Hybrid vesicles against helicobacter pylori infection and methods of making same

Abstract

The present application belongs to the field of biological medicine, and particularly relates to a bifunctional hybrid vesicle for resisting Helicobacter pylori (HP) infection and repairing mucosal damage and a preparation method thereof. The present application will transform probiotics through gene editing technology, so that the probiotics synthesize and release probiotic vesicles with a targeting peptide (HTP) and an antibacterial peptide (AMP), and fuse the probiotic vesicles with milk exosomes (mEVs) to form functionally integrated hybrid vesicles (Hybrid Vesicles). The design integrates multiple functions such as targeted antibacterial, mucosal repair and flora regulation, and is used for preventing and removing Helicobacter pylori and repairing gastric mucosal damage.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to heterozygous vesicles for combating Helicobacter pylori infection and their preparation method. Background Technology

[0002] Helicobacter pylori (HP) is a widespread Gram-negative spirochete primarily transmitted through oral-oral or fecal-oral routes. According to the World Health Organization (WHO), approximately 50% of the global population is infected with HP, with even higher infection rates in developing countries. In China, approximately 700 million people are infected, with an infection rate of 59%, and infection rates are significantly higher in rural areas and among adults than in urban areas and among children.

[0003] Helicobacter pylori (H. pylori) infection can cause a series of serious health problems. Almost all H. pylori-infected individuals have chronic active gastritis, and some patients experience indigestion symptoms such as early satiety, abdominal pain, upper abdominal burning sensation, and nausea. Severe infection can lead to peptic ulcers, causing long-term chronic abdominal pain, and even serious complications such as gastrointestinal bleeding and perforation; prolonged infection can also lead to duodenal stricture. In China, approximately 340,000 new cases of gastric cancer each year are caused by H. pylori infection. Long-term H. pylori infection can cause gastric mucosal atrophy and intestinal metaplasia, thus increasing the probability of gastric cancer by 5.9 times. Furthermore, H. pylori infection is also closely related to mucosa-associated lymphoid tissue lymphoma (MALToma) and diseases of the cardiovascular, respiratory, endocrine, and hematologic systems, such as coronary heart disease, chronic obstructive pulmonary disease, type 2 diabetes, idiopathic thrombocytopenic purpura, and urticaria.

[0004] The current standard clinical treatment for *Helicobacter pylori* infection is triple or quadruple therapy containing a proton pump inhibitor, bismuth, and two antibiotics (such as clarithromycin or amoxicillin). This regimen faces significant challenges:

[0005] 1) Soaring drug resistance: Antibiotic overuse has led to a continuous increase in H. pylori resistance rates (especially clarithromycin and metronidazole) globally and in China, significantly reducing eradication success rates;

[0006] 2) Side effects: Antibiotics can easily cause intestinal flora imbalance, diarrhea, nausea, allergic reactions, etc., reducing patient compliance.

[0007] 3. High recurrence rate: There is a certain recurrence rate after eradication, and some patients need multiple treatments.

[0008] 4. Disruption of gastric microecology: Broad-spectrum antibiotics kill indiscriminately, severely disrupting the balance of normal flora in the stomach.

[0009] In addition, antibiotic treatment has limited effect on repairing the gastric mucosal barrier and is insufficient to resolve post-infection mucosal damage.

[0010] Antimicrobial peptides (AMPs), as natural immune molecules, have attracted attention due to their broad-spectrum antibacterial activity and unique bacterial membrane disruption mechanism. Their mechanism of action mainly includes disrupting the integrity of the bacterial cell membrane, causing leakage of cell contents, thereby leading to bacterial death; they can also interfere with bacterial nucleic acid and protein synthesis processes, inhibiting bacterial growth and reproduction. The advantages of antimicrobial peptides compared to traditional antibiotics include:

[0011] 1) Low drug resistance: Its mechanism of action is relatively diverse, making it difficult for bacteria to develop drug resistance through a single gene mutation, thus reducing the risk of drug resistance;

[0012] 2) Broad-spectrum antibacterial properties: It has inhibitory or bactericidal effects on a variety of bacteria, including Helicobacter pylori;

[0013] 3) Low side effects: Antimicrobial peptides usually have good biocompatibility and low toxicity, and cause less damage to normal human cells.

[0014] 4) Immune regulation: Some AMPs have the ability to regulate the host's immune response.

[0015] However, the broad-spectrum antibacterial properties of antimicrobial peptides also present problems. Due to their lack of selectivity, when inhibiting or eliminating Helicobacter pylori, they indiscriminately suppress both beneficial and harmful bacteria in the gastrointestinal tract, which may also lead to intestinal flora imbalance and affect normal digestive and immune functions of the gastrointestinal tract, thus limiting the widespread application of antimicrobial peptides in the treatment of Helicobacter pylori.

[0016] To address this issue, research on Helicobacter pylori-targeting peptides has gradually become a focus. Helicobacter pylori-targeting peptides (HTPs) can specifically recognize surface proteins of Helicobacter pylori (such as urease and adhesins), achieving precise killing and reducing the impact on symbiotic bacteria. Conjugating the targeting peptide with antimicrobial peptides can significantly improve selective antibacterial efficacy and reduce side effects. Experiments show that the AMP-HTP fusion protein increases the inhibition efficiency of HP by 3-5 times, while reducing the killing rate of symbiotic bacteria to 1%. Combining antimicrobial peptides with targeting peptides can construct an anti-Helicobacter pylori system that combines selectivity and high efficiency: HTP-HTP specifically accumulates on the surface of Helicobacter pylori, enhancing bactericidal efficiency while reducing interference with beneficial bacteria.

[0017] Milk-derived extracellular vesicles (mEVs), as natural nanovesicles, are rich in various bioactive components, such as miRNAs, growth factors (e.g., EGF, TGF-β), and anti-inflammatory proteins. Studies have confirmed that mEVs have the following multiple functions:

[0018] 1) Mucosal repair: mEVs can be taken up by gastric mucosal cells, transmit repair signals, promote epithelial cell proliferation and migration, accelerate ulcer healing, enhance the tight connection of the mucus layer, and strengthen the mucosal barrier function.

[0019] 2) Microbial balance: The active ingredients carried by mEVs can directly regulate the proliferation of probiotics, and can also indirectly promote the stability of beneficial microbiota in the stomach and inhibit the excessive growth of pathogens by regulating the immune microenvironment or competitive inhibition, thus helping to restore the health of the microecology.

[0020] However, due to the effect of gastric emptying, milk exosomes have a short retention time in the stomach after oral administration, which affects their ability to promote the repair and regeneration of gastric mucosal damage.

[0021] Therefore, providing heterozygous vesicles that resist Helicobacter pylori infection and their preparation methods is of great practical significance. Summary of the Invention

[0022] In view of this, the present invention provides a highly efficient, safe and antibiotic-free heterozygous vesicle for combating Helicobacter pylori infection and a method for preparing the same, thereby achieving targeted clearance of HP and repair of the gastric mucosal barrier.

[0023] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0024] In a first aspect, the present invention provides the design of a fusion protein comprising domains of a Helicobacter pylori targeting peptide (HTP) and an antimicrobial peptide (AMP);

[0025] The Helicobacter pylori targeting peptide targets characteristic proteins of Helicobacter pylori, such as VacA and UreB. Preferably, the Helicobacter pylori targeting peptide is Multimerin-1 (MM1) [SEQ ID NO:1].

[0026] In some specific embodiments of the present invention, the Helicobacter pylori targeting peptide (HTP) includes, but is not limited to, one or more of the following: human thrombin protein multimerin-1 (UniProt ID: Q13201), urease B subunit binding peptide (UBP) (UniProt ID: P69996), cytotoxic protein A binding peptide (CagA-BP) (UniProt ID: P80200), adhesin binding peptide (BabA / B-BP) (UniProt ID: Q6DSV8), or Lpp20 protein epitope peptide (UniProt ID: Q6DSV8);

[0027] In some specific embodiments of the present invention, the antimicrobial peptides (AMPs) include, but are not limited to: PGa-AM1 [UniProt ID: P84385], Tilapia piscidins (TP4 / TP3) [UniProt ID: L0CKG3], LL-37 [UniProt ID: P49913], antimicrobial peptide-1 (Epinecidin-1) [UniProt ID: Q6JWQ9], Cathelicidins [UniProt ID: P49913], Defensins (HNP-1) [UniProt ID: P59665], Bicarinalin [UniProt ID: W8GNV3], Odorranain-HP [UniProt ID: E7EKD9], Nisin A [UniProt ID: P13068], and Alyteserin [UniProt ID: One or more of the following: P0DQW5, laterosporulin [UniProt ID: H1ZZ98], or CRAMP [UniProt ID: P51437].

