CN114225025A - Use of probiotic-containing formulations for the treatment of gastrointestinal disorders - Google Patents

Abstract

The invention relates to the use of preparations containing probiotics for the treatment of gastrointestinal disorders. The invention provides a monoclonal antibody specifically aiming at MAdCAM-1, the antibody can be specifically combined with MAdCAM-1 to block the interaction of alpha 4 beta 7 and MAdCAM-1 so as to treat ulcerative enteritis, and meanwhile, corresponding treatment effect can be synergistically promoted by adding probiotics in the treatment process, so that the monoclonal antibody has a good application prospect.

Description

Use of probiotic-containing formulations for the treatment of gastrointestinal disorders

Technical Field

The present invention relates to the field of biology, more specifically to the use of probiotic containing formulations for the treatment of gastrointestinal disorders.

Background

Ulcerative Colitis (UC) is an inflammatory intestinal disease which is chronic, idiopathic and alternately appears in attack and remission stages, and the incidence and prevalence of UC in China tend to increase year by year. UC usually occurs in the rectum and distal colon, but may affect the entire colon, and the disease may occur at any age, but is more prevalent between 15 and 30 years of age, and is relatively low between 50 and 7O. UC mainly invades the colonic mucosa and submucosa and manifests as inflammation and ulceration. The existing data show that the incidence of diseases is different in all regions, developed regions are relatively consistent, and great differences exist in Asia Africa and south America.

At present, the ulcerative colitis is mainly treated by medicines, and at present, the following main medicine types are mainly adopted. The 5-aminosalicylic acid (5-ASA) formulation is the main drug for the treatment of mild and moderate UC, which began in the 4O's of the 20 th century. There are a large number of 5-ASA formulations available clinically for oral administration, including drugs containing azo bonds, such as sulfasalazine, olsalazine, balsalazide and mesalamine in controlled release dosage forms. The medicine has good treatment effect, but the clinical compliance is lower because the medicine needs to be taken for a long time. The safety of the 5-ASA formulation was generally good according to a number of clinical trials. However, the use of sulfasalazine has been limited by its adverse effects (nausea, vomiting, abdominal pain, fever, rash, agranulocytosis, neutropenia, male infertility, folate deficiency, autoimmune hemolysis and rare nephrotoxicity, hepatotoxicity or pancreatitis) and higher intolerance rates. Corticosteroid drugs can be divided into systemic and topical 2 types. Corticosteroids are the first line UC medications that treat moderate to severe and other conservative treatments are poorly effective, with a 50% remission rate on severe UC. The sudden severe UC fatality rate can be obviously reduced. In the latter half of the 20 th century, glucocorticoids were widely used to treat active UC, reducing the higher fatality rate of the disease from 75% to less than 1%. Corticosteroid hormones with systemic action, such as prednisone (0.25-0.75 mg/(kg. d)) or methylprednisolone (48mg), are continuously applied for about 17 weeks, and the remission rate is 60% -80%. Topically acting glucocorticoid budesonide, which is orally or enema-administered for the treatment of distal UC, is effective in delaying or controlling recurrence and has fewer adverse effects. Beclomethasone dipropionate is a novel corticosteroid medicine with local effect, the medicine has better safety, is mainly used for treating mild to moderate active-period UC, 434 patients with the UC are treated by oral beclomethasone dipropionate (5mg/d) by adopting a retrospective multi-center method, the average taking time is 6.2 weeks, and the remission rate and the effective rate are respectively 44.4 and 22.3 percent.

The anti-tumor necrosis factor (infliximab) infliximab is a monoclonal antibody which specifically blocks tumor necrosis factor IX. For patients with moderate to severe UC who are refractory to treatment with corticosteroids, infliximab may be used. Although infliximab has good treatment effect on a specific type of UC, serious adverse reaction exists, so that the clinical application needs to closely observe the reaction after taking the medicine. The role of infliximab in treating UC disease studied by Wilhelm et al is believed to have some therapeutic effect in treating UC disease, but further clinical trials are needed to evaluate the effect of this drug on disease treatment and progression. Probiotics are active nonpathogenic microorganisms that can improve the microenvironment balance, and particularly have more pronounced effects in the gastrointestinal tract.

