Human helicobacter pylori UreB antigen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a human helicobacter pylori UreB antigen as well as a preparation method and application thereof. The human helicobacter pylori UreB antigen has an amino acid sequence as shown in SEQ ID NO: 2; or an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 2 and has the same function as the amino acid sequence as shown in SEQ ID NO: 2. UreB glycoprotein of a main epidemic virus strain NCTC11637 of human helicobacter pylori is selected as antigen protein, through biological information analysis, amino acids of a full-length target sequence 1-572 of the UreB antigen are selected, the target sequence is synthesized into a target sequence, a GST tag is inserted, a recombinant plasmid expression vector is constructed, the recombinant plasmid expression vector is expressed and purified in an escherichia coli prokaryotic system and verified at the same time, and the UreB antigen is obtained. The obtained human helicobacter pylori UreB antigen has the characteristic of high expression, can be specifically combined with human helicobacter pylori, and has important clinical significance.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a human Helicobacter pylori UreB antigen and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori ( Helicobacter Pylori HP) is a Gram-negative bacterium, spiral and slightly anaerobic, mainly existing on the host's gastric mucosa, and multiplying in a parasitic manner. Human Helicobacter pylori is mainly transmitted through oral-oral, fecal-oral and iatrogenic routes. The clinical course of Helicobacter pylori infection is: HP settles and infects the gastric mucosa after reaching it through the mouth, causing chronic, superficial gastritis after weeks or months, and developing into duodenal ulcers, gastric ulcers, lymphoproliferative gastric lymphoma, chronic atrophic gastritis, etc. after several years or decades, and the latter is the most dangerous factor leading to gastric cancer. HP can cause related diseases such as gastric ulcers, duodenal ulcers, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. The International Agency for Research on Cancer has classified HP as a Class I carcinogen. As Helicobacter pylori's drug resistance increases, the eradication rate of traditional treatment options has dropped significantly.

[0003] After the body is infected by HP, IgM, IgG and IgA antibodies can be produced. Studies have shown that the main immunogenic proteins of HP include cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), urease, heat shock protein (Hsp) and oxygen-insensitive NADPH nitroreductase. Urease is expressed in all HP strains and is the most expressed protein in Helicobacter pylori. It is an essential factor for the survival of HP. Urease consists of two structural subunits, Urease A and Urease B, and 5 auxiliary proteins. It can decompose urea into CO2 and NH3, forming ammonia clouds in the stomach, protecting HP from gastric acid damage and facilitating its colonization. Ammonia can also cause various changes in cells, such as hindering the cell cycle, acidic swelling, and causing epithelial damage. Urease can also stimulate platelet activation and promote angiogenesis, leading to accelerated progression of gastric cancer.

[0004] Human Helicobacter pylori urease B (UreB) is a subprotein of urease, and its gene is highly conserved among different strains. UreB antibodies can be detected in HP-infected patients, especially those with symptoms. The antibody level is correlated with the severity of the disease. UreB antigen can be used as the main target protein of HP neutralizing antibodies and is also a major candidate protein for vaccine research and antibody development. Therefore, it is necessary to develop a human Helicobacter pylori UreB antigen. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a human Helicobacter pylori UreB antigen and a preparation method and application thereof. The human Helicobacter pylori UreB antigen uses the UreB glycoprotein of the main prevalent virus strain NCTC11637 of human Helicobacter pylori as the antigen protein, selects the full-length target sequence 1-572 amino acids of the UreB antigen through biological information analysis, synthesizes the target sequence and inserts a GST tag, constructs a recombinant plasmid expression vector, expresses and purifies it in an Escherichia coli prokaryotic system, and verifies it at the same time. The obtained human Helicobacter pylori UreB antigen has the characteristics of high expression, can specifically bind to human Helicobacter pylori, and has important clinical significance.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The first object of the present invention is to provide a human Helicobacter pylori UreB antigen having an amino acid sequence as shown in SEQ ID NO:2; or, having an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids as shown in SEQ ID NO:2.

[0007] Furthermore, the human Helicobacter pylori UreB antigen specifically binds to human Helicobacter pylori antibodies.

[0008] The second object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned human Helicobacter pylori UreB antigen, which has a nucleotide sequence as shown in SEQ ID NO:1.