[0028] In some specific embodiments of the present invention, the Helicobacter pylori targeting peptide (HTP) has the following characteristics:

[0029] (I) The amino acid sequence as shown in SEQ ID NO:1;

[0030] (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or

[0031] (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

[0032] In some specific embodiments of the present invention, the antimicrobial peptide (AMP) has the following characteristics:

[0033] (I) The amino acid sequence shown in SEQ ID NO:2;

[0034] (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or

[0035] (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

[0036] In some specific embodiments of the present invention, the fusion protein further includes one or more of an anchoring sequence, a secreted peptide, and a linker.

[0037] In some specific embodiments of the present invention, the source of the anchoring sequence includes, but is not limited to, one or more of the following: multidrug-resistant ABC transporter (LmrA) [UniProt ID: P97046], cell wall hydrolase (AcmA) [UniProt ID: P0C2T5], ABC transporter family (OpuA [UniProt ID: P46920][P46921][P46922], GlnP [UniProt ID: P0AEQ6], mlaE [UniProt ID: P64606]), oligopeptide transporter (oppA) [UniProt ID: P06202], lactose transporter (LacF) [UniProt ID: P29823], transmembrane protein (Holin) [UniProt ID: P03705], and cell wall peptidoglycanase (Lysin) [UniProt ID: P15057].

[0038] In some specific embodiments of the present invention, the source of the anchoring sequence includes the multidrug resistance ABC transporter protein (LmrA).

[0039] In some specific embodiments of the present invention, the secreted peptide includes, but is not limited to, Usp45.

[0040] In some specific embodiments of the present invention, the linker peptide includes, but is not limited to, flexible peptide linker sequences (SGGGGS) derived from scFv. n , where n = 1~3.

[0041] In some specific embodiments of the present invention, the anchoring sequence includes the 2nd to 6th transmembrane regions M2-6 of the multidrug resistance ABC transporter (LmrA).

[0042] In some specific embodiments of the present invention, the anchoring sequence has:

[0043] (I) The amino acid sequence shown in SEQ ID NO:3;

[0044] (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or

[0045] (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

[0046] In some specific embodiments of the present invention, the secretory peptide has:

[0047] (I) The amino acid sequence shown in SEQ ID NO:4;

[0048] (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or

[0049] (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

[0050] In some specific embodiments of the present invention, the linked peptide has:

[0051] (I) An amino acid sequence as shown in any of SEQ ID NO:5~7;

[0052] (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or

[0053] (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

[0054] In some specific embodiments of the present invention, the structure of the fusion protein includes: Usp45-(SGGGGS)1-MM1-(SGGGGS)2-TP4-(SGGGGS)3-LmrA(M2-6).

[0055] In some specific embodiments of the present invention, the fusion protein has:

[0056] (I) The amino acid sequence shown in SEQ ID NO:8;

[0057] (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or

[0058] (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

[0059] Secondly, the present invention also provides a nucleotide sequence encoding the fusion protein or a component thereof.

[0060] In some specific embodiments of the present invention, the nucleotide sequence encoding the antimicrobial peptide has:

[0061] (I) A nucleotide sequence as shown in SEQ ID NO:9; or

[0062] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0063] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0064] In some specific embodiments of the present invention, the nucleotide sequence encoding the antimicrobial peptide has:

[0065] (I) A nucleotide sequence as shown in SEQ ID NO:10; or

[0066] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0067] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0068] In some specific embodiments of the present invention, the nucleotide sequence encoding the anchoring sequence has the following characteristics:

[0069] (I) A nucleotide sequence as shown in SEQ ID NO:11; or

[0070] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0071] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0072] In some specific embodiments of the present invention, the nucleotide sequence encoding the secretory peptide has:

[0073] (I) A nucleotide sequence as shown in SEQ ID NO:12; or

[0074] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0075] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0076] In some specific embodiments of the present invention, the nucleotide sequence encoding the linked peptide has:

[0077] (I) A nucleotide sequence as shown in any of SEQ ID NO: 13-15; or

[0078] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0079] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0080] In some specific embodiments of the present invention, the nucleotide sequence encoding the fusion protein has:

[0081] (I) A nucleotide sequence as shown in SEQ ID NO:16; or

[0082] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0083] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0084] Thirdly, the present invention also provides an expression vector comprising the aforementioned nucleotide sequence.

[0085] In some specific embodiments of the present invention, the expression vector further includes sgRNA.

[0086] In some specific embodiments of the present invention, the sgRNA includes htrAsgRNA-1 targeting the hrtA gene promoter and htrA sgRNA-2 targeting the 5' coding region of the hrtA gene;

[0087] The htrA sgRNA-1 has:

[0088] (I) A nucleotide sequence as shown in SEQ ID NO:17; or

[0089] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0090] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0091] In some specific embodiments of the present invention, the htrA sgRNA-2 has:

[0092] (I) A nucleotide sequence as shown in SEQ ID NO:18; or

[0093] (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or

[0094] (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

[0095] Fourthly, the present invention also provides biological materials for transforming or transducing the expression vector.

[0096] In some specific embodiments of the present invention, the biomaterial includes, but is not limited to, cells or microorganisms.

[0097] In some specific embodiments of the present invention, the microorganisms include, but are not limited to, probiotics.

[0098] In some specific embodiments of the present invention, the probiotics include, but are not limited to, lactic acid bacteria.

[0099] In some specific embodiments of the present invention, the lactic acid bacteria include, but are not limited to, one or more of Lactococcuslactis, Lactobacillus reuteri, or Lactobacillus gasseri.

[0100] Fifthly, the present invention also provides probiotic vesicles, wherein the biological material is cultured, the supernatant is collected, and centrifuged to obtain the probiotic vesicles.

[0101] In a sixth aspect, the present invention also provides heterozygous vesicles, including milk exosomes (mEVs) and the probiotic vesicles described above.

[0102] In some specific embodiments of the present invention, the protein ratio of the milk exosomes (mEVs) to the probiotic vesicles as described in claim 10 includes, but is not limited to, 2:1.

[0103] In some specific embodiments of the present invention, the fusion of the milk exosomes (mEVs) and the probiotic vesicles is performed by microporous extrusion, with a pressure of 0.8 to 1.2 MPa and a cycle of 10 to 25 times.

[0104] In a seventh aspect, the present invention also provides the use of the said heterozygous vesicles in the preparation of medicaments for the prevention, improvement, treatment or adjunctive treatment of Helicobacter pylori infection.

[0105] In an eighth aspect, the present invention also provides a medicament comprising the said heterozygous vesicles and pharmaceutically acceptable excipients.

[0106] In a ninth aspect, the present invention also provides a pharmaceutical combination comprising the said heterozygous vesicles and other active ingredients.

[0107] In some specific embodiments of the present invention, the other active ingredients include, but are not limited to, probiotic preparations.

[0108] In some specific embodiments of the present invention, the heterozygous vesicles are administered sequentially to the probiotic preparation.

[0109] This invention utilizes gene editing technology to construct probiotic engineered strains containing targeting peptides (HTP) and antimicrobial peptides (AMP), preparing probiotic vesicles with both HTP and AMP. These vesicles are then fused with milk exosomes (M-EVs) to form functionally integrated hybrid vesicles. This design integrates multiple functions, including targeted antibacterial activity, mucosal repair, and gut microbiota regulation, for the prevention and eradication of Helicobacter pylori and the repair of gastric mucosal damage. Attached Figure Description

[0110] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0111] Figure 1 A schematic diagram showing the preparation process of hybrid vesicles;

[0112] Figure 2 Transmission electron microscopy images showing hybrid vesicles;

[0113] Figure 3 The inhibition rate of heterozygous vesicles against Helicobacter pylori in in vitro experiments is shown (90% inhibition rate compared with the control PBS group).

[0114] Figure 4 The hepatic vesicle load in the gastric mucosa of the animal model was shown (immunohistochemical staining quantification showed that heterozygous vesicles reduced the hepatic load by 92% compared with the control PBS group).

[0115] Figure 5 Hybrid vesicles showed that they promoted the expression levels of tight junction proteins (Claudin-1 and Occludin) in the gastric mucosa (mEVs and hybrid-EVs significantly increased tight junction protein expression levels compared with the control PBS group). Detailed Implementation

[0116] This invention discloses heterozygous vesicles against Helicobacter pylori infection and their preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of protection of this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0117] This patent constructs probiotic engineered strains using gene editing technology to create targeted peptides and antimicrobial peptides, prepares probiotic vesicles with targeted peptides (HTP) and antimicrobial peptides (AMP), and fuses them with milk exosomes (M-EVs) to form functionally integrated hybrid vesicles.

[0118] The heterozygous vesicles are formed by the fusion of gene-edited probiotic-derived exosome-like vesicles (proELVs) and milk extracellular vesicles (mEVs).

