Active super-antigen compound biological injection preparation G for treating muscle and bone joint diseases and preparation method of active super-antigen compound biological injection preparation G

Abstract

The invention relates to the technical field of biological pharmacy, and discloses an active super antigen compound biological injection preparation G for treating muscle and joint diseases. The preparation comprises the following components in percentage by mass: 18%-28% of the super antigen compound, 35%-45% of the amino acid mixture and 12%-18% of the bioactive nanoparticle carrier, wherein the super antigen compound accounts for 18%-28% of the total mass of the preparation; the amino acid mixture accounts for 35%-45% of the total mass of the preparation; and the bioactive nanoparticle carrier accounts for 12%-18% of the total mass of the preparation. According to the active super antigen compound biological injection preparation G for treating the muscle and bone joint diseases and the preparation method, a super antigen compound comprises a staphylococcus aureus enterotoxin variant modified by gene engineering and a recombinant human interleukin-1 receptor antagonist fusion protein, and inflammation is reduced without damaging cartilage by precisely regulating and controlling immune response; l-homocysteine and L-hydroxyproline in the amino acid mixture have a synergistic effect, so that not only can free radicals be eliminated, but also cartilage repair and regeneration can be promoted, the problem of cartilage degeneration caused by long-term use of a traditional medicine is effectively solved, and the self-repair capability of tissues is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly to a compound biological injection preparation G for treating bone and joint diseases with active superantigen and a preparation method thereof. Background Art

[0002] The immune system is the core defense network of the body to recognize and eliminate pathogens and maintain the stability of the internal environment. The imbalance of its function can lead to chronic inflammatory diseases, such as osteoarthritis and rheumatoid arthritis. In bone and joint diseases, the release of inflammatory factors caused by the over-activation of immune cells, such as IL-1β and TNF-α, not only destroys the cartilage matrix but also inhibits the repair function of chondrocytes. Existing treatment methods are difficult to achieve the dual goals of precisely regulating the immune response and promoting tissue regeneration simultaneously.

[0003] The existing technology has the following core problems: Traditional anti-inflammatory drugs, such as glucocorticoids or non-steroidal anti-inflammatory drugs, can inhibit inflammation, but long-term use leads to accelerated cartilage degeneration and lack of repair function; Single-target biological agents, such as anti-TNF-α monoclonal antibodies, rely on the level of inflammatory factors and cannot solve the synergistic requirements of immune regulation and tissue repair; Existing compound preparations, such as hyaluronic acid combined with growth factors, have low bioavailability and short action time due to the lack of an efficient delivery carrier and antigen-drug co-design, and are difficult to meet the long-term treatment needs of chronic joint diseases. Therefore, a compound biological injection preparation G for treating bone and joint diseases with active superantigen and a preparation method thereof are proposed. Summary of the Invention

[0004] (1) Technical Problems to be Solved

[0005] Aiming at the deficiencies of the existing technology, the present invention provides a compound biological injection preparation G for treating bone and joint diseases with active superantigen and a preparation method thereof, which has the advantages of ensuring the anti-inflammatory and cartilage repair synergistic functions of the preparation by precisely defining the component ratio, innovating the connection technology and the delivery system, and solves the problems that traditional anti-inflammatory drugs, such as glucocorticoids or non-steroidal anti-inflammatory drugs, can inhibit inflammation, but long-term use leads to accelerated cartilage degeneration and lack of repair function; Single-target biological agents, such as anti-TNF-α monoclonal antibodies, rely on the level of inflammatory factors and cannot solve the synergistic requirements of immune regulation and tissue repair; Existing compound preparations, such as hyaluronic acid combined with growth factors, have low bioavailability and short action time due to the lack of an efficient delivery carrier and antigen-drug co-design, and are difficult to meet the long-term treatment needs of chronic joint diseases.