[0009] The third object of the present invention is to provide an expression vector, comprising the above-mentioned nucleic acid molecule. In addition to the above-mentioned nucleic acid molecule, the expression vector may also contain an appropriate promoter or control sequence. The vector can be used to transform an appropriate host cell to enable it to express protein.

[0010] The fourth object of the present invention is to provide a host cell, comprising the above nucleic acid molecule or the above expression vector. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells such as Escherichia coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc. The host cell particularly suitable for the present invention is a prokaryotic host cell, especially Escherichia coli, such as TOP10 strain, Rosetta strain.

[0011] The fifth object of the present invention is to provide a composition comprising an immunologically effective amount of the human Helicobacter pylori UreB antigen, or the expression vector, or the host cell.

[0012] The sixth object of the present invention is to provide a method for preparing human Helicobacter pylori UreB antigen, comprising the following steps: (1) Selection of anti-human Helicobacter pylori UreB antigen and construction of recombinant plasmid: Take the full-length target sequence of UreB antigen (amino acids 1-572) and insert the GST tag to construct a recombinant plasmid expression vector; (2) Expression and purification of human Helicobacter pylori UreB antigen: The Escherichia coli containing the recombinant plasmid expression vector of UreB is activated, expanded, induced with IPTG, and ultrasonically disrupted to obtain the primary UreB antigen. The obtained antigen solution is then incubated with beads and eluted 3-4 times to obtain the UreB antigen.

[0013] At the same time, relevant verification methods are also provided, as follows: SDS-PAGE verification of antigen protein: SDS-PAGE gel electrophoresis was performed on samples during the expression and purification of UreB antigen, and the purified UreB-GST band was visible at 95KD; Western blot verification of antigen protein: Load different volumes of UreB antigen protein and GST-tagged protein and perform Western blot experiments to verify that UreB antigen can specifically bind to human Helicobacter pylori antibodies, while GST cannot; Elisa verification of antigen protein: 96 plates were coated with UreB antigen protein and GST tag protein respectively, and Elisa experiments were performed using human Helicobacter pylori antibody as the primary antibody to verify that UreB antigen can specifically bind to human Helicobacter pylori antibody, while GST cannot.

[0014] The seventh object of the present invention is a use of the human Helicobacter pylori UreB antigen or the human Helicobacter pylori UreB antigen or its binding fragment prepared by the above method or the above nucleic acid molecule in the preparation of a drug for treating diseases related to human Helicobacter pylori infection. Specifically, diseases related to human Helicobacter pylori infection are gastric ulcer, duodenal ulcer, lymphoproliferative gastric lymphoma, chronic atrophic gastritis and gastric cancer.

[0015] The eighth object of the present invention is to provide a use of the human Helicobacter pylori UreB antigen as described above or the human Helicobacter pylori UreB antigen or its binding fragment prepared by the above method or the above nucleic acid molecule in the preparation of a vaccine for preventing human Helicobacter pylori infection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention selects the key protein UreB protein of NCTC11637 of the main prevalent virus strain of human Helicobacter pylori as the antigen protein, and takes the full length 1-571 amino acids of the UreB antigen to construct a recombinant plasmid expression vector UreB, and performs protein expression, purification and verification on it; the antigen has the characteristics of high expression, can specifically bind to human Helicobacter pylori, and can be used to develop drugs, antibodies, vaccines, etc. for the prevention and treatment of diseases related to human Helicobacter pylori infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a vector map of the HP-UreB-pGEX antigen protein in one embodiment of the present invention; Figure 2 The electrophoresis result diagram of SDS-PAGE electrophoresis for detecting the expression and purification of the recombinant antigen protein UreB-GST in one embodiment of the present invention. Among them, M is the protein molecule standard; 1 is the bacterial fluid before induction; 2 is the bacterial fluid after induction; 3 is the supernatant; 4 is the inclusion body; 5 is the flow-through liquid; 6, 7, 8 are washing liquids; 9, 10, 11 are the protein sample eluents; Figure 3 The figure is a result diagram for verifying the specific binding and sensitivity of antigen and antibody in one embodiment of the present invention; wherein A is the Western blot detection of the specific binding ability of antigen and antibody; B is the Elisa detection of the sensitivity of UreB-GST binding antibody; C is the Elisa detection of the binding of GST and antibody. DETAILED DESCRIPTION

[0019] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0020] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution.