[0119] Genetically edited probiotic vesicles: obtained from genetically modified lactic acid bacteria, preferably *Lactococcus lactis*. Lactic acid bacteria include, but are not limited to, strains that can survive in human gastric juice, such as *Lactococcus lactis*, *Lactobacillus reuteri*, *Lactobacillus gasseri*, *Streptococcus thermophilus*, *Lactobacillus acidophilus*, *Bifidobacterium bifidum*, and *Lactobacillus casei*, with *L. Lactis NZ9000 being the preferred variety.

[0120] The membrane of probiotic vesicles contains Helicobacter pylori targeted peptides (HTPs) and antimicrobial peptides (AMPs). HTPs and AMPs are anchored to the outer membrane of probiotic vesicles by fusing with vesicle membrane protein sequences.

[0121] The fusion protein consists of a secretory peptide, HTP, AMP, and a membrane protein anchoring sequence. After expression, the secretion pathway is initiated first through the N-terminal secretion signal peptide, which guides the functional targeting peptide and antimicrobial peptide to the outside of the probiotic membrane. The C-terminal membrane protein sequence anchors the fusion protein to the vesicle membrane.

[0122] 1. Sequence design of functional elements: targeting peptides, antimicrobial peptides, vesicle membrane proteins

[0123] 1) HTP:

[0124] Including, but not limited to, human thrombin protein multiplyin-1 (Multimerin-1), urease B subunit binding peptide (UBP), cytotoxin-associated protein A (CagA) binding peptide, adhesin BabA / B binding peptide, Lpp20 protein epitope peptide, etc., with Multimerin-1 (MM1) [SEQ ID NO:1] being preferred. MM1 can bind to Helicobacter pylori virulence factor VacA, guiding vesicles to target and bind to HP.

[0125] 2) AMP:

[0126] This includes, but is not limited to, PGa-AM1, Tilapia piscidins (TP4 and TP3), LL-37, Pexigaran (MSI-78), Epinecidin-1, Cathelicidins, Defensins, HNP-1, Bicarinalin, Odorranain-HP, Bacteriocins, Nisin A, alyteserin, laterosporulin, and CRAMP. Tilapia piscidin 4 (TP4) [SEQ ID NO:2] is preferred. AMPs exert their bactericidal effect by disrupting the synthesis of Helicobacter pylori cell walls. TP4 (MIC = 1.5-3 µg / mL) is the most active AMP and has a rapid onset of action; TP4 at twice the MIC concentration can reduce Helicobacter pylori by 99.9% within 60 minutes. Furthermore, antibiotic resistance does not affect the bactericidal effect of TPs against Helicobacter pylori.

[0127] 3) Vesicle membrane anchoring sequence:

[0128] The sources of vesicle membrane anchoring sequences include, but are not limited to, multidrug-resistant ABC transporters (LmrA), cell wall hydrolases (AcmA), the ABC transporter family (OpuABC, GlnP, mlaE), oligopeptide transporters (oppA), lactose transporters (LacF), transmembrane proteins (Holin), PrtP proteases, and cell wall peptidoglycanase (Lysin). Membrane anchoring sequences derived from LmrA are preferred. The LmrA protein is 590 amino acids in length, with 6 transmembrane regions in its N-terminal hydrophobic domain and a hydrophilic ATP-binding cassette (ABC) domain at its C-terminus. Both the N-terminus and C-terminus are located intracellularly. To express the functional peptide HTP-AMP on the outer surface of the membrane, the second to sixth transmembrane regions M2-6 (61 to 350 amino acids) of LmrA were selected as the anchoring sequence for the vesicle membrane [SEQ ID NO:3], and the Usp45 secretion signal peptide was fused to the N-terminus to guide the HTP-AMP functional peptide to be localized to the outer side of the vesicle membrane.

[0129] 4) Linker peptides between secreted peptides and functional peptides:

[0130] To improve the efficiency of HTP-AMP functional peptide expression for transport and localization to the vesicle membrane surface, a 27-amino acid secretion signal peptide [SEQ ID NO:4] of the major secretory protein Usp45 from Lactococcus lactis was fused to the N-terminus of the functional peptide sequence. To avoid potential interactions between different elements of the functional peptide, flexible peptide linker sequences from scFv [(SGGGGS)n, n=1~3, SEQ ID NO:5~7] were used to connect the different elements.

[0131] 5) Fusion proteins:

[0132] The complete fusion protein is composed of the fusion expression of the above functional elements. The structure of the fusion protein is Usp45-(SGGGGS)1-MM1-(SGGGGS)2-TP4-(SGGGGS)3-LmrA(M2-6) [SEQ ID NO:8].

[0133] 1) SEQ ID NO:1 (MM1)

[0134] MQKMTDQVNYQAMKLTLLQK

[0135] 2)SEQ ID NO:2 (TP4)

[0136] FIHHIIGGLFSAGKAIHRLIRRRRR

[0137] 3)SEQ ID NO:3 (LmrA(M2-6))

[0138] PLINSFGHGVNGGKVALVIALYIGSAAVSAIAAIVLGIFGESVVKNLRTRVWDKMIHLPVKYFDEVKTGEMSSRLANDTTQVKNLIANSIPQAFTSILLLVGSIIFMLQMQWRLTLAMIIAVPIVMLIMFPIMTFGQKIGWTRQD SLANFQGIASESLSEIRLVKSSNAEKQASKKAENDVNALYKIGVKEAVFDGLMSPVMMLSMMLMIFGLLAYGIYLISTGVMSLGTLLGMMMYLMNLIGVVPTVATFFTELAKASGSTGRLTELLDEEQEVLHQGDSLDLEGKTLS

[0139] 4)SEQ ID NO:4 (Usp45)

[0140] MKKKIISAILMSTVILSAAAPLSGVYA

[0141] 5)SEQ ID NO:5 ((SGGGS)1)

[0142] SGGGS

[0143] 6)SEQ ID NO:6 ((SGGGS)2)

[0144] SGGGSSGGGS

[0145] 7)SEQ ID NO:7 ((SGGGS)3)

[0146] SGGGSSGGGSSGGGS

[0147] 8) SEQ ID NO: 8 (Usp45-(SGGGGS)1-MM1-(SGGGGS)1-TP4-(SGGGGS)3-LmrA(M2-6)):

[0148] MKKKIISAILMSTVILSAAAPLSGVYASGGGSMQKMTDQVNYQAMKLTLLQKSGGGSSGGGSFIHHIIGGLFSAGKAIHRLIRRRRRSGGGSSGGGSSGGGSPLINSFGHGVNGGKVALVIALYIGSAAVSAIAAIVLGIFGESVVKNLRTRVWDKMIHLPVKYFDEVKTGEMSSRLANDTTQVKNLIANSIPQAF TSILLLVGSIIFMLQMQWRLTLAMIIAVPIVMLIMFPIMTFGQKIGWTRQDSLANFQGIASESLSEIRLVKSSNAEKQASKKAENDVNALYKIGVKEAVFDGLMSPVMMLSMMLMIFGLLAYGIYLISTGVMSLGTLLGMMMYLMNLIGVVPTVATFFTELAKASGSTGRLTELLDEEQEVLHQGDSLDLEGKTLS

[0149] The stability of codons and mRNA sequences was optimized using LinearDesign software to improve the expression efficiency of functional peptides in *L. lactis*. The optimized codon sequences are [SEQ ID NO: 9-15]. The promoter for fusion protein gene expression was the constitutively highly expressed promoter P32, and the transcription termination sequence was the common T1 transcription termination sequence. The complete gene expression frame sequence is [SEQ ID NO: 16].

