Application of human TSHR A subunit in preventing Graves' disease

Abstract

The present invention discloses the application of human TSHR A subunit in preventing Graves' disease. The present invention orally administers the key pathogenic autoantigen human TSHR A subunit of Graves' disease to neonatal Balb / c mice to induce neonatal oral tolerance to Graves' disease, and explores the dosage of TSHR A subunit. It is found that oral administration of a high dose of TSHR A subunit in the neonatal period can successfully induce immune tolerance of Balb / c female mice to Graves' disease, which provides a new idea and effective basis for the treatment and prevention of Graves' disease and other autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of Graves' disease treatment, and relates to the application of human TSHR A subunit in preventing Graves' disease. Background Art

[0002] Diffuse toxic goiter, also known as Graves' disease (GD), is the most common cause of hyperthyroidism. It is characterized by excessive synthesis and secretion of thyroid hormones and diffuse goiter. Clinical manifestations mainly include thyrotoxicosis, diffuse goiter, eye signs and pretibial myxedema. The etiology of Graves' disease is still unclear, but it is generally recognized that it is related to the autoimmune reaction of the thyroid gland. Its prominent feature is the presence of autoantibodies in the patient's blood that can react with their own thyroid tissue, mainly including thyroid stimulating hormone (TSH) receptor (TSH receptor, TSHR) antibodies (TSH receptorantibody, TRAb), thyroid thyroglobulin antibodies (TgAb) and thyroid peroxidase antibodies (thyroid peroxidase antibodies, TPOAb). Among them, TRAb plays a major role in the pathogenesis of Graves' disease.

[0003] There are several common treatments for Graves' disease: antithyroid drugs, radioactive 131I and surgical treatment. Drug treatment mainly inhibits thyroid peroxidase, which is related to the synthesis of thyroid hormones. The latter two treatments are mainly aimed at reducing the amount of functional thyroid tissue, each with its own advantages and disadvantages. Generally, the appropriate plan is carefully selected based on the patient's gender, age, severity of the disease, duration of the disease, complications, comorbidities, combined with the patient's own situation, local medical conditions and the physician's experience level. Although the treatment methods for Graves' disease are now relatively mature, the above three main treatment measures are all treatments for thyrotoxicosis. Their mechanism of action is mainly to inhibit the synthesis of thyroid hormones or reduce functional thyroid cells, in order to control thyroid function while expecting the patient's own immune abnormalities to alleviate and correct themselves. There is no treatment for etiology, that is, autoimmune abnormalities. There is currently no better way to focus on the autoimmune process for treatment, actively correct or prevent the existence or occurrence of autoimmune abnormalities.

[0004] Appropriate animal models are one of the important tools for studying the pathogenesis of diseases and evaluating the efficacy of treatment. The research on animal models of Graves' disease has also gone through a long development process, especially in recent years, and very important progress has been made. Now a relatively mature method for preparing animal models of Graves' disease has been obtained. The latest transgenic animal models are also in the process of development, but the several existing transgenic mouse models still have certain problems. The initial attempt at an animal model of Graves' disease was to use the human TSHR extracellular segment complex protein produced from a prokaryotic or eukaryotic expression system, but the result was the induction of antibodies that had an inhibitory effect on thyroid function. In 1994, Shimojo et al. prepared a Graves' disease model with hyperthyroidism for the first time. The method was to co-inject TSHR and MCH class II molecules expressed by fibroblasts into the abdominal cavity of mice, and the results successfully induced hyperthyroidism and TSHR stimulating antibodies. Subsequently, researchers gradually began to use plasmid vectors expressing the extracellular segment of TSHR or viral vectors to infect mice to induce Graves' disease animal models. In 2003, Chen et al. successfully induced TSAb and hyperthyroidism by immunizing Balb / c mice with adenovirus encoding human TSHR A subunit. More importantly, compared with the previous Graves' disease model induced by the full-length human TSHR, the incidence of the mouse model immunized with adenovirus encoding only the A subunit was significantly increased. This finding once again proved that the TSHR A subunit has a stronger pathogenicity and also made the animal model of Graves' disease mature. In recent years, inducing immune tolerance has become a new way to try to treat or prevent autoimmune diseases from an etiological perspective. However, there is no consensus on the way, timing and influencing factors that can successfully induce immune tolerance, and they are not uniform in different animal models. In the previous study of our research group, we successfully induced an immune tolerance model of Graves' disease in Balb / c mice by intramuscular injection of adenovirus expressing human TSHR A subunit in the neonatal period. Although this animal model has been successfully induced, its application in humans is greatly limited due to safety issues.

[0005] In recent years, oral immune tolerance has been increasingly used to prevent or treat autoimmune diseases due to its high safety, simple operation and many other advantages. It has also become a new direction for inducing immune tolerance models for Graves' disease. In the early stage, the TSHR A subunit gene was expressed by intramuscular injection, and the TSHR A subunit protein was directly orally administered. The antigen can be cleaved into peptide fragments by proteolytic enzymes in the gastrointestinal tract. Although studies have confirmed that some TSHR peptides can induce the occurrence of adult immune tolerance in Graves' disease, it is unknown what kind of peptides will be generated after the oral TSHR A subunit protein is cleaved by proteolytic enzymes in the gastrointestinal tract, and whether these peptides are antigenic is difficult to predict. The induction of oral immune tolerance is related to many factors, such as the body's own immune system status, antigen type, antigen properties, antigen dose, antigen use time, etc. These will also increase many unknowns in the success of tolerance.

