CN113846080B

CN113846080B - Helicobacter pylori composite antigen, antibody, preparation method and application

Info

Publication number: CN113846080B
Application number: CN202111039944.6A
Authority: CN
Inventors: 唐小军; 张秀芳
Original assignee: Youlikang Jiangsu Biological Pharmaceutical Co ltd
Current assignee: Youlikang Jiangsu Biological Pharmaceutical Co ltd
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2022-06-28
Anticipated expiration: 2041-09-06
Also published as: WO2023029666A1; CN113846080A; AU2022336191A1

Abstract

The invention provides a helicobacter pylori composite antigen, which consists of full-length or partial sequences of five antigen proteins of urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA; the amino acid sequences of the five antigen proteins are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5. The anti-Hp yolk antibody prepared by the composite antigen has stronger activity of neutralizing Hp, can effectively block the colonization of Hp and inhibit the activity of Hp, and further effectively prevents and treats gastrointestinal diseases caused by Hp infection.

Description

Helicobacter pylori composite antigen, antibody, preparation method and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a helicobacter pylori composite antigen, an antibody, a preparation method and application.

Background

Helicobacter pylori (Hp) is a spiral, gram-negative microaerophilic bacterium with stringent growth conditions, the only microbial species that has been found to be viable in the human stomach, and was first isolated by Barry Marshall and Robin Warren in 1982. Hp can cause various digestive tract diseases and even gastric cancer, the global infection rate exceeds 50 percent, and the drug resistance of Hp is continuously increased to cause the eradication rate to be continuously reduced, thereby promoting a large amount of refractory infection to occur and seriously threatening the health of human beings.

Hp is continuously colonized on human gastric and duodenal mucosa, and directly damages gastric epithelial cells such as vacuolar degeneration, abnormal proliferation and differentiation and the like by releasing virulence factors such as vacuolar toxin (VacA) and cytotoxin-related gene A protein (CagA) and the like, and induces gastric mucositis to cause indirect damages, thereby causing related gastric diseases.

The World Health Organization (WHO) has listed Hp as a class I carcinogen in 1994. With the continuous and intensive understanding of Hp, in 2015, the global consensus on Kyoto clarified that Hp gastritis is a chronic infectious disease, and it is suggested that Hp infectors should be eradicated unless there are counter factors (advanced age, basic disease, reinfection, etc.).

The drugs currently used to treat Hp infection are mainly classified into 3 classes: antibiotics, Proton Pump Inhibitors (PPI) and bismuth agents. The current clinical common treatment scheme is to use the 3 types of medicines in a compatible way according to a proper proportion and set a proper administration interval so as to achieve an ideal elimination effect.

The H.pyriri is mainly planted in human stomach, and the urease on the outer membrane surface of the H.pyriri can decompose urea in the environment into ammonia and carbon dioxide, so that an ammonia cloud is formed to surround the H.pyriri surface, a nearly neutral microenvironment around the H.pyriri is created, and the H.pyriri is protected from being corroded by gastric acid. At the same time, most antibacterial drugs are less active or even inactive in the extremely strong acid environment in the stomach.

In addition to being able to combat the pH environment of extreme acids, h. Because h.pylori can move continuously depending on its helical form and powerful flagella, and penetrate the gastric mucosal layer of approximately 200 μm and then anchor to the surface of the epithelial cells of the gastric mucosa (a neutral pH environment), not only is gastric acid erosion effectively avoided, but also the influence of gastric emptying is significantly reduced. Conversely, regular gastric emptying makes the presence of the antimicrobial agent in the stomach difficult, reducing its cumulative concentration at the site of infection. Because H.pyrori is fixedly planted in the deep of the mucous layer, the effective contact of the antibacterial drug to the thallus is mechanically isolated, so that the drug is difficult to exert the drug effect, and the difficulty of eradicating the H.pyrori in the stomach is further increased.

