 As shown.

[0064] 3. Using overlap PCR, with PF1 and PR2 as primers and PCR products T1 and T2 as templates, the purified DNA fragments at positions 64-1248 / 2035-2292 were PCR-assembled to obtain PCR product (T4). PCR product T4 was subjected to 1% agarose gel electrophoresis, purified and recovered using a gel extraction kit, and identified. The identification results are as follows: Figure 2 As shown.

[0065] 4. Using overlap PCR, with PF1 and PR3 as primers and PCR recovered products T3 and T4 as templates, the gene sequence of 64-1248bp / 2035-2292bp was PCR-assembled with the human antibody constant region sequence to obtain PCR product (T5). PCR product T5 was subjected to 1% agarose gel electrophoresis, purified and identified using a gel extraction kit; the identification results are as follows: Figure 3 As shown, the desired segment is obtained.

[0066] 5. Take 2 μL of the purified and recovered linear vector and target fragment, and dilute with sterile water to a concentration of 0.1 μg / μL for later use.

[0067] 6. The purified target fragment and linear vector were assembled using Gibson assembly buffer (5.6 mU / μL T5 exonuclease; 33.3 mU / μL phusion polymerase; 5.3 U / μL Taq ligase; 0.27 mM dNTP). The molar ratio of the target fragment to the linear vector was controlled at 4:1. The system was as follows: linear vector fragment: 1 μL; DNA fragment tandemly bound to the constant region of the human antibody at positions 64-1248 / 2035-2292 of hTSHR (target fragment): 4 μL; Gibson assembly buffer: 5 μL. The mixture was vortexed and incubated at 50°C for 1 h. The ligation system was then transformed into E. coli competent cells Trans5α (purchased from Kangti Life Insurance Co., Ltd.). Positive and negative controls were set up during transformation. The positive control was an undigested plasmid used to detect transformation efficiency; the negative control was empty E. coli competent cells used to detect whether the competent cells were contaminated. The transformation product was plated on LB agar containing ampicillin (100 μL / mL) and incubated overnight at 37°C. Multiple single colonies were randomly picked from the LB plates containing the recombinant plasmid and used as templates for PCR amplification using universal primers. The presence of the target band was detected by 1% agarose gel electrophoresis. Simultaneously, the picked single colonies were cultured separately, and plasmids were extracted according to the plasmid extraction kit instructions. The plasmids were then digested with the corresponding restriction endonucleases, and the digestion products were detected by electrophoresis.

[0068] 7. Samples that tested positive by both bacterial PCR and double enzyme digestion were sent to an external sequencing facility to confirm the correctness of plasmid construction.

[0069] 8. Preparation of high-purity recombinant plasmids

[0070] The plasmid extraction kit from TIANGEN was used for the procedure.

[0071] 1) For plasmids with high copy numbers, collect the bacterial cells using 5 mL of overnight LB culture, centrifuge at 8000 rpm for 5 min, and discard the supernatant;

[0072] 2) Add 250 μL of P1 (with added Rnase A) to resuspend the bacterial cells and mix thoroughly by pipetting.

[0073] 3) Add 250ul of P2 and gently invert to mix 7-8 times (do not vortex);

[0074] 4) Add 350ul of P3 and immediately and gently invert the container 7-8 times (do not vortex);

[0075] 5) Centrifuge at 12000 rpm at room temperature for 10 min;

[0076] 6) Transfer the supernatant from the centrifugation to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the centrifuged liquid;

[0077] 7) Add 600 μL of PW washing solution to the column, centrifuge at 12,000 rpm for 1 min, discard the centrifuged liquid, and repeat the washing operation once.

[0078] 8) Centrifuge the empty column at 12000 rpm for 1 min, transfer the column to a new centrifuge tube, place it in a dry constant temperature heater, and dry it at 50℃ for 10 min;

[0079] 9) Add 50 μL of ddH2O to the adsorption column, let stand for 2 min, and centrifuge at 12000 rpm for 2 min;

[0080] 10) Take 2 μL of the centrifuged liquid, dilute it 50 times with sterile water, and calculate the concentration of the vector DNA using a DU730 spectrophotometer for later use.

[0081] Target protein expression and identification (baculovirus-insect expression system)

[0082] 1. Baculovirus preparation:

[0083] (1) Before transfection, SF9 cells (insect cells) in the logarithmic growth phase were seeded into 24-well culture plates, with each well containing 2.5-2.8×10^5 cells. The serum-free SIMHF medium volume was 500ul, and the cells were placed in a humidified incubator at 27℃ for 30min.

