An OMV vaccine capable of preventing Helicobacter pylori infection and preparation method thereof

Abstract

The present invention relates to an OMV vaccine that can prevent Helicobacter pylori infection and a preparation method thereof. The vaccine uses a conserved trisaccharide (Trio) in Helicobacter pylori lipopolysaccharide as a core antigen epitope, and its structure is α-DD-Hep-(1→3)-α-D-Fuc-(1→3)-β-D-GlcNAc-. The epitope is biosynthesized by recombinant Escherichia coli, and a non-reducing end of a core oligosaccharide on the Escherichia coli outer membrane vesicle (OMV) is used as a connection anchor. The oligosaccharide is catalyzed and synthesized by glycosyltransferases from Helicobacter pylori and Escherichia coli. Trio can be efficiently displayed on the surface of OMV using this oligosaccharide on the surface of bacteria. Animal experiments have shown that the vaccine has the ability to activate the body's immune response and protective efficacy, and can be developed as a vaccine to prevent Helicobacter pylori infection. It has important application value and significance for effectively preventing and treating diseases such as peptic ulcers, chronic inflammation, and even gastric cancer caused by Helicobacter pylori infection.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and synthetic biology, and specifically relates to heterologous expression of sugars and antigen presentation based on an OMV platform. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative bacterium closely associated with the development of gastric and duodenal ulcers in humans. It is a major pathogenic factor and initiator of chronic gastritis and gastric cancer. H. pylori infection rates are high, and drug resistance is a serious threat to human health. Therefore, the development of a safe and effective new vaccine to combat persistent H. pylori infection is crucial.

[0003] Polysaccharides are typically located on the outermost surface of bacteria, with the most representative capsular polysaccharides and O antigens considered ideal protective antigens. Lipopolysaccharide (LPS) is composed of lipid A, core oligosaccharides, and O antigen. According to Li et al. (Li H et al, 2017) on the structure of Helicobacter pylori LPS, the main structural units of the O antigen, starting from the reducing end, are trio, glucan, heptose, and Lewis antigen. Because the Lewis antigen is structurally similar to antigens on the surface of human gastric epithelial cells, natural polysaccharides containing this structure cannot be directly used as vaccine antigen candidates. Currently, studies have attempted to develop vaccines using heptose or glucan as antigens, but both are poorly conserved in H. pylori and similarly lack the qualities to be ideal antigens. In contrast, trio is a highly conserved structure at the reducing end of the H. pylori O antigen, with N-acetylglucosamine (GlcNAc), fucose (Fuc), and heptose (D,D-Hep) linked in the order from the reducing end to the non-reducing end. In addition, Trio is also associated with the colonization of pathogens in the host, suggesting that Trio can serve as a potential protective epitope.

[0004] Currently, the production of saccharide antigens from Helicobacter pylori faces bottlenecks. First, H. pylori cannot be cultured at high densities in liquid environments, resulting in low yields of the target product. Furthermore, H. pylori is a pathogenic bacterium, and large-scale cultivation poses safety risks. Therefore, the development of a simple and efficient method for preparing Helicobacter pylori vaccines using saccharides as core antigens is urgently needed.

[0005] Outer membrane vesicles (OMVs) are composed of lipids, LPS, outer membrane proteins, and periplasmic proteins, and are typically secreted by Gram-negative bacteria with a diameter of 20-300 nm. Studies have shown that the non-replicative, stable, and natural adjuvant properties of OMVs make them potential candidates for development as ideal antigen delivery platforms. The introduction of genetic engineering technology has enabled OMVs to be loaded with homologous or heterologous antigens while increasing yield and reducing endotoxic activity. Currently, there are no reports on the development of a carbohydrate vaccine for the prevention of Helicobacter pylori infection using Trio as the core antigen and the OMV technology route. Summary of the Invention

[0006] The purpose of the present invention is to provide an OMV vaccine capable of preventing Helicobacter pylori infection and a preparation method thereof.

[0007] One aspect of the present invention is to provide an OMV vaccine that can prevent Helicobacter pylori infection, characterized in that the core antigen epitope is a conserved trisaccharide (Trio) in Helicobacter pylori lipopolysaccharide, which is biosynthesized by recombinant Escherichia coli and uses Escherichia coli outer membrane vesicles (OMV) as a presentation vector.

[0008] The vaccine of the present invention, the recombinant Escherichia coli is constructed using Escherichia coli BL21 (DE3) ΔompAΔmsbBΔpagP as a chassis strain and is named HpTrEc.

[0009] The vaccine of the present invention, the conserved trisaccharide (Trio) has a structure of α-DD-Hep-(1→3)-α-D-Fuc-(1→3)-β-D-GlcNAc-.

[0010] In the vaccine of the present invention, the recombinant Escherichia coli HpTrEc oligosaccharide is synthesized by catalysis of glycosyltransferases from Helicobacter pylori and Escherichia coli, and the core oligosaccharide D,D-Hep is connected to the D-GlcNAc of the conserved trisaccharide (Trio).

[0011] The present invention further provides a method for preparing the Helicobacter pylori OMV vaccine of the present invention, characterized in that the method includes: constructing recombinant Escherichia coli HpTrEc, culturing the recombinant Escherichia coli HpTrEc to obtain a vaccine stock solution, and preparing the obtained vaccine stock solution into a preparation form such as injection, oral administration, and nasal spray.

[0012] The preparation method of the present invention and the construction method of recombinant Escherichia coli HpTrEc are as follows:

[0013] Knockout the enteric bacterial common antigen synthesis gene cluster, clarithromycin synthesis gene cluster, O antigen synthesis gene cluster, and core oligosaccharide synthesis gene cluster in the genome of the Escherichia coli BL21 (DE3) chassis strain;

[0014] The core oligosaccharide synthesis gene and Trio synthesis gene on the Helicobacter pylori genome were integrated into the Escherichia coli genome;

[0015] Then the guanosine diphosphate-fucose synthesis related genes of the Escherichia coli chassis strain: cpsG, cpsB, fcl and gmd;

[0016] The O-antigen flippase and polymerase genes: rfbX and wzy were complemented, and the corresponding proteins of the genes have the NCBI accession numbers "ACT43810.1", "ACT43809.1", "ACT43806.1", "ACT43805.1", "ACT43789.1" and "ACT43794.1", respectively.

