Preparation method of probiotics and application thereof in treating gastrointestinal diseases

Abstract

The present invention relates to a preparation method of probiotics and application thereof in treating gastrointestinal diseases. The present invention optimizes and prepares a specific probiotic composition of Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1, which has the effect of inhibiting Helicobacter pylori. The probiotics and Helicobacter pylori monoclonal antibodies are used together to better inhibit Helicobacter pylori and its adhesion function. Moreover, the combination also has good safety and is suitable for treating gastrointestinal diseases caused by Helicobacter pylori interference.

Description

Technical Field

[0001] The present application relates to the biological field, and more specifically to a method for preparing probiotics and an application thereof in treating gastrointestinal diseases. Background Art

[0002] The intestinal microbial ecosystem is the largest and most complex micro-ecosystem in the human body, in which there are a large number of various bacteria of different contents and types, most of which are acquired in childhood. The balance of the digestive tract micro-ecosystem is achieved through antagonism and symbiosis between different species of bacteria, as well as the interaction between microorganisms and hosts, which is very important for preventing intestinal disorders and pathogenic bacteria infections. Once this balance is broken, it is easy to cause dysbiosis, providing opportunities for the growth of intestinal pathogens and conditional pathogens, resulting in the formation of intestinal diseases. Chronic gastritis, gastric ulcers, diarrheal diseases, chronic enteritis and other diseases caused by intestinal pathogens cause about two million deaths worldwide each year. Antibiotics are usually used clinically to treat such diseases, which will increase the drug resistance of pathogens and aggravate the condition. Therefore, in view of the problem of pathogenic bacteria infection and its resistance to multiple antibiotics, probiotic preparations with no residue, no drug resistance and no toxic side effects undoubtedly have broad development and application prospects. Currently, many probiotics have been used in clinical practice to prevent or treat various gastrointestinal diseases, including infectious diarrhea, Helicobacter pylori infection, neonatal enteritis, chronic colitis, irritable bowel syndrome and inflammatory bowel disease.

[0003] As the most widely used probiotic, Lactobacillus inhibits pathogens in vivo mainly by limiting the growth environment of pathogens, mainly by producing a large amount of short-chain fatty acids (lactic acid, acetic acid, propionic acid, etc.), fatty acids (phenyllactic acid, p-hydroxyphenyllactic acid), hydrogen peroxide, bacteriocins or peptides, etc., which inhibit the growth of pathogens, thereby playing a biological barrier role and inhibiting the adhesion of pathogens to intestinal epithelial cells. Bacteriocins are currently hot antibacterial substances. Several bacteriocins produced by lactobacilli have been reported, but the antibacterial activity of these bacteriocins is different. Nisin is a natural food preservative recognized as safe in the world; to date, more than 50 countries and regions have applied it in the food industry. However, Nisin can only inhibit Gram-positive bacteria, and generally has no inhibitory effect on Gram-positive bacteria, yeast, mold and virus. It is powerless against enteric pathogens such as Escherichia coli, Salmonella and Shigella. In addition, it has low solubility and instability in neutral and alkaline environments, which greatly reduces the antibacterial effect. With the deepening of research, people's research goals are mainly focused on the development of broad-spectrum bacteriocins. For example, the antimicrobial peptides produced by the probiotic L. salivarius subsp. salivarius UCC118 have broad-spectrum antibacterial effects and have inhibitory effects on Bacillus, Staphylo-coccus, Enterococcus, Listeria and Salmonella bacteria; the bacteriocin produced by Lactococcus lactis subsp. can effectively inhibit the growth of C. diffi-cile. Lactic acid is an important antibacterial compound produced by lactic acid bacteria. Undissociated lactic acid has high lipid solubility and can be integrated into the cell membrane and penetrate into the cytoplasm of specific intestinal pathogens in the form of undissociated lactic acid molecules, acidifying the cytoplasm and destroying the substrate transport, energy supply, and macromolecular synthesis of the cell membrane to achieve an antibacterial effect. Lactobacillus and Bifidobacterium can metabolize oligosaccharides and amino acids that are not absorbed by humans in the intestine to produce this organic acid, mainly including lactic acid, acetic acid, propionic acid, succinic acid, phenyllactic acid, and hydroxyphenyllactic acid. The pH value in the intestine is lowered by producing organic acids. In a low-acid environment, the pH difference between the inside and outside of the pathogenic bacterial cell membrane can enhance the cytotoxicity of undissociated organic acids.

