Helicobacter pylori composite antigen, antibody, preparation method and use

Abstract

A Helicobacter pylori composite antigen, which is composed of full-length or partial sequences of five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, wherein the amino acid sequences of the five antigen proteins are respectively as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5. The composite antigen has a higher immunogenicity, and an anti-Hp egg yolk antibody prepared by the composite antigen has a higher Hp-neutralizing activity, and can efficiently block the colonization of Hp and inhibit the activity of the Hp at the same time, thereby efficiently preventing and treating gastrointestinal diseases caused by Hp infections.

Description

Technical Field The present invention belongs to the technical field of biological medicines, and more particularly relates to a Helicobacter pylori composite antigen, an antibody and a preparation method and use. Background Art Helicobacter pylori (Hp) is a spiral Gram-negative microaerophilic bacillus with strict growth condition requirements, is the unique microorganism species that can survive in the human stomach so far, and was first isolated by Barry Marshall and Robin Warren in 1982. The Hp may cause many digestive tract diseases even gastric carcinoma, and has a global infection rate exceeding 50%. Due to the continuously increasing drug resistance, a radical cure rate is continuously decreased, thereby further causing stubborn infections and severely threatening human health. The Hp permanently colonizes in the human stomach and duodenal mucosa, causes direct damage of gastric epithelial cells such as vacuolar degeneration and abnormal proliferation and differentiation by releasing vacuolating cytotoxin (VacA), cytotoxin gene A (CagA) and other virulence factors, and induces gastric mucosal inflammation to cause indirect damage, thereby causing related gastric diseases. The Hp has been listed in class I carcinogens by World Health Organization (WHO) in 1994. With the deeper understanding of the Hp, it was confirmed by Kyoto Global Consensus in 2015 that, Hp gastritis was an infectious chronic disease. Unless there are counterbalance factors (such as advanced age, underlying diseases and reinfection), infected persons should be subjected to eradicative treatment. At present, drugs used for treating Hp infections are mainly divided into 3 classes: antibiotics, proton-pump inhibitors (PPI) and bismuth reagents. A current common clinical treatment plan is as follows: the 3 classes of drugs are in compatible use according to an appropriate ratio; and a suitable dosing interval is set, thereby achieving an ideal clearance effect. H. pylori colonizes in the human stomach mainly; urea in the environment can be decomposed into ammonia and carbon dioxide by urease on the outer membrane surface of H. pylori; and “ammonia cloud” is formed surrounding the surface of the H. pylori, thereby creating a nearly neutral microenvironment around the H. pylori and protecting the H. pylori from being attacked by gastric acid. At the same time, most of the antibacterial drugs have decreased activity even are inactivated in an extremely strong acid environment of the stomach. In addition to resisting an extremely acidic pH environment, the H. pylori can cope with periodic gastric emptying, this is because the H. pylori can continuously move by virtue of spiral morphology and powerful flagella and arrest in the surface of gastric mucosa epithelial cells (a neutral pH environment) after penetrating through a gastric mucus layer close to 200 um. Thus, gastric acid attack is effectively avoided; and the effect of gastric emptying is significantly decreased. On the contrary, due to regular gastric emptying, the antibacterial drugs difficultly exist in the stomach, thereby decreasing an accumulated concentration of the drugs at infection sites. Since the H. pylori colonizes in the deep mucous layer, effective contact of the antibacterial drugs with the bacillus is mechanically isolated. Thus, effects of the drugs are difficultly achieved, thereby further increasing difficulty of eradicating the H. pylori in the stomach. In addition, DNA is often exchanged among H. pylori, and then a high gene recombination probability and a high mutation rate are caused, so that highly variable strains

[10] are produced by the H. pylori in persistent infections. H. pylori gene mutation is a significant cause of producing drug resistance. Therefore, even if the antibacterial drugs can achieve a concentration threshold of conventional effective treatment at gastric infection sites, the antibacterial drugs cannot achieve an ideal bactericidal effect due to sensitivity decrease of a drugresistant strain on the antibacterial drugs. In addition, H. pylori infection is generally chronic and persistent; and bacterial biofilm is easily formed due to long-term H. pylori infection. It was first verified by Stark et al.

[11] in 1999 that, the H. pylori had capacity of forming the bacterial biofilm. While sensing an adverse outside environment, the H. pylori will embed into auto-secretory extracellular polymeric substances (EPS) to form a multi-cellular three-dimensional structure, namely the bacterial biofilm. Researches of Yonezawa et al.