Furthermore, H.pyri strains often exchange DNA with each other, resulting in a high probability of gene recombination and a high rate of mutation, thereby causing H.pyri to produce highly variable strains in persistent infection [10 ]. The pyrori gene mutation is an important reason for generating drug resistance. Therefore, even if the concentration of the antibacterial drug in the stomach can reach the concentration threshold for conventional effective treatment, the reduction of the sensitivity of the drug-resistant strain to the antibacterial drug also makes the antibacterial drug unable to achieve the ideal bactericidal effect. In addition, h.pyri infection is generally chronic and persistent, with long-term infections prone to biofilm formation. H.pyrori was first demonstrated to have the ability to form biofilms in 1999. When h.pyri senses an external adverse environment, it embeds into self-secreted Extracellular Polymeric Substrates (EPS) to form a multicellular three-dimensional structure, i.e., a biofilm. Yonezawa et al [12] found that H.pyrori, after forming a biofilm, increases its resistance to clarithromycin: after h.pyri forms a mature biofilm, the Minimum Inhibitory Concentration (MIC) of clarithromycin may increase 16-fold. Residual pathogenic bacteria can lead to recurrent and persistent infections, as pharmacological treatment has difficulty ensuring complete kill of h. Under normal conditions, immune cells, antibodies and the like are involved in immune recognition and response of an organism to H.pyrori infection, and can eliminate a small amount of residual bacteria in the body. Pyrori, however, can successfully evade the host's immune response by modifying its envelope proteins to evade recognition by the body, down-regulate migration and uptake of immune cells, promote apoptosis of macrophages, suppress T cell immune responses, and the like, resulting in long-term colonization and causing persistent infection. Thus, restoring the body's immune response to h.pyri is of paramount importance to increase the eradication rate of h.pyri. (progress of research on anti-helicobacter pylori drug delivery strategy, Chen nan, Vanmei Gu).

Therefore, a safe and effective solution for resisting Hp infection is urgently needed clinically and in the market. The research at home and abroad shows that the Hp-resistant specific yolk immunoglobulin (IgY) prepared by using helicobacter pylori or recombinant protein of a certain pathogenic factor can achieve the Hp-resistant effect and has no drug resistance problem. The Yolk Immunoglobulin (IgY) is the only Immunoglobulin in Yolk, and the IgY has the advantages of fast production, high yield, high titer, stable physicochemical property, wide source and no toxic or side effect. Anti-Hp-IgY can resist the degradation of pepsin in the stomach, and can generate immune response to Hp infection at the level of mucous membranes after entering the body, thereby reducing inflammation and ulcer caused by the Hp-IgY; can also specifically aim at Hp infection, can neutralize Hp protein toxin after being taken orally, block the toxin activity of Hp protein toxin on epithelial cells, eradicate or lighten Hp infection and improve the immunity of human bodies.

Egg yolk antibodies against H.pylori have been reported for more than 15 years and the recombinant antigens selected include UreB, Hsp60, CagA, VacA, NAP, OMP18, HpaA, HspA and the like. The applicant also researches an Hp tetravalent antigen and has applied for Chinese invention patent with the publication number of CN111793137A and the name of "an Hp tetravalent antigen and a preparation method and application thereof", which is characterized in that corresponding nucleotides of four antigen proteins of UreB, VacA, CagA and HpaA are optimized and then cloned into pET28a, pET30 or pColdII prokaryotic expression vectors, and the antigen is obtained by prokaryotic expression bacteria BL21, Rosetta or OrigamiB expression, and the egg containing the yolk antibody of the Hp tetravalent antigen and the yolk antibody capable of efficiently blocking the Hp colonization and infection are prepared by the Hp tetravalent antigen; compared with the existing product, the yolk antibody has good solubility and high product purity, and the novel yolk antibody containing tetravalent antigen Hp can block the permanent planting of Hp, effectively inhibit the activity of Hp, and can effectively prevent and treat gastrointestinal diseases caused by Hp infection. However, the tetravalent antigen mainly targets the toxin factor and HpaA of helicobacter pylori, and has poor colonization inhibition effect on helicobacter pylori which does not completely or independently exert adhesion effect on HpaA, so that the effect of treating different helicobacter pylori infections is still to be improved.