[0006] In summary, Graves' disease is an autoimmune disease, but the existing treatments are mostly functional treatments, and there is no treatment or prevention method targeting the etiology, namely autoimmunity, to cure Graves' disease. Summary of the invention

[0007] (I) Purpose of the invention

[0008] The purpose of the present invention is to provide a method for orally administering the key pathogenic autoantigen human TSHR A subunit of Graves' disease to neonatal Balb / c mice, thereby inducing neonatal oral tolerance to Graves' disease and exploring new approaches for the etiology of Graves' disease treatment and prevention. It was found that oral administration of a high dose of TSHR A subunit in the neonatal period can successfully induce immune tolerance to Graves' disease in Balb / c female mice, which provides new ideas and effective basis for the treatment and prevention of Graves' disease and other autoimmune diseases.

[0009] (II) Technical solution

[0010] To solve the above problems, the first aspect of the present invention provides a use of human TSHR A subunit in the preparation of an oral drug for preventing Graves' disease.

[0011] Furthermore, the human TSHR A subunit is a recombinant human TSHR A subunit produced and purified using a yeast expression system.

[0012] Furthermore, the dosage of the recombinant human TSHR A subunit is 100-1000 μg, preferably 500 μg-1000 μg, and more preferably 1000 μg.

[0013] According to another aspect of the present invention, an oral pharmaceutical composition for preventing Graves' disease is provided, wherein the active ingredient of the composition is human TSHR A subunit.

[0014] Furthermore, the human TSHR A subunit is a recombinant human TSHR A subunit produced and purified using a yeast expression system.

[0015] Furthermore, the content of the recombinant human TSHR A subunit in the composition is 100-1000 μg.

[0016] Furthermore, the content of recombinant human TSHR A subunit in the recombinant human TSHR A subunit solution in the composition is 500-1000 μg, preferably 1000 μg.

[0017] According to another aspect of the present invention, there is provided a method for inducing an animal model of de novo oral tolerance to Graves' disease using human TSHR A subunit, the method comprising the following steps:

[0018] S1. Within 24 hours after birth, inject the human TSHR A subunit solution into the animal's pharynx several times, and stimulate the animal's palate to induce swallowing reflex;

[0019] S2. The animals injected with the recombinant human TSHR A subunit are placed back into the breeding space, and breastfeeding is continued. After weaning, the animals are continued to be raised until they reach adulthood.

[0020] Furthermore, the human TSHR A subunit is a recombinant human TSHR A subunit produced and purified using a yeast expression system.

[0021] Furthermore, the content of recombinant human TSHR A subunit in the recombinant human TSHR A subunit solution is 100 μg-1000 μg, preferably 500 μg-1000 μg, and more preferably 1000 μg.

[0022] Furthermore, the animal is an immunocompetent rodent, preferably a mouse.

[0023] According to another aspect of the present invention, provided is an animal model induced by the method provided in the third aspect, and application of the animal model in a study of oral immune tolerance of Graves' disease.

[0024] (III) Beneficial effects

[0025] The above technical solution of the present invention has the following beneficial technical effects:

[0026] 1. In previous studies, TSHR crudely extracted from animal thyroid tissue does not have the immune activity of TSHR in the human body, and the A subunit produced by the E. coli system lacks glycosylation and has no immune activity; although the A subunit expressed by mammalian cells has partial immune activity, the production cost is high and the yield is low. In the present invention, a yeast expression system is used to produce purified recombinant human TSHR A subunit, and experiments have proved that the purified protein has the immune activity of TSHR A subunit in the human body.

[0027] 2. The present invention induces oral tolerance to Graves' disease in the neonatal period. Balb / c female mice are given different doses (1000μg high-dose tolerance group, 500μg medium-dose tolerance group and 100μg low-dose tolerance group) of yeast expression system to produce purified recombinant human TSHR A subunit orally in the neonatal period, and the Graves' disease model is induced in adulthood. The results show that the incidence rate of the high-dose tolerance group is 62.5% (10 / 16), which is lower than the incidence rate of the model group 95.2% (20 / 21), and there is a statistical difference between the two (P<0.05); the serum TT4 level and TRAb level of the mice in the high-dose tolerance group are significantly lower than those in the model group (P<0.05), and the degree of follicular epithelial cell hyperplasia in the thyroid tissue pathology is lighter than that in the model group, that is, the Graves' disease in the high-dose tolerance group is lighter than that in the model group. It can be seen that oral administration of 1000μg A subunit in the neonatal period can successfully induce immune tolerance to Graves' disease in Balb / c female mice. It can be seen that whether oral tolerance can be successfully induced in the neonatal period is related to the antigen dose. In this study, high-dose antigen is more likely to successfully induce oral tolerance in the neonatal period than medium and low doses. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation are briefly introduced below.

[0029] Figure 1 This is an immunoblot identification diagram of the purified protein. Lane 1 is the protein marker, lane 2 is the purified protein, and lane 3 is the purified protein after deglycosylation.