[0021] Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

[0022] Example 1: Selection of anti-human Helicobacter pylori UreB protein antigen and construction of recombinant plasmid UreB is one of the most studied glycoproteins at home and abroad. It is highly conservative and immunogenic. Therefore, NCTC11637 strain UreB was selected as the antigen protein. The full-length target sequence of the antigen, amino acids 1-572 (SEQ ID NO: 2), was taken and the target nucleotide sequence shown in SEQ ID NO: 1 was obtained after fragment optimization. The target sequence was compared with Figure 1 The PGEX-4T-3 vector plasmid containing the GST tag was recombined to obtain the recombinant plasmid expression vector UreB.

[0023] * (SEQ ID NO:2) Example 2: Expression and purification of human Helicobacter pylori UreB protein antigen 1. Expression of antigen protein (1) Preparation of LB medium: weigh broth powder in a ratio of 10 g:500 mL into a 500 mL conical flask and two 100 mL conical flasks, add ddH2O to make the volume to 200 mL and 20 mL respectively. Autoclave at 121°C for 15 min. After sterilization, place the conical flasks in a 4°C refrigerator for later use.

[0024] (2) Activation of bacterial strains: Take 1 mL of LB culture medium from each of the two 100 mL conical flasks and place them in a 1.5 mL sterile EP tube. Pipet 20 µL of the target bacterial solution into one of the EP tubes, and set the other EP tube as a blank control without adding bacterial solution. Place the two EP tubes in a shaker and shake at 37°C, 150 rpm for 3 h. After shaking, add 20 µL of ampicillin (100 mg / mL, 1:1000) to each of the two conical flasks with the remaining 19 mL of LB culture medium, then add 1 mL of LB culture medium from the EP tube back to the corresponding conical flask, place them in a shaker and shake at 37°C, 150 rpm for 12 h. (3) Expanded culture: On the second day, observe the turbidity of the LB culture medium in the two conical flasks. If the experimental group culture medium is turbid and the control group culture medium is clear, it proves that the target bacteria are growing and there is no interference from other bacteria, and the next step of the experiment can be continued. Add 200µL of ampicillin antibiotics to the sterilized 200mL LB culture medium, and then add 5mL of activated bacterial solution, shake the culture at 37℃ and 200rpm for 2h, and measure the OD value using a UV spectrophotometer (detection wavelength is 595nm). If the OD value is between 0.6 and 0.8, stop shaking the culture, take 1mL of the pre-induction bacterial solution, prepare a sample, mark it as before induction, and store it in a -20℃ refrigerator. If the OD value does not reach 0.6, continue shaking the culture until it reaches this range.

[0025] (4) IPTG induction: Add 200 µL IPTG (0.8 mM, 1:1000) to 200 mL of bacterial solution for induction. Place the culture in a shaker and shake at 37°C, 150 rpm for 4 h. After shaking, take 1 mL of the induced bacterial solution, prepare a sample, mark it as induced, and store it in a -20°C refrigerator. (5) Ultrasonic disruption: Place the induced bacterial solution in a centrifuge cup, balance it, and centrifuge it at 4700rpm for 20min. Discard the supernatant, add 20mL PBS to resuspend the bacterial precipitate in a 50mL centrifuge tube, centrifuge it at 5000rpm for 5min, and remove the residual culture medium. Add another 20mL PBS to mix the bacterial precipitate, place the centrifuge tube in an ice-water mixture, and disrupt it on an ultrasonic disruptor (supersonic for 4s, stop for 8s, power 20%). When the bacterial solution is clear, centrifuge it at 4℃, 5000g for 20min. Filter the supernatant with a 0.22µm filter membrane and transfer it to a new 50mL centrifuge tube. Add an equal amount of PBS to resuspend the remaining precipitate, mark it as inclusion body, take 1mL of supernatant and inclusion body samples respectively, and store them in a -20℃ refrigerator.

