CN115073566B - Helicobacter pylori specific immunogenic peptide fragments - Google Patents

Abstract

The invention relates to a helicobacter pylori specific immunogenic peptide comprising an amino acid sequence as shown in SEQ ID No. 1. The antigens of the invention are useful in the preparation of novel antigensH.pyloriVaccines and other usesH.pyloriActive or passive immunotherapy of treatment, forH.pyloriThe prevention and the treatment of infection have important significance.

Description

Helicobacter pylori-specific immunogenic peptides

technical field

The invention relates to a Helicobacter pylori specific immunogenic peptide.

Background technique

More than 50% of the world's population is infected with Helicobacter pylori (H. pylori), which is more common in developing countries such as Asia and Africa. Almost all patients with H. pylori infection will have chronic gastritis, about 10% of them will develop peptic ulcer, and 1% will progress to gastric cancer in the next ten to several decades. Therefore, the prevention and treatment of H. pylori infection is an important means to prevent and treat peptic ulcer, chronic gastritis and gastric cancer.

The clinical treatment for H. pylori infection mainly relies on antibiotic-based triple or quadruple therapy, but there are many problems, such as high cost, increasing drug-resistant strains, destroying normal intestinal flora, easy recurrence and Reinfection etc. In particular, the problem of rising drug resistance has made the treatment of Helicobacter pylori infection more and more difficult.

During H. pylori infection, some membrane proteins or bacterial proteins can cause human immune response, which is closely related to H. pylori adhesion colonization, persistent infection and disease severity. At present, in addition to antibiotic treatment, immunotherapy methods targeting membrane proteins and/or other bacterial proteins and preparing related vaccines are also used to treat H. pylori infection. For example, some progress has been made in the research of H. pylori vaccines prepared by using H. pylori recombinant urease and its subunits, cytotoxin-associated protein (CagA), vacuolar toxin-associated protein (VacA), and adhesin A (HpaA).

However, none of the existing H.pylori vaccines can eradicate H.pylori. It is urgent to find new immunogenic protein targets to provide the basis for the preparation of effective H.pylori vaccines.

Contents of the invention

The purpose of the present invention is to provide a novel immunogenic Helicobacter pylori-specific immunogenic peptide.

The present invention adopts following technical scheme:

A Helicobacter pylori-specific immunogenic peptide, which comprises the amino acid sequence shown in SEQ ID No.1.

A nucleic acid molecule encoding the above-mentioned Helicobacter pylori-specific immunogenic peptide, which comprises the nucleotide sequence shown in SEQ ID No.2.

A vector comprising the nucleic acid molecule described above.

A monoclonal antibody prepared by using the above-mentioned Helicobacter pylori-specific immunogenic peptide.

A vaccine prepared by using the above Helicobacter pylori-specific immunogenic peptide, the vaccine is used to stimulate the immune response of the host organism.

A Helicobacter pylori-specific immunogenic protein, which comprises the membrane protein of the amino acid sequence shown in SEQ ID No.1.

Further, the Helicobacter pylori-specific immunogenic protein is a membrane protein pgbB comprising the amino acid sequence shown in SEQ ID No.3.

The beneficial effect of the present invention is that: the antigen of the present invention can be used to prepare new H. pylori vaccines and other active or passive immunological therapies for H. pylori treatment, and is of great significance for the prevention and treatment of H. pylori infection.

Description of drawings

Figure 1 shows the detection of H. pylori milk antibody immune membrane protein target by two-dimensional electrophoresis and western blot.

Figure 2 is the immune agar two-way diffusion test H. pylori milk antibody immune membrane protein target.

Figure 3 is the detection of H. pylori milk antibody immune membrane protein target by IP test.

Fig. 4 is an ELISA verification antigenicity experiment.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

1. Culture of Helicobacter pylori

The Helicobacter pylori clinical isolate HP435 was inoculated in a modified Campylobacter medium (Campy-BAP) containing 10% defibrated sheep blood with a diameter of 9 cm, at 37°C, microaerophilic (5% O ₂ , 10% CO ₂ , 85 % N ₂ , humidity 95%) for 3-4 days.

2. Extraction of Helicobacter pylori membrane protein

The H. pylori cells were washed three times with sterile 0.1 mol/L phosphate buffered saline (PBS), suspended in 20% glycerol broth and frozen at -80°C for future use. For strains producing membrane proteins, H.pylori cells should be washed 3 times with sterile 0.1mol/L PBS. Prepare EZ-Link Sulfo-NHS-SS-biotin biotin reaction solution with a concentration of 250μg/ml to label freshly washed H.pylori cell membrane proteins. The labeled bacterial cells were ultrasonically disrupted (3s on/7s off, intensity 20%, 5min). Centrifuge at 12,000g for 30min at 4°C to collect the supernatant rich in H.pylori membrane protein. Then, the membrane protein was purified using an avidin protein chromatography column.

