A kind of antigen for preparing anti-helicobacter pylori and its preparation method and application

Abstract

The application discloses an antigen for resisting helicobacter pylori and a preparation method and application thereof, and belongs to the technical field of resisting helicobacter pylori infection. The bivalent antigen combination provided by the application can ensure broad spectrum coverage of the antigen, can avoid epitope competition and non-specific immune consumption among the antigens, and has low production cost; the vaccine provided by the application can induce strong humoral immune response without an adjuvant, can express endogenous antigens in a chicken body, and can simulate a natural infection process; the antibody preparation process using the antigen provided by the application is simple, has a short cycle, has better batch consistency, is easy to standardize and enlarge production, has a clear immune cycle, and has high antibody titer; the helicobacter pylori bivalent IgY product provided by the application can be directly made into an oral preparation (such as egg yolk powder, microcapsules, tablets and the like), is used for preventing or assisting in treating helicobacter pylori infection, is especially suitable for children, pregnant women and antibiotic-resistant patients, and has wide market application value.

Description

Technical Field

[0001] This invention relates to the field of anti-Helicobacter pylori infection technology, and in particular to an antigen for preparing anti-Helicobacter pylori, its preparation method and application. Background Technology

[0002] Helicobacter pylori is a spiral-shaped, microaerophilic, Gram-negative bacillus with extremely demanding growth requirements. It is the only known microorganism capable of surviving in the human stomach. Diseases caused by Helicobacter pylori infection include gastritis, peptic ulcers, and gastric lymphoma. A poor prognosis for Helicobacter pylori infection is gastric cancer.

[0003] Helicobacter pylori is a major pathogenic bacterium that colonizes the human gastric mucosa and is closely related to the occurrence of chronic gastritis, peptic ulcers, and gastric cancer. Currently, combination antibiotic therapy (such as triple and quadruple therapy) is mainly used clinically, but drug resistance rates are increasing year by year, and gastrointestinal adverse reactions are also observed. In recent years, passive immunization using specific egg yolk antibodies (IgY) has become a new strategy for combating Helicobacter pylori infection. IgY is a polyclonal antibody extracted from the egg yolk of immunized laying hens, and it has advantages such as high yield, convenient collection, and no animal ethics controversies.

[0004] Existing technical solutions for preparing anti-Helicobacter pylori IgY products typically include the following steps: Antigen preparation. Most methods use whole-cell inactivated Helicobacter pylori antigen or recombinant proteins (such as urease and adhesin) expressed through genetic engineering as immunogens. The genes are cloned into prokaryotic expression vectors, transformed into E. coli for protein expression, purified by nickel ion affinity chromatography, and then lyophilized for preservation.

[0005] Emulsification and Immunization. The purified protein antigen is emulsified with Freund's complete or incomplete adjuvant and administered to healthy laying hens aged 10-40 weeks via subcutaneous or intramuscular injection at multiple sites. The immunization dose is typically 10-120 micrograms of protein per hen, with booster immunizations every two weeks after the initial immunization, for a total of 2-4 immunizations.

[0006] Antibody collection and detection. Egg yolks from immunized hens were collected, and IgY was extracted using water dilution, salt precipitation, or chromatography. The antibody titer was then determined using an indirect ELISA method.

[0007] However, existing technical solutions have the following defects and shortcomings: First, antigen screening lacks systematicity and optimization. Current technologies often rely on empirically listing multiple known virulence factors (e.g., using five antigens simultaneously) or directly using whole-bacterial inactivated antigens. Whole-bacterial antigens contain numerous non-protective or immunosuppressive components, easily inducing non-specific immune responses and reducing the proportion of specific antibodies. While multi-antigen complexes (e.g., pentavalent mixtures) cover multiple targets, epitope competition and immune interference between different antigens may weaken the immune response to key protective epitopes. Furthermore, multi-antigen approaches significantly increase production costs, purification difficulty, and quality control complexity.

[0008] Second, there are significant drawbacks to the immunogen delivery methods. Current technologies generally use recombinant proteins expressed in prokaryotes as immunogens, which must be emulsified with Freund's adjuvant to effectively induce an immune response. However, complete Freund's adjuvant can cause severe granulomas, sterile abscesses, and even ulceration at the injection site, affecting not only the welfare of laying hens but also potentially leading to a decrease in egg production. While incomplete Freund's adjuvant reduces stimulation, its immune-enhancing effect is limited. The protein antigens themselves are easily degraded in vivo, requiring repeated booster immunizations to maintain high antibody levels. This not only prolongs the immunization cycle but also increases labor costs and stress on the chickens. Summary of the Invention

[0009] The purpose of this invention is to provide an antigen for preparing anti-Helicobacter pylori, its preparation method, and its application, in order to solve the problems of existing antigens containing a large number of non-protective or immunosuppressive components, which easily induce non-specific immune responses; epitope competition and immune interference; the need to use Freund's adjuvant to effectively induce immune responses, affecting laying hen welfare and egg production rate; and the high cost, complicated process, difficult purification, long cycle, and difficult quality control in the preparation of specific egg yolk antibody IgY.

