CN115073566B

CN115073566B - Helicobacter pylori specific immunogenic peptide fragments

Info

Publication number: CN115073566B
Application number: CN202210721363.9A
Authority: CN
Inventors: 单保恩; 赵连梅; 李志荣; 胡代伦; 张璁; 戴素丽
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: 2021-08-25
Filing date: 2022-06-24
Publication date: 2023-05-05
Anticipated expiration: 2042-06-24
Also published as: CN115073566A; CN113896775A

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 peptide fragments

Technical Field

The invention relates to a helicobacter pylori specific immunogenic peptide.

Background

Over 50% of the population worldwide is infected with helicobacter pylori (h.pyri), which is common in developing countries such as asia and africa. Almost all h.pyri infected patients have chronic gastritis, of which about 10% develop peptic ulcers and 1% develop gastric cancer in the next decade to decades. Thus, prevention and treatment of h.pyri infection is an important means of preventing and treating peptic ulcers and chronic gastritis, and preventing the occurrence of gastric cancer.

The treatment of H.pyri infection in clinic mainly depends on triple or quadruple therapy based on antibiotics, but has a plurality of problems such as high cost, continuous increase of drug-resistant strains, destruction of normal intestinal flora, easy recurrence and reinfection after drug withdrawal, and the like. Particularly the problem of increased resistance, results in an increasing difficulty in the treatment of helicobacter pylori infections.

During h.pyri infection, part of the membrane or bacterial proteins can elicit an immune response in humans, closely related to h.pyri adhesion colonization, persistent infection, and severity of disease. Currently, in addition to antibiotic treatment, immunotherapeutic approaches to prepare related vaccines are also used to treat h.pyri infection, targeting membrane proteins and/or other mycoproteins. For example, H.pyri vaccine studies have been partially developed using H.pyri recombinant urease and subunits thereof, cytotoxin-related proteins (CagA), vacuolated toxin-related proteins (VacA), adhesin A (HpaA), and the like.

However, none of the currently available h.pyri vaccines has been able to eradicate h.pyri, and there is an urgent need to search for new targets of immunogenic proteins, which provide the basis for the preparation of effective h.pyri vaccines.

Disclosure of Invention

The invention aims at providing a novel helicobacter pylori specific immunogenic peptide segment with immunogenicity.

The invention adopts the following technical scheme:

an helicobacter pylori-specific immunogenic peptide comprising the amino acid sequence as shown in SEQ ID No. 1.

A nucleic acid molecule encoding the above helicobacter pylori-specific immunogenic peptide comprising a nucleotide sequence as shown in SEQ ID No. 2.

A vector comprising the above nucleic acid molecule.

A monoclonal antibody prepared by using the helicobacter pylori specific immunogenic peptide.

A vaccine prepared using the helicobacter pylori-specific immunogenic peptide described above, for stimulating an immune response in a host organism.

An helicobacter pylori-specific immunogenic protein comprising a membrane protein having the amino acid sequence shown in SEQ ID No. 1.

Further, the helicobacter pylori specific immunogenic protein is a membrane protein pgbB comprising an amino acid sequence shown as SEQ ID No. 3.

The invention has the beneficial effects that: the antigen of the invention can be used for preparing new H.pyri vaccine and other active or passive immunology therapies for H.pyri treatment, and has important significance for preventing and treating H.pyri infection.

Drawings

FIG. 1 shows dielectrophoresis and immunoblotting to detect the H.pyri milk antibody immunomembrane protein target.

FIG. 2 shows the immune agar bi-directional diffusion assay H.pyri milk antibody immunomembrane protein target.

FIG. 3 shows the detection of the H.pyrri milk antibody immunomembrane protein target by IP assay.

FIG. 4 is an ELISA validation antigenicity experiment.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

1. Cultivation of helicobacter pylori

Helicobacter pylori clinical isolate HP435 was inoculated in a modified campylobacter medium (Campy-BAP) 9cm in diameter containing 10% defibrinated sheep blood, microaerophilic (5% O) at 37 ℃ ₂ ，10％ CO ₂ ，85％ N ₂ Humidity 95%) for 3-4 days.

2. Extraction of helicobacter pylori membrane protein

H.pyri cells were washed 3 times with sterile 0.1mol/L Phosphate Buffer (PBS) and suspended in 20% glycerol broth for frozen storage at-80 ℃. For strains producing membrane proteins, H.pyrri cells should be washed 3 times with sterile 0.1mol/L PBS. EZ-Link Sulfo-NHS-SS-biotin reaction solution with the concentration of 250 mug/ml is prepared, and the freshly washed H.pyri cell membrane protein is marked. The labeled somatic cells were sonicated (3 s on/7s off, intensity 20%,5 min). The supernatant enriched in H.pyri membrane protein was collected by centrifugation at 12,000g for 30min at 4 ℃. The membrane proteins were then purified using an avidin column.