[0084] (2) Preparation of liposome / DNA complex

[0085] Solution 1: Take 1 μL of purified hTSHR / pAcGP67-B recombinant plasmid and 1 μL of linearized Baculovirus DNA and dilute them in 50 μL Medium.

[0086] Solution 2: Dilute 2ul of Lipofectamine™ 2000 in 50ul of Medium.

[0087] Mix Solution 1 and Solution 2 thoroughly to obtain the liposome / DNA complex, and incubate at room temperature for 30 minutes.

[0088] (3) Transfection: Discard the original culture medium in the 24-well plate, rinse once with 1 ml of serum-free SIMHF medium, and finally add 500 μl of serum-free SIMHF medium. Slowly add the prepared liposome / DNA complex into the 24-well plate, gently mix, and incubate overnight at 27°C in a humidified incubator. The next day, add 1 ml of SIMHF medium containing FBS to the 24-well plate and continue incubation for 3 days. The virus reaches generation P0. Transfect the empty plasmid pAcGP67-B as a negative control using the same method.

[0089] (4) P1 generation virus amplification: 72 h after transfection, the cells from the 24-well plate were transferred to a 10 cm plate with a cell density of 1×10^6 cells / mL and incubated in a humidified incubator at 27℃ for 72 h.

[0090] (5) P2 generation virus amplification: After 72 h of adherent culture, the adherent cells on a 10 cm plate were resuspended with an electric pipette, and the culture medium was enriched in a sterile tube. 2 ml of the culture medium was transferred to a 100 ml Erlenmeyer flask containing 50 ml of cells at a density of 1×10^6 cells / mL. The flask was then placed in a constant temperature shaker at 27 °C for 96 h of suspension culture. The virus was P2 generation. The remaining enriched culture medium (P1 generation virus) was stored at 4 °C in the dark.

[0091] (6) P3 generation virus amplification: Take 2 ml of P2 generation culture medium and transfer it to a 100 ml Erlenmeyer flask containing 50 ml of cells with a density of 1×10^6 cells / mL. After suspension culture for 96 h, take 20 μl of culture medium, add 20 μl of trypan blue staining, monitor the cytopathic effect of cells with a cell counter, enrich the culture medium in sterile tubes, store at 4℃ in the dark, and use it for protein expression preparation.

[0092] 2. Expression of the target protein in High-Five (H5) cells (insect cells):

[0093] H5 cells were diluted to a density of 3.0 × 10^6 cells / mL, with a total volume of 500 mL. Cell viability was greater than 98%, and cell diameter was 17-17.5 μm. 5 mL of P3 generation virus containing the target gene was added, and the cells were incubated at 27°C in a shaker. After 72 h, 20 μL of culture medium was added and stained with 20 μL of trypan blue. Cellular pathogenesis was monitored using a cell counter. Results before and after pathogenesis are shown below. Figure 4 , 5 As shown, the diameter distribution of normal cells before expression was statistically analyzed using Countstar (purchased from ALIT) at 25x magnification. When the virus effectively infected the cells, the cell diameter increased significantly. After the cell viability decreased to a certain level, the culture supernatant was collected for activity identification and protein purification.

[0094] 3. Activity identification of expression products in culture supernatant:

[0095] A chemiluminescent double-antibody sandwich assay using acridinium ester, constructed from a TSHR C-terminal mouse antibody 10F8 and a human-stimulated TRAb, was used to detect the expression in the cell supernatant. The results showed that the expression product exhibited high activity. The specific detection method is shown below:

[0096] 3.1 10F8 magnetic bead coating

[0097] (1) Equilibrate the magnetic microparticles and EDC to room temperature, take magnetic microparticles with a concentration of 4 mg / ml, add a certain concentration of activator EDC, and place at room temperature for 30 min to activate.

[0098] (2) Take the activated magnetic microparticles, collect the magnets using a magnetizing device, remove the supernatant, and resuspend them in MES buffer solution.

[0099] (3) Add a certain amount of anti TSHR 10F8 antibody, mix well and react at room temperature for 3 hours.

[0100] (4) After collecting the magnets using a magnetic rack, remove the supernatant, add an equal volume of phosphate buffer containing a certain amount of protein, and react at room temperature for 3 hours.