[0010] To achieve the above objectives, the present invention provides an antigen for preparing anti-Helicobacter pylori, said antigen being composed of coding sequences of UreB and HpaA.

[0011] Preferably, the coding sequences of UreB and HpaA are further fused with the coding sequence of a chicken-derived leader peptide at their 5' ends.

[0012] Preferably, the coding sequence of the chicken-derived leader peptide is shown in SEQ ID NO.8; the coding sequence of UreB is shown in SEQ ID NO.5; and the coding sequence of HpaA is shown in SEQ ID NO.6.

[0013] The method for preparing the above-mentioned antigen for preparing Helicobacter pylori comprises the following steps: S1. Codon optimization of the amino acid sequences of UreB and HpaA, adding chicken-derived leader peptide to the front of the optimized codon, sending the chicken-derived leader peptide + optimized codon sequence to the company for sequence synthesis, linking the synthesized sequence with the cloning vector respectively, and verifying the correct recombinant vector to be transformed into engineered bacteria. S2. After the received engineered bacteria are propagated, plasmids are extracted, linearized, transcribed in vitro, and capped and tailed. After purification, the antigen used to prepare anti-Helicobacter pylori is obtained.

[0014] Preferably, the optimized coding sequence of the UreB codon in S1 is shown in SEQ ID NO.5, and the optimized coding sequence of the HpaA codon is shown in SEQ ID NO.6.

[0015] Preferably, the cloning vector in S1 is pUC57, pUC19, or pBluescript SK(+); the cloning vector contains restriction enzyme sites, Kozak sequences, or transcription terminators.

[0016] Preferably, the sequences synthesized in S1 are respectively linked with the cloning vector to form two recombinant vectors, or the two sequences synthesized in S1 are linked with the same vector to form one recombinant vector.

[0017] The application of the antigen used to prepare anti-Helicobacter pylori as described above in the preparation of chicken anti-Helicobacter pylori vaccine, wherein the vaccine is a naked mRNA solution or mRNA encapsulated with lipid nanoparticles.

[0018] A specific egg yolk antibody IgY against Helicobacter pylori, wherein the specific egg yolk antibody IgY against Helicobacter pylori is prepared by immunizing laying hens with the antigens used to prepare anti-Helicobacter pylori antibodies as described above.

[0019] The application of the anti-Helicobacter pylori specific egg yolk antibody IgY as described above in the preparation of oral formulations for the prevention or adjuvant treatment of Helicobacter pylori infection.

[0020] Therefore, the present invention provides an antigen for preparing anti-Helicobacter pylori, a method for preparing the antigen, and its application, the specific technical effects of which are as follows: (1) The antigens used are immunogenic and redundancy and interference can be avoided. The bivalent antigen combination provided by the present invention abandons the practice of empirically using five or more antigens in the prior art. The bivalent combination not only ensures broad-spectrum coverage (targeting different pathogenic links), but also avoids epitope competition and non-specific immune consumption among multiple antigens, reducing production costs and quality control difficulties.

[0021] (2) Advanced delivery method, no harm to chickens. This invention uses mRNA vaccine to replace the traditional protein antigen + Freund's adjuvant regimen. mRNA expresses endogenous antigens in chickens, mimicking the natural infection process, and can induce a strong humoral immune response without adjuvants. It completely avoids the problems of injection site granuloma, sterile abscess and decreased egg production caused by Freund's adjuvant, and meets animal welfare requirements.

[0022] (3) The preparation process is simple and the cycle is short. The present invention only requires five steps from antigen synthesis to mRNA acquisition: gene synthesis, plasmid extraction, linearization, in vitro transcription and purification. Compared with the cumbersome steps of prokaryotic expression, bacterial cell lysis, affinity chromatography and renaturation in the prior art, the operation process is greatly simplified, the batch consistency is better, and it is easy to standardize and scale up production.

[0023] (4) Clear immunization cycle and high antibody titer. This invention provides complete immunization cycle data (antibody growth curves on days 21, 28, 34, and 42 post-immunization) through systematic time-point sampling and dynamic ELISA monitoring, clarifying the optimal immunization dose and immunization site. Experimental data show that after immunization with the bivalent mRNA vaccine provided by this invention, the specific IgY titer in egg yolk can reach over 1:160,000, significantly higher than the 1:6400~1:12800 reported in the prior art.