[12] find out that, the H. pylori will enhance the resistance degree to clarithromycin after the bacterial biofilm is formed, namely a minimal inhibitory concentration (MIC) of the clarithromycin may be increased by 16 times after a mature bacterial biofilm is formed by the H. pylori. Since it is difficultly ensured that the H. pylori is completely killed during drug treatment, residual pathogenic bacteria may cause recurrent and stubborn infections. Normally, immune cells and antibodies participate in immune recognition and response of the body to H. pylori infection, and thus a small quantity of residual bacterial in the body may be removed. However, the H. pylori may successfully avoid immune response of the host in many ways of avoiding recognition of the body by modifying an outer membrane protein, down-regulating migration and uptake of immune cells, promoting macrophage apoptosis and inhibiting immune response of T cells, thereby causing long-term colonization and persistent infection. Therefore, restoring the immune response of the body to the H. pylori is extremely important for increasing an eradication rate of the H. pylori. (Research Progress of Anti-H. pylori Drug Delivery strategy, Chen Xiaonan, Fan Mogu) Therefore, a safe and effective anti-Hp infection solution is urgently needed in clinical treatment and in the market at present. Domestic and foreign researches have shown that, when an anti-Hp specific immunoglobulin of yolk (IgY) is prepared by a recombinant protein of H. pylori or a certain pathogenic factor, an effect of resisting the Hp infection may be achieved; and the problem of drug resistance may be solved. The IgY is the unique immunoglobulin in yolk, and is fast in production, high in yield, high in titer, stable in physicochemical property, wide in source and zero in toxic or side effect. Anti-Hp-IgY can resist degradation of pepsase in the stomach, and can produce immune response to mucosal level Hp infection after entering the body, thereby decreasing inflammations and ulcers. Further, the Anti-Hp-IgY can be specified at the Hp infection, may neutralize Hp protein toxins after orally taken, and may block toxin activity of the Hp to epithelial cells, eradicate or alleviate the Hp infection and increase human immunity. At present, an existing anti-Hp yolk antibody has been reported for more than 15 years. Selected recombinant 2022336191   05 Jun 2026 antigens include UreB, Hsp60, CagA, VacA, NAP, OMP18, HpaA and HspA. The applicant researches a quadrivalent Hp antigen, and has applied for a Chinese invention patent with a publication number of CN111793137A and an invention name of “Quadrivalent Hp Antigen and Preparation Method and Use thereof”. The quadrivalent Hp antigen is an antigen prepared by optimizing nucleotides corresponding four antigen 5 proteins of UreB, VacA, CagA and HpaA, cloning the nucleotides into a pET28a, pET30 or pColdII prokaryotic expression vector, and expressing the nucleotides through prokaryotic expression bacteria BL21, Rosetta or OrigamiB. By utilizing the quadrivalent Hp antigen, an egg containing a yolk antibody of the quadrivalent Hp antigen and a yolk antibody that can efficiently block Hp colonization and infection are prepared. Compared with existing products, the yolk antibody has excellent dissolving property and higher product purity. The novel 10 yolk antibody containing the quadrivalent Hp antigen can block the Hp colonization, effectively inhibit Hp activity and efficiently prevent and treat gastrointestinal diseases caused by the Hp infection. However, the quadrivalent antigen is mainly specified at toxin factors of the Hp and the HpaA, and has a poor colonization inhibition effect on Hp that achieves an adherence effect by incompletely depending on HpaA or without depending on the HpaA. Thus, an effect of treating different Hp infections needs to be improved. 15 An important factor of colonizing the Hp in the stomach is that the bacterium has the characteristic of adhering to the gastric mucosa. Such an adherence characteristic reflects that, some adherence factors exist on the Hp, and corresponding specific receptors exist on the gastric epithelial cells. Due to specific binding of these adherence factors and the corresponding receptors, the Hp stably adheres to and colonizes on the gastric mucosa surface for a long time, thereby providing a precondition for a pathogenic effect thereof (Boyle EC. Finlay BB. 20 Bacterial pathogenesis: exploiting cellular adherence [J].CurrOpin Cell Biol, 2003; 15(5): 633-639). Although adhesin plays an important role in the Hp infection process, different Hp may adhere to the gastric epithelial cells by utilizing different adhesins. Therefore, a solution is urgently needed for effectively inhibiting the Hp colonization effect and effectively inhibiting Hp activity, thereby further preventing and treating gastrointestinal diseases caused by Hp infections. 25 Summary of the Invention The present invention provides a Helicobacter pylori (Hp) composite antigen, an antibody and a preparation method and use. Compared with an existing quadrivalent antigen, the antigen in the present invention desirably has a higher immunogenicity. An anti-Hp yolk antibody prepared through the composite antigen desirably has 30 more excellent broad-spectrum property and higher Hp-neutralizing activity, and desirably can efficiently block the colonization of Hp, inhibit the activity of the Hp and neutralize toxins of the Hp at the same time, thereby desirably efficiently preventing and treating gastrointestinal diseases caused by Hp infections. Thus, the present invention provides the Hp composite antigen, which is composed of sequences of five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and 35 adhesin BabA, wherein the amino acid sequences of the five antigen proteins are as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 respectively, wherein, the five antigen proteins, 2022336191   05 Jun 2026 namely the urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are antigen proteins expressed by performing sequential optimization on corresponding nucleotides of the antigen proteins, wherein the optimized nucleotide sequences are as shown in SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 respectively. 5 The present invention further provides a preparation method of the Hp composite antigen. The preparation method includes the following steps: (1) constructing expression plasmids of five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA; (2) transforming the expression plasmids into competence bacteria for expression; collecting the bacteria; and 10 extracting the antigen proteins; and (3) purifying the five antigen proteins by a nickel ion affinity chromatographic column; and lyophilizing and preserving the composite antigen. Preferably, in the step (1), nucleotides corresponding to the five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are subjected to 15 sequential optimization and then cloned into prokaryotic expression vectors; and the expression plasmids are constructed. Preferably, the sequential optimization includes increase of a polypeptide sequence CTB at the N end or C end of the antigen protein, wherein the sequence CTB is as shown in SEQ ID No. 11. The sequential optimization further includes increase of a protein expression tag at the N end or C end of the antigen protein, and selection 20 of a hydrophilic region and a conserved region of the antigen protein. The present invention further provides an anti-Hp yolk antibody. The yolk antibody is prepared from eggs laid by immunized hens through the Hp composite antigen. The present invention further provides a preparation method of the anti-Hp yolk antibody. The preparation method includes the following steps: 25    (1) emulsifying the Hp composite antigen according to claim 1 or 2 with an adjuvant; (2) immunizing laying hens: separately raising healthy laying hens at an age of 10-40 weeks; performing subcutaneous injection on the laying hens; selecting 2-6 injection points for each hen; determining an antigen dose for immunization as 80-200 ug per hen; and enhancing the immunization once every two weeks after the first immunization; 30    (3) collecting eggs laid in the last immunization, namely obtaining eggs containing the yolk antibody resisting five Hp antigens; and (4) isolating and purifying the eggs containing the yolk antibody resisting five Hp antigens, thereby obtaining the anti-Hp yolk antibody. Preferably, the step (4) specifically includes: cleaning and disinfecting the eggs containing the anti-Hp yolk 35 antibody, and opening the eggs to let egg white flow out; rolling over the yolk on ordinary filter paper for several times, removing the egg white, breaking yolk membranes, and collecting the yolk; adding distilled water in an amount of 5 times that of the volume of a yolk solution for dilution; fully stirring the solution; freezing the solution at -40°C for 24 hours; slowly melting the frozen solution at 16°C after taken out; filtering the solution by a sterile filter membrane of 400 meshes while melting; filtering the filtered supernatant by ceramic membranes of 8 pM, 1 pM and 0.2 pM sequentially; collecting a yolk antibody solution; determining absorbance of 280 nm; calculating content of the yolk antibody; and identifying purity of the