Non-naturally occurring amino acid modified proteins and related biomaterials, methods of making and uses

Abstract

This invention provides a non-natural amino acid-modified protein and related biomaterials, preparation methods, and applications. The non-natural amino acid-modified protein provided by this invention is obtained by modifying a soluble BAFF variant protein with p-nitrophenylalanine; the modification site of the p-nitrophenylalanine is located at at least one of positions 91, 100, 139, or 158 in the soluble BAFF variant protein. This invention, by introducing non-natural amino acids at specific sites in the soluble BAFF variant protein, controls the alteration of the protein's antigenic structure, thereby effectively breaking the body's immune tolerance to endogenous BAFF, inducing a humoral immune response against BAFF, and achieving a neutralizing effect on endogenous BAFF. This provides a new technical approach for the prevention and treatment of related autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a non-natural amino acid-modified protein and related biomaterials, preparation methods and applications. Background Technology

[0002] B cell activating factor (BAFF) is a member of the tumor necrosis factor superfamily and plays a crucial regulatory role in maintaining the development, maturation, and survival of peripheral B cells. Numerous studies have shown that abnormally elevated BAFF levels are closely associated with the development of various autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and collagen-induced arthritis (CIA). Elevated serum BAFF levels in patients are positively correlated with disease activity; its persistent overexpression can promote autoantibody production, the formation of abnormal germinal centers, and B cell differentiation imbalance. Therefore, BAFF is widely recognized as an important molecule regulating humoral immune homeostasis and a key target for the treatment of autoimmune diseases.

[0003] Currently, passive immunotherapy drugs targeting BAFF have been approved for clinical treatment, such as the anti-BAFF monoclonal antibody belimumab and the soluble receptor fusion protein atacicept that binds to BAFF / APRIL. These drugs can neutralize circulating BAFF and inhibit its mediated B cell signaling pathways, thereby reducing autoantibody production and improving disease symptoms. However, passive immunotherapy generally suffers from problems such as the need for long-term, continuous administration, high drug costs, strong treatment dependence, and the development of drug resistance or immunogenicity due to exogenous proteins. Furthermore, since BAFF plays an irreplaceable role in maintaining physiological B cell homeostasis, its long-term suppression is often accompanied by varying degrees of immunosuppression, which may increase the risk of infection and affect the body's immune surveillance function. Therefore, developing an active immunization strategy that can induce a durable immune response, is less costly, and has a higher safety profile has become a research focus.

[0004] In recent years, active immunization targeting cytokines in the body has become a new research direction. Related studies have shown that inducing the body to produce neutralizing antibodies against its own cytokines through vaccination can achieve relatively long-term therapeutic effects without repeated drug administration. However, unlike pathogen-associated antigens, autoantigens are strictly controlled by central and peripheral immune tolerance mechanisms, making it difficult to induce effective autoantigen-specific humoral immune responses in their natural state, greatly limiting the application of active immunization strategies in the field of autoimmune diseases. Existing attempts include enhancing antigen immunogenicity through the fusion of immune-enhancing fragments, chemical modification, or epitope optimization, but significant controversies remain regarding specificity, neutralizing capacity, and safety. In recent years, with the advent of genetic code extension technology, it has become possible to modify proteins with non-natural amino acids to disrupt their immune tolerance. Related literature shows that introducing non-natural amino acids with significantly different chemical structures at specific sites on proteins can alter antigen presentation and generate new epitopes, thereby inducing cross-reactive antibodies against natural proteins. While this strategy has shown potential to induce autoimmune responses in some models, its application in key immunomodulatory molecules such as BAFF remains limited, particularly lacking studies that systematically validate its efficacy and safety in disease models such as autoimmune arthritis. Therefore, new technological solutions are needed to effectively break BAFF immune tolerance, achieve controllable neutralization of endogenous BAFF, and exert therapeutic effects on autoimmune diseases while maintaining immune homeostasis. Summary of the Invention

[0005] This invention provides a non-natural amino acid-modified protein and related biomaterials, preparation method and application.