[0024] (5) It has good prospects for industrialization and application. The anti-Helicobacter pylori bivalent IgY product provided by this invention can be directly made into oral preparations (such as egg yolk powder, microcapsules, lozenges, etc.) for the prevention or adjuvant treatment of Helicobacter pylori infection. It is especially suitable for children, pregnant women and antibiotic-resistant patients, and has broad market application value.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is the SDS-PAGE result of IgY prepared in Example 4 of this invention; Figure 2 These are the experimental results of drug administration at different sites in Example 4 of the present invention; Figure 3 These are the experimental results of different drug dosages in Example 4 of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0031] Example 1 The specific steps for synthesizing the coding sequences of UreB and HpaA are as follows: (1) Perform codon optimization.

[0032] Codon optimization was performed on the amino acid sequences of UreB (WP_000732218.1 urease subunit beta [Helicobacter pylori], amino acid sequence as shown in SEQ ID NO.1, corresponding nucleotide sequence in NCBI as shown in SEQ ID NO.3) and HpaA (WP_000389035.1 HpaA family protein [Helicobacter pylori], amino acid sequence as shown in SEQ ID NO.2, corresponding nucleotide sequence in NCBI as shown in SEQ ID NO.4).

[0033] Without altering the amino acid sequence, reverse translation and synonym substitution optimization were performed independently based on the codon usage frequency table of chicken (Gallus gallus) to eliminate rare codons used infrequently in chickens. Optimization principles included: selecting synonymous codons corresponding to high-abundance tRNAs in chickens; avoiding consecutive occurrences of rare codons; increasing the CAI (codon fit index) to above 0.8; and controlling the sequence GC content within the range of 50%-60%.

[0034] The optimized encoding sequence of UreB is shown in SEQ ID NO.5, with GC=54.11% and CAI=0.93. The optimized encoding sequence of HpaA is shown in SEQ ID NO.6, with GC=48.53% and CAI=0.93.