[0030] Figure 2Figure 1 shows the validation of the purified protein's partial immune function of the TSHR A subunit in humans. (A) Elisa assay to evaluate the immune activity of the purified protein. Control, healthy human serum; Graves'sera, TRAb-positive serum from a newly diagnosed, untreated female Graves' disease patient; Ab 6048, specific positive control. OD450 values ​​are expressed as Mean±SE. (B) TRAb assay to validate the binding of purified protein to serum components. Normal sera, normal human serum; Graves'sera, serum from a Graves' patient positive in the Elisa assay; Before is a 1:400 diluted serum stock solution, and After is the supernatant recovered after incubation with the purified protein at the same dilution multiple. ***, P<0.001.

[0031] Figure 3 Serological indicators of thyroid function in each group of mice. (A) TT4 level in mouse serum was detected by radioimmunoassay. TT4 value is expressed as Mean±SE. **, P<0.01. (B) TRAb level in mouse serum was detected by radioimmunoassay. TRAb value is expressed as Mean±SE. *, P<0.05. Control (n=13), control group; Model (n=21), model group; 1000μg (n=16), high-dose tolerance group; 500μg (n=11), medium-dose tolerance group; 100μg (n=17), low-dose tolerance group.

[0032] Figure 4 The thyroid tissue pathological examinations of mice in each group were performed under light microscopy (HE). (A, B) Thyroid follicular cells of mice in the control group (×100, ×400, respectively), the follicles were round or oval, the follicular cavity was filled with homogeneous eosinophilic colloid, and the follicular epithelial cells were single-layer cubic; (C, D) Thyroid follicular cells of mice in the model group (×100, ×400, respectively), the follicular epithelial cells were obviously proliferated, columnar or cubic, and papillary structures were visible; (E, F) Thyroid follicular cells of mice in the high-dose tolerance group (×100, ×400, respectively), the follicular epithelial cells were single-layer cubic, the follicles were round or oval, and the follicles were The cavity was filled with homogeneous eosinophilic colloid; (G, H) Thyroid follicular cells of mice in the high-dose tolerance group (×100, ×400, respectively), the follicular epithelial cells were slightly proliferative, but without papillary structures; (I, J) Thyroid follicular cells of mice in the medium-dose tolerance group (×100, ×400, respectively), the follicular epithelial cells were moderately proliferative, showing low columnar shapes, and papillary structures were visible; (K, L) Thyroid follicular cells of mice in the low-dose tolerance group (×100, ×400, respectively), the follicular epithelial cells were significantly proliferative, showing columnar or cubic shapes, and papillary structures were visible.

[0033] Figure 5Flow cytometry was used to detect the proportion of splenic Tregs cells in each group of mice. (A) The proportion of CD25+FoxP3+ cells in splenic CD4+ monocytes of mice in the high-dose tolerance group; (B) The proportion of CD25+FoxP3+ cells in splenic CD4+ monocytes of mice in the medium-dose tolerance group; (C) The proportion of CD25+FoxP3+ cells in splenic CD4+ monocytes of mice in the low-dose tolerance group; (D) The proportion of CD25+FoxP3+ cells in splenic CD4+ monocytes of mice in the model group; (E) The proportion of CD25+FoxP3+ cells in splenic CD4+ monocytes of mice in the control group.

[0034] Figure 6 Comparison of the proportion of Tregs in splenic CD4+ monocytes of mice in each group. Control, control group; Model, model group; 1000μg, high-dose tolerance group; 500μg, medium-dose tolerance group; 100μg, low-dose tolerance group. *, P<0.05; **, P<0.01. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0036] definition

[0037] The "TSHR" gene described in the present invention is located on chromosome 14q31, including 10 exons and 9 introns, encoding a total of 764 amino acids, of which the first 21 amino acid residues are signal peptides. Human TSHR is a member of the G protein-coupled receptor family, consisting of 764 amino acid residues, and its molecular mass is 84.5kDa. TSHR belongs to a seven-transmembrane protein, consisting of an extracellular segment, a transmembrane region and an intracellular segment. The most mysterious region of TSHR in terms of structure and function is the hinge region between the extracellular segment and the transmembrane region, which is composed of amino acid residues at positions 277 to 418. There is a region of about 50 amino acid residues in the hinge region that is not present in other similar glycoprotein receptors, which is called the C-peptide region. Under physiological conditions, the C-peptide region is spontaneously degraded after translation, and TSHR is split from the molecule, dividing TSHR into two parts, the 289 amino acid residues at the amino terminal constitute the A subunit, and the remaining amino acid residues at the carboxyl terminal constitute the B subunit. The A subunit and the B subunit are bound by a disulfide bond.

[0038] The "TSHR A" subunit of the present invention is composed of 289 amino acid residues of the "TSHR" gene, and the specific sequence is shown in SEQ ID NO:1.