[0150] 9)SEQ ID NO:9 (MM1)

[0151] ATGCAAAAAATGACAGATCAAGTTAATTATCAAGCTATGAAACTTACTCTTCTTCAAAAA

[0152] 10)SEQ ID NO:10 (TP4)

[0153] TTTATTCATCACATTATCGGAGGTCTTTTCTCAGCTGGTAAAGCTATTCATCGTCTTATTCGTCGTCGTAGACGT

[0154] 11)SEQ ID NO:11 (LmrA(M2-6))

[0155] CCATTAATTCTTTTGGACATGGTGTTAATGGCGGAAAAGTTGCACTTGTAATTGCTTTGTATATTGGAAGTGCAGCTGTTTCTGCCATTGCCGCCATTGTCCTT GGAATTTTTGGTGAATCAGTTGTCAAAAAATTTGCGTACGCGAGTTTGGGACAAAATGATTCACCTTCCAGTTAAATATTTTGAAGTTAAAACAGGAGAAATGTCGT CACGGTTGGCTAATGACACCACTCAAGTTAAGAATTTGATTGCAAATTCTATTCCAAGCTTTTACTAGCATTCTCTTGTTAGTTGGAAGCATAATTTTTTTGTTACA AATGCAATGGCGATTAACATTAGCTATTATTGCCGTTCCAATTGTAATGTTAATTTGTTTCCAATTATGACTTTTGGTCAAAAATTGGCTGGACTAGACAAGAT AGTTTAGCCAATTTTCAAGGGATTGCTAGTGAAAGCTTGAGTGAATTAGACTGTTAAATCAAGTAATGCCGAAAAGCAAGCATCTAAAAAAGCTGAAAATGATGTT AATGCATTATAAAATGGTGTTAAAGAAGCCGTTTTTGATGGCTTAATGTCACCAGTTATGCTTAGCATGATGCTTATGATTTTTGGATTGCTTGCTTATGGTA TTTATTTGATTTCAACAGGTGTAATGTCATTAGGTACATTATTGAATGATGTATTTAATGAATTTGATTGGTGTCGTGCCAACCGTGGCGACATTTTTCACTGA ATTAGCTAAAGCATCAGGATCAACTGGAAGATTAACTGAACTCCTTGATGAAGAACAAGAAGTTCTTCATCAAGGAGATTCACTTGATCTTGAAGGAAAAACTCTTTCA

[0156] 12)SEQ ID NO:12 (Usp45)

[0157] ATGAAAAAAAAAATTATTTCAGCTATTCTTATGTCTACAGTTATCCTTTCAGCTGCAGCTCCACTTTCAGGAGTTTATGCT

[0158] 13) SEQ ID NO:13 ((SGGGS)1)

[0159] TCAGGTGGAGGTTCA

[0160] 14) SEQ ID NO:14 ((SGGGS)2)

[0161] TCAGGTGGAGGTTCATCAGGTGGTGGATCT

[0162] 15)SEQ ID NO:15 ((SGGGS)3)

[0163] TCAGGTGGAGGTTCATCAGGTGTGTGGATCTTCTGGTGGTGGATCT

[0164] 16) SEQ ID NO:16

[0165] (P32-Usp45-(SGGGGS)1-MM1-(SGGGGS)2-TP4-(SGGGGS)3-LmrA(M2-6)-T1)

[0166]

[0167] 2. Gene Editing Construction of Engineered Probiotics

[0168] Vectors for gene editing include:

[0169] 1) pEV68: Constructed based on the pLH01 (Addgene plasmid #117261) vector. The Plp_0640 promoter before the RecT gene of the recombinase in the pLH01 vector was replaced with the PnisA promoter, and the expression of the recombinase was regulated by the nisin-induced peptide.

[0170] 2) pEV69-1a / b: A vector backbone based on pHSP02 (Addgene plasmid #117262), containing the following elements: a) P23-cas9: Expression of Cas9 protein initiated by the P23 promoter. b) P11-sgRNA: Transcription of sgRNA initiated by the P11 promoter. c) Complete expression cassette of the fusion protein (p32 promoter - fusion protein coding region - T1 terminator). d) Recombinant arms: Two homologous recombination arm sequences at the htrA gene locus at both ends of the fusion protein expression cassette, with deletion of the endogenous htrA gene promoter and the coding sequence of the N-terminus of the HtrA protein to remove the expression of the endogenous HtrA protein.

[0171] Using RecT-assisted CRISPR-Cas9 gene editing technology, the complete fusion protein coding sequence, containing both a promoter and transcription termination sequence, was knocked into the genome of the probiotic *L. Lactis* NZ9000. The knock-in genomic site was the *htrA* gene, which encodes the *HtrA* protease, which binds to the extracellular surface of the cell membrane and participates in both the degradation of aberrant secretory proteins and the maturation of secretory protein precursors. Deleting this protease gene (Δ *htrA*) improves the stability of the heterologous secretory protein. The complete gene expression coding sequence of the fusion protein was inserted into the 5' end of the *htrA* gene, while simultaneously deleting the endogenous promoter sequence preceding the translation start codon and the first 65 bp of the *htrA* gene sequence. This method knocks in the fusion expression gene sequence while simultaneously knocking out the expression of the endogenous *htrA* gene. The resulting engineered probiotic strain was used to produce *proELVs*.

[0172] 3. Preparation of heterozygous vesicles

[0173] The proELVs and mEVs were fused using a microporous extrusion method with the following parameters: a mixing ratio (protein mass ratio) of 1:2, and repeated extrusion through a 0.1 μm polycarbonate membrane 15 times; the heterozygous vesicles were purified by sucrose density gradient centrifugation [sucrose concentrations were 60% → 45% → 30% → 15% → 8% (w / w)].

[0174] Methods for preparing mEVs include:

[0175] This invention provides a method for preparing milk exosomes, comprising the following steps:

[0176] Step 1: Take milk and pre-treat it to obtain crude whey;

[0177] Step 2: The crude whey is clarified by microfiltration, concentrated by the first ultrafiltration, and then concentrated by the second ultrafiltration with the addition of an anti-aggregation reagent to obtain crude exosome extract;

[0178] Byproduct recovery: The filtrate is collected and concentrated by a third ultrafiltration to obtain whey protein components and prepare whey protein powder;

[0179] Step 3: Dialyze the crude exosome extract to remove impurities and / or salts, and obtain the dialysis product;

[0180] Step 4: The dialysis product is freeze-dried to obtain lyophilized exosomes.

[0181] Preferably, the pretreatment in step 1 includes extracting fat and / or extracting casein.

[0182] Preferably, the extraction speed for fat is 6000-8000 r / min, and skim milk is collected.

[0183] Preferably, the casein extraction process involves taking the skim milk, hydrolyzing it with rennet, separating the precipitate, collecting the squeezed liquid, and obtaining crude whey.

[0184] Preferably, the rennet has an enzyme activity of 20,000 IMCU / g and is added in an amount of 2,000-10,000 IMCU / L.

[0185] Preferably, the enzymatic hydrolysis temperature is 30-35°C and the time is 60 minutes.

[0186] Preferably, the microfiltration clarification in step 2 uses a 0.45 μm pore size microfiltration membrane, a pressure of 0.1~0.2 MPa, and a temperature of 20~25℃.

[0187] Preferably, in step 2, the membrane used for the first ultrafiltration concentration has a molecular weight cutoff of 300 kDa, a pressure of 0.2~0.3 MPa, and a temperature of 30~35°C, and is ultrafiltration concentrated to 50 times (1 / 50 of the initial volume) based on the initial whey volume.

[0188] Preferably, the anti-agglomeration agent in step 2 includes trehalose, which is added when the initial whey volume has been concentrated 50 times (to 1 / 50 of the initial volume) by the first ultrafiltration.

[0189] Preferably, the final concentration of trehalose is 5-10% (w / v); more preferably, the final concentration of trehalose is 6% (w / v).

[0190] Preferably, the membrane used for the second ultrafiltration concentration has a molecular weight cutoff of 300 kDa, a pressure of 0.2~0.5 MPa, and a temperature of 25~35°C, resulting in an ultrafiltration concentration of 100 times (1 / 100 of the initial whey volume).

[0191] Preferably, the filtrate (containing <300kDa components) produced by the second ultrafiltration concentration is further concentrated by the third ultrafiltration to obtain whey protein components for use in the production of whey protein powder.

[0192] Preferably, the membrane molecular weight cutoff of the third ultrafiltration concentration is 10-20 kDa; more preferably, the membrane molecular weight cutoff of the third ultrafiltration concentration is 20 kDa.

[0193] Preferably, the addition of an anti-aggregation reagent for the second ultrafiltration concentration specifically involves: based on the initial whey volume, after the first ultrafiltration concentration to 50 times (1 / 50 of the initial volume), adding trehalose to a final concentration of 6% (w / v), stirring evenly (100-200 r / min), and continuing ultrafiltration to 100 times (1 / 100 of the initial volume) to obtain crude exosome extract.

[0194] Preferably, the microfiltration clarification, the first ultrafiltration concentration, the second ultrafiltration concentration, and the third ultrafiltration concentration each employ a spiral wound membrane independently.

[0195] Preferably, the spiral wound film is in series.

[0196] Preferably, the series connection mode is: clarified whey → filtrate from a 300 kDa membrane → ultrafiltration from a 10 kDa membrane.

[0197] Preferably, the preservation solution for dialysis in step 3 includes 1.5% trehalose, 6% mannitol and 0.5% human serum albumin; the dialysis time is 8-12 hours.

[0198] Preferably, the freeze-drying temperature in step 4 is -50℃ to -60℃, and the vacuum degree is 1 to 10 Pa.

[0199] Specifically, taking a single batch processing of 10 tons of fresh milk as an example, the preparation process of mEVs will be explained in detail:

[0200] ① 10 tons of fresh milk are separated using a disc separator (8000 r / min) to obtain 1.2 tons of fat fraction (for butter production) and 8.8 tons of skim milk fraction;

[0201] ② Add 4.4 kg (0.05% w / v) of rennet to skim milk, react at 35°C for 45 minutes, and obtain 1.5 tons of crude cheese (for cheese production) and 7.3 tons of primary whey by static sedimentation and extrusion.