[0017] The preparation method of the present invention,

[0018] Among them, the intestinal bacterial common antigen synthesis gene cluster (wzzE-rffM) contains 11 genes, namely wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE and rffM, and the proteins corresponding to the genes have accession numbers of "ACT45456.1", "ACT45457.1", "ACT45458.1", "ACT45459.1", "ACT45460.1", "ACT45461.2", "ACT45462.1", "ACT45463.1", "ACT45464.1", "ACT45465.1" and "ACT45466.1" on NCBI;

[0019] The carboxylic acid synthesis gene cluster (wcaM-wza) contains 20 genes, namely wcaM, wcaL, wcaK, wzxC, wcaJ, cpsG, cpsB, wcaI, gmm, fcl, gmd, wcaF, wcaE, wcaD, wcaC, wcaB, wcaA, wzc, wzb and wza. The accession numbers of the proteins corresponding to the genes on NCBI are "ACT43800.1", "ACT43801.1", "ACT43802.1", "ACT43803.1", "ACT43804.1", "ACT43805.1", "ACT43806.1", "ACT43807.1", "ACT43808.1", "ACT43809.1", "ACT43810.1", "ACT43811.1", "ACT43812.1", "ACT43813.1", "ACT43814.1", "ACT43815.1", "ACT43816.1", "ACT43817.1", "ACT43818.1", "ACT43819.1", "ACT43810.1", "ACT43811.1", "ACT43811.1", "ACT43812.1", "ACT43813.1", "ACT43814.1" "43804.1", "ACT43805.1", "ACT43806.1", "ACT43807.1", "ACT43808.1", "ACT43809.1", "ACT43810.1", "ACT43811.1", "ACT43812.1", "ACT43813.1", "ACT43814.1", "ACT43815.1", "ACT43816.1", "ACT43817.1", "ACT43818.1" and "ACT43819.1";

[0020] The O antigen synthesis gene cluster (gnd-galF) contains 16 genes, namely gnd, manB, manC, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, rfbX, rfbC, rfbA, rfbD, rfbB and galF. The accession numbers of the proteins corresponding to the genes on NCBI are "ACT43782.1", "ACT43783.1", "ACT43784.1" and "ACT43785.1". ", "ACT43787.1", "ACT43788.1", "ACT43789.1", "ACT43790.1", "ACT43791.1", "ACT43792.1", "ACT43793.1", "ACT43794.1", "ACT43795.1", "ACT43796.1", "ACT43797.1", "ACT43798.1" and "ACT43799.1";

[0021] The core oligosaccharide synthesis gene cluster (waaL-waaQ) of the Escherichia coli chassis strain contains 9 genes, namely waaL, waaV, waaW, waaY, waaT, waaO, waaP, waaG and waaQ, and the accession numbers of the proteins corresponding to the genes on NCBI are "ACT45278.1", "ACT45279.1", "ACT45280.1", "ACT45281.1", "ACT45282.1", "ACT45284.1", "ACT45285.2", "ACT45286.1" and "ACT45287.1" respectively;

[0022] The core oligosaccharide synthesis genes on the Helicobacter pylori genome include HP_1284, HP_0805, and HP_1416, and the proteins corresponding to the genes have accession numbers "AAD08327.1", "AAD07853.1", and "AAD08459.1" on NCBI, respectively.

[0023] The Trio synthetic genes on the Helicobacter pylori genome include HP_1039, HP_0102, and HP_0479, and the proteins corresponding to the genes have accession numbers "AAD08086.1", "AAD07172.1", and "AAD07549.1" on NCBI, respectively.

[0024] Among them, the Helicobacter pylori is 26695 strains;

[0025] Among them, HP_1284, HP_0805, HP_1416, HP_1039, HP_0102 and HP_0479 are polypeptides having at least 50% sequence identity with "AAD08327.1", "AAD07853.1", "AAD08459.1", "AAD08086.1", "AAD07172.1" and "AAD07549.1", respectively, and homologs retaining the same biological activity.

[0026] The preparation method of the present invention comprises the steps of constructing recombinant Escherichia coli HpTrEc, culturing the recombinant Escherichia coli HpTrEc to obtain a vaccine stock solution, and preparing the obtained vaccine stock solution into a preparation form such as injection, oral administration, and nasal spray.

[0027] The vaccine of the present invention is prepared in the form of a pharmaceutical preparation.

[0028] The present invention further provides use of the vaccine of the present invention in preparing a vaccine for preventing Helicobacter pylori infection.

[0029] The application of the present invention is that the vaccine has the effect of preventing diseases such as gastric ulcer, duodenal ulcer, chronic gastritis, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer.

[0030] As a preferred embodiment, the construction method of the OMV high-yield engineered strain HpTrEc capable of presenting Helicobacter pylori Trio of the present invention is as follows:

[0031] The enterobacterial common antigen (ECA) synthesis gene cluster and core oligosaccharide synthesis gene cluster were partially deleted from the E. coli chassis strain genome, while the colanic acid synthesis gene cluster and O-antigen synthesis gene cluster were completely deleted. Furthermore, the core oligosaccharide synthesis gene and Trio synthesis gene from Helicobacter pylori were integrated into the corresponding positions of the waaL-waaQ and gnd-galF gene clusters, respectively. The O-antigen flippase gene rfbX and O-antigen polymerase gene wzy from the E. coli chassis strain were complemented into the gnd-galF gene cluster locus, and the genes involved in guanosine diphosphate-fucose synthesis (cpsG, cpsB, fcl, and gmd) were complemented into the wcaM-wza gene cluster locus.

