CN112402600B

CN112402600B - Helicobacter pylori tetravalent virulence factor GEM particle vaccine, preparation method and application

Info

Publication number: CN112402600B
Application number: CN202011088805.8A
Authority: CN
Inventors: 刘昆梅; 郭乐; 杨华; 王淑娥; 张帆; 姜中佳
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2022-11-18
Anticipated expiration: 2040-10-13
Also published as: CN112402600A

Abstract

The invention provides a helicobacter pylori tetravalent virulence factor GEM particle vaccine, a preparation method and application thereof, belonging to the field of biomedicine and bioengineering. The application provides a helicobacter pylori tetravalent virulence factor GEM particle vaccine GEM-SAM ^E FVpE, which will contain mainly the core component SAM ^E And recombinant antigen SAM of helicobacter pylori virulence factor multi-epitope peptide FVpE ^E the-FVPE display is prepared on the GEM particle surface and has good antigen surface display property. Helicobacter pylori tetravalent virulence factor GEM particle vaccine GEM-SAM ^E The FVpE has good M cell targeting of the gastrointestinal tract and can efficiently recombine the antigen SAM ^E Targeted delivery of FVpE to gastrointestinal M cells stimulates the gastrointestinal tract to mount a specific mucosal immune response against a number of virulence factors of helicobacter pylori. GEM-SAM (GEM-S-M) particle vaccine of helicobacter pylori tetravalent virulence factor ^E the-FVPE can be applied to preventing and treating helicobacter pylori related gastropathy.

Description

Helicobacter pylori tetravalent virulence factor GEM particle vaccine, preparation method and application

Technical Field

The application relates to the field of biomedicine and bioengineering, in particular to a tetravalent virulence factor GEM particle vaccine of helicobacter pylori.

Background

Helicobacter pylori (Hp) is an important pathogenic factor of gastritis, gastric ulcer and gastric cancer, the world infection rate exceeds 50 percent, the Hp infection rate in China reaches 58.07 percent, and the helicobacter pylori has obvious family aggregation and more severe form. At present, the method for clinically treating the Hp infectious gastropathy is mainly a multi-antibiotic therapy, but the method faces the problems of Hp drug resistance caused by antibiotic, reinfection after treatment, damage of intestinal microecological balance by antibiotics, toxic and side effects of medicines, poor patient compliance and the like, is difficult to popularize and use in Hp infected people on a large scale, and has good application prospect in research and development of effective Hp vaccines.

Oral vaccines are generally weak in immunogenicity, require multiple vaccinations with a large amount of antigen, have short duration of immune response, and are prone to immune tolerance. The microfold cells (M cells) are specialized antigen transport cells in the gastrointestinal mucosal immune system, are dispersed among mucosal epithelial cells, can transport antigens from the gastrointestinal cavity to the lymphatic tissues under the epithelium, and induce mucosal immune response. M cells are the "entry" to the gastrointestinal mucosal immune system, and their uptake of antigen is the first step critical to the initiation of the gastrointestinal mucosal immune response. Therefore, whether the antigen can be massively taken by M cells is the key point of the immune success or failure of the oral vaccine. The antigen is presented to the M cells in a targeted way by using the M cell targeted peptide combined with the M cell surface receptor, the antigen uptake and transport efficiency of the M cells is increased, the high-level mucosal immune response is stimulated, and the method is an effective strategy for improving the immune effect of the oral vaccine.

The oral vaccine has certain advantages in stimulating gastrointestinal mucosal immune response and specific cellular immune response, but the vaccine effect is usually very little when the oral vaccine is directly taken orally, wherein the main reasons are as follows: the presence of large amounts of acids and proteolytic enzymes in the gastrointestinal tract can destroy vaccine antigens. Therefore, the research of safe and effective mucosal immune delivery system is a hot spot in the research field of oral vaccine. The lactobacillus is a group of common probiotic bacteria in human intestinal tracts, is used as a mucous membrane immune delivery system of oral vaccines, and has the unique advantages of high safety and the like. In addition, the antigen displayed on the surface of the lactobacillus is more suitable for the requirement of mucosal vaccine. The dockerin is a core component in bacterial surface display technology. N-acetylmuramidase (AcmA) is a dockerin commonly used in lactobacillus surface display systems. However, acmA expressed intracellularly tends to have weak transmembrane transport activity. The design of a more effective lactobacillus cell surface display system based on AcmA is still a work to be researched.

Disclosure of Invention

In the first aspect of the application, a tetravalent virulence factor GEM particle vaccine of helicobacter pylori is disclosed, and the GEM particle vaccine comprises a recombinant antigen SAM displayed on the surface of GEM particles ^E -FVpE; recombinant antigen SAM ^E FVpE contains the core component SAM ^E And a helicobacter pylori virulence factor polyepitope peptide FVpE.

The GEM particle surface display technology is a novel inactive and non-genetically modified lactobacillus surface display technology based on gram-positive enhanced matrix particles (namely GEM particles), is called as GEM particle surface display technology and is also called as bacteria-like particle surface display technology. The GEM particle surface display technology mainly utilizes hot acid treatment lactobacillus to remove macromolecular substances such as protein, nucleic acid and the like, and hollow particles only containing cell wall peptidoglycan skeletons, called GEM particles, are prepared; the high-purity vaccine antigen is mixed with GEM particles, the vaccine antigen is displayed on the surfaces of the GEM particles through the anchoring protein coupled with the vaccine antigen, and the prepared vaccine is called as GEM particle vaccine and bacterial-like particle vaccine.

The helicobacter pylori mainly adheres to gastric mucosal cells through adhesion factors, and then can release some virulence factors to destroy the gastric mucosal cells, so that diseases such as chronic gastritis, peptic gastric ulcer and gastric cancer are caused. The virulence factors of helicobacter pylori are mainly: urease, cytotoxin-associated protein A (CagA), vacuolating toxin-associated protein A (VacA), neutrophil activating protein NAP, etc. The Hp virulence factor multi-epitope vaccine FVpE mainly contains dominant antigen epitopes or sections from Hp key virulence factors (urease, cagA and VacA) and neutrophil activating protein NAP, and researches prove that the FVpE has good immune effect for preventing and treating Hp infection.

Helicobacter pylori tetravalent virulence factor GEM particle vaccine GEM-SAM ^E -FVpE characteristics as follows: (1) GEM particle vaccine GEM-SAM ^E FVpE displays the recombinant antigen SAM on the surface of the GEM particles ^E -FVpE; recombinant antigen SAM ^E FVpE contains the core component SAM ^E And a helicobacter pylori virulence factor polyepitope peptide FVpE. (2) GEM particle vaccine GEM-SAM ^E -FVPE surface-displayed recombinant antigen SAM ^E The FVpE has the property of M cell targeting and can increase the SAM of M cells in the gastrointestinal tract to recombinant antigen ^E -uptake and transport efficiency of FVpE, enhancing gastrointestinal mucosa-specific immune responses. (3) GEM particle vaccine GEM-SAM ^E The FVPE stimulates the body to produce specific mucosal immune responses against a number of key virulence factors of H.pylori.

The helicobacter pylori virulence factor multi-epitope peptide FVpE mainly contains dominant antigen epitopes or sections from Hp key virulence factors (urease, cagA and VacA) and neutrophil activating protein NAP, and researches prove that the FVpE has good immune effect on preventing and treating Hp infection.

In some embodiments of the first aspect of the foregoing, the amino acid sequence of the virulence factor polyepitope peptide FVpE is shown as SEQ ID No.1 and the nucleotide sequence encoding the virulence factor polyepitope peptide FVpE is shown as SEQ ID No. 2.