[0006] In a first aspect, the present invention provides a non-naturally modified amino acid protein, said protein being any one of the following A1)-A2):

[0007] A1) A protein whose amino acid sequence is SEQ ID NO:1, positions 1-178;

[0008] A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus and / or amino terminus of the protein shown in A1);

[0009] The non-natural amino acid mentioned is p-nitrophenylalanine;

[0010] The modification site of the p-nitrophenylalanine is located at at least one of the 91st, 100th, 139th and 158th positions of the protein in A1).

[0011] As described above, for proteins with non-natural amino acid modifications, the protein tag refers to a polypeptide or protein expressed by fusing it with a target protein using in vitro DNA recombination technology, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0012] The non-natural amino acid modified protein as described above, wherein the modification site of the non-natural amino acid is located at position 91 of the protein in A1).

[0013] Secondly, the present invention provides a biomaterial, said biomaterial being any one of the following A1)-A9):

[0014] A1) Nucleic acid molecules that encode the above proteins;

[0015] A2) The nucleic acid molecule obtained by mutating the codons of the amino acids corresponding to the modification sites of the protein described in A1) to TAG;

[0016] A3) An expression cassette containing the nucleic acid molecules described in A1) or A2);

[0017] A4) A recombinant vector containing the nucleic acid molecules described in A1) or A2);

[0018] A5) A recombinant vector containing the expression cassette described in A3);

[0019] A6) Recombinant microorganisms containing the nucleic acid molecules described in A1) or A2);

[0020] A7) Recombinant microorganisms containing the expression cassette described in A3);

[0021] A8) Recombinant microorganisms containing the recombinant vector described in A4);

[0022] A9) Recombinant microorganisms containing the recombinant vector described in A5).

[0023] In the biological material described above, the nucleic acid molecule described in A1) is selected from one of B1)-B2):

[0024] B11) The nucleotide sequence is the DNA molecule consisting of positions 1-534 of SEQ ID NO:2;

[0025] B12) The nucleotide sequence is the DNA molecule of SEQ ID NO:2;

[0026] DNA molecules defined by B13 (B11) or B12 (B12) that share more than 80% identity with each other and encode the same protein are DNA molecules.

[0027] In the biological materials described above, the 80% or more of identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0028] The nucleic acid molecule described in A2) of the above-mentioned biological material is specifically selected from one of B14)-B17):

[0029] B14) The nucleic acid molecule obtained by mutating nucleotides (TAT) at positions 271-273 in the nucleic acid molecule shown in B11) to TAG;

[0030] B15) The nucleic acid molecule obtained by mutating nucleotides (TAC) at positions 298-300 in the nucleic acid molecule shown in B11) to TAG;

[0031] B16) The nucleic acid molecule obtained by mutating nucleotides (TAC) at positions 415-417 in the nucleic acid molecule shown in B11) to TAG;

[0032] B17) The nucleic acid molecule obtained by mutating nucleotides (CGG) at positions 472-474 in the nucleic acid molecule shown in B11) to TAG.

[0033] As described above, the recombinant vector described in A3)-A4) can be constructed based on existing vectors to contain the protein-coding gene. Further, the recombinant vector is named pET21-DmBAFF, which is obtained by inserting the aforementioned protein-coding gene between the NdeI and XhoI sites of the pET-28a(+) vector.

[0034] The recombinant microorganisms described in the above-mentioned biological materials (A5)-A8) can specifically be at least one of yeast, bacteria, algae, and fungi. Further, the recombinant microorganism is *Escherichia coli*.

[0035] Thirdly, the present invention provides a method for preparing a protein with non-natural amino acid modification, comprising:

[0036] A recombinant vector expressing the protein was constructed, and the codons corresponding to the modified sites in the recombinant vector were replaced with TAGs to obtain the mutated recombinant vector;

[0037] The mutated recombinant vector and a plasmid containing genes encoding orthogonal-tRNA and orthogonal-tRNA synthetase were simultaneously transferred into a host microorganism to obtain a recombinant microorganism.

[0038] The recombinant microorganisms were cultured in a medium containing p-nitrophenylalanine to obtain proteins with non-natural amino acid modifications.

[0039] Thirdly, the present invention provides a pharmaceutical composition comprising any of the above-described non-natural amino acid-modified proteins and a pharmaceutically acceptable carrier.

