Preparation method and application of a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein

Abstract

The present invention discloses a preparation method and application of a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. The present invention belongs to the field of biomedical technology. The preparation method includes the following steps: cloning the coding sequences of the extracellular region of BAFF-R3 and the adiponectin globular domain; connecting the extracellular region of BAFF-R3 and the adiponectin globular domain through a peptide chain, and inserting the expression sequence of the eBR3-gAD fusion protein into a plasmid using restriction enzymes and DNA ligase; transfecting the above plasmid into cells and screening out a cell line with stable expression; culturing the above cell line, collecting the supernatant, and purifying by gel filtration and chromatography to obtain the soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. The preparation method of this novel fusion protein has the advantages of high specificity, high expression level, and simple purification.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a preparation method and application of a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic autoimmune disease that affects multiple joints. In China, the incidence rate of RA is approximately 0.28%, and the disability rate caused by RA is as high as 48%, imposing a serious burden on patients' families and society. The pathogenesis of RA is closely related to the combined action of environmental factors and genetic factors, resulting in the disruption of the immune tolerance balance in the body, abnormal activation of immune cells such as T cells, B cells, macrophages, and dendritic cells, production of various pro-inflammatory cytokines, matrix degrading enzymes, and autoantibodies against autoantigens, ultimately leading to the destruction of bone and cartilage. Research shows that compared with bone tissue destruction, joint cartilage tissue damage has a more direct relationship with disability and work ability impairment caused by RA. Currently, the clinical treatment of RA mainly includes non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatic drugs, corticosteroids, and other cell-targeted therapies, including T, B, and monocyte / macrophage-targeted therapies. These treatment methods are mainly used to reduce joint inflammation, slow down the disease progression rate, and maintain joint function as much as possible. Joint cartilage destruction is one of the main factors leading to disability and work ability impairment in RA patients, but the mechanism of RA cartilage destruction is still unclear, and current drug treatments cannot fundamentally reverse cartilage destruction caused by RA. Therefore, further exploration of the key mechanisms mediating RA cartilage injury will provide a theoretical basis for the development of new, highly targeted, safe, and effective therapeutic drugs. Summary of the Invention

[0003] In order to overcome the above-mentioned drawbacks of the prior art, the present invention provides a preparation method and application of a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein.

[0004] A drug, comprising a soluble B lymphocyte stimulator receptor 3 and an adiponectin globular domain fusion protein; the sequence of the fusion protein includes one or more of SEQ ID No.1 or SEQ ID No.2.

[0005] Use of a soluble B lymphocyte stimulator receptor 3 and an adiponectin globular domain fusion protein in the preparation of a drug for treating rheumatoid arthritis; the sequence of the fusion protein includes one or more of SEQ ID No.1 or SEQ ID No.2.

[0006] Use of soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein in the preparation of drugs for preventing rheumatoid arthritis; the sequence of the fusion protein includes one or more of SEQ ID No.1 or SEQ ID No.2.

[0007] Preferably, the adiponectin includes a protein hormone.

[0008] Preferably, the adiponectin is derived from adipose tissue.

[0009] A method for preparing a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein, comprising the following steps:

[0010] Clone the extracellular region coding sequence SEQ ID No.3 of BAFF-R3 and the adiponectin globular domain coding sequence SEQ ID No.4;

[0011] Connect the extracellular region of BAFF-R3 and the adiponectin globular domain through a peptide chain, and insert the eBR3-gAD fusion protein expression sequence into a plasmid using restriction enzymes and DNA ligase;

[0012] Transfect the above plasmid into cells, and screen out a cell line with stable expression;

[0013] Culture the above cell line, collect the supernatant, perform gel filtration, and purify by chromatography to obtain a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein.

[0014] Preferably, the restriction enzymes include Xhol, Xbal and EcoRV.

[0015] Preferably, the plasmid is pET28a(+)-His.

[0016] Preferably, the cells transfected with the plasmid include one or more of CHO cells or HEK293 cells.