[0004] Helicobacter pylori (Hp) is a Gram-negative bacterium that colonizes the gastrointestinal tract for a long time. It is also a pathogenic factor for gastrointestinal diseases ranging from chronic active gastritis to peptic ulcer, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. At present, the main method for the treatment of Hp is triple or quadruple therapy with PPI plus antibiotics. However, the use of antibiotics to treat Hp infection can cause intestinal flora imbalance and mucosal barrier damage in the digestive system, causing many adverse reactions. Intestinal probiotics play an important role in human health, such as providing essential nutrients, synthesizing vitamin K, assisting in the digestion of cellulose, promoting angiogenesis and intestinal nerve function, etc. Studies have shown that some probiotic strains can regulate intestinal flora and mucin expression, enhance the gastric mucosal barrier, and protect it from the adhesion of H. pylori. Therefore, the use of probiotics to eradicate Hp can maintain the balance of intestinal flora and protect the mucosal barrier, providing a new idea for the treatment of H. pylori.

[0005] Currently, although the ideal treatment for eradication of H. pylori has not yet been determined, probiotics (Lactobacillus, Bifidobacterium, and yeast) have been suggested as adjuncts to antibiotics for the treatment of H. pylori. Among more than 700 wild-type strains of the genus Lactobacillus, Lactobacillus reuteri DSM17648 was found to be a highly specific binding antagonist to H. pylori. Strain DSM17648 coaggregates pathogens in vivo and in vitro, and it undergoes specific copolymerization reactions with different H. pylori strains and serotypes, as well as H. heilmannii, but not with Campylobacter jejuni or other commensal oral and intestinal bacteria. In a single-blind, randomized, placebo-controlled pilot study, it was shown that Reuteri DSM17648 significantly reduced the load of healthy adults who were not yet infected with H. pylori. Reducing the amount of H. pylori in the stomach through selective bacteria-bacterial cell interactions may be an effective and novel approach to combat gastric pathogens. Reuteri DSM17648 may prove to be used as an adhesion blocker in antibiotic-free H. pylori therapy. The effect of probiotics on the changes in intestinal microbiota after H.pylori eradication was studied. The experiment was divided into two groups: the antibiotic group received general treatment, and the probiotic group received general treatment and probiotic supplementation. The results showed that before treatment, Firmicutes, Bacteroidetes, and Proteobacteria were dominant; after treatment, the relative abundance of Firmicutes decreased, while the relative abundance of Proteobacteria increased in both groups. However, the proportion of intestinal microbiota in the antibiotic group changed more than that in the probiotic group, and the increase in the level of antibiotic-resistant bacteria in the antibiotic group was higher than that in the probiotic group. The results showed that probiotic supplementation can reduce the changes and imbalances in the composition of intestinal microbiota caused by antibiotics. This effect may limit the growth of antibiotic-resistant bacteria in the intestine and improve the success rate of H.pylori eradication.

[0006] However, at present, there are not enough types of drugs to treat Helicobacter pylori, especially the treatment method using probiotics combined with biological drugs needs further research and development. Summary of the invention

[0007] The present invention provides a monoclonal antibody specifically targeting Helicobacter pylori.

[0008] Specifically, the Helicobacter pylori monoclonal antibody is 8D14.

[0009] Furthermore, the variable region sequences of the Helicobacter pylori monoclonal antibody 8D14 are:

[0010] The amino acid sequence of the light chain variable region is:

[0011] AYQMTQSPSSVSASVGDRVTITCDLEAPIAVDTMWYQQKPGKAPKLLIYDRGEDIPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCWMYMETRRKDTWIFGGGTKVEIK;

[0012] The amino acid sequence of the heavy chain variable region is:

[0013] EVQLVESGGGLVQPGGSLRLSCAASGFSLSGEFWLWVRQAPGKGLEWVGGALWNFVLWFAGCFQIRFTISKDNSKNTLYLQMNSLRAEDTAVYYCARHPRNSLSCCHDRMWGQGTLVTVSS.

[0014] Furthermore, the specific light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2, or a variable region sequence with 90% homology thereto.