[0006] (2) Technical Solutions

[0007] To achieve the above object of precisely defining the component ratios, innovating connection technologies and delivery systems to ensure the synergistic anti-inflammatory and cartilage repair functions of the preparation, the present invention provides the following technical solutions: a compound biological injection preparation G for treating tendon, bone and joint diseases with active superantigen, and the preparation contains the following components and their ratio parameters:

[0008] The superantigen complex accounts for 18% to 28% of the total mass of the preparation, and includes a genetically engineered variant of Staphylococcus aureus enterotoxin and a recombinant human interleukin-1 receptor antagonist fusion protein;

[0009] The amino acid mixture accounts for 35% to 45% of the total mass of the preparation, and contains L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine. The molar ratio of homocysteine to hydroxyproline is 1:1.2, and the mass ratio of N-acetylcysteine is 0.8%;

[0010] The bioactive nanoparticle carrier accounts for 12% to 18% of the total mass of the preparation, and is composed of a chondrocyte growth factor encapsulated by a poly(lactic-co-glycolic acid) copolymer with a molecular weight of 15 kDa, surface-modified with polyethylene glycol, with a particle size D50 of 120 ± 15 nm and an encapsulation efficiency of not less than 90%.

[0011] Preferably, the amino acid sequence of the Staphylococcus aureus enterotoxin variant is replaced with alanine at the 45th position and glutamine at the 80th position, and its pro-inflammatory property is reduced by more than 80% compared with the wild-type Staphylococcus aureus enterotoxin.

[0012] Preferably, the recombinant human interleukin-1 receptor antagonist fusion protein is linked to the 100th lysine residue at the C-terminus of the Staphylococcus aureus enterotoxin variant through a disulfide bond, and the molar ratio of the recombinant human interleukin-1 receptor antagonist to the Staphylococcus aureus enterotoxin variant is 1:2.5.

[0013] Preferably, N-acetylcysteine in the amino acid mixture acts synergistically with homocysteine, and the scavenging rate of hydroxyl radicals is not less than 75%.

[0014] A preparation method of the compound biological injection preparation G for treating tendon, bone and joint diseases with active superantigen, and the preparation method prepares the compound biological injection preparation G for treating tendon, bone and joint diseases with active superantigen, including the following preparation steps:

[0015] S1. Preparation of the superantigen complex: Cultivate recombinant Escherichia coli containing the pET-28a plasmid in LB medium, and after fermentation, purify the target protein by affinity chromatography and ion exchange chromatography; Mix the Staphylococcus aureus enterotoxin variant and the recombinant human interleukin-1 receptor antagonist in a buffer solution and form a fusion protein through a disulfide bond;

[0016] S2. Preparation of Amino Acid Mixture: Dissolve L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine in a buffer solution in a specific ratio; mix them through a high-pressure homogenizer to ensure the uniformity of the components;

[0017] S3. Preparation of Nanoparticle Carrier: Dissolve poly(lactic-co-glycolic acid) copolymer and chondrocyte growth factor in an organic solvent, add polyethylene glycol aqueous phase after forming an emulsion; remove the unencapsulated growth factor by ultrasonic treatment and centrifugation, and detect the particle size and encapsulation efficiency;

[0018] S4. Assembly of the Final Product of the Preparation: Mix the superantigen complex, amino acid mixture and nanoparticles, and covalently connect them through click chemistry reaction; obtain the final product through ultrafiltration and filtration sterilization.

[0019] Preferably, in the preparation process of the nanoparticle carrier in S3, the encapsulation efficiency of chondrocyte growth factor is monitored in real time by a confocal laser scanning microscope to ensure that the encapsulation efficiency is ≥90%.

[0020] Preferably, in S4, the connection between the superantigen complex and the nanoparticles is achieved through click chemistry reaction:

[0021] Introduce an azide group on the surface of the antigen, NaN3 with a final concentration of 0.1 M, pH 9.0, and react for 1 hour;

[0022] Introduce an alkyne group on the surface of the nanoparticles, propiolic anhydride with a final concentration of 0.5 M, pH 5.0, and react for 2 hours;

[0023] Form a stable triazole bond under the catalysis of 0.1 mM CuSO4, and the connection efficiency is ≥95%.

[0024] Preferably, in S4, the pH value of the final product of the preparation is 7.2 - 7.4, the osmotic pressure is 280 - 320 mOsm / kg, it is sterile and the endotoxin content is ≤0.125 EU / mL.