[0035] SEQ ID NO.1: MKKLDYVNTYGPTKGDKVRLGDTDLWAEVEHDYTIYGEELKFGAGKTIRECMGQSNSHDENTLDLVITNALIIDYTGIYKADIGIKNGKIAGIGKAGNKDMQDGVSPNLVVGVGTEALAGEGMIVTAGGIDSHTHFLSPQQFPTALANGVTTMFGGGTGPVDGTNATTITPGEWNIHRMLRAAEEYAMNVGFLGKGNSSSKTQLVEQIEAGIVGFKLHEDWGTTPSAIDTCLSVADEYDVQVCIHTDTVNEAGYVEDTLNAMNGRAIHAYHIEGAGGGHSPDV ITMAGEENILPSSTTPITIPYTINTVAEHLDMLMTCHLDKKIREDLQFSQSRIRPGSIAAEDVLHDNGMIAMTSSDSQAIGRAGEVVPRTWQTADKNKKEFGPLKEYDQNGNDFRIKRYISKYTINPAITHGVSEYIGSVEAGKIADLVVWNPAFFGVKPKIIIKGLVVFSEMGDSNASVTPQPVYYREMFGHGGAKFDTSITFVNKLAYEKGIKEKLGLERQVLPIKNVRNITKKDFKFNNTIGKLTVDPKTFEVFLDGKLCTSKPASELPLAQRYTFF SEQ ID NO.2: MECSFIFKKVRVYSKMLVALGLSSVLIGCAMNPSAETKKPNDAKNQQPVQTHERIQTSSEHVTPLDFNYPVHIVQAPQNHHVVGILMPRIQVSDNLKPYIDKFQDALANQIQTIFEKRGYQVLRFQDEKALSEQDKKKIFS VLDLKGWVGILEDLKMNLKDPNSPNLDTLVDQSSGSVWFNFYEPESNRVVHDFAVEVGTFQAITYTYTSTNNASGGFNSSKNVIHENLDKNREDAIHKILNRMYAVVMKKAVTELTKENIAKYRDAIDRMKGFKSSMPQKK SEQ ID NO.3: SEQ ID NO.4: CTACTTTTTTTGAGGCATAGAACTTTTAAAGCCTTTCATTCTATCAATAGCGTCTCTGTATTTGGCGATATTTTCTTTTGTAAGTTCTGTGTTACAGCTTTTTTCATGACAACCGCATACATTCTGTTTAAAATCTTGTGTATCGCGTCTTCTCTATTCTTATCCAAATTTTCATGGATAACGTTTTTTGAAGAATTAAACCCTCCTGAAGCGTTATTAGTAGAAGTGTATGTGTATGTTATTGCTTGAAAAGTTCCTACTTCCACAGCAAAATCATGGACGACACGATTGCTTTCTGGCTCATAAAAATTAAACCACACAGAGCCTGAGCTTTGATCCACTAGCGTGTCTAAATTGGGACTATTGGGATCTTTTTAAATTCATTTTCAAATCTTCTAAGATTCCTACCCACCCTTTCAAATCCAAA ACGGAAAAAATCTTTTTCTTATCTGCTCGCTCAAAGCCTTTTCATCTTGAAAACGCAACACTTGATAGCCTCTTTTTTCAAAAATAGTCTGGATTTGATTGGCTAAAGCGTCTTGAAACTTATCAATATAGGGTTTTAGATTATCGCTCACTTGAATGCGTGGCATTAAAATACCTACAACATGATGGTTTTGTGGGGCTTGAACAATATGCACCGGGTAATTAAAATCTAGTGGCGTAACATGTTCAGAGCTTGTTTGTATTCTTTCATGAGTTTGAACTGGTTGTTGGTTTTTGGCGTCATTTGGTTTTTTTGTCTCAGCGCTTGGATTCATCGCGCAACCGATCAACACGCTTGAAAGCCCTAAAGCCACCAACATTTTAGAATAAACCCTAACTTTTTTAAAAATAAACGAACACTCCAT SEQ ID NO.5: SEQ ID NO. 6: CTGCTGTTCCTGAGACACAGGACGTTCAAGGCTTTTCACAGCATCAACTCCGTGTCAGTGTTCGGCGATATTTTCTTCTGCAAGTTTTGCTACAGCTTTTTCCACGACAACAGGATCCACTCTGTGTAAAACCTGGTGTATCGGGTGTTCTCCATCCTGATCCAGATCTTTATGGACAACGTGTTCTGAAGGATCAAGCCAAGCTGATCCGTGATCAGCCGCTCTGTGTGCGTGTGCTACTGCCTGAAATCTTCATACTTCCACAGTAAGATCATGGATGACACGATCGCTTTCTGGCTGATCAAGATTAAGCCACACAGGGCCTGAGCTCTGATCCATTGAAGGGTGTAAATCGGCACAATCGGGATCTTCTGAATACACTTCCAGATCTTTTGAGATAGTTACCCACCATTCCAGATCCAGAACGGAAAGAACCTGTTTCTGATCCTGCTGGCTCAGAGCCTGTTCATCCTGAAGACTCAGCACCTGATCGCCTCCTTTTTCAAGAACTCCCTGGACCTGATTGGCTGATCCGTGCTGAAACTGATCAACATCGGCTTTTGAATCATTGCTCACCTGAACGCTTGGCACTGAAATACCTACAACATGATGGTGCTGTGGGGACTGAACAATATGCATAGAGTGATCAAGATCTAATGGCGGAACATGTTCAGGGCTTGCCTGTACAGCTTCATGAGCCTGAACTGGCTGCTGGTGTTCGGCGTGATCTGGTTCTTCTGCCTGTCAGCTTGGATCCACAGGGCAACAGACCAGCATGCATAGAAACCCTGATCACACCAGCACTTCAGGATCAACCCTAACTTTTTCAAGAACAAGCGCACCCTGCAC (2) Adding a chicken leader peptide.

[0036] The coding sequence of a chicken-derived leader peptide (amino acid sequence as shown in SEQ ID NO. 7) was added to the 5' end of the optimized coding sequence (sequence as shown in SEQ ID NO. 8, located in D74, GenBank accession number of D74: >NP_001001613.1, amino acid sequence as shown in SEQ ID NO. 9) to promote the secretory expression of the antigen protein in chicken cells.

[0037] SEQ ID NO.7: MAEEQRDLISSDGSSGVLPIGNSERS SEQ ID NO.8: ATGGCAGAAGAACAGCGAGACCTCATCAGCAGCGACGGCAGCAGCGGTGTGCTCCCAATCGGCAACAGCGAGAGAAGC SEQ ID NO.9: MAEEQRDLISSDGGSSGVLPIGNSERSSLGRRTALSALSILVALLIAGQAVTIYYVYQQSGQISKLTKTSQTLKLESLQRKMPIGTQPANKMSMSTMNMPMAMKVLPLAPSVGDMPMEAMKPRSNKTEDQIRHLLLKSDPRKTF PDLKDDMLGNLKRLKKTMSAMDWQDFETWMHKWLLFEMAKGPKMEEQNTIPAEKVQTKCQAEASFGGVHPGRFRPECDENGDYLPKQCYASTGYCWCCYKNGTRIEGTATRGQLDCSAPAPTQPPSAEPEEVIFSGVDMVKAK (3) Gene synthesis.

