CN112048008A

CN112048008A - Helicobacter pylori B cell tolerance epitope and antibody prepared from same

Info

Publication number: CN112048008A
Application number: CN202010806364.4A
Authority: CN
Inventors: 单保恩; 史中立; 赵连梅; 胡代伦
Original assignee: Hebei Medical University; Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
Current assignee: Hebei Medical University; Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2020-12-08
Anticipated expiration: 2040-08-12
Also published as: AU2021100512A4; CN112048008B

Abstract

The invention discloses a helicobacter pylori B cell tolerance epitope and a monoclonal antibody prepared by the epitope, which can be used for treating an infected person with helicobacter pylori natural immune tolerance and preventing children with maternal vertical transmission but no established infection. Has the advantages of small side effect, good patient compliance, easy large-scale production and the like.

Description

Helicobacter pylori B cell tolerance epitope and antibody prepared from same

Technical Field

The invention relates to a helicobacter pylori B cell tolerance epitope and a monoclonal antibody prepared by the helicobacter pylori B cell tolerance epitope.

Background

In 1983, Warren and Marshall successfully isolated helicobacter pylori from biopsy of gastric mucosa of patients with chronic gastritis (Helicobacter pyloriHp), it has been found through further studies that Hp infection is closely related to the occurrence of diseases such as chronic gastritis, peptic ulcer, gastric cancer, gastric MALT lymphoma, etc., and with the extensive and intensive research on Hp, some researchers believe that Hp infection is also related to the onset of diseases such as allergic purpura, coronary heart disease, diabetic complications, Parkinson's disease, senile dementia, etc. According to the latest epidemic data, about more than 50% of the world population is infected with the bacterium, the Chinese infection rate is 58.07%, and about more than 50% of the world pregnant women are infected with Hp.

Prevention and treatment of Hp infection is a key issue in the field of Hp research at present, and eradication of Hp is an important means for preventing recurrence of gastric ulcer, treating chronic gastritis, and preventing occurrence of gastric cancer. With the emergence of helicobacter pylori drug-resistant strains, the treatment of helicobacter pylori infection is more and more difficult now, the one-time radical treatment rate of the original antibiotics with stronger sensitivity can reach more than 90 percent, the radical treatment rate is reduced to 40 to 60 percent by using the same drugs at present, and the longer the treatment time is, the smaller the cure hope is. The research on the drug resistance rate of 600 Hp strains covering Beijing, Shanghai, Wuhan and Guangzhou shows that the overall drug resistance rate is very high and multiple drug resistance is presented, and the antibiotic drug resistance rates in different areas have no obvious difference. Therefore, the search for new therapeutic methods is urgent.

Disclosure of Invention

We have found in our studies that H.pylori can colonize the stomach wall of the host for long periods of time without being cleared by the immune system, because of natural immune tolerance, i.e. maternal pre-pregnancy infection with H.pylori, can be transmitted vertically to the foetus during pregnancy, and that the substance mediating vertical transmission is its exosome, i.e. Outer Membrane Vesicles (OMVs). Hp-secreted Outer Membrane Vesicles (OMVs) of 20-300 nm in size. OMVs are considered to be a special secretion vector which provides a means of transporting bacterial material to the bacterial outer space, in such a way that the bacteria are in signal communication with the external environment. OMVs of Hp contain various lipids and at least 100 different proteins, such as adhesins, lipopolysaccharides, peptidoglycans, VacA, CagA, etc. The Hp-infected mother is established before pregnancy, Hp-secreting OMVs carry biologically active substances across the gastric mucosa into the blood circulation and across the placental barrier into the embryo during pregnancy, and part of the content carried by OMVs is the "tolerogen" of the offspring immune system. Pathological tolerance to pathogen antigens may prevent the effective exertion of normal immune defenses, leading to the development of chronic persistent infections. This may be a major cause of the difficulty of helicobacter pylori clearance within the body's stomach wall, and a major cause of its potential for human evolution.