[0025] (III) Beneficial Effects

[0026] Compared with the prior art, the present invention provides a compound biological injection preparation G for treating bone and joint diseases with active superantigen and its preparation method, which has the following beneficial effects:

[0027] 1. The compound biological injection preparation G for treating bone and joint diseases with active superantigen and its preparation method adopt a superantigen complex and an amino acid mixture in a specific ratio. Among them, the superantigen complex includes a genetically engineered variant of Staphylococcus aureus enterotoxin and a recombinant human interleukin-1 receptor antagonist fusion protein, which can precisely regulate the immune response to reduce inflammation without damaging cartilage. The L-homocysteine and L-hydroxyproline in the amino acid mixture act synergistically, not only scavenging free radicals but also promoting cartilage repair and regeneration, effectively solving the problem of cartilage degeneration caused by long-term use of traditional drugs and improving the tissue self-repair ability.

[0028] 2. The compound biological injection preparation G for treating bone and joint diseases with active superantigen and its preparation method also include bioactive nanoparticle carriers composed of chondrocyte growth factors encapsulated by poly(lactic-co-glycolic acid), surface-modified with polyethylene glycol, and the particle size is controlled at D50 of 120 ± 15 nm to ensure efficient delivery to the target site. This design not only enhances the bioavailability of the drug and prolongs the action time, but also realizes the synergistic effect of antigen and drug through the nanoparticle carrier, overcoming the problem of short-term efficacy caused by the lack of an efficient delivery system in existing compound preparations, and is particularly suitable for the long-term management of chronic joint diseases. Description of the Drawings

[0029] Figure 1 It is a block diagram of the preparation method of the compound biological injection preparation G of the present invention. Detailed Embodiments

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments and drawings 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] Please refer to Figure 1 , the compound biological injection preparation G for treating bone and joint diseases with active superantigen, which preparation contains the following components and their ratio parameters:

[0032] The superantigen complex accounts for 18% to 28% of the total mass of the preparation, including a genetically engineered variant of Staphylococcus aureus enterotoxin and a recombinant human interleukin-1 receptor antagonist fusion protein;

[0033] The amino acid mixture accounts for 35% to 45% of the total mass of the preparation, containing L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine, wherein the molar ratio of homocysteine to hydroxyproline is 1:1.2, and the mass ratio of N-acetylcysteine is 0.8%;

[0034] The bioactive nanoparticle carrier accounts for 12% to 18% of the total mass of the preparation, and is composed of chondrocyte growth factor encapsulated by poly(lactic-co-glycolic acid) with a molecular weight of 15 kDa, surface-modified with polyethylene glycol, with a particle size D50 of 120 ± 15 nm and an encapsulation efficiency of not less than 90%.

[0035] A preparation method of the compound biological injection preparation G of active superantigen for treating bone and joint diseases. The preparation method prepares the compound biological injection preparation G of active superantigen for treating bone and joint diseases, including the following preparation steps:

[0036] S1. Preparation of superantigen complex: Recombinant Escherichia coli containing pET-28a plasmid is cultured in LB medium, and the target protein is purified by affinity chromatography and ion exchange chromatography after fermentation; Staphylococcal enterotoxin variant and recombinant human interleukin-1 receptor antagonist are mixed in a buffer solution and form a fusion protein through disulfide bond connection;

[0037] S2. Preparation of amino acid mixture: L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine are dissolved in a buffer solution according to a specific ratio; Mixed by a high-pressure homogenizer to ensure uniform components;

[0038] S3. Preparation of nanoparticle carrier: Poly(lactic-co-glycolic acid) and chondrocyte growth factor are dissolved in an organic solvent, and polyethylene glycol aqueous phase is added after forming an emulsion; Unencapsulated growth factor is removed by ultrasonic treatment and centrifugation, and the particle size and encapsulation efficiency are detected;

[0039] S4. Assembly of the final product of the preparation: The superantigen complex, amino acid mixture and nanoparticles are mixed and covalently connected through click chemical reaction; The final product is obtained by ultrafiltration and filtration sterilization.