[0038] The coding sequences of UreB and HpaA designed in step (2) were sent to the company for whole-gene synthesis. Then, they were cloned into the modified pUC57 vector (the target sequence is inserted into the T7 promoter and contains a single BspQ1 restriction endonuclease recognition sequence, as shown in SEQ ID NO.10) to obtain two recombinant vectors. The two recombinant vectors were transformed into Escherichia coli DH5α competent cells. After the sequencing verification was correct, glycerol bacteria were prepared and stored at -80℃.

[0039] SEQ ID NO.10: GCTCTTC Example 2 The specific steps for synthesizing mRNA-LNP are as follows: (1) A small amount of bacterial culture was picked from the glycerol bacteria obtained in Example 1 (containing recombinant plasmids pUC57-HP-UreB and pUC57-HP-HpaA, respectively) and inoculated into LB liquid medium containing 100 μg / mL kanamycin (components: 10 g / L soybean peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar). The medium was cultured at 37°C and 200 rpm for 12 hours with shaking. The recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit. The concentration and A260 / A280 ratio of the obtained samples were determined (should be 1.8~2.0), and the plasmid conformation was confirmed by agarose gel electrophoresis.

[0040] (2) Linearize the recombinant plasmid obtained in step (1) by restriction endonuclease BspQ1. The digestion system and digestion conditions are carried out according to the instructions attached to the enzyme. The obtained reaction solution is concentrated and replaced in a 100KD ultrafiltration tube (to remove ions and other impurities in the reaction system). The obtained linear template is stored in nuclease-free water.

[0041] (3) Preparation of co-transcribed capped and tailed mRNA.

[0042] S31. In vitro transcription (IVT) reaction: Set up independent transcription reaction systems for each of the two linearized DNA templates. After gentle mixing, react in a 37°C water bath for 2-3 hours.

[0043] Each reaction system (20 μL) contains: 4 μL of 5× transcription buffer, 1.5 μL of NTP (1.5 μL each of ATP, CTP, GTP and N1-methylpseudouridine triphosphate), 1.2 μL of cap analog, 1 μg of linearized DNA template, 0.8 μL of T7 RNA polymerase, 0.5 μL of ribonuclease inhibitor and 1 μL of inorganic pyrophosphatase.

[0044] S32, DNase digestion: After the in vitro transcription reaction in step S31 is completed, add 2 μL of DNase I (RNase-free) to each system and incubate at 37°C for 15 minutes to degrade the DNA template. Then, add 2 μL of 0.5 MEDTA to each reaction system to terminate the reaction.

[0045] (4) mRNA purification and sample processing.

[0046] S41. Column Preparation: Use an OligodT affinity chromatography column. Pre-equilibrate with a high-salt binding buffer (0.5M NaCl, 10mM Tris-HCl, 1mM EDTA, pH 7.5) before loading the sample to ensure column stability and easy binding of the Poly(A) tail to OligodT.

[0047] Sample loading: Load the sample onto the chromatography column at a relatively low flow rate (5 mL / min). Complete mRNA containing a Poly(A) tail will specifically bind to the OligodT ligand on the column through base complementarity pairing, while impurities such as RNA fragments lacking a Poly(A) tail, DNA, proteins, and unbound nucleotides will flow through directly.

[0048] S42. Washing: After loading the sample, use 3 column volumes (CV) of high-salt binding buffer to wash at a flow rate of 10 mL / min to further and thoroughly wash away non-specifically bound impurities (unbound RNA fragments, proteins, and free nucleotides).

[0049] S43, Elution: Elute with enzyme-free water at a flow rate of 6 mL / min. The low ionic strength environment disrupts the hydrogen bond between the Poly(A) tail and OligodT, thereby specifically eluting high-purity mRNA. Prepare a 50 mL centrifuge tube, observe the computer-generated chromatogram, and when the peak is about to emerge, insert the collector into the 50 mL centrifuge tube to collect the elution peak.

[0050] S44. Column regeneration and storage: After elution, rinse the column with high-salt binding buffer to regenerate it, and then store it at room temperature.

[0051] S45. Dilute the obtained mRNA sample to 150-190 ng / μL and use microfluidics to synthesize mRNA-LNP solution (final concentration after concentration is 200 ng / μL) for later use.

[0052] Example 3 The specific steps for immunizing laying hens and collecting samples are as follows: (1) Experimental animals and grouping.

