A method and system for preparing an antibacterial peptide complex and its application in the killing of helicobacter pylori

Abstract

The application discloses a kind of antibacterial peptide complex preparation and its application method and system in Helicobacter pylori killing, it is related to biological medicine technical field.The antibacterial peptide complex is a kind of intelligent precursor protein, and its amino acid sequence includes proton sensitive domain, pepsin cleavage domain and functional domain.The precursor protein is in locked conformation in neutral environment, conformation conversion occurs in gastric acid environment and exposes enzyme cutting site, is released and self-assembled into active antibacterial unit after specific cleavage by pepsin.The application further provides a preparation method for expressing the precursor protein using silkworm baculovirus system, an application method for activating and killing Helicobacter pylori under simulated gastric acid conditions, and a system comprising the foregoing method units.The application provides a new strategy for anti-Helicobacter pylori, which is low in cost, stable in gastric environment and can be precisely activated.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method and system for preparing an antimicrobial peptide complex and its application in the eradication of Helicobacter pylori. Background Technology

[0002] Helicobacter pylori infection is a common chronic bacterial infection worldwide, closely associated with gastritis, peptic ulcers, and gastric cancer. Currently, standard treatment relies on the combined use of proton pump inhibitors and multiple antibiotics. However, the increasing prevalence of antibiotic resistance, persistent treatment side effects, and high recurrence rates make the development of non-antibiotic therapies an urgent need. Antimicrobial peptides, due to their broad-spectrum antibacterial activity and low resistance-inducing properties, are considered a potential alternative. However, their use in treating gastric infections faces two major challenges: first, the highly acidic environment and abundant proteases in the stomach easily inactivate most antimicrobial peptides; second, overcoming the gastric mucosal barrier and achieving effective local concentrations often requires high-dose administration, while the high cost of producing antimicrobial peptides through chemical synthesis or traditional recombinant technologies limits their clinical application.

[0003] In the prior art, patent CN107573409A discloses a method for producing antimicrobial peptide fusion proteins using a silkworm expression system to reduce costs, and proposes the idea of ​​co-expressing enterokinase for cleavage. However, this method relies on the addition of enterokinase to cleave and release the active peptide in vitro or in vivo. The activity of enterokinase may be affected in the acidic environment of the stomach, and the cleaved active peptide may still be rapidly degraded by pepsin in the complex environment of the stomach. Another patent, CN117624299A, discloses a method to improve the antimicrobial activity of the original antimicrobial peptide by chemically modifying it (such as by linking fatty acid chains). Although this method improves the penetration and activity of the antimicrobial peptide, it relies on expensive solid-phase chemical synthesis, making it difficult to achieve the large-scale, low-cost production required for treating Helicobacter pylori infection. Furthermore, the stability and metabolic pathways of the chemically modified peptide in the gastric environment need further verification. Therefore, in response to the above problems, a method and system for preparing antimicrobial peptide complexes and their application in the eradication of Helicobacter pylori are proposed. Summary of the Invention

[0004] To overcome the aforementioned deficiencies of the prior art, embodiments of the present invention provide a method and system for preparing an antimicrobial peptide complex and its application in the eradication of Helicobacter pylori. The aim is to solve the problem of how to keep the antimicrobial peptide stable in the strong acid and high protease environment of the stomach and to enable it to be specifically activated and release its activity, while realizing the low-cost and large-scale production of the antimicrobial peptide complex, so as to overcome the problems of easy degradation of active peptides, high production costs, and dependence on complex post-processing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a Helicobacter pylori-disinfecting antimicrobial peptide complex.

[0006] The Helicobacter pylori-disinfecting and antimicrobial peptide complex is a fusion protein containing a proton-sensitive domain, an enzyme recognition domain, and a functional domain.

[0007] The proton-sensitive domain contains 7 to 12 consecutive histidine residues, which are used to respond to changes in environmental pH. The enzyme recognition domain contains the amino acid sequence Leu-Leu-Phe-Ala, serving as a specific cleavage site for pepsin.