[0101] (5) After collecting magnets using a magnetizing device, remove the supernatant and add an equal volume of cleaning solution to wash repeatedly 4 times.

[0102] (6) Store the cleaned magnetic microparticles in a magnetic bead dilution solution at 2-8℃.

[0103] 3.2 Human-stimulated TRAb acridine ester labeling

[0104] (1) Add acridine ester to the stimulating TRAb antibody containing phosphate buffer, mix well, and react at room temperature in the dark for 30 min.

[0105] (2) Add the stop buffer, mix well, and react at room temperature in the dark for 30 minutes.

[0106] (3) After dialyzing the labeled acridine ester, add an equal volume of glycerol, mix well, and store at -20°C in the dark.

[0107] 3.3 Activity detection of supernatant expression products

[0108] H5 cell expression supernatant was diluted to prepare a series of samples. First, the recombinant hTSHR in the samples reacted with magnetically coated 10F8 for 15 min, forming a 10F8-coated magnetic microparticle-recombinant hTSHR complex. After magnetic separation and washing, other substances not bound to the magnetic microparticles were removed. Next, acridil ester-labeled stimulating TRAb was added and reacted with the recombinant hTSHR in the samples for 10 min, forming a "10F8-coated magnetic microparticle-recombinant hTSHR-acridil ester-labeled stimulating TRAb" complex. After magnetic separation and washing, other substances not bound to the magnetic microparticles were removed. Finally, pre-activation and activation solutions were added, and the complex emitted a light signal of detectable intensity, expressed as a relative luminescence intensity (RLU). The mean light signal intensity was proportional to the amount of recombinant hTSHR in the sample. The results of the serially diluted H5 expression supernatant samples showed that the expression product in the cell supernatant had high activity (Table 1).

[0109] Table 1

[0110]

[0111] Purification and identification of target protein:

[0112] 4.1 Purification of the target protein

[0113] The enriched H5 cell culture medium was centrifuged at 8000 rpm for 10 min, and the supernatant was dialyzed into 20 mM PBS buffer. The pH of the supernatant after dialysis was measured to be around 7.2. Protein purification was performed directly using a Protein A column with citric acid gradient elution. The elution peak of 70% citric acid was collected, and the target protein was harvested after dialysis.

[0114] 4.2 Western blot analysis of cell supernatant and purified protein activity

[0115] (1) Sample preparation: Take 50 μL of H5 cell culture supernatant, 50 μL of purified protein, and 50 μL of unrelated protein fused with human FC expression, add 10 μL of 6×SDS gel loading buffer, mix, boil in boiling water for 10 min, and centrifuge at 10000g for 10 min.

[0116] (2) Electrophoresis: Load the above protein samples and perform 12% SDS-PAGE at a constant voltage of 80V;

[0117] (3) Transfer: Transfer the protein onto a nitrocellulose membrane at 35 mA / gel for 45 min.

[0118] (4) Sealing: Slowly shake in 5% skim milk powder sealing solution for 1 hour;

[0119] (5) Primary antibody reaction: Dilute hTSHR C-terminal antibody 10F8 with 5% skim milk powder to 10ug / ml, prepare 10ml, put the above-transferred nitrocellulose membrane into the prepared primary antibody, and react for 30min.

[0120] (6) Washing: Wash the above nitrocellulose membrane with 11×TNT 5 times, 3-5 minutes each time;

[0121] (7) Secondary antibody incubation: Dilute KPL GAM-AP with 5% skim milk powder at a ratio of 1:5K to prepare 10ml. Place the above-transferred nitrocellulose membrane into the prepared primary antibody and react for 30min.

[0122] (8) Washing: Wash the above nitrocellulose membrane with 1×TNT 5 times, 3-5 minutes each time;

[0123] (9) Color development and termination: Add 10ml of diluted BCIP / NBT color development solution, and perform the color development reaction in the dark for 15min. Discard the color development solution, rinse once with deionized water, take a picture, and store in the dark.

[0124] SDS-PAGE and WB identification results as follows Figure 6 As shown, the target protein band is clearly visible in the expression supernatant. After purification, the purity of the target protein can reach more than 90%, indicating that the protein has a high level of soluble expression, i.e., high yield. The expression cell supernatant can be directly run into a gel without concentration to see a relatively obvious target protein band.