The probiotic bacteria mainly comprise Saccharomyces cerevisiae or lactic acid bacteria such as Lactobacillus and Bifidobacterium, which can be used as dietary supplement or food. Sang et al assessed the UC treatment effect of probiotics under the guidelines of 13 relevant clinical randomized trials, performed meta analysis, and the results suggest that UC-treating probiotics were more efficacious than placebo. Hegazy et al randomized 3O mild to moderate UC into 2 groups and performed clinical observations over a period of 8 weeks, one group taking sulfasalazine (2400g/d) orally and the other group taking sulfasalazine containing probiotics at the same dose orally. They considered oral probiotic-containing supplements to be helpful in maintaining patients in remission and preventing relapse.

Vedolizumab (Vedolizumab) approved 3 months in 2020 is a novel integrin antagonist, is an intestinal selective anti-lymphocyte migration drug, has the advantages of short-term symptom improvement, rapid disease control and the like, and is used for treating adult patients with moderate severe active UC/Crohn Disease (CD) with insufficient response, no response or intolerance of traditional therapies or TNF-alpha inhibitors. A large number of real world studies have confirmed that vederlizumab has good efficacy and safety for long-term treatment, and has low incidence of severe infection, infusion-related reactions and malignant tumors. 11 and 9 days in 2020, two cases of severe patients with UC with infliximab failure response successfully complete the infusion of the veulizumab in the digestive department of drugstore hospital affiliated to the medical college of Nanjing university, and 11 and 18 days in the third case of UC patients successfully infuse the veulizumab injection, thereby providing a brand-new choice for patients with Inflammatory Bowel Disease (IBD) in the region and being expected to benefit more IBD patients. Compared with systemic biological agents, the vederlizumab does not affect the systemic immune function because it can specifically bind to the alpha 4 beta 7 integrin, thereby accurately and selectively inhibiting intestinal inflammation.

Monoclonal antibodies directed against the integrin binding to α 4 β 7 have been shown to act to treat ulcerative enteritis due to the interaction of α 4 β 7 and MAdCAM-1, but there is currently no monoclonal antibody directed against MAdCAM-1 and use of the monoclonal antibody in the treatment of ulcerative enteritis.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a medicine box for specifically treating ulcerative enteritis.

Specifically, the kit consists of a monoclonal antibody specific for MAdCAM-1 and probiotics.

Further, the light chain variable region of the monoclonal antibody of MAdCAM-1 is as shown in SEQ ID NO: 1, the heavy chain variable region sequence is shown as SEQ ID NO: 2, respectively.

Further, the invention provides a health product, which consists of the monoclonal antibody specifically aiming at the MAdCAM-1 and probiotics.

In certain embodiments, the antibodies provided herein encompass any antigen binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that includes one or more CDRs or any other antibody fragment that binds to an antigen but does not include the entire native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, fabs, Fab ", F (ab") 2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv "), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), diabodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding fragment is capable of binding to the same antigen as the parent antibody.

By "Fab" of an antibody is meant the portion of the antibody consisting of a single light chain (variable and constant regions) joined by disulfide bonds to the variable and first constant regions of a single heavy chain.

"Fab'" refers to a Fab fragment that contains a portion of the hinge region.

"F (ab ') 2" refers to a dimer of Fab'.

"Fc" with respect to an antibody (e.g., of the IgG, IgA or IgD isotype) refers to the portion of the antibody consisting of the second and third constant domains of the first heavy chain that are bound to the second and third constant domains of the second heavy chain by disulfide bonds. The Fc for IgM and IgE isotype antibodies further comprises a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

"Fv" with respect to an antibody refers to the smallest fragment of an antibody that carries an intact antigen binding site. The Fv fragment consists of the variable region of a single light chain joined to the variable region of a single heavy chain.

As described herein, minor variations in the amino acid sequence of the antibodies of the invention, provided that the amino acid sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% variation. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those occurring in the amino acid family, which are related to their side chains. Genetically encoded amino acids are generally classified into the following families: (1) the acidic amino acid is aspartic acid or glutamic acid; (2) the basic amino acid is lysine, arginine and histidine; (3) the nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and (4) the amino acids without electric polarity are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are a family of amides; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, it is reasonable to expect that an isolated substitution of an isoleucine or valine for a leucine, an isolated substitution of a glutamic acid for an aspartic acid, an isolated substitution of a serine for a threonine, or a similar substitution of a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid in a framework position. Whether an amino acid change results in a functional peptide can be readily determined by determining the specific activity of the polypeptide derivative. The measurement method is not particularly limited, and measurement can be performed by a method known in the art. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one skilled in the art. Preferably, the amino-and carboxy-termini of the fragment or analog occur near the boundaries of the functional domains. Domains and functional domains can be identified by comparing nucleotide and/or amino acid sequence data to public or private sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains (which occur in other proteins of known structure and/or function). Methods for identifying protein sequences that fold into known three-dimensional structures are known.