[0008] The functional domain includes membrane-interacting peptides and anti-Helicobacter pylori antimicrobial peptides linked by a linker sequence.

[0009] The C-terminus of the proton-sensitive domain contains a first anchoring sequence consisting of three consecutive lysine residues, and the anti-Helicobacter pylori antimicrobial peptide contains a second anchoring sequence consisting of three consecutive aspartic acid residues at the position corresponding to the first anchoring sequence after spatial folding.

[0010] In a solution with pH 7.0 to 7.4, the first anchoring sequence and the second anchoring sequence bind through electrostatic interaction, causing the Leu-Leu-Phe-Ala sequence of the enzyme recognition domain to be buried inside the protein, thereby rendering it inactive. In solutions with pH 2.0 to 3.5, the electrostatic interactions are disrupted, and the Leu-Leu-Phe-Ala sequence is exposed on the protein surface, transforming into a pre-activated state that can be cleaved.

[0011] In one optional embodiment, a Pro-Gly-Pro tripeptide sequence is attached to each side of the enzyme cleavage recognition domain to form a flexible linkage structure; the C-terminus of the membrane interaction peptide contains the amino acid sequence Asn-Gln-Gly, and the N-terminus of the anti-Helicobacter pylori antimicrobial peptide contains the amino acid sequence Lys-Leu-Val. After the enzyme cleavage recognition domain is cleaved, the Asn-Gln-Gly sequence and the Lys-Leu-Val sequence can interact through hydrogen bonds to form an antiparallel β-sheet structure, thereby driving the recombination of the active fragment.

[0012] In another alternative embodiment, in the anti-Helicobacter pylori antimicrobial peptide fragment, the isoleucine residue at position 8 is replaced by a glutamic acid residue. This substitution helps to reduce the spontaneous membrane affinity of the antimicrobial peptide fragment when the fusion protein is in a neutral environment.

[0013] Secondly, the present invention provides a method for preparing the Helicobacter pylori-disinfecting antimicrobial peptide complex.

[0014] The preparation method includes the following steps: First, synthesize the nucleic acid sequence encoding the fusion protein and clone it into the multiple cloning site of the baculovirus transfer vector to construct a recombinant transfer plasmid containing the target gene.

[0015] Next, the recombinant transfer plasmid and the baculovirus backbone plasmid were co-transfected into insect cells to obtain a recombinant baculovirus through homologous recombination. Then, silkworm larvae or pupae were infected with the recombinant baculovirus, and protein expression was performed using a silkworm expression system. Finally, 96 to 120 hours post-infection, hemolymph from the silkworms was collected to obtain a Helicobacter pylori-disinfecting antimicrobial peptide complex solution containing the fusion protein.

[0016] The baculovirus transfer vector used in the steps can be selected from the pFastBac series vectors; the insect cells can be selected from Sf9 cells or BmN cells.

[0017] Thirdly, the present invention provides a method for eliminating Helicobacter pylori.

[0018] The disinfection method includes: mixing the aforementioned Helicobacter pylori disinfection antimicrobial peptide complex, or a solution containing the fusion protein obtained by its preparation method, with a pepsin solution with a pH of 2.0 to 3.5, and incubating at 37°C for 10 to 60 minutes to simulate a gastric acid environment to trigger conformational change of the fusion protein and complete enzymatic cleavage, forming an activation reaction solution; then, contacting the activation reaction solution with Helicobacter pylori to allow the released active antimicrobial components to exert a bactericidal effect.

[0019] In specific implementations, the concentration of the pepsin solution can be 0.05 to 0.5 mg / mL; the concentration of Helicobacter pylori can be 1 × 10^6 to 5 × 10^6 colony-forming units per mL. The contact process with Helicobacter pylori is preferably carried out under microaerophilic conditions, such as a mixed gas environment of 5% oxygen, 10% carbon dioxide, and 85% nitrogen.

[0020] Fourthly, the present invention provides a Helicobacter pylori eradication system.