In some embodiments of the foregoing first aspect, the core component SAM ^E The amino acid sequence of (A) is shown as SEQ ID NO.3, and the coding core component SAM ^E The nucleotide sequence of (A) is shown in SEQ ID NO. 4.

In some embodiments of the foregoing first aspect, the recombinant antigen SAM ^E The amino acid sequence of FVpE is shown in SEQ ID NO. 5; recombinant antigen SAM ^E The nucleotide sequence of-FVpE is shown in SEQ ID NO. 6.

In the examples, recombinant antigen SAM ^E In the FVpE, by inserting the sequence of the virulence factor FVpE into the core component SAM ^E Is obtained in (1).

The second aspect of the application discloses the application of the helicobacter pylori tetravalent virulence factor GEM particle vaccine in preparing the medicine for preventing and treating diseases caused by the helicobacter pylori.

In a third aspect of the application, a preparation method of the helicobacter pylori tetravalent virulence factor GEM particle vaccine comprises the following steps:

synthesizing the core component SAM shown as a sequence SEQ ID NO.4 ^E Gene fragment, SAM ^E The gene fragment is cloned to a pCzn1 carrier through enzyme digestion and connection to obtain the PESAM ^E A recombinant vector;

amplifying to obtain a virulence factor multi-epitope peptide FVpE gene fragment shown as a sequence SEQ ID NO.2, and carrying out double enzyme digestion on the FVpE fragment and the pepSAM ^E Mixing the recombinant vectors, and cloning the FVpE fragment into the pepSAM by a one-step seamless cloning technology ^E Recombinant vector to obtain a PESAM ^E -an FVpE recombinant vector;

mixing the pepSAM ^E Transforming the-FVpE recombinant vector into a host cell, and obtaining a recombinant antigen SAM through culture, induction expression by an inducer and purification ^E -FVpE；

Preparing GEM granules of lactic acid bacteria by trichloroacetic acid, preparing the GEM granules and recombinant antigen SAM ^E Mixing and resuspending-FVpE to obtain GEM particle vaccine GEM-SAM ^E -FVpE。

In some embodiments of the foregoing third aspect, the inducing agent is IPTG and the working concentration of the inducing agent is 0.5mmol/L.

In the embodiment, the recombinant expression strain can rapidly express the SAM of the recombinant antigen in a large quantity through the induction of IPTG ^E -FVpE。

In some embodiments of the foregoing third aspect, the expression host cell is e.coli;

preferably, the E.coli is selected from Arcticexpress (DE 3), DH5 α, TOP10, BL21 (DE 3), BL21 (DE 3) pLysS or Rosetta;

further preferably, the escherichia coli is ArcticExpress (DE 3).

In the examples, there are various host cells for protein expression, and ArcticExpress (DE 3) strain in Escherichia coli is preferable for expression in the examples.

The target protein can be expressed rapidly and efficiently through an expression system of escherichia coli, and the yield and the quality of the recombinant protein are improved.

In some embodiments of the foregoing third aspect, purifying the recombinant antigen comprises chromatography and dialysis; the column chromatography adopts Ni-IDA-Sepharose CL-6B affinity chromatography column; the dialyzed dialysate was 20 mM Tris-HCl, 0.10M NaCl, pH 8.0.

In the examples, not only the concentration and quality of the recombinant protein can be improved by affinity chromatography and dialysis, but also some toxins in the protein can be removed by dialysis and the like. The concentration of the protein is improved, the immune effect of the protein can be improved, the toxin is removed by dialysis, and the safety of the protein can be improved.

In some embodiments of the foregoing third aspect, the flow rate of the chromatography buffer is 0.5-1mL/min, and the dialyzed dialysate is a solution of 20 mmol/L Tris-HCl,0.10 mol/L NaCl, pH 8.0.

In the examples, the protein is preferably purified by washing with a buffer at a suitable flow rate. The toxin and the like can be better removed without damaging the structure and the function of the protein through the dialysis of the dialysate with proper concentration.

Compared with the prior art, the beneficial effect of this application is: the application provides a helicobacter pylori tetravalent virulence factor GEM particle vaccine GEM-SAM ^E FVpE, which will contain mainly the core component SAM ^E And recombinant antigen SAM of helicobacter pylori virulence factor multi-epitope peptide FVpE ^E the-FVPE is prepared on the surface of GEM particles and has good antigen surface display property. GEM-SAM (GEM-S-M) particle vaccine of helicobacter pylori tetravalent virulence factor ^E The FVpE has good M cell targeting of the gastrointestinal tract and can efficiently recombine the recombinant antigen SAM ^E Targeted delivery of FVpE to M cells of the gastrointestinal tract provokes the gastrointestinal tract to mount a specific mucosal immune response against multiple virulence factors of helicobacter pylori. GEM-SAM (GEM-S-M) particle vaccine of helicobacter pylori tetravalent virulence factor ^E the-FVpE can be applied to preventing and treating helicobacter pylori related gastropathy.

Drawings

FIG. 1 is a schematic view of an embodimentColi plasmid pesAM in example 1 ^E A schematic diagram of (a);

FIG. 2 is the E.coli plasmid pesAM of example 1 ^E Double digestion electrophorogram;

FIG. 3 is the E.coli plasmid PESAM of Experimental example 2 ^E -double-restriction electrophoretogram of FVpE;

FIG. 4 shows Escherichia coli in Experimental example 3E. coli-peSAM ^E -graphs of the induced expression results of FVpE;

FIG. 5 shows the GEM-SAM vaccine for GEM particle in Experimental example 4 ^E -an antigen surface display profile of FVpE;

FIG. 6 is the GEM particle vaccine GEM-SAM of Experimental example 5 ^E -an M cell targeting profile of FVpE;

FIG. 7 is GEM particle vaccine GEM-SAM of Experimental example 6 ^E -a graph of efficacy of FVpE immunotherapy of helicobacter pylori infection;

FIG. 8 is the GEM particle vaccine GEM-SAM of Experimental example 7 ^E Schematic representation of the induction of antibody production in mice after FVpE immunotherapy.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Material

Iptg solution: 1.2g of IPTG is weighed and placed in a 50mL centrifuge tube, 40mL of sterile water is added, and after the sterile water is fully mixed and dissolved, the volume is determined to be 50mL. Filtering with 0.22 μm filter, sterilizing, packaging, and storing at-20 deg.C.

2. Ampicillin (Amp) stock (100 mg/mL): weighing 100mg of ampicillin (Amp) and dissolving the ampicillin (Amp) in 1mL of sterile water to prepare a stock solution with the concentration of 100mg/mL, filtering and sterilizing the stock solution through a 0.22 mu m bacterial filter, and subpackaging and storing the solution in a refrigerator at the temperature of-20 ℃.

LB medium: (1) LB liquid Medium: 10g tryptone, 5g yeast extract and 5g NaCl were weighed, distilled water was added to 1000mL, pH was adjusted to 7.4, and autoclaved. (2) LB solid Medium: 1.5g agar powder per 100ml LB medium, autoclaved, and poured onto plates.

DNA electrophoresis buffer (50 XTAE): 242g Tris, 37.2g Na2EDTA.2H2O and 57.1mL glacial acetic acid are weighed, and water is added to 1000mL for dilution by 50 times when in use.

SDS-PAGE running buffer (5X): weighing 15.1g of Tris powder, 94g of glycine and 5.0g of SDS; adding about 800mL of deionized water, and stirring for dissolving; adding deionized water to a constant volume of 1L, and storing at room temperature; note that: the water should be allowed to flow down slowly along the wall when added to avoid the formation of a lot of foam due to SDS.