[0040] Example 1:

[0041] This example provides a compound biological injection preparation G of active superantigen for treating bone and joint diseases, and the preparation contains the following components and their ratio parameters:

[0042] The superantigen complex accounts for 18% of the total mass of the preparation, and includes a genetically engineered Staphylococcal enterotoxin variant and a recombinant human interleukin-1 receptor antagonist fusion protein;

[0043] The amino acid mixture accounts for 35% of the total mass of the preparation, and contains L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine, wherein the molar ratio of homocysteine to hydroxyproline is 1:1.2, and the mass ratio of N-acetylcysteine is 0.8%;

[0044] The bioactive nanoparticle carrier accounts for 12% of the total mass of the preparation, and is composed of chondrocyte growth factor encapsulated by poly(lactic-co-glycolic acid) with a molecular weight of 15 kDa, surface-modified with polyethylene glycol, with a particle size D50 of 120 ± 15 nm and an encapsulation efficiency of not less than 90%.

[0045] This example provides a preparation method for the compound biological injection preparation G of active superantigen for treating bone and joint diseases. The preparation method prepares the compound biological injection preparation G of active superantigen with the above component ratios, including the following preparation steps:

[0046] S1. Preparation of superantigen complex: Recombinant Escherichia coli containing the pET-28a plasmid is cultured in LB medium, and the target protein is purified by affinity chromatography and ion exchange chromatography after fermentation; in a buffer solution, staphylococcal enterotoxin variant is mixed with recombinant human interleukin-1 receptor antagonist, and a fusion protein is formed by disulfide bond connection;

[0047] S2. Preparation of amino acid mixture: L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine are dissolved in a buffer solution in a specific ratio; mixed by a high-pressure homogenizer to ensure uniform components;

[0048] S3. Preparation of nanoparticle carrier: Poly(lactic-co-glycolic acid) and chondrocyte growth factor are dissolved in an organic solvent, and after forming an emulsion, polyethylene glycol aqueous phase is added; unencapsulated growth factor is removed by ultrasonic treatment and centrifugation, and the particle size and encapsulation efficiency are detected;

[0049] S4. Assembly of the final product of the preparation: 18% of the superantigen complex, 35% of the amino acid mixture and 12% of the nanoparticles are mixed and covalently connected by click chemical reaction; the final product is obtained by ultrafiltration and filtration sterilization.

[0050] Specifically, the ratios in this example have the following effects:

[0051] The superantigen complex accounts for 18% of the total mass of the preparation, ensuring that the complex can effectively activate the immune system without causing an excessive inflammatory response.

[0052] The amino acid mixture accounts for 35%, and the molar ratio of L-homocysteine to L-hydroxyproline is 1:1.2. Such a combination not only helps to scavenge free radicals, but also promotes the repair and regeneration of chondrocytes; the mass ratio of N-acetylcysteine is 0.8%, and its synergistic effect significantly improves the scavenging rate of hydroxyl free radicals, thereby reducing the damage of oxidative stress to joint tissues.

[0053] The bioactive nanoparticle carrier accounts for 12% of the total mass of the preparation. The chondrocyte growth factor is encapsulated by poly(lactic-co-glycolic acid), and the surface is modified with polyethylene glycol, enabling the nanoparticles to have good biocompatibility and stability and effectively delivering drugs to the target site.

[0054] Example 2:

[0055] This example provides a compound biological injection preparation G for treating bone and joint diseases with active superantigen, which contains the following components and their ratio parameters:

[0056] The superantigen complex accounts for 23% of the total mass of the preparation, including a genetically engineered variant of Staphylococcus aureus enterotoxin and a recombinant human interleukin-1 receptor antagonist fusion protein;

[0057] The amino acid mixture accounts for 40% of the total mass of the preparation, containing L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine. The molar ratio of homocysteine to hydroxyproline is 1:1.2, and the mass ratio of N-acetylcysteine is 0.8%;

[0058] The bioactive nanoparticle carrier accounts for 15% of the total mass of the preparation, and is composed of chondrocyte growth factor encapsulated by poly(lactic-co-glycolic acid) with a molecular weight of 15 kDa. The surface is modified with polyethylene glycol, the particle size D50 is 120 ± 15 nm, and the encapsulation efficiency is not less than 90%.