obtained yolk antibody through SDS-PAGE electrophoresis. The present invention further provides use of the anti-Hp yolk antibody in preparation of foods, food additives, medicines and health foods. Beneficial effects are as follows: The Hp composite antigen in the present invention is composed of the five antigen proteins, namely the urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA. Thus, the concentration and purity of an effective antigen are increased, and immune response of the hens is further increased, so that the titer of the produced anti-Hp yolk antibody is significantly increased. Moreover, in the present invention, antigens that can be expressed by more than 95% of clinical strains are selected as immunizing antigens, and then the Hp composite antigen having high immunogenicity is obtained. The pentavalent Hp composite antigen in the present invention has the adhesin BabA, so that an antigen adhesin yolk antibody prepared by inducing the composite antigen may effectively block the colonization of the Hp in the body, thereby achieving a clearance effect. Through detection, in combination with the anti-HpaA and BabA yolk antibody, the pentavalent composite antigen in the present invention may have an effect of inhibiting adherence of more than 95% of Hp. In addition, in combination with protein differences of different clinical strains, the composite antigen in the present invention further optimizes the amino acid sequences of the antigen proteins, so that the prepared antiHp yolk antibody has the broad-spectrum property. By selecting an antigen region, the expression and purity of the antigen may be increased. In addition, the induced yolk antibody can well recognize and neutralize extracelluar proteins of the Hp, thereby efficiently eradicating the Hp. Brief Description of the Drawings To easily clearly understand the content of the present invention, the present invention will be further described below in detail according to specific embodiments and in combination with drawings. Fig. 1 is a schematic diagram of SDS-PAGE detection results of five antigens prepared in embodiment 1 of the present invention; Fig. 2 is a schematic diagram of a result of an inhibiting effect of a yolk antibody produced from a pentavalent composite antigen prepared in embodiment 1 of the present invention and a researched quadrivalent composite antigen on Helicobacter pylori (Hp); and Fig. 3 is a schematic diagram of in-vivo treatment safety and effect evaluation results of an anti-Hp yolk antibody prepared in embodiment 2 of the present invention. Detailed Description of the Invention To describe technical contents, achieved purposes and effects of the present invention in detail, the present invention is described below in combination with implementation modes. Unless otherwise specified, reagents and competence bacteria used in the present description are all commercially available products. Related diseases caused by Helicobacter pylori (Hp) are mainly caused by gastric cells destroyed by the Hp. The current experiments and theories confirm that, the diseases are mainly related to three in-vivo expression proteins of the Hp, namely urease (Ure), vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA). The Ure is an enzyme enriched in Helicobacter, may change a local strong acid environment in the stomach, and plays an important role in parasitism and pathogensis of the Helicobacter in the stomach. At present, all the discovered Hp bacteria contain the Ure. UreB is an active center of forming the Ure, is excellent in conservative property and antigenicity, and is an ideal Hp vaccine target. The VacA exists in genome of all Hp strains. An expression product of the VacA is the unique protein toxin secreted by the Hp, is an important virulence factor, and may change cell membranes, form a transmembrane anion channel, release acid hydrolase, cause cellular swelling accompanying with cell membrane fusion, and finally produce vacuolating degeneration of the epithelial cells. The CagA is one of the virulence markers of the Hp, and is related to pathogenicity of the Hp and severity of related clinical diseases. The study on the Hp shows that, colonization of the Hp on the gastric mucosa is realized by virtue of specific binding between a surface ligand of the Hp and a gastric mucosa epithelial cell receptor. HpaA is a flagellar sheath membrane protein of the Hp, is one of the most important adhesins, almost exists on surfaces of all the clinically isolated Hp strains, and has characteristics of surface exposure and high antigen conservation. In addition, an HpaA vaccine immunized animal can induce protectiveness. BabA is one of the discovered Hp adhesins, and can be bound to a fucosylated Lewis blood group antigen on the surface of gastric epithelial cells to mediate adherence of the Hp and the gastric epithelial cells. The BabA is coded by a gene babA2. The study finds out that, the gene BabA2 is closely related to occurrence of duodenal ulcer and gastric carcinoma. The full length of the BabA includes 746 amino acids; and homology of different Hp strains is about 85% only. In the present invention, the protein sequence is subjected to optimized screening; an extracellular domain (573-746) of the protein is selected as an antigen; and it is determined that homology of the domain in different Hp strains is more than 98%. Cholera toxin (CT) is an important virulence factor of Vibrio cholera, and contains two subunits A and B. Toxicity of the CT is removed by CTB. The CTB has mucosal adjuvant activity and can effectively stimulate the body to secrete an antitoxin antibody; and the most basic receptor GM1 of the CTB exists on the surfaces of most of the mammalian cells. Therefore, the CTB may serve as an excellent adjuvant. The CTB may: 1) help to enhance a presentation effect of DC and other APCs to the antigen, and promote the antigen to cross a mucosal barrier; 2) induce the body to secrete a specific antibody; 3) stimulate receptor expression on the surfaces of T cells, B cells and APCs and secretion of cytokines. Many expression vectors of the CTB and the antigens or antigenic determinant fusion proteins have been successfully constructed by many domestic and foreign scholars, and expressed in host bacteria. Therefore, enhancing immunogenicity of the specific antigen through the fusion proteins is a feasible way. In the present invention, the anti-HpaA titer of the yolk antibody is significantly enhanced by increasing the CTB sequence for the HpaA antigen. According to the pentavalent composite antigen of the UreB, VacA, CagA, adhesin HpaA and adhesin BabA researched and designed in the present invention, through specific antigen composition, sequence optimization and selection of the antigen region, the expression and purity of the antigen are increased; and antigen preparation cost is decreased. Further, the induced yolk antibody can well recognize and neutralize the extracelluar proteins of the Hp, and efficiently block the colonization of Hp in the body, thereby efficiently eradicating the Hp. In addition, the prepared yolk antibody has broad-spectrum property. Embodiment 1 The present embodiment provides a pentavalent Hp composite antigen composed of five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, wherein the amino acid sequences of the five antigen proteins are as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 respectively after optimization. Sequential optimization includes optimization according to codon preference of Escherichia coli and sequence conservation of the Hp, and increase of a polypeptide sequence CTB at the N end or C end of an HpaA antigen protein, wherein the sequence CTB is as shown in SEQ ID No. 11. The sequential optimization further includes increase of a protein expression tag at the N end or C end of each antigen protein. A specific preparation method is as follows: S1. Expression of an Hp composite antigen Nucleotides corresponding to five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, were subjected to sequential optimization and then cloned into prokaryotic expression vectors; expression plasmids were constructed; the expression plasmids were transformed into competence bacteria BL21, Rosetta or OrigamiB; the bacteria were induced by IPTG at 37°C for 4 h and then centrifugally collected; the bacteria were lysed through ultrasonication; and the lysed bacterium supernatant was centrifugally collected; wherein the competence bacteria BL21, Rosetta or OrigamiB may be purchased. S2. Purification of the Hp composite antigen Purification of the antigen by a nickel ion affinity chromatographic column: the antigen-expressing lysed bacterium supernatant was filtered; the sample was added onto the nickel ion affinity column; the supernatant was washed with a buffer solution till a baseline state and then eluted with an eluent; an elution peak was collected; the eluted protein was dialyzed and concentrated; and the antigen was lyophilized and preserved. S3. SDS-PAGE detection of the Hp composite antigen (1) The five prepared Hp antigen protein samples were uniformly mixed with 5xloading buffer according to a volume ratio of 1:4; and the mixture was denatured in a metal bath at 95°C for 5 min and then preserved in a refrigerator at -80°C for later use. (2) Gel preparation: separation gel and