[0015] In the description of the present invention, at least 90% sequence similarity should be understood to include at least 95%, more preferably at least 99% sequence similarity. In this case, "sequence similarity" is a combination of the degree of identity and the degree of conservative change. The "sequence similarity" percentage is the percentage of identical or conservatively changed amino acids or nucleotides, i.e., "sequence similarity" = sequence identity percentage + conservative change percentage. Therefore, for the purposes of the present invention, "conservative change" and "identity" are considered to be broad "similarity" types. Therefore, whenever the term sequence "similarity" is used, it includes sequence "identity" and "conservative change". According to certain embodiments, conservative changes are not taken into account, and the sequence similarity percentage refers to the sequence identity percentage. In certain embodiments, the changes in the sequence allowed by the sequence identity percentage mentioned are all or almost all conservative changes. That is, when a sequence is 90% identical, the remaining 10% are all or almost all conservative changes. In this article, the term "almost all" means that at least 75% of the allowed sequence changes are conservative changes, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

[0016] Furthermore, the present invention also provides the use of the Helicobacter pylori monoclonal antibody in preparing a pharmaceutical composition for inhibiting Helicobacter pylori.

[0017] Furthermore, the present invention also provides the use of the Helicobacter pylori monoclonal antibody in preparing a pharmaceutical composition for inhibiting the adhesion of Helicobacter pylori.

[0018] Furthermore, the pharmaceutical composition for preventing or treating Helicobacter pylori according to the present invention may further comprise a pharmaceutical carrier, excipient or diluent, in addition to the above-described active ingredients, for administration. The above-mentioned carrier, excipient and diluent may be exemplified by lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0019] Further, the pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injections according to conventional methods. Specifically, commonly used fillers, weighting agents, adhesives, wetting agents, disintegrants, surfactants and other diluents or excipients can be used for formulation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., but are not limited thereto. Such solid preparations can be mixed with at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in the compound or pharmaceutical salt represented by the above chemical formula 1. In addition, lubricants such as magnesium stearate and talc can also be used in addition to simple excipients. For oral liquids, various excipients, wetting agents, sweeteners, aromatics, preservatives, etc. can be added in addition to liquid paraffin. Non-oral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and granules. As non-aqueous solvents and suspensions, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate can be used. As the main agent of suppositories, semi-synthetic fatty acid esters (witepsol), polyethylene glycol (macrogol), Tween 61, cocoa butter, laurin, glyceryl gelatin, etc. can be used.

[0020] Furthermore, the present invention also provides use of a probiotic composition in preparing a drug for inhibiting Helicobacter pylori; the probiotic is prepared from Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1 in a ratio of 1:1.

[0021] Furthermore, the present invention also provides the use of a monoclonal antibody combined with a probiotic composition against Helicobacter pylori in the preparation of a medicine kit for inhibiting Helicobacter pylori; the probiotics are prepared from Lactobacillus acidophilus ATCC4356 and Clostridium butyricum CGMCC0313.1 in a ratio of 1:1.

[0022] The kit of the invention preferably administers the strain or composition at least once a week over a treatment period of at least 4 weeks, and preferably at least 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18 or 20 weeks. Preferably, the probiotic composition is administered several times a week, and ideally once a day. The composition of the invention typically comprises between 10 g of dry weight of the composition. 3 cfu with 10 12 cfu of the strain of the present invention. In one embodiment, the composition comprises 10 3 and 10 12 cfu, or 10 4 and 1012 cfu, or 10 6 and 10 10 The daily dose usually includes between 103 and 10 12 In one embodiment, the daily dose includes 10 3 and 10 12 cfu, or 10 4 and 10 12 cfu, or 10 6 and 10 10 cfu of the strain of the present invention.

[0023] The term "treating" of the present invention is generally understood to mean alleviating or managing the symptoms of a pathology, curing the pathology, preventing the development of the pathology, or inhibiting the development of the pathology.

[0024] In various embodiments, the medicine or medicine box also includes an excipient or carrier. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol and water. The selection of pharmaceutical carriers, excipients or diluents can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable adhesive, lubricant, suspending agent, coating agent, solubilizing agent as a carrier, excipient or diluent or in addition to a carrier, excipient or diluent. Examples of suitable adhesives include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, -lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and parabens. Antioxidants and suspending agents may also be used.