[0039] Experimental methods and steps

[0040] 1. Yeast culture

[0041] 1) Yeast resuscitation: (1) Take out the stored yeast strain from the -80℃ refrigerator, thaw it on ice, and then pipette 200μL into a 50mL centrifuge tube containing 10mL YPD medium. At the same time, add 10μL of the antibiotic Zeocine (final concentration is 100μg / mL), place it in a shaker, loosen the lid half a turn, and culture it at 28℃, 250rpm for 16-18 hours. When the bacterial solution becomes turbid and opaque, take it out and store it briefly at 4℃ for the next step of amplification. (2) All related operations are performed in a clean bench, and attention should be paid to aseptic operation.

[0042] 2) Yeast culture amplification: (1) Take several 2L large conical flasks, add 200mL of BMGY medium (PH6.0) to each flask, and then take 1mL of the yeast solution revived in the previous step and add it to each flask; place in a shaker, 28°C, 250rpm, and culture for 24 hours; (2) After 24 hours, the bacterial solution will be completely turbid, and the OD600 value will reach 10-20, then it can proceed to the next step of expression culture; within a certain range, the higher the concentration of the strain, the higher the protein yield in the future, but if the culture time is too long and the number of culture passes is too large, the yeast will age and affect the yield.

[0043] 2. Expression of recombinant human TSHR A subunit

[0044] 1) Concentrate the yeast amplified in the previous step, pour the bacterial solution into a 50 mL sterile centrifuge tube, and centrifuge at 8000g for 5 minutes. When the bacteria are completely precipitated and the supernatant is clear, discard the supernatant;

[0045] 2) Pour BMMY medium (PH 7.4) into the centrifuge tube, resuspend the bacteria, add the same volume of BMMY medium as in the previous amplification culture, mix well and pour into 2L large triangular conical flasks, 200mL per bottle; add 3mL of methanol to each bottle to make the final concentration reach 1.5%, and induce the yeast to express the recombinant human TSHR A subunit;

[0046] 3) Place the conical flask in a shaker at 28°C and 250 rpm, add 3 mL of methanol to each conical flask every 24 hours, and culture for a total of 5 days.

[0047] 3. Isolation and purification of recombinant human TSHR A subunit

[0048] 1) Collect the supernatant: After 5 days, centrifuge the yeast culture at 12000g for 10 minutes and collect the supernatant. Keep the temperature low to prevent degradation of the A subunit. Add an appropriate amount of 1M NaOH solution to the precipitated yeast to inactivate the yeast before discarding. Do not discharge it directly into the sewer to prevent environmental pollution.

[0049] 2) Ultrafiltration concentration: (1) Wash and balance the ultrafiltration column and pipeline system with distilled water and PBS solution respectively; (2) Pump the recovered protein supernatant containing subunit A into the ultrafiltration column with a peristaltic pump, maintaining an appropriate flow rate and pressure. Since the target protein molecule is larger than the pore size of the ultrafiltration membrane, the subunit A can be retained in the filter membrane, while the solvent and small molecule impurities are filtered out of the membrane; (3) After the loading is completed, continue to pump a large amount of PBS solution with a peristaltic pump, the total volume is 5 to 10 times the volume of the supernatant, remove the small molecule impurities in the supernatant as much as possible, and replace the solvent to dissolve the subunit A in the PBS solution; (4) Recover the liquid in the ultrafiltration column, which is the PBS solution of subunit A. At this time, the total volume of the protein solution is about 1 / 10 of the original supernatant volume, and temporarily store it at 4°C; (5) Wash the ultrafiltration column system with distilled water; store the ultrafiltration column in 0.1M NaOH solution;

[0050] 3) Purification: Use the AKAT protein purification system and nickel ion affinity column to separate and purify the A subunit. (1) Turn on the machine and clean the system with filtered distilled water until the conductivity of the effluent drops below 0.003; (2) Connect the nickel column to the purification system and clean the nickel column with filtered distilled water to wash out the 20% ethanol solution in the column bed; (3) Use PBS solution to balance the system and the nickel column, and the total balance liquid volume is 4 column volumes; (4) Load the protein and slowly pass the ultrafiltered and concentrated A subunit solution through the column while observing the changes in conductivity and UV280. If it rises too high and too fast, it indicates that the column bed may have reached the maximum load, and the sample can be loaded and eluted in batches; (5) Use PBS solution to balance the system and the nickel column again, and the total balance liquid volume is 4 column volumes; (6) Use 50 mM imidazole solution to elute the impurities in the column bed; (7) Use 250 mM imidazole solution to pass the column and closely observe the UV280 value. When UV280 rises rapidly, collect the flow-through until the entire flow-through peak ends. This flow-through is the target protein recombinant human TSHR. A subunit, temporarily stored at 4°C; (8) Pass 250 mM imidazole solution through the column to clean the residual protein on the column bed; (9) Wash the system and nickel column with filtered distilled water until the conductivity of the effluent drops below 0.003; (10) Pass 20% ethanol solution for a total of 4 column volumes, and store the system and nickel column in a 20% ethanol environment; (11) Remove the nickel column and store at 4°C; turn off the system;

[0051] 4) Dialysis concentration: Add the collected flow-through into an ultrafiltration concentration centrifuge tube, centrifuge at 3000 g, continuously add PBS solution and centrifuge repeatedly to elute the imidazole in the flow-through and concentrate the A subunit solution. Finally, recover the remaining liquid in the upper chamber to obtain the concentrated recombinant human TSHR A subunit PBS solution, which is stored at -80°C.

