CN115873121A - High-activity biological fermentation method and pharmaceutical composition containing probiotics - Google Patents

Abstract

The invention relates to a high-activity biological fermentation method and a pharmaceutical composition containing probiotics. Based on the characteristic of Caspase-1 processable precursor IL-1 beta, the epitope peptide is specifically screened and a specific monoclonal antibody is prepared by immunizing a mouse, the antibody can effectively inhibit the expression of IL-1 beta in an in vitro cell model, and simultaneously can be combined with probiotics to restrictively reduce the inflammation index in the mouse model so as to treat gout, so that the Caspase-1 processable precursor IL-1 beta has a good effect.

Description

High-activity biological fermentation method and pharmaceutical composition containing probiotics

Technical Field

The application relates to the field of biology, in particular to a high-activity biological fermentation method and a pharmaceutical composition containing probiotics.

Background

Gout is a common and complex type of arthritis, which can be suffered by all ages, with higher incidence in men than women. Patients with gout often have sudden joint pain at night, the joint is in urgent attack, pain, edema, red swelling and inflammation appear at the joint, and the pain is slowly relieved until the pain disappears and lasts for several days or weeks. Gout attack is related to the concentration of uric acid in the body, and gout can form urate deposition in joint cavities and the like, thereby causing acute joint pain.

Gout is a group of metabolic diseases caused by purine metabolic disorder, and the continuous increase of blood uric acid concentration causes urate crystal to deposit soft tissues. The pathological mechanism of gout and hyperuricemia is mainly 3 aspects as follows: (1) Congenital abnormalities of several uric acid transporters of the renal tubule may lead to the occurrence of hyperuricemia and are associated with the mechanisms of promoting uric acid elimination drugs and inhibiting uric acid excretion; (2) Epidemiological studies have shown that hyperuricemia and gout are both associated with death from cardiovascular disease, even hyperuricemia has been shown to be associated with chronic nephropathy; (3) The TO 11-like receptor (TLR-2, TLR-4) and NALP3 inflammasome in innate immunity receive danger signals of uric acid, and then cause inflammatory response of gout through activation of Bai Jiesuo IL-l, and in addition, transforming growth factor TGF-beta may be related TO automatic relief of the inflammatory response.

Gout treatment can be divided into three parts: (1) early control and relief of the onset of acute arthritis; (2) preventing further deposition of uric acid in the tissue by reducing the uric acid level in the blood; (3) prevent uric acid calculus formation, and reduce severe joint injury and renal function damage caused by uric acid calculus formation. Anti-inflammatory and analgesic treatment is recommended early (generally within 24 h) in the acute gout attack stage, and non-steroidal anti-inflammatory drugs (NSAIDs), colchicine and glucocorticoid can effectively resist inflammation and relieve pain and improve the life quality of patients. The acid reduction treatment is not carried out in the acute attack stage, but the patient who takes the acid reduction medicine does not need to stop taking the acid reduction medicine so as to avoid causing fluctuation of blood uric acid, and the attack time is prolonged or the attack is carried out again.

Colchicine and non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used drugs for the treatment of acute gout. Colchicine is the first choice drug for treating acute gouty arthritis, and can inhibit the aggregation of white blood cells at the joint inflammation part, weaken the action of the white blood cells on phagocytizing uric acid, and relieve the inflammation reaction caused by local white blood cell destruction, thereby achieving the purpose of quickly diminishing inflammation. But it often causes gastrointestinal reaction and impairment of liver and kidney functions in clinic. The non-steroidal anti-inflammatory drug inhibits the activity of cyclooxygenase, blocks the synthesis of Prostaglandin (PG), and has antipyretic, anti-inflammatory and analgesic effects. The NSAIDs for treating acute gout mainly comprise acetaminophen, diclofenac sodium, indomethacin and the like. The urate crystals induce giant cells to activate aspartate proteolytic enzyme, catalyzing the conversion of interleukin-1 p precursor (Pro-IL-1 β) to IL-1 β. IL-1 beta is recognized as a key cytokine causing gouty arthritis and has important effects on the treatment of gout. Any drug that directly or indirectly blocks the binding of IL-1 β to a receptor may block the action of IL-1 β.

Current research on interleukin-1 beta inhibitors for treating acute phase gout has yielded promising results. Three drugs, canakiumab (ilakinumab, ilaris), linaclocept (irinotect) and anakinra (anakinra, kinereto, are approved by the FDA for the treatment of periodic fever syndrome (CAPS), and anakinra are also approved by the FDA for the treatment of rheumatoid arthritis.