Further, the present invention provides a kit for maintaining clinical remission of ulcerative enteritis in a human patient, wherein the composition is administered to the human patient at a dose of 300mg once every 4 weeks or 8 weeks.

Further, the antibody is in the form of a liquid formulation, which may have a pH between about 6.3 and about 7.0. The pH of the formulation may be between about 6.5 and about 6.8. The formulation may have a pH between about 6.1 and about 7.0 or between about 6.2 and 6.8.

In some embodiments, a stable liquid pharmaceutical formulation contains an antibody, a buffer, and at least about 10mM citrate. The buffer may be a histidine buffer.

In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising at least about 100mg/ml to about 200mg/ml of an antibody, a buffer, and at least about 5mM citrate. The buffer may be a histidine buffer.

In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising an antibody and at least about 10mM citrate. The formulation may further comprise polysorbate 80.

In another aspect, the invention relates to a stable liquid pharmaceutical formulation comprising an antibody and at least about 5mM citrate. The formulation may further comprise polysorbate 80.

In another aspect, the present invention relates to a stable liquid pharmaceutical formulation comprising a mixture of an antibody, citrate, histidine, arginine and polysorbate 80. The formulation may be present in a container (e.g., a vial, cartridge, syringe, or auto-injector).

According to another preferred embodiment, the composition of the probiotic is: lactobacillus plantarum CGMCC No.1258 and Bifidobacterium animalis CGMCC No. 9273. The probiotic bacteria may be prepared for use in the form of microcapsules.

Advantageous effects

The invention provides a monoclonal antibody specifically aiming at MAdCAM-1, the antibody can be specifically combined with MAdCAM-1 to block the interaction of alpha 4 beta 7 and MAdCAM-1 so as to treat ulcerative enteritis, and meanwhile, corresponding treatment effect can be synergistically promoted by adding probiotics in the treatment process, so that the monoclonal antibody has a good application prospect.

Drawings

FIG. 1 is a graph showing the results of protein detection by SDS-PAGE

FIG. 2 is a graph showing the results of expression levels of hsp70 and JNK

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of MAdCAM-1

Design of primers, F: 5'-TGGATCCATGCAAAGCTTGCAGGTGAAG-3', R: 5'-AAGGATCCTTAGACGGGGATGGCCTGG-3', using human DNA as a template, using F and R as primers, and adopting PCR for amplification under the conditions of 94 ℃ lmin, 49 ℃ 30s and 72 ℃ lmin) multiplied by 5; (94 ℃ 1min, 65 ℃ 30s, 72 ℃ 1 min). times.25, for a total of 30 cycles. And recovering a PCR product, cloning the PCR product into a pGEM-T vector, identifying the PCR product correctly, performing double enzyme digestion to obtain MAdCAM-1, connecting the MAdCAM-1 to a pQE30 vector, recombining the successfully plasmid, performing double enzyme digestion, and performing electrophoresis to generate two bands which are respectively an MAdCAM-1 insertion fragment and an enzyme digestion product of the PQE30 vector. And (4) taking the positive clones, respectively sending the positive clones to Invitrogen company for sequencing, and comparing the sequences to ensure that the sequencing result is consistent with the MAdCAM-1 sequence in GenBank.