[0025] In some embodiments, the medicament or kit further comprises maltodextrin, inulin, inositol, trehalose, microcrystalline cellulose (MCC), calcium lactate, magnesium stearate, fructooligosaccharides (FOS) or glucose oligosaccharides (GOS).

[0026] In certain embodiments, the medicament or kit further comprises a surfactant or an emulsifier.

[0027] In some embodiments, the surfactant includes polysorbate 20 and / or polysorbate 80.

[0028] Other non-limiting examples of surfactants include lecithin, monoglycerides, sorbitan esters, sorbitol esters, sucrose esters, glycolipids, fatty alcohols, fatty acids, benzalkonium chloride, cetylpyridinium chloride, sodium dodecylbenzene sulfonate, polyethoxylated octylphenol, n-dodecylpyridinium chloride, lauryl monoethanol, sorbitan monoesters, dimethyl tetradecylphosphonate, diglycerides, dodecyl betaine, anionic surfactants, cationic surfactants, nonionic surfactants, zwitterionic surfactants and gemini surfactant ethoxylates.

[0029] In certain embodiments, the medicament or kit comprises a lyophilized powder-based composition.

[0030] Beneficial Effects

[0031] The present invention optimizes and prepares a specific probiotic composition of Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1, which has the effect of inhibiting Helicobacter pylori. The probiotics are used together with Helicobacter pylori monoclonal antibodies to effectively inhibit Helicobacter pylori and its adhesion function. Moreover, the combination also has good safety and is suitable for the treatment of gastrointestinal diseases caused by Helicobacter pylori interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Antibacterial effect of different probiotics

[0033] Figure 2 The inhibitory effect of Helicobacter pylori monoclonal antibody on Helicobacter pylori

[0034] Figure 3 Effects of probiotics and / or monoclonal antibodies on Helicobacter pylori adhesion to GES-1 cells DETAILED DESCRIPTION

[0035] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conditions recommended by normal conditions or manufacturers. The reagents used or the instruments not specified by the manufacturer are all conventional products that can be purchased from the market.

[0036] Example 1 Preparation of probiotics and anti-Helicobacter pylori experiment

[0037] The activated Lactobacillus acidophilus ATCC 4356 strain (Fuxiang Biotechnology) and Clostridium butyricum CGMCC0313.1 (Ataining, Donghai Pharmaceutical) were inoculated into a 250ml sealed triangular flask containing 200ml fermentation medium at 5% and then cultured. The fermentation medium consisted of 1.5% glucose, 1.5% motherwort powder, 0.5% beef extract, 0.2% KH2PO4, 0.1% MgSO4, 0.1% cysteine, 0.1% NaHCO3, and natural pH. The culture temperature was 36°C, the inoculation amount was 5%, and the culture time was 24h. By cell counting, the concentrations of the two bacterial solutions were adjusted to 1×10 8 CFU / ml for future use.

[0038] Helicobacter pylori ATCC43504 was inoculated into nutrient broth and cultured overnight at 37°C in a shaking incubator. The bacterial concentration was adjusted to 1×10 7 cfu / mL, and applied to Skirrow blood agar plates containing 4% TTC. After there were no obvious water droplets, sterile Oxford cups were placed equidistantly on each plate, and 240 μL of bacterial suspension from different experimental groups was added to each Oxford cup. Experimental group 1 used ATCC4356 alone, experimental group 2 used CGMCC0313.1 alone, and experimental group 3 used ATCC4356 and CGMCC0313.1 in a 1:1 ratio. Liquid MRS was used as a negative control, and aspirin with a final concentration of 5 mmol / L was used as a positive control. Two parallel experiments were performed. The blood agar plates were cultured in a 37°C microaerobic incubator for 40 hours, and the size of the inhibition ring was measured. The results are as follows Figure 1 shown.

[0039] from Figure 1 The results show that the experimental group and the positive control group are significantly different from the negative control group (P<0.01). And from the experimental results, it can be seen that the size of the inhibition zone when using ATCC4356 or CGMCC0313.1 alone is significantly different from the size of the inhibition zone when using ATCC4356 and CGMCC0313.1 at the same time, which shows that the killing substances secreted by ATCC4356 and CGMCC0313.1 can synergistically inhibit and kill Helicobacter pylori, with a better effect.