Helicobacter pylori is required to resist gastric motility, gastric emptying and the constant shedding and regeneration of gastric mucus layers, so helicobacter pylori must adhere strongly to the cell surface by means of adhesion molecules, while the molecules exerting adhesion are mainly located on the surface of the bacteria. Thus if antibodies against adhesion molecules are available, the binding of the antibodies to the adhesion molecules of the bacteria will cause the bacteria to lose their ability to adhere and thus colonize, and the ideal vaccine candidate antigen should be on the surface of the pathogen. The molecules located on the surface of H.pylori are mainly outer membrane proteins and Lipopolysaccharides (LPS). The H.pylori genome encodes most of the outer membrane proteins (about 64 outer membrane proteins) with approximately 4% of the genes, and these unique outer membrane proteins fulfill the need for H.pylori to adapt to the unique gastric environment. The Outer Membrane Protein (OMP) spectrum of H.pylori does not have a predominant content of proteins, but rather contains a number of outer membrane proteins of low abundance, compared to the outer membrane protein spectrum of other gram-negative bacteria. These outer membrane proteins are divided into five gene families based on gene similarity. Family 1 includes the hop family (21 members) and the hor family (12 members); family 2 includes hof family (8 members); family 3 includes the hom family (4 members); family 4 includes iron ion-regulated outer membrane proteins (6 members); family 5 includes pump efflux proteins (3 members), and 10 members are not included in the above classification. Among these outer membrane proteins, only a small part of them are confirmed for their functions, and the functions of many outer membrane proteins are not yet clear. Our earlier studies found that the OMV content contained outer membrane proteins, and therefore a portion of the outer membrane proteins contained within OMVs were "tolerogens" of the progeny immune system.

The invention provides a helicobacter pylori B cell tolerance epitope and an antibody prepared from the same, which can be used for preventing and treating helicobacter pylori infection by breaking natural immune tolerance and improving the Hp removing capability of an organism.

The invention adopts the following technical scheme:

a helicobacter pylori B cell tolerance epitope, which comprises an amino acid sequence shown as SEQ ID No. 1. The method comprises the following steps: YYGFFSYNGASVGFR are provided.

A nucleic acid molecule encoding the helicobacter pylori B cell tolerance epitope.

A vector comprising the nucleic acid molecule described above.

A monoclonal antibody prepared by using the helicobacter pylori B cell tolerance epitope.

Further, the above monoclonal antibody is used for the treatment of an infected person having natural tolerance to helicobacter pylori.

A vaccine prepared by using the helicobacter pylori B cell tolerance epitope, and the vaccine is used for stimulating the immune response of a host organism.

Further, the vaccine is used for preventing children who have maternal vertical transmission but have not yet established infection.

The screening method of the helicobacter pylori B cell tolerance epitope and the method for obtaining the monoclonal antibody thereof are as follows:

(1) separating and purifying helicobacter pylori OMV, quantifying protein by BCA method, preparing into 100 μ g/ml concentration, injecting BALB/c pregnant mouse from tail vein in 8, 12, 16 days, respectively, each time 0.1ml OMV; injection of 0.1ml PBS was used as a control group;

(2) after the offspring mice were born, they were grown to 8 weeks of age and then immunized with OMVs. Mixing OMV solution and Freund's complete adjuvant at a ratio of 1:1 (v/v), emulsifying, and performing primary immunization with subcutaneous injection at a dose of 100 μ g per rat; on week 3, the OMV solution and Freund's incomplete adjuvant were mixed at 1:1 (v/v) and emulsified thoroughly to boost the immune response at a dose of 50. mu.g. Boost was performed every 14d interval;

(3) coating an enzyme label plate with 100 mul OMV (0.1 mg/ml), detecting the serum antibody titer of the submouse by indirect ELISA, taking the submouse with the serum titer of the experimental group being more than 1: 8000 and the serum titer of the control group being more than 1: 12000, respectively carrying out 1-time boosting immunization, and taking the submouse serum and spleen B cells which are 3d after boosting immunization for later use;

(4) preparing two-dimensional glue of Hp total protein by using a two-dimensional electrophoresis technology, then transferring a membrane, detecting binding sites of an immune mouse serum antibody by using a WB technology, comparing the difference of the binding sites of the antibody with a control group, and then digging a protein spectrometer on the difference points for identification to find out the antigen-tolerant protein;

(5) selecting a tolerant membrane protein according to the screened out tolerant original protein, designing 5-10 peptide segments according to a protein sequence, artificially synthesizing the peptide segments by 15-25 amino acids of each peptide segment, coating the peptide segments on a carboxymethylated glucan substrate chip which is fixed with streptavidin in advance after biotinylation, detecting the spleen B cells of the mice by using a Biacore molecular interaction technology, and confirming the tolerant peptide segments;

(6) taking spleen B cells of a control group rat and fusing the spleen B cells with Sp2/0 myeloma cells to form hybridoma cells according to a classical PEG method.