[0202] ③ Primary whey is filtered through a 0.45μm microfiltration spiral wound membrane (0.15 MPa, 25℃) to obtain 7.0 tons of clarified whey; the concentrate retained during the clarification process (≈0.3 tons) is used for cheese preparation;

[0203] ④ The clarified whey was filtered through an ultrafiltration membrane (wound membrane) with a molecular weight cutoff of 300 kDa at 0.3 MPa and 25°C. When the concentration was increased to 50 times (140 L), 8.4 kg of trehalose (final concentration 6% w / v) was added, and ultrafiltration was continued to increase to 100 times (70 L) to obtain crude exosome extract.

[0204] ⑤ The ultrafiltration filtrate (6.86 tons) was concentrated using a 10 kDa nanofiltration membrane (wound membrane) to obtain 0.5 tons of whey protein concentrate, which was used for whey protein powder production;

[0205] ⑥ The crude exosome extract was dialyzed (preservation solution: 1.5% trehalose + 6% mannitol + 0.5% HSA) for 12 hours, then lyophilized to obtain 12.8 kg of exosome powder and 1.2 × 10⁻⁶ milk exosomes. 17 (1.2×10 13 / L), purity 92%.

[0206] 4. Application Scenarios

[0207] ①Preventive application

[0208] High-risk individuals who are not infected with H. pylori (such as those with a family history of gastric cancer or those infected through shared meals);

[0209] Dosage: 1×10 11 Particles / day, orally, for 14 consecutive days.

[0210] ②Therapeutic applications:

[0211] HP infected individuals (e.g., those with a positive C13 uric acid breath test)

[0212] To eradicate HP using only: 2 × 10 11 Particles / time, twice daily, for a course of 14 days;

[0213] When used in combination with antibiotics: antibiotic dosage is reduced by 50%.

[0214] ③ Combination strategy:

[0215] Sequential therapy: First, administer heterozygous vesicles orally to clear HP, then supplement with probiotics (such as L. reuteri, L. lactis, etc.) to restore the gut microbiota;

[0216] Combined formulation: heterozygous vesicles, freeze-dried probiotic powder, and mEV are combined into a compound capsule.

[0217] 5. Beneficial effects

[0218] ① Targeting: HTP guides vesicles to specifically bind to HP, improving sterilization efficiency and reducing side effects such as bacterial imbalance;

[0219] ② Synergistic effect: AMP directly kills HP, and mEVs repair the gastric mucosal barrier and regulate the immune response;

[0220] ③ Safety: It does not rely on antibiotics, avoiding drug resistance problems, and both probiotics and mEVs are natural ingredients with high biocompatibility.

[0221] This design integrates multiple functions such as targeted antibacterial activity, mucosal repair, and flora regulation. Its advantages are:

[0222] Highly efficient delivery (hybrid vesicles): The vesicle structure provides a stable carrier while improving targeted bactericidal and repair efficacy, prolonging the residence time of milk exosomes on damaged mucosa, and increasing the repair effect;

[0223] Precise and selective killing (HTP+AMP): Targeted peptides guide vesicles to accumulate in the Helicobacter pylori infection area, and antimicrobial peptides act precisely on Helicobacter pylori, avoiding the side effects of non-selective antibacterial treatment;

[0224] Microecological balance (targeted killing + mEVs): Targeted killing maximizes the protection of symbiotic flora in the stomach, while mEVs further synergistically maintain the microecological balance.

[0225] Mucosal barrier (mEVs): Eliminate Helicobacter pylori, simultaneously repair damaged gastric mucosa, and rebuild and strengthen mucosal barrier function.

[0226] Safety: Biocompatible materials avoid the toxicity risks of chemically synthesized carriers and are suitable for use in food or adjunctive therapeutic preparations.

[0227] This hybrid vesicle, which integrates "targeted clearance, gut microbiota balance, and barrier repair," not only holds promise as a novel therapeutic agent for effectively eradicating H. pylori and reducing recurrence rates, but its excellent biocompatibility and gastric protective properties also give it great potential as a "functional food ingredient" or "adjunctive therapy" for preventing and maintaining gastric health, providing a new approach to addressing the global challenge of H. pylori infection and improving gastric health.

[0228] The raw materials and reagents used in the heterozygous vesicles against Helicobacter pylori infection and their preparation method provided by this invention are all commercially available.

[0229] The present invention will be further illustrated below with reference to the embodiments:

[0230] Example 1: Preparation of engineered probiotic vesicles

[0231] 1.1. Probiotic culture:

[0232] Lactococcus lactis NZ9000 was selected as the host strain and statically cultured at 30°C in GM17 medium (M17 containing 0.5% (w / v) glucose). Electroporation recovery medium was GM17 medium containing 2 mM CaCl2 and 20 mM MgCl2, and the solid medium contained 1.5% agar.

[0233] 1.2. Gene Editing:

[0234] An engineered strain was constructed by knocking the fusion gene into the L. Lactis NZ9000 genome using CRISPR-Cas9 technology.

[0235] 1) Construction of gene editing vectors pEV68, pEV69a, and pEV69b:

[0236] The PnisA promoter was synthesized by Beijing Qingke Biotechnology Co., Ltd., and cloned into the pLH01 vector before the RecT gene of the recombinase. The original Plp_0640 promoter was replaced and deleted to obtain the pEV68 vector.

[0237] Two sgRNAs targeting the promoter and 5' coding region of the hrtA gene were designed using the online prediction software CRISPOR for Lactococcus lactis strain NZ9000 [SEQ ID NO:17-18].

[0238] 17)SEQ ID NO:17 (htrA sgRNA-1)

[0239] AGGATAAGTGGATATTTAAG;

[0240] 18)SEQ ID NO:18 (htrA sgRNA-2)

[0241] GGTGGAGCTATCGCACTCGG.

[0242] Two sgRNA targeting sequences were inserted before the sgRNA backbone region of the pHSP02 vector using a point mutation PCR kit (KOD Plus Mutagenesis Kit (SMK-101), Toyobo (Shanghai) Biotechnology Co., Ltd.), resulting in pEV69-sg-1 and pEV69-sg-2. The point mutation PCR parameters and procedures were followed according to the kit instructions. The point mutation PCR primers are as follows:

[0243] htrA-sg-1F: (as shown in SEQ ID NO:19) TCATCTGATCATATTCCGAGGTTTTAGAGCTAGAAATAGCAAGT;

[0244] htrA-sg-2F: (as shown in SEQ ID NO:20) GCTGAAGCATCAATGGCTGGGTTTTAGAGCTAGAAATAGCAAGT;

[0245] LL-sg-R: CAATATATCATAGTATGTCCATTCTGT (as shown in SEQ ID NO:21).

[0246] The complete gene expression coding sequence of the fusion protein synthesized by Beijing Qingke Biotechnology Co., Ltd. Using the NEBuilder Assembly kit, two homologous recombination arms and the fusion gene sequence were cloned into the vector containing the inserted sgRNA. PCR amplification and recovery of the synthesized gene fragments yielded PCR product #1. The 5' and 3' homologous recombination arm sequences knocked into the CRISPR-cas9 gene were amplified by PCR from the L. Lactis NZ9000 genome, yielding PCR products #2 and #3, respectively. A pair of reverse PCR primers were designed flanking the 0537-1 and 0537-2 sequences of the pEV69-sg-1 and pEV69-sg-2 vectors, respectively. The vector sequences were amplified and recovered, yielding PCR products #4a / #4b. PCR primers were designed using the NEBuilder Assembly Tool online software, and the four PCR products were assembled in the order of fragment #2-#1-#3-#4a / 4b to obtain pEV69a and pEV69b (a and b are pEV69 vectors containing sgRNA-1 and sgRNA-2 sequences, respectively).

[0247] The PCR primers for PCR amplification of the synthesized gene are as follows (PCR product 1):

[0248] F1-F: CAACATACTAGCTTGATAAATCTCCCTACGGGAGTTT (as shown in SEQ ID NO:22);

[0249] F1-R: GTTAGCATTTCCTACTAGTCTAGGGCGGCGGATTTGTC (as shown in SEQ ID NO:23).

[0250] The primers used for PCR amplification of the 5' homologous recombination arm sequence from the NZ9000 genome are as follows (PCR product 2):

[0251] F2-F: ACACACCAATCATCTTCAACCAACTTTGCCC (as shown in SEQ ID NO:24);

[0252] F2-R: TTTATCAAGCTAGTATGTTGATAAATCATGCTTTCATCTTG (as shown in SEQ ID NO:25).