[0021] The system includes: a preparation module configured to perform the aforementioned preparation method to produce the antimicrobial peptide complex; an activation module configured to convert the complex into an activation reaction solution by adjusting the pH and incubating it with pepsin; and a disinfection module configured to mix and contact the activation reaction solution with Helicobacter pylori under set conditions to complete the disinfection process and evaluate the effect.

[0022] Fifthly, the present invention provides a pharmaceutical composition.

[0023] The pharmaceutical composition comprises the aforementioned Helicobacter pylori-disinfecting antimicrobial peptide complex as the active ingredient, and pharmaceutically acceptable excipients. The excipients include lyophilization protectants for maintaining protein stability and enteric coating materials for achieving intestinal-specific release after oral administration.

[0024] The technical effects and advantages of this invention are as follows: The technical advantages of this invention are primarily reflected in providing a smart antimicrobial peptide precursor protein with gastric environment responsiveness. This precursor protein integrates a proton-sensitive domain, a specific cleavage domain, and a functional domain through a precisely designed amino acid sequence. Under near-neutral production or storage conditions, it maintains a locked conformation through intramolecular electrostatic interactions, shielding the cleavage sites and thus remaining stable and inactive. Upon entering the gastric acid environment, protonation disrupts the original electrostatic balance, triggering a conformational transition and exposing the specific cleavage sites of pepsin. Subsequently, the functional peptides released by pepsin cleavage self-assemble into the final active antimicrobial unit through pre-defined intermolecular interactions (such as the formation of specific secondary structures). This mechanism ensures that the active antimicrobial unit is generated only under the specific environment of the stomach, helping to prevent non-specific degradation by pepsin before reaching the site of action, thus improving targeting and stability.

[0025] Secondly, this invention provides a low-cost preparation method matching this intelligent precursor protein. This method utilizes a silkworm-baculovirus expression system as a production platform. The gene encoding the precursor protein is integrated into the baculovirus genome, and then silkworm larvae are infected with the recombinant virus. The silkworm, acting as a bioreactor, can efficiently express and secrete the precursor protein into its hemolymph. The collected hemolymph only requires simple filtration to obtain a crude extract containing the target precursor protein. The entire production process is based on feeding silkworms with inexpensive mulberry leaves or artificial feed, without the need for complex protein purification or in vitro chemical modification steps. This large-scale breeding and production model based on an insect expression system has potential advantages in reducing the production cost of antimicrobial peptide drugs compared to chemical synthesis or traditional microbial fermentation systems.

[0026] Finally, the final active antibacterial unit generated by the activation of the aforementioned intelligent precursor protein primarily operates by physically disrupting the bacterial cell membrane. This membrane perforation mechanism differs from the target sites of most antibiotics (such as inhibition of protein synthesis or cell wall synthesis), making it difficult for Helicobacter pylori to develop effective resistance through a single gene mutation. Therefore, the antimicrobial peptide complex provided by this invention helps to slow down or prevent the development of bacterial resistance in long-term or repeated use scenarios. Furthermore, because the activation of this precursor protein strictly depends on the unique low pH and pepsin-rich environment of the stomach, its active release is site-specific, providing a design safeguard to reduce potential impacts on the body's normal flora and host cells. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the entire process from preparation to application of the intelligent antimicrobial peptide complex of the present invention. Figure 2 This is a flowchart of the intelligent activation mechanism of the antimicrobial peptide complex of the present invention; Figure 3 This is a flowchart illustrating the preparation process of the silkworm-based bioreactor according to the present invention. Figure 4 This is a modular workflow diagram of the Helicobacter pylori eradication system of the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] As attached Figures 1 to 4 The preparation method and system of an antimicrobial peptide complex and its application in the eradication of Helicobacter pylori are shown below: In the following embodiments, unless otherwise stated, the following terms have the following meanings: "Lock-up conformation" refers to the state of fusion protein F in... In the solution, the protein is in a stable conformational state in which its enzyme recognition sites are shielded and it has no antibacterial activity.