[0032] The present invention further provides a method for preparing a vaccine, which, as a preferred embodiment, comprises the following steps:

[0033] The recombinant Escherichia coli HpTrEc was inoculated on a non-resistant LB solid medium and cultured at 37°C overnight. A single colony was picked and inoculated into a non-resistant LB liquid medium and cultured until the OD 600 The concentration of the supernatant is 0.8-1.2. The supernatant is transferred to 1 L of resistance-free LB liquid medium at 1% (v / v) and cultured overnight at 37°C until the end of the logarithmic phase. The bacterial solution is collected and centrifuged at 6000 rpm at 4°C for 15 minutes. The supernatant is filtered through a 0.45 μm filter membrane and collected in an ultracentrifuge tube. Ultracentrifuged at 200,000 × g at 4°C for 2 hours, resuspended in PBS, and filtered through a 0.22 μm filter membrane to obtain OMVs carrying Helicobacter pylori Trio, i.e., the vaccine stock solution. The obtained vaccine stock solution is processed, such as purification, filtration, formulation, canning, and sterilization, to prepare it into injection, oral, nasal spray, and other formulations.

[0034] Another aspect of the present invention is to provide an OMV high-yielding engineered strain HpTrEc capable of presenting Helicobacter pylori Trio.

[0035] The present invention uses CRISPR / Cas9 gene editing technology to construct recombinant strains; uses transmission electron microscopy (TEM) observation and dynamic light scattering (DLS) to characterize the morphological structure of OMVs; uses SDS-PAGE, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), nuclear magnetic resonance spectroscopy ( 1 The carbohydrate antigen structure was identified using H NMR spectroscopy (H NMR spectroscopy) technology; the vaccine immunogenicity and protective efficacy were evaluated based on antibody titer determination and mouse challenge protection test.

[0036] In the process of preparing the OMV high-yield engineered strain HpTrEc that can present Helicobacter pylori outer membrane surface oligosaccharide Trio, all raw materials used in the present invention, including strains, genes, enzymes, various reagents, etc., are all publicly available existing technologies. All materials can be purchased or prepared according to existing literature.

[0037] The following is an explanation of the terminology of the present invention:

[0038]

[0039]

[0040]

[0041]

[0042] Beneficial effects

[0043] (1) The present invention provides an OMV vaccine that can prevent Helicobacter pylori infection. It is prepared from recombinant Escherichia coli HpTrEc that can heterologously synthesize Helicobacter pylori Trio. Specifically, some core oligosaccharide synthesis genes and Trio synthesis genes derived from Helicobacter pylori are integrated into the E. coli genome in which four gene clusters have been knocked out. Genes involved in guanosine diphosphate-fucose synthesis, O-antigen flippase, and polymerase genes derived from the E. coli chassis strain are then back-filled into the same E. coli genome to obtain the recombinant strain HpTrEc. The present invention modifies the outer membrane structure of E. coli to enable it to be used more efficiently as a polysaccharide presentation carrier.

[0044] (2) The present invention is characterized by SDS-PAGE, MALDI-TOF mass spectrometry and nuclear magnetic resonance ( 1 H NMR analysis confirmed the LPS structure of the HpTrEc strain OMV: an E. coli-H. pylori constitutive core oligosaccharide, linked to Trio at its non-reducing end. Leveraging the advantages of OMVs, such as their non-replicative properties, stability, and natural adjuvant activity, the present invention is the first to express the conserved H. pylori saccharide epitope Trio onto E. coli OMVs for vaccine development.

[0045] (3) The present invention confirmed through immune efficacy testing that the vaccine provided by the present invention has a good protective effect against Helicobacter pylori strains with different O antigen structures.

[0046] (4) The Trio-deliverable OMV obtained in the present invention has the function of a Helicobacter pylori vaccine and has important application and promotion value for the prevention and control of Helicobacter pylori and the treatment of diseases caused by Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 :a. The structure of the gene cluster for the synthesis of core oligosaccharides, O antigens and exopolysaccharides of Escherichia coli BL21 (DE3); b. The structure of the gene cluster for the synthesis of core oligosaccharides and O antigens of recombinant Escherichia coli HpTrEc; c. Schematic diagram of the LPS structure of HpTrEc.

[0048] Figure 2 : PCR verification of recombinant strain construction.

[0049] Figure 3 : Growth kinetics determination of chassis strains and recombinant Escherichia coli HpTrEc.

[0050] Figure 4 :OMV characterization: a. OMV content determination (protein quantification method); b. Transmission electron microscopy morphology observation results; c. OMV particle size determination results.

[0051] Figure 5 : SDS-PAGE detection of LPS in OMVs secreted by HpTrEc.

[0052] Figure 6 : MALDI-TOF mass spectrometry was used to determine the composition of oligosaccharide products in OMVs secreted by HpTrEc.

[0053] Figure 7 : Trio structure determination by nuclear magnetic resonance spectroscopy.

[0054] Figure 8 : Determination of Trio content by HPLC.

[0055] Figure 9 : Detection of specific antibodies in mouse serum and feces.

[0056] Figure 10 : Cytokine detection in mouse spleen cells.

[0057] Figure 11 : Analysis of Helicobacter pylori colonization in mouse stomach.

[0058] Figure 12 : Analysis of gastric inflammation in mice (HE staining). DETAILED DESCRIPTION

[0059] The present invention will be further illustrated below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the art and can be purchased commercially. The methods used in the present invention can be obtained by consulting technical literature.

[0060] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0061] The culture medium involved in the following examples is as follows:

[0062] LB medium (g / L): 5 g yeast extract powder, 10 g tryptone, 10 g sodium chloride, dilute to 1 L with ddH2O, and sterilize at 121°C for 20 min after fully dissolving.

[0063] The strains, plasmids and primer sequences involved in the following examples are as follows:

[0064] Table 1 Strains and plasmids involved in the examples

[0065]

[0066]

[0067] Table 2 Primers involved in the examples

[0068]

[0069] Example 1. Construction of recombinant Escherichia coli HpTrEc

[0070] CRISPR / Cas9 gene editing technology was used to construct the recombinant strain. The gene cluster structure and LPS structure in the chassis strain and the recombinant strain HpTrEc are shown in Figure 2. Figure 1 As shown. The pEcCas plasmid contains the kanamycin resistance gene (kan R ), expressing the nuclease Cas9 and λ-Red recombinase; the pEcgRNA plasmid contains the spectinomycin resistance gene (spc R ), carrying the gRNA sequence. The working concentrations of kanamycin and spectinomycin were both 50 μg / mL, and the culture medium contained the above two antibiotics at Kan + and Spc + express.