[0253] The primers used for PCR amplification of the 3' homologous recombination arm sequence from the NZ9000 genome are as follows (PCR product 3):

[0254] F3-F: GACTAGTAGGAAATGCTAACCGCTCAAATAC (as shown in SEQ ID NO:26);

[0255] F3-R: GAAGAATTTGACATTTGCTGTGGCTGATTTAC (as shown in SEQ ID NO:27).

[0256] The PCR primers for PCR amplification of the sgRNA vector are as follows (PCR product 4):

[0257] F4-F: CAGCAAATGTCAAATTCTTCTACGGCTTTTTCCATATATAC (as shown in SEQ ID NO:28)

[0258] F4-R: GTTGAAGATGATTGGTGTGTGTGTCTTGTTGTTAGTAT (as shown in SEQ ID NO: 29).

[0259] 2) Gene editing of Lactococcus lactis NZ9000:

[0260] Preparation of NZ9000 electrocompetent cells: 4 mL of overnight cultured NZ9000 cells were transferred to 50 mL of fresh GM17 medium (M17 medium containing 0.5 M sucrose and 0.8% glycine). When the OD600 reached 0.4–0.6, the cells were centrifuged at 4000 × g for 10 min at 4 °C and recovered. The cells were resuspended in Solution III [100 mM lithium acetate dihydrate, 10 mM dithiothreitol, 0.6 M sucrose, 1 M Tris-HCl (pH 7.5)]. After 30 min, the cells were centrifuged at 4,000 g for 10 min at 4 °C, and the supernatant was discarded. The cell pellet was washed with Solution I [10% glycerol (vol / vol) and 0.5 M sucrose] and Solution II [10% glycerol (vol / vol), 0.5 M sucrose and 0.05 M EDTA]. The cells were then resuspended in Solution I. 1.0 μg of the pEV68 recombinase expression plasmid was introduced into *Lactococcus lactis* NZ9000 via electroporation. Electroporation was performed using a GenePulser Xcell (Bio-Rad, USA) and a 2 mm electroporation cuvette (BTX, USA) with the following parameters: 2.5 kV, 200 Ω, 25 μF. After electroporation, the cells were resuspended in 1 mL of pre-warmed electroporation recovery medium (GM17, containing 2 mM CaCl2 and 20 mM MgCl2), and the mixture was incubated at 30°C for 2–3 hours. Transformants were then screened by inoculating the cells onto GM17 plates supplemented with 10 μg / mL chloramphenicol.

[0261] The edited plasmid was introduced into lactic acid bacteria NZ9000 via electroporation. First, transformants carrying pEV68 were prepared into competent cells: 4 mL of overnight culture was transferred to 50 mL of fresh GM17 medium (containing 10 μg / mL chloramphenicol). When OD600 reached 0.3, nisin-inducible peptide at a final concentration of 10 ng / mL was added to induce RecT expression. When OD600 reached 0.4–0.5, the cells were recovered and prepared into competent cells using the same method as above. NZ9000 / pEV68 competent cells were electroporated with 1.0 μg of pEV69a and pEV69b plasmids (0.5 μg each of pEV69a and pEV69b), using the same electroporation parameters as above. After recovery at 30 °C for 2–3 h, the cells were seeded onto GM17 culture plates supplemented with 10 μg / mL erythromycin and cultured at 30 °C for 24–36 h to screen transformants. Colony PCR was performed using primers flanking the homologous arms of the target gene to screen strains that had undergone correct gene editing. To confirm gene editing in lactic acid bacteria NZ9000, all PCR fragments were sequenced for verification.

[0262] 1.3. Vesicle extraction:

[0263] 1) Bacterial culture and supernatant collection

[0264] ① Seed culture preparation: Inoculate Lactococcus into 5 mL of MRS liquid medium and incubate at 30℃ for 12-16 hours until the logarithmic growth phase.

[0265] ② Expanded culture: Transfer 1 mL of seed culture to 500 mL of MRS liquid medium (containing 1 mM PMSF and 5 mM EDTA), and culture at 30℃ with shaking at 200 rpm until OD. 600 ≈0.8 (late logarithmic growth phase).

[0266] ③ Centrifugation to remove bacteria: Transfer the culture to a centrifuge tube, centrifuge at 4℃ and 8,000 g for 30 minutes, discard the precipitate (bacterial cells), and retain the supernatant.

[0267] 2) Supernatant pretreatment

[0268] ① Filtration: The supernatant is filtered through a 4.5 μm microfiltration membrane to remove residual bacterial fragments and large molecular impurities.

[0269] ②Dispensing: Dispense the filtrate into 50 mL centrifuge tubes, each tube not exceeding 40 mL, and label the batch.

[0270] 3) ProELVs ultracentrifugation

[0271] First round of ultracentrifugation:

[0272] ① Transfer the filtrate to an ultracentrifuge tube (such as an OptiSeal tube) and centrifuge at 4°C and 100,000 g for 2 hours.

[0273] ② Carefully aspirate the supernatant; a white or transparent gelatinous precipitate will be visible at the bottom of the tube (crude ProELVs extraction).

[0274] Washing and resuspension:

[0275] ① Add 2 mL of pre-cooled PBS (containing 1 mM PMSF and 5 mM EDTA) to the precipitate, and gently resuspend it by pipetting, avoiding the formation of air bubbles.

[0276] ② Transfer the suspension to a new ultracentrifuge tube and centrifuge again at 4°C and 100,000 g for 2 hours.

[0277] ③ Final resuspension: Discard the supernatant, resuspend the precipitate in 100-200 μL of pre-cooled PBS, transfer to a 1.5 mL centrifuge tube, and store at 4°C for later use.

[0278] Example 2: Preparation of hybrid vesicles (hybrid-EVs)

[0279] 2.1 Determination of mEV (preparation method as described above) and proELV protein concentration:

[0280] ① Follow the instructions of the BCA protein quantification kit, take appropriate amounts of the self-prepared mEV and proELV samples, and determine the protein concentration. Set up 3 parallel wells for each sample to ensure data accuracy.

[0281] ② Using PBS as a blank control, the absorbance at 562 nm was measured on an ELISA reader, and the protein concentrations (mg / mL) of mEV and proELV were calculated based on the standard curve.

[0282] 2.2 mEV and proELV are mixed in proportion:

[0283] ① Based on the measured protein concentration, mix the bacterial outer membrane vesicles and mEV protein in a 1:2 ratio. [For example, if the proELV protein concentration is 1 mg / mL and the mEV protein concentration is 2 mg / mL, mix 1 mL of proELV and 1 mL of mEV in a 1.5 mL centrifuge tube, for a total volume of 2 mL.]

[0284] ② Place the centrifuge tube on a vortex mixer and vortex at low speed for 1 minute to ensure thorough mixing.

[0285] 2.3 Preparation of heterozygous vesicles:

[0286] ① Assemble the micro extruder and install the 400 nm pore size polycarbonate filter membrane. Ensure the filter membrane is installed correctly and tightly to avoid leakage.

[0287] ② Rinse the micro-squeezer and filter membrane three times with PBS to remove any possible impurities.

[0288] ③ Transfer the mixed mEV and proELV suspension to the syringe of the micro-squeezer, and slowly push the syringe to squeeze the suspension through a 400 nm pore size filter membrane into another syringe. During this process, it is important to maintain a constant speed and avoid generating air bubbles.

[0289] ④ Repeat the above squeezing operation for a total of 15 times. After each squeeze, briefly vortex the suspension in the syringe to ensure that the vesicles are fully mixed and deformed.

[0290] 2.4 Collection and preservation of heterozygous vesicles:

[0291] ① After squeezing, transfer the suspension containing heterozygous vesicles to a new 1.5 mL centrifuge tube.

[0292] ② If not used temporarily, the hybrid vesicle suspension can be stored in a 4°C refrigerator for short-term (1-2 days) use; if long-term storage is required, it can be stored in a -80°C refrigerator to avoid repeated freeze-thaw cycles, so as not to affect the stability of the vesicles.

[0293] Example 3 In vitro antibacterial experiment

[0294] 3.1 Helicobacter pylori resuscitation and pre-culture

[0295] ①Strain resuscitation: Remove the HP26695 cryovial from -80℃, thaw rapidly in a 37℃ water bath, spread 100μL of bacterial solution onto an agar plate, and incubate in a microaerophilic environment at 37℃ for 48-72 hours until grayish-white, semi-transparent colonies are formed.

[0296] ② Subculture: Pick a single colony and inoculate it onto a fresh blood agar plate. Incubate at 37°C for 24-48 hours in a microaerophilic environment to obtain bacteria in the logarithmic growth phase.