concentration gel were prepared by referring to the instruction of a SDS-PAGE gel kit. (3) Loading: a glass plate was cleaned; leakage was detected; the gel was filled; after setting of the concentration gel, a gel preparation plate was transferred into an electrophoresis tank; a comb was removed after the electrophoresis tank was filled with an electrophoretic buffer solution; and the denatured protein samples were added into loading wells of the gel. The total protein content of each well should be consistent; and a marker was filled in another well. (4) Electrophoresis: an appropriate amount of the electrophoretic buffer solution was added into an electrophoresis box; an electrophoresis apparatus was assembled; electrophoresis was conducted at a constant voltage of 80 V; and the electrophoresis was stopped when the samples flowed to the bottom of the gel. (5) Coomassie blue staining: SDS-PAGE gel was put in Coomassie blue staining apparatus for staining for 1 h. (6) Destaining: the stained SDS-PAGE gel was destained with a destaining solution until protein bands were clear. (7) Test results were shown as Fig. 1; and purity of each of the five antigens was higher than 85%. Experimental case of obtaining antigen expressions: In the present embodiment, the expression of an antigen BabA in truncated expression is higher than that of a full-length antigen BabA. Expression plasmids that expressed the full-length BabA and a BabA-OPM region were respectively transformed into BL21 Escherichia coli; after the IPTG induced expression, the bacteria were lysed ultrasonically; the proteins were purified through affinity chromatography (a specific method was the same as that in embodiments 1 and 2); and protein concentrations of the purified proteins were determined by a BCA method. The results showed that, the full-length BabA had a plasmid expression of 0.42 mg / L; the BabA-OPM had a plasmid expression of 1.34 mg / L; and the expression of a BabA antigen-soluble protein in truncated expression was 3.2 times that of the full-length antigen. Contrast case of an inhibition effect of a yolk antibody produced from the pentavalent composite antigen prepared in the present embodiment and the researched quadrivalent composite antigen (as shown in a Chinese patent application CN111793137A) on the Hp: Cells MGC-803 in a logarithmic phase were digested through pancreatin; a cell concentration was regulated as 2x104 cells / ml; the cells were added into a 6-well plate on which a sterile cover glass was placed; 2 ml of the cells were added into each well; the cells were cultured in a cell incubator at 37°C for 24 h; after cell adherence, 1 ml of culture medium containing a pentavalent anti-Hp yolk antibody and a quadrivalent anti-Hp yolk antibody was respectively added into an experimental group, wherein the yolk antibodies had concentrations of 20 ^g / ml, 10 ^g / ml and 5 ^g / ml; 20 ^g / ml of a common yolk antibody was added into a control group; 1 ml of Hp strain A (high expression of HpaA and low expression of BabA) or Hp strain B (low expression of HpaA and high expression of BabA) was continuously added into each well at 4*106 cfu / ml (4 compound wells were made for each concentration); the cells were continuously cultured in the cell incubator at 37°C for 2 h; the cells were washed with PBS for 3 times; the cover glass was taken out and aired; the cells were immobilized with methanol for 15 minutes and washed with the PBS once; then the cells were stained by a methylene blue method; bacterial count of the Hp adsorbed on each cell was observed and recorded under an oil immersion lens; 15 cells were counted in each group; and the mean of bacteria adhered onto each cell was calculated. Referring to Fig. 2, test results show that, inhibition effects of the pentavalent anti-Hp yolk antibody on the tested Hp A and Hp B are significantly better than those of the quadrivalent anti-Hp yolk antibody. Therefore, inhibition of the pentavalent anti-Hp yolk antibody in the present invention on the Hp A and Hp B is significantly increased. Embodiment 2 The present embodiment provides an anti-Hp yolk antibody. Immunization of hens is induced by the pentavalent composite antigen prepared in embodiment 1, and then a yolk antibody containing five anti-Hp antigens is obtained. Specific preparation steps are as follows: (1) Five Hp combined antigens were emulsified with an adjuvant, wherein the adjuvant was a Freund's complete adjuvant. (2) Immunization of laying hens: healthy laying hens at an age of 10-40 weeks were separately raised; subcutaneous injection was performed on the laying hens; 2-6 injection points were selected for each hen; an antigen dose for immunization was determined as 80-200 ng per hen, preferably 80-200 ug per hen; the antigen dose of 100 ug per hen was adopted in the present embodiment; and the immunization was enhanced once every two weeks after the first immunization. (3) Eggs laid in the last immunization were collected, namely eggs containing the yolk antibody resisting five Hp antigens were obtained. (4) The eggs containing the yolk antibody resisting five Hp antigens were isolated and purified, thereby obtaining the anti-Hp yolk antibody. Specific procedures were as follows: the eggs containing the anti-Hp yolk antibody were cleaned and disinfected, and the eggs were opened to let egg white flow out; the yolk was rolled over on ordinary filter paper for several times, the egg white was removed, yolk membranes were broken, and the yolk was collected; distilled water in an amount of 5 times that of the volume of a yolk solution was added for dilution; the solution was fully stirred; the solution was frozen at -40°C for 24 hours; the frozen solution was slowly melted at 16°C after taken out; the solution was filtered by a sterile filter membrane of 400 meshes while melting; the filtered supernatant was filtered by ceramic membranes of 8 uM, 1 uM and 0.2 uM sequentially; a yolk antibody solution was collected; absorbance of 280 nm was determined; content of the yolk antibody was calculated; and purity of the obtained yolk antibody was identified through SDS-PAGE electrophoresis. Experimental case of identifying immunization effect of obtained antibody 1. ELISA titer of the prepared anti-Hp yolk antibody (1) The lyophilized yolk antibody was dissolved with sterile water to 1 mg / ml. (2) UreB (2 gg / ml), CagA (0.5 gg / ml), VacA (0.5 gg / ml), HpaA (1 gg / ml) and BabA (0.5 gg / ml) were diluted with a carbonate buffer solution or a PBS having a pH value of 9.6; the diluted antigens were added into wells of an ELISA plate (100 gl per well); a sealing film was adhered; and the antigens were placed at 4°C overnight. (3) A coating solution was removed; and the ELISA plate was washed with TBST containing 0.05% Tween 20 for 3 times. (4) 300 gl of a TBS blocking solution containing 3% BSA was added into each well; the antigens were incubated at 37°C for 1 h; and the ELISA plate was washed with the TBST for 3 times. (5) After diluted with PBS by 10000 times, the yolk antibody (1 mg / ml) was subjected to doubling dilution. The diluted yolk antibody was added into the ELISA plate coated with different antigens and then incubated at 37°C for 2 h. The ELISA plate was washed with the TBST for 5 times. (6) A horse radish peroxidase (HRP)-labeled anti-chicken IgY antibody diluted with TBS at 1:5000 was added at a dose of 100 gl per well; the solution was incubated at 37°C for 1 h; and the ELISA plate was washed with the TBST for 5 times. (7) A TMB coloring solution was added at a dose of 100 gl per well; the solution was incubated at room temperature for 5-15 min; a color reaction was terminated with 2N sulfuric acid; a value OD of 450 nm was read by a microplate reader; the antibody was positive when the value OD was 1.6 times higher than that of negative control; and a maximum dilution ratio of the positive-value wells was the titer of the antibody. (8) The results showed that, when the laying hens were immunized by the composite antigen provided by the present invention, titers of the prepared anti-Hp yolk antibody (1 mg / ml) were as follows: the anti-UreB ELISA titer was 1:640000; the anti-CagA ELISA titer was 1:640000; the anti-VacA ELISA titer was 1:320000; the anti-HpaA ELISA titer was 1:640000; and the anti-BabA ELISA titer was 1:320000. Therefore, the yolk antibody obtained from the laying hens immunized by the pentavalent composite antigen in the present invention has a higher titer. 2. Enhancement of immunization effect of HpaA through CTB fusion protein After the laying hens were immunized by HpaA and HpaA-CTB, a yolk antibody prepared from egg white was collected, and a concentration of the yolk antibody was regulated as 1 mg / ml; the HpaA antigen was diluted to1 gg / ml with a carbonate buffer solution having a pH value of 9.6; and the antigen was added into an ELISA plate according to 100 gl / well and then incubated at 4°C overnight. Test steps were as shown in embodiment 5. Results showed that, the anti-HpA ELISA titer of the yolk antibody after HpaA immunization was 1:160000, and the anti-HpA ELISA titer of the yolk antibody after HpaA-CTB immunization was 1:640000. 