[0071] 1. Construction of HpTrEc-1

[0072] Using E. coli BL21(DE3)ΔompAΔmsbBΔpagP as the chassis strain, the wzzE-rffM gene cluster, which synthesizes common enteric bacterial antigens, was deleted. The sequences of the gRNA and primers required for homology arm amplification are shown in Table 2.

[0073] Preparation of E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP / pEcCas electroporation competent cells: The pEcCas plasmid was transformed into the chassis strain to obtain E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP / pEcCas. The activated recombinant strain was inoculated into 10 mL LB (Kan + ) medium, and cultured at 37°C and 200 rpm until OD 600 = 0.2, add arabinose to a final concentration of 10 mM and further culture until OD 600 = 0.5, ice bath for 30 min, centrifuge at 4000 rpm for 5 min at 4°C to collect the cells, wash twice with pre-cooled sterile water and 10% glycerol solution, add 500 μL 10% glycerol to resuspend the cells, and distribute them in 100 μL / tube.

[0074] E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP / pEcCas was electroporated into competent cells, 100 ng pEcgRNA- wzzE-rffM Plasmid and 400 ng of the synthetic donor DNA fragment UD-Δ(wzzE-rffM) were added to a pre-chilled and washed 75% ethanol cuvette. Electroporation was performed at 2.5 kV. After a 3-minute ice bath, the bacterial solution was transferred to a new sterile 1.5 mL EP tube and incubated at 37°C, 200 rpm, for 1 hour. The tube was then evenly plated on LB (Kan + and Spc + ) plates and incubated upside down in a 37°C biochemical incubator for 12 h.

[0075] The transformants were verified by colony PCR using the forward primer of the upstream homology arm and the reverse primer of the downstream homology arm, with the chassis strain E coli BL21 (DE3) ΔompA ΔmsbB ΔpagP as the negative control. Figure 2 As shown in a, lane 1 is the control band of the chassis strain, and lane 2 is the band of the transformant knocked out of the gene cluster for the synthesis of common enteric bacterial antigens. Comparison of lanes 1 and 2 indicates successful knockout of the gene cluster. DNA sequencing of the positive clones ultimately confirmed the gene knockout.

[0076] Plasmid pEcgRNA- wzzE-rffM Elimination: Transfer the positive clones to 5 mL of LB (Kan +) medium, 37 ℃, 200 rpm shaking culture for 12 hours. Dilute the bacterial solution and spread it on LB (Kan + ) plates and cultured at 37℃ for 12h. + and Spc + ) and LB(Kan + ) plates and screened out spectinomycin-sensitive single colonies, thus obtaining the elimination plasmid pEcgRNA- wzzE-rffM The strain HpTrEc-1 was inoculated into 5 mL LB (Kan + ) medium, 37 ° C, 200 rpm shaking culture, when the bacterial density OD 600 When the concentration reaches 0.8-1.2, the strain is preserved and continuous gene knockout is performed.

[0077] 2. Construction of HpTrEc-2

[0078] The HpTrEc-2 strain was constructed based on the HpTrEc-1 strain. The clarithromycin biosynthesis gene cluster wcaM-wza was deleted, and the genes cpsG, cpsB, fcl, and gmd involved in guanosine diphosphate-fucose biosynthesis were complemented at the corresponding genomic locations from the chassis strain. The sequences of the gRNAs and primers required for homology arm amplification are shown in Table 2.

[0079] Prepare HpTrEc-1 electroporation competent cells according to the steps of preparing electroporation competent cells in the construction of HpTrEc-1 strain in Example 1-1. wcaM-wza Plasmid and 400ng of artificially synthesized donor DNA fragment UD-Δ(wcaM-wza)::(cpsG-cpsB-fcl-gmd) were added to the electroporation cuvette and electroporation conditions were set at 2.5kV. After the cells were activated, they were evenly spread on LB (Kan + and Spc + ) plates and inverted culture in a 37°C biochemical incubator for 12 h.

[0080] The transformants were verified by colony PCR using the chassis strain E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP as a negative control. Figure 2 As shown in b, lane 1 is a control band for the chassis strain, and lane 2 is a transformant band in which the guanosine diphosphate-fucose synthesis-related genes cpsG, cpsB, fcl, and gmd replaced the clarithromycin synthesis gene cluster. Comparison of lanes 1 and 2 indicates successful genome integration of the gene cluster. DNA sequencing of the positive clones ultimately confirmed gene knockout and integration.

[0081] Plasmid pEcgRNA-wcaM-wza Elimination: According to the construction of the plasmid pEcgRNA- wzzE-rffM Eliminate the step of eliminating the plasmid pEcgRNA- wcaM-wza The strain HpTrEc-2 was obtained. The strain was inoculated into 5 mL LB (Kan + ) medium, 37 ° C, 200 rpm shaking culture, when the bacterial density OD 600 When the concentration reaches 0.8-1.2, the strain is preserved and subsequent gene knockout is performed.

[0082] 3. Construction of HpTrEc-3

[0083] The HpTrEc-3 strain was constructed based on the HpTrEc-2 strain. The O antigen synthesis gene cluster gnd-galF was deleted, and the O antigen flippase gene rfbX and polymerase gene wzy from the chassis strain were complemented. The Trio synthesis genes HP_1039, HP_0102, and HP_0479 from Helicobacter pylori were integrated into the corresponding genomic locations. The sequences of the gRNAs and primers required for homology arm amplification are shown in Table 2.

[0084] Prepare HpTrEc-2 electroporation competent cells according to the steps of preparing electroporation competent cells in the construction of HpTrEc-1 strain in Example 1-1. gnd-galF Plasmid and 400ng of artificially synthesized donor DNA fragment UD-Δ(gnd-galF)::(wzy-rfbX-HP_1039-HP_0102-HP_0479) were added to the electroporation cuvette and electroporation conditions were set at 2.5kV. After the cells were activated, they were evenly spread on LB (Kan + and Spc + ) plates and inverted culture in a 37°C biochemical incubator for 12 h.