[0059] This example provides a preparation method for the compound biological injection preparation G for treating bone and joint diseases with active superantigen. This preparation method prepares the compound biological injection preparation G for treating bone and joint diseases with active superantigen with the above component ratios, including the following preparation steps:

[0060] S1. Preparation of the superantigen complex: Recombinant Escherichia coli containing the pET-28a plasmid is cultured in LB medium, and the target protein is purified by affinity chromatography and ion exchange chromatography after fermentation; in a buffer solution, the Staphylococcus aureus enterotoxin variant is mixed with the recombinant human interleukin-1 receptor antagonist, and a fusion protein is formed by disulfide bond connection;

[0061] S2. Preparation of the amino acid mixture: Dissolve L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine in a buffer solution according to a specific ratio; mix them with a high-pressure homogenizer to ensure uniform components;

[0062] S3. Preparation of the nanoparticle carrier: Dissolve poly(lactic-co-glycolic acid) and chondrocyte growth factor in an organic solvent, add the polyethylene glycol aqueous phase after forming an emulsion; remove the unencapsulated growth factor by ultrasonic treatment and centrifugation, and detect the particle size and encapsulation efficiency;

[0063] S4. Assembly of the final pharmaceutical product: Mix 23% of the superantigen complex, 40% of the amino acid mixture, and 15% of the nanoparticles, and covalently link them through click chemistry; obtain the final product through ultrafiltration and filtration sterilization.

[0064] Specifically, the proportions in this example have the following effects:

[0065] The proportion of the superantigen complex is increased to 23%, which enhances the anti-inflammatory ability and at the same time reduces the usage amount of pro-inflammatory variants, thereby further reducing the risk of side effects.

[0066] The proportion of the amino acid mixture is increased to 40%. This adjustment optimizes the nutritional support. Especially for damaged joint tissues that require additional repair, it provides more sufficient raw materials, which is beneficial to accelerating the recovery process; in addition, the increased amino acid content also helps to improve the stability and solubility of the overall formulation.

[0067] The proportion of the bioactive nanoparticle carrier is correspondingly adjusted to 15%. This can maintain a balance while ensuring efficient delivery, avoiding the influence of excessive carriers on the effects of other components; the design of this proportion aims to maximize the advantages of each component, form a synergistic effect, and jointly act on the treatment of diseases.

[0068] Example 3:

[0069] This example provides an active superantigen compound biological injection preparation G for treating bone and joint diseases. The preparation contains the following components and their ratio parameters:

[0070] The superantigen complex accounts for 28% of the total mass of the preparation, including a genetically engineered variant of Staphylococcus aureus enterotoxin and a recombinant human interleukin-1 receptor antagonist fusion protein;

[0071] The amino acid mixture accounts for 45% of the total mass of the preparation, containing L-homocysteine, L-hydroxyproline, L-asparagine, L-serine, and N-acetylcysteine. The molar ratio of homocysteine to hydroxyproline is 1:1.2, and the mass ratio of N-acetylcysteine is 0.8%;

[0072] The bioactive nanoparticle carrier accounts for 18% of the total mass of the preparation and is composed of chondrocyte growth factor encapsulated by poly(lactic-co-glycolic acid) with a molecular weight of 15 kDa, surface-modified with polyethylene glycol, with a particle size D50 of 120 ± 15 nm and an encapsulation efficiency of not less than 90%.

[0073] This embodiment provides a preparation method of a compound biological injection preparation G of active superantigen for treating bone and joint diseases. The preparation method prepares the compound biological injection preparation G of active superantigen with the above component ratios, including the following preparation steps:

[0074] S1. Preparation of superantigen complex: Recombinant Escherichia coli containing pET-28a plasmid is cultured in LB medium, and the target protein is purified by affinity chromatography and ion exchange chromatography after fermentation; In a buffer solution, staphylococcal enterotoxin variant is mixed with recombinant human interleukin-1 receptor antagonist, and a fusion protein is formed through disulfide bond connection;

[0075] S2. Preparation of amino acid mixture: L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine are dissolved in a buffer solution according to a specific ratio; Mix through a high-pressure homogenizer to ensure uniform components;

[0076] S3. Preparation of nanoparticle carrier: Poly(lactic-co-glycolic acid) copolymer and chondrocyte growth factor are dissolved in an organic solvent, and polyethylene glycol aqueous phase is added after forming an emulsion; Unencapsulated growth factor is removed by ultrasonic treatment and centrifugation, and the particle size and encapsulation efficiency are detected;

[0077] S4. Assembly of the final product of the preparation: 28% of the superantigen complex, 45% of the amino acid mixture and 18% of the nanoparticles are mixed and covalently linked through click chemistry reaction; Ultrafiltration and filtration sterilization are carried out to obtain the final product.