[0040] Example 2 Preparation of monoclonal antibody against Helicobacter pylori ATCC43504

[0041] Helicobacter pylori ATCC43504 was inactivated, emulsified with Freund's complete adjuvant, and then intraperitoneally immunized three mice for the first time. The inoculation amount of inactivated bacteria was 10 7 / mouse; the second and third intraperitoneal immunizations were performed with inactivated bacteria emulsified with Freund's incomplete adjuvant, with an interval of 2 weeks and the immunization doses as before. After the third immunization, the serum titer was measured, and the mouse No. 2 with the highest titer was selected to obtain spleen cells at a ratio of 1:20000 for cell fusion. Mouse myeloma Sp2 / 0 cells in the logarithmic growth phase were fused with spleen cells of immunized mice under the mediation of PEG4000, and specific hybridoma cells were screened by indirect ELISA. A total of 59 cell strains were cloned by the limiting dilution method, and these 59 positive cells were subcloned 4 times. The cloned cell lines were continuously passaged for 3 months to detect the titer of the culture supernatant, and the 7 monoclonal antibodies with the highest supernatant titer were selected. According to a similar method in Example 1, the amount of supernatant added was also 240μL, and the results are as follows Figure 2 shown.

[0042] from Figure 2 It can be seen that the inhibition ring size of the two monoclonal antibodies 1A15 and 8D14 is the best. Therefore, these two monoclonal antibodies were selected for subsequent experiments. In addition, the two monoclonal antibodies were respectively expanded and prepared. Specifically, four BALB / c mice were taken, and hybridoma cells in the logarithmic growth phase were collected. Each mouse was intraperitoneally injected with 10 7 cells until the mouse abdomen swells for about 10 days, then the ascites is extracted and centrifuged (4°C, 3000r / min, 15min). The supernatant is the mouse ascites monoclonal antibody, which is purified by column purification and stored in aliquots at -70°C.

[0043] The titer of the purified antibody was determined by indirect ELISA. The inactivated Helicobacter pylori was coated on an ELISA plate, and the ascites mAb was used as the primary antibody for 2×10 4 , 4×10 4 , 8×10 4 , 1.6×10 5 , 3.2×10 5 , 6.4×10 5 , 1.28×10 6 , 2.56×10 6 , 5.12×10 6 The OD value was measured after OPD color development using Sigma's horseradish peroxidase-labeled goat anti-mouse antibody as the secondary antibody. 492nm The results are shown in Table 1.

[0044] Table 1 Titers of monoclonal antibodies against two strains of Helicobacter pylori

[0045] mAbs Ascites titer 1A15 <![CDATA[2.56×10 6 ]]> 8D14 <![CDATA[5.12×10 6 ]]>

[0046] From the results in Table 1, we can see that the titers of the two monoclonal antibodies 1A15 and 8D14 are relatively high, both reaching 106 level.

[0047] Example 3 Characterization of 8D14 Monoclonal Antibody

[0048] Flow cytometer detection specificity: Helicobacter pylori ATCC43504, Escherichia coli, GES-1 human gastric mucosal cells, and HepG2 human liver cancer cells were digested with trypsin and the cell concentration was adjusted to 3×104 / ml; 100ul of cell suspension was added to each tube, 20ul of purified 8D14 monoclonal antibody was added, and after reacting at room temperature for 0.5h, FITC-labeled goat anti-mouse secondary antibody was added, and after washing 3 times, the cells were detected by flow cytometer, and immune mouse serum was used as positive control, and PBS was used as blank control. The results are shown in Table 2.

[0049] Table 2 Cell binding rate (%)

[0050] 8D14 monoclonal antibody Cell binding rate (%) Helicobacter pylori ATCC43504 99.7±0.09 Escherichia coli 0.14±0.02 GES-1 human gastric mucosal cells 0.20±0.03 HepG2 human hepatoma cells 0.10±0.01

[0051] From the flow cytometry test results in Table 2, it can be seen that the 8D14 monoclonal antibody reacts positively with Helicobacter pylori with a binding rate of more than 99%, but does not bind to other cells, and has good specificity.

[0052] Subclass identification: Inactivated Helicobacter pylori was used to coat the ELISA plate, 100 μl / well. The monoclonal antibody was used as the primary antibody and serially diluted at 1:4000. SouthernBiotech's goat anti-mouse IgG1, IgG2a, IgM, and IgA (horseradish peroxidase labeled) were used as the secondary antibody (1:5000) for indirect ELISA. According to the results of ELISA, 8D14 could react with HRP-labeled goat anti-mouse IgG1 and belonged to the IgG1 subclass.