[0085] The transformants were verified by colony PCR using the chassis strain E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP as a negative control. Figure 2 As shown in c, lane 1 represents the control band of the chassis strain, while lane 2 represents the transformant band in which the Helicobacter pylori Trio synthesis gene, the chassis strain O-antigen flippase gene rfbX, and the polymerase gene wzy were replaced with the chassis strain O-antigen synthesis gene cluster. Comparison of lanes 1 and 2 indicates successful integration of the gene cluster into the genome. DNA sequencing of the positive clones ultimately confirmed gene knockout and integration.

[0086] Plasmid pEcgRNA- gnd-galFElimination: According to the construction of the plasmid pEcgRNA- wzzE-rffM Eliminate the step of eliminating the plasmid pEcgRNA- gnd-galF The strain HpTrEc-3 was obtained. The strain was inoculated into 5 mL LB (Kan + ) culture medium and then cultured in the culture medium for preservation and subsequent gene knockout.

[0087] 4. Construction of HpTrEc-4

[0088] The HpTrEc-4 strain was constructed based on the HpTrEc-3 strain. The core oligosaccharide biosynthesis gene cluster waaL-waaQ was deleted, and the core oligosaccharide biosynthesis genes HP_1284, HP_0805, and HP_1416 from Helicobacter pylori were integrated into the corresponding genomic locations. The sequences of the gRNAs and primers required for homology arm amplification are shown in Table 2.

[0089] Prepare HpTrEc-3 electroporation competent cells according to the steps of preparing electroporation competent cells in the construction of HpTrEc-1 strain in Example 1-1. waaL-waaQ Plasmid and 400ng of artificially synthesized donor DNA fragment UD-Δ(waaL-waaQ)::(HP_1284-HP_0805-HP_1416) were added to the electroporation cuvette and electroporation conditions were set at 2.5kV. After the cells were activated, they were evenly spread on LB (Kan + and Spc + ) plates and inverted culture in a 37°C biochemical incubator for 12 h.

[0090] The transformants were verified by colony PCR using the chassis strain E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP as a negative control. Figure 2 As shown in Figure d, lane 1 represents the control band of the chassis strain, and lane 2 represents the band of the transformant in which the core oligosaccharide synthesis gene cluster of the chassis strain was replaced with the core oligosaccharide synthesis gene of H. pylori. Comparison of lanes 1 and 2 indicates that the gene cluster was successfully integrated into the genome. DNA sequencing of the positive clones ultimately confirmed gene knockout and integration.

[0091] Plasmid pEcgRNA- waaL-waaQ Elimination with pEcCas: According to the construction of Example 1-1HpTrEc-1 strain, pEcgRNA- wzzE-rffM Plasmid elimination steps to eliminate pEcgRNA- waaL-waaQ Plasmid. pEcgRNA- waaL-waaQThe mutant strain of the plasmid was inoculated into LB medium containing 5 mg / mL glucose and cultured at 37°C with shaking at 200 rpm for 12 hours. The bacterial solution was diluted and evenly spread on LB plates containing 5 mg / mL glucose and 10 mg / mL sucrose and cultured at 37°C for 12 hours. Several single colonies were randomly selected and plated on LB (Kan + ) and non-resistant LB plates, and single colonies sensitive to kanamycin were screened to obtain the mutant strain HpTrEc-4, i.e., HpTrEc, which eliminated pEcCas.

[0092] Example 2. Determination of the growth curve of the recombinant strain HpTrEc

[0093] With the chassis strain E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP as the control, the chassis strain and HpTrEc were inoculated into 200 mL of non-resistant LB liquid medium, and the initial OD 600 =0.05, 37°C, 200 rpm. Every 2 hours, take 1 mL of bacterial solution to a cuvette, and OD 600 The absorbance was measured using a UV-visible spectrophotometer under 40 °C and repeated 3 times to draw a growth curve. Figure 3 As shown, HpTrEc and the chassis strain have similar growth kinetics curves, that is, the genetic modification of the chassis strain has no effect on its physiological activity.

[0094] Example 3. Preparation of OMV

[0095] Centrifuge 1 L of the chassis strain and HpTrEc strain grown to late logarithmic phase at 6,000 rpm at 4°C for 15 minutes. Filter the supernatant through a 0.45 μm filter, collect it in an ultracentrifuge tube, and ultracentrifuge it at 200,000 × g at 4°C for 2 hours. Repeat the above method for multiple fermentations and collect the precipitate. Resuspend it in 5 mL of PBS and filter it through a 0.22 μm filter to obtain OMVs.

[0096] Example 4. Characterization of OMVs

[0097] 1. OMV content determination (Bradford protein content determination method)

[0098] Add 10 μL of serially diluted standard (BSA) or sample to be tested to a 96-well plate, and add 300 μL of Pierce detergent-compatible Bradford assay reagent to each well. Incubate at room temperature for 10 minutes. Measure the absorbance at 595 nm using a plate reader. Figure 4a The BSA standard curve was drawn and used to determine that the OMV release amounts of the chassis strain and HpTrEc were 10.2 mg / L and 10.6 mg / L, respectively, indicating that the modification of the chassis strain did not significantly affect the secretion level of OMVs.

[0099] 2. OMV morphological observation

[0100] Dilute OMV with PBS to a final concentration of 80 μg / mL, take 5 μL and apply it to a hydrophilic carbon / formaldehyde copper grid (200 square mesh) treated with Q150RS carbon rope coating. After 30 seconds, remove the excess solution with Whatman filter paper No. 1. Negatively stain with 1% phosphotungstic acid (pH = 7.4) for 2 minutes, quickly remove the phosphotungstic acid with filter paper, and dry the copper grid under a 65°C lamp. Observe and obtain photos using a transmission electron microscope (final magnification of 120,000×). Figure 4 As shown in b, OMVs from both samples were similar in size and morphology, with outer membrane vesicles approximately 75 nm in diameter and no visible contamination or aggregates.

[0101] 3. Dynamic light scattering (DLS) determination of OMV particle size distribution and ξ-potential

[0102] Set the instrument parameters as follows: Measurement type = size, material = protein, dispersant = PBS buffer, general options = Mark Houwink parameters (default settings), temperature = 25°C, equilibration time = 120 s, measurement angle = 173° backscatter (NIBS default), measurement duration = automatic, measurement value = 3, delay between measurements = 30 s, data processing = general analysis model (normal resolution). Dilute the OMV sample to 50 μg / mL with PBS, transfer the sample to a DLS cuvette, place it in the instrument, and measure. Figure 4 The OMVs from the chassis strain (c) have a size of 70 nm and a ζ-potential of -11.24 mV, while those from the HpTrEc strain have a size of 70 nm and a ζ-potential of -11.08 mV. The similar OMV sizes and ζ-potentials of the two samples indicate that knockout and integration of the LPS synthesis-related gene cluster do not affect OMV size or dispersion.