[0053] Healthy Hy-Line Brown laying hens (weighing 1.5 ± 0.2 kg) aged 23 weeks were selected and isolated for one week to acclimatize to the environment. They were then immunized with the mRNA-LNP prepared in Example 2 according to the following groups, with 3 hens in each group.

[0054] Different injection sites: The groups were divided into two groups with different injection sites for comparison. Group 1: Intramuscular injection on the lateral leg, administered once on day 0. Group 2: Subcutaneous injection in the neck, administered once on day 0. Group 3: Intramuscular injection in the chest, administered once on day 0. The dosage for all groups was 50 μg (i.e., 250 μL of the mRNA-LNP prepared in Example 2).

[0055] Different dosage groups: Group 4, subcutaneous injection in the neck, on day 0, 10 μg (i.e., 50 μL of mRNA-LNP prepared in Example 2), 50 μg (i.e., 250 μL of mRNA-LNP prepared in Example 2), and 120 μg (i.e., 600 μL of mRNA-LNP prepared in Example 2) were injected respectively.

[0056] (2) Immunization procedures.

[0057] The mRNA-LNP samples obtained after purification and aliquoting in Example 2 were injected into different sites and at different doses, including the lateral leg muscles, subcutaneous neck muscles, or chest muscles, using a 1mL syringe with a 26G needle, according to the grouping and dosage described above.

[0058] (3) Sample collection.

[0059] Collect eggs laid that day at the following times and mark them accordingly: Different sites of administration: Eggs from each group were collected on days 21, 28, 34 and 42 after administration.

[0060] Different dosage groups: Eggs from each group were collected on days 21, 28, 34 and 42 after administration.

[0061] Example 4 The samples taken in Example 3 were subjected to ELISA detection. The specific steps are as follows: (1) Extraction of egg yolk antibodies (water dilution method).

[0062] IgY was extracted using the water dilution method, and the specific steps are as follows: Break the egg, separate the yolk from the egg white, blot the yolk membrane dry with sterile filter paper, puncture the membrane, and collect the yolk fluid. Take 1 mL of yolk fluid, add 9 mL of pre-cooled sterile distilled water, and vortex vigorously for 1 minute. Adjust the pH to 5.0-5.2 with 0.1 M HCl, and incubate overnight at 4°C. Centrifuge at 12,000 × g for 20 minutes at 4°C, and collect the supernatant, which is the water-soluble component (containing IgY). Filter the supernatant through a 0.45 μm filter membrane, precipitate with 50% saturated ammonium sulfate, and reconstitute the precipitate with PBS. Determine the IgY concentration (9 mg / mL) using a UV spectrophotometer, and analyze the purity using SDS-PAGE.

[0063] The results of IgY SDS-PAGE are as follows: Figure 1 As shown, a heavy chain of approximately 67 kDa and a light chain of approximately 25 kDa can be observed.

[0064] (2) Double antigen coated ELISA.

[0065] The specific IgY titers against two antigens (the recombinant antigen protein was purchased from Wuhan Huamei Biotechnology Co., Ltd.) were detected using a double-antigen-coated ELISA. The specific steps are as follows: S41. Coating: Dilute each antigen to 2 μg / mL with carbonate buffer (pH 9.6), add 100 μL to each well of a 96-well microplate, and coat overnight at 4°C. Coat each antigen independently on one plate, and detect the antibody titer against antigen A and antigen B separately.

[0066] S42. Blocking: Discard the coating solution, add 5% skim milk powder-PBST (PBS containing 0.05% Tween-20), and block at 37°C for 2 hours.

[0067] S43. Primary antibody incubation: Serially dilute the IgY sample to be tested obtained in step (1) starting from 1:100 (e.g., 1:100, 1:200, 1:400...1:51200), 100 μL per well, and incubate at 37°C for 1 hour. At the same time, set up a blank control group (unimmunized chicken IgY) and a positive control group (known positive serum or standard).

[0068] S44. Secondary antibody incubation: Discard the primary antibody and wash the plate 5 times with PBST. Add 100 μL of HRP-labeled rabbit anti-chicken IgY secondary antibody (1:5000 dilution) to each well and incubate at 37°C for 1 hour.

[0069] S45. Development and Termination: Wash the plate 5 times, add 100 μL of TMB development solution to each well, and develop at room temperature in the dark for 10-15 minutes. Add 50 μL of 2M H2SO4 stop solution to each well.

[0070] The absorbance (OD) at a wavelength of 450 nm was measured using an ELISA reader. 450 Potency determination: based on sample OD. 450 Value / Negative Control OD 450 The maximum dilution factor with a value ≥ 2.1 is taken as the antibody titer of the sample.