[0053] Affinity identification: The affinity constant (Ka) was determined by non-competitive ELISA. During coating, Helicobacter pylori ATCC43504 was diluted with carbonate buffer. The 8D14 monoclonal antibody was diluted in multiples starting from 100 μg / mL, and 4 coating original concentrations were added respectively. The other steps were the same as the determination of monoclonal antibody titer. The absorbance value at a wavelength of 450nm was measured. Referring to the method of Raghava et al., the affinity constant Ka value was calculated according to the formula Ka=(n-1) / 2(n[Ab']t-[Ab]t). The results showed that the affinity constant Ka of 8D14 monoclonal antibody was 7.9×109L / mol.

[0054] Monoclonal antibody gene sequencing: Total RNA was extracted from the monoclonal antibody cell line and reverse transcribed into cDNA. The heavy chain variable region and light chain variable region of the antibody were then PCR amplified using cDNA as a template. Total RNA was extracted using the RNA extraction kit from Invitrogen according to its instructions. Then, Takara's 5'RACE FULL kit was used, with total RNA as a template and the random primers in the kit reverse transcribed into the first-strand cDNA. The heavy chain was then PCR amplified using the constant region designed primer CTCAGGGAARTARCCYTTGAC and the adapter primer in the kit. The light chain was PCR amplified using the constant region designed primer TCACTGCCATCAATCTTCCAC and the adapter primer in the kit. The PCR fragment was recovered using the agarose gel recovery kit for TA cloning and then adjusted to a single clone for PCR identification. The identification primers were M13-F and M13-R. The identified correct strain was sent to Invitrogen for sequencing. The light and heavy chain variable region sequences of the 8D14 monoclonal antibody were finally determined to be: the light chain variable region protein sequence was SEQ ID NO: 1, and the heavy chain variable region protein sequence was SEQ ID NO: 2.

[0055] Example 4 Urease inhibition test of 8D14 monoclonal antibody and probiotics on Helicobacter pylori adhesion to GES-1 cells

[0056] Helicobacter pylori ATCC43504 was inoculated into nutrient broth and cultured overnight at 37°C in a shaking incubator. The bacterial concentration was adjusted to 1×10 7 GES-1 cells were cultured in 96-well cell plates for 2 days to form a monolayer and then washed 3 times with PBS.

[0057] Add test bacteria and antibodies to the cell wells according to the following groups:

[0058] Experimental group 1: 50 μl of Helicobacter pylori ATCC43504, 50 μl of Lactobacillus acidophilus ATCC4356, and 50 μl of Clostridium butyricum CGMCC0313.1. The concentration of the latter two bacterial solutions was 1×10 8 CFU / ml;

[0059] Experimental group 2: 50 μl of Helicobacter pylori ATCC43504, 50 μl of Lactobacillus acidophilus ATCC 4356, and 50 μl of Clostridium butyricum CGMCC0313.1. The concentration of the latter two bacterial solutions was 1×10 8 CFU / ml; then add 50μg / mL of 8D14 monoclonal antibody

[0060] Experimental group 3: 50 μl of Helicobacter pylori ATCC43504 and 50 μg / mL of 8D14 monoclonal antibody Experimental group 4: 50 μl of Helicobacter pylori ATCC43504 and 100 μg / mL of 8D14 monoclonal antibody

[0061] Experimental group 5: 50 μl of Helicobacter pylori ATCC43504 and 50 μl of Lactobacillus acidophilus ATCC 4356, the latter of which had a bacterial concentration of 1×10 8 CFU / ml;

[0062] Experimental group 6: 50 μl of Helicobacter pylori ATCC43504 and 50 μl of Clostridium butyricum CGMCC0313.1, the latter bacterial solution concentration was 1×10 8 CFU / ml;

[0063] Each group was directly incubated for 2 hours and then washed 3 times with PBS. Each reaction well was repeated 3 times, and then 100 μL of urease reagent was added to each well for 30 minutes. The absorbance at 540 nm was detected on a microplate reader. GES-1 containing only Helicobacter pylori was used as a positive control. The adhesion calculation formula was: Helicobacter pylori adhesion = (OD positive control-OD experimental) / OD positive control.