[0103] Example 5. Preparation of carbohydrate antigens

[0104] 1. Extraction of LPS

[0105] OMVs were prepared according to the procedures described in Example 3. The OMV pellet was thoroughly resuspended in 20 mL of purified water. Then, 20 mL of 90% phenol preheated at 68°C was added. The pellet was shaken in a 68°C water bath for 1 hour, cooled to room temperature, and centrifuged for 20 minutes. The supernatant was aspirated into a centrifuge tube. After standing at 4°C for 12 hours, the supernatant was transferred to a dialysis bag and dialyzed for 24 hours. The crude LPS sample was then concentrated by vacuum centrifugation.

[0106] 2. Purification of LPS

[0107] To the crude LPS sample, add 9 mL of ddH2O, 1 mL of reaction buffer (100 mM Tris-HCl, 25 mM MgCl2, 1 mM CaCl2, pH 7.5), and appropriate amounts of DNase I and RNase A (1 μg per 1 mg LPS sample) and incubate at 37°C for 4 h. Add an appropriate amount of proteinase K (1 μg per 1 mg LPS sample) to the reaction solution and incubate at 37°C for 12 h. To remove residual protein, add 5 mL of saturated phenol to the centrifuge tube, mix thoroughly, and centrifuge for 30 min. The upper phase is then transferred to a dialysis bag and dialyzed against ddH2O for 24 h, with the ddH2O replaced every 4 h to remove any residual phenol. The liquid in the dialysis bag is freeze-dried under vacuum to obtain semi-pure LPS. The semi-pure LPS is then re-dissolved in a mixture of chloroform and methanol (2:1, v / v), centrifuged at 12,000 rpm for 20 min, and the supernatant discarded. Repeat the above steps and concentrate by vacuum centrifugation to obtain pure LPS.

[0108] 3. Separation of carbohydrate antigens

[0109] 1% acetic acid was added to the purified LPS sample, and the mixture was incubated at 100°C for 2 h. The heating was stopped and the pH was adjusted to neutral. The supernatant was collected after centrifugation and vacuum freeze-dried to collect the sugar product.

[0110] Example 6: Detection and Analysis of Target Antigens

[0111] 1. SDS-PAGE detection

[0112] According to the method described in Example 5, LPS was extracted from the chassis strain and the recombinant strain HpTrEc, 10 μg of each was taken, dissolved in 10 μL of distilled water, mixed with 10 μL of freshly prepared 2× solubilization buffer, and heated in a boiling water bath for 5 minutes. The samples were injected into the gel channels respectively. The samples migrated at 9 mA in the stacking gel and at 12 mA in the separation gel. When the indicator reached the bottom of the separation gel, the electrophoresis was stopped, the gel was peeled off the plate and stained. Figure 5As shown, lane 1 is the LPS band of the chassis strain, which has one structure corresponding to the core-lipid A structure. Lane 2 is the LPS band of the recombinant strain HpTrEc, which has two structures. The faster-migrating band corresponds to the core-lipid A structure, while the slower-migrating band corresponds to the Trio-core-lipid A structure.

[0113] 2. MALDI-TOF mass spectrometry analysis

[0114] According to the method described in Example 5, LPS was extracted from the chassis strain and the recombinant strain HpTrEc, and the oligosaccharide composition was identified and analyzed using MALDI-TOF mass spectrometry. Figure 6 As shown, there are two oligosaccharide structures in the sample (one carries Trio and the other does not), the left peak has an m / z of 1393.24, indicating that the structure is core-lipid A, and the right peak has an m / z of 1934.76. According to the molecular weight analysis, the oligosaccharide structure is composed of GlcNAc, Fuc and Hep trisaccharides.

[0115] 3. Nuclear magnetic resonance spectroscopy analysis

[0116] According to the method described in Example 5, sugar antigens were extracted from the recombinant strain HpTrEc and samples were prepared according to the sample loading ratio (0.50 mL D2O plus 6 mg sample); the samples were treated with CHELEX-100 resin, filtered, and transferred to 5 mm NMR tubes. 1 H NMR analysis of the structure of carbohydrate antigens. Figure 7 of 1 H NMR spectrum analysis showed that the Trio structure started from the reducing end and consisted of D-GlcNAc, D-Fuc and D,D-Hep.

[0117] Example 7: Quantification of carbohydrate antigens

[0118] High performance liquid chromatography was used to quantitatively analyze Trio heterologously expressed in the recombinant strain HpTrEc.

[0119] 1. Extraction of polysaccharides

[0120] According to the OMV preparation, LPS extraction and purification, and carbohydrate antigen separation steps in Examples 3 and 5, freeze-dried sugar samples from HpTrEc strain OMVs were obtained and analyzed using fucose as the only detection substance.

[0121] 2. Preparation of standards and samples

[0122] Take a clean 15mL EP tube, add 8mL of sterile water, add 100mg of fucose, and after fully dissolving, dilute to 10mL with a volumetric flask to prepare a 10mg / mL standard stock solution; add water to dilute stepwise to obtain fucose reference solutions with concentrations of 5.00, 2.50, 1.25, 0.63, 0.31, 0.16, 0.08, and 0.04mg / mL, respectively.

[0123] Accurately weigh 5 mg (±0.05 mg) of polysaccharide sample, dissolve in sterile water, and add 2 mL of 4 mol / L trifluoroacetic acid dropwise. Vortex to dissolve. Place in a constant temperature drying oven at 110°C for 3 h. Cool to room temperature, neutralize with 8 mol / L NaOH solution until neutral, vortex mix, and centrifuge at 4000 rpm for 10 min. Collect the supernatant. Transfer 450 μL of the supernatant to a 15 mL centrifuge tube, add 450 μL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution and 450 μL of 0.3 mol / L sodium hydroxide solution, vortex mix, and react in a 70°C water bath for 70 min. Remove and cool to room temperature, add 450 μL of 0.3 mol / L hydrochloric acid solution, vortex mix, add 2 mL of chloroform solution, vortex thoroughly, let stand to separate, and discard the chloroform layer. The extraction was repeated twice, and the supernatant was filtered through a 0.45 μm filter membrane before detection.