3. In the present invention, affinity of IgY (IgY-opm) produced by BabAOPM region immunization to the antigen was obviously higher than affinity of IgY (IgY-BabA) produced by full-length BabA immunization to the antigen. Affinity of the antibody was detected by BLITZ: The yolk antibody was diluted to 25 gg / ml with a buffer solution (1^PBS containing 0.02% Tween and 0.1% BSA), 200 gl per tube; BabA-OPM and BabA antigens were respectively diluted to 50 gg / ml, 25 gg / ml, 12.5 gg / ml, 6.25 gg / ml, 3.125 gg / ml and 0, 20gl per tube; the antigens were added into a probe; the probe was activated in PBS for 10 min; the antibody was incubated at normal temperature for 1 h; operations were conducted on a machine; 200 ul of the buffer solution was filled in a 500 ul brown EP tube; and the EP tube was placed on the machine for detection. Data analysis was conducted; and the probe was recovered. The detection results showed that, the affinity of the yolk antibody obtained through BabA-OPM immunization to the BabA was KD=2.3*10-8, while the affinity of the yolk antibody obtained through full-length BabA immunization to the BabA was KD=5.2*10-7. It can be seen that, the affinity of IgY (IgY-opm) produced by BabAOPM region immunization to the antigen was obviously higher than the affinity of IgY (IgY-BabA) produced by full-length BabA immunization to the antigen. 4. Protective effect of the prepared anti-Hp yolk antibody on mice A mouse Hp infection model is established in the reference; and the test is divided into a model group, a normal IgY control group, and an anti-Hp IgY group prepared through immunization of five antigens. The results show that, compared with the model group and the normal IgY control group, the anti-Hp IgY group significantly improves gastric mucosa pathology and the number of the Hp. Experimental procedures are as follows: 40 BALB / c mice were averagely divided into 5 groups (namely, a blank group, an infection group, a normal yolk antibody group (that is, a yolk antibody extracted from non-specific immune eggs), a quadrivalent yolk antibody group, and a pentavalent yolk antibody group); within 24 h after fasting, mice in the blank group were subjected to gavage with 0.2 ml brucella broth, mice the other groups were subjected to gavage with 0.1 ml 3% NaHCO3, and mice in the yolk antibody groups were subjected to gavage with 0.2 ml yolk antibody having a concentration of 1 mg / ml; 15 minutes later, except for the blank group, the mice in each group were subjected to gavage with a 0.2 ml Hp B bacterium solution having a concentration of 1*109 CFU / ml; the same gavage operation was conducted once every two days, totaling 3 times; the mice were killed in the 8th of the last gavage; stomachs were taken under sterile conditions and placed on sterilized filter paper; forestomachs were removed; glandular stomachs were cut off along the stomach outline; residual foods in the stomachs were washed with a sterile saline solution; and then the glandular stomachs were averagely divided into two parts longitudinally, wherein one part was used for rapid urease test of bacteria, and the other part was used for culture of the bacteria. Mice determined as positive both in the rapid urease test and in the culture of the bacteria were determined as Hp infection positive. Urease test: mouse gastric mucosa suspension was added into a rapid urease determination kit, and it was determined as positive if the reagent turned from yellow to red within 30 minutes. Hp culture test: urease-positive gastric tissues were placed on an Hp selective medium; the mucosa surface was repeatedly applied with the medium downwards and then marked in different areas; and the gastric tissues were cultured in 5% O2, 10% CO2 and 85% N2 at 37°C for 3-4 days. Calculation of an infection rate: the infection rate=(positive infected mice / 8) *100%. Test results were subjected to statistical analysis by a Fisher exact test, and analysis results were shown as Table 1 below. Table 1 Group Number of infected mice Infection rate Blank group 0 0 Infection group 8 100% Normal yolk antibody group 7 87.5% Quadrivalent yolk antibody group 2 25% Pentavalent yolk antibody group 0 0 2022336191   05 Jun 2026 The results showed that, the protective effect of the pentavalent yolk antibody on the Hp strain B was better than that of the quadrivalent yolk antibody, and significantly better than that of the normal yolk antibody. 5. In-vivo treatment safety and effect evaluation of the prepared anti-Hp yolk antibody 5 The laying hens were immunized by the composite antigen; the eggs were collected; a yolk antibody was prepared; a concentration of the yolk antibody was regulated as 1 mg / ml; through ELISA, an anti-UreB titer was more than 1:640000, an anti-CagA titer was more than 1:640000, an anti-VacA titer was more than 1:320000, an anti-HpaA titer was more than 1:640000, and an anti-BabA titer was more than 1:320000; then the eggs were collected; an anti-Hp yolk antibody was prepared; and the antibody was processed and prepared into oral 10 capsules (250 mg per capsule, namely, each oral capsule contained 250 mg of the antibody) by using an existing technology. 39 Hp-positive subjects with recurrence after subjected to standard quadruple treatment were recruited, wherein 16 subjects had different clinical symptoms including stomachache, gastro spasm and sour regurgitation; after signing informed consent, each subject continuously took the anti-Hp yolk antibody for 2 weeks (once in every morning and evening) and orally took 1 capsule within half an hour after meal each time. 15 A value DOB during C13 detection was detected within 1 week and 2 weeks before and after the medicine was taken. At the end of the treatment, the subjects were subjected to evaluation of the clinical symptoms and recording of adverse events. The value DOB during C13 detection before and after the anti-Hp yolk antibody treatment of the subjects was subjected to statistical analysis by using paired T test; mapping was conducted by Graphad8.0.1 software; and it was judged that the value DOB had statistic difference when P <0.05. 20 Test results were as shown in Fig. 3. The results showed that, none of the 39 subjects had adverse reaction after the treatment; the value DOB of 34 subjects during C13 detection was decreased, and effectiveness was 88.2%; and the value DOB of 8 subjects (20.5%) after treatment was lower than 4, and a curative effect of clinically eradicating the Hp was achieved. Apparently, the above embodiments are merely for clearly describing listed examples, rather than limiting the 25 implementation modes. Other changes or modifications of different forms may be made by those ordinary skilled in the art on the basis of the above descriptions. All the implementation modes do not need to and cannot be exhaustive. Obvious derived changes or modifications still fall within the protection scope of the present invention. 2022336191   05 Jun 2026 10 The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge. It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied. In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to “at least one of” a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c. 15 Sequence listing <110> Unik (Jiangsu) Biomedicine Co., Ltd. <120> Helicobacter pylori composite antigen, antibody and preparation method and use <130> 211231AP <160> 11 <170>PatentIn version 3.3 <210> 1 <211> 569 <212> PRT <213> Artificial sequence <400> 1 Met Lys Lys Ile Ser Arg Lys Glu Tyr Val Ser Met Tyr Gly Pro Thr 1               5                    10                   15 ThrGly Asp Lys Val Arg Leu Gly Asp Thr Asp Leu Ile Ala Glu Val 20                25              30 Glu His Asp Tyr Thr Ile Tyr Gly Glu Glu Leu Lys Phe Gly Gly Gly 35              40                 45 Lys Thr Leu Arg Glu Gly Met Ser Gln Ser Asn Asn Pro Ser Lys Glu 50                55                60 Glu Leu Asp Leu Ile Ile Thr Asn Ala Leu Ile Val Asp Tyr Thr Gly 65                70               75                80 Ile Tyr Lys Ala Asp Ile Gly Ile Lys Asp Gly Lys Ile Ala Gly Ile 85               90               95 Gly Lys Gly Gly Asn Lys Asp Met Gln Asp Gly Val Lys Asn Asn Leu 100                105                110 Ser Val Gly Pro Ala Thr Glu Ala Leu Ala Gly Glu Gly Leu Ile Val 115                120                125 Thr Ala Gly Gly Ile Asp Thr His Ile His Phe Ile Ser Pro Gln Gln 130               135               140 Ile Pro Thr Ala Phe Ala Ser Gly Val Thr Thr Met Ile Gly Gly Gly 145              150               155              160 Thr Gly Pro Ala Asp Gly Thr Asn Ala Thr Thr Ile Thr Pro Gly Arg 165                 170               175 Arg Asn Leu Lys Trp Met Leu Arg Ala Ala Glu Glu Tyr Ser Met Asn 180               185               190 Leu Gly Phe Leu Ala Lys Gly Asn Ala Ser Asn Asp Ala Ser Leu Ala 195               200                205 Asp Gln Ile Glu Ala Gly Ala Ile Gly Phe Lys Ile His Glu Asp Trp 210             215              220 Gly Thr Thr Pro Ser Ala Ile Asn His Ala Leu Asp Val Ala Asp Lys 225             230              235              240 Tyr Asp Val Gln Val Ala Ile His Thr Asp Thr Leu Asn Glu Ala Gly 245            250               255 Cys Val Glu Asp Thr Met Ala Ala Ile Ala Gly Arg Thr Met His Thr 260               265             270 Phe His Thr Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Ile Lys 275                 280          285 Val Ala Gly Glu His Asn Ile Leu Pro Ala Ser Thr Asn Pro Thr Ile 290              295             300 Pro