[0026] 2. Purification of Antigen Protein (1) Remove ethanol and wash beads (GST agarose gel): Take 1 mL of beads in a 15 mL centrifuge tube, centrifuge at 3500g for 5 min, and discard the supernatant. Add 10 mL of PBS and mix on a four-dimensional rotating mixer at 4°C for 10 min. After mixing, centrifuge at 4000g for 5 min, discard the supernatant, and wash twice with PBS as above.

[0027] (2) Add the supernatant containing the target protein to the washed beads, pipette to mix, and place in a four-dimensional rotating mixer at 4°C for 4 h.

[0028] (3) After mixing, centrifuge at 4°C, 4000 g, for 5 min. Aspirate the supernatant into a new 15 mL centrifuge tube and mark it as flow-through (sample preparation).

[0029] (4) Add 10 mL of PBS to the centrifuge tube and mix it in a four-dimensional rotating mixer at 4°C for 10 min. After mixing, centrifuge it at 4°C, 4000 g for 5 min. Pipette the supernatant into a new 15 mL centrifuge tube and mark it as Wash 1 (Sample Preparation).

[0030] (5) Add 10 mL of PBS to the centrifuge tube and place it in a four-dimensional rotating mixer at 4°C for 10 min. After mixing, centrifuge at 4°C, 4000 g for 5 min. Pipette the supernatant into a new 15 mL centrifuge tube and mark it as Wash 2 (Sample Preparation).

[0031] (6) Add 10 mL of PBS to the centrifuge tube and place it in a four-dimensional rotating mixer at 4°C for 10 min. After mixing, centrifuge at 4°C, 4000 g for 5 min. Pipette the supernatant into a new 15 mL centrifuge tube and mark it as Wash 3 (Sample Preparation).

[0032] (7) Resuspend the precipitate with 500 µL of GSH in a new 1.5 mL EP tube, and then add 500 µL of GSH to resuspend the remaining precipitate again, adding a total of 1 mL of GSH. Place the tube in a four-dimensional rotating mixer and mix at 4°C for 10 min.

[0033] (8) Centrifuge at 4000 g for 5 min at 4°C, aspirate the supernatant into a new 1.5 mL EP tube and label it as deionized water 1 (sample preparation). Add 1 mL of GSH to the precipitate and mix it in a four-dimensional rotating mixer at 4°C for 10 min. (9) Centrifuge at 4000 g for 5 min at 4°C, aspirate the supernatant into a new 1.5 mL EP tube and label it as Deionized 2 (sample preparation). Add 1 mL of GSH to the precipitate and mix it in a four-dimensional rotating mixer at 4°C for 10 min. (10) Centrifuge at 4000 g for 5 min at 4°C. Aspirate the supernatant and transfer it to a new 1.5 mL EP tube and label it as Deionized Water 3 (sample preparation).

[0034] (11) After the remaining beads are dissolved in 1 mL of GSH, they are placed on a four-dimensional rotating mixer and mixed for 10 min at 4°C. Centrifuge at 4000 g for 5 min at 4°C and wash twice with GSH as above. The final beads are sealed with 20% ethanol and stored at 4°C until use.

[0035] (12) Sample preparation: Mix the samples before induction, after induction, inclusion bodies, supernatant, flow-through, wash 1, wash 2, wash 3, stripping 1, stripping 2, and stripping 3 with 2× loading buffer, boil them in a metal bath at 100°C for 10 min, cool them, and store them in a -20°C refrigerator. (13) Mix the collected deionized water 1, deionized water 2, and deionized water 3 together and put them into a dialysis bag. Clamp the dialysis bag and throw it into 4 L PBS and stir overnight.

[0036] (14) Protein concentration measurement: BCA protein concentration measurement, the measured protein solution was divided into aliquots, and then quickly frozen in liquid nitrogen overnight, and then transferred to -80 °C for storage.