[0124] 3. Sample testing

[0125] Agilent TC-C18 (2) (250 mm × 4.6 mm, 5 μm) column was used, the mobile phase was phosphate buffer (pH = 6.8) / acetonitrile (85:15, v / v), the flow rate was 1.00 mL / min, the column temperature was 35 ° C, the injection volume was 10 μL, and the UV detection wavelength was 307 nm. The peak position of the sample to be tested was determined according to the peak position of the standard fucose, and the fucose concentration in the sample was calculated according to the peak area, which corresponded to the fucose content per unit mass of OMV. Figure 8 As shown, HPLC detection showed that the fucose content in OMV was 17.41 μg / mg, and the corresponding Trio sugar content was 57.45 μg / mg.

[0126] Example 7. Evaluation of antigen immunogenicity and vaccine protective efficacy

[0127] Vaccines were prepared using chassis strain OMVs and recombinant E. coli HpTrEc OMVs, respectively. To verify whether the above immunogens can effectively activate the immune system to produce high-titer antibodies, in this example, 6- to 8-week-old BALB / c mice were divided into 6 groups, with 10 mice in Groups 1-5 and 7 mice in Group 6. They were immunized on days 0, 14, and 28, respectively. They were fasted and deprived of water for 12 hours before each immunization. 0.2 mL of 0.1 mol / L NaHCO3 was gavaged 0.5 hours before immunization to neutralize gastric acid. They were fasted and deprived of water for 2 hours after immunization.

[0128] Group 1: Oral immunization with HpTrEc OMV, the single immunization OMV dose was 50 μg, corresponding to a Trio dose of 2.87 μg;

[0129] Group 2: Oral immunization with HpTrEc OMV, the single immunization OMV dose was 100 μg, corresponding to a Trio dose of 5.74 μg;

[0130] Group 3: Oral immunization with HpTrEc OMV, the single immunization OMV dose was 200 μg, corresponding to a Trio dose of 11.48 μg;

[0131] Group 4: Oral immunization with chassis strain OMV, the OMV dose for a single immunization was 200 μg (same as Group 3);

[0132] Group 5: oral immunization with PBS buffer (challenge group);

[0133] Group 6: Oral immunization with PBS buffer (non-challenged group).

[0134] The weight changes of mice were recorded every 7 days. On the 41st day, 3 mice in each group 1-5 were randomly sacrificed. They were fasted and deprived of water for 12 hours before sacrifice. The specific antibody levels were detected. On the 42nd, 44th and 46th days, 0.2 mL of 1×10 8 The mice in groups 1-5 were gavaged with Helicobacter pylori 26695 strains at a concentration of CFU / mL. They were fasted and deprived of water for 12 hours before each infection, and 0.2 mL of 0.1 mol / L NaHCO3 was gavaged in the first 0.5 hours to neutralize gastric acid. They were fasted and deprived of water within 2 hours after infection. Subsequently, feces and tail vein blood were collected from all mice in groups 1-6 every 5 days, and the levels of specific antibodies in serum and feces were detected by ELISA. They were fasted and deprived of water for 12 hours before sampling. All mice were killed on the 76th day. They were fasted and deprived of water for 12 hours before being killed. ELISA was used to detect the antibody levels in serum and feces and the expression of cytokines in mouse spleen cells. The colonization of Helicobacter pylori in the mouse stomach was detected by Helicobacter pylori culture and counting, and the degree of gastritis was observed by HE staining of mouse stomach tissue.

[0135] 1. Determination of the titer of specific antibodies induced by vaccine immunity

[0136] Sample preparation: Fecal samples were weighed and suspended in PBS at a ratio of 0.5 mL / 100 mg feces. Vortex at room temperature for 5 min, centrifuge at 8000 × g for 10 min, collect the supernatant, and add 1% protease inhibitors.

[0137] ELISA test: Before the infection, the antigen-specific IgG in the mouse serum and the sIgA titer in the fecal supernatant were detected. The sugar antigen (100 μL, 0.1 mg / mL) obtained in Example 5 was used as the coating antigen and incubated at 4°C overnight. After washing with PBST three times, the blocking buffer (PBST containing 1% BSA) was added at 37°C for 1 hour. The blocking solution was discarded, and the serially diluted serum samples or fecal supernatant were added to the ELISA plate, incubated at 37°C for 1 hour, and washed 3 times with PBST. Horseradish peroxidase (HRP)-coupled anti-mouse IgG and sIgA antibodies (100 μL / well, 1 μg / mL) were added, incubated at 37°C for 1 hour, and then washed 3 times with PBST. Subsequently, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well, and after 15 minutes of reaction in the dark, 50 μL of 2M H2SO4 was added to terminate the reaction, and A was measured. 450 Analysis showed that HpTrEc OMV immunization induced significantly higher levels of mucosal and humoral immunity in mice than in the chassis strain OMV group and the negative control group. Furthermore, mice immunized with high-dose OMVs showed stronger immune responses. The IgG antibody titers in the 50μg, 100μg, and 200μg OMV immunization groups were 1936, 3173, and 4569, respectively, and the sIgA antibody titers were 238, 314, and 354, respectively.

[0138] 2. Evaluation of vaccine protective efficacy

[0139] Changes in mouse weight showed no animal mortality or signs of illness in any group before challenge. The mice maintained good vitality, appetite, and appearance. Weight increased over time in all groups, with no significant difference in the average weight between the experimental group and the negative control group. Six weeks after challenge, the weight of mice in the negative control group and the chassis strain OMV-immunized group decreased slightly, while the weight of mice in the experimental group did not differ significantly from that in the non-challenged group.