[0030] "Activated conformation" refers to the state of fusion protein F in... In the solution, its enzyme recognition sites are exposed, and it is in a conformational state that can be cleaved by specific proteases.

[0031] "Active complex" refers to the final product with anti-Helicobacter pylori activity formed by recombination of the peptide released from the fusion protein F after specific cleavage by a protease.

[0032] The functional activation of the fusion protein F is designed as a cascade response mechanism sequentially coupled with gastric environmental signals. This process comprises three sequentially occurring stages: The first stage is triggered by a low pH signal provided by the gastric acid environment, which leads to conformational changes in protein molecules. In the second stage, the aforementioned conformational changes expose the pepsin-specific cleavage sites and cause hydrolysis. In the third stage, the functional peptides released after hydrolysis spontaneously recombine through pre-defined molecular interactions to form a final structure capable of membrane damage. This stepwise activation mechanism aims to target the antibacterial activity to a specific site with the appropriate enzymatic environment.

[0033] Example 1: Design and Sequence Synthesis of Antimicrobial Peptide Complex This embodiment specifically illustrates the Helicobacter pylori eradication and antimicrobial peptide complex, i.e., the fusion protein. Molecular design and nucleic acid synthesis that encodes its amino acid sequence.

[0034] 1. Amino acid sequence design of fusion proteins Target fusion protein For proton sensitive region Enzyme recognition domain and functional domain A single polypeptide chain formed by sequential covalent linkage.

[0035] Proton Sensitive Domain Design: From the core histidine sequence and the first anchoring sequence composition. It consists of 10 consecutive histidine residues, and its amino acid sequence is "HHHHHHHHHH". It consists of three consecutive lysine residues, and its amino acid sequence is "KKK". Therefore, The complete amino acid sequence is "HHHHHHHHHHKKK".

[0036] Enzyme recognition domain Design: By flexible connection sequence and core cleavage site composition. It consists of two "proline-glycine-proline" tripeptides, located in... Both sides. The amino acid sequence is "leucine-leucine-phenylalanine-alanine". Therefore, The complete amino acid sequence is “PGPLLFAPGP”.

[0037] Functional domain Design: Membrane-interacting peptides Connector sequence Second anchoring sequence Antimicrobial peptides against Helicobacter pylori They are connected sequentially to form a whole. The amino acid sequence is “GLFRILLFSVNQG”. The amino acid sequence is "GGGGSGGGGS". It consists of three consecutive aspartic acid residues, and its amino acid sequence is "DDD". Modification based on the known antimicrobial peptide sequence “GVLSNVIGYLKKLGTGALNAVLKQ”: The isoleucine residue at position 8 is replaced with a glutamic acid residue, and the sequence "lysine-leucine-valine" is added to its amino terminus. Therefore, The modified amino acid sequence is “KLVDDDGVLSNVEGYLKKLGTGALNAVLKQ”.

[0038] 2. Complete fusion protein Sequence and conformation verification Based on the above design, the fusion protein The complete amino acid sequence is: - - - - - , namely "HHHHHHHHHHKKKKPGPLLFAPGPGLFRILLFSVNQGGGGGSGGGGSKLVDDDGVLSNVEGYLKKLGTGALNAVLKQ".

[0039] To verify whether the protein possesses the designed pH-dependent conformational change and enzymatic cleavage characteristics, the following verification experiment was performed: The protein, prepared and purified using the E. coli recombinant expression system, was dissolved in phosphate buffer at pH 7.4 to prepare a solution with a concentration of [missing information]. Protein solution.

[0040] Take two portions of the above protein solution, each with a volume of [volume value missing]. Take the pH value of one portion and use... Adjust the hydrochloric acid to 2.5, and record this as the sample. The other sample was kept at pH 7.4 and designated as the final sample. .

[0041] To the sample and Pepsin solution was added to each sample to ensure that the final concentration of pepsin in each sample was the same. It is known that pepsin has higher activity at pH 2.5, while its activity decreases significantly at pH 7.4.