[0037] Example 3: Validation of Antigen Protein 1. SDS-PAGE verification of antigen protein (1) Glue preparation: Slowly add the prepared separation glue to the assembled and fixed rubber plate, and then add appropriate Flatten the gel surface with a certain amount of isopropanol and let it stand at room temperature for about 30 minutes. After the separation gel solidifies, discard the upper isopropanol and wash away the residual isopropanol with ddH2O. Then add the prepared concentrated gel to the gel plate and slowly insert the comb. Let it stand at room temperature for about 30 minutes. After the gel is completely solidified, pull out the comb. (2) Sample processing: Dissolve the sample at -20°C at room temperature. After complete dissolution, vortex the sample for 60 seconds and centrifuge at 5000 rpm for 1 minute. (3) Sample loading and electrophoresis: Load the sample in a certain volume from left to right, and add a protein marker to the leftmost well. Pour in 1× electrophoresis buffer, plug in the electrodes, run at a constant voltage of 60V for 40 minutes, and then increase the voltage to 110V and run for about 1.5 hours. (4) Staining: After electrophoresis, carefully remove the gel block, cut off the concentrated gel, put the separation gel into the gel box and add Coomassie Brilliant Blue staining solution until the gel surface is completely covered. Incubate on a shaker at room temperature for 30 minutes, recover the staining solution, wash twice with 1× destaining solution, each time for 15 minutes, and then wash three times with ddH2O, each time for 10 minutes. (5) Exposure imaging: Place the destained separation gel into the gel imaging system for imaging and analysis. Figure 2 As shown, a purified UreB-GST band was visible at 95KD, indicating that the UreB antigen protein was successfully prepared.

[0038] 2. Western blot verification of antigen protein (1) Glue preparation: slowly add the prepared separation gel to the assembled and fixed gel plate, then add an appropriate amount of isopropanol to flatten the gel surface, and let it stand at room temperature for about 30 minutes. After the separation gel solidifies, discard the upper isopropanol and wash away the residual isopropanol with ddH2O. Then add the prepared concentrated gel to the gel plate, slowly insert the comb, and let it stand at room temperature for about 30 minutes. After the gel is completely solidified, pull out the comb. (2) Sample processing: Dissolve the sample at -20°C at room temperature. After complete dissolution, vortex the sample for 60 seconds and centrifuge at 5000 rpm for 1 minute. (3) Sample loading and electrophoresis: Samples are loaded from left to right in a certain volume in the following order: Marker, UreB-GST (5µL, 10µL, 20µL, 30µL), GST (5µL, 10µL). Pour in 1× electrophoresis buffer, insert electrodes, run at a constant voltage of 60V for 40min, then increase the voltage to 110V and run for about 1.5h. (4) Transfer: Soak a 0.45µm PVDF membrane of appropriate size in methanol for 1 min. Stack the sponge pad, filter paper, separation gel, PVDF membrane, filter paper, and sponge pad in a "sandwich" pattern from the negative electrode (black board) to the positive electrode (white board), pour in 1× Transfer buffer, plug in the electrodes, and transfer at a constant current of 280mA for 90 min. (5) Blocking: Place the PVDF membrane in the prepared blocking solution and shake slowly at room temperature for 2 hours.

[0039] (6) Primary antibody incubation: After blocking, wash the membrane three times with 1× TBST solution, 10 min each time. Add the pre-prepared primary antibody (Human Human cytomegalovirus gB Antibody) and incubate overnight at 4°C on a shaker. (7) Secondary antibody incubation: Recover the primary antibody and wash the membrane three times with 1×TBST solution, 10 min each time. Add the pre-prepared secondary antibody (Goat-anti mouse HRP secondary antibody), incubate at room temperature for 1.5 h, and then wash the membrane three times with 1×TBST solution, 10 min each time. (8) Exposure and development: Soak the PVDF membrane in the developer for 30 seconds, image it using the Tianneng gel imaging system, and analyze its grayscale value. Figure 3As shown in A, with the increase of the amount of UreB-GST protein loaded, the target band at 95KD became significantly darker, while the tag protein GST did not bind to the antibody, indicating that the prepared antigen protein can effectively bind to the specific antibody and can be used for subsequent monoclonal antibody screening.

[0040] 3. Elisa verification of antigen protein Coating: Take out the UreB-GST antigen protein and GST tag protein of known concentration from the -80℃ refrigerator, wait for them to dissolve completely, add them to the coating solution at 100ng / well, mix thoroughly, add them to the 96-well ELISA plate with a dispenser, 50µL per well, gently tap the four sides of the ELISA plate, and then place it in a 4℃ refrigerator for overnight coating. The specific coating layout is shown in Table 1.