3. Dairy cow immunization and preparation of antibodies

3.1 Immunization of cows

Choose 3-4 years old, 6-7 months pregnant, healthy Holstein cattle. Prepare 5mg of membrane protein plus 4× ¹⁰⁹ whole bacterial cells, dilute to 5ml with 0.1mol/LPBS, shake and mix with 5ml of aluminum hydroxide adjuvant (alum adjuvant), and perform 5 subcutaneous and rib treatment on cows Subcutaneous injection of immunization in subiliac lymph nodes. The interval between the first and second immunizations was 20 days, and the next immunization was carried out on the 10th day until 10 days before calving.

3.2 Antibody preparation

Collect the colostrum within one week after calving, centrifuge at 3500g at 4°C for 20min, add appropriate amount of hydrochloric acid to adjust the pH to 4.6, centrifuge at 12000rpm at 4°C for 30min to remove the casein precipitate, and keep the clarified supernatant (whey). Use the GE protein purification instrument to connect the Hitrap Protein G HP 1ml purification column to purify the extracted antibody-rich whey. Antibody titer detection was performed on the obtained purified antibody by ELISA method. Purified antibodies with an antibody titer of 1:15000 were obtained.

4. Identification of Immunogenic Membrane Proteins Using Three Methods

4.1 Two-dimensional electrophoresis and immunoblotting to detect H. pylori membrane protein positive for immunogenicity with the milk antibody. Two copies (150 μg protein each) of HP435 strain membrane protein samples were labeled with the same Cy dye, and two-dimensional electrophoresis was performed simultaneously. The PAGE gels after two-dimensional electrophoresis on both plates were imaged and positioned using a Typhoon multicolor fluorescence imaging scanner. Then, use the purified antibody prepared by the present invention as the primary antibody, and use the goat anti-bovine secondary antibody labeled with Cy5 or Cy3 (distinguished from the dye of the labeled protein sample) to perform immunoblotting on one of the PAGE gels. Detection of immunogenic protein spots.

As a result, it was found that 6-8 immunoreactive positive protein spots could be identified (see Figure 1). Corresponding to the results of the western blot test and the position of the immunoreactive positive protein spot on the original gel, dig out the protein spot on the corresponding position on the other plate of PAGE gel for protein spectrum identification. As a result, it was found that 112 proteins could be identified.

4.2 Immuno-agar two-way diffusion method to detect H.pylori membrane protein with positive immunogenicity of milk antibody The whole bacterial suspension of the HP435 strain was subjected to the immuno-agar two-way diffusion test with the blank group antibody and the purified antibody after immunization (see Figure 2) .

It was found that an obvious antigen-antibody precipitation line appeared between the purified antibody and the whole bacterial suspension. The precipitate line was cut off, boiled and denatured with 5× protein loading buffer, and then subjected to SDS-PAGE electrophoresis. After electrophoresis, the PAGE gel was stained with Coomassie Brilliant Blue, and the colored protein bands were excised, enzymatically digested in the gel, and identified by mass spectrometry. As a result, it was found that 501 proteins could be identified.

4.3 IP test and protein spectrum identification

IP test was performed on the extracted H.pylori membrane protein and purified antibody. The protein after IP was subjected to SDS-PAGE electrophoresis, stained with Coomassie brilliant blue, and the colored protein bands were subjected to in-gel digestion and mass spectrometry identification (see Figure 3). As a result, it was found that 403 proteins could be identified.

5. Further analysis of identification results

5.1 Intersection analysis was performed on the protein identification results obtained by the three immunological identification methods, and 13 common proteins were found. Among them, pgbB whose amino acid sequence is shown in SEQ ID No.3 is a newly discovered membrane protein with immunogenicity.

SEQ ID No.3:

MNKPFLILLIALIVFSGCNMKKYFKPAKHQIKGEAYFPNHLQESIVSSNRYGAILKNGAVIGD

KGLTQLRIGKNFNYESSFLNESQGFFILAQDCLNKIDKKTSKSKVAKTEETELKLKGVEAEV

QDKVCHQVELISNNPASQQSIIIPLETFALSASVKGNLLAVVLADNSANLYDITSQKLLFSE

KGSPSTTINSLMAMPIFMDTVVVFPMLDGRLLVVDYVHGNPTPIRNIVISSDKFFNNITYLIV

DGNNMIASTGKRILSVVSGQEFNYDGDIVDLLYDKGTLYVLTLDGQILQMDKSLRELNSVK

LPFASLNTIVLNHNKLYSLEKRGYVIEVDLNDFNSYNVYKTPTIGSFKFFSSNRLDKGVFYD

KNRVYYDRYYLDYNNFKPKLYPVVEKPASKKSQKGEKGNAPIYLQERHKAKEKPLEENKV

KPRNSGFEEDEVKANQRGMEPINNQNNANDENKVGNENNAIQRGENKNAPVSKESNAFKEAPKLSPKEEKRRLKEEKKKAKAEQRAREFEQRAREQQERDEKELEERRKALKTK.