[0040] In the pharmaceutical composition described above, the carrier includes at least one of an excipient, a buffer solution, and an immune adjuvant.

[0041] The dosage form of the pharmaceutical composition described above can be determined according to actual needs, and may be selected from, for example, injections, tablets, capsules, lozenges, oral liquids, granules, powders, pills, powders, ointments, syrups, mixtures, elixirs, effervescent tablets, pastes, emulsions, teas, pills, suspensions, powders, implants, ointments, plasters, creams, sprays, drops, and patches. Further, the pharmaceutical composition is an injection. Injection sites include, but are not limited to, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and spinal injection.

[0042] Fourthly, the present invention provides the use of the above-mentioned non-natural amino acid-modified protein, the above-mentioned biological material, or the above-mentioned pharmaceutical composition, wherein the use is selected from at least one of C1)-C2):

[0043] C1) Use in the preparation of medicines to alleviate and / or treat BAFF-related autoimmune diseases;

[0044] C2) Application in the preparation of drugs that modulate the immune response of subjects to BAFF.

[0045] In the application described above, the autoimmune disease is at least one of rheumatoid arthritis, collagen-induced arthritis, systemic lupus erythematosus, and Sjögren's syndrome.

[0046] As described above, the autoimmune disease is collagen-induced arthritis.

[0047] Fifthly, the present invention provides a method for alleviating and / or treating BAFF-related autoimmune diseases, comprising administering to a subject the aforementioned protein containing non-natural amino acid modifications or the aforementioned pharmaceutical composition.

[0048] As described above, the dosage of the drug used to alleviate and / or treat BAFF-related autoimmune diseases in the subject is variable, depending on the method of administration, route of administration, individual age and / or weight, and the individual's condition, and is ultimately determined by the attending physician. The dosage administered to the individual, in the case of this invention, should be sufficient for a period of time to elicit a beneficial response in the individual.

[0049] In the method described above, the subject can be a mammal, which can be selected from bovine, equine, feline, canine, lagomorph, suidae, camel, rodent, and primate animals, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, and orangutans), and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys, and humans.

[0050] This invention introduces the non-natural amino acid p-nitrophenylalanine into a specific site of a soluble BAFF variant protein, thereby controllingly altering the protein's antigenic structure and effectively breaking the body's immune tolerance to endogenous BAFF. Compared to natural BAFF or unmodified BAFF variants, the non-natural amino acid-modified protein provided by this invention can more stably expose or form new helper T cell epitopes, enabling the body to produce antibodies that specifically recognize endogenous BAFF. Therefore, when used as an immunogen, this variant protein can induce a humoral immune response against BAFF and neutralize endogenous BAFF.

[0051] The non-natural amino acid modification strategy provided by this invention can maintain the overall stability of the protein structure while enabling modifications at different sites to produce differentiated immunogenicity. This allows the resulting antibodies to recognize different natural epitopes of BAFF, achieving a broader or more targeted immune response. When this protein is prepared into a vaccine composition, it can be used to regulate BAFF-related signaling pathways in the body, effectively controlling immune responses associated with BAFF overexpression or dysfunction. The vaccine composition of this invention has shown effects in animal models of reducing inflammatory responses, inhibiting disease progression, and reducing tissue damage, while no significant adverse effects on peripheral immune cell composition were observed, demonstrating a good safety profile. In summary, this invention achieves controllable neutralization of endogenous BAFF, providing a new technical approach for the prevention and treatment of related autoimmune diseases. Attached Figure Description

[0052] Figure 1This diagram illustrates the construction of DmBAFF and the pNO2-Phe introduction site. A shows agarose gel electrophoresis images of the soluble fragment of wild-type mouse BAFF (WT-mBAFF) and the mutant BAFF protein (DmBAFF). B shows agarose gel electrophoresis images of DmBAFF before and after pNO2-Phe modification, obtained by culturing recombinant bacteria on media containing or without pNO2-Phe. - indicates DmBAFF without pNO2-Phe modification, + indicates DmBAFF modified with pNO2-Phe, and 158, 139, 100, and 91 represent different protein variants.