[0041] Table 1

[0042] (2) Blocking: Take the coated ELISA plate out of the 4°C refrigerator, wash the plate five times with detergent, tap the plate twice, add blocking solution (10% BSA in PBS), 200 µL per well, and incubate in a 37°C oven for 2 h. (3) Primary antibody incubation: After blocking, wash the plate and add the corresponding primary antibody (Human Human cytomegalovirus UreB Antibody) in a gradient dilution from 400 ng / mL to 3.125 ng / mL in sequence, 50 µL per well, and incubate in a 37°C oven for 1 h. (4) Secondary antibody incubation: After primary antibody incubation, wash the plate and add the corresponding secondary antibody (Goat-anti mouse HRP secondary antibody) at 50 µL per well and incubate in a 37°C oven for 45 min. (5) TMB color development: After incubation with the secondary antibody, wash the plate and add 50 µL of the prepared color development solution to each well. Incubate the plate in room temperature in the dark for 30 min. (6) Stop: When the solution turns blue, quickly add 100 µL of the prepared stop solution to each well. (7) OD value detection: Place the 96-well ELISA plate on an ELISA reader and detect the OD value of each well at a wavelength of 450nm. Figure 3 As shown in Figures B and C, even if the standard, human anti-cytomegalovirus immunoglobulin IgG, is diluted 2-fold stepwise, from the highest concentration of 1000 ng / mL to the lowest concentration of 7.8125 ng / mL, it can specifically bind to the UreB-GST recombinant antigen protein and show an obvious gradient change. On the contrary, the GST-tagged protein has no specific binding to the standard, indicating that the UreB-GST antigen protein can bind to the specific antibody with high sensitivity.

[0043] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A human Helicobacter pylori UreB antigen, characterized in that: It has the amino acid sequence shown in SEQ ID NO: 2; or, has the amino acid sequence shown in SEQ ID NO: 2 with equivalent function formed by replacing, deleting or adding one or more amino acids.

2. The human Helicobacter pylori UreB antigen according to claim 1, characterized in that The human Helicobacter pylori UreB antigen specifically binds to the human Helicobacter pylori antibody.

3. The nucleic acid molecule encoding the human Helicobacter pylori UreB antigen according to claim 1 or 2, characterized in that: It has a nucleotide sequence as shown in SEQ ID NO:

1.

4. An expression vector, characterized in that: Comprising the nucleic acid molecule as described in claim 2.

5. A host cell, characterized in that Comprising the expression vector as claimed in claim 4.

6. A composition, characterized in that The method comprises an immunologically effective amount of the human Helicobacter pylori UreB antigen according to any one of claims 1 or 2, or the expression vector according to claim 4, or the host cell according to claim 5.

7. The method for preparing the human Helicobacter pylori UreB antigen according to claim 1 or 2, characterized in that: The following steps are involved: (1) Selection of anti-human Helicobacter pylori UreB antigen and construction of recombinant plasmid: Take the full-length target sequence of UreB antigen (amino acids 1-572) and insert the GST tag to construct a recombinant plasmid expression vector; (2) Expression and purification of human Helicobacter pylori UreB antigen: The Escherichia coli containing the recombinant plasmid expression vector of UreB is activated, expanded, induced with IPTG, and ultrasonically disrupted to obtain the primary UreB antigen. The obtained antigen solution is then incubated with beads and eluted 3-4 times to obtain the UreB antigen.

8. Use of the human Helicobacter pylori UreB antigen as claimed in claim 1 or 2, or the human Helicobacter pylori UreB antigen or its binding fragment prepared by the method of claim 7, or the nucleic acid molecule as claimed in claim 3 in the preparation of drugs for the treatment of diseases related to human Helicobacter pylori infection.

9. The use according to claim 8, characterized in that: The human Helicobacter pylori infection-related diseases are gastric ulcer, duodenal ulcer, lymphoproliferative gastric lymphoma, chronic atrophic gastritis and gastric cancer.

10. Use of the human Helicobacter pylori UreB antigen according to claim 1 or 2, or the human Helicobacter pylori UreB antigen or its binding fragment prepared by the method according to claim 7, or the nucleic acid molecule according to claim 3 in preparing a vaccine for preventing human Helicobacter pylori infection.

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