An important factor for the colonization of Hp in the stomach is the adhesion of bacteria to the gastric mucosa, which reflects the existence of some adhesion factors for Hp and the existence of corresponding specific receptors for gastric epithelial cells, and the specific binding of these adhesion factors to the corresponding receptors, so that Hp can be stably colonized on the surface of gastric mucosa for a long time, providing a precondition for its pathogenic effect (Boyle EC. FinlayBB. bacteriological pathogenesis: proliferating cellular adherence [ J. Curropin Cell Biol, 2003; 15(5): 633-. Although adhesins play an important role in the course of H.pylori infection, different H.pylori can adhere to gastric mucosal epithelial cells using different adhesins. Therefore, a solution is needed to achieve an effective effect of inhibiting the colonization of Hp and at the same time to effectively inhibit the activity of Hp, so as to prevent and treat gastrointestinal diseases caused by Hp infection.

Disclosure of Invention

The invention provides a helicobacter pylori composite antigen, an antibody, a preparation method and an application, the antigen has higher immunogenicity than the existing 4-valent antigen, and an anti-Hp yolk antibody prepared by the composite antigen has better broad spectrum and stronger Hp neutralizing activity, can simultaneously and efficiently block the colonization of Hp, inhibit the activity of Hp and neutralize the toxin of Hp, and further efficiently prevent and treat gastrointestinal diseases caused by Hp infection.

Therefore, the invention provides a helicobacter pylori composite antigen which consists of full-length or partial sequences of five antigen proteins of urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA; the amino acid sequences of the five antigen proteins are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5.

Preferably, five antigen proteins of urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA are antigen proteins obtained by expression after the corresponding nucleotide sequences are optimized, and the optimized nucleotide sequences are respectively shown as SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10.

The invention also provides a preparation method of the helicobacter pylori composite antigen, which comprises the following steps:

(1) constructing expression plasmids of five antigen proteins of urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA;

(2) transforming the cells into competent bacteria for expression, collecting the bacteria, and extracting antigen protein;

(3) purifying five antigen proteins by using a nickel ion affinity chromatography column, and freeze-drying and storing.

Preferably, in the step (1), the nucleotide corresponding to five antigen proteins, urease (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are subjected to sequence optimization and then cloned into a prokaryotic expression vector to construct an expression plasmid.

Preferably, the sequence optimization comprises adding a polypeptide sequence CTB at the N end or the C end of the antigen protein, wherein the CTB sequence is shown as SEQ ID No. 11; also includes adding protein expression label at N-terminal or C-terminal of antigen protein; and selecting a hydrophilic region and a conserved region of the antigen protein.

The invention also provides a yolk antibody for resisting helicobacter pylori, which is obtained by laying eggs after the helicobacter pylori composite antigen is immunized by hens.

The invention also provides a preparation method of the helicobacter pylori resistant yolk antibody, which comprises the following steps:

(1) emulsifying the helicobacter pylori complex antigen of claim 1 or 2 with an adjuvant;

(2) immunizing egg-laying hens: healthy laying hens of 10-40 weeks old are selected for isolated feeding, subcutaneous injection is carried out, 2-6 injection points are selected for injection of each hen, the antigen amount for immunization is 80-200 mug per chicken, and after the first immunization, the immunization is strengthened once every two weeks;

(3) Collecting the eggs laid by the last immunization to obtain eggs containing yolk antibodies for resisting five Hp antigens;

(4) and (3) separating and purifying the eggs containing the yolk antibodies for resisting the five Hp antigens to obtain the Hp-resistant yolk antibodies.

Preferably, the step (4) is specifically: cleaning and sterilizing egg with anti-Hp yolk antibody, and opening to allow egg white to flow out; turning and rolling the yolk on common filter paper for several times, removing egg white, and breaking yolk membrane to collect yolk; adding distilled water with volume 5 times of the volume of the yolk solution for dilution, fully stirring, freezing at-40 ℃ for 24 hours, taking out, slowly thawing at 16 ℃, and filtering with a 400-mesh sterile filter membrane while thawing. Filtering the supernatant with ceramic membranes of 8 μ M, 1 μ M and 0.2 μ M, collecting egg yolk antibody solution, measuring absorbance at 280nm, and calculating egg yolk antibody content; the purity of the obtained yolk antibody is identified by SDS-PAGE electrophoresis.

The invention also provides the application of the helicobacter pylori resistant yolk antibody in the preparation of foods, food additives, medicines and health-care foods.