Phe Thr Val Asn Thr Glu Ala Glu His Met Asp Met Leu Met Val 305              310              315               320 Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp 325             330              335 Ser Arg Ile Arg Pro Gln Thr Ile Ala Ala Glu Asp Thr Leu His Asp 340             345               350 Met Gly Ile Phe Ser Ile Thr Ser Ser Asp Ser Gln Ala Met Gly Arg 355             360             365 Val Gly Glu Val Ile Thr Arg Thr Trp Gln Thr Ala Asp Lys Asn Lys 370            375              380 Lys Glu Phe Gly Arg Leu Lys Glu Glu Lys Gly Asp Asn Asp Asn Phe 385              390               395               400 Arg Ile Lys Arg Tyr Leu Ser Lys Tyr Thr Ile Asn Pro Ala Ile Ala 405              410             415 His Gly Ile Ser Glu Tyr Val Gly Ser Val Glu Val Gly Lys Val Ala 420              425             430 Asp Leu Val Leu Trp Ser Pro Ala Phe Phe Gly Val Lys Pro Asn Met 435             440              445 Ile Ile Lys Gly Gly Phe Ile Ala Leu Ser Gln Met Gly Asp Ala Asn 450             455              460 Ala Ser Ile Pro Thr Pro Gln Pro Val Tyr Tyr Arg Glu Met Phe Ala 465            470              475              480 His His Gly Lys Ala Lys Tyr Asp Ala Asn Ile ThrPhe Val Ser Gln 485              490             495 Ala Ala Tyr Asp Lys Gly Ile Lys Glu Glu Leu Gly Leu Glu Arg Gln 500              505              510 Val Leu Pro Val Lys Asn Cys Arg Asn Ile Thr Lys Lys Asp Met Gln 515               520              525 Phe Asn Asp Thr Thr Ala His Ile Glu Val Asn Pro Glu Thr Tyr His 530               535             540 Val Phe Val Asp Gly Lys Glu Val Thr Ser Lys Pro Ala Asn Lys Val 545               550              555                560 Ser Leu Ala Gln Leu Phe Ser Ile Phe 565 <210> 2 <211> 240 <212> PRT <213> Artificial sequence <400> 2 His Tyr Trp Val Lys Gly Gly Gln Trp Asn Lys Leu Glu Val Asp Met 1               5                    10                   15 Lys Asp Ala Val Gly Thr Tyr Lys Leu Ser Gly Leu Ile Asn Tyr Thr 20               25              30 Gly Gly Asp Leu Asp Val Asn Met Gln Lys Ala Thr Leu Arg Leu Gly 35                 40                45 Gln Phe Asn Gly Asn Ser Phe Thr Ser Phe Lys Asp Ser Ala Asp Arg 50                 55                60 Thr Thr Arg Val Asp Phe Asn Ala Lys Asn Ile Ser Ile Asp Asn Phe 65                70                75               80 Leu Glu Ile Asn Asn Arg Val Gly Ser Gly Ala Gly Arg Lys Ala Ser 85                90                95 Ser Thr Val Leu Thr Leu Gln Ala Ser Glu Gly Ile Thr Ser Gly Lys 100                105               110 Asn Ala Glu Ile Ser Leu Tyr Asp Gly Ala Thr Leu Asn Leu Ala Ser 115               120                 125 Asn Ser Val Lys Leu Met Gly Asn Val Trp Met Gly Arg Leu Gln Tyr 130               135               140 Val Gly Ala Tyr Leu Ala Pro Ser Tyr Ser Thr Ile Asn Thr Ser Lys 145               150              155              160 Val Thr Gly Glu Val Asn Phe Asn His Leu Thr Val Gly Asp Arg Asn 165                170                175 Ala Ala Gln Ala Gly Ile Ile Ala Ser Lys Lys Thr Tyr Ile Gly Thr 180              185              190 Leu Asp Leu Trp Gln Ser Ala Gly Leu Asn Ile Ile Ala Pro Pro Glu 195               200              205 Gly Gly Tyr Lys Asp Lys Pro Asn Asn Thr Thr Ser Gln Ser Gly Ala 210              215              220 Lys Asn Asp Lys Asn Glu Ser Ala Lys Asn Asp Lys Gln Asp Ser Asn 225               230              235              240 <210> 3 <211> 654 <212> PRT <213> Artificial sequence <400> 3 Met Thr Asn Glu Ala Ile Asn Gln Gln Pro Gln Thr Glu Ala Ala Phe 1                 5                    10                   15 Asn Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Ile Lys Val 20                25               30 Asp Asn Val Val Ala Ser Phe Asp Pro Asn Gln Lys Pro Ile Val Asp 35               40                45 Lys Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Lys Ile Ser Gln Leu 50                 55                 60 Arg Glu Glu Phe Ala Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn 65                70                75                80 Gln Tyr Phe Ser Ser Phe Ile Ser Lys Ser Asn Asp Leu Ile Asp Lys 85               90                95 Asp Asn Leu Ile Asp Thr Gly Ser Ser Ile Lys Ser Phe Gln Lys Phe 100               105               110 Gly Thr Gln Arg Tyr Gln Ile Phe Met Asn Trp Val Ser His Gln Asn 115               120                 125 Asp Pro Ser Lys Ile Asn Thr Gln Lys Ile Arg Gly Phe Met Glu Asn 130              135               140 Ile Ile Gln Pro Pro Ile Ser Asp Asp Lys Glu Lys Ala Glu Phe Leu 145             150               155               160 Arg Ser Ala Lys Gln Ala Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile 165               170               175 Arg Ser Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu 180               185               190 Arg Gln Glu Ala Glu Lys Asn Gly Glu Pro Asn Gly Asp Pro Thr Gly 195                200               205 Gly Asp Trp Leu Asp Ile Phe Leu Ser Phe Val Phe Asn Lys Lys Gln 210             215              220 Ser Ser Asp Leu Lys Glu Thr Leu Asn Gln Glu Pro Val Pro His Val 225              230               235             240 Gln Pro Asp Val Ala Thr Thr Thr Thr Asp Ile Gln Ser Leu Pro Pro 245             250              255 Glu Ala Arg Asp Leu Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr 260               265               270 Leu Gly Asp Met Asn Met Leu Asp Val Glu Gly Val Ala Asp Ile Asp 275               280              285 Pro Asn Tyr Lys Phe Asn Gln Leu Leu Ile His Asn Asn Ala Leu Ser 290               295             300 Ser Val Leu Met Gly Ser His Asn Gly Ile Glu Pro Glu Lys Val Ser 305              310             315              320 Leu Leu Tyr Gly Asn Asn Gly Gly Pro Glu Ala Arg His Asp Trp Asn 325               330              335 Ala Thr Val Gly Tyr Lys Asn Gln Arg Gly Asp Asn Val Ala Thr Leu 340              345              350 Ile Asn Val His Met Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly 355               360             365 Glu Lys Gly Ile Asn Asn Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln 370             375              380 Leu Thr Gly Ser Gln Arg Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys 385              390              395              400 Val Asp Phe Met Glu Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Asn 405              410               415 Leu Ser Lys Lys Glu Lys Glu Lys Phe Gln Asn Glu Ile Glu Asp Phe 420                 425              430 Gln Lys Asp Ser Lys Ala Tyr Leu Asp Ala Leu Gly Asn Asp His Ile 435             440               445 Ala Phe Val Ser Lys Lys Asp Lys Lys His Leu Ala Leu Val Ala Glu 450             455              460 Phe Gly Asn Gly Glu Leu Ser Tyr Thr Leu Lys Asp Tyr Gly Lys Lys 465               470              475              480 Ala Asp Lys Ala Leu Asp Arg Glu Ala Lys ThrThr Leu Gln Gly Ser 485               490              495 Leu Lys His Asp Gly Val Met Phe Val Asp Tyr Ser Asn Phe Lys Tyr 500               505              510 Thr Asn Ala Ser Lys Ser Pro Asp Lys Gly Val Gly Ala Thr Asn Gly 515              520               525 Val Ser His Leu Glu Ala Gly Phe Ser Lys Val Ala Val PheAsn Leu 530              535             540 Pro Asn Leu Asn Asn Leu Ala Ile Thr Ser Val Val Arg Gln Asp Leu 545               550                555           560 Glu Asp Lys Leu Ile Ala Lys Gly Leu Ser Pro Gln Glu Ala Asn Lys 565               570              575 Leu Val Lys Asp Phe Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala 580               585               590 Leu Asn Phe Asn Lys Ala Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr 595              600              605 Asp Glu Val Lys Gln Ala Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys 610             615               620 Arg Glu Arg Leu Glu Lys Asp Val Ala Lys Asn Leu Glu Ser Lys Ser 625               630             635              640 GlyAsn Lys Asn Lys Met Glu Ala Lys Ser Gln Ala Asn Ser 645              650 <210> 4 <211> 260 <212> PRT <213> Artificial sequence <400> 4 Met Lys Ala Asn Asn His Phe Lys Asp Phe Ala Trp Lys Lys Cys Leu 1               5                    10                   15 Leu Gly Ala Ser Glu Val Ala Leu Leu Val GlyCys Ser Pro His Ile 20              25              30 Ile Glu Thr Asn Glu Val Ala Leu Lys Leu Asn Tyr His Pro Ala Ser 35                40                45 Glu Lys Val Gln Ala Leu Asp Glu Lys Ile Leu Leu Leu Arg Pro Ala 50                55               60 Phe Gln Tyr Ser Asp Asn Ile Ala Lys Glu Tyr Glu Asn Lys Phe Lys 65                70                75                80 Asn Gln Thr Ala Leu Lys Val Glu Gln Ile Leu Gln Asn Gln Gly Tyr 85                90                95 Lys Val Ile Thr Leu Asp Thr Ser Asp Lys Asp Asp Phe Ser Phe Ser 100                105                 110 Gln Lys Lys Glu Gly Tyr Leu Ala Leu Ala Met Asn Ala Glu Ile Val 115                120                125 Leu Arg Pro Asp Pro Lys Arg Thr Ile Gln Lys Lys Ser Glu Pro Gly 130              135               140 Leu Leu Phe Ser Thr Gly Leu Asp Lys Met Glu Gly Val Leu Ile Pro 145                150                 155               160 Ala Gly Phe Ile Lys Val Thr Ile Leu Glu Pro Met Ser Gly Glu Ser 165                170                175 Leu Asp Ser Phe Thr Met Asp Leu Ser Glu Leu Asp Ile Gln Glu Lys 180                 185               190 Phe Leu Lys Thr Thr His Ser Ser His Ser Gly Gly Leu Val Ser Thr 195               