3. Milk cow immunization and antibody preparation

3.1 cow immunization

Selecting 3-4 years old, and gestating for 6-7 months, and keeping healthy Holstein cow. Preparation of 5mg Membrane protein plus 4X 10 ⁹ The whole cells were fixed to 5ml with 0.1mol/LPBS, mixed with 5ml of aluminum hydroxide adjuvant (alum adjuvant) by shaking, and immunized by subcutaneous injection in the cervical subcutaneous and the subiliac lymph nodes of the cows 5 times. Wherein the first and second immunizations are separated by 20 days, and the immunization is completed from day 10 to day 10 before calving of the cow.

3.2 preparation of antibodies

Collecting colostrum of milk cow within one week after calving, centrifuging at 4deg.C for 20min, adding appropriate amount of hydrochloric acid to adjust pH to 4.6, centrifuging at 12000rpm 4deg.C for 30min to remove casein precipitate, and collecting clear supernatant (whey). The extracted antibody-enriched whey was subjected to antibody purification using a GE protein purifier, followed by connection of Hitrap Protein G HP ml purification column. The obtained purified antibody was subjected to antibody titer detection using an ELISA method. Purified antibodies were obtained with an antibody titer of 1:15000.

4. Identification of immunogenic Membrane proteins Using three methods

4.1 two-dimensional electrophoresis and immunoblotting detection of H.pyri membrane protein positive for immunogenicity with milk antibody two (150. Mu.g protein each) HP435 strain membrane protein samples were labeled with the same Cy dye, and two-dimensional electrophoresis was performed simultaneously. And (3) using a Typhoon multicolor fluorescence imaging scanner to image and position the PAGE gel after two-dimensional electrophoresis. Then, one of the plate PAGE gels was subjected to immunoblotting using the purified antibodies prepared according to the present invention as primary antibodies, with Cy5 or Cy3 labeled (as distinguished from the dye labeling the protein sample) goat anti-bovine secondary antibodies. Detecting the immunogenic protein spots.

As a result, it was found that 6 to 8 immunoreactive-positive protein spots were identified (see FIG. 1). And (3) corresponding to the immunoblotting test result and the position of the immunoreactive positive protein spot on the original gel, digging a protein spot at the corresponding position on the other plate PAGE gel, and carrying out protein mass spectrum identification. As a result, it was found that 112 proteins could be identified.

4.2 immune agar two-way diffusion assay detection of H.pyrri membrane protein positive for immunogenicity with milk antibodies the whole bacterial suspension of HP435 strain was subjected to an immune agar two-way diffusion assay with a blank antibody and purified antibodies after immunization, respectively (see FIG. 2).

As a result, it was found that a clear antigen-antibody precipitation line appeared between the purified antibody and the whole bacterial suspension. The pellet was excised, denatured by boiling with 5 Xprotein loading buffer, and subjected to SDS-PAGE. And (3) after electrophoresis, carrying out coomassie brilliant blue staining on the PAGE gel, cutting off the developed protein bands, and carrying out mass spectrum identification after enzymolysis in the gel. As a result, it was found that 501 proteins could be identified.

4.3IP assay and protein Mass Spectrometry identification

The extracted h.pyri membrane protein was IP tested with purified antibodies. The proteins after IP were subjected to SDS-PAGE, stained with Coomassie blue, and the developed protein bands were subjected to in-gel enzymolysis and mass spectrometry (see FIG. 3). As a result, it was found that 403 protein could be identified.

5. Further analysis of the authentication results

5.1 carrying out intersection analysis on the protein identification results obtained by the detection of the three immunological identification methods, and finding 13 common proteins. Wherein, the pgbB with the amino acid sequence shown in SEQ ID No.3 is a newly discovered membrane protein with immunogenicity.