6. Coomassie brilliant blue protein staining reagent: (1) Coomassie brilliant blue R-250 dye liquor: 0.29g of Coomassie Brilliant blue R-250 was dissolved in 250ml of destaining solution. (2) fixing liquid: 500mL of ethanol and 100mL of glacial acetic acid were diluted to 1000mL with distilled water. (3) decolorizing liquid: 250mL of ethanol and 80mL of glacial acetic acid were diluted to 1000mL with distilled water. (4) preserving fluid: 25mL 87% glycerol was dissolved in 225mL destaining solution.

7. Experimental animals: BALB/c mice were SPF grade, male, 8-10 weeks old, purchased from Ningxia medical university laboratory animal center.

ELISA reagent: (1) coating liquid: 1.6g Na ₂ CO ₃ ，2.9g NaHCO ₃ ，0.2g NaN ₃ Adding double distilled water to 1L, and adjusting pH to 9.6. (2) washing solution: respectively weighing 0.2g KH ₂ PO ₄ ，2.9g Na ₂ HPO ₄ ∙12H ₂ O,8.0g NaCl,0.2g KCl,0.5mL Tween-20, ddH ₂ O to 1000mL (PBST). (3) sealing liquid: 3.0g of BSA was weighed and dissolved in 100mL of washing buffer, filtered and sterilized, and then stored at 4 ℃. (4) substrate solution: soluble mono-component TMB substrate solution. (5) stop solution: 178.3mL of distilled water were measured out and 21.7mL (1M H) of concentrated sulfuric acid was added dropwise ₂ SO ₄ ）。

Bhi blood plates: weighing 3.5g of BHI dry powder, adding 93mL of distilled water, 1.5g of agar powder, sterilizing at 121 ℃ for 13min, cooling to below 60 ℃, adding 7mL of defibrinated sheep blood, polymyxin B (with the final concentration of 5 mu g/mL), vancomycin (with the final concentration of 10 mu g/mL) and trimethoprim (with the final concentration of 5 mu g/mL), subpackaging to a culture dish, and cooling for later use.

The features and properties of the present application are described in further detail below with reference to examples.

Example 1: escherichia coli plasmid PESAM ^E Construction of

SAM according to core component ^E The amino acid sequence of SEQ ID NO.3, the coding gene thereof is designed and optimized and the core component SAM is synthesized ^E The gene sequence of (A) is shown in SEQ ID NO. 4.

Extracting pCzn1 carrier plasmid, and double cutting pCzn1 carrier plasmid and core component SAM with Nde I/Hind III enzyme ^E (ii) a At 37 ℃ for 2 h; SAM recovery by DNA recovery kit ^E Gene and pCzn1 carrier double enzyme digestion products. The recovered pCzn1 linearized vector and SAM ^E The gene segments are connected into a closed circular DNA molecule through complementary cohesive ends under the action of T4 DNA ligase, namely the escherichia coli plasmid PESAM is formed ^E 。

The plasmid structure is shown in FIG. 1, the ligated plasmid is detected by double digestion and electrophoresis, and the result is shown in FIG. 2, wherein lane 1 is the E.coli plasmid PESAM ^E Lane 2 is the result of double digestion; the double digestion results are consistent with the actual results. And (3) the sample is sent for sequencing, the sequencing result is accurate, and no frameshift mutation exists.

Example 2: escherichia coli recombinant plasmid PESAM ^E Construction of the-FVpE

The obtained Escherichia coli recombinant plasmid PESAM ^E The mixture is subjected to double enzyme digestion with Kpn I and Xba I at 37 ℃ for 2 hours, and the PESAM is recovered by using a DNA recovery kit ^E Carrying out double digestion on linearized fragments; amplifying according to the sequence shown in the sequence SEQ ID NO.2 to obtain the FVpE gene segment (two ends of which are provided with the linear cloning vector peSAM) expressing the amino acid sequence shown in the sequence SEQ ID NO.1 ^E 15bp homologous sequence with perfect agreement at both ends). The recovered pesAM ^E Mixing the linearized vector and the FVPE gene, connecting into a closed circular DNA molecule by a one-step seamless cloning technology,namely, the Escherichia coli recombinant plasmid PESAM is formed ^E -FVpE。

The Escherichia coli recombinant plasmid pesAM ^E The results of electrophoresis after double digestion of the-FVPE are shown in FIG. 3, in which lane 1 is the E.coli recombinant plasmid PESAM ^E -FVpE; lane 2 is the NdeI and HindIII digested recombinant E.coli plasmid PESAM ^E -FVpE; in line with the practice. The obtained recombinant Escherichia coli recombinant plasmid PESAM ^E Sequencing verification is carried out on the-FVpE, and the result shows that the constructed Escherichia coli recombinant plasmid peSAM ^E the-FVPE sequence is accurate and free of frameshift mutations.

Example 3: recombinant antigen SAM ^E Expression, purification and detection of-FVPE

The obtained Escherichia coli recombinant plasmid PESAM ^E -FVpE transforming host cells for protein expression and purification.

（1）E.coli-peSAM ^E Inducible expression of FVpE: the correct recombinant expression plasmid, PESAM, will be verified ^E -FVPE was transformed into E.coli ArcticExpress (DE 3). The positive clones were inoculated into 3mL LB medium tubes containing 50. Mu.g/mL AMP, shaken overnight at 37 ℃ and 220 rpm; the next day, according to 1:100 was inoculated into 30 mL of LB medium containing 50. Mu.g/mL of AMP, and cultured at 37 ℃ and 220 rpm with shaking for about 2 hours until the OD of the cells became ₆₀₀ 0.6-0.8; then, IPTG was added to a final concentration of 0.5mM, 11 ℃ and 220 rpm, and shaken overnight to induce recombinant protein expression.

(2) SDS-PAGE detectionE.coli-peSAM ^E Expression of FVpE:E.coli-peSAM ^E after the inducible expression of-FVPE, taking out 1mL of culture, centrifuging at 10000g of room temperature for 2 min, discarding the supernatant, and resuspending the bacterial pellet with 100 μ L of loading buffer; centrifuging the residual culture at 4000g for 10min, discarding the supernatant, and resuspending the thallus precipitate with PBS; after the heavy suspension is subjected to ultrasonic disruption, respectively taking the supernatant and the precipitation solution, adding a loading buffer solution for heavy suspension, and performing SDS-PAGE detection analysis.

(3) Recombinant antigen SAM ^E Purification of the FVPE: the inclusion body solution was loaded to Ni-IDA Binding-Buffer pre-equilibrated Ni-IDA-Sepharose CL-6B affinity chromatography column; flushing with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL/min until the effluent OD280 value reaches the baseline; washing with Ni-IDA Washing-Buffer at a flow rate of 1mL/min until the effluent OD ₂₈₀ The value reached the baseline; eluting the target protein by using Ni-IDA Elution-Buffer at the flow rate of 1mL/min, and collecting the effluent liquid; the protein solution collected above was added to a dialysis bag and dialyzed overnight using 20 mM Tris-HCl, 0.10M NaCl, pH8.0, and subjected to 12% SDS-PAGE and Western Blot identification analysis.

The result of SDS-PAGE is shown in FIG. 4a, after the induction of expression, SDS-PAGE detection shows that:E. coli-peSAM ^E the FVpE shows a target protein band at about 101KD, and the recombinant antigen SAM ^E Theoretical size of-FVpE (fig. 4 a); after Ni-IDA protein purification, high-purity recombinant antigen SAM can be obtained ^E -FVpE, purity about 98.86%, purification was better (fig. 4 b); mouse anti-FVpE recognizable SAM ^E FVpE, but murine Normal serum is not a recognition SAM ^E -FVpE (fig. 4 c), the expressed protein can be recognized by the corresponding antibody.