[0297] ③ Preparation of bacterial suspension: Wash the bacterial colonies on the plate with sterile PBS, collect the bacterial suspension into a centrifuge tube, centrifuge at 1200×g for 5 minutes, discard the supernatant, resuspend in PBS, and adjust the concentration to 0.5 McFarland turbidity (approximately 1×10⁻⁶) using a McFarland turbidimeter. 8 (CFU / mL), then diluted to 1×10⁻⁶ with Brucella broth. 6 CFU / mL for later use.

[0298] 3.2. Grouping and Processing (96-well plate method)

[0299] ①Each group has 6 replicates, and the edge wells are filled with sterile PBS to reduce evaporation error.

[0300] ② Add 100 μL of 1×10 to each well 6 CFU / mL HP26695 bacterial suspension.

[0301] ③ Add treatment reagents according to the groups (final volume 200 μL per well):

[0302] mEVs group: Add 100 μL 2×10 11 cells / mL mEVs (final concentration 1×10⁻⁶) 11 (pcs / mL).

[0303] proELVs group: Add 100 μL 2×10 11 cells / mL proELVs (final concentration 1×10⁻⁶)11 (pcs / mL).

[0304] hybrid-EVs group: Add 100 μL 2×10 11 1 / mL mEV / proELV hybrid vesicle (hybrid-EVs, i.e., the mEV / proELV hybrid vesicles prepared in Example 2) (final concentration 1×10⁻⁶) 11 (pcs / mL).

[0305] Amoxicillin group: Add 100 μL of 1 μg / mL amoxicillin (final concentration 0.5 μg / mL).

[0306] PBS group: Add 100 μL of sterile PBS.

[0307] 3.3. Cultivation and Antibacterial Effect Detection

[0308] ① Culture conditions: Place the 96-well plate in a 37℃ microaerophilic incubator and incubate statically for 24 hours. ② OD 600 Measurement: After the culture was completed, the OD of each well was measured using a microplate reader. 600 The value is used to assess the inhibition of bacterial growth.

[0309] ③ Plate count verification (CFU counting):

[0310] Take 100 μL of bacterial culture from each treatment group and perform 10-fold serial dilutions with PBS (10⁻¹ to 10⁻¹). 6 Take 100 μL of diluent (10⁻) 4 10⁻ 5 10⁻ 6 Spread on Columbia blood agar plates and incubate at 37°C for 48 hours under microaerophilic conditions.

[0311] Count the number of colonies and calculate the CFU value per milliliter of bacterial culture (CFU / mL = number of colonies × dilution factor × 10).

[0312] 3.4. Calculation of Antibacterial Rate

[0313] Formula: Inhibition rate (%) = [(Mean CFU of negative control group - Mean CFU of experimental group) / Mean CFU of negative control group] × 100%

[0314] If OD value is used for calculation: Antibacterial rate (%) = [(Mean OD value of negative control group - Mean OD value of experimental group) / Mean OD value of negative control group] × 100%.

[0315] 3.5 Experimental Results

[0316] The antibacterial rates were: mEV 19.78%, proELVs 90%, hybrid-EVs 89%, and Amoxicillin 95%.

[0317] Example 4: In vivo antibacterial and repair

[0318] 4.1. Mouse model of Helicobacter pylori infection

[0319] ①Preparation of bacterial suspension: HP26695 was cultured microaerophilically on Columbia blood agar plates (Solepro, LA3540) for 48 h, the colonies were washed with PBS, and the concentration was adjusted to 1×10⁻⁶ using a McFarland turbidimeter. 9 CFU / mL.

[0320] ② Mouse pretreatment: Fasting (free access to water) was performed for 12 hours before the experiment to reduce interference from stomach contents.

[0321] ③ Infection with *H. pylori*: Each mouse was given 0.2 mL of *H. pylori* suspension orally via gavage (containing 2 × 10⁻⁶ bacteria). 8 CFU was administered via gavage for 3 consecutive days. Control group mice were administered an equal volume of sterile PBS via gavage.

[0322] ④ Infection verification: On day 21 post-infection, three mice were randomly sacrificed, and their gastric tissue homogenate was cultured. If CFU > 1 × 10⁻⁶, the infection was verified. 4 The / g organization indicates that the model has been successfully established.

[0323] 4.2. Grouping and Dosing

[0324] Mice infected with *Helicobacter pylori* were randomly divided into 5 groups of 8 mice each, with 3 uninfected mice serving as a blank control.

[0325] mEVs group: 2×10¹¹ mEVs / mL by gavage, 0.2 mL / animal / day.

[0326] proELVs group: 2×10¹¹ proELVs / mL by gavage, 0.2 mL / animal / day.

[0327] hybrid-EVs group: 2×10¹¹ hybrid-EVs / mL were administered by gavage, 0.2 mL / animal / day.

[0328] Amoxicillin group: 1 μg / mL amoxicillin solution was administered by gavage, 0.2 mL / animal / day.

[0329] PBS group: 0.2 mL of sterile PBS was administered by gavage to each animal per day.

[0330] Administration time: Start administration on the 22nd day after infection, and administer via gavage for 7 consecutive days, at 9:00 am every day.

[0331] 4.3. Sample Collection

[0332] Sacrifice time: 24 h after the last administration, mice were weighed and then euthanized by dislocation of the neck.

[0333] Gastric tissue processing: The entire stomach was dissected and harvested. The gastric contents were rinsed with pre-cooled PBS, and the excess water was blotted dry with filter paper. The stomach was cut along the greater curvature and divided into three parts:

[0334] 1 / 3 of the gastric tissue: immediately fixed with 4% paraformaldehyde (Beyotime, P0099), and embedded in paraffin after 24 hours (for immunohistochemistry).

[0335] 1 / 3 of the stomach tissue: Place it in a homogenization tube, add 1 mL of pre-cooled RIPA cell lysis buffer (Beyotime, P0013), homogenize on ice at 3000 rpm for 30 s × 3 times (for HP conformation).

[0336] 1 / 3 of the stomach tissue: frozen at -80°C for ELISA detection of cytokines.

[0337] 4.4 Evaluation Indicators

[0338] 1) Gastric tissue HP load

[0339] ① Blocking: Dry the frozen sections at room temperature for 15 minutes, then block them with 10% blocking goat serum (Beyotime, C0265) for 1 hour to reduce nonspecific binding.

[0340] ② Primary antibody incubation: Place the blocked sections in a humidified chamber, add an appropriate amount of diluted anti-H. pylori specific primary antibody (Abcam, ab20459), and incubate overnight at 4°C.

[0341] ③ Warming and washing: The next day, move the humidified chamber to room temperature for 1 hour to warm it again, and rinse the slides with PBS to remove unbound primary antibody.

[0342] ④ Secondary antibody incubation: Add fluorescently labeled secondary antibody (Boster, SA1022) and incubate in the dark for 1 hour to allow the secondary antibody to specifically bind to the primary antibody, and the location of HP can be indicated by the fluorescence signal.

[0343] ⑤ Nuclear staining: After washing the sections with PBS, add DAPI staining solution (Solepro, D8200) and incubate at room temperature for 20 minutes to stain the cell nuclei, which facilitates the localization of cell structures.

[0344] ⑥ Mounting: Add anti-fluorescence quencher (Solebo, S2110) and seal the slide with nail polish to prevent fluorescence decay and slide drying.

[0345] ⑦ Fluorescence imaging: The slides were scanned using an automated fluorescence microscope to acquire the fluorescence signal (e.g., red) corresponding to H. pylori and the nuclear staining signal (blue) of DAPI.

[0346] ⑧ Quantitative analysis: The mean fluorescence intensity (MFI) of H. pylori was calculated using image analysis software to quantify the H. pylori load in the gastric mucosa and assess the degree of infection or the effectiveness of intervention.

[0347] 2) Gastric mucosal pathology and immunohistochemistry:

[0348] HE staining: Paraffin sections were dewaxed to water, stained with HE, and the degree of gastric mucosal inflammation (such as inflammatory cell infiltration and glandular damage) was observed and assessed according to the inflammation scoring criteria (0-4 points).

[0349] Immunohistochemistry:

[0350] ① After antigen retrieval, anti-Claudin-1 (Beyotime, AG4424) and Occludin primary antibody (Beyotime, AG5376) (1:200 dilution) were added to the slides and incubated overnight at 4°C.

[0351] ② Add secondary antibody (Beyotime, A0210), incubate at room temperature for 30 min, develop with DAB, and counterstain with hematoxylin.

[0352] ③ Observe the protein expression location under a microscope, and use Image-Pro Plus software to quantitatively analyze the average optical density (IOD / Area) to reflect the protein expression level.

[0353] 3) Inflammatory cytokine ELISA detection:

[0354] ① Sample processing: Cryopreserved gastric tissue was lysed in RIPA lysis buffer (Beyotime, P0013) on ice for 30 min, centrifuged at 4℃ and 12000 g for 15 min, and the supernatant was collected.