[0052] 4. Raising and breeding mice

[0053] 1) Mouse feeding: The Balb / c mice used in this experiment were all raised in an SPF environment. Each mouse cage contained about 6 mice, with males and females separated. Most mice in the same cage were from the same litter or were raised in the same cage after weaning. The feed was sterile feed for mice and rats, and the drinking water was sterile distilled water. The bedding was changed every four days, and the room was kept at a constant temperature and humidity, with 8 to 10 hours of light per day.

[0054] 2) Reproduction of mice: When mice are mature (about 60-90 days old), adult mice are caged together in the evening at a ratio of 3:1 for females. The next morning, observe whether the female mice have vaginal plugs and take vaginal epithelial cell smears to determine whether the female mice are pregnant. The pregnant female mice are placed alone, and after 2 weeks, the pregnancy can be observed to verify whether they are pregnant. Female mice can give birth 3 weeks after conception, mostly at night. Before giving birth, female mice can be observed to be restless and constantly tidying up the birthing nest. The lactation period is about 18 to 23 days. During pregnancy and lactation, attention should be paid to strengthening the nutrition of the mother mice. If necessary, they can be fed with melon seeds to prevent the mother mice from cannibalizing the pups. Three weeks after giving birth, the mother mice are separated from the pups, and the male and female pups are separated into cages and continue to be raised.

[0055] 5. Inducing neonatal oral tolerance

[0056] 1) After the mother mouse gave birth, the recombinant human TSHR A subunit stored in the -80°C refrigerator was taken out and placed on ice. After melting, it was diluted to 13 μL with PBS solution, so that the content of A subunit was 100 μg, 500 μg and 1000 μg respectively; it was freshly prepared before use and used immediately to prevent degradation of A subunit;

[0057] 2) Within 24 hours after birth, the A subunit solution with different contents or the same volume of PBS solution was slowly injected into the pharynx of the newborn mice with the tip of a gun in batches according to different groups, and the upper palate of the newborn mice was gently stimulated to induce swallowing reflex, ensure oral efficiency, and prevent accidental inhalation into the trachea; before oral administration, the liquid was placed for half an hour to equilibrate to room temperature to prevent the newborn mice from being harmed by low temperature; at the same time, pay attention to odor protection to prevent mother mice from eating their offspring. Among them, the tolerance group took different doses of A subunit orally, and the model group and the control group took the same volume of PBS solution orally;

[0058] 3) Place the newborn mice back into their nests and continue to feed them with breast milk. Continue to raise them after weaning until they reach adulthood.

[0059] 6. Induced Graves' disease model

[0060] 1) Dilute Ad-TSHR289 adenovirus and Ad-null adenovirus with sterile PBS solution to a final concentration of 10 8 pfu / 50μL;

[0061] 2) Graves' disease was induced by intramuscular injection of Ad-TSHR 289 adenovirus three times at 6, 9, and 12 weeks after birth. The injection site was the posterior buttocks muscle. Each mouse in the tolerance group and the model group was injected with 10 8 pfu / 50μL, and the control group was injected intramuscularly with Ad-null adenovirus 10 8 pfu / 50μL;

[0062] 3) Process the mice four weeks after the third virus injection.

[0063] 7. Handling of mice

[0064] 1) Remove one eyeball of the mouse with sterile ophthalmic forceps, collect blood, let stand at room temperature for 30 minutes, centrifuge at 3000 rpm for 10 minutes, collect serum, and store at -80°C;

[0065] 2) Kill the mice by dislocating the neck, cut the hair on the chest and abdomen of the mice with ophthalmic scissors, and soak them in 75% alcohol for 2-3 minutes;

[0066] 3) Take the mouse out of the alcohol, fix it on a foam board with thumbtacks, and cut the mouse's abdominal skin with sterile tissue scissors to expose the abdominal cavity;

[0067] 4) Use sterile ophthalmic scissors and toothed forceps to free and remove the mouse spleen, cut the spleen into two pieces, and place them in a sterile 3 cm culture dish and a sterile RNase-free cryopreservation tube respectively. The cryopreservation tube is immediately stored in liquid nitrogen;

[0068] 5) Cut the mouse neck skin along the anterior midline, bluntly separate the anterior cervical muscles and fascia, fully expose and free the trachea, cut the trachea from both ends, and immerse the trachea and attached thyroid tissue in 10% formalin solution.

[0069] 8. Detection of mouse serum TT4 and TRAb levels by radioimmunoassay

[0070] 1) Serum TT4 level detection: According to the instructions of the radioimmunoassay kit, add 200 μL 125I-T4 and 200 μL anti-T4 serum to the γ-counting tubes containing the standard, quality control or 50 μL mouse serum, and incubate in a 37°C water bath for 45 minutes; add 500 μL separation agent, mix thoroughly, centrifuge at 3500 rpm for 20 minutes, discard the supernatant, and use a γ counter to measure the radioactivity count of the precipitate in each tube, and find out the concentration of TT4 in the serum of the mouse to be tested based on the dose-response standard curve;

[0071] 2) Detection of serum TRAb levels: According to the instructions of the radioimmunoassay kit, add 50 μL of receptor and 100 μL of 125I-TSH marker to the γ-counting tube containing the standard, quality control or 50 μL of mouse serum, and incubate at room temperature for 2 hours; add 1000 μL of precipitant, mix thoroughly, centrifuge at 3500 rpm for 30 minutes, discard the supernatant, and use a γ-meter to measure the radioactivity count of each tube of precipitate. According to the dose-response standard curve, the concentration of TRAb in the serum of the mouse to be tested is determined.