[0071] (3) The representative test results obtained from the actual experiment are as follows. The final titer is summarized here, and the unit is expressed as a dilution of 10,000 times.

[0072] ① Different administration sites (50μg, n=3, time points 21, 28, 34, 42 days): On day 21 post-immunization, the serum antibody titers of the three chickens in the lateral leg intramuscular injection group were 55,000, 58,000, and 57,000, respectively, with a mean of 56,700 and a standard deviation of 1,500; the titers of the three chickens in the neck subcutaneous injection group were 95,000, 100,000, and 98,000, respectively, with a mean of 97,700 and a standard deviation of 2,500; and the titers of the three chickens in the breast intramuscular injection group were 48,000, 50,000, and 49,000, respectively, with a mean of 49,000 and a standard deviation of 1,000.

[0073] On day 28 post-immunization, the titers in the lateral leg intramuscular injection group were 59,000, 61,000, and 60,000, with a mean of 60,000 and a standard deviation of 1,000; the titers in the neck subcutaneous injection group were 102,000, 105,000, and 103,000, with a mean of 103,300 and a standard deviation of 1,500; and the titers in the chest intramuscular injection group were 51,000, 53,000, and 52,000, with a mean of 52,000 and a standard deviation of 1,000.

[0074] On day 34 post-immunization, the potencies in the lateral leg intramuscular injection group were 60,000, 63,000, and 61,000, respectively, with a mean of 61,300 and a standard deviation of 1,500; the potencies in the neck subcutaneous injection group were 105,000, 108,000, and 106,000, respectively, with a mean of 106,300 and a standard deviation of 1,500; and the potencies in the chest intramuscular injection group were 52,000, 54,000, and 53,000, respectively, with a mean of 53,000 and a standard deviation of 1,000.

[0075] On day 42 post-immunization, the titers in the lateral leg intramuscular injection group were 61,000, 64,000, and 62,000, respectively, with a mean of 62,300 and a standard deviation of 1,500; the titers in the neck subcutaneous injection group were 108,000, 110,000, and 109,000, respectively, with a mean of 109,000 and a standard deviation of 1,000; and the titers in the chest intramuscular injection group were 53,000, 55,000, and 54,000, respectively, with a mean of 54,000 and a standard deviation of 1,000.

[0076] ② Different dosage groups (subcutaneous injection in the neck, 10μg, 50μg, 120μg, n=3, time points 21, 28, 34, 42 days).

[0077] In the 10μg dosage group, the antibody titers of the three chickens were 0.9,000, 1.0,000, and 1.1,000 on day 21 post-immunization, with a mean of 1.00 million and a standard deviation of 0.10 million; on day 28, the titers were 1.0,000, 1.1,000, and 1.2,000, with a mean of 1.10 million and a standard deviation of 0.10 million; on day 34, the titers were 1.1, 1.2,000, and 1.3,000, with a mean of 1.20 million and a standard deviation of 0.10 million; and on day 42, the titers were 1.2, 1.3,000, and 1.4,000, with a mean of 1.30 million and a standard deviation of 0.10 million. It can be seen that the titers in this dosage group remained at a low level, with a slight increase over time.

[0078] In the 50μg dose group, the antibody titers of the three chickens were 75,000, 78,000, and 77,000 on day 21 post-immunization, with a mean of 76,700 and a standard deviation of 0.15 million; on day 28, the titers were 80,000, 82,000, and 81,000, with a mean of 81,000 and a standard deviation of 0.10 million; on day 34, the titers were 83,000, 85,000, and 84,000, with a mean of 84,000 and a standard deviation of 0.10 million; and on day 42, the titers were 85,000, 87,000, and 86,000, with a mean of 86,000 and a standard deviation of 0.10 million. The titers in this dose group were significantly higher than those in the 10μg group, and showed a slow upward trend over time.

[0079] The 120 μg dose group showed the following antibody titers in the three chickens on day 21 post-immunization: 140,000, 145,000, and 142,000, with a mean of 142,300 and a standard deviation of 0.25; on day 28, the titers were 150,000, 155,000, and 153,000, with a mean of 152,700 and a standard deviation of 0.25; on day 34, the titers were 155,000, 160,000, and 158,000, with a mean of 157,700 and a standard deviation of 0.25; and on day 42, the titers were 160,000, 165,000, and 163,000, with a mean of 162,700 and a standard deviation of 0.25. This dose group exhibited the highest antibody titers at all time points, with a significant increase over time, indicating that the 120 μg dose induced the strongest and continuously rising humoral immune response.