Gout is clinically mainly manifested as arthritic symptoms, high-level uric acid in blood in an acute stage can directly stimulate and release a large amount of proinflammatory cytokines such as IL-1, IL-6, IL-8, IL-12 and the like and tumor necrosis factors, and the imbalance of the proinflammatory and anti-inflammatory cytokines is an important reason for gouty arthritis. Research shows that the lactobacillus can regulate the mRNA expression level of the cytokine in the spleen of the diabetic mouse and reduce the proinflammatory factors IL-6 and IL-8. When kefir fermented milk is used for interfering mouse breast cancer cells, the anticancer activity of the kefir fermented milk is realized by reducing the expression of IL-6 through the regulation of an immune system and an endocrine system by lactic acid bacteria. The barley extract fermented by lactic acid bacteria was also confirmed to be capable of reducing the secretion of rat inflammatory factors IL-1 beta, IL-6 and TNF-alpha, and causing the reduction of the expression of pro-inflammatory factors by inhibiting the expression of NF-. Kappa.B protein. Therefore, lactic acid bacteria have the functions of participating in cellular immune response, reducing inflammatory reaction of gout, particularly in the acute gout attack stage, and reducing adverse symptoms such as local red swelling and hot pain caused by inflammatory reaction. The high-concentration uric acid causing gout diseases mainly comes from purine metabolism, a reasonable mode is adopted to intervene in purine metabolic pathways, and the generation of uric acid end products is reduced, so that the method is a method for preventing and treating gout or hyperuricemia. The intestinal lactobacillus is a beneficial flora in the gastrointestinal tract of the organism, has the capability of decomposing and generating nutrient substances, can provide more nutrient substances for host cells, and can bring more probiotic effects to the organism through the absorption and transformation functions of the intestinal lactobacillus. Three aspects of purine absorption, purine metabolic intermediate inosine, guanosine absorption and uric acid absorption are also proved. Japanese has found that a strain of lactic acid bacteria PA-3 decomposes and absorbs purines, making them difficult to absorb directly by the human body. Domestic scholars have also demonstrated that orally administered lactic acid bacteria can break down purines ingested by food in the gut, and can reduce purine absorption and serum uric acid levels.

However, at present, there are few pharmaceutical compositions for using these probiotics and other therapeutic drugs, and few drugs with better effects are needed to be developed.

Disclosure of Invention

Previous studies have demonstrated that Caspase-1, also known as IL-l convertase, can process precursor IL-1 β, produce mature IL-1 β, and is secreted extracellularly. IL-1 β can activate nodulin MyD88 by interacting with IL-1 β receptors on synoviocytes, endothelial cells, fibroblasts, etc., which in turn initiates NF-. Kappa.B, triggers the transcription of secondary inflammatory cytokines, producing large amounts of cytokines including IL-1 β, TNF-. Alpha., IL-6, and neutrophil chemokines. Therefore, the development of drugs inhibiting Caspase-1 activity can be used for treating gout.

In one aspect of the invention, a monoclonal antibody specific to Caspase-1 is provided.

In one aspect, the Caspase-1 monoclonal antibody is Cas-5H14, and the light chain variable region sequence is

DIVITQRPALMAASPGEKVTITCGARFAIPPVQNTWYQQKSGISPKPWIYVMIFEIPGVPARFSGSGSGTSYSLTITSMEAEDAATYYCFWCDVRKSEFGAGTKLELK

The heavy chain variable region sequence is

EVQLEESATELARPGASVKLSCKASGYIFSDICAHWIKQRPGQGLEWIGCLNKPKFARIDYVRFPGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDWASKSIWGLGTTLAVSS。

On one hand, the invention provides the application of the Caspase-1 monoclonal antibody in preparing a medicament for inhibiting the expression of IL-1 beta.

In another aspect of the invention, the invention provides an application of the Caspase-1 monoclonal antibody in preparation of a medicament for treating gout.

Previous researches show that lactobacillus casei ZM15 can efficiently degrade four purine substances, namely adenosine, guanylic acid, adenosine and guanosine, the degradation rate reaches 100%, uric acid and allantoin are not produced after degradation, the amount of free purine bases (xanthine, hypoxanthine and guanine) produced is low and is only 1.40mmol/L (accounting for 0.10% of total degradation products), the in vivo uric acid generation can be effectively reduced, and the blood uric acid level can be reduced. Therefore, the lactobacillus is used as a drug of the composition.