Transforming the pQE30-MAdCAM-1 recombinant plasmid into M15 competent cells, and inoculating the competent cells into an LB solid culture medium; after incubation overnight at 37 ℃, picking a single colony and inoculating the single colony in an LB liquid culture medium; the cells were cultured overnight at 37 ℃ with shaking. Inoculating 60 mu L of recombinant bacteria cultured overnight to each of 3mL of LB culture medium containing Amp and kana; shaking and culturing at 37 deg.C until OD600 is about 0.5; under the optimal induction condition (0.5 mmol. L-¹IPTG (isopropyl thiogalactoside), induction is carried out for 6h) at 28 ℃, 200mL of bacterial liquid is induced, the precipitate is suspended by 10mL of LPBS and then is subjected to ultrasonic crushing, centrifugation is carried out for 10000r/min for 25min, the precipitate is collected, the precipitate is washed for 3 times by 10mL of inclusion body washing liquid, then washed by 9 times of TE buffer solution (containing 20mmol/Ltris-HC1, 1mmol 1/L EDTA and pH8.0) containing 4mol/L urea, the supernatant and the precipitate are respectively kept overnight, the precipitate is dissolved by buffer solution (containing 0.2mol/L of NaC1, 0.1mol/L of Tris-HCl, 1mmol/L of EDTA, 1mmol/L of DTT and pH8.0) containing 8 mol/g of urea according to the proportion of 10mL/g, the supernatant is stirred overnight at 4 ℃, and the supernatant is centrifuged at low temperature at 4 ℃ and is the target protein. Diluting the above inclusion body lysate to 0.2mg/mL with a buffer solution containing 8mol/L urea (containing 20mmol/Ltris-HC1, 1mmol/LEDTA, pH8.0), adding DTT to a final concentration of 1mmol/L, standing at room temperature for 4h, performing gradient dialysis renaturation at 4 deg.C for 24h with an external dialyzing solution containing 4, 3, 2mol/L urea (containing 20mmol/Ltris-HC1, 1mmol/LEDTA, 3mmol/LGSH, 0.5mmol/L GSSG, pH8.0), affinity purifying the renatured solution, and detecting the target egg by SDS-PAGEWhite, the results are shown in FIG. 1.

As can be seen from FIG. 1, lane 3 is the target protein eluate, which is of good purity; lanes 1 and 2 are flow-through solutions, which are substantially free of target protein, and lane 1 has only weak target protein, indicating that the purification is cleaner.

EXAMPLE 2 preparation of MAdCAM-1 monoclonal antibody

1) BALB/c mice were immunized. First immunization 1, 100. mu.L of CFA was added to 0.5mg/mL of the recombinant MAdCAM-1 protein prepared in example 1, and emulsified, and 6 to 8-week-old mice were immunized with the above emulsion. ② 2 nd immunization, namely after 2-3 weeks of the 1 st immunization, adding 100 uL IFA into 100 uL 0.5mg/mL recombinant MAdCAM-1 protein for emulsification, and immunizing the mice with the age of 6-8 weeks for 2 nd time by using the emulsion. And thirdly, 3 rd immunization, namely performing 3 rd immunization after 2-3 weeks of the 2 nd immunization. The method is the same as the 2 nd immunization. Tail vein bleeds were performed on day 10 after completion of the 3 rd immunization. 2) Antibody titers were determined. Coating a 96-hole enzyme label plate with 1 mu g/mL antigen solution; diluting the mouse serum with the ratio of 1: 100 in a 96-hole enzyme label plate in a multiple ratio, measuring the antibody titer in the mouse serum, and screening the cell fusion used by the mouse with the serum antibody titer of more than 1: 10000. 3) And (4) fusing the cells. The spleen of the immunized mouse was removed and 4mL of cell culture medium (containing 1.64% RPMI1640, 0.2% NaHCO) was added to a sterile tissue culture dish ₃1% Penicilin-streptomycin, 10% inactivated newborn bovine serum), placing the spleen into the tissue culture dish and crushing; mixing NS-1 myeloma cells and mouse spleen cells in a 50mL centrifuge tube according to the ratio of 1: 10, centrifuging at 1500r/min for 5min, removing supernatant, and slowly adding 1mL 50% PEG 3000; washing off PEG3000 with RPMI1640 medium; screening hybridoma cells by adopting a semi-colloid culture medium cloning method; hybridoma cells were cultured in 96-well cell culture plates (37 ℃, 5% CO)₂) (ii) a After culturing for 3 days, screening positively grown cell cloning wells by using an indirect ELISA method, selecting 4 strains with higher reading numbers for cloning and subcloning, and finally obtaining 2 hybridoma cell strains secreting anti-MAdCAM-1 monoclonal antibodies, wherein the hybridoma cell strains are named as 2A4 and 4C7 respectively.

The 2A4 hybridoma cells are injected into the abdominal cavity of a mouse, the abdominal cavity of the mouse is waited to be obviously enlarged, and ascites of the mouse is collected. Purifying with protein A column, placing purified monoclonal antibody solution into dialysis bag, scattering water absorbent PEG 20000 around the dialysis bag, standing at room temperature for more than 4 hr to allow water in the solution to exude; the protein concentration was 1.55mg/mL by Lowry method.