[0017] The beneficial effects of the present invention are as follows:

[0018] The rigidly linked eBR3-gAD fusion protein and the flexibly linked eBR3-gAD fusion protein of the present invention significantly reduce the arthritis score and swelling degree of AA rats, improve the arthritis symptoms and bone destruction degree of AA rats, and have a good effect in treating rheumatoid arthritis. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 Modeling of eBR3-gAD fusion protein and prediction of its binding energy with BAFF.

[0021] Figure 2 Schematic diagram and restriction digestion results of eBR3-gAD-pET28a(+)-His recombinant plasmid.

[0022] Figure 3 Results of identification of eBR3-gAD fusion protein expression.

[0023] Figure 4 Results of SDS-PAGE of affinity purification of eBR3-gAD fusion protein.

[0024] Figure 5 Results of SDS-PAGE of dialysis of eBR3-gAD fusion protein.

[0025] Figure 6 Effect of eBR3-gAD on arthritis swelling in AA rats.

[0026] Figure 7 Effect of eBR3-gAD on arthritis score and number of swollen joints in AA rats.

[0027] Figure 8 Effect of eBR3-gAD on degree of bone destruction in AA rats.

[0028] Figure 9 Effect of eBR3-gAD on joint pathological damage in AA rats.

[0029] Figure 10 Effect of eBR3-gAD on organs of AA rats. Specific implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] Example 1: Modeling of eBR3-gAD fusion protein and prediction of its binding energy with BAFF

[0032] The following coding sequences were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ):

[0033] Coding sequence of the extracellular part of BAFFR SEQ ID No.3:

[0034] MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLL.

[0035] Coding sequence of the globular domain of adiponectin SEQ ID No.4:

[0036] AYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYF

[0037] AYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQ VWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN.

[0038] Successfully constructed the eBR3-gAD fusion protein (rigidly linked with EAAAK2) using Alphafold v2.3.1 ( Figure 1 A) and the eBR3-gAD fusion protein (flexibly linked with GGGGS) model ( Figure 1 B). Further, Pymol and PRODIGY were used to analyze and predict the binding energy of the multimer formed between the predicted eBR3-gAD fusion protein and BAFF protein. It was found that the binding constant of the eBR3-gAD fusion protein (rigidly linked with EAAAK2) and BAFF protein was 6.9e-10, and the binding constant of the eBR3-gAD fusion protein (flexibly linked with GGGGS) and BAFF protein was 9.2e-10, both belonging to strong binding.

[0039] Example 2: Design and restriction enzyme digestion results of the eBR3-gAD recombinant plasmid

[0040] The eBR3-gAD fusion protein sequence was inserted into the pET28a(+)-His plasmid using the restriction enzymes Xhol, Xbal, EcoRV and DNA ligase, and the restriction enzyme digestion effect was detected by gel electrophoresis. The expression sequences of the eBR3-gAD fusion protein (flexibly linked with GGGGS) and the eBR3-gAD fusion protein (rigidly linked with EAAAK2) are as follows:

[0041] (1) The expression sequence of the eBR3-gAD fusion protein (flexible linker GGGGS) is SEQ ID No.1: MHHHHHHAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTNGGGGSMRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLL.

[0042] (2) The expression sequence of the eBR3-gAD fusion protein (rigid linker EAAAK2) is SEQ ID No.2: MHHHHHHAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTNEAAAKEAAAKMRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLL.

[0043] The digestion result showed DNA fragments of the expected size were observed ( Figure 2 )

[0044] Example 3: Identification of the expression of the eBR3-gAD fusion protein