Furthermore, the invention also provides the application of the monoclonal antibody of Caspase-1 and lactobacillus casei ZM15 in preparing a kit for inhibiting the expression of IL-1 beta.

In another aspect of the invention, the use of a Caspase-1 monoclonal antibody and Lactobacillus casei ZM15 in the preparation of a kit for treating gout is provided.

The viable cell count of the Lactobacillus casei ZM15 is preferably 1.0X 10 to 3.0X 10 1CFU/g, more preferably 2.0X 10 to 1.0X 10 1CFU/g.

The dosage of the medicine box for treating gout is preferably 1-20 g/day, and more preferably 10 g/day.

Further, the compositions of the present invention utilize a buffering agent to adjust the pH. Suitable buffers for the composition are not limited, and examples thereof include gluconate, histidine, citrate, phosphate [ e.g., sodium or potassium ], succinate [ e.g., sodium ], acetate, tris (hydroxymethyl) aminomethane, glycine, arginine, and combinations thereof, and can be used individually depending on the pH to be adjusted. The pharmaceutical composition of the present invention preferably contains acetate, histidine, or phosphate as a buffer, more preferably histidine as a buffer. The concentration of the buffer is not particularly limited as long as it is pharmaceutically acceptable, and is preferably 1mM to 150mM, more preferably 5mM to 100mM, and still more preferably 10mM to 50mM. In addition, the concentration of the buffer is preferably about 1mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM. From the viewpoint of using a salt, particularly an inorganic salt buffer, it is preferable to use a concentration of less than 30mM, particularly 10mM or less.

Histidine (e.g., at a concentration of 5mM to 50mM, e.g., 10mM to 50mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50 mM) is particularly beneficial as a buffer in the pharmaceutical compositions or kits of the invention. In one embodiment, the stable pharmaceutical composition contains 5mM to 20mM histidine. The pH of the pharmaceutical composition may be in the range of 4.0 to 8.0, pH in the range of 4.5 to 7.5, such as 5.0 to 7.0, 5.2 to 6.8, such as about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.8, about 7.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.7.7, about 7.8 is common. The pharmaceutical composition of the present invention has an increased turbidity in long-term storage at pH4 or pH 8. In addition, when the pharmaceutical composition of the present invention has pH4 or pH8, pH drift is significant in long-term storage. Therefore, in one embodiment, the pH of the stable antibody-containing pharmaceutical composition is greater than 4 and less than 8, preferably 5 or more and 7 or less, more preferably 5.5 or more and 6.5 or less, and most preferably 6.0.

The pharmaceutical composition or kit of the present invention preferably contains a surfactant. Suitable surfactants for pharmaceutical compositions are not limited and include nonionic surfactants, ionic surfactants, zwitterionic surfactants, and combinations thereof. Typical surfactants for use in the present invention are not limited, and include sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol fatty acid esters (e.g., glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol fatty acid esters (e.g., decaglycerol monostearate, decaglycerol distearate, decaglycerol monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glycerol monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), polyoxyethylene fatty acid amides (e.g., polyoxyethylene stearamide), C10-C18 alkylsulfates (e.g., sodium hexadecylsulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkylether sulfates (e.g., sodium polyoxyethylene lauryl sulfate) and C1-C18 alkylsulfosuccinate salts (e.g., sodium lauryl sulfosuccinate) obtained by adding an average of 2 to 4 moles of ethylene oxide units, and natural surfactants such as lecithin, glycerophospholipids, sphingomyelin (e.g., sphingomyelin) and sucrose esters of C12-C18 fatty acids.

The pharmaceutical composition or kit of the present invention may contain 1 or more of the above surfactants. Preferred surfactants are nonionic surfactants (e.g., sorbitan fatty acid esters, sorbitan trioleate, glycerin fatty acid esters, polyglycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene hydrogenated castor oil, polyoxyethylene beeswax derivatives, polyoxyethylene lanolin derivatives, polyoxyethylene fatty acid amides), more preferably polyoxyethylene alkyl ethers (e.g., poloxamer 188) or polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60 or 80. The concentration of the surfactant may be any concentration commonly used in the art, and may be used, for example, at a concentration of about 0.01% (w/v) to about 0.1% (w/v), for example, about 0.01% (w/v) to about 0.04% (w/v), for example, about 0.01% (w/v), about 0.02% (w/v), about 0.04% (w/v), about 0.06% (w/v), about 0.08% (w/v), or about 0.1% (w/v). Among the surfactants, polysorbate 80 (tween 80) is particularly advantageous. In one embodiment, the stable pharmaceutical composition comprises about 0.02% (w/v) polysorbate 80. In one embodiment, the stable pharmaceutical composition contains about 0.02% (w/v) polysorbate 20.