[0125] Example 2 Performance evaluation of recombinant thyroid-stimulating hormone receptor protein

[0126] Using the acrid ester chemiluminescence double antibody sandwich method combined with a competitive method mentioned in Example 1, a prototype reagent for TRAb detection of the target protein was further constructed. The constructed TRAb reagent was placed on a fully automated chemiluminescence analyzer Caris 200, and the samples were tested according to the standard operating procedures of the Caris 200 fully automated chemiluminescence analyzer. First, TRAb was serially diluted into a series of samples, and the serially diluted samples of TRAb were tested strictly according to the standard operating procedures of the Caris 200 fully automated chemiluminescence analyzer. The detected relative luminescence intensity (RLU) was fitted with the corresponding concentration in an appropriate manner (data shown in Table 2). The results showed that the correlation coefficient r of linear fitting in the range of 1-40 IU / L was 0.9972 (e.g., ...). Figure 7This indicates that the reagent exhibits good linearity within the range of 1-40 IU / L. The commercially available Roche TRAb electrochemiluminescence immunoassay reagent was selected as a control reagent. Following strict adherence to the instructions for the commercial reagent, the constructed TRAb prototype assay was used to perform parallel assays on 30 clinical samples with the commercially available TRAb assay. The correlation coefficient r between the two assays was 0.975 (e.g., ...). Figure 8 The results indicate that this reagent and the control reagent have a good quantitative correlation within the detection range, proving that this recombinant protein can be effectively used in the development of TRAb detection reagents and applied to industrial production. In summary, the above experimental results demonstrate that the purified protein has high activity.

[0127] Table 2

[0128] Concentration IU / L RLU 0.00 812326 0.63 796432 1.25 758456 2.50 727718 5.00 633856 10.00 463529 20.00 312326 40.00 171960

[0129] Example 3: Stability evaluation of recombinant thyroid-stimulating hormone receptor protein:

[0130] The purified recombinant protein was aliquoted and subjected to repeated freeze-thaw activity testing after storage at -20°C. Signal values ​​were measured before freezing, after one thaw, two thaws, three thaws, four thaws, and five thaws. Duplicate wells were tested, and the mean values ​​were analyzed. Using the mean signal value before freezing as the standard, the activity change corresponding to the signal value deviation after 1-5 freeze-thaw cycles was calculated to assess the freeze-thaw stability of the recombinant protein. The specific testing method is as follows: The recombinant hTSHR antigen to be evaluated was diluted to a certain concentration and added to magnetically coated 10F8 polymer solution. The reaction was carried out for 15 min to form a 10F8-coated magnetic microparticle-recombinant hTSHR complex. After magnetic separation and washing, other substances not bound to the magnetic microparticles were removed. Next, acrid ester-labeled stimulating TRAb was added and reacted with the recombinant hTSHR in the sample for 10 min to form a "10F8-coated magnetic microparticle-recombinant hTSHR-acrididyl ester-labeled stimulating TRAb" complex. After magnetic separation and washing, other substances not bound to the magnetic microparticles were removed. Finally, the pre-activation solution and activation solution are added, and the complex can emit a light signal of detectable intensity by the luminescence analyzer, expressed as the relative luminescence value (RLU). The results are shown in Table 3. That is, even if the recombinant protein prepared by this method is repeatedly frozen and thawed 5 times, the detection activity deviation can be controlled within 10%, that is, freezing and thawing does not significantly affect the activity of the protein. This shows that the recombinant antigen prepared by this method has good freeze-thaw stability and can further support industrial production.

[0131] Table 3

[0132] Number of thawing times Activity deviation thaw 1 5％ thaw 2 6％ 3 thaw 9％ thaw 4 4％ thaw 5 -4％