[0080] Results of drug administration experiments at different sites, such as Figure 2 As shown, at the same dose of 50 μg, the antibody titers at each time point in the subcutaneous neck injection group (9.77 ± 0.25 million at day 21, 10.33 ± 0.15 million at day 28, 10.63 ± 0.15 million at day 34, and 10.90 ± 0.10 million at day 42) were significantly higher than those in the ( P<0.01) The lateral leg muscle group (21 days 5.67±0.15 million, 42 days 6.23±0.15 million) and the chest muscle group (21 days 4.90±0.10 million, 42 days 5.40±0.10 million) suggest that the subcutaneous neck is a better immunization route.

[0081] Experimental results at different doses, such as Figure 3 As shown, after subcutaneous injection of 10μg, 50μg, and 120μg of antigen into the neck, the antibody titer increased in a dose-dependent manner. The 120μg group had the highest titer (162,700 ± 2,500 at 42 days), followed by the 50μg group (86,000 ± 1,000 at 42 days), and the 10μg group had the lowest titer (13,000 ± 1,000 at 42 days). Moreover, the titer of each dose group increased slightly over time.

[0082] Therefore, the bivalent antigen combination provided by this invention not only ensures broad-spectrum antigen coverage but also avoids epitope competition and non-specific immune consumption between antigens, resulting in low production costs. The provided vaccine can induce a strong humoral immune response without adjuvants, expressing endogenous antigens in chickens and mimicking the natural infection process. The antibody preparation process using the antigens provided by this invention is simple, has a short cycle, better batch-to-batch consistency, and is easy to standardize and scale up production. The immunization cycle is clear, and the antibody titer is high. The anti-Helicobacter pylori bivalent IgY product provided by this invention can be directly formulated into oral preparations (such as egg yolk powder, microcapsules, lozenges, etc.) for the prevention or adjuvant treatment of Helicobacter pylori infection, especially suitable for children, pregnant women, and antibiotic-resistant patients, and has broad market application value.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antigen for preparing anti-Helicobacter pylori, characterized in that: The antigen consists of coding sequences for UreB and HpaA.

2. The antigen for preparing anti-Helicobacter pylori according to claim 1, characterized in that: The coding sequences of UreB and HpaA also incorporate the coding sequence of a chicken-derived leader peptide at their 5' ends.

3. The antigen for preparing anti-Helicobacter pylori according to claim 2, characterized in that: The coding sequence of the chicken-derived leader peptide is shown in SEQ ID NO.8; the coding sequence of UreB is shown in SEQ ID NO.5; and the coding sequence of HpaA is shown in SEQ ID NO.

6.

4. A method for preparing an antigen against Helicobacter pylori as described in any one of claims 1-3, characterized in that, The steps are as follows: S1. Codon optimization of the amino acid sequences of UreB and HpaA, adding chicken-derived leader peptide to the front of the optimized codon, sending the chicken-derived leader peptide + optimized codon sequence to the company for sequence synthesis, linking the synthesized sequence with the cloning vector respectively, and verifying the correct recombinant vector to be transformed into engineered bacteria. S2. After the received engineered bacteria are propagated, plasmids are extracted, linearized, transcribed in vitro, and capped and tailed. After purification, the antigen used to prepare anti-Helicobacter pylori is obtained.

5. The method for preparing antigens for preparing anti-Helicobacter pylori according to claim 4, characterized in that: The optimized coding sequence of the UreB codon in S1 is shown in SEQ ID NO.5, and the optimized coding sequence of the HpaA codon is shown in SEQ ID NO.

6.

6. The method for preparing antigens for preparing anti-Helicobacter pylori according to claim 4, characterized in that: The cloning vector in S1 is pUC57, pUC19, or pBluescript SK(+); the cloning vector contains restriction enzyme sites, Kozak sequences, or transcription terminators.

7. The method for preparing antigens for preparing anti-Helicobacter pylori according to claim 4, characterized in that: The sequences synthesized in S1 are respectively linked with the cloning vector to form two recombinant vectors, or the two sequences synthesized in S1 are linked with the same vector to form one recombinant vector.

8. The use of the antigen for preparing anti-Helicobacter pylori as described in any one of claims 1-3 in the preparation of a chicken anti-Helicobacter pylori vaccine, characterized in that: The vaccine is a naked mRNA solution or mRNA encapsulated with lipid nanoparticles.

9. A specific egg yolk antibody IgY against Helicobacter pylori, characterized in that: The anti-Helicobacter pylori specific egg yolk antibody IgY is prepared by immunizing laying hens with the antigen used to prepare anti-Helicobacter pylori as described in any one of claims 1-3.

10. The use of the anti-Helicobacter pylori specific egg yolk antibody IgY as described in claim 9 in the preparation of an oral formulation for the prevention or adjunctive treatment of Helicobacter pylori infection.

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