[0045] Select monoclonal colonies and culture them in 5 tubes of LB medium at 37°C until the OD600 of the bacteria reaches 0.6 - 0.8. Add IPTG to a final concentration of 0.5 mM, culture at 37°C for 4 h, then centrifuge to collect the bacteria and prepare samples for SDS-PAGE and Western Blot analysis. Add IPTG to a final concentration of 0.2 mM and 1 mM respectively to 4 tubes of the culture after inoculating and culturing the conserved bacteria until the OD600 of the bacteria reaches 0.6 - 0.8, and culture at 37°C and 15°C at 220 rpm for 4 h and 16 h respectively to induce the expression of the fusion protein. Prepare samples for SDS-PAGE analysis under each condition. Take the bacterial liquid under each condition in the previous step, centrifuge to collect the bacteria, break them (Tris-NaCl buffer), and prepare samples for the supernatant and precipitate respectively for SDS-PAGE analysis. The results of the identification of the expression of the eBR3-gAD fusion protein show that after induction of the samples, the eBR3-gAD fusion protein (flexible linker GGGGS) can be observed at 26 KD ( Figure 3 A) and the eBR3-gAD fusion protein (rigid linker EAAAK2) ( Figure 3 B) were expressed.

[0046] Example 4: Affinity purification of eBR3-gAD fusion protein

[0047] The optimal expression clone strain was expanded and cultured in 2 L of LB medium at 37°C until OD600 = 0.6 - 0.8, and the bacteria were collected after induction at 15°C for 16 h; the bacteria were collected by centrifugation, resuspended, and ultrasonically broken; the optimal expression clone strain was expanded and cultured in 2 L of LB medium at 37°C until OD600 = 0.6 - 0.8, and the bacteria were collected after induction at 15°C for 16 h; protein purification was carried out using a HisTrap protein purification column equipped with Ni 2+ . The equilibration buffer: PBS (pH 7.4), the washing buffer: PBS (pH 7.4 with 20 mM and 50 mM imidazole), the elution buffer: PBS (pH 7.4 with 500 mM imidazole), and the liquid flow rate was 1 mL / min. Take an appropriate amount of the effluent for SDS-PAGE analysis. The SDS-PAGE results of the affinity purification of the eBR3-gAD fusion protein show that the eBR3-gAD fusion protein (flexible linker GGGGS) can be observed at 26 KD ( Figure 4 A) and the eBR3-gAD fusion protein (rigid linker EAAAK2) ( Figure 4 B) were expressed, and the protein was soluble.

[0048] Example 5: Concentration of eBR3-gAD fusion protein

[0049] The eluted sample was added to an ultrafiltration tube (retaining 10KD proteins) and centrifugally concentrated (4°C, 3500×g, 20 min), filtered through bacteria, and stored at -80°C. The SDS-PAGE results of the dialysis of the eBR3-gAD fusion protein showed that, detected by SDS-PAGE, the affinity purification could obtain the eBR3-gAD fusion protein (flexible linker GGGGS)( Figure 5 A) and the eBR3-gAD fusion protein (rigid linker EAAAK2)( Figure 5 B), and the purity of the protein product was >90%.

[0050] Comparative Example 6:

[0051] Experimental method: After male SD rats (180±20 g) were adaptively fed for one week, they were randomly divided into: a normal group and an AA control group, with 7 rats in each group. The BCG vaccine was inactivated at 80°C and then added to Freund's incomplete adjuvant and fully emulsified and mixed to prepare Freund's complete adjuvant (10 mg / ml). Each rat in the AA control group was subcutaneously injected with 0.1 mL of Freund's complete adjuvant on the left plantar surface to induce arthritis and construct AA rats.

[0052] Example 6: Effect of eBR3-gAD on the arthritis manifestation of AA rats

[0053] Experimental method: After male SD rats (180±20 g) were adaptively fed for one week, they were randomly divided into: an eBR3-gAD-F treatment group, an eBR3-gAD-R treatment group, and a methotrexate (MTX) treatment group, with 7 rats in each group. The BCG vaccine was inactivated at 80°C and then added to Freund's incomplete adjuvant and fully emulsified and mixed to prepare Freund's complete adjuvant (10 mg / ml). Each rat in the treatment group was subcutaneously injected with 0.1 mL of Freund's complete adjuvant on the left plantar surface to induce arthritis and construct AA rats. On the 11th, 14th, 17th, 20th, 23rd, and 26th days after the AA rats in the control group were modeled, the rats in the eBR3-gAD fusion protein treatment group were treated by intra-articular injection with eBR3-gAD-F or eBR3-gAD-R, and the rats in the positive control group were treated by gavage with 0.75 mg / kg MTX. On the 29th day after administration, the secondary joints of the AA rats were photographed, and the arthritis symptoms were macroscopically observed and images were taken.