The mouse monoclonal antibody typing kit is adopted for identification, the operation steps are strictly carried out according to the instruction, and 2A4 is Ig2a immunoglobulin through detection.

The 2A4 monoclonal antibody prepared by the invention is determined to be only combined with MAdCAM-1 through Western blot detection, but not combined with other proteins such as BSA and plasma, and has better specificity.

Example 32A 4 antibody characterization

(1) Determination of monoclonal antibody affinity: and (3) coating an enzyme label plate with MAdCAM-1 at the concentration of 1 mu g/mL by using an indirect ELISA method, sealing, adding a purified monoclonal antibody diluted in a doubling ratio for incubation, taking goat anti-mouse IgG marked by HRP as a secondary antibody, and reading the light absorption value of OD450nm by using an enzyme label instrument. The OD450nm readings at several consecutive dilutions were no longer increasing and were taken as 100% binding of antigen antibody, a scatter plot was made with antibody concentration (mol/L) as abscissa and OD450nm absorbance as ordinate, and the antigen-antibody binding rate at half the maximum reading was 50% to generate a logarithmic trend line and formula. The half of the maximum value of OD450nm was substituted into the formula to determine the antibody concentration at that time, which was the affinity dissociation constant (Kd). The results showed that the affinity relay constant of the 2A4 monoclonal antibody was 3.31X 10^-10(M) has a good binding effect.

(2) And (3) identifying the sequence of the monoclonal antibody variable region:

RNA was extracted from single cell sorted B cell cultures using Qiagen RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Human antibody genes were amplified using a Qiagen one-step RT-PCR kit (Qiagen, Cat: 210212). RT-PCR primers were designed based on published prior art. The RT-PCR products were used as templates in nested PCR for amplification of antibody variable regions with Invitrogen (Invitrogen) pfx50 DNA polymerase, and forward and reverse nested PCR primers were designed based on the sequences at the beginning of framework 1 regions of the human IgG heavy and light chain variable regions as described previously. Subsequently, the nested PCR products were used as templates in an overlapping PCR for ligating antibody light and heavy chain PCR products with linker sequences and cloned into plasmid vectors for sequencing with an infusion HD cloning kit (Clontech, Cat: 639649). The sequences of the light chain variable region and the heavy chain variable region of the antibody were identified and obtained as follows, respectively.

Light chain variable region sequence (SEQ ID NO: 1):

DIVITQRPALMAASPGEKVTITCELQLNMFGCYAPWYQQKSGISPKPWIYSAKRRVRGVPARFSG SGSGTSYSLTITSMEAEDAATYYCWLYFHFWREFGAGTKLELK

heavy chain variable region sequence (SEQ ID NO: 2):

EVQLEESATELARPGASVKLSCKASGYIFSKGAMMWIKQRPGQGLEWIGWKGHPKLPWRTCKEIN GKATLTADKSSSTAYMQLSSLASEDSAVYYCAGHWNHTYMWGLGTTLAVSS

example 4 preparation of probiotic microcapsules

The probiotic consists of: the preparation method of the lactobacillus plantarum CGMCC No.1258 and the bifidobacterium animalis CGMCC No.9273 comprises the following steps:

(1) preparation of bacterial sludge

Respectively culturing Lactobacillus plantarum CGMCC No.1258 and Bifidobacterium animalis CGMCC No.9273 at 37 deg.C for 24 hr to obtain fermentation broth; wherein the components of the culture medium are as follows: angel yeast extract powder FM 8085 g/L, glucose 20g/L, sodium acetate 5g/L, citric acid diamine 2g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, Angel yeast peptone 10g/L, use food-grade sodium hydroxide with concentration of 2mol/L to adjust pH to 7.0 +/-0.1; centrifuging the fermentation broth liquid at 4 deg.C and 4000rpm for 10min, and discarding supernatant to obtain bacterial mud.

(2) Preparation of probiotic microcapsules

5.0g of dextran 40 was weighed out and added to 100.0mL of distilled water to dissolve. Then 2.0g of sodium alginate powder is weighed and dissolved in the prepared dextran 40 solution, and the solution is kept stand and dissolved. Then, the obtained solution is autoclaved at 121.0 ℃ for 15min, and after standing and cooling, the solution is prepared by mixing the following components in a mass ratio of 4: 1, forming liquid drops through a microcapsule granulator under the vibration frequency of a peristaltic pump with the pumping speed of 15 ml.min < -1 > and 2000Hz, vertically dropping the liquid drops into 0.3M CaCl2 solution, solidifying for 40min, washing and filtering for later use.