[0064] from Figure 3 The results show that Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1 can effectively inhibit the adhesion of Helicobacter pylori to GES-1 cells. In particular, after the monoclonal antibody is used together with Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1 to act on Helicobacter pylori, its adhesion rate is only (4.8±0.3)%, while the adhesion rate of 8D14 monoclonal antibody used alone at 100μg / mL also reaches (8.1±1.1)%, indicating that the monoclonal antibody itself also has a good inhibitory effect on adhesion.

[0065] Example 5 Toxicity experiment in mice

[0066] Twenty 18-22g BALB / c mice were randomly divided into a normal group, a bacterial solution group, and an 8D14 monoclonal antibody group, with 10 mice in each group, and half of each group was male and half was female. Each mouse in the normal group was orally gavaged with 0.5mL of normal saline (0.85%), and each mouse in the bacterial solution group was orally gavaged with 0.5mL of fresh Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1 bacterial solution (1.0×10 10 CFU / mL), and then intraperitoneally injected with monoclonal antibody 100ug / mouse for 3 consecutive days, and the body weight was weighed every day. The mice were observed for 1 week, and the liver, heart, kidney, lung, stomach, and spleen were weighed after being killed, and the organ index was calculated according to the following formula: Organ index = organ weight / mouse body weight.

[0067] Table 3 Organ index

[0068]

[0069]

[0070] During the entire experiment, the mice in the saline group, the bacterial solution group, and the 8D14 monoclonal antibody group did not show obvious activity or behavioral changes, and no disease or death occurred. At the same time, there was no statistically significant difference in body weight between the saline group and the bacterial solution and 8D14 monoclonal antibody group mice (P>0.05, Table 3). In addition, the organ weights and organ indexes of the bacterial solution and monoclonal antibody group mice were not statistically significant compared with the saline group (P>0.05, Table 3). This shows that the 8D14 monoclonal antibody and probiotics of the present invention have good safety.

[0071] It should be understood that the present invention is not necessarily limited to the details of the construction and arrangement of the components described in the following description and / or illustrated in the accompanying drawings in its application. The present invention can have embodiments other than those embodiments described and practiced or performed in different ways. Moreover, it should be understood that the phrases and terms and abstracts adopted herein are for descriptive purposes and should not be considered restrictive. Although the present invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use the present invention, various substitutions, modifications and improvements should be apparent without departing from the spirit and scope of the present invention. The embodiments provided herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Those skilled in the art will think of modifications and other uses therein. These modifications are included in the spirit of the present invention and are limited by the scope of the claims.

[0072] Unless otherwise stated, the use of "or" herein means "and / or." Similarly, "comprising," "including," "having," "containing," and "having" are interchangeable and not limiting.

[0073] It will be further understood that, while the description of various embodiments uses the terms "comprising" / "including", those skilled in the art will understand that in some specific cases, the language "consisting essentially of" or "consisting of..." may alternatively be used to describe such embodiments.

[0074] Thus, those skilled in the art will appreciate that the concepts based on this disclosure can be readily used as a basis for designing other structures, methods and systems for carrying out the several purposes of the present invention. Therefore, it is important that the claims be deemed to include such equivalent structures to the extent that they do not depart from the spirit and scope of the present invention.

Claims

1. A Helicobacter pylori monoclonal antibody 8D14, characterized in that The amino acid sequence of the light chain variable region of the Helicobacter pylori monoclonal antibody 8D14 is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

2. Use of the Helicobacter pylori monoclonal antibody 8D14 as claimed in claim 1 in the preparation of a pharmaceutical composition for inhibiting Helicobacter pylori.

3. Use of the Helicobacter pylori monoclonal antibody 8D14 according to claim 1 in the preparation of a pharmaceutical composition for inhibiting the adhesion of Helicobacter pylori.

4. Use of the Helicobacter pylori monoclonal antibody 8D14 combined with a probiotic composition according to claim 1 in preparing a drug kit for inhibiting Helicobacter pylori; the probiotics are prepared from Lactobacillus acidophilus ATCC 4356 and Clostridium butyricum CGMCC0313.1 in a ratio of 1:

1.

5. The method according to any one of claims 2 to 4, characterized in that The pharmaceutical composition or medicine kit contains a pharmaceutically acceptable carrier.

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