[0054] Experimental results: The typical image results of the secondary joints of AA rats are shown in Figure 6 . Compared with the normal group, the joints of the rats in the AA model group were significantly swollen, while compared with the rats in the AA control group, the joint swelling of the rats in the eBR3-gAD-F, eBR3-gAD-R, and MTX treatment groups was significantly improved.

[0055] Comparative Example 7:

[0056] Experimental method: After male SD rats (180±20 g) were adaptively fed for one week, they were randomly divided into a normal group and an AA control group, with 7 rats in each group. The BCG vaccine was inactivated at 80 °C and then added to Freund's incomplete adjuvant and fully emulsified and mixed to prepare Freund's complete adjuvant (10 mg / ml). Each rat in the control group was subcutaneously injected with 0.1 mL of Freund's complete adjuvant into the left plantar surface to induce arthritis and construct AA rats.

[0057] Example 7: Effects of eBR3-gAD on arthritis index and number of swollen joints in AA rats

[0058] Experimental method: The AA rats were modeled and grouped in the same way as in Example 6. On the 9th, 12th, 15th, 18th, 21st, 23rd, 25th, 27th, and 29th days after the AA rats were modeled, the arthritis index score and the number of swollen joints score were performed every 2 - 3 days to observe the secondary lesions of each group of rats. The scoring criteria for the arthritis index score and the number of swollen joints are as follows:

[0059] (1) Scoring criteria for arthritis index: Normal without redness and swelling is 0 point; Erythema and slight swelling of the ankle joint is 1 point; Erythema and slight swelling from the ankle joint to the metatarsophalangeal joint or metacarpophalangeal joint is 2 points; Erythema and moderate swelling from the ankle joint to the metatarsophalangeal joint or metacarpophalangeal joint is 3 points; Erythema and severe swelling from the ankle joint to the toe joint is 4 points.

[0060] (2) Scoring criteria for the number of swollen joints: No swelling is 0 point; Slight swelling of the toe joint is 1 point; Swelling of the little toe joint and toes is 2 point; Swelling of the paw below the ankle joint is 3 points; Swelling of the entire paw including the ankle joint is 4 points.

[0061] Experimental results: The results are shown in Figure 7 , at different time points, eBR3-gAD-F, eBR3-gAD-R, and MTX treatments reduced the arthritis index and the number of swollen joints in AA rats. Compared with the AA model group rats on the 29th day, the arthritis index and the number of swollen joints in the eBR3-gAD-F treatment group rats were significantly reduced (P < 0.01 or P < 0.001), and the arthritis index and the number of swollen joints in the eBR3-gAD-R and MTX treatment group rats were also significantly reduced (P < 0.05 or P < 0.01). The above results indicate that: Intra-articular injection of the eBR3-gAD fusion protein can significantly improve the arthritis index and the number of swollen joints in AA rats, that is, effectively relieve the symptoms of secondary joint lesions in AA rats.

[0062] Comparative Example 8:

[0063] Experimental method: After male SD rats (180±20 g) were adaptively fed for one week, they were randomly divided into a normal group and an AA control group, with 7 rats in each group. The BCG vaccine was inactivated at 80 °C and then added to Freund's incomplete adjuvant and fully emulsified and mixed to prepare Freund's complete adjuvant (10 mg / ml). 0.1 mL of Freund's complete adjuvant was subcutaneously injected into the left plantar surface of each rat in the control group to induce arthritis and construct AA rats.

[0064] Example 8: Effect of eBR3-gAD on the degree of bone destruction in AA rats

[0065] Experimental method: The AA rats were modeled and grouped in the same way as in Example 6, and Micro-CT was used to take pictures of the bones of the secondary lateral ankle joints of the rats.