(7) The enzyme-labeled microporous plate is coated with artificially synthesized tolerance peptide segments, and positive clone cells are screened out by an indirect ELISA method to prepare the monoclonal antibody.

The invention has the beneficial effects that: the B cell tolerance peptide segment can be used for preparing vaccines and monoclonal antibodies, can be used for treating infected persons with helicobacter pylori natural immune tolerance, and can also be used for preventing children with maternal vertical transmission but no established infection. Avoids the problem of drug resistance of bacteria, and has the advantages of small side effect, good patient compliance, easy large-scale production and the like.

Drawings

FIG. 1 is an electron micrograph of H.pylori OMV after extraction and purification.

FIG. 2 shows the bidimensional electrophoresis display of the helicobacter pylori whole protein antigen spectrum.

FIG. 3 is a secondary mass spectrum of one peptide fragment of the WB derived differential protein.

FIG. 4 is an IgG SDS electrophoretogram.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

1. Culture of helicobacter pylori

A standard strain of H.pylori 26695 was inoculated into brain-perfused broth (5 mg/L trimethoprim, 5mg/L polymyxin-B, 5mg/L amphotericin-B and 10mg/L vancomycin) and placed in a three-atmosphere incubator (10% CO)₂，85% N₂，5% O₂) Shaking culture is carried out at the temperature of 37 ℃ and 80 rpm for 3-10 days.

2. Extraction of OMV

(ii) crude extraction of helicobacter pylori OMV

Centrifuging 10000 Xg of helicobacter pylori liquid cultured to logarithmic phase for 10min, collecting supernatant, filtering with a 0.22 μm bacterial filtering membrane, concentrating to 1/10 of the original volume with an ultrafiltration centrifugal tube with the relative molecular mass cut-off of about 100KD, collecting liquid, centrifuging 39000 Xg for 60min, discarding supernatant, and suspending precipitate in 180 μ L of 50mM/L HEPES buffer solution (4-hydroxyethyl piperazine ethanesulfonic acid, N- (2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid, pH 6.8) to obtain crude extracted OMV.

② purification of OMV

The crude extracted OMVs were added to the bottom of a 15ml ultracentrifuge tube and the following densities of OptiPrep gradient centrifugate were added from bottom to top, respectively: 1.5ml 50%, 1ml 45%, 1ml 40%, 1ml 35%, 1ml 30% and 1.5ml 25% (OptiPrep/50 mM/L, HEPES-150 mM/LNaCl), centrifugation at 100000 Xg for 16h at 4 ℃, taking 0.5ml of the liquid from top to bottom, diluting 10-fold with 50mM HEPSE-150 mM/L, NaCl, ultracentrifugation at 4 ℃ 150000 Xg for 1h, resuspending the pellet in 50mM/L HEPES, i.e., purified OMV, and storing at 4 ℃.

(iii) OMV's Electron microscope Observation

And (3) taking a purified OMV sample, dropwise adding the OMV sample to a carbon-coated copper mesh, dropwise adding 2% uranyl acetate for dyeing, drying at room temperature, and performing morphological identification by using a microscope (TEM, JEOL 2100F). As shown in FIG. 1, the arrows indicate outer membrane vesicles secreted by H.pylori.

Identification of OMV protein

The total protein concentration of OMVs was determined according to the BCA method protein quantification kit instructions and then formulated to 100. mu.g/ml. OMV content was identified using a Q exact HF mass spectrometer (Thermo Fisher Scientific).

Treating OMV with proteinase K to remove impure proteins

50 μ g of purified OMV was incubated with 100 μ g/ml proteinase K at 37 ℃ for 30min, centrifuged at 150000 Xg for 30min, the pellet resuspended in sterile PBS and centrifuged again as above for 3 more times to remove excess proteinase K. Storing at 4 ℃.