(3) Chitosan hydrochloride coating

Diluting chitosan hydrochloride to 7% (m/v), autoclaving at 121.0 deg.C for 15min, and cooling. And (3) adding the product obtained in the step (2) into chitosan hydrochloride solution, magnetically stirring for 60min, washing and filtering to obtain the microcapsule.

(4) Freeze drying

And (3) adding the microcapsule obtained in the step (3) into a dextran 40 solution with the volume fraction of 5.0%, balancing for 30min, then placing the mixture into a refrigerator with the temperature of minus 45.0 ℃ for pre-freezing for 6h, and then carrying out vacuum freeze drying for 36h at the temperature of minus 40.0 ℃ to obtain the probiotic microcapsule preparation.

Example 5 colitis mouse treatment experiment

Animal grouping and treatment: male Balb/c mice were randomly divided into 7 groups, which were a normal group, a model group, a negative control (normal saline, NS) group, a monoclonal antibody group, a probiotic group, and a probiotic-combined monoclonal antibody-treated group, 10 mice per group. The normal group freely drinks double distilled water, and the other 5 groups freely drink 2.5% DSS 7d to establish an acute UC animal model. Starting from 2d before the model building, the negative group, the monoclonal antibody group, the probiotic group and the probiotic combined monoclonal antibody group are respectively administrated with sterile physiological saline 0.2ml each time, and are perfused for 2 times/d, monoclonal antibodies (20 mg/kg of the monoclonal antibody of the embodiment 2 is injected in the abdominal cavity, and 1 time/2 d), probiotic microcapsules 10mg/d and monoclonal antibodies (20 mg/kg of the monoclonal antibody of the embodiment 2 is injected in the abdominal cavity, and 1 time/2 d) are perfused with probiotic microcapsules 10 mg/d. And (3) killing the mice by a 8 th morning neck-bending method, taking out the colon, taking part of the far-end colon tissue, fixing with formaldehyde, embedding in paraffin, slicing, and carrying out HE staining. The body weight, stool characteristics and occult blood condition of the mice were recorded daily during the experiment. The DAI integration was performed on the basis of the combination of mouse body mass, stool characteristics and occult blood, and the results are shown in table 1.

Table 1 enteritis mice groups DAI scores

Group of	n	DAI score
			Normal group	10	0.00±0.00
Model set	10	6.90±2.01
			Negative control group	10	6.88±1.98
Monoclonal antibody group	10	2.38±0.85
			Probiotic group	10	3.84±0.90
Probiotic combined monoclonal antibody treatment group	10	1.29±0.32

As can be seen from Table 1, the normal control group mice were normal in diet, activity and stool characteristics, and had increased quality to various degrees. The remaining 5 groups of mice developed weight loss, dull hair, decreased activity, listlessness, mucous stool at 3d of molding, and severe cases with visual bloody stool accompanied by various degrees of quality loss. The DAI integral of the mice in the normal group is always maintained at a zero level, and the difference compared with each group has statistical significance (P < 0.05); the DAI score of mice in the model group is relatively higher than that of other groups, the DAI score of the monoclonal antibody group, the probiotic group and the probiotic combined monoclonal antibody treatment group is maintained at a relatively low level, and compared with the model group and the negative group, the difference is statistically significant (P < 0.05); the DAI integral of the mice in the model group is up to 6.90, the minimum value of the combined treatment group reaches 1.29 +/-0.32, and the DAI integral is obviously reduced.

Second, histological lesion scoring: according to the Dielemen et al scoring criteria. Inflammation: none (score 0), mild (score 1), severe (score 2); lesion depth: none (0 min), submucosa (1 min), muscularis (2 min), serosa (3 min); crypt disruption: none (score 0), basal 1/3 crypts were destroyed (score 1), basal 2/3 crypts were destroyed (score 2), only intact surface epithelium (score 3), all crypts and epithelium were destroyed (score 4); lesion range (%): 0-25 for 1min, 26-50 for 2 min, 50-75 for 3 min, and 76-100 for 4 min. More than 10 high power fields (400 times) are randomly selected for each section to be scored, and the average value is taken as the score of histological damage. The results are shown in Table 2.