Example 4: GEM particle vaccine GEM-SAM ^E Preparation of FVpE and characterization of the antigen surface display Properties

Preparing GEM granules of lactic acid bacteria by trichloroacetic acid, and mixing the obtained GEM granules with purified recombinant antigen SAM ^E Mixing and resuspending-FVpE, and preparing GEM particle vaccine GEM-SAM ^E -FVpE。

The GEM-SAM ^E -FVpE、GEM-SAM ^E 、SAM ^E the-FVPE and GEM particles were coated separately onto ELSIA plates at 4 ℃ overnight. After washing 4 times, 300. Mu.L/well blocking solution was added at 37 ℃ for 2h. After 4 washes, mouse anti-FVPE (1. After 4 washes, HRP-labeled goat anti-mouse IgG (1 10000), 100 μ L/well, 37 ℃,60 min was added. Washing for 4 times, adding TMB developing solution, standing at room temperature for 10min, adding 100 μ L stop solution, and measuring OD ₄₅₀ The value is obtained.

The results are shown in FIG. 5, GEM-SAM ^E -FVPE and recombinant antigen SAM ^E the-FVPE can react specifically with Mouse anti-FVPE, but GEM-SAM ^E And GEM cannot be associated withThe specificity reaction of Mouse anti-FVpE occurs, which indicates that GEM-SAM ^E the-FVPE surface exhibits SAM ^E -FVPE antigen, GEM-SAM ^E Surface without SAM ^E -a FVpE antigen.

Example 5: GEM particle vaccine GEM-SAM ^E Identification of M cell targeting of FVpE

BALB/c mice were fasted overnight (about 12 h), anesthetized, the abdomen opened, the mid-ileum section ligated about 2cm in length to form a closed loop, and 100. Mu.L of the GEM particulate vaccine GEM-SAM ^E Injecting the FVPE and GEM particles into the ilex, excising the ilex after about 1h, washing with PBS three times, and fixing with 4% paraformaldehyde. Tissue sections were blocked with 3% Goat serum and then separately combined with Rabbit anti-FVpE polyclonal antibody and Goat anti-Rabbit IgG H&L (Alexa Fluor 647) antibody. M cells on the Pi lymph nodes are marked by Mouse anti-GP2 mAb (Alexa Fluor 488); nuclei were stained by DAPI.

The results are shown in FIG. 6, GEM particle vaccine GEM-SAM, compared to control GEM particles ^E -FVpE is able to bind to target cells with good M cell targeting properties; none of the controls had a targeting result.

Example 6: GEM particle vaccine GEM-SAM ^E Identification of the Effect of FVpE in the treatment of Hp infection

(1) Mouse experimental procedure

Grouping experiments: GEM-SAM ^E -FVPE immunomer, GEM-SAM ^E An immunization group and a GEM particle immunization group; each group had 10 mice, 30 mice in total.

Preparation of Hp-infected mouse model: the mice in each group were perfused with helicobacter pylori for 1, 3, 5, and 7 days for a total of 4 times, each time at 300 μ L/mouse, at a concentration of 1 × 10 ¹⁰ CFU/mL。

Mouse immunization: immunizing GEM particle vaccine GEM-SAM at 15, 22, 29 and 36 days respectively ^E -FVpE、GEM-SAM ^E And GEM at a concentration of

1X

10, 4 times per 300. Mu.L/mouse ¹⁰ CFU/mL (i.e., 3X 10) ⁹ CFU）。

Mice were sacrificed: mice were sacrificed on day 50 and samples were taken for testing.

(2) Quantitative culture detection of Hp in mouse stomach

A certain weight of mouse stomach tissue was weighed, the contents were rinsed out with normal saline, a tissue homogenate was prepared in 0.5mL of normal saline, 20 μ L of the homogenate was aspirated, and diluted according to 1. mu.L of each dilution was uniformly applied to BHI blood plates and cultured at 37 ℃ for 3 to 5 days under microaerophilic conditions. The Hp colony is identified by methods such as bacterial gram staining and hydrogen peroxide catalase. The positive Hp colonies were counted and the colony counts were converted to colony forming units per gram of stomach tissue (CFU/g) and calculated as: hp colonization density = number of Hp colonies x dilution/stomach weight.

(3) Histopathological examination of the mouse stomach:

HE staining: stomach tissue of mice fixed with 10% formaldehyde was embedded with paraffin, cut into tissue sections about 6 μm thick, observed for inflammation of stomach tissue by HE staining, and histopathological scoring was performed.

The scoring criteria were as follows:

no obvious visible leukocyte (lymphocyte and neutrophil) infiltration is 0 point;

a little of the medicine is dispersed in the deep part of the inherent layer of the gastric mucosa and infiltrated by the white blood cells to be 1 minute;

the infiltration of moderate leucocytes in the lamina propria from the deep part to the middle part of the gastric mucosa is 2 minutes;

a large amount of white blood cells infiltrate the inherent layer from the deep part to the middle part of the gastric mucosa, and a small amount of micro-abscess is 3 minutes;

severe disseminated leukocyte infiltration exists from the whole layer of the inherent layer of the gastric mucosa to the submucosa, and the number of micro-abscesses is 4.

Immunohistochemical staining: paraffin wax is cut into slices and dewaxed, and then sealed by normal goat serum for 10min at room temperature; dripping Rabbit anti-Hp diluted in a proper proportion at 37 ℃ for 1h; after washing, adding dropwise goat anti-rabbit IgG-HRP at 37 ℃ for 10min; DAB color development, washing, counterdyeing and sealing.

The results are shown in FIG. 7, after GEM-SAM ^E Hp quantitative culture and urease activity detection after Hp infected mice were immunotherapy with FVpE, the results show that: GEM-SAM ^E Stomach of-FVPE-immunized miceA significant reduction in Hp colonization, a reduction in urease activity (fig. 7a and 7 b); the histopathology experiment results of the stomach show that: GEM-SAM ^E The stomach mucosa and the stomach submucosa of the mice in the GEM immune group are infiltrated by a large amount of white blood cells, the stomach inflammation is obvious, and the Hp colonization is obvious; while GEM-SAM ^E Gastric inflammation was significantly reduced in-FVpE immunized mice without significant Hp colonization (fig. 7c, 7d, and 7 e).

Example 7: GEM particle vaccine GEM-SAM ^E Detection of mouse-specific antibodies after FVPE immunotherapy

Hp-infected mice are treated with GEM particle vaccine GEM-SAM ^E after-FVpE immunotherapy, sacrifice, serum 1 dilution, gastric mucus and intestinal fluid 1-fold dilution. 100 μ L/well of ELISA plate coated with Hp lysate was added at 37 ℃ for 60 min. After washing 4 times, HRP-labeled goat anti-mouse IgG or IgA (1 10000) was added at 100. Mu.L/well, 37 ℃ for 60 min. Washing for 4 times, adding TMB developing solution, keeping away from light for 10min at room temperature, adding 100 μ L stop solution, and measuring OD ₄₅₀ The value is obtained.

The results are shown in fig. 8, where fig. 8a is serum IgG antibody detection and fig. 8b is mucosal sIgA antibody detection. The Elisa experiment result shows that GEM-SAM ^E -the FVpE immunised group of mice is capable of producing Hp-specific IgG, sIgA; however, GEM-SAM ^E And GEM immunization group mice were unable to produce Hp-specific IgG, sIgA (fig. 8a, fig. 8 b).