3. OMV injection pregnant mouse

Each group of sexually mature female 2-3 BALB/c mice and 1-2 male BALB/c mice was housed at about 4-5 pm in the previous day, female BALB/c mice were taken out about 8-9 am every other day, and then pregnant was judged as the formation of vaginal emboli and counted as 0d, and they were marked and individually housed. Injecting BALB/c pregnant mouse from tail vein at 8, 12, and 16 days, respectively, 0.1ml OMV (100 μ g/ml) each time; injection of 0.1ml PBS was used as a control group; the abdomen of the mice was observed again at 12d to confirm pregnancy, and the non-pregnant mice were discarded.

4. OMV immunized mice

OMVs were diluted to a concentration of 0.5g/L in PBS buffer and emulsified thoroughly in 1:1 (v/v) with Freund's complete adjuvant. Taking BALB/c child mice growing to 8 weeks old, and adopting an immunization method of subcutaneous injection to carry out primary immunization at a dose of 100 mu g per mouse; on week 3, the OMV solution and Freund's incomplete adjuvant were mixed at 1:1 (v/v) and emulsified thoroughly, and booster immunization was performed at a dose of 50. mu.g, once every 14 days. Mouse sera were collected at the first 1d of each immunization and tested for total anti-OMV antibody titers in the mouse sera by indirect ELISA.

5. Indirect ELISA for detecting mouse serum antibody titer

Pretreating a 96-pore plate by using 150 mu l/pore of 2.5% glutaraldehyde solution for 1 hour, washing the 96-pore plate by using ultrapure water at 37 ℃ for 4 times, and wiping the plate dry each time; coating an enzyme label plate with 100 mul OMV (0.1 mg/ml), drying at 37 ℃, washing the plate 3 times with PBST (PBS buffer solution containing 0.5ml/L Tween 20), and drying after throwing away completely each time for 3min each time; blocking with 3% BSA in PBST for 1 hour, washing as above; adding immune mouse serum with different dilutions, 100 mu l/well and 3 multiple wells per sample, and incubating for 1 hour at 37 ℃; after washing for 3 times, adding rabbit anti-mouse IgG-HRP (1: 8000), 100 mul/hole, and incubating for 1 hour at 37 ℃; adding 100 mu l of tetramethylbenzidine substrate working solution after washing for 3 times, and adding 50 mu l H per hole after reacting for 15 minutes at 37 DEG C₂SO₄(2 mol/L) the reaction was terminated, and the absorbance value was measured at a wavelength of 450nm with an enzyme-linked immunosorbent assay (OD)₄₅₀The maximum dilution of 0.05 was the antibody titer. Then taking the serum of the submouse with the serum titer of the experimental group being more than 1: 8000 and the serum titer of the control group being more than 1: 12000 and reserving the serum and the spleen cells for later use.

6. Antigen site detection by two-dimensional electrophoresis-WB technology

Carrying out two-dimensional electrophoresis: hp total protein was extracted according to the bacterial total protein kit, and the total protein sample was then adjusted to about 2mg/ml with aqueous solution and supplemented with DTT, IPG buffer and bromophenol blue for aqueous loading. Adopting 24cm adhesive tapes, loading each adhesive tape with 450 mu l, and performing equal electric focusing on IPGphor, wherein the procedure is as follows: 1) low-pressure desalting, namely setting six gradients of 100V, 200V, 300V, 500V, 800V, 1000V and the like, wherein each gradient is 1 h; 2) gradient boosting, wherein the voltage is increased from 1000V to 8000V within 4 h; 3) isoelectric focusing, constant voltage 8000V, focusing 70000 Vh; 4) constant voltage is maintained, 1000V voltage, 12 h. And each rubber strip is subjected to flow limitation of 50 muA in the whole process. After focusing, balancing the gel strips with a DTT equilibrium solution (75 mmol/L Tris-HCl, pH8.8, 6mol/L urea, 2% SDS, 8% glycerol, 1% DTT) and an IAA equilibrium solution (75 mmol/L Tris-HCl, pH8.8, 6mol/L urea, 2% SDS, 8% glycerol, 2.5% IAA) respectively for 15min, loading the gel strips on 12.5% gel, sealing with low-melting point agarose, performing second-direction vertical electrophoresis, and performing electrophoresis under the conditions: the constant power was initially 1W/gel, and after about 40min, the power was increased by 13-15W/gel until the bromophenol blue indicator was about 1.5cm from the bottom edge, the gel was removed and stained with Coomassie Brilliant blue, followed by scanning with Image Scanner III. The results are shown in FIG. 2.