[0133] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention. SEQUENCE LISTING <110> Xiamen Imbio Biotechnology Co., Ltd. <120> A recombinant thyroid-stimulating hormone receptor protein, its preparation method and application <130> 2021 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 2295 <212> DNA <213> Artificial sequence <400> 1 atgaggccgg cggacttgct gcagctggtg ctgctgctcg acctgcccag ggacctgggc 60 ggaatggggt gttcgtctcc accctgcgag tgccatcagg aggaggactt cagagtcacc 120 tgcaaggata ttcaacgcat ccccagctta ccgcccagta cgcagactct gaagcttatt 180 gagactcacc tgagaactat tccaagtcat gcattttcta atctgcccaa tatttccaga 240 atctacgtat ctatagatgt gactctgcag cagctggaat cacactcctt ctacaatttg 300 agtaaagtga ctcacataga aattcggaat accaggaact taacttacat agaccctgat 360 gccctcaaag agctccccct cctaaagttc cttggcattt tcaacactgg acttaaaatg 420 ttccctgacc tgaccaagt ttattccact gatatattct ttatacttga attacagac 480 aacccttaca tgacgtcaat ccctgtgaat gctttcagg gactatgca tgaaccttg 540 acactgaagc tgtacaacaa tggctttact tcagtccaag gatatgcttt caatgggaca 600 aagctggatg ctgtttacct aaacaagaat aaatacctga cagttattga caagatgca 660 tttggagg tatacagtgg accaagcttg ctggacgtgt ctcaaccag tgtcactgcc 720 cttccatcca aaggcctgga gcacctgaag gaacgatag caaacacc ctggactctt 780 aagaaacttc cactttcctt gagttctctt cacctcacac gggctgacctt ttcttacca 840 agccactgct gtgcttta gatcagaag aaatcagag gatccttga gtccttgatg 900 tgtaatgaga gcagtagca gagcttgcgc cagagaaaat ctgtgaatgc cttgaatagc 960 cccctccacc aggaatatga agagaatctg ggtgacagca tgttggta caggaaag 1020 tccaagttcc aggatactca taacaacgct cattattacg tctctttga agaacagag 1080 gatgagatca tggttttgg aaaaaccccc aggagagac tctacaagct 1140 tttgacagcc attatgacta caccatatgt ggggacagtg aagacatggt gtgtaccccc aagtccgatg agttcaaccc gtgtgaagac ataatgggct acaagttcct gagaattgtg gtgtggttcg ttagtctgct ggctctcctg ggcaatgtct ttgtcctgct tattctcctc 1320 accagccact acaaactgaa cgtcccccgc tttctcatgt gcaacctggc ctttgcggat 1380 ttctgcatgg ggatgtacct gctcctcatc gcctctgtag acctctacac tcactctgag 1440 tactacaacc atgccatcga ctggcagaca ggccctgggt gcaaccggc tggtttcttc actgtctttg caagcgagtt atcggtgtat acgctgacgg tcatcaccct ggagcgctgg 1560 tatgccatca ccttcgccat gcgcctggac cggaagatcc gcctcaggca cgcatgtgcc atcatggttg ggggctgggt ttgctgcttc cttctcgccc tgcttccttt ggtgggaata 1680. agtagctg ccaaagtcag tatctgcctg cccatggaca ccgagacccc tcttgctctg gcatatattg tttttgttct gacgctcaac atagttgcct tcgtcatcgt ctgctgctgt 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860 aaaattgcca agaggatggc tgtgttgatc ttcaccgact tcatatgcat ggccccaatc 1920 tcattctatg ctctgtcagc aattctgaac aagcctctca tcactgttag caactccaaa 1980 atcttgctgg tactcttcta tccacttaac tcctgtgcca atccattcct ctatgctatt 2040 ttcaccaagg ccttccagag ggatgtgttc atcctactca gcaagtttgg catctgtaaa 2100 cgccaggctc aggcataccg ggggcagagg gttcctccaa agaacagcac tgatattcag 2160 gttcaaaagg ttacccacga gatgaggcag ggtctccaca acatggaaga tgtctatgaa 2220 ctgattgaaa actcccatct aaccccaaag aagcaaggcc aaatctcaga agagtatatg 2280 caaacggttt tgtaa 2295 <210> 2 <211> 681 <212> DNA <213> Artificial Sequence <400> 2 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgttagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggacga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 3 <211> 48 <212> DNA <213> Artificial sequence <400> 3 tctgcctttg cggcggatct tggatccatg ggctgcagca gtccgccg 48 <210> 4 <211> 42 <212> DNA <213> Artificial sequence <400> 4 gatattatgg gttataaatt tgcaattttt accaaagcct tt 42 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <400> 5 gaatatatgc agaccgttct ggaattcgac aaaactcaca catgccca 48 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <400> 6 aaatttataa cccataatat cttcacacgg att 33 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <400> 7 gaattccaga acggtctgca tatattcttc 30 <210> 8 <211> 57 <212> DNA <213> Artificial Sequence <400> 8 gtcccaggaa aggatcagat ctgcagcggc cgcttattta cccggagaca gggagag 57 <21着> 9 <211> 1185 <212> DNA <213> Artificial Sequence <400> 9 atgggctgca gcagtccgcc gtgtgaatgt catcaggaag aagattttcg tgtgacctgt 60 aaagatattc agcgtattcc gagcctgccg ccgagcaccc agaccttaaa actgattgaa 120 It should be noted that there seems to be an error in the "21着" in line . It should be corrected to "210" for a more accurate translation. The above translation is based on the corrected content.acccatctgc gtaccattcc gagtcatgca ttttctaatc tgccgaatat tagccgcatc 180 tatgttagca ttgatgttac cctgcagcag ctggaaagcc atagctttta taatctgagc 240 aaagtgaccc atattgaaat tcgcaatacc cgtaatctga cctatattga tccggatgca 300 ctgaaagaac tgccgctgct gaaatttctg ggcattttta ataccggtct gaaaatgttt 360 ccggatctga ccaaagttta tagtaccgat attttcttta tcctggaaat taccgataac 420 ccgtatatga ccagcattcc ggtgaatgcc tttcagggtc tgtgcaatga aaccctgacc 480 ctgaaactgt ttttaccagt gtgcaggct atgcctttaa tggcaccaaa 540 ctggatgccg tgtatctgaa tagagaataag tatctgaccg tgattgataa agatgccttt 600 ggcggcgtgt atagcggtcc gagtctgctg gatgttagtc agaccagtgt taccgcactg 660 ccgagcaaag gtctggaaca tctgaaagaa ttaattgcac gtaatacctg gaccctgaaa 720 aaactgccgc tgagcctgag ctttctgcat ctgacccgtg ccgatctgag ttatccgagc 780 cattgttgtg cctttaaaaa tcagaaaaag atccgtggta tcctggaaag tctgatgtgc 840 aatgaaagca gcatgcagag cctgcgtcag cgcaaaagcg tgaatgcact gaatagcccg 900 ctgcatcagg aatatgaaga aaatctgggc gatagtattg tgggctataa agaaaaaagt 960 aagttccagg atacccataa taatgcccat tattatgttt tcttcgagga acaggaagat 1020 gaaattattg gttttggtca ggaactgaaa aatccgcagg aagaaaccct gcaggccttt 1080 gatagccatt atgattatac catttgcggc gatagtgaag atatggtgtg caccccgaaa 1140 agtgatgagt ttaatccgtg tgaagatatt atgggttata aattt 1185 <210> 10 <211> 258 <212> DNA <213> Artificial sequence <400> 10 gcaattttta ccaaagcctt tcagcgtgat gtttttattc tgctgagtaa atttggtatc 60 tgtaaacgcc aggcccaggc atatcgtggc cagcgcgttc cgccgaaaaa tagcaccgat 120 attcaggtgc agaaagttac ccatgaaatg cgtcagggtc tgcataatat ggaagatgtg 1八十 tatgaactga ttgaaaatag tcatctgacc ccgaaaaaac agggccagat tagcgaagaa 240 tatatgcaga ccgttctg 258 <210> 11 <211> 764 <212> PRT <213> artificial sequence <400> 11 Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro 1 5 10 15 Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His 20 25 30 Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro 35 40 45 Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu 50 55 60 Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg 65 70 75 80 Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser 85 90 95 Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg 100 105 110 Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu 115 120 125 Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu 130 135 140 Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp 145 150 155 160 Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys 165 170 175 Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val 180 185 190 Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn 195 200 205 Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val 210 215 220 Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala 225 230 235 240 Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn 245 250 255 Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu 260 265 270 Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn 275 280 285 Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser 290 295 300 Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser 305 310 315 320 Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly 325 330 335 Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr 340 345 350 Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln 355 360 365 Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His 370 375 380 Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro 385 390 395 400 Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe 405 410 415 Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn 420 425 430 Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val 435 440 445 Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly 450 455 460 Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu 465 470 475 480 Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr 485 490 495 Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu 500 505 510 Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg 515 520 525 Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly 530 535 540 Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile 545 550 555 560 Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr 565 570 575 Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val 580 585 590 Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val 595 600 605 Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys 610 615 620 Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile 625 630 635 640 Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val 645 65Q 655 Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys 660 665 670 Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp 675 680 685 Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln 690 695 700 Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln 705 710 715 720 Val Gln Lys Val Thr His Glu Met Arg Gln Gly Leu His Asn Met Glu 725 730 735 Asp Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln 740 745 750 Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu 755 760 <210> 12 <211> 395 <212> PRT <213> Artificial Sequence It should be noted that there is a "65Q" in the original text which might be a typo. I translated it as it is. If it's incorrect, please correct the original text for a more accurate translation. <400> 12 Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His Gln Glu Glu Asp Phe 1 5 10 15 Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro Ser Leu Pro Pro Ser 20 25 30 Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu Arg Thr Ile Pro Ser 35 40 45 His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg Ile Tyr Val Ser Ile 50 55 60 Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser Phe Tyr Asn Leu Ser 65 70 75 80 Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg Asn Leu Thr Tyr Ile 85 90 95 Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu Lys Phe Leu Gly Ile 100 105 110 Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu Thr Lys Val Tyr Ser 115 120 125 Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp Asn Pro Tyr Met Thr 130 135 140 Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys Asn Glu Thr Leu Thr 145 150 155 160 Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val Gln Gly Tyr Ala Phe 165 170 175 Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn Lys Asn Lys Tyr Leu 180 185 190 Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val Tyr Ser Gly Pro Ser 195 200 205 Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala Leu Pro Ser Lys Gly 210 215 220 Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn Thr Trp Thr Leu Lys 225 230 235 240 Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu Thr Arg Ala Asp Leu 245 250 255 Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn Gln Lys Lys Ile Arg 260 265 270 Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser Ser Met Gln Ser Leu 275 280 285 Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser Pro Leu His Gln Glu 290 295 300 Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly Tyr Lys Glu Lys Ser 305 310 315 320 Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr Tyr Val Phe Phe Glu 325 330 335 Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln Glu Leu Lys Asn Pro 340 345 350 Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His Tyr Asp Tyr Thr Ile 355 360 365 Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro Lys Ser Asp Glu Phe 370 375 380 Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe 385 390 395 <210> 13 <211> 86 <212> PRT <213> Artificial Sequence <400> 13 Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp Val Phe Ile Leu Leu Ser 1 5 10 15 Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln Ala Tyr Arg Gly Gln Arg 20 25 30 Val Pro Pro Lys Asn Ser Thr Asp Ile Gln Val Gln Lys Val Thr His 35 40 45 Glu Met Arg Gln Gly Leu His Asn Met Glu Asp Val Tyr Glu Leu Ile 50 55 60 Glu Asn Ser His Leu Thr Pro Lys Lys Gln Gly Gln Ile Ser Glu Glu 65 70 75 80 Tyr Met Gln Thr Val Leu 85