In conclusion, the GEM particle vaccine GEM-SAM provided by the invention ^E FVpE displaying the recombinant antigen SAM on the surface of the GEM particles ^E FVpE, and the recombinant antigen SAM ^E The FVpE is delivered to M cells of gastrointestinal tracts in a targeted mode, the uptake and transport efficiency of the M cells to vaccine antigens is improved, specific mucosal immune response aiming at multiple key virulence factors of Hp is induced to an organism, and the potential of preventing and treating stomach diseases related to helicobacter pylori infection is achieved.

The embodiments described above are some, but not all embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.

SEQUENCE LISTING

<110> Ningxia medical university

<120> helicobacter pylori tetravalent virulence factor GEM particle vaccine, preparation method and application

<130> AAA

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 671

<212> PRT

<213> Artificial sequence

<400> 1

Met Glu Thr Phe Glu Ile Leu Lys His Leu Gln Ala Asp Ala Ile Val

1 5 10 15

Leu Phe Met Lys Val His Asn Phe His Trp Asn Val Lys Gly Thr Asp

20 25 30

Phe Phe Asn Val His Lys Ala Thr Glu Glu Ile Tyr Glu Glu Phe Ala

35 40 45

Asp Met Phe Asp Asp Leu Ala Glu Arg Ile Val Gln Leu Gly His His

50 55 60

Pro Leu Val Thr Leu Ser Glu Ala Ile Lys Leu Thr Arg Val Lys Glu

65 70 75 80

Glu Thr Lys Thr Ser Phe His Ser Lys Asp Ile Phe Lys Glu Ile Leu

85 90 95

Glu Asp Tyr Lys Tyr Leu Glu Lys Glu Phe Lys Glu Leu Ser Asn Thr

100 105 110

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

115 120 125

Leu Ala Lys Leu Gln Lys Ser Ile Trp Met Leu Gln Ala His Leu Ala

130 135 140

His Met Ile His Asn Asn Ala Leu Ser Ser Val Leu Met Gly Ser His

145 150 155 160

Asn Gly Ile Glu Pro Glu Lys Val Ser Leu Leu Tyr Gly Gly Asn Gly

165 170 175

Gly Pro Glu Ala Arg His Asp Trp Asn Ala Thr Val Gly Tyr Lys Asn

180 185 190

Gln Gln Gly Asp Asn Val Ala Thr Leu Ile Asn Val His Met Lys Asn

195 200 205

Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn Asn Pro

210 215 220

Ser Phe Tyr Leu Tyr Lys Glu Asp Gln Leu Thr Gly Ser Gln Arg Ala

225 230 235 240

Leu Ser Gln Glu Glu Ile Gln Asn Lys Val Asp Phe Met Glu Phe Leu

245 250 255

Ala Gln Asn Asn Ala Lys Leu Asp Asn Leu Ser Lys Lys Glu Lys Glu

260 265 270

Lys Phe Gln Asn Glu Ile Glu Asp Phe Gly Ser Ala Phe Phe Thr Thr

275 280 285

Val Ile Ile Pro Ala Ile Val Gly Gly Ile Ala Thr Gly Thr Ala Val

290 295 300

Gly Thr Val Ser Gly Leu Leu Gly Trp Gly Leu Lys Gln Ala Glu Glu

305 310 315 320

Ala Asn Lys Thr Pro Asp Lys Pro Asp Asn Ser Thr Gln Lys Thr Glu

325 330 335

Val Gln Pro Thr Gln Val Ile Asp Gly Pro Phe Ala Gly Gly Lys Asp

340 345 350

Thr Val Val Asn Ile Asp Arg Ile Asn Thr Lys Ala Asp Gly Thr Ile

355 360 365

Lys Val Gly Gly Phe Lys Ala Ser Leu Thr Thr Asn Ala Ala His Leu

370 375 380

Asn Ile Gly Lys Gly Gly Val Asn Leu Ser Asn Gln Ala Ser Gly Arg

385 390 395 400

Thr Leu Leu Val Glu Asn Leu Thr Gly Asn Ile Thr Val Asp Gly Pro

405 410 415

Leu Arg Val Asn Asn Gln Val Gly Gly Tyr Ala Leu Ala Gly Ser Ser

420 425 430

Ala Asn Phe Glu Phe Lys Ala Gly Val Asp Thr Lys Asn Gly Thr Ala

435 440 445

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

450 455 460

Asp Ala His Thr Ala Asn Phe Lys Gly Ile Asp Thr Gly Asn Gly Gly

465 470 475 480

Phe Asn Thr Leu Asp Phe Ser Gly Val Thr Asn Lys Lys Leu Asp Pro

485 490 495

Arg Val Pro Ser Ser Val Ala Ser Met Ile His Glu Val Gly Ile Glu

500 505 510

Ala Met Phe Pro Asp Gly Lys Lys Val Ala Ser Met Ile His Glu Val

515 520 525

Gly Ile Glu Ala Met Phe Pro Asp Gly Lys Lys Ser Ala Ile Asn His

530 535 540

Ala Leu Asp Val Ala Asp Lys Tyr Asp Val Gln Val Ala Ile His Thr

545 550 555 560

Asp Thr Lys Lys Ser Ala Ile Asn His Ala Leu Asp Val Ala Asp Lys

565 570 575

Tyr Asp Val Gln Val Ala Ile His Thr Asp Thr Lys Lys Ser Val Glu

580 585 590

Leu Ile Asp Ile Gly Gly Asn Arg Arg Ile Phe Gly Phe Asn Ala Leu

595 600 605

Val Asp Gly Ser Ser Val Glu Leu Ile Asp Ile Gly Gly Asn Arg Arg

610 615 620

Ile Phe Gly Phe Asn Ala Leu Val Asp Gly Ser Ser Ile Lys Glu Asp

625 630 635 640

Val Gln Phe Gly Ser Ser Ile Lys Glu Asp Val Gln Phe Gly Ser Ser

645 650 655

Ile Lys Glu Asp Val Gln Phe Leu Glu His His His His His His

660 665 670

<210> 2

<211> 2013

<212> DNA

<213> Artificial sequence

<400> 2

atggagacct ttgaaatcct gaaacacctg caagccgacg ccattgtcct gtttatgaaa 60

gtccacaact tccattggaa cgtgaaaggt acggactttt tcaacgtgca taaagccacc 120

gaagaaatct acgaagaatt cgctgatatg ttcgatgacc tggcggaacg cattgttcag 180

ctgggccatc acccgctggt cacgctgagc gaagcgatca aactgacccg tgttaaagaa 240

gaaaccaaga cgagctttca ctctaaagac atcttcaagg aaatcctgga agattacaag 300

tacctggaaa aggaattcaa ggaactgagc aataccgccg aaaaagaagg tgataaggtg 360

accgttacgt atgctgatga ccagctggcg aaactgcaaa agtctatttg gatgctgcag 420

gcacatctgg ctcatatgat tcacaacaac gcactgtcct ctgtcctgat gggctctcac 480

aatggtatcg aaccggaaaa agtctccctg ctgtatggtg gcaacggcgg tccggaagca 540

cgtcatgatt ggaacgctac cgtcggctat aaaaaccagc aaggtgacaa tgtggcgacg 600

ctgattaacg ttcacatgaa aaatggcagt ggtctggtga ttgccggcgg tgaaaaaggc 660

atcaacaatc cgtcctttta tctgtacaaa gaagatcagc tgaccggtag ccaacgcgcg 720

ctgtctcagg aagaaatcca aaacaaagtt gatttcatgg aattcctggc gcagaacaat 780