[0042] Place the two samples Incubation under the conditions .

[0043] After incubation, protein cleavage was detected using protein analysis methods such as high-performance liquid chromatography or mass spectrometry. In the sample... In (pH 7.4), intact protein was detected without significant degradation; in the sample At pH 2.5, the intact protein was effectively cleaved, and the expected cleavage product was detected. This result indicates that the protein can be effectively cleaved by pepsin at pH 2.5, while remaining stable at pH 7.4.

[0044] To assess the stability of the "lock-in conformation," the protein was placed in a buffer solution at pH 7.4. Pre-incubation Then, proceed with... The same pepsin treatment was applied. The cleavage pattern was similar to that of the unincubated sample. No significant difference was observed, indicating that the protein is conformationally stable at physiological pH.

[0045] The above experimental results were compared. The protein's cleavage sensitivity to the same protease under different pH conditions verified its conformational dependence on pH. The cleavage resistance at pH 7.4 and the efficient cleavage at pH 2.5 together indicate... The accessibility of protein cleavage sites is pH-regulated. Combined with the pre-defined pH response and cleavage elements in the protein's sequence, this result provides experimental evidence for the existence of "lock-in conformations" and "activated conformations."

[0046] 3. Synthesis of nucleic acid coding sequences The above The amino acid sequence of the protein was optimized based on the codons preferred by silkworm cells. A BamHI restriction endonuclease recognition sequence “GGATCC” was added to the 5' end of the optimized DNA coding sequence, and an XbaI restriction endonuclease recognition sequence “TCTAGA” was added to the 3' end. This DNA fragment was chemically synthesized and cloned into a universal sequencing vector. After verification by nucleotide sequencing, a plasmid was obtained, denoted as [insert plasmid name here]. .

[0047] Example 2: Preparation of antimicrobial peptide complex This embodiment describes in detail the antimicrobial peptide complex, i.e., the complex containing the fusion protein. The solution and its preparation method.

[0048] 1. Construction of recombinant transfer plasmids The purpose of this step is to encode... The protein gene was cloned into a baculovirus expression vector to construct an intermediate recombinant plasmid.

[0049] Main input materials: containing Gene plasmids Baculovirus transfer vector pFastBac1; BamHI and XbaI restriction endonucleases; T4 DNA ligase; Escherichia coli DH5α Competent cells.

[0050] Procedure: BamHI and XbaI were used to treat the plasmids respectively. The enzyme was double-digested with the vector pFastBac1. The resulting product was purified by agarose gel electrophoresis. Gene fragments and a linearized pFastBac1 vector backbone were obtained. The two were ligated using T4 DNA ligase, and the ligation product was transformed into *E. coli* DH5α. competent cells.

[0051] The transformed cells were coated onto a substrate containing... LB agar plates containing ampicillin. Incubate overnight. Pick single colonies and preliminarily screen positive clones using colony polymerase chain reaction (using M13 universal primers). After plasmid extraction, verify the correct recombinant transfer plasmid by BamHI / XbaI double digestion and DNA sequencing. The plasmid middle, The gene is under transcriptional control of the very late PH promoter of baculoviruses.

[0052] 2. Preparation of recombinant baculovirus genome The purpose of this step is to use site-specific transposition to... The gene was integrated from the transfer plasmid into the baculovirus shuttle genome.

[0053] Main input material: recombinant transfer plasmid Escherichia coli DH10Bac competent cells (the genome of this strain contains a baculovirus shuttle vector and an auxiliary plasmid that provides transposases).

[0054] Procedure: Plasmid Transformed into DH10Bac competent cells.

[0055] The conversion mixture was coated onto a surface containing... Kanamycin, Gentamicin, tetracycline, X-gal and On IPTG's LB board Culture in the dark White colonies were picked and inoculated into LB broth medium containing the appropriate antibiotic. A large baculovirus shuttle plasmid was extracted from the culture and denoted as [insert plasmid name here]. ,by Use as a template Gene-specific primers were used for polymerase chain reaction verification.