② Western Blot: and cutting the protein two-dimensional gel and the PVDF membrane according to the size of a membrane transferring mold, and transferring the protein to the PVDF membrane by using a Berle transfer printing groove. The PVDF membrane was taken out and immersed in a blocking solution (3% bovine serum albumin), and then blocked for 1 hour at room temperature with shaking. Then primary antibody (immune mouse serum, diluted 500 times for use) was added and incubated at room temperature for 1h with shaking. The membrane was removed and washed 4 times with PBS for 10min each. The membrane was then immersed in a secondary antibody solution (goat anti-mouse IgG-Cy5, 1: 5000 dilution) and incubated on a shaker for 1h with slow shaking. The membrane was removed and washed 4 times with PBS for 10min each. Differential protein spots were identified by analysis software using a Typhoon 9500 Image Quant TL scan.

Third, mass spectrum identification: differential protein spots from Coomassie blue stained gels were pipetted into 1.5mL centrifuge tubes and 1:1 mixed 30mM K was added₄Fe(CN)₆And 100mM Na₂S₂O₃(liquid submerges the gel), and the gel is decolored by shaking at room temperature for about 6 hours. The reaction was stopped by washing twice with MilliQ water. Adding 300uL 100% acetonitrile into each tube, standing at room temperature for 10min to dehydrate and whiten the colloidal particles in the tube, and vacuum-drying for 10 min. 300uL of 10mM DTT/50mM NH was added₄HCO₃And (3) carrying out water bath on the buffer solution at 56 ℃ for 1h, removing the buffer solution, adding 300uL of 100% acetonitrile until colloidal particles are dehydrated and whitened, and carrying out vacuum drying for 10 min. 300uL of 60mM DTT/50mM NH was added₄HCO₃The buffer solution was placed in a dark room for 30min and removed. Adding 300uL 100% acetonitrile until the colloidal particles become white after dehydration, and vacuum-drying for 10 min. 100uL of 50mM NH was added to each tube₄HCO₃And 1 μ g of pancreatin, the gel was broken up and digested overnight at 37 ℃. The digestion solution was collected, quenched by the addition of 0.1% trifluoroacetic acid and lyophilized. Dissolving the mixture in 0.2 percent TFA solution to prepare C₁₈Desalting with small column, freeze drying, dissolving with 0.1% FA, and detecting.

HPLC Easy-nLC1200 (Thermo Fisher Scientific) was used for HPLC analysis at a flow rate of 300 nl/min. Buffer A was 0.1% FA and Buffer B was 80% ACN with 0.1% FA. The liquid phase gradient was set as follows: 2% -8% of B for 1 min; 8-28% of B for 60 min; 28% -37% of B14 min; 37% -100% of B5 min; 100% B10 min.

The Mass spectrometry was performed using a Q active HF Mass spectrometer (Thermo Fisher Scientific) set to cation mode with Mass range 350-. MS1 is set to resolution 60000, AGC target 3e6, maximum IT 20MS, top 20 is selected for MS2, resolution 15000, AGC target 1e5, maximum IT 45 MS. The other settings are fixed first mess 110.0m/z, Isolation window 1.6m/z, NCE 27%, dynamic exclusion time 45 ms. The results are shown in FIG. 3.

7. Molecular interaction technology for confirming tolerance peptide fragment

Selecting tolerant membrane protein according to the selected tolerant original protein, and designing 5-10 peptide segments according to the protein sequence, wherein each peptide segment is 15-25 amino acids. According to the operating guidelines of the Biacore instrument, the artificially synthesized tolerizing peptide is biotinylated and then the molecules are coated on a carboxymethylated dextran matrix chip on which streptavidin has been immobilized beforehand, the B-cell concentration being adjusted to 5X 10⁵And/ml, detecting the combination of the polypeptide-B lymphocyte by using a Biacore biomacromolecule interaction analyzer, comparing the difference between an experimental group and a control group, and confirming the tolerance peptide segment.