[0355] ②ELISA detection: According to the ELISA kit instructions, add the standard, sample and detection antibody in sequence, incubate at 37℃ for 30 min, wash the plate and add the color development solution, and measure the absorbance at 450 nm using an ELISA reader.

[0356] ③ Calculation: Calculate the concentrations of IL-8, TNF-α, and IL-1β (pg / mg protein, corrected for protein concentration using the BCA method) based on the standard curve.

[0357] 4) Test results:

[0358] ①HP load (expressed as MFI) was as follows: mEV group 76%, proELVs group 18%, hybrid-EVs group 8%; PBS group 100%;

[0359] ② HE staining: Gastric mucosal damage was reduced in both the mEV and hybrid-EVs groups, with the hybrid-EVs group showing better repair than the mEVs group; compared with the PBS group, the proELVs group showed some improvement, but it was not significant and was significantly lower than the mEVs and hybrid-EVs groups.

[0360] ③ Immunohistochemistry: Claudin-1 and Occludin expression were increased in both the mEV and hybrid-EVs groups, with no significant difference between the two groups.

[0361] The inhibition rates of Helicobacter pylori in in vitro experiments are shown in Table 1 and 2. Figure 3 (The antibacterial rate was 90% compared with the control group).

[0362] Table 1. Inhibition rate of heterozygous vesicles against Helicobacter pylori

[0363]

[0364] The hepatic globulin (HP) load in the gastric mucosa of the animal models is shown in Table 2 and... Figure 4 (Immunohistochemical staining and quantitative analysis showed that heterozygous vesicles reduced HP load by 92%).

[0365] Table 2. Helicobacter pylori load in the gastric mucosa

[0366]

[0367] Hybrid vesicles promoted the expression levels of tight junction proteins (Claudin-1 and Occludin) in the gastric mucosa (mEVs and hybrid-EVs significantly increased tight junction protein expression levels) (see Tables 3-4). Figure 5 .

[0368] Table 3. Expression levels of the tight junction protein Occludin in the gastric mucosa

[0369]

[0370] Table 4. Expression level of Claudin-1, a tight junction protein in the gastric mucosa

[0371]

[0372] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein that specifically targets and kills Helicobacter pylori, characterized in that, It contains domains that contain Helicobacter pylori targeted peptides (HTP) and antimicrobial peptides (AMP); The Helicobacter pylori targeting peptide targets characteristic proteins of Helicobacter pylori, preferably VacA and / or UreB.

2. The fusion protein as described in claim 1, characterized in that, The Helicobacter pylori targeting peptide (HTP) includes, but is not limited to, one or more of the following: human thrombin protein multiplyin-1 (Multimerin-1), urease B subunit binding peptide (UBP), cytotoxin-associated protein A binding peptide (CagA-BP), adhesin binding peptide (BabA / B-BP), or Lpp20 protein epitope peptide. The antimicrobial peptides (AMPs) include, but are not limited to, one or more of the following: PGa-AM1, Tilapia piscidins (TP4 / TP3), LL-37, Pexigaran (MSI-78), Epinecidin-1, Cathelicidins, Defensins (HNP-1), Bicarinalin, Odorranain-HP, Bacteriocins, Nisin A, Alyteserin, laterosporulin, or CRAMP.

3. The fusion protein as described in claim 1 or 2, characterized in that, The Helicobacter pylori targeting peptide (HTP) has the following characteristics: (I) The amino acid sequence as shown in SEQ ID NO:1; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II); The antimicrobial peptide (AMP) has the following characteristics: (I) The amino acid sequence shown in SEQ ID NO:2; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

4. The fusion protein according to any one of claims 1 to 3, characterized in that, The fusion protein also includes one or more of the following: anchoring sequences, secreted peptides, and linkers; Preferably, the source of the anchoring sequence includes, but is not limited to, one or more of the following: multidrug-resistant ABC transporter (LmrA), cell wall hydrolase (AcmA), ABC transporter family (OpuABC, GlnP, mlaE), oligopeptide transporter (oppA), lactose transporter (LacF), transmembrane protein (Holin), PrtP protease, or cell wall peptidoglycanase (Lysin). More preferably, the source of the anchoring sequence includes the multidrug resistance ABC transporter protein (LmrA); and / or The secreted peptides include, but are not limited to, Usp45; and / or The linker peptides include, but are not limited to, flexible peptide linker sequences (SGGGGS) derived from scFv. n , where n = 1~3.

5. The fusion protein as described in claim 4, characterized in that, The anchoring sequence includes the 2nd to 6th transmembrane regions M2-6 of the multidrug resistance ABC transporter (LmrA); Preferably, the anchoring sequence has: (I) The amino acid sequence shown in SEQ ID NO:3; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II); and / or The secretory peptide has the following characteristics: (I) The amino acid sequence shown in SEQ ID NO:4; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II); and / or The linked peptide has: (I) An amino acid sequence as shown in any of SEQ ID NO:5~7; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

6. The fusion protein as described in claim 4 or 5, characterized in that, The structure of the fusion protein includes: Usp45-(SGGGGS)1-MM1-(SGGGGS)2-TP4-(SGGGGS)3-LmrA(M2-6); Preferably, the fusion protein has the following characteristics: (I) The amino acid sequence shown in SEQ ID NO:8; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substitution, deletion, or addition of one or more amino acids; or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence described in (I) or (II).

7. A nucleotide sequence encoding the fusion protein or a component thereof as described in any one of claims 1 to 6; Preferably, the nucleotide sequence encoding the antimicrobial peptide has: (I) A nucleotide sequence as shown in SEQ ID NO:9; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence shown in (I) or (ii); and / or Preferably, the nucleotide sequence encoding the antimicrobial peptide has: (I) A nucleotide sequence as shown in SEQ ID NO:10; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence shown in (I) or (ii); and / or Preferably, the nucleotide sequence encoding the anchoring sequence has the following characteristics: (I) A nucleotide sequence as shown in SEQ ID NO:11; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence shown in (I) or (ii); and / or Preferably, the nucleotide sequence encoding the secretory peptide has: (I) A nucleotide sequence as shown in SEQ ID NO:12; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence shown in (I) or (ii); and / or Preferably, the nucleotide sequence encoding the linked peptide has: (I) A nucleotide sequence as shown in any of SEQ ID NO: 13-15; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequence shown in (I) or (ii); and / or Preferably, the nucleotide sequence encoding the fusion protein has: (I) A nucleotide sequence as shown in SEQ ID NO:16; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

8. An expression carrier, characterized in that, Includes the nucleotide sequence as described in claim 7; As a preferred option, sgRNA is also included; More preferably, the sgRNA includes htrA sgRNA-1 targeting the hrtA gene promoter and htrA sgRNA-2 targeting the 5' coding region of the hrtA gene; The htrA sgRNA-1 has: (I) A nucleotide sequence as shown in SEQ ID NO:17; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii); The htrA sgRNA-2 has: (I) A nucleotide sequence as shown in SEQ ID NO:18; or (II) A nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in (I), and whose function is the same as or similar to that of (I); or (III) A nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the nucleotide sequence shown in (I) or (ii).

9. A biomaterial, characterized in that, Transformation or transduction of the expression vector as described in claim 8; Preferably, the biomaterial includes, but is not limited to, cells or microorganisms; Preferably, the microorganisms include, but are not limited to, probiotics; More preferably, the probiotics include, but are not limited to, lactic acid bacteria; More preferably, the lactic acid bacteria include, but are not limited to, one or more of Lactococcus lactis, Lactobacillus reuteri, or Lactobacillus gasseri.

10. Probiotic vesicles, characterized in that, Cultivate the biological material as described in claim 9, collect the supernatant, centrifuge, and obtain the probiotic vesicles.

11. A heterozygous vesicle, characterized in that, This includes milk exosomes (mEVs) and probiotic vesicles as described in claim 10.

12. The heterozygous vesicle as described in claim 11, characterized in that, The protein ratio of the milk exosomes (mEVs) to the probiotic vesicles as described in claim 10 includes, but is not limited to, 2:1; and / or The fusion of the milk exosomes (mEVs) and the probiotic vesicles is achieved by microporous extrusion at a pressure of 0.8 to 1.2 MPa for 10 to 25 cycles.

13. Use of the heterozygous vesicles as described in claim 11 or 12 in the preparation of medicaments for the prevention, improvement, treatment or adjunctive treatment of Helicobacter pylori infection.

14. A drug, characterized in that, Includes the heterozygous vesicles as described in claim 11 or 12, and pharmaceutically acceptable excipients.

15. A drug combination, characterized in that, Includes the hybrid vesicles as described in claim 11 or 12 and other active ingredients; Preferably, the other active ingredients include, but are not limited to, probiotic preparations; Preferably, the heterozygous vesicles are administered sequentially to the probiotic preparation.

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