[0072] 9. Mouse thyroid tissue pathology

[0073] 1) Making sections: (1) Fixation: Place mouse thyroid tissue in 10% formalin solution for 24 hours, then take it out and rinse it with running water; (2) Dehydration, transparency, wax immersion and embedding: Immerse the thyroid tissue attached to the trachea in 95% ethanol for 2-4 hours, 95% ethanol for 2 hours, anhydrous ethanol for 1.5 hours, anhydrous ethanol for 1 hour, xylene + anhydrous ethanol (1:1) for 20 minutes, xylene for 10 minutes, xylene for 10 minutes, soft wax (50°C) for 30 minutes, soft wax (50°C) for 1 hour, hard wax (58°C) for 30 minutes, hard wax (58°C) for 30 minutes, and embed; (3) Sectioning: Use a rotary microtome to evenly slice the paraffin block to a thickness of 3-5 μm, and spread the sections flat on a slide; bake the sections at 60°C for 30 minutes;

[0074] 2) HE staining: (1) Soak the sections in xylene for 10 minutes to dewax, repeat once; (2) Soak in anhydrous ethanol for 5 minutes to wash off the xylene, repeat once; (3) Soak in 95% ethanol solution for 10 minutes and 80% ethanol solution for 10 minutes, wash with tap water for 1 minute, and wash with distilled water for 1 minute; (4) Soak in hematoxylin for 4 minutes, and wash with tap water for 2 minutes; (5) Soak in 1% hydrochloric acid ethanol for 20 seconds, and wash with running water until it turns blue; (6) Soak in 0.5% eosin solution for 90 seconds, and quickly rinse with distilled water for 2 seconds; (7) Soak in 80% ethanol solution for 10 seconds, 95% ethanol solution for 10 seconds, and anhydrous ethanol for 5 minutes to dehydrate; (8) Soak in anhydrous ethanol for 10 minutes; (9) Soak in xylene for 10 minutes, repeat once; (10) Seal the sections with neutral gum;

[0075] 3) Observe the pathological changes of thyroid tissue under a microscope.

[0076] 10. Preparation of Splenic Mononuclear Cell Suspension

[0077] 1) Take out the isolated mouse spleen tissue from the 3 cm culture dish, put it into a 6 cm sterile culture dish with a 200 mesh steel mesh, and add 4 mL of complete 1640 medium containing 10% fetal calf serum (FCS);

[0078] 2) Use tissue scissors to cut the spleen tissue into small pieces the size of millet grains; use the needle of a large glass syringe to gently grind the spleen fragments, and add a small amount of 1640 culture medium during the grinding process to obtain a cell suspension;

[0079] 3) Pipette the spleen cell suspension in the 6 cm culture dish into a 7 mL sterile centrifuge tube and centrifuge at 1200 rpm for 5 minutes; discard the supernatant;

[0080] 4) Add 2 mL of red blood cell lysis buffer to the centrifuge tube, resuspend the pellet, let stand at room temperature for 3 minutes, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant;

[0081] 5) Wash cells: Add 3 mL of 1640 medium to the centrifuge tube, resuspend the pellet, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and repeat 3 times;

[0082] 6) Add 3 mL of 1640 culture medium to the centrifuge tube and resuspend the pellet to make a cell suspension;

[0083] 7) Cell counting (1) Take 10 μL of cell suspension and add it to a 1.5 mL EP tube, then add 1640 culture medium to dilute to 1 mL; (2) Take 15 μL of the diluted cell suspension and gently drip it along the edge into the cell counting plate covered with a coverslip so that the cell suspension evenly covers the cell counting plate; (3) Place the cell counting plate under a microscope, count the cells, and mark the concentration. Cell density (cells / ml) = (∑ number of cells in four large grids / 4)*10 4 .

[0084] 11. Flow cytometry

[0085] 1) Dilute the cell suspension: dilute the spleen mononuclear cell suspension obtained in the previous step with 1640 complete medium to a final concentration of 2×107 / mL, and pipette 500 μL into two 2 mL EP tubes respectively;