5.2 Using Bepipred Linear Epitope Prediction 2.0 to analyze the immunogenicity sequence of the above membrane protein, it is found that SEQ ID No.1 (QERHKAKEKPLE) is a peptide with strong immunogenicity (highest score), and its corresponding nucleotide sequence is SEQ ID No. 2: CAAGAAAGGCATAAAGCTAAAGAAAAGCCTTTAGAA.

5.3 In order to further verify the immunogenicity of the peptide, 5 mg of the peptide shown in SEQ ID No.1 (purity 98%) was synthesized by a peptide synthesis company; 5 mg of a non-immunogenic peptide was synthesized as a control.

5.4 ELISA Verification

(1) Peptide pretreatment: First, dissolve 100mg BSA in 5mL water, 100mg EDC in 3mL water, then add the BSA solution dropwise to the EDC with stirring, react at room temperature for 10min, dissolve 5mg synthetic peptide in 200μL DMSO, and then Add 800 μL of PBS and mix well, then add activated BSA (stirring state) drop by drop, react at room temperature for 1 hour, then add the mixed solution into the dialysis bag, dialyze overnight at 4°C, and take the peptide out of the dialysis bag the next day.

(2) ELISA coated plate: first irradiate the polystyrene plate with ultraviolet light for 2 hours, then dilute the synthetic peptide to 0.1 mg/ml with coating buffer (coating buffer: Na ₂ CO ₃ and NaHCO ₃ buffer), Add 50 μL to the reaction wells of the styrene plate, overnight at 4°C, discard the solution in the wells the next day, and wash once with 1×TBST washing buffer at 180 μL per well.

(3) Blocking: add 60 μL of 1% BSA (prepared in TBST) to each well for blocking, and incubate at 37°C for 1 hour. Then discard the blocking solution.

(4) Adding samples: after serially diluting the collected immune whey (see step 3.2), 50 μL/well, incubate at 37°C for 1 hour, then discard the blocking solution and wash 2 with 1×TBST washing buffer at 180 μL per well. Second-rate.

(5) Adding enzyme-labeled antibody: add freshly diluted goat anti-bovine secondary antibody-HRP (diluted to 1:5000 with 1% BSA) into the wells of the enzyme-labeled plate at 50 μL/well, and incubate at 37°C for 45 minutes, then Discard the blocking solution and wash 3 times with 180 μL per well of 1× TBST buffer.

(6) Color development by adding substrate solution: Add 100 μL of temporarily prepared TMB substrate solution to each reaction well, and react at 37° C. for 5 minutes, as shown in FIG. 4 .

(7) Termination of the reaction: 90 μL of 2M sulfuric acid was added to each reaction well.

(8) Measure the absorbance value at a wavelength of 450nm on a microplate reader, and take the maximum dilution factor of OD ₄₅₀ 0.5 as the antibody titer (more than 2.1 times the OD value of the negative well). The antibody titer of the strongly immunogenic peptide (1:15000) was 2.5 times that of the control peptide (1:6000).

5.4 Mouse Validation

Fully emulsify the polypeptide shown in SEQ ID No.1+BSA+Freund's complete adjuvant, and subcutaneously inject 50 μg of polypeptide at multiple points on the limbs and back of BALB/c mice; perform the second immunization after 2 weeks, and use SEQ ID No. The polypeptide shown in 1+BSA+Freund's incomplete adjuvant was fully mixed, and 25 μg/mouse was injected subcutaneously at multiple points on the limbs and back. After 2 weeks, the third immunization was the same as the second time. 7-10 days after the third immunization, blood was collected from eyeballs to measure serum antibody titer (1:4000) by indirect ELISA (the experimental method was the same as that of milk antibody). The control group was injected with PBS.

SEQUENCE LISTING

<110> The Fourth Hospital of Hebei Medical University, Hebei Medical University

<120> Helicobacter pylori-specific immunogenic peptides

<130> 2021

<160> 3

<170> PatentIn version 3.3

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<211> 12

<212> PRT

<213> Synthetic

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Gln Glu Arg His Lys Ala Lys Glu Lys Pro Leu Glu

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<210> 2

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<212>DNA

<213> Synthetic

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caagaaaggc ataaagctaa agaaaagcct ttagaa 36

<210> 3

<211> 548

<212> PRT

<213> Helicobacter pylori

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Met Asn Lys Pro Phe Leu Ile Leu Leu Ile Ala Leu Ile Val Phe Ser

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Gly Cys Asn Met Lys Lys Tyr Phe Lys Pro Ala Lys His Gln Ile Lys