TABLE 2 histological scoring of enteritis mice in each group

Group of	n	Histological scoring
			Normal group	10	0.00±0.00
Model set	10	10.56±1.89
			Negative control group	10	10.38±1.69
Monoclonal antibody group	10	5.20±0.77
			Probiotic group	10	6.93±0.56
Probiotic combined monoclonal antibody treatment group	10	2.01±0.21

As can be seen from the results in Table 2, the normal mice had intact colonic mucosa, well-arranged glands, normal crypts, no reduction in goblet cells, no mucosal erosion, no bleeding, and no inflammatory cell infiltration. The model control group and the negative control group have colonic mucosa loss, incomplete glands, inflammatory cell infiltration and typical inflammatory changes. The monoclonal antibody group, the probiotic group and the probiotic combined monoclonal antibody group have light mucosal injury degree, the infiltration of inflammatory cells is reduced, the histological score is reduced, the difference between the histological score and the model and the negative control group has statistical significance (P is less than 0.05), particularly, the histological score of the probiotic combined monoclonal antibody treatment group is only 2.01 +/-0.21, and a better treatment effect is achieved.

Immunohistochemical staining method: immunohistochemical SABC method for detecting expression of mouse colonic mucosa hsp70 and JNK was performed according to the procedures of the SABC staining kit instructions, in which PBS was used as a negative control instead of primary antibody. And (5) judging the result to be positive by the brown yellow particles, and automatically recording the average optical density of the positive cells by a computer image analysis system. The results are shown in FIG. 2.

As can be seen from FIG. 2, the HSP70 content in the normal group is higher, the content in each experimental group is reduced, the expression level of the HSP70 can be increased after the treatment by the monoclonal antibody and the probiotics, the difference has statistical significance of less than 0.05), and particularly the expression level of the HSP70 in the probiotics and monoclonal antibody combined treatment group reaches 0.57 +/-0.04. The expression of JNK is obviously increased in a model group and a negative control group when the JNK is expressed in a normal group in a micro manner, the expression of the JNK can be inhibited after the monoclonal antibody and the probiotics are applied for treatment, the difference has statistical significance (P is less than 0.05), and particularly the expression level of the JNK reaches 0.08 +/-0.005 after the probiotics and the monoclonal antibody combined treatment group are used for treatment. The research result shows that the monoclonal antibody and the probiotics have better treatment effect, and one of the action mechanisms of the monoclonal antibody and the probiotics is probably to achieve the treatment effect on the UC by inhibiting a JNK signal conduction pathway and increasing the expression of heat shock protein.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

<110> Beijing Chuangshi Biotech Co., Ltd

<120> use of probiotic-containing preparations for treating gastrointestinal disorders

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85 90 95

Ala Gly His Trp Asn His Thr Tyr Met Trp Gly Leu Gly Thr Thr Leu

100 105 110

Ala Val Ser Ser

115

Claims (5)

1. A kit for the treatment of enteritis, consisting of monoclonal antibody 2a4 specific for MAdCAM-1 and probiotic bacteria; wherein the variable region of the light chain of the monoclonal antibody 2A4 of the MAdCAM-1 is shown as SEQ ID NO: 1, the heavy chain variable region sequence is shown as SEQ ID NO: 2 is shown in the specification; the probiotics are microcapsules prepared from lactobacillus plantarum CGMCC No.1258 and bifidobacterium animalis CGMCC No. 9273.

2. Use of a monoclonal antibody specific for MAdCAM-1 and a probiotic for the preparation of a kit for the treatment of enteritis; wherein the variable region of the light chain of the monoclonal antibody 2A4 of the MAdCAM-1 is shown as SEQ ID NO: 1, the heavy chain variable region sequence is shown as SEQ ID NO: 2 is shown in the specification; the probiotics are microcapsules prepared from lactobacillus plantarum CGMCC No.1258 and bifidobacterium animalis CGMCC No. 9273.

3. Use according to claim 2, characterized in that the monoclonal antibody 2a4 is in the form of a liquid formulation having a pH between 6.3 and 7.0.

4. The use of claim 3, wherein the stable liquid pharmaceutical formulation comprises an antibody, a buffer, and at least about 10mM citrate.

5. Use according to claim 4, characterized in that the formulation is present in a container vial, cartridge, syringe or auto-injector.

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