[0053] Figure 2 Mass spectrometry results of 158-DmBAFF protein after modification of amino acid 158 of DmBAFF;

[0054] Figure 3 The titer of anti-BAFF antibody pNO2-Phe-DmBAFF in mice is shown in Figure 1. A represents the antibody titer detection results at different time points; B is a bar chart of antibody titers at 3 weeks.

[0055] Figure 4 The table shows the arthritis scores, incidence rates, and imaging results in the CIA mouse model; where A represents the disease symptom scores of each group of mice, B represents the disease incidence rates of each group of mice, and C represents the imaging results of the paws of each group.

[0056] Figures 3-4 In this context, ns represents p > 0.05, * represents p ≤ 0.05, ** represents p ≤ 0.01, and *** represents p ≤ 0.001. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Example 1: Preparation of pNO2-Phe-DmBAFF

[0060] 1. The soluble fragment sequence of mouse BAFF was modified by replacing two glycines in the amino acid region from position 115 to 122 to obtain the inactivated mutant DmBAFF. The amino acid sequence of the inactivated mutant DmBAFF is SEQ ID NO:1.

[0061] SEQ ID NO:1:

[0062] MAFQGPEETEQDVDLSAPPAPCLPGCRHSQHDDNGMNLRNIIQDCLQLIADSDTPTIRKGTYTFVPWLLSFKRGNALEEKENKIVVRQTG Y FFIYSQVL Y TDPIFAMGHVIQRKKGGSLVTLFRCIQNMPKTLPNNSC Y SAGIARLEEGDEIQLAIP R ENAQISRNGDDTFFGALKLLHHHHHH.

[0063] The coding sequence of the above-mentioned inactivated mutant DmBAFF (nucleotide sequence SEQ ID NO:2) was synthesized and inserted into the NdeI and XhoI sites of the pET21-a(+) vector to construct the recombinant expression vector pET21-DmBAFF. After the recombinant expression vector was verified to be correct by sequencing, the codon (TAT) encoding tyrosine (Y) at position 91 of the target gene was mutated to the stop codon TAG using site-directed mutagenesis, resulting in the recombinant expression vector pET21-DmBAFF-91 (i.e., expressing the protein with the amino acid sequence of SEQ ID NO:1, and with p-nitrophenylalanine modified at position 91, named 91-DmBAFF). Alternatively, the codon (TAC) encoding tyrosine (Y) at position 100 of the target gene can be mutated to the stop codon TAG to obtain the recombinant expression vector pET21-DmBAFF-100 (which expresses the protein with the amino acid sequence SEQ ID NO:1, and whose amino acid position 100 is modified with p-nitrophenylalanine, named 100-DmBAFF). Alternatively, the codon (TAC) encoding tyrosine (Y) at position 139 of the target gene can be mutated to the stop codon TAG to obtain the recombinant expression vector pET21-DmBAFF-139 (which expresses the protein with the amino acid sequence SEQ ID NO:1, and whose amino acid position 139 is modified with p-nitrophenylalanine, named 139-DmBAFF). Alternatively, the codon (CGG) encoding arginine (R) at position 158 in the target gene can be mutated to the stop codon TAG to obtain the recombinant expression vector pET21-DmBAFF-158 (which expresses the protein with the amino acid sequence SEQ ID NO:1, and whose amino acid position 158 is modified with p-nitrophenylalanine, named 158-DmBAFF). pET21-DmBAFF-91, pET21-DmBAFF-100, pET21-DmBAFF-139, and pET21-DmBAFF-158 are hereinafter referred to as the series of recombinant expression vectors pET21-DmBAFF-TAG. The resulting series of recombinant expression vectors were verified to have correct mutations by sequencing.