20 25 30

Gly Glu Ala Tyr Phe Pro Asn His Leu Gln Glu Ser Ile Val Ser Ser

35 40 45

Asn Arg Tyr Gly Ala Ile Leu Lys Asn Gly Ala Val Ile Gly Asp Lys

50 55 60

Gly Leu Thr Gln Leu Arg Ile Gly Lys Asn Phe Asn Tyr Glu Ser Ser

65 70 75 80

Phe Leu Asn Glu Ser Gln Gly Phe Phe Ile Leu Ala Gln Asp Cys Leu

85 90 95

Asn Lys Ile Asp Lys Lys Thr Ser Lys Ser Lys Val Ala Lys Thr Glu

100 105 110

Glu Thr Glu Leu Lys Leu Lys Gly Val Glu Ala Glu Val Gln Asp Lys

115 120 125

Val Cys His Gln Val Glu Leu Ile Ser Asn Asn Pro Asn Ala Ser Gln

130 135 140

Gln Ser Ile Ile Ile Pro Leu Glu Thr Phe Ala Leu Ser Ala Ser Val

145 150 155 160

Lys Gly Asn Leu Leu Ala Val Val Leu Ala Asp Asn Ser Ala Asn Leu

165 170 175

Tyr Asp Ile Thr Ser Gln Lys Leu Leu Phe Ser Glu Lys Gly Ser Pro

180 185 190

Ser Thr Thr Ile Asn Ser Leu Met Ala Met Pro Ile Phe Met Asp Thr

195 200 205

Val Val Val Phe Pro Met Leu Asp Gly Arg Leu Leu Val Val Asp Tyr

210 215 220

Val His Gly Asn Pro Thr Pro Ile Arg Asn Ile Val Ile Ser Ser Asp

225 230 235 240

Lys Phe Phe Asn Asn Ile Thr Tyr Leu Ile Val Asp Gly Asn Asn Met

245 250 255

Ile Ala Ser Thr Gly Lys Arg Ile Leu Ser Val Val Ser Gly Gln Glu

260 265 270

Phe Asn Tyr Asp Gly Asp Ile Val Asp Leu Leu Tyr Asp Lys Gly Thr

275 280 285

Leu Tyr Val Leu Thr Leu Asp Gly Gln Ile Leu Gln Met Asp Lys Ser

290 295 300

Leu Arg Glu Leu Asn Ser Val Lys Leu Pro Phe Ala Ser Leu Asn Thr

305 310 315 320

Ile Val Leu Asn His Asn Lys Leu Tyr Ser Leu Glu Lys Arg Gly Tyr

325 330 335

Val Ile Glu Val Asp Leu Asn Asp Phe Asn Ser Tyr Asn Val Tyr Lys

340 345 350

Thr Pro Thr Ile Gly Ser Phe Lys Phe Phe Ser Ser Asn Arg Leu Asp

355 360 365

Lys Gly Val Phe Tyr Asp Lys Asn Arg Val Tyr Tyr Asp Arg Tyr Tyr

370 375 380

Leu Asp Tyr Asn Asn Phe Lys Pro Lys Leu Tyr Pro Val Val Glu Lys

385 390 395 400

Pro Ala Ser Lys Lys Ser Gln Lys Gly Glu Lys Gly Asn Ala Pro Ile

405 410 415

Tyr Leu Gln Glu Arg His Lys Ala Lys Glu Lys Pro Leu Glu Glu Asn

420 425 430

Lys Val Lys Pro Arg Asn Ser Gly Phe Glu Glu Asp Glu Val Lys Ala

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Asn Gln Arg Gly Met Glu Pro Ile Asn Asn Gln Asn Asn Ala Asn Asp

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Glu Asn Lys Val Gly Asn Glu Asn Asn Ala Ile Gln Arg Gly Glu Asn

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Lys Asn Ala Pro Val Ser Lys Glu Ser Asn Ala Phe Lys Glu Ala Pro

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Lys Leu Ser Pro Lys Glu Glu Lys Arg Arg Leu Lys Glu Glu Lys Lys

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Lys Ala Lys Ala Glu Gln Arg Ala Arg Glu Phe Glu Gln Arg Ala Arg

515 520 525

Glu Gln Gln Glu Arg Asp Glu Lys Glu Leu Glu Glu Arg Arg Lys Ala

530 535 540

Leu Lys Thr Lys

545

Claims (3)

1. A Helicobacter pylori-specific immunogenic peptide, characterized in that its amino acid sequence is as shown in SEQ ID No .1. 2. A nucleic acid molecule encoding the Helicobacter pylori-specific immunogenic peptide as claimed in claim 1, characterized in that its nucleotide sequence is as shown in SEQ ID No.2. 3. A vector comprising the nucleic acid molecule of claim 2.

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