200              205 Met Val Lys Gly Thr Asp Asn Ser Asn Asp Ala Met Lys Ser Ala Leu 210              215              220 Asn Lys Ile Phe Ala Asn Ile Met Gln Glu Ile Asp Lys Lys Leu Thr 225             230              235              240 Gln Lys Asn Leu Glu Ser Tyr Gln Lys Asp Ala Lys Glu Leu Lys Lys 245              250              255 Lys Arg Asn Arg 260 <210> 5 <211> 174 <212> PRT <213> Artificial sequence <400> 5 Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp 1               5                    10                   15 Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys 20               25                30 Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly 35               40                45 Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu 50                55               60 Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Ala Leu 65                70                 75               80 Ala GlyThr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val 85                90                95 Asn Asn Val Tyr Asn Ala Lys Ile Asn Thr Ala Asn Phe Gln Phe Leu 100             105                110 Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly 115                 120                   125 Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro 130               135              140 Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr 145               150                155                160 Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 165              170 <210> 6 <211> 1710 <212> DNA <213> Artificial sequence <400> 6 atgaaaaaga tatcaaggaa agaatatgta tccatgtatg gtccaacgac cggtgataaa 60 gttcgtctgg gcgatactga cctgatcgct gaagttgagc acgactatac catttacggc 120 gaagagttga agtttggtgg tggtaagacg ctgcgtgaag gcatgagcca aagcaataat 180 ccgagcaagg aggagttaga tttgatcatc acaaacgcgt tgattgttga ttacaccggt 240 atttacaaag ccgacatcgg cattaaggat ggtaagatcg ccggtatcgg caaaggtgga 300 aacaaggata tgcaagatgg cgtaaaaaac aacctgagcg ttggcccggc gacggaggca 360 ttggcgggtg aaggcctcat cgtcacggcg ggtggcatcg atacccatat tcactttatc 420 agcccgcagc aaattccgac ggcgtttgcg tcgggtgtta ccaccatgat tggtggcggt 480 acaggcccag cggacggcac gaacgccacc accatcaccc cgggtagacg taacttgaag 540 tggatgctgc gcgcagcgga agagtacagc atgaatctgg gtttcctggc aaagggtaat     600 gcgtctaacg acgcttcgct ggccgatcag attgaagcgg gtgcgattgg cttcaaaatc      660 catgaagact ggggtacgac cccatccgca atcaaccatg ctctggacgt ggccgacaag    720 tacgacgtgc aggtggcgat ccacaccgat accttgaatg aagcggggtg cgttgaggac    780 accatggccg ctatcgctgg tcgcaccatg cacacctttc ataccgaggg tgcaggcggt     840 ggccatgcac cggatattat caaagtggct ggtgaacaca atatcctgcc ggcgtccacc      900 aacccgacca ttccgtttac agttaatacc gaggcggagc acatggatat gttaatggtt       960 tgtcaccacc tggacaaaag catcaaggag gacgtccaat tcgccgactc ccgtattcgt      1020 ccgcaaacca ttgcggcgga ggacaccctg catgatatgg gcattttcag cattacttcg 1080 agcgatagcc aggctatggg acgtgttggt gaggtgatca cccgtacctg gcagactgcg 1140 gataagaaca aaaaggagtt cggccgcctt aaggaggaaa aaggcgacaa cgataatttc 1200 cgcattaagc gctatctgag caaatacacc attaatccgg cgatcgcgca tggtatcagc 1260 gaatacgtgg gtagcgttga agtgggcaaa gttgcagacc tggtgctatg gtcaccggcg 1320 tttttcggtg tgaaacctaa tatgatcatc aagggcggct tcatcgcgtt gtcccaaatg 1380 ggagacgcaa acgcgtctat cccgaccccg cagccggtgt attatcgtga aatgtttgcc     1440 caccacggca aagctaaata cgatgcgaac attacgttcg ttagccaggc agcgtatgat     1500 aagggtatta aagaggagct gggtctggaa cgtcaggtcc tgccggtgaa aaactgccgt 1560 aacataacca aaaaagacat gcagtttaac gacacgaccg ctcacattga agtcaacccg 1620 gaaacctatc atgtgttcgt ggacggcaag gaggttactt caaaaccggc gaacaaggtc 1680 tccctcgcac agctgttttc tatcttctaa                                          1710 <210> 7 <211> 717 <212> DNA <213> Artificial sequence <400> 7 tattgggtca aaggcgggca atggaacaag cttgaagtgg atatgaaaga tgctgtaggg 60 acttataagc tttcagggct aataaactac actggtgggg atttagatgt caatatgcaa 120 aaagccactt tgcgtttggg acaattcaat ggcaattctt tcacaagctt taaggatagt 180 gctgatcgca ccacgagagt ggatttcaac gctaaaaata tctcaattga taatttttta        240 gaaatcaata atcgtgtagg ttctggagcc gggaggaaag ccagctctac ggttttaact 300 ttgcaagctt cagaagggat cactagcggt aaaaacgctg aaatttctct ttatgatggc 360 gccacgctta atttggcttc aaacagcgtt aaattaatgg gtaatgtgtg gatgggtcgt 420 ttgcaatatg tgggagcgta tttggctcct tcatacagca cgataaacac ttcaaaagtg 480 acaggggaag tgaattttaa ccacctcact gtgggcgata gaaacgccgc tcaagcaggg 540 attattgcca gtaaaaagac ttatattggc acactggatt tgtggcaaag cgctgggtta 600 aacattatcg cccctccaga aggtggttat aaggataaac ctaataatac cacttctcaa 660 agtggtgcta aaaacgacaa aaatgaaagt gctaaaaacg acaaacaaga tagtaac 717 <210> 8 <211> 1962 <212> DNA <213> Artificial sequence <400> 8 atgacaaatg aagctataaa ccagcaaccc caaaccgaag cggcatttaa tccgcagcag 60 tttatcaaca acctgcaggt tgctttcatc aaagttgaca acgtagtggc gagcttcgat 120 ccgaaccaaa aaccgattgt tgataaaaac gaccgcgata atcgtcaggc gtttgaaaag     180 atcagccagc tgcgcgaaga gtttgcaaat aaagctatta aaaacccgac caaaaaaaac    240 caatacttca gcagctttat cagcaagtct aacgacctga tcgacaagga caacttgatc 300 gacaccggtt ctagcattaa atccttccaa aaatttggca cccagcgtta tcagattttc        360 atgaattggg tttcccacca gaacgatcca agcaaaatca atacccagaa aatccgcggt 420 ttcatggaaa atattatcca accaccgatt tctgatgata aagagaaagc tgaatttctg 480 cgtagcgcaa agcaagcctt tgctggcatc atcattggta atcaaattcg ctccgatcag 540 aagttcatgg gggtgttcga cgagagcttg aaggagcgtc aagaagccga gaagaacggc 600 gaaccgaatg gtgacccaac gggtggtgat tggctggaca tctttctgtc ttttgttttt 660 aataagaagc agtccagtga tctgaaagaa accttgaatc aagagccggt tccgcatgtt 720 cagccggatg ttgctacgac cactactgac atccaatccc tgcctccgga ggcgagagat    780 ctcctggatg aacgcggtaa ctttagcaag ttcaccttgg gcgacatgaa catgctggac      840 gttgaaggtg tcgcggatat cgatccgaat tacaaattca accagttgct gattcacaat 900 aacgcgctct cctctgttct gatgggtagc cacaacggca ttgaaccgga aaaggtgagc 960 ctgctgtacg gcaacaacgg cggtccggaa gcccgtcatg attggaatgc tacggttggc 1020 tataaaaacc aacgtggcga caacgtcgcg acccttatca acgtgcatat gaaaaacggc 1080 tccggtttgg ttattgcggg tggcgaaaag ggcattaaca atccgagctt ctatctctac 1140 aaggaggacc agttgacggg ttctcagcgc gcattgtcgc aagaggaaat tcaaaacaaa 1200 gtggatttca tggaattcct ggcgcagaat aacgcgaagc tggacaacct gtcaaagaag 1260 gagaaggaga aattccagaa tgaaatcgaa gatttccaga aggacagcaa ggcgtatctt 1320 gatgcattag gtaatgacca tattgctttc gtgagcaaga aggataaaaa gcacctggca 1380 ttggtggcag agttcggcaa cggcgagctg agctataccc tgaaagacta cggtaaaaag 1440 gctgataaag cgctagaccg tgaagctaaa accactctgc aaggtagcct gaaacacgat 1500 ggtgtgatgt ttgtggacta cagcaacttc aaatacacca atgccagcaa atcgccggat 1560 aaaggtgtgg gtgcgacgaa tggcgtgagc cacctggagg ccggttttag caaggtcgcg 1620 gtttttaacc tgccgaacct gaacaacctg gcgattacct ccgtggttcg tcaagatctc 1680 gaggacaaat tgatcgcgaa aggcctgtct ccgcaggagg cgaacaagct cgtaaaggac 1740 ttcttatcca gtaataagga gctcgtcgga aaagctttaa attttaacaa agctgtggcg 1800 gaggcgaaga acaccggcaa ctatgatgaa gtgaaacagg cccagaagga cctggagaag 1860 tcccttaaaa aacgtgaacg tctggagaaa gacgtggcca agaacttgga gtccaagagc 1920 ggtaataaaa acaagatgga ggcgaagtcg caagcaaaca gc                      1962 <210> 9 <211> 780 <212> DNA <213> Artificial sequence <400> 9 atgaaggcta ataaccactt taaagatttc gcgtggaaaa aatgtctgct cggcgcttcc 60 gaggtggcgt tgctggttgg ttgcagcccg cacattattg aaaccaatga agtagccttg 120 aagctgaact atcatccggc ttccgagaag gtgcaggcac tggacgaaaa gatcctgttg 180 ttgcgtccgg cgtttcagta tagcgacaac atcgcgaaag aatacgagaa caaattcaaa 240 aaccagaccg cattaaaagt ggaacagatt ctgcaaaacc aaggttacaa agtgatcacc 300 ctggatacca gcgacaagga tgattttagc ttctctcaga aaaaggaggg ctatctggcg 360 cttgccatga atgcagaaat tgttctgcgt ccagatccga aacgcactat ccaaaaaaag 420 agcgagccgg gtctgttatt ttctacgggt ctggacaaga tggaaggtgt cctgattccg 480 gcgggtttca tcaaagtcac cattctggaa ccgatgagcg gcgaaagcct ggactcgttc 540 accatggatc tgagcgagct ggacatccaa gagaagttcc tgaagaccac ccattcgagc 600 cactccggcg gtttggttag cacgatggtt aaaggcaccg ataactccaa tgatgccatg      660 aaaagcgcgt tgaacaaaat ctttgcgaat attatgcagg agatcgacaa aaagctcacg      720 cagaagaacc tggagtctta ccaaaaggac gctaaagagc tcaagaagaa gcgcaatcgt 780 <210> 10 <211> 522 <212> DNA <213> Artificial sequence <400> 10 gggatacaag taggatataa acagtttttc ggccagaaac gtaagtgggg tgctcgctat 60 tacggctttt tcgactataa ccacgcgttt atcaaaagca gcttcttcaa ctctgcaagc        120 gacgtttgga cctatggctt cggcgctgat gcgctgtaca acttcatcaa tgataaagca 180 accaacttct tgggcaagaa caacaaactg agcgttggtt tgtttggtgg gatcgcgctg 240 gcgggtacga gctggctgaa ctctgagtac gtgaacctcg cgactgtaaa taacgtgtat 300 aacgccaaaa tcaacaccgc taatttccaa tttttattta atatgggtgt tcgtatgaat         360 ctggcgcgtt ccaaaaagaa gggtagcgac cacgccgcac agcatggtat tgagctgggt 420 ctgaagattc cgaccattaa caccaattac tactcgttca tgggcgcgga attgaagtac 480 cgccgtctgt actccgtgta cctgaattat gtctttgcgt at                            522 <210> 11 <211> 174 <212> RRT <213> Artificial sequence <400> 11 Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr 1               5                    10                   15 Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu 20               25              30 Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr 35               40               45 Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys 50               55               60 Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu 65                70                75               80 Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala 85               90                 95 Ile Ala Ala Ile Ser Met Ala Asn 100