Further, the pharmaceutical compositions or kits disclosed herein are intended for oral administration to humans and animals in unit dosage form or multiple dosage forms, such as capsules, caplets, powders, pellets, granules, tablets, microtablets, sachets or stick packs. The pharmaceutical composition may comprise compound (a) or further comprise compound (b) as described above. Preferably, the unit dosage form or multiple dosage forms are, for example, capsules, tablets, microtablets, sachets or stick packs. More preferably, the pharmaceutical composition is in the form of a capsule or tablet. This may be achieved by mixing the pharmaceutical composition as defined herein with diluents, lubricants, binders, disintegrants and/or absorbents, colouring, flavouring and sweetening agents.

Capsules comprising the particles or compositions of the invention as defined herein may be prepared using techniques known in the art. Suitable capsules may be selected from soft gelatin capsules, hard shell capsules, hard gelatin capsules, plant based shell capsules, hypromellose (HPMC) based capsules or mixtures thereof. A pharmaceutical composition, as described herein, may be presented in a hard gelatin capsule, a soft gelatin capsule, a hard or plant shell capsule, a Hypromellose (HPMC) capsule, wherein the pharmaceutical composition is further mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or cellulose-based excipients. Hard gelatin capsules are made from a two-piece outer gelatin shell called a body and a cap. The shell may comprise vegetable or animal gelatin (e.g. pork, beef or fish-based gelatin), water, one or more plasticizers, and possibly some preservatives. The capsules may hold a dry mixture in the form of a powder, tiny granules or granules, comprising a non-bile acid FXR agonist, e.g., compound (a), at least one binder, and optionally excipients. The outer shell may be transparent, opaque, colored or flavored. Capsules containing the particles may be coated with enteric and/or gastric soluble or delayed release coating materials using techniques well known in the art to achieve, for example, better stability in the gastrointestinal tract or to achieve a desired release rate. Hard gelatin capsules of any size (e.g., 000-5 in size) may be prepared.

Advantageous effects

Based on the characteristic that Caspase-1 can process precursor IL-1 beta, the epitope peptide is specifically screened and a specific monoclonal antibody is prepared by immunizing a mouse, the antibody can effectively inhibit the expression of the IL-1 beta in an in vitro cell model, and simultaneously can reduce the inflammation index in the mouse model in a limited manner after being combined with probiotics so as to treat gout, so that the invention has better effect.

Drawings

FIG. 1 is a diagram showing the result of the specific identification of monoclonal antibody

FIG. 2 Effect of monoclonal antibodies on IL-1 beta content in cell models

FIG. 3 is a graph showing the result of IL-1. Beta. Content in serum after treatment with mAb and/or probiotic

Detailed Description

The present invention may be understood more readily by reference to the following description of certain embodiments of the invention and the detailed description of the examples included therein. Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

EXAMPLE 1 preparation of Caspase-1 monoclonal antibody

The DNASAR software is used for predicting secondary structures, surface characteristics (such as hydrophilicity, surface property and antigenicity) and the like of Caspase-1 protein sequences of human and mice, an online IEDB analysis tool is used for analyzing, and according to an analysis result, a conserved immunogenic peptide Cas with better immunogenicity is selected: pehktsdstflvfmshgi. The jieji peptide organisms were committed to synthesis.

Coupling an immunogenic peptide to a macromolecule KLH to form a Cas-KLH; cas peptides were additionally combined with a large molecule GGG, known as coat Immunoglobulin G, to form Cas-GGG for use in ELISA screening for antibody production.