SEQ ID No.3：

MNKPFLILLIALIVFSGCNMKKYFKPAKHQIKGEAYFPNHLQESIVSSNRYGAILKNGAVIGD

KGLTQLRIGKNFNYESSFLNESQGFFILAQDCLNKIDKKTSKSKVAKTEETELKLKGVEAEV

QDKVCHQVELISNNPNASQQSIIIPLETFALSASVKGNLLAVVLADNSANLYDITSQKLLFSE

KGSPSTTINSLMAMPIFMDTVVVFPMLDGRLLVVDYVHGNPTPIRNIVISSDKFFNNITYLIV

DGNNMIASTGKRILSVVSGQEFNYDGDIVDLLYDKGTLYVLTLDGQILQMDKSLRELNSVK

LPFASLNTIVLNHNKLYSLEKRGYVIEVDLNDFNSYNVYKTPTIGSFKFFSSNRLDKGVFYD

KNRVYYDRYYLDYNNFKPKLYPVVEKPASKKSQKGEKGNAPIYLQERHKAKEKPLEENKV

KPRNSGFEEDEVKANQRGMEPINNQNNANDENKVGNENNAIQRGENKNAPVSKESNAFKEAPKLSPKEEKRRLKEEKKKAKAEQRAREFEQRAREQQERDEKELEERRKALKTK。

5.2 analysis of the above membrane proteins by using Bepipred Linear Epitope Prediction 2.0.2.0 for immunogenicity sequence, SEQ ID No.1 (QERHKAKEKPLE) was found to be a peptide fragment with strong immunogenicity (highest score), and the corresponding nucleotide sequence was SEQ ID No.2: CAAGAAAGGCATAAAGCTAAAGAAAAGCCTTTAGAA.

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

5.4ELISA verification

(1) Polypeptide pretreatment: firstly, 100mg of BSA is dissolved in 5mL of water, 100mg of EDC is dissolved in 3mL of water, then the BSA solution is stirred and dropwise added with EDC, the mixture is stirred and reacted at room temperature for 10min, 5mg of synthetic polypeptide is dissolved in 200 mu L of DMSO, 800 mu L of PBS is added and uniformly mixed, activated BSA (stirred state) is added dropwise and reacted at room temperature for 1h, then the mixed solution is added into a dialysis bag, the dialysis is carried out at 4 ℃ for overnight, and the polypeptide is taken out from the dialysis bag the next day.

(2) ELISA plates: the polystyrene plate was first irradiated with ultraviolet rays for 2 hours, and then with a coating buffer (coating buffer: na) ₂ CO ₃ And NaHCO ₃ Buffer) the synthetic polypeptide was diluted to 0.1mg/ml, 50. Mu.L was added to each reaction well of the polystyrene plate, overnight at 4℃for the next day, the intra-well solution was discarded, and the buffer was washed 1 time with 1 XTBE at 180. Mu.L per well.

(3) Closing: each well was blocked with 60. Mu.L of 1% BSA (TBST formulation) and incubated at 37℃for 1 hour. After which the blocking solution is discarded.

(4) Sample adding: after gradient dilution of the collected immune whey (see step 3.2), 50. Mu.L/well was incubated at 37℃for 1 hour, after which the blocking solution was discarded and washed 2 times with 1 XTBE buffer at 180. Mu.L/well.

(5) Adding enzyme-labeled antibody: freshly diluted goat anti-bovine secondary antibody-HRP (diluted 1:5000 with 1% BSA) was added to the wells of the microplate at 50. Mu.L/well, incubated at 37℃for 45min, after which the blocking solution was discarded and washed 3 times with 180. Mu.L/well of 1 XTBST buffer.

(6) Adding a substrate solution for color development: 100. Mu.L of the TMB substrate solution prepared temporarily was added to each reaction well, and the mixture was allowed to react at 37℃for 5 minutes, as shown in FIG. 4.

(7) Terminating the reaction: to each reaction well was added 90. Mu.L of 2M sulfuric acid.

(8) Measuring absorbance at 450nm wavelength on enzyme-labeled instrument with OD ₄₅₀ The maximum dilution factor of 0.5 is 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 verification

Full emulsification of the polypeptide shown in SEQ ID No.1, BSA and Freund's complete adjuvant, and subcutaneous multipoint injection of 50 mug/mouse BALB/c in four limbs and back; immunization 2 was performed after 2 weeks, and the polypeptide shown in SEQ ID No. 1+BSA+Freund's incomplete adjuvant was thoroughly mixed, and four limbs and back were subcutaneously injected at 25. Mu.g/dose, followed by immunization 3 rd after 2 weeks, and the same as 2 nd. Serum antibody titers (1:4000) were determined by indirect ELISA (experimental procedure was as with the pre-milk antibodies) from 7-10d eyeballs after the 3 rd immunization. Control groups were injected with PBS.

SEQUENCE LISTING

<110> fourth Hospital of university of Hebei medical science, university of Hebei medical science

<120> helicobacter pylori-specific immunogenic peptide fragment

<130> 2021

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> Synthesis

<400> 1

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

1 5 10

<210> 2

<211> 36

<212> DNA

<213> Synthesis

<400> 2

caagaaaggc ataaagctaa agaaaagcct ttagaa 36

<210> 3

<211> 548

<212> PRT

<213> Helicobacter pylori

<400> 3

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

1 5 10 15

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

1. A helicobacter pylori specific immunogenic peptide fragment is characterized in that the amino acid sequence is shown as SEQ ID No. 1.

2. A nucleic acid molecule encoding an immunogenic peptide specific for helicobacter pylori according to claim 1, characterized in that it has the nucleotide sequence shown in SEQ ID No. 2.

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