[0078] Specifically, the ratios in this embodiment have the following functions:

[0079] The proportion of the superantigen complex reaches the highest value of 28%, which is designed for more severe or refractory cases; A high proportion of the superantigen complex can more strongly regulate the immune response and is suitable for patients who need a more potent treatment plan.

[0080] The proportion of the amino acid mixture also reaches the highest level of 45%, providing the richest nutritional basis for joint tissues and supporting the maximum degree of repair and regeneration; This ratio is particularly suitable for patients with a long chronic course and severe injuries, helping them recover normal function as soon as possible.

[0081] The proportion of the bioactive nanoparticle carrier is 18%, ensuring high drug delivery efficiency, enabling all active ingredients to accurately reach the lesion site and exert the maximum therapeutic potential; This ratio strategy comprehensively considers efficacy, safety and patient individual differences, and strives to provide the most suitable treatment plan for each patient.

[0082] In summary, for the compound biological injection preparation G for treating bone and joint diseases with active superantigen and its preparation method, a superantigen complex and an amino acid mixture in a specific ratio are adopted. Among them, the superantigen complex includes a genetically engineered variant of staphylococcal enterotoxin and a recombinant human interleukin-1 receptor antagonist fusion protein, which can reduce inflammation by precisely regulating the immune response without damaging cartilage. The L-homocysteine and L-hydroxyproline in the amino acid mixture act synergistically to not only scavenge free radicals but also promote cartilage repair and regeneration, effectively solving the problem of cartilage degeneration caused by long-term use of traditional drugs and improving the tissue self-repair ability.

[0083] Moreover, the compound biological injection preparation G for treating bone and joint diseases with active superantigen and its preparation method further includes a bioactive nanoparticle carrier composed of a chondrocyte growth factor encapsulated by poly(lactic-co-glycolic acid), with polyethylene glycol surface modification, and the particle size is controlled such that D50 is 120 ± 15 nm, ensuring efficient delivery to the target site. This design not only enhances the bioavailability of the drug and prolongs the action time, but also realizes the synergistic effect of the antigen and the drug through the nanoparticle carrier, overcoming the problem of short-term efficacy caused by the lack of an efficient delivery system in existing compound preparations, and is particularly suitable for the long-term management of chronic joint diseases, solving the problems of traditional anti-inflammatory drugs, such as glucocorticoids or non-steroidal anti-inflammatory drugs, which can inhibit inflammation but accelerate cartilage degeneration and lack repair function during long-term use; single-target biological agents, such as anti-TNF-α monoclonal antibodies, which rely on the level of inflammatory factors and cannot solve the synergistic requirements of immune regulation and tissue repair; and existing compound preparations, such as hyaluronic acid combined with growth factors, which have low bioavailability and short action time due to the lack of an efficient delivery carrier and antigen-drug synergistic design, making it difficult to meet the long-term treatment needs of chronic joint diseases.

[0084] All relevant modules involved in this system are hardware system modules or functional modules that combine computer software programs or protocols in the prior art with hardware. The computer software programs or protocols themselves involved in this functional module are well-known technologies to those skilled in the art and are not the improvements of this system. The improvement of this system lies in the interaction relationship or connection relationship between the modules, that is, the overall structure of the system is improved to solve the corresponding technical problems to be solved by this system.

[0085] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The compound biological injection preparation G for treating tendon, bone and joint diseases with active superantigen, characterized in that, The preparation contains the following components and their ratio parameters: The superantigen complex accounts for 18% to 28% of the total mass of the preparation, including a genetically engineered Staphylococcus aureus enterotoxin variant and a recombinant human interleukin-1 receptor antagonist fusion protein; The amino acid mixture accounts for 35% to 45% of the total mass of the preparation, containing L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine, wherein the molar ratio of homocysteine to hydroxyproline is 1:1.2, and the mass ratio of N-acetylcysteine is 0.8%; The bioactive nanoparticle carrier accounts for 12% to 18% of the total mass of the preparation, and is composed of chondrocyte growth factor encapsulated by a poly(lactic-co-glycolic acid) copolymer with a molecular weight of 15 kDa, surface-modified with polyethylene glycol, with a particle size D50 of 120 ± 15 nm and an encapsulation efficiency of not less than 90%.