[0066] (1) μCT scanning

[0067] After all the sampled specimens were fixed, Micro-CT scanning was performed. The scanning parameters were as follows: tube current 200 μA, voltage 85 KV, scanning the entire object, scanning resolution 10.133799 μm, exposure time 384 ms, scanning angle 180 degrees. The phantom was scanned under the same conditions for calibration. The original images were obtained after scanning.

[0068] (2) μCT reconstruction

[0069] The three-dimensional reconstruction software Bruker SkyScan was used to reconstruct the selected areas of the original images. Before reconstruction, the reconstructed images were previewed first. In order to reduce image artifacts and achieve the best reconstruction effect, the reconstruction parameters were adjusted and set to the following values: Smoothing = 3 (smoothness), Beam-hardening = 5 (light intensity), Ring artifacts = 30% (annular tail shadow), etc. After the parameters were set, the specified folder was edited and selected to start image reconstruction.

[0070] Experimental results: See details in Figure 8, compared with the normal group, the ankle joints of the rats in the AA control group were significantly swollen, the bones of the joints were severely eroded, and the bone mass was damaged; compared with the AA control group, the treatments with eBR3-gAD-F, eBR3-gAD-R, and MTX could effectively eliminate the swelling of the ankle joints and inhibit bone erosion and bone mass damage. Further analysis of the percent bone volume (BV / TV), bone surface / volume ratio (BS / BV), bone surface density (BS / TV), trabecular pattern factor (Tb.Pf), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) found that, compared with the normal group, the percent bone volume (BV / TV), bone surface density (BS / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) of the rats in the AA control group were significantly decreased (P < 0.05 or P < 0.001), while the bone surface / volume ratio (BS / BV), trabecular pattern factor (Tb.Pf), and trabecular separation (Tb.Sp) were significantly increased (P < 0.001); compared with the AA control group, the treatments with eBR3-gAD-F, eBR3-gAD-R, and MTX could significantly increase the percent bone volume (BV / TV), bone surface density (BS / TV), and trabecular number (Tb.N) (P < 0.001), significantly decrease the bone surface / volume ratio (BS / BV), trabecular pattern factor (Tb.Pf), and trabecular separation (Tb.Sp) (P < 0.001), and the treatments with eBR3-gAD-F and MTX could significantly increase the trabecular thickness (Tb.Th) (P < 0.05 or P < 0.001). The above results indicate that the eBR3-gAD fusion protein can effectively reduce the degree of bone damage in AA rats.

[0071] Comparative Example 9:

[0072] Experimental method: After one week of adaptive feeding of male SD rats (180 ± 20 g), they were randomly divided into a normal group and an AA control group, with 7 rats in each group. The BCG vaccine was inactivated at 80 °C and then added to Freund's incomplete adjuvant and fully emulsified and mixed to prepare Freund's complete adjuvant (10 mg / ml). Each rat in the control group was subcutaneously injected with 0.1 mL of Freund's complete adjuvant on the left plantar surface to induce arthritis and construct AA rats.

[0073] Example 9: Effect of eBR3-gAD on the joint pathological damage of AA rats

[0074] Experimental method: The AA rats were modeled and grouped in the same way as in Example 6. On the 29th day, the rats were anesthetized and the ankle joints were collected. After being fixed with 4% paraformaldehyde for 48 h, they were decalcified with EDTA for 4 weeks, and finally embedded in paraffin and sectioned. The paraffin sections of the ankle joints were stained with hematoxylin-eosin (H&E) by trained observers in a blind manner.

[0075] Steps of H&E staining: After baking the paraffin sections at 56 °C for 2 h, first dewax and hydrate them, that is, remove the paraffin in the sections through xylene, then through alcohol from high concentration to low concentration, and finally into distilled water; then perform H&E staining. The sections that have been put into distilled water are stained in hematoxylin solution for 10 min, differentiated with 1% hydrochloric acid alcohol for 10 s, rinsed with running water for 15 min, then stained with eosin for 2 min, and rinsed with running water 3 times; then dehydrate and clarify, repeat the dehydration treatment with 90% alcohol 2 times, 1 min each; repeat the dehydration treatment with 95% alcohol 2 times, 1 min each; repeat the dehydration treatment with 100% alcohol 2 times, 1 min each; clarify with xylene for 3 min, repeat 3 times; finally, seal with gum, air dry and observe under a microscope.