Selecting SPF-grade 6-week BALB/c mice, female mice, 3 mice, immunizing 3 mice once, taking 80 mu g Cas-KLH conjugate each time, uniformly mixing the Cas-KLH conjugate with Freund's complete adjuvant according to the proportion of 1:1, then adopting a mouse subcutaneous multi-point injection method, separating for 3 weeks, taking 80 mu g Cas-KLH conjugate from 2 to 3 times, uniformly mixing the Cas-KLH conjugate with the Freund's incomplete adjuvant according to the proportion of 1:1, then adopting the mouse subcutaneous multi-point injection method, separating for 3 weeks from 2 and 3 times, taking blood after 3 weeks of immunization, measuring the titer, selecting a No. 2 mouse (1 25000) with the highest titer to carry out boosting immunization on 100 mu g Cas-KLH conjugate for immunization, and preparing cell fusion after 3 days of boosting immunization. According to the traditional monoclonal antibody preparation technology, hybridoma cell strains Cas-1D23 and Cas-5H14 with stable monoclonal antibody secretion are obtained through cloning culture by a 4-time limiting dilution method.

Preparing ascites from 8-week-old BALB/c mice by a conventional method, and treating the ascites with a single antibody of I: the dilution ratio is increased by 100, the reaction titer is detected by indirect ELISA, and the result shows that the titers of the Cas-1D23 monoclonal antibody and the Cas-5H14 monoclonal antibody are both above 1. And meanwhile, the subclass identification of the monoclonal antibody in the purified ascites is carried out by using an HRP-labeled goat anti-mouse antibody subclass identification kit, and the result shows that 2 monoclonal antibodies are IgG1 and light chains are kappa chains.

Example 2 identification of biological Functions of Cas-5H14 monoclonal antibodies

(1) Monoclonal antibody relative affinity identification: cas-GGG (l. Mu.g/well) was coated, monoclonal antibody concentrations were l00, 50, 25, l2.5, 6.25 and 3.125. Mu.g/ml, incubated at 37 ℃ for l hours, washed 3 times, then enzyme-labeled secondary antibody (l: 5000) was added, washed after 1 hour at 37 ℃, and then subjected to chromogenic assay with substrate, and the relative affinities were calculated as 50% of the maximum bound monoclonal antibodies, as shown in Table 1.

TABLE 1 Cas-5H14 monoclonal antibody relative affinities

Name of antibody	Relative affinity
		Cas-5H14 monoclonal antibody	(2.19±0.10)μg/mL

As can be seen from the results in Table 1, the Cas-5H14 monoclonal antibody has better affinity.

(2) Cas-5H14 monoclonal antibody specificity identification: cas-GGG, BSA, recombinant human Caspase-3 protein (Biovision, cat # 1083-10) and recombinant human Caspase-1 protein (Biovision cat # 1081-25) were each coated with antigen at l. Mu.g/well and left overnight at 4 ℃. The concentration of the Cas-5H14 monoclonal antibody is 1 mug/mL, the goat anti-mouse Ig marked by AP is diluted according to 1.

As can be seen from the results of FIG. 1, the Cas-5H14 monoclonal antibody prepared by the invention shows positive effects only against Cas-GGG and recombinant human Caspase-1 protein, but has no obvious combination effect against recombinant human Caspase-3 protein, BSA, GGG and the like, which indicates that the monoclonal antibody prepared by the invention has better specificity.

The light chain sequence and the heavy chain sequence of the Cas-5H14 monoclonal antibody are amplified by adopting a kit and sequenced to obtain a light chain variable region sequence of DIVITQRPALMAASPGEKVTITCGARFAIPPVQNTWYQQKSGISPKPWIYVMIFEIPGVPARFSGSGSGTSYSLTITSMEAEDAATYYCFWCDVRKSEFGAGTKLELK and a heavy chain variable region sequence of EVQLEESATELARPGASVKLSCKASGYIFSDICAHWIKQRPGQGLEWIGCLNKPKFARIDYVRFPGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDWASKSIWGLGTTLAVSS.

Example 3 Effect of Cas-5H14 monoclonal antibodies on gout model cells

CO ₂ After the mice were sacrificed, the femur and tibia were removed under sterile conditions, the muscle and other tissues were thoroughly removed, holes were punched at both ends of the femur and tibia with a 7-gauge needle, all bone marrow cells were washed out with PBS, and then a cell suspension was prepared with a 4-gauge needle, centrifuged at 1000 Xg for 5min at room temperature, after washing, the number of bone marrow cells was counted, and the cell concentration was adjusted to 1X 10 by using a DMEM medium containing 20% macrophage colony stimulating factor, 2mmol/L of glutamate, 0.1mmol/L of non-essential amino acids, 100mg/mL of streptomycin, and 10% of fetal bovine serum inactivated at high temperature ⁶ one/mL, and 4 mL/well in sterile 6-well plastic plates, at 37 ℃ in CO ₂ Culturing in 5% culture box for 3d, changing culture solution to remove suspended cells, adding the same culture solution, and culturing until the blood cover sheet at the bottom of the culture plate is fully covered with elliptical macrophages. When the cell concentration reaches 2X 10 ⁶ at/mL, mononuclear macrophages were transferred to serum-free tissue culture plates and the experimental groups first induced cells for 10h with 250. Mu.g/mL MSU crystals. Dividing the cells into blank control group (cells not treated), MSU induced group (MSU treated cells alone), and positive control diacerein + MSU group (diacerein used for MSU induced cells)Treatment is carried out, the concentration is 10 mug/mL), a Cas-5H14 monoclonal antibody + MSU group (cells induced by MSU are treated by the Cas-5H14 monoclonal antibody, the concentration is 10 mug/mL), the IL-1 beta content is detected, and blank control cells are not treated. The results are shown in FIG. 2.

Compared with a blank control group, the level of IL-1 beta in the MSU induction group is obviously increased (difference is obvious, P is less than 0.01), compared with a positive control group, after the treatment by the Cas-5H14 monoclonal antibody, the content of IL-1 beta is only (83.5 +/-4.0) pg/mL, and the effect is better than that of the positive control group. This fully suggests that the Cas-5H14 monoclonal antibody can be used for treating gout by inhibiting the processing precursor IL-1 beta and further inhibiting the content of mature IL-1 beta.

Example 4 preparation of probiotics for the treatment of gout

Taking out original strain ZM15 (preservation number is CGMCC No. 13980) of lactobacillus casei strain from-80 ℃, picking out the original strain with sterilized toothpick, then marking on YE plate, culturing at constant temperature of 37 ℃ for 2d, picking out a single clone with toothpick, inoculating the single clone into 3mL of liquid sterile MRS culture medium for overnight culture, transferring 1% of inoculum size into 100mL of sterilized liquid MRS culture medium, standing at 37 ℃ for 12h, then culturing at 150r/min in a shaking way for 28h, centrifuging at 10000r/min for 50min, collecting precipitate, adjusting the viable count to 1.0 x 10 ¹⁰ CFU/g, 10g and 10g maltodextrin are taken and stirred for 10min at the rotating speed of 1000r/min, and the probiotic composition, namely the probiotic activity product is obtained.

Wherein the MRS culture medium comprises (g/L): 10.0 parts of peptone, 8.0 parts of beef extract, 4.0 parts of yeast powder, 20.0 parts of glucose, 2.0 parts of dipotassium phosphate, 2.0 parts of diammonium hydrogen citrate, 5.0 parts of sodium acetate, 0.2 part of magnesium sulfate, 0.04 part of manganese sulfate, 801.0 part of tween-801.0, 5.7 +/-0.2 parts of pH value and sterilization at 118 ℃ for 25min. YE culture medium: 15g of yeast powder, 20g of glucose, 1000mL of distilled water, sterilizing the yeast powder and the glucose respectively, cooling and mixing, adding 1.5% of agar into the solid YE culture medium, and 2% of CaCO ₃ (CaCO ₃ Sterilized separately from the medium).

EXAMPLE 5 therapeutic testing of drugs on mouse models

Male Kunming mice of 6 weeks old are selected, a model group is inserted into the inner side of a tibial tendon from the 45-degree direction of the back side of the right hind limb ankle joint of a tested rat by using a No. 6 injection needle, 0.05mL of MSU solution (50 mg/mL) is injected into the ankle joint cavity, and the acute GA model is prepared by once more administration at an interval of 3d.