[0140] Specific antibody detection: The levels of specific IgG in the serum and specific sIgA in the feces of each group of mice after challenge were detected by ELISA according to the steps described in Example 7-1. The results are expressed as ELISA units relative to the standard serum. One ELISA unit is equal to the reciprocal of the dilution of the standard serum with an OD405nm-490nm of 1 in the standard test. Figure 9 As shown, at 76 days, all three groups of HpTrEc OMV-immunized mice produced specific IgG and sIgA antibodies, and the mice in the high-dose OMV group showed a stronger immune response.

[0141] Cytokine detection in mouse spleen cells: On day 76, mice were sacrificed and spleens were removed aseptically. The spleens were pressed through a fine nylon mesh with a syringe to prepare single cell suspensions. 2×10 6 Splenocytes were cultured in 2 mL of RPMI 1640 medium containing 2 mM L-glutamine, 1% penicillin-streptomycin, and 10% FBS. After incubation at 37°C, 5% CO2 for 48 h, 100 μg of OMVs were added, and after 72 h, the supernatant was collected. The levels of gamma interferon (IFN-γ), interleukin-12 (IL-12), interleukin-17 (IL-17), and interleukin-4 (IL-4) in the supernatant were measured according to the manufacturer's instructions. The test results are shown in Figure 2. Figure 10 As shown, the cytokine levels produced by spleen cells of mice in the HpTrEc OMV immunization group were significantly higher than those in the negative control group and the chassis strain OMV immunization group, among which the upregulation of IFN-γ and IL-12 indicated the activation of Th1 cells, the upregulation of IL-4 indicated the activation of Th2 cells, and the upregulation of IL-17 indicated the activation of Th17 cells, which is more beneficial for the elimination of Helicobacter pylori infection.

[0142] Helicobacter pylori gastric colonization detection: Aseptically remove the mouse stomach, cut it along the greater curvature from the pylorus to the cardia, and place it flat in a sterile culture dish with the mucosal surface facing up. Rinse the gastric mucosal surface with sterile saline. Take half of the gastric tissue and weigh it, transfer it to a homogenizing tube containing Brucella broth (ratio 1:10), and homogenize it thoroughly. Dilute the gastric homogenate 10 times and spread it on a Campylobacter agar plate (containing 10μg / mL amphotericin B, 10μg / mL vancomycin, 20U / mL bacitracin, 10μg / mL polymyxin B and 5μg / mL trimethoprim) and culture it for 7 days. Count the colonies and express the data as CFU / g stomach. Figure 11 As shown in the figure, the colony counts in the gastric tissues obtained from the three groups of mice immunized with HpTrEc OMV were significantly lower than those in the negative control group (challenge group) and the chassis strain OMV immunization group, indicating that Trio-OMV can induce effective immune protection.

[0143] Histopathological examination: The other half of the gastric tissue sample was fixed in 10% formalin, processed and embedded in paraffin. After sectioning, it was stained with hematoxylin-eosin and the degree of lymphocyte infiltration was observed under a microscope. The degree of gastritis was graded as follows: 0: None; 1: A small amount of lymphocyte infiltration in the lamina propria; 2: A moderate amount of lymphocyte infiltration in the lamina propria; 3: Dense lymphocyte infiltration in the lamina propria, and some of the lymphocytes entered the submucosa; 4: Dense and diffuse lymphocyte infiltration in the lamina propria and submucosa. Figure 12As shown in the figure, there were no significant abnormalities in the gastric tissue structure in the 6 groups of experimental animals. The negative control group (challenge group) and the chassis strain OMV immunization group showed a wider range of inflammation and the most severe lesions. The low-dose OMV immunization group showed milder inflammation than the control group. The high-dose OMV immunization group had the mildest inflammation, with only 2 of the 7 mice showing mild local chronic inflammation. The HE staining results are shown in the figure. Figure 12 The inflammation scores of all mice are shown in Table 3. The above results show that the protection efficacy of HpTrEc OMV challenge is significantly higher than that of the negative control group (challenge group) and the chassis strain OMV immunization group, and the challenge protection effect is best when the OMV immunization dose is 200 μg.

[0144] Table 3 Gastric inflammation scores in mice

[0145]

[0146] Nucleotide sequence of the gene

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[0148]

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[0151]

Claims

1. An OMV vaccine for preventing Helicobacter pylori infection, characterized in that: The core antigen epitope is Trio in Helicobacter pylori lipopolysaccharide, which is biosynthesized by recombinant Escherichia coli and expressed on Escherichia coli OMV using Escherichia coli OMV as a presentation vector. The Trio structure is α-DD-Hep-(1→3)-α-D-Fuc-(1→3)-β-D-GlcNAc, and the E. coli OMV is OMV isolated from recombinant E. coli HpTrEc, wherein the recombinant E. coli HpTrEc is constructed using a chassis strain that can efficiently secrete low-toxic OMVs, and the chassis strain is E. coli BL21(DE3)ΔompAΔmsbBΔpagP.

2. A method for preparing the vaccine according to claim 1, characterized in that: The method comprises the steps of constructing a recombinant Escherichia coli, culturing the recombinant Escherichia coli to obtain a vaccine stock solution, and preparing the obtained vaccine stock solution into an injection, oral or nasal spray preparation; The construction method of the recombinant Escherichia coli is as follows: the intestinal bacterial common antigen synthesis gene cluster, the clarithromycin synthesis gene cluster, the O antigen synthesis gene cluster, and the core oligosaccharide synthesis gene cluster on the chassis strain genome are knocked out; the core oligosaccharide synthesis gene and the Trio synthesis gene on the Helicobacter pylori genome are integrated into the E. coli genome; the guanosine diphosphate-fucose synthesis-related genes: cpsG, cpsB, fcl, and gmd; and the O antigen flippase and polymerase genes: rfbX and wzy of the E. coli chassis strain are complemented, and the chassis strain is E. coli BL21 (DE3) ΔompA ΔmsbB ΔpagP; the core oligosaccharide synthesis genes on the Helicobacter pylori genome are HP_1284, HP_0805, and HP_1416; wherein, the Trio synthesis genes on the Helicobacter pylori genome are HP_1039, HP_0102, and HP_0479.

3. The vaccine according to claim 1, characterized in that The vaccine is prepared in the form of a pharmaceutical preparation.

4. Use of the vaccine according to claim 1 in the preparation of a vaccine for preventing Helicobacter pylori infection.

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