2. The compound biological injection preparation G for treating bone and joint diseases with active superantigen according to claim 1, wherein, The amino acid sequence of the Staphylococcus aureus enterotoxin variant is replaced with alanine at position 45 and glutamine at position 80, and its pro-inflammatory property is reduced by more than 80% compared with the wild-type Staphylococcus aureus enterotoxin.

3. The compound biological injection preparation G for treating bone and joint diseases with active superantigen according to claim 1, wherein, The recombinant human interleukin-1 receptor antagonist fusion protein is linked to the lysine residue at the 100th position of the C-terminus of the Staphylococcus aureus enterotoxin variant through a disulfide bond, and the molar ratio of recombinant human interleukin-1 receptor antagonist to Staphylococcus aureus enterotoxin variant is 1:2.

5.

4. The compound biological injection preparation G for treating diseases of muscles, bones and joints with active superantigen according to claim 1, characterized in that, N-acetylcysteine in the amino acid mixture synergistically acts with homocysteine, and the scavenging rate of hydroxyl radicals is not less than 75%.

5. Preparation method of compound biological injection preparation G for treating bone and joint diseases with active superantigen, characterized in that, The preparation method prepares the active superantigen therapeutic compound biological injection preparation G described in any one of claims 1-4, including the following preparation steps: S1. Preparation of the superantigen complex: Cultivate recombinant Escherichia coli containing the pET-28a plasmid in LB medium, and purify the target protein by affinity chromatography and ion exchange chromatography after fermentation; Mix the Staphylococcus aureus enterotoxin variant and the recombinant human interleukin-1 receptor antagonist in a buffer solution, and form a fusion protein through disulfide bond linkage; S2. Preparation of the amino acid mixture: Dissolve L-homocysteine, L-hydroxyproline, L-asparagine, L-serine and N-acetylcysteine in a buffer solution according to a specific ratio; Mix through a high-pressure homogenizer to ensure uniform components; S3. Preparation of the nanoparticle carrier: Dissolve the poly(lactic-co-glycolic acid) copolymer and the chondrocyte growth factor in an organic solvent, form an emulsion and then add the polyethylene glycol aqueous phase; Remove the unencapsulated growth factor by ultrasonic treatment and centrifugation, and detect the particle size and encapsulation efficiency; S4. Assembly of the final product of the preparation: Mix the superantigen complex, the amino acid mixture and the nanoparticles, and covalently link them through click chemistry; Obtain the final product through ultrafiltration and filtration sterilization.

6. The preparation method of the compound biological injection preparation G for treating bone and joint diseases with active superantigen, characterized in that, In S3, during the preparation of the nanoparticle carrier, the encapsulation efficiency of the chondrocyte growth factor is monitored in real time by a laser confocal microscope to ensure that the encapsulation efficiency ≥ 90%.

7. The preparation method of the compound biological injection preparation G of active superantigen for treating bone and joint diseases according to claim 5, characterized in that, In S4, the connection between the superantigen complex and the nanoparticles is achieved through click chemistry: Introduce an azide group on the antigen surface with NaN3 at a final concentration of 0.1 M, pH 9.0, and react for 1 hour; Introduce an alkyne group on the nanoparticle surface with propiolic anhydride at a final concentration of 0.5 M, pH 5.0, and react for 2 hours; Form a stable triazole bond under the catalysis of 0.1 mM CuSO4, and the ligation efficiency is ≥95%; 8. The preparation method of the compound biological injection preparation G for treating bone and joint diseases with active superantigen, characterized in that, For the said S4, the pH value of the final product of the preparation is 7.2 - 7.4, the osmotic pressure is 280 - 320 mOsm / kg, it is sterile and the endotoxin content is ≤0.125 EU / mL.

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