[0056] 3. Obtaining recombinant baculovirus The purpose of this step is to produce recombinant baculovirus particles in insect cells.

[0057] Main input material: recombinant baculovirus shuttle plasmid Sf9 insect cells; liposome transfection reagent.

[0058] Procedure: Culture Sf9 cells to an appropriate density in six-well plates. Using liposome transfection reagent, follow the manufacturer's recommended operating procedures to transfect the cells. The plasmid was transfected into Sf9 cells.

[0059] After transfection, at Continue to cultivate Collect the culture supernatant, and then... Centrifugation After removing cell debris, the resulting supernatant is the P1 generation virus stock solution containing recombinant baculovirus, denoted as [missing information]. It can be detected by the plaque assay method. Virus titer.

[0060] 4. Expression and complex collection in silkworms The purpose of this step is to use silkworm larvae as a live bioreactor to express and preliminarily purify the fusion protein in large quantities. .

[0061] Main input material: P1 generation recombinant baculovirus stock solution Healthy fifth-instar silkworm larvae; sterile phosphate buffer; containing ( Pre-cooled phosphate buffer (anticoagulant) for phenylthiourea.

[0062] Procedure: Dilute the virus stock solution appropriately with sterile phosphate buffer. Using a micro-syringe, approximately [amount missing] per silkworm larva was injected. Dilute the virus solution.

[0063] Place the injected silkworms in Raising them under suitable humidity conditions, after infection Silkworms were hypothermiac, their bodies disinfected, and their abdominal legs severed to collect hemolymph, which was then added directly to a pre-cooled anticoagulant. The mixed hemolymph was then... , Centrifugation Collect the supernatant, and then... Microporous membrane filters with small pore sizes are used for filtration and sterilization.

[0064] The resulting clear filtrate contains the fusion protein. The antimicrobial peptide complex solution is denoted as solution. The Bradford method was used to determine the solution. The total protein concentration can be determined by taking a portion of the solution. Western blot analysis of proteins was performed using antihistamine-tagged monoclonal antibodies to validate the results. Protein expression.

[0065] Example 3: Application of Helicobacter pylori eradication methods This embodiment specifically illustrates how to use the antimicrobial peptide complex prepared in Example 2 to evaluate the in vitro eradication of Helicobacter pylori.

[0066] 1. In vitro activation of antimicrobial peptide complex This step simulates the acidic environment of the stomach, allowing the fusion protein to... It undergoes a conformational change and is cleaved by enzymes to generate the active product.

[0067] Main input material: Antimicrobial peptide complex solution ; Hydrochloric acid; pepsin; glycine-hydrochloric acid buffer solution at pH 2.5.

[0068] Process: Take solution Add dropwise to the centrifuge tube. Add hydrochloric acid and monitor the pH with a pH meter until the solution pH stabilizes at 2.5. Add freshly prepared pepsin solution in pH 2.5 buffer to bring the final concentration of pepsin in the reaction system to [value missing]. Place the mixture in Incubate in a constant temperature water bath .

[0069] Immediately after incubation, place the reaction tube on ice, and record the resulting liquid as the activating solution. .

[0070] 2. In vitro eradication experiment of Helicobacter pylori This step evaluates the activator. Direct killing effect on Helicobacter pylori.

[0071] Main input material: Activation solution Helicobacter pylori standard strain ATCC43504; Brucella broth medium; containing Büssler agar plates of defibrinated sheep blood; mixed gas environment ( , , ).

[0072] process: a. In the mixed gas environment, Helicobacter pylori is placed... Under controlled conditions, the culture was revived and passaged to late logarithmic growth. The bacterial concentration was adjusted to approximately [missing value] using Brunner's broth. The working bacterial solution is then further diluted. To make its concentration approximately The concentration was calibrated using the plate coating method.

[0073] b. Set up experimental and control groups in 24-well cell culture plates. Add [amount] to each well of the experimental group. Activation solution and Working bacterial solution The control group added to each well Treated with the same pH adjustment and pepsin but without Protein buffer and Working bacterial solution .