The experiment was performed using the following grouping pattern, with 10 mice per group:

the blank control group (group A) and the model group (group B) were administered with physiological saline;

positive control group (group C): 1mg of diacerein per 100g of mouse body weight, and carrying out intraperitoneal injection administration;

mab treatment group (group D): cas-5H14 monoclonal antibody 1mg/100g mouse weight, intraperitoneal injection administration;

probiotic group (group E): probiotic composition 4.0 x 10 prepared according to example 4 ⁹ CFU/day, administration by gavage;

mab combined probiotic group (group F): 1mg of Cas-5H14 monoclonal antibody per 100g of mouse body weight, and carrying out intraperitoneal injection administration; meanwhile, the probiotic composition prepared according to example 4 was 4.0 × 10 ⁹ CFU/day, administration by gavage;

dosing was started once daily for 3 days in each group after the start of molding. After 3d, the inflammation index, the grading standard of the inflammation index: normally 0 (score 0); erythema of the articular skin, mild swelling, bony signs visible as grade 1 (score 2); the joint is obviously red and swollen, the bony mark disappears, but the swelling is limited to the joint part and is grade 2 (4 points); swelling of the limbs other than the joints was grade 3 (score 6). Meanwhile, the joint cavity is internally used by 0.5mL. The solution is washed by physiological saline, and 0.05mL of washing solution is reserved. The leukocytes were immediately counted under microscopic examination. The results are shown in table 2 below.

TABLE 2 inflammation index and leukocyte count in the groups of mice

As can be seen from the results in table 2, the inflammation index of each experimental group has a significant statistical significance (P < 0.01) compared to the model group. According to observation, the swelling degree of the joints of rats in each group is increased after model building, the inflammation indexes are reduced to different degrees after treatment, particularly, the monoclonal antibody combined probiotic group has a remarkable down-regulation effect, the inflammation indexes and the white blood cell number show a good treatment effect, the swelling of the joints is also remarkably relieved, and the good treatment effect is reflected.

72h after molding, taking blood from heart of an anesthetized rat with 0.8mL of chloral hydrate of 100g/L, injecting 1mL of the blood into an EDTA anticoagulation tube, taking serum to detect the amount of IL-1 beta, and detecting by adopting an ELISA method strictly according to the kit instructions. The results are shown in FIG. 3.

As can be seen from the results of fig. 3, the amount of IL-1 β was significantly increased in the model group relative to the blank control group (P < 0.01). The amount of IL-1 beta can be reduced by adopting probiotic treatment, but the reduction amplitude is not particularly high relative to the Cas-5H14 monoclonal antibody or a positive control, but the probiotic and the Cas-5H14 monoclonal antibody have better reduction effect after being combined, and the amount of IL-1 beta is only (22.03 +/-0.52) mu g/L, so that the effect is better.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims (6)

1. Caspase-1 monoclonal antibody Cas-5H14, characterized in that: the light chain variable region sequence is shown as SEQ ID NO:1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO:2, respectively.
2. The application of the Caspase-1 monoclonal antibody in preparing a pharmaceutical composition for treating gout, wherein the Caspase-1 monoclonal antibody is Cas-5H14, and the light chain variable region sequence of the Caspase-1 monoclonal antibody is shown as SEQ ID NO:1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO:2, respectively.
3. 3. The probiotic composition prepared by a high-activity biological fermentation method and the application of a Caspase-1 monoclonal antibody in preparing a kit for treating gout, wherein the Caspase-1 monoclonal antibody is Cas-5H14, and the light chain variable region sequence of the Caspase-1 monoclonal antibody is shown as SEQ ID NO:1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO:2 is shown in the specification; the probiotic composition prepared by the high-activity biological fermentation method specifically comprises the steps of taking out a starting strain of lactobacillus casei ZM15 with the preservation number of CGMCC No.13980 from minus 80 ℃, picking the starting strain by using a sterilized toothpick, then marking the selected starting strain on a YE flat plate, culturing the selected starting strain at the constant temperature of 37 ℃ for 2d, picking a single clone by using the toothpick, inoculating the single clone into a 3mL liquid sterile MRS culture medium for overnight culture, transferring the selected bacterial strain into 100mL of sterilized liquid MRS culture medium by using the inoculation amount of 1%, standing the obtained culture medium at the temperature of 37 ℃ for 12h, then culturing the obtained culture medium by using a 150r/min shaking table to 28h, centrifuging the obtained culture medium for 50min by using 10000r/min, collecting precipitates, adjusting the viable count to be 1.0 x 1010CFU/g, and stirring 10g and 10g of maltodextrin at the rotating speed of 1000r/min to obtain the probiotic composition.
4. 4. Use according to claim 2 or 3, wherein the pharmaceutical composition or kit comprises a suitable buffer.
5. 5. Use according to claim 2 or 3, wherein the pharmaceutical composition or kit comprises a suitable surfactant.
6. 6. The use of claim 4, wherein the buffering agent is histidine.

Images