Has the beneficial effects that:

the helicobacter pylori composite antigen consists of five antigen proteins, including urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesion HpaA and adhesion BabA, and has raised effective antigen concentration and purity, raised chicken's immune reaction and obviously raised yolk antibody titer against helicobacter pylori. In addition, more than 95% of antigens which can be expressed by clinical strains are selected as immunogens to obtain the Hp composite antigen with high immunogenicity.

The Hp pentavalent composite antigen of the invention contains adhesin BabA, so that the antigen adhesin yolk antibody induced by the composite antigen can effectively prevent the colonization of helicobacter pylori in vivo, thereby achieving the effect of clearing. Through detection, the pentavalent composite antigen of the present invention combined with yolk antibody produced by HpaA and BabA can inhibit the adhesion of pyloric helicobacter in over 95%.

In addition, the composite antigen of the invention combines the protein difference of different clinical strains, further optimizes the amino acid sequence of the antigen protein, and ensures that the prepared yolk antibody for resisting helicobacter pylori has wider spectrum. Through the selection of the antigen region, the expression quantity and purity of the antigen can be improved, and in addition, the induced egg yolk antibody can better identify and neutralize the extracellular protein of the helicobacter pylori, so that the helicobacter pylori can be more efficiently eliminated.

Drawings

In order that the present invention may be more readily and clearly understood, reference will now be made in detail to the present invention, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a diagram showing the SDS-PAGE results of five antigens prepared in example 1 of the present invention;

FIG. 2 is a graph showing the results of the inhibitory effect of yolk antibody against helicobacter pylori, produced by the pentavalent complex antigen prepared in this example 1 of the present invention and the tetravalent complex antigen which has been studied;

FIG. 3 is a schematic diagram showing the evaluation results of the safety and effect of the anti-Hp yolk antibody in vivo therapy prepared in example 2 of the present invention.

Detailed Description

The following description will be given with reference to the embodiments in order to explain the technical contents, the objects and the effects of the present invention in detail.

The reagents and competent bacteria used in the present specification were commercially available products except for the specific instructions.

The related diseases caused by helicobacter pylori are mainly caused by the destruction of gastric cells by Hp, and the current experiments and theories prove that the related diseases are mainly related to three proteins of Hp in vivo expression urease (urea, Ure), vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (cagA).

Ure is an enzyme rich in helicobacter, which changes the local strongly acidic environment in the stomach and plays an important role in the gastric parasitism and pathogenesis of this bacterium, and all Hp bacteria found so far contain urease. UreB is an active center forming Urea, has good conservation and antigenicity and is an ideal Hp vaccine target.

VacA exists in the genome of all Hp strains, the expression product of the VacA is the only protein toxin secreted by Hp, and the VacA is an important virulence factor and can change cell membranes to form a transmembrane anion channel, so that acid hydrolase is released to cause cell swelling, and the vacuole-like degeneration of epithelial cells is finally generated along with cell membrane fusion.

CagA is one of Hp virulence markers and is related to Hp pathogenicity and clinical related disease severity.

Studies on Hp have shown that Hp colonizes the gastric mucosa by specific binding of its surface ligands to the gastric mucosal epithelial cell receptors. HpaA is the flagellium membrane protein of Hp, is one of important adhesins, exists on the surface of almost all clinically isolated Hp strains, and has the characteristics of surface exposure, high antigen conservation and the like. In addition, the HpaA vaccine can induce protective effects in animals immunized with the HpaA vaccine.

BabA is one of the discovered adhesins of helicobacter pylori, which is capable of binding to fucosylated lewis blood group antigens on the surface of gastric epithelial cells to mediate adhesion of helicobacter pylori to gastric epithelial cells. The BabA is coded by a babA2 gene, and researches show that the BabA2 gene is closely related to the occurrence of duodenal ulcer and gastric cancer. The full length of BabA is 746 amino acids, the homology of helicobacter pylori of different strains is only about 85%, the invention carries out optimization screening on the protein sequence, selects the membrane outer region (573-746) of the protein as an antigen, and determines that the homology of the region in different helicobacter pylori is more than 98%.

Cholera enterotoxin (CT) is an important virulence factor of Vibrio cholerae and contains two subunits, A and B. CTB removes the toxicity of CT, has mucosal adjuvant activity, can effectively stimulate the