[0074] c. Quickly transfer the 24-well plate into an incubator with the pre-equilibrated mixed gas environment, and... static culture .

[0075] After cultivation, samples were taken from each well in both the experimental and control groups and serially diluted tenfold with Bourg's broth. The appropriate dilution of the bacterial culture was then selected. Spread onto Buchner blood agar plates and incubate under the same gaseous conditions. The colony count was performed 3 days later.

[0076] Calculate the sterilization rate using the following formula. : ; In the formula, In order to activate the solution Average concentration of surviving Helicobacter pylori after co-culture ( ), The average concentration of surviving Helicobacter pylori after co-culturing with control buffer ( This experiment should be repeated independently three times, and the results should be taken as follows: The arithmetic mean is used as the final evaluation result.

[0077] Example 4: Operation of the disinfection system and preparation of the drug composition This embodiment illustrates the implementation scheme of the disinfection system and the final product pharmaceutical composition based on the aforementioned method.

[0078] 1. Operation of the disinfection system The Helicobacter pylori eradication system consists of three functionally connected units: Preparation Unit: This unit is configured to perform all the processes described in Example 2. Its inputs are silkworm larvae and a stock solution of recombinant baculovirus. After viral infection, feeding management, hemolymph collection and filtration, the output is an antimicrobial peptide complex solution. .

[0079] Activation Unit: This unit is configured to perform the "In Vitro Activation of Antimicrobial Peptide Complex" procedure in Example 3. Its input is a solution. After precise pH adjustment, pepsin addition, and incubation, the output is an activation solution. .

[0080] Disinfection Unit: This unit is configured to perform the "In Vitro Disinfection Experiment of Helicobacter pylori" procedure in Example 3. Its input is an activation solution. and standardized Helicobacter pylori working bacterial solution Co-culturing was carried out in a controlled mixed gas environment, and the sterilization rate was calculated and output by colony counting. This is used to evaluate the effectiveness of disinfection.

[0081] 2. Preparation of pharmaceutical compositions This section describes the antimicrobial peptide complex solution. Methods for converting to a stable oral dosage form.

[0082] Main input material: Antimicrobial peptide complex solution Mannitol; Trehalose; Enteric-coated gelatin empty capsule shell.

[0083] Process: Adding solution Mannitol and trehalose were added as freeze-drying protectants in a 2:1 mass ratio and stirred until completely dissolved. The mixture was then subjected to... The solution is then aseptically filtered through a membrane. The filtrate is subsequently dispensed into suitable containers and freeze-dried. The freeze-drying process typically includes pre-freezing, primary drying, and secondary drying stages, ultimately yielding a loose, freeze-dried powder.

[0084] In a humidity-controlled environment, a predetermined amount of lyophilized powder is filled into an enteric-coated gelatin capsule shell to obtain a pharmaceutical composition of oral enteric-coated capsule form. The enteric-coated capsule form can remain intact in the pH environment of the stomach and disintegrate and release the contents after delivery to the duodenum and subsequent intestinal sites.

[0085] This delivery strategy and fusion protein Activation logic adaptation: Capsule protection Proteins are protected from premature and excessive cleavage in the stomach; after entering the intestines, they are protected under suitable local pH and protease conditions. Proteins can be activated according to a designed mechanism, thereby exerting their effects at the target site.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Helicobacter pylori-disinfecting antimicrobial peptide complex, characterized in that, It is a fusion protein containing a proton-sensitive domain, an enzyme recognition domain, and a functional domain; The proton-sensitive domain comprises 7 to 12 histidine residues arranged in a continuous sequence; The enzyme recognition domain contains the amino acid sequence Leu-Leu-Phe-Ala; The functional domain includes membrane-interacting peptides and anti-Helicobacter pylori antimicrobial peptides linked by a linker sequence. The C-terminus of the proton-sensitive domain contains a first anchoring sequence consisting of three consecutive lysine residues, and the anti-Helicobacter pylori antimicrobial peptide contains a second anchoring sequence consisting of three consecutive aspartic acid residues at the position corresponding to the first anchoring sequence after spatial folding. In a solution with pH 7.0 to 7.4, the first anchoring sequence and the second anchoring sequence bind through electrostatic interaction, embedding the Leu-Leu-Phe-Ala sequence of the enzyme recognition domain inside the protein; in a solution with pH 2.0 to 3.5, the electrostatic interaction is disrupted, and the Leu-Leu-Phe-Ala sequence is exposed on the protein surface.