[0064] SEQ ID NO:2:

[0065] 5'-ATGGCTTTCCAGGGACCAGAGGAAACAGAACAAGATGTAGACCTCTCAGCTCCTCCTGCACCATGCCTGCCTGGATGCCGCCATTCTCAACATGATGATAATGGAATGAACCTCAGAAACATCATTCAAGACT GTCTGCAGCTGATTGCAGACAGCGACACGCCGACTATACGAAAAGGAACTTACACATTTGTTCCATGGCTTCTCAGCTTTAAAGAGGAAATGCCTTGGAGGAGAAAGAGAACAAAATAGTGGTGAGGCAAACAGGC TAT TTCTTCATCTACAGCCAGGTTCTA TAC ACGGACCCATCTTTGCTATGGGGTCATGTCATCCAGAGGAAGAAAGGCGGAAGCCTGGTGACCCTGTTCCGATGTATTCAGAATATGCCCAAAACACTGCCCAACAATTCCTGC TAC TCGGCTGGCATCGCGAGGCTGGAAGAAGGAGATGAGATTCAGCTTGCAATTCCT CGG GAGAATGCACAGATTTCACGCAACGGAGACGACACCTTCTTTGGTGCCCTAAAACTGCTGCACCATCACCATCACCATTAA-3'.

[0066] 2. Construct a plasmid pEVOL-pNO2-Phe containing an orthocyanin-tRNA expression cassette and an orthocyanin-tRNA synthetase encoding gene. The nucleotide sequence corresponding to the orthocyanin-tRNA expression cassette is SEQ ID NO:3 (including the promoter proK prom (situations 1-107 of SEQ ID NO:3), the corresponding tRNA sequence (situations 108-184 of SEQ ID NO:3), and the terminator proK-Term (situations 185-224 of SEQ ID NO:3)). The nucleotide sequence of the orthocyanin-tRNA synthetase encoding gene is SEQ ID NO:4.

[0067] SEQ ID NO:3:

[0068] 5’-GTGCACGGCTAACTAAGCGGCCTGCTGACTTTCTCGCCGATCAAAAGGCATTTTGCTATTAAGGGATTGACGAGGGCGTATCTGCGCAGTAAGATGCGCCCCGCATTCCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCAGGGGTTCAAATCCCCTCCGCCGGACCAAATTCGAAAAGCCTGCTCAACGAGCAGGCTTTTTTGCATG-3’。

[0069] SEQ ID NO:4:

[0070] 5’-ATGGACGAGTTCGAAATGATTAAACGCAACACCAGCGAAATTATCTCTGAAGAAGAGCTGCGCGAGGTGCTGAAGAAAGACGAGAAGAGCGCGCTGATTGGCTTTGAGCCGTCCGGTAAAATTCACCTGGGTCACTACCTGCAAATCAAGAAGATGATTGATCTGCAAAACGCTGGTTTTGACATCATTATCCTGCTGGCGGACCTGCACGCCTACCTGAATCAAAAGGGCGAGCTGGATGAGATTCGCAAGATCGGCGACTACAATAAGAAAGTCTTCGAAGCCATGGGTTTGAAGGCTAAATACGTCTACGGTAGCAGCTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCGTTGAACTATGAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGAGATTATAA-3’。

[0071] Escherichia coli Origami B (DE3) was selected as the host strain, and a series of recombinant expression vectors pET21-DmBAFF-TAG and plasmids pEVOL-pNO2-Phe were transformed into the host strain. The transformed bacteria were inoculated into LB medium containing ampicillin and chloramphenicol and cultured overnight at 37°C with shaking. Subsequently, the bacterial culture was inoculated into GMML minimal medium containing appropriate amounts of antibiotics and cultured at 37°C until the OD600 reached 0.5. Then, pNO2-Phe (final concentration 1 mM), followed by IPTG (final concentration 0.5 mM) and L-arabinose (final concentration 0.1%) were added to the medium to induce protein expression. The culture temperature was adjusted to 25°C, and induction continued for 12–16 hours.

[0072] The collected bacterial cells were resuspended in His-Binding buffer and lysed using a high-pressure homogenizer, with the mixture treated twice at 1200 bar. After centrifugation to remove cell debris, the supernatant was mixed with Ni-NTA metal affinity resin and incubated at 4°C for 2 hours.