[0086] 2) Cell staining: (1) Resuspend cells: Centrifuge the cell suspension in the above EP tube at 400g for 5 minutes, discard the supernatant, flick the precipitated cells, and add 100μL Staining Buffer to resuspend; (2) Surface staining: Add 0.3μL FITC anti-mouse CD4 and 0.3μL APC anti-mouse CD25 to each tube, vortex to mix, and incubate at 4℃ in the dark for 30 minutes; (3) Wash cells: Add 1.5mL Staining Buffer to each tube, mix, centrifuge at 400g for 5 minutes, discard the supernatant, and repeat twice; (4) Permeabilize cells: Add 1mL freshly prepared Fixation / Permeabilization working solution to each tube, resuspend cells, vortex to mix, incubate at 4℃ in the dark for 30 minutes; centrifuge at 400g for 5 minutes, and discard the supernatant; (5) Wash cells: Add 1.5mL Permeabilization Buffer, mix well, centrifuge at 400g for 5 minutes, discard the supernatant, repeat twice; (6) Blocking: add 100μL Permeabilization Buffer to each tube to resuspend the cells, add 2μL anti-mouse CD16 / 32 respectively, incubate at 4℃ in the dark for 15 minutes; (7) Intracellular staining: add 2.5μL PE anti-mouse FOXP3 and 2.5μL anti-mouse FOXP3 isotype control to each tube, mix well, incubate at 4℃ in the dark for 30 minutes; (8) Wash cells: add 1.5mL Permeabilization Buffer to each tube, mix well, centrifuge at 400g for 5 minutes, discard the supernatant, repeat twice; (9) Fix: add 1mL 4% paraformaldehyde to each tube and fix at room temperature for 20 minutes; (10) Wash cells: add 1.5mL Staining Buffer to each tube, mix well, centrifuge at 400g for 5 minutes, discard the supernatant; (11) Prepare for loading: add 500μL Resuspend cells in Staining Buffer, mix well, protect from light, and prepare for loading.

[0087] 3) Detection of Treg cells by flow cytometry: The cell suspension in the previous step was analyzed by FAcscan flow cytometer, gated by CD4 and SSC, and CD4+ cells were selected to analyze the expression of CD25 and FOXP3. The results were calculated as the percentage of positive cells.

[0088] 12. Statistical analysis

[0089] SPSS 21.0 software was used for statistical analysis. The χ2 test of row lists was used to compare the incidence of Graves' disease in mice in different groups. Dunnett-t test was used to compare the TT4 level, TRAb level, spleen Tregs ratio and stimulation index of each group. P<0.05 indicated statistically significant differences.

[0090] Example 1 The purified protein is a recombinant human TSHR A subunit protein

[0091] We first used the BCA method to detect the concentration of the purified protein after concentration. According to the standard curve, the concentration of the highly concentrated purified protein was calculated to be 79.5 mg / mL. A total of 500 μL was dispensed, and the total output of 15 batches was 39.75 mg. Western Blot tests were performed on each batch of purified protein and the mixed purified protein to verify whether the purified protein was the target protein, recombinant human TSHR A subunit protein. The results showed that the purified protein had a long tail and could specifically bind to the anti-human TSHR antibody Ab6048 (specifically binds to the amino acid residues 22-35 at the amino terminus of the A subunit), which is consistent with the previous experimental results. Figure 1 As can be seen, the purified protein is the recombinant human TSHR A subunit protein produced by yeast expression.

[0092] Example 2 The purified protein has some immune functions of the TSHR A subunit in humans

[0093] In order to detect whether the purified target protein has the same immunogenicity as the TSHR A subunit in the human body, or whether the immune function is similar, this study collected 15 TRAb(+) serum samples from newly diagnosed and first-treated female Graves' disease patients from the clinic, and used the Elisa method to detect whether the purified protein can specifically bind to the patient's serum. The OD value was 2.1 times greater than that of the negative control group, which was considered positive to verify the function of the purified protein. The results showed that 8 of the 15 patient serum samples were positive ( Figure 2Figure 2 (A), P = 0.066), indicating that the immune function of the purified protein partially overlaps with the immune function of the TSHR A subunit in the human body, and the recombinant human TSHR A subunit purified from the yeast expression system has some active immune epitopes. In order to further verify that the patient serum component that binds to the substrate purified protein in the Elisa test is indeed TRAb rather than other, this study collected the supernatant after the positive group in the Elisa test was incubated with the primary antibody, and compared it with the patient serum stock solution of the same dilution multiple, and detected the TRAb value by radioimmunoassay, and compared the changes in TRAb in the serum (supernatant) before and after binding with the substrate. The results showed that compared with normal human serum, the TRAb content of the serum of Graves' disease patients was significantly reduced after binding with the substrate (Figure 2 (B), P < 0.001), confirming that the patient serum component that binds to the purified protein in the above Elisa test is indeed TRAb, indicating that the purified protein has the immune activity of the TSHR A subunit in the human body.

[0094] Example 3 Oral administration of high doses of A subunit in neonates can induce immune tolerance in Graves' disease

[0095] To investigate whether oral administration of recombinant human TSHR A subunit protein during the neonatal period can induce immune tolerance to Graves' disease in Balb / c mice in adulthood, this study gave Balb / c female mice oral administration of high-dose (1000 μg), medium-dose (500 μg) and low-dose (100 μg) A subunit within 24 hours after birth, and the model group and control group orally administered the same volume of PBS solution at the same time point. After the mice became adults, each tolerance group and the model group were immunized with Ad-TSHR adenovirus for multiple times to induce Graves' disease, and the control group was injected with the same dose of Ad-null adenovirus at the same time point. The thyroid function (TT4, TRAb) and thyroid tissue pathology of each group of mice were observed to evaluate the pathogenesis of Graves' disease and assess whether the tolerance of each group was successful. The normal range of serum TT4 level is the Mean±2SD of the control group's serum TT4 level, that is, 3.10-62.07μg / L is the normal range of thyroid function; serum TRAb level is >10 U / L for TRAb positive; mouse thyroid tissue pathology is judged as positive based on whether there is thyroid follicular epithelial cell hyperplasia and papilla formation. The results of each group are expressed as Mean±SE. Figure 3 As shown in Table 1.