Claims

1. A recombinant thyroid-stimulating hormone receptor protein, characterized in that, The recombinant thyroid-stimulating hormone receptor protein is composed, from N-terminus to C-terminus, of the extracellular segment of TSHR protein as shown in SEQ ID NO: 12, the intracellular segment of TSHR protein as shown in SEQ ID NO: 13, the amino acid sequence encoded by the GAATTC sequence in primer PF3 as shown in SEQ ID NO: 5, and the amino acid sequence encoded by the human antibody constant region DNA sequence as shown in SEQ ID NO:

2.

2. A method for preparing the recombinant thyroid-stimulating hormone receptor protein according to claim 1, characterized in that, Includes the following steps: S1, construct a recombinant plasmid, the recombinant plasmid comprising an extracellular segment gene fragment of TSHR protein and an intracellular segment gene fragment of TSHR protein; the C-terminus of the extracellular segment of TSHR protein is connected to the N-terminus of the intracellular segment of TSHR protein; S2, the recombinant plasmid is transferred into a eukaryotic expression system for expression and identification of the target protein; the eukaryotic expression system is selected from the rod-insect expression system; S3, Purify the target protein.

3. The method according to claim 2, characterized in that, The method for constructing the recombinant plasmid involves first using overlap PCR to connect the extracellular and intracellular gene fragments of the TSHR protein into the target gene fragment, and then using the Gibson cloning method to construct the target gene fragment into the plasmid vector.

4. The method according to claim 2, characterized in that, The method for constructing the recombinant plasmid involves first chemically synthesizing a target gene fragment including an extracellular segment of the TSHR protein and an intracellular segment of the TSHR protein, and then constructing the target gene fragment into a plasmid vector.

5. The use of the recombinant thyroid-stimulating hormone receptor protein according to claim 1 in the preparation of a thyroid-stimulating hormone receptor antibody detection kit.

6. A thyroid-stimulating hormone receptor antibody detection kit, characterized in that, Includes the recombinant thyroid-stimulating hormone receptor protein as described in claim 1.

Images