gccaaactgg acaacctgag caaaaaagaa aaagaaaaat tccagaacga aatcgaagac 840

ttcggatccg ccttctttac cacggtaatc atcccagcaa ttgtgggcgg cattgctacc 900

ggcaccgccg tgggcacagt tagtggcctg ttaggatggg gtctgaagca ggcggaagaa 960

gcgaacaaga ccccagataa accggataat tcaacccaga aaacggaagt gcagcccacg 1020

caagtcatcg atggtccgtt tgccggggga aaggataccg tagtcaacat tgatcgcatc 1080

aacacgaaag ccgacggcac cattaaagta ggaggcttca aagcctcact gacaacgaac 1140

gcggcgcatc tgaatatcgg caaaggcgga gtaaaccttt caaatcaggc ctcggggcgt 1200

accttattag tggaaaatct gaccggcaac atcaccgtag acggcccgtt gcgcgttaac 1260

aatcaagtgg gcggttatgc actcgccggt tcgtccgcta acttcgaatt taaagccggc 1320

gtagatacca aaaacggcac cgcgaccttt aataacgata tttcgctggg ccgtttcgtt 1380

aacttgaagg tggacgcgca tactgcaaat tttaaaggca ttgacacggg taacggaggt 1440

ttcaataccc tggatttcag cggtgtgacc aacaaaaagc ttgatccgcg ggtaccgagc 1500

agcgtggcga gcatgattca tgaagtgggc attgaagcga tgtttccgga tggcaaaaaa 1560

gtggcgagca tgattcatga agtgggcatt gaagcgatgt ttccggatgg caaaaaaagc 1620

gcgattaacc atgcgctgga tgtggcggat aaatatgatg tgcaggtggc gattcatacc 1680

gataccaaaa aaagcgcgat taaccatgcg ctggatgtgg cggataaata tgatgtgcag 1740

gtggcgattc ataccgatac caaaaaaagc gtggaactga ttgatattgg cggcaaccgc 1800

cgcatttttg gctttaacgc gctggtggat ggcagcagcg tggaactgat tgatattggc 1860

ggcaaccgcc gcatttttgg ctttaacgcg ctggtggatg gcagcagcat taaagaagat 1920

gtgcagtttg gcagcagcat taaagaagat gtgcagtttg gcagcagcat taaagaagat 1980

gtgcagtttc tcgagcacca ccaccaccac cac 2013

<210> 3

<211> 264

<212> PRT

<213> Artificial sequence

<400> 3

Met Asn His Lys Val His His His His His His Met Thr Thr Tyr Thr

1 5 10 15

Val Lys Ser Gly Asp Thr Leu Trp Gly Ile Ser Gln Arg Tyr Gly Ile

20 25 30

Ser Val Ala Gln Ile Gln Ser Ala Asn Asn Leu Lys Ser Thr Ile Ile

35 40 45

Tyr Ile Gly Gln Lys Leu Val Leu Thr Gly Ser Ala Ser Ser Thr Asn

50 55 60

Ser Gly Gly Ser Asn Asn Ser Ala Ser Thr Thr Pro Thr Thr Ser Val

65 70 75 80

Thr Pro Ala Lys Pro Thr Ser Gln Thr Thr Val Lys Val Lys Ser Gly

85 90 95

Asp Thr Leu Trp Ala Leu Ser Val Lys Tyr Lys Thr Ser Ile Ala Gln

100 105 110

Leu Lys Ser Trp Asn His Leu Ser Ser Asp Thr Ile Tyr Ile Gly Gln

115 120 125

Asn Leu Ile Val Ser Gln Ser Ala Ala Ala Ser Asn Pro Ser Thr Gly

130 135 140

Ser Gly Ser Thr Ala Thr Asn Asn Ser Asn Ser Thr Ser Ser Asn Ser

145 150 155 160

Asn Ala Ser Ile His Lys Val Val Lys Gly Asp Thr Leu Trp Gly Leu

165 170 175

Ser Gln Lys Ser Gly Ser Pro Ile Ala Ser Ile Lys Ala Trp Asn His

180 185 190

Leu Ser Ser Asp Thr Ile Leu Ile Gly Gln Tyr Leu Arg Ile Lys Gly

195 200 205

Thr Thr Ser Ser Arg Cys Lys Ser Thr His Pro Leu Ser Cys Ser Phe

210 215 220

His Gln Leu Pro Ala Arg Ser Pro Leu Pro Ser Leu Asp Ala Gly Gln

225 230 235 240

Tyr Val Leu Val Met Lys Ala Asn Ser Ser Tyr Ser Gly Asn Tyr Pro

245 250 255

Tyr Ser Ile Leu Phe Gln Lys Phe

260

<210> 4

<211> 795

<212> DNA

<213> Artificial sequence

<400> 4

atgaatcaca aagtgcatca tcatcatcat catatgacta cttataccgt caaatctggt 60

gatactcttt ggggaatctc acaaagatat ggaattagtg tcgctcaaat tcaaagtgcg 120

aataatctta aaagtaccat tatctacatt ggtcaaaaac ttgtactgac aggttcagct 180

tcttctacaa attcaggtgg ttcaaacaat tccgcaagca ctactccaac cacttctgtg 240

acacctgcaa aaccaacttc acaaacaact gttaaggtta aatccggaga taccctttgg 300

gcgctatcag taaaatataa aactagtatt gctcaattga aaagttggaa tcatttaagt 360

tcagatacca tttatattgg tcaaaatctt attgtttcac aatctgctgc tgcttcaaat 420

ccttcgacag gttcaggctc aactgctacc aataactcaa actcgacttc ttctaactca 480

aatgcctcaa ttcataaggt cgttaaagga gatactctct ggggactttc gcaaaaatct 540

ggcagcccaa ttgcttcaat caaggcttgg aatcatttat ctagcgatac tattttaatt 600

ggtcagtatc tacgaataaa aggtaccact agttctagat gtaaatcaac acatccatta 660

tcatgttcat ttcatcaatt accagctcgt tcaccattac catcattaga tgctggtcaa 720

tatgttttag ttatgaaagc taattcatca tattcaggta attatccata ttcaatttta 780

tttcaaaaat tttga 795

<210> 5

<211> 933

<212> PRT

<213> Artificial sequence

<400> 5

Met Asn His Lys Val His His His His His His Met Thr Thr Tyr Thr

1 5 10 15

Val Lys Ser Gly Asp Thr Leu Trp Gly Ile Ser Gln Arg Tyr Gly Ile

20 25 30

Ser Val Ala Gln Ile Gln Ser Ala Asn Asn Leu Lys Ser Thr Ile Ile

35 40 45

Tyr Ile Gly Gln Lys Leu Val Leu Thr Gly Ser Ala Ser Ser Thr Asn

50 55 60

Ser Gly Gly Ser Asn Asn Ser Ala Ser Thr Thr Pro Thr Thr Ser Val

65 70 75 80

Thr Pro Ala Lys Pro Thr Ser Gln Thr Thr Val Lys Val Lys Ser Gly

85 90 95

Asp Thr Leu Trp Ala Leu Ser Val Lys Tyr Lys Thr Ser Ile Ala Gln

100 105 110

Leu Lys Ser Trp Asn His Leu Ser Ser Asp Thr Ile Tyr Ile Gly Gln

115 120 125

Asn Leu Ile Val Ser Gln Ser Ala Ala Ala Ser Asn Pro Ser Thr Gly

130 135 140

Ser Gly Ser Thr Ala Thr Asn Asn Ser Asn Ser Thr Ser Ser Asn Ser

145 150 155 160

Asn Ala Ser Ile His Lys Val Val Lys Gly Asp Thr Leu Trp Gly Leu

165 170 175

Ser Gln Lys Ser Gly Ser Pro Ile Ala Ser Ile Lys Ala Trp Asn His

180 185 190

Leu Ser Ser Asp Thr Ile Leu Ile Gly Gln Tyr Leu Arg Ile Lys Gly

195 200 205

Thr Met Glu Thr Phe Glu Ile Leu Lys His Leu Gln Ala Asp Ala Ile

210 215 220

Val Leu Phe Met Lys Val His Asn Phe His Trp Asn Val Lys Gly Thr

225 230 235 240

Asp Phe Phe Asn Val His Lys Ala Thr Glu Glu Ile Tyr Glu Glu Phe

245 250 255

Ala Asp Met Phe Asp Asp Leu Ala Glu Arg Ile Val Gln Leu Gly His