Claims

1. A Helicobacter pylori (Hp) composite antigen, which is composed of five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, wherein the amino acid sequences of the five antigen proteins are as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 respectively,wherein the five antigen proteins, namely the urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are antigen proteins expressed by performing sequential optimization on corresponding nucleotides of the antigen proteins, and the optimized nucleotide sequences are as shown in SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 respectively.

2. A preparation method of the Hp composite antigen according to claim 1, comprising the following steps:(1) constructing expression plasmids of five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA;(2) transforming the expression plasmids into competence bacteria for expression; collecting the bacteria; and extracting the antigen proteins; and(3) purifying the five antigen proteins by a nickel ion affinity chromatographic column; and lyophilizing and preserving the composite antigen.

3. The preparation method of the Hp composite antigen according to claim 2, wherein in the step (1), nucleotides corresponding to the five antigen proteins, namely urease (UreB), vacuolating cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are subjected to sequential optimization and then cloned into prokaryotic expression vectors; and the expression plasmids are constructed.

4. The preparation method of the Hp composite antigen according to claim 3, wherein the sequential optimization comprises increase of a polypeptide sequence CTB at the N end or C end of the antigen protein, and the sequence CTB is as shown in SEQ ID No. 11; the sequential optimization further comprises increase of a protein expression tag at the N end or C end of the antigen protein, and selection of a hydrophilic region and a conserved region of the antigen protein.

5. An anti-Hp yolk antibody, which is prepared from eggs laid by immunized hens through the Hp composite antigen according to claim 1.

6. A preparation method of the anti-Hp yolk antibody according to claim 5, comprising the following steps:(1) emulsifying the Hp composite antigen according to claim 1 or 2 with an adjuvant;(2) immunizing laying hens: separately raising healthy laying hens at an age of 10-40 weeks; performing subcutaneous injection on the laying hens; selecting 2-6 injection points for each hen; determining an antigen dose for immunization as 80-200 Lig per hen; and enhancing the immunization once every two weeks after the first immunization;(3) collecting eggs laid in the last immunization, namely obtaining eggs containing the yolk antibody resisting2022336191   05 Jun 2026five Hp antigens; and(4) isolating and purifying the eggs containing the yolk antibody resisting five Hp antigens, thereby obtaining the anti-Hp yolk antibody.

7. The preparation method of the anti-Hp yolk antibody according to claim 6, wherein the step (4) specifically comprises: cleaning and disinfecting the eggs containing the anti-Hp yolk antibody, and opening the eggs to let egg white flow out; rolling over the yolk on ordinary filter paper for several times, removing the egg white, breaking yolk membranes, and collecting the yolk; adding distilled water in an amount of 5 times that of the volume of a yolk solution for dilution; fully stirring the solution; freezing the solution at -40°C for 24 hours; slowly melting the frozen solution at 16°C after taken out; filtering the solution by a sterile filter membrane of 400 meshes while melting; filtering the filtered supernatant by ceramic membranes of 8 ^M, 1 liM and 0.2 liM sequentially; collecting a yolk antibody solution; determining absorbance of 280 nm; calculating content of the yolk antibody; and identifying purity of the obtained yolk antibody through SDS-PAGE electrophoresis.

8. Use of the anti-Hp yolk antibody according to claim 5 in preparation of foods, food additives, medicines and health foods.

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