8. Preparation of hybridoma cells

Taking a control group of mice with the serum titer of more than 1: 12000, carrying out 1-time boosting immunization, and taking the splenocytes of the mice 3d after boosting immunization for cell fusion. Spleen cells of mice were fused with Sp2/0 cells according to the classical PEG method. After cell fusion, the cells were resuspended in 2% HAT in RPMI1640 medium, and the cell suspension was dropped into 96-well plates at 37 ℃ with 5% CO₂Culturing in an incubator for 3 d; the culture was continued for 2 weeks by supplementing RPMI1640 medium containing 2% HAT, and the medium was replaced with RPMI1640 medium containing 2% HT. And (3) taking the synthesized B cell tolerance peptide segment as a coating antigen, and screening strong positive clones by using an indirect ELISA method (the specific method refers to the step 5). Such asThis was repeated several times until positive clones were 100%. Continuously passaging the detected positive hybridoma cells in vitro for more than 3 months, taking cell supernatant every 1 week, and detecting the antibody titer. And (3) repeatedly freezing and storing the cell strain, recovering for 2-3 times, and taking the supernatant after culturing to detect the antibody titer. The analysis was performed using the classical colchicine method. Selecting 5 non-overlapped split phases with scattered chromosomes for counting each cell, and averaging, wherein when the number is 98-101, the hybridoma cell is formed by fusing spleen cells and myeloma cells, and part of chromosomes are lost; when the value is 102-108, the hybridoma cell is formed by fusing spleen cells and myeloma cells, and no chromosome is lost.

9. Purification of monoclonal antibodies

The abdominal cavity of an 8-week-old BALB/c male mouse was injected with 0.5ml of liquid paraffin 1 week before hybridoma inoculation. Collecting well-grown hybridoma cells, and adjusting cell density to 2 × 10⁶Each cell/ml was injected intraperitoneally with 0.5ml of cell suspension per mouse. After 7-12 days, ascites is extracted when the abdomen of the mouse is obviously enlarged. Centrifuging at 3000g for 10min, discarding upper layer oil, sucking yellowish ascites, and storing at-20 deg.C. The collected ascites fluid was slowly added to an equal volume of saturated ammonium sulfate solution, centrifuged at 12000g for 10min, and the precipitate was dissolved with a certain volume of PBS buffer (pH 7.4). 12000g 4 ℃ centrifugal 10min, collect the supernatant to the dialysis bag, then its dialysis balance to 20 times the volume of binding buffer (pH 7.4) to remove high concentration of ions. And (4) carrying out protein G affinity chromatography on the dialyzed crude monoclonal antibody. Adding 5ml of binding buffer solution to pre-neutralize the protein G affinity column, loading 1ml of crude dialyzed monoclonal antibody, controlling the flow rate to be 1ml/min, rinsing with 25ml of binding buffer solution, then eluting with 10ml of elution buffer solution (0.1 mol/L glycine, pH 3.0), collecting the antibody purified by affinity chromatography, filling a dialysis bag, and dialyzing and balancing to PBS buffer solution (pH 7.4). The results of SDS-PAGE electrophoresis of the mouse ascites and the purified antibody are shown in FIG. 4.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

SEQUENCE LISTING

<110> Hospital fourth Hospital of Hebei medical university (tumor Hospital, Hebei province), Hebei medical university

<120> helicobacter pylori B cell tolerance epitope and antibody prepared by the same

<130> 2020

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<400> 1

Tyr Tyr Gly Phe Phe Ser Tyr Asn Gly Ala Ser Val Gly Phe Arg

1 5 10 15

Claims

1. A helicobacter pylori B cell tolerance epitope, which is characterized by comprising an amino acid sequence shown as SEQ ID No. 1.

2. A nucleic acid molecule encoding the H.pylori B-cell tolerance epitope of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A monoclonal antibody prepared using the H.pylori B-cell tolerance epitope of claim 1.

5. The monoclonal antibody according to claim 4, for use in the treatment of an infected person who is naturally immune-tolerant to helicobacter pylori.

6. A vaccine prepared using the B-cell tolerant epitope of helicobacter pylori according to claim 1 for stimulating an immune response in a host organism.

7. The vaccine of claim 6, wherein the vaccine is for the prevention of a child with maternal vertical transmission but no established infection.