260 265 270

His Pro Leu Val Thr Leu Ser Glu Ala Ile Lys Leu Thr Arg Val Lys

275 280 285

Glu Glu Thr Lys Thr Ser Phe His Ser Lys Asp Ile Phe Lys Glu Ile

290 295 300

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

305 310 315 320

Thr Ala Glu Lys Glu Gly Asp Lys Val Thr Val Thr Tyr Ala Asp Asp

325 330 335

Gln Leu Ala Lys Leu Gln Lys Ser Ile Trp Met Leu Gln Ala His Leu

340 345 350

Ala His Met Ile His Asn Asn Ala Leu Ser Ser Val Leu Met Gly Ser

355 360 365

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

370 375 380

Gly Gly Pro Glu Ala Arg His Asp Trp Asn Ala Thr Val Gly Tyr Lys

385 390 395 400

Asn Gln Gln Gly Asp Asn Val Ala Thr Leu Ile Asn Val His Met Lys

405 410 415

Asn Gly Ser Gly Leu Val Ile Ala Gly Gly Glu Lys Gly Ile Asn Asn

420 425 430

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

435 440 445

Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys Val Asp Phe Met Glu Phe

450 455 460

Leu Ala Gln Asn Asn Ala Lys Leu Asp Asn Leu Ser Lys Lys Glu Lys

465 470 475 480

Glu Lys Phe Gln Asn Glu Ile Glu Asp Phe Gly Ser Ala Phe Phe Thr

485 490 495

Thr Val Ile Ile Pro Ala Ile Val Gly Gly Ile Ala Thr Gly Thr Ala

500 505 510

Val Gly Thr Val Ser Gly Leu Leu Gly Trp Gly Leu Lys Gln Ala Glu

515 520 525

Glu Ala Asn Lys Thr Pro Asp Lys Pro Asp Asn Ser Thr Gln Lys Thr

530 535 540

Glu Val Gln Pro Thr Gln Val Ile Asp Gly Pro Phe Ala Gly Gly Lys

545 550 555 560

Asp Thr Val Val Asn Ile Asp Arg Ile Asn Thr Lys Ala Asp Gly Thr

565 570 575

Ile Lys Val Gly Gly Phe Lys Ala Ser Leu Thr Thr Asn Ala Ala His

580 585 590

Leu Asn Ile Gly Lys Gly Gly Val Asn Leu Ser Asn Gln Ala Ser Gly

595 600 605

Arg Thr Leu Leu Val Glu Asn Leu Thr Gly Asn Ile Thr Val Asp Gly

610 615 620

Pro Leu Arg Val Asn Asn Gln Val Gly Gly Tyr Ala Leu Ala Gly Ser

625 630 635 640

Ser Ala Asn Phe Glu Phe Lys Ala Gly Val Asp Thr Lys Asn Gly Thr

645 650 655

Ala Thr Phe Asn Asn Asp Ile Ser Leu Gly Arg Phe Val Asn Leu Lys

660 665 670

Val Asp Ala His Thr Ala Asn Phe Lys Gly Ile Asp Thr Gly Asn Gly

675 680 685

Gly Phe Asn Thr Leu Asp Phe Ser Gly Val Thr Asn Lys Lys Leu Asp

690 695 700

Pro Arg Val Pro Ser Ser Val Ala Ser Met Ile His Glu Val Gly Ile

705 710 715 720

Glu Ala Met Phe Pro Asp Gly Lys Lys Val Ala Ser Met Ile His Glu

725 730 735

Val Gly Ile Glu Ala Met Phe Pro Asp Gly Lys Lys Ser Ala Ile Asn

740 745 750

His Ala Leu Asp Val Ala Asp Lys Tyr Asp Val Gln Val Ala Ile His

755 760 765

Thr Asp Thr Lys Lys Ser Ala Ile Asn His Ala Leu Asp Val Ala Asp

770 775 780

Lys Tyr Asp Val Gln Val Ala Ile His Thr Asp Thr Lys Lys Ser Val

785 790 795 800

Glu Leu Ile Asp Ile Gly Gly Asn Arg Arg Ile Phe Gly Phe Asn Ala

805 810 815

Leu Val Asp Gly Ser Ser Val Glu Leu Ile Asp Ile Gly Gly Asn Arg

820 825 830

Arg Ile Phe Gly Phe Asn Ala Leu Val Asp Gly Ser Ser Ile Lys Glu

835 840 845

Asp Val Gln Phe Gly Ser Ser Ile Lys Glu Asp Val Gln Phe Gly Ser

850 855 860

Ser Ile Lys Glu Asp Val Gln Phe Leu Glu His His His His His His

865 870 875 880

Ser Arg Cys Lys Ser Thr His Pro Leu Ser Cys Ser Phe His Gln Leu

885 890 895

Pro Ala Arg Ser Pro Leu Pro Ser Leu Asp Ala Gly Gln Tyr Val Leu

900 905 910

Val Met Lys Ala Asn Ser Ser Tyr Ser Gly Asn Tyr Pro Tyr Ser Ile

915 920 925

Leu Phe Gln Lys Phe

930

<210> 6

<211> 2802

<212> DNA

<213> Artificial sequence

<400> 6

atgaatcaca aagtgcatca tcatcatcat catatgacta cttataccgt caaatctggt 60

gatactcttt ggggaatctc acaaagatat ggaattagtg tcgctcaaat tcaaagtgcg 120

aataatctta aaagtaccat tatctacatt ggtcaaaaac ttgtactgac aggttcagct 180

tcttctacaa attcaggtgg ttcaaacaat tccgcaagca ctactccaac cacttctgtg 240

acacctgcaa aaccaacttc acaaacaact gttaaggtta aatccggaga taccctttgg 300

gcgctatcag taaaatataa aactagtatt gctcaattga aaagttggaa tcatttaagt 360

tcagatacca tttatattgg tcaaaatctt attgtttcac aatctgctgc tgcttcaaat 420

ccttcgacag gttcaggctc aactgctacc aataactcaa actcgacttc ttctaactca 480

aatgcctcaa ttcataaggt cgttaaagga gatactctct ggggactttc gcaaaaatct 540

ggcagcccaa ttgcttcaat caaggcttgg aatcatttat ctagcgatac tattttaatt 600

ggtcagtatc tacgaataaa aggtaccatg gagacctttg aaatcctgaa acacctgcaa 660

gccgacgcca ttgtcctgtt tatgaaagtc cacaacttcc attggaacgt gaaaggtacg 720

gactttttca acgtgcataa agccaccgaa gaaatctacg aagaattcgc tgatatgttc 780

gatgacctgg cggaacgcat tgttcagctg ggccatcacc cgctggtcac gctgagcgaa 840

gcgatcaaac tgacccgtgt taaagaagaa accaagacga gctttcactc taaagacatc 900

ttcaaggaaa tcctggaaga ttacaagtac ctggaaaagg aattcaagga actgagcaat 960

accgccgaaa aagaaggtga taaggtgacc gttacgtatg ctgatgacca gctggcgaaa 1020

ctgcaaaagt ctatttggat gctgcaggca catctggctc atatgattca caacaacgca 1080

ctgtcctctg tcctgatggg ctctcacaat ggtatcgaac cggaaaaagt ctccctgctg 1140

tatggtggca acggcggtcc ggaagcacgt catgattgga acgctaccgt cggctataaa 1200

aaccagcaag gtgacaatgt ggcgacgctg attaacgttc acatgaaaaa tggcagtggt 1260

ctggtgattg ccggcggtga aaaaggcatc aacaatccgt ccttttatct gtacaaagaa 1320

gatcagctga ccggtagcca acgcgcgctg tctcaggaag aaatccaaaa caaagttgat 1380

ttcatggaat tcctggcgca gaacaatgcc aaactggaca acctgagcaa aaaagaaaaa 1440

gaaaaattcc agaacgaaat cgaagacttc ggatccgcct tctttaccac ggtaatcatc 1500

ccagcaattg tgggcggcat tgctaccggc accgccgtgg gcacagttag tggcctgtta 1560

ggatggggtc tgaagcaggc ggaagaagcg aacaagaccc cagataaacc ggataattca 1620