[0073] The resin was washed with washing buffer (containing 20 mM–30 mM imidazole) to remove non-specifically bound proteins, followed by elution of the target protein with elution buffer containing 300 mM imidazole. The elution buffer was diluted 20-fold and loaded onto a SOURCE15Q anion exchange column, and eluted using a linear gradient of 0–30% NaCl. The target protein elution peak was collected, and the buffer was replaced using ultrafiltration centrifuge tubes to finally obtain the recombinant DmBAFF variant protein in PBS.

[0074] The purity of the obtained protein was determined by SDS-PAGE, and the results are as follows: Figure 1 As shown. Mass spectrometry analysis was performed on 158-DmBAFF, and the results are as follows. Figure 2 As shown, this proves that the synthesis was successful.

[0075] Example 2: Immunization of animals

[0076] Eight- to ten-week-old DBA / 1 mice were selected, and each mouse underwent subcutaneous immunization. Proteins 91-DmBAFF, 100-DmBAFF, 139-DmBAFF, or 158-DmBAFF obtained in Example 1 were respectively suspended in a 1:1 ratio with complete Freund's adjuvant to prepare a 200 μL mixture containing 5 μg of antigen, which served as the initial immunization. Booster immunizations were performed on days 7 and 14 using the same dose of antigen without adjuvant; PBS buffer and protein DmBAFF were used as controls. Serum was collected via orbital plexus blood sampling on days 7, 14, and 21 to detect IgG antibody levels. The results are as follows: Figure 3As shown, proteins 91-DmBAFF, 100-DmBAFF, 139-DmBAFF, and 158-DmBAFF can all elicit an immune response in mice.

[0077] After the above immunization procedure was completed, a collagen-induced arthritis model was established on day 15 by injection of bovine type II collagen (100 μg, CFA emulsified). A booster immunization was performed on day 36 by injection of the same dose of collagen (IFA emulsified). The degree of joint swelling in the mice was scored, with scores for each of the four paws added together, up to a maximum of 16 points. The scoring criteria are as follows:

[0078] 0 points: Normal;

[0079] 1 point: Mild redness and swelling: Redness of the ankle (no obvious swelling) or swelling of one toe bone;

[0080] 2 points: Moderate redness and swelling: swelling of the ankle or 3-4 toe bones;

[0081] 3 points: Moderate redness and swelling: Severe swelling of the ankle or swelling of the bones of all 5 toes;

[0082] 4 points: Severe redness and swelling: The entire sole of the foot is swollen.

[0083] The results are as follows Figure 4 As shown, both 91-DmBAFF and 139-DmBAFF can reduce the joint swelling score and incidence in mice, and have good therapeutic effects on collagen-induced arthritis, demonstrating good clinical application results.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of non-naturally modified amino acid proteins in the preparation of medicaments for relieving and / or treating BAFF-related rheumatoid arthritis, characterized in that, The protein is any one of the following A1)-A2): A1) A protein whose amino acid sequence is SEQ ID NO:1, positions 1-178; A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus and / or amino terminus of the protein shown in A1); The non-natural amino acid mentioned is p-nitrophenylalanine; The modification site of p-nitrophenylalanine is located at position 91 or 139 of the protein described in A1).

2. The application according to claim 1, characterized in that, The modification site of the non-natural amino acid is located at position 91 of the protein described in A1).

3. The method for preparing the non-natural amino acid-modified protein according to any one of claims 1-2, characterized in that, include: A recombinant vector expressing the protein was constructed, and the codons corresponding to the modified sites in the recombinant vector were replaced with TAGs to obtain the mutated recombinant vector; The mutated recombinant vector and a plasmid containing genes encoding orthogonal-tRNA and orthogonal-tRNA synthetase were simultaneously transferred into a host microorganism to obtain a recombinant microorganism. The recombinant microorganisms were cultured in a medium containing p-nitrophenylalanine to obtain proteins with non-natural amino acid modifications.

4. A pharmaceutical composition, characterized in that, This includes the non-natural amino acid-modified protein and pharmaceutically acceptable carrier described in any one of claims 1-2.

5. The pharmaceutical composition according to claim 4, characterized in that, The carrier includes at least one of an excipient, a buffer solution, and an immune adjuvant.

6. The pharmaceutical composition according to claim 4 or 5, characterized in that, The pharmaceutical composition is an injectable preparation.

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