[0076] The results of H&E staining were pathologically scored from five aspects: synoviocyte hyperplasia, inflammation, pannus, bone erosion, and cell erosion. See Table 1 for details.

[0077] Table 1 Ankle joint pathological scoring table

[0078]

[0079]

[0080] Experimental results: The pathological results of the ankle joint tissues of rats detected by H&E staining are as Figure 10 shown. In the normal group, there were no any inflammations and cartilage damages in the ankle joints of rats. In AA rats, the excessive proliferation of synoviocytes led to the stenosis of the joint cavity, a large number of immune cells eroded into the joint capsule, accompanied by a large area of pannus formation sites, obvious cartilage erosion and a large number of leukocyte aggregations. However, the treatments with eBR3-gAD-F and eBR3-gAD-R could significantly inhibit the abnormal proliferation of synoviocytes, joint tissue inflammation and inflammatory cell infiltration, pannus formation and damage to bone tissue. Through pathological scoring, it was found that compared with the AA group, the treatments with eBR3-gAD-F and eBR3-gAD-R could significantly inhibit synovial hyperplasia, inflammation, pannus, bone erosion and cell erosion (P < 0.01 or P < 0.001). The above results suggest that intra-articular injection of eBR3-gAD fusion protein can significantly inhibit the abnormal proliferation of synovial fibroblasts, reduce bone destruction, and effectively relieve the condition of AA rats.

[0081] Comparative Example 10:

[0082] Experimental method: After male SD rats (180±20 g) were adaptively fed for one week, they were randomly divided into a normal group and an AA control group, with 7 rats in each group. The BCG vaccine was inactivated at 80 °C and then added to Freund's incomplete adjuvant and emulsified and mixed thoroughly to prepare Freund's complete adjuvant (10 mg / ml). 0.1 mL of Freund's complete adjuvant was subcutaneously injected into the left plantar surface of each rat in the control group to induce arthritis and construct AA rats.

[0083] Example 10: Effects of eBR3-gAD on organs of AA rats

[0084] Experimental method: The AA rats were modeled and grouped in the same way as in Example 6. After the experiment, the rats in each group were sacrificed, and the heart, liver, spleen, lung, and kidney tissues were gently removed with ophthalmic scissors. Each organ was fixed with 4% paraformaldehyde for 48 h, and then paraffin-embedded and sectioned. The paraffin sections of each organ were stained with hematoxylin-eosin (H&E) by trained observers in a blind method, and typical images were taken. The specific steps are shown in Example 9.

[0085] Experimental results: The results are as Figure 5 shown. Intra-articular injection of eBR3-gAD-F and eBR3-gAD-R had no obvious effects on the heart, liver, spleen, lung, kidney and other organs of AA rats.

[0086] In summary, the above results show that intra-articular injection of the eBR3-gAD fusion protein can significantly improve the arthritis index, joint swelling number, arthritis symptoms and bone destruction degree of AA rats, and has no obvious effect on other organs, and it is a potential drug for the treatment of rheumatoid arthritis.

[0087] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0088] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A drug, characterized in that The drug consists of soluble B lymphocyte stimulating factor receptor 3 and adiponectin globular fusion protein; the sequence of the fusion protein is SEQ ID No.1 or SEQ ID No.2; and the drug is used for treating rheumatoid arthritis.

2. Use of soluble B lymphocyte stimulator receptor 3 and adiponectin globular fusion protein in the preparation of a drug for treating rheumatoid arthritis; the sequence of the fusion protein is SEQ ID No.1 or SEQ ID No.

2.

3. Use of soluble B lymphocyte stimulator receptor 3 and adiponectin globular fusion protein in the preparation of a drug for preventing rheumatoid arthritis; the sequence of the fusion protein is SEQ ID No.1 or SEQ ID No.2.

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