2. The Helicobacter pylori eradication and antimicrobial peptide complex according to claim 1, characterized in that, Each side of the enzyme cleavage recognition domain is connected to a Pro-Gly-Pro tripeptide sequence; the C-terminus of the membrane interaction peptide contains the amino acid sequence Asn-Gln-Gly, and the N-terminus of the anti-Helicobacter pylori antimicrobial peptide contains the amino acid sequence Lys-Leu-Val. After the enzyme cleavage recognition domain is cleaved, the Asn-Gln-Gly sequence and the Lys-Leu-Val sequence can form an antiparallel β-sheet structure through hydrogen bond interaction.

3. The Helicobacter pylori eradication and antimicrobial peptide complex according to claim 1 or 2, characterized in that, In the anti-Helicobacter pylori antimicrobial peptide fragment, the isoleucine residue at position 8 is replaced by a glutamic acid residue.

4. A method for preparing the Helicobacter pylori eradication and antimicrobial peptide complex according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Synthesize the nucleic acid sequence encoding the fusion protein and clone it into the multiple cloning site of the baculovirus transfer vector to construct a recombinant transfer plasmid; S2. The recombinant transfer plasmid and the baculovirus backbone plasmid are co-transfected into insect cells to obtain recombinant baculovirus; S3. Infect silkworm larvae or pupae with the recombinant baculovirus; S4. 96 to 120 hours after infection, collect the hemolymph of the silkworm to obtain a Helicobacter pylori-disinfecting and antimicrobial peptide complex solution containing the fusion protein.

5. The method for preparing the Helicobacter pylori-disinfecting antimicrobial peptide complex according to claim 4, characterized in that, In step S1, the baculovirus transfer vector is a pFastBac series vector; in step S2, the insect cells are Sf9 cells or BmN cells.

6. A method for eliminating Helicobacter pylori, characterized in that, include: The Helicobacter pylori eradication antimicrobial peptide complex according to any one of claims 1 to 3, or the solution containing the fusion protein prepared according to the method of claim 4 or 5, is mixed with a pepsin solution with a pH of 2.0 to 3.5 and incubated at 37°C for 10 to 60 minutes to form an activation reaction solution; the activation reaction solution is then contacted with Helicobacter pylori.

7. The method for eliminating Helicobacter pylori according to claim 6 is characterized in that, The concentration of the pepsin solution is 0.05 to 0.5 mg / mL; the concentration of Helicobacter pylori is 1 × 10^6 to 5 × 10^6 colony-forming units per mL.

8. The method for eradicating Helicobacter pylori according to claim 6 or 7, characterized in that, The contact with Helicobacter pylori was carried out under microaerophilic conditions, which were a mixed gas environment of 5% oxygen, 10% carbon dioxide and 85% nitrogen.

9. A Helicobacter pylori eradication system, characterized in that, include: A preparation module, configured to perform the preparation method according to claim 4 or 5; An activation module configured to perform the steps of claim 6 or 7 to form the activation reaction solution; The disinfection module is configured to perform the steps of claim 6 or 8 to bring the activation reaction solution into contact with Helicobacter pylori and complete the disinfection.

10. A pharmaceutical composition, characterized in that, The product comprises a Helicobacter pylori-disinfecting antimicrobial peptide complex according to any one of claims 1 to 3, and a pharmaceutically acceptable lyophilization protectant and / or enteric coating material.

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