acccagaaaa cggaagtgca gcccacgcaa gtcatcgatg gtccgtttgc cgggggaaag 1680

gataccgtag tcaacattga tcgcatcaac acgaaagccg acggcaccat taaagtagga 1740

ggcttcaaag cctcactgac aacgaacgcg gcgcatctga atatcggcaa aggcggagta 1800

aacctttcaa atcaggcctc ggggcgtacc ttattagtgg aaaatctgac cggcaacatc 1860

accgtagacg gcccgttgcg cgttaacaat caagtgggcg gttatgcact cgccggttcg 1920

tccgctaact tcgaatttaa agccggcgta gataccaaaa acggcaccgc gacctttaat 1980

aacgatattt cgctgggccg tttcgttaac ttgaaggtgg acgcgcatac tgcaaatttt 2040

aaaggcattg acacgggtaa cggaggtttc aataccctgg atttcagcgg tgtgaccaac 2100

aaaaagcttg atccgcgggt accgagcagc gtggcgagca tgattcatga agtgggcatt 2160

gaagcgatgt ttccggatgg caaaaaagtg gcgagcatga ttcatgaagt gggcattgaa 2220

gcgatgtttc cggatggcaa aaaaagcgcg attaaccatg cgctggatgt ggcggataaa 2280

tatgatgtgc aggtggcgat tcataccgat accaaaaaaa gcgcgattaa ccatgcgctg 2340

gatgtggcgg ataaatatga tgtgcaggtg gcgattcata ccgataccaa aaaaagcgtg 2400

gaactgattg atattggcgg caaccgccgc atttttggct ttaacgcgct ggtggatggc 2460

agcagcgtgg aactgattga tattggcggc aaccgccgca tttttggctt taacgcgctg 2520

gtggatggca gcagcattaa agaagatgtg cagtttggca gcagcattaa agaagatgtg 2580

cagtttggca gcagcattaa agaagatgtg cagtttctcg agcaccacca ccaccaccac 2640

tctagatgta aatcaacaca tccattatca tgttcatttc atcaattacc agctcgttca 2700

ccattaccat cattagatgc tggtcaatat gttttagtta tgaaagctaa ttcatcatat 2760

tcaggtaatt atccatattc aattttattt caaaaatttt ga 2802

Claims

1. A helicobacter pylori tetravalent virulence factor GEM particle vaccine is characterized in that the GEM particle vaccine comprises a recombinant antigen SAM displayed on the surface of GEM particles ^E -FVpE; recombinant antigen SAM ^E FVpE contains the core component SAM ^E And helicobacter pylori virulence factor multi-epitope peptide FVpE; the amino acid sequence of the virulence factor multi-epitope peptide FVpE is shown in SEQ ID NO. 1; the nucleotide sequence of the virulence factor multi-epitope peptide FVpE is shown in SEQ ID NO. 2; the core component SAM ^E Has the amino acid sequence shown as SEQ ID NO.3, encodes the core component SAM ^E The nucleotide sequence of (A) is shown as SEQ ID NO. 4; recombinant antigen SAM ^E The amino acid sequence of FVpE is shown in SEQ ID NO. 5; recombinant antigen SAM ^E The nucleotide sequence of-FVPE is shown in SEQ ID NO. 6.

2. The use of the helicobacter pylori tetravalent virulence factor GEM particle vaccine of claim 1 in the preparation of a medicament for preventing and treating gastropathy related to helicobacter pylori infection.

3. The method for preparing the helicobacter pylori tetravalent virulence factor GEM particle vaccine of claim 1, wherein the preparation method comprises the following steps:

synthesis of SAM shown in SEQ ID NO.4 ^E Gene fragment, SAM thereof ^E The gene fragment is cloned to a pCzn1 carrier through enzyme digestion and connection to obtain the PESAM ^E A recombinant vector;

obtaining a virulence factor multi-epitope peptide FVpE gene segment shown as a sequence SEQ ID NO.2 through amplification, and carrying out double enzyme digestion on the FVpE gene segment and the double enzyme digestion on the pepSAM ^E Mixing the recombinant vectors, and cloning the FVpE fragment into the pepSAM by a one-step seamless cloning technology ^E Recombinant vector to obtain a PESAM ^E -an FVpE recombinant vector;

transforming the recombinant vector of the peSAM-FVpE into host cells, culturing, inducing expression by an inducer, and purifying to obtain recombinant antibodyOriginal SAM ^E -FVpE；

Preparing GEM particles of lactic acid bacteria by trichloroacetic acid, and preparing the GEM particles and the recombinant antigen SAM ^E Mixed re-suspension of-FVpE to obtain GEM particle vaccine GEM-SAM ^E -FVpE。

4. The method for preparing the helicobacter pylori tetravalent virulence factor GEM particle vaccine according to claim 3, wherein the inducer is IPTG, and the working concentration of the inducer is 0.5mmol/L.

5. The method for preparing the helicobacter pylori tetravalent virulence factor GEM particle vaccine according to claim 3, wherein the host cell is escherichia coli.

6. The method for preparing granular vaccine of tetravalent virulence factor GEM of helicobacter pylori according to claim 5, characterized in that the escherichia coli is selected from ArcticExpress (DE 3), DH5 α, TOP10, BL21 (DE 3) pLysS or Rosetta.

7. The method for preparing the helicobacter pylori tetravalent virulence factor GEM particle vaccine according to claim 6, wherein the escherichia coli is ArcticExpress (DE 3).

8. The method for preparing the helicobacter pylori tetravalent virulence factor GEM particle vaccine according to claim 3, wherein the recombinant protein purification comprises chromatography and dialysis; the chromatography adopts a Ni-IDA-Sepharose CL-6B affinity chromatography column; the dialyzed dialysate is 20 mM Tris-HCl, 0.10M NaCl, pH8.0 dialysate.

9. The method for preparing the particle vaccine of the helicobacter pylori tetravalent virulence factor GEM according to claim 8, wherein the flow rate of the chromatography buffer is 0.5-1mL/min, and the dialyzed dialysate is 20 mmol/L Tris-HCl,0.10 mol/L NaCl, ph8.0 solution.