[0096] Table 1 Evaluation of the positive rate of thyroid function related indicators in each group of mice

[0097]

[0098] Control, control group; Model, model group; 1000μg, high-dose tolerance group; 500μg, medium-dose tolerance group; 100μg, low-dose tolerance group; incidence of hyperthyroidism, serum TT4 level higher than the normal range; TRAb positivity rate, serum TRAb positive; thyroid tissue hyperplasia positivity rate, thyroid follicular epithelial cell hyperplasia.

[0099] Thyroid function test, i.e. serum TT4 result ( Figure 3 A, Table 1) showed that the serum TT4 level of mice in the model group was 143.101±9.755μg / L, of which 1 / 21 mice had serum TT4 levels within the normal range; the serum TT4 level of mice in the high-dose tolerance group, i.e., the 1000μg oral group, was 6.542±13.774μg / L, which was significantly lower than that in the model group (P<0.01), of which 6 / 16 mice had serum TT4 levels within the normal range; the serum TT4 level of mice in the medium-dose tolerance group, i.e., the 500μg oral group, was 155.479±25.051μg / L, and the serum TT4 level of mice in the low-dose tolerance group, i.e., the 100μg oral group, was 141.999±14.334μg / L, and there was no statistical difference between the two groups and the model group, of which 3 / 11 mice in the medium-dose group had serum TT4 levels within the normal range, and 2 / 17 mice in the low-dose group had serum TT4 levels within the normal range.

[0100] Serum TRAb results ( Figure 3 (B, Table 1) showed that the serum TRAb level of mice in the model group was 197.253±20.837U / L, and all the mice were positive for serum TRAb; the serum TRAb level of mice in the high-dose tolerance group was 122.734±17.574U / L, which was lower than that of mice in the model group (P<0.05), and one mouse was negative for serum TRAb; the serum TRAb level of mice in the medium-dose tolerance group was 171.661±19.673U / L, and the serum TRAb level of mice in the low-dose tolerance group was 194.133±20.837U / L, and there was no statistical difference between the two groups and the model group.

[0101] Thyroid tissue pathological HE staining light microscopy results ( Figure 4, Table 1) showed that the thyroid follicular epithelial cells of the mice in the model group significantly proliferated, showing columnar or cubic shapes, forming papillary structures; in the high-dose tolerance group, 8 / 16 mice had no hyperplasia of the thyroid follicular epithelial cells, similar to the control group, and 8 / 16 mice had mild hyperplasia of the thyroid follicular epithelial cells, but no papillary structures; in the medium-dose tolerance group, the thyroid follicular epithelial cells of the mice proliferated, showing low columnar shapes, with papillary structures formed, but the degree of hyperplasia was slightly lighter than that of the model group; in the low-dose tolerance group, the thyroid follicular epithelial cells of the mice proliferated significantly, showing columnar shapes, with papillary structures formed, and the degree of hyperplasia was similar to that of the model group.

[0102] Example 4 Increased proportion of spleen Tregs cells in mice in the high-dose tolerance group

[0103] To evaluate the relationship between Tregs and immune tolerance induction, this study used flow cytometry to detect the proportion of CD25+FoxP3+ cell subsets in the CD4+ monocyte population in mouse spleen, and the values ​​were expressed as Mean±SE. Figure 5 , 6 ) showed that the proportion of splenic Tregs cells in the control group was 10.22±0.41%, and the proportion of splenic Tregs cells in the model group was 9.66±0.31%; the proportion of splenic Tregs cells in the high-dose tolerance group was 11.63±0.27%, which was higher than that in the control group (P<0.05) and the model group (P<0.01); the proportion of splenic Tregs cells in the medium-dose tolerance group was 10.04±0.31%, and the proportion of splenic Tregs cells in the low-dose tolerance group was 10.03±0.51%, and there was no statistical difference between these two groups and the control group and the model group.

[0104] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention to be protected.

Claims

1. Use of human TSHR A subunit in the preparation of an oral drug for preventing Graves' disease, characterized in that: The human TSHRA subunit is a recombinant human TSHR A subunit purified by yeast expression system. The dosage of the recombinant human TSHR A subunit is 1000 μg. The oral drug is administered during the neonatal period.

2. A method for inducing an animal model of de novo oral tolerance to Graves' disease using human TSHR A subunit, characterized in that: The method comprises the following steps: S1. Within 24 hours after birth, a solution containing 1000 μg of human TSHR A subunit is injected into the pharynx of the animal in batches, and the upper palate of the animal is stimulated to induce swallowing reflex. The human TSHR A subunit is a recombinant human TSHR A subunit purified by yeast expression system; S2. The animals injected with the recombinant human TSHR A subunit are placed back into the breeding space, and breastfeeding is continued. After weaning, the animals are continued to be raised until they reach adulthood.

3. The method according to claim 2, characterized in that The animal is a mouse.

4. Use of the animal model induced by the method according to any one of claims 2-3 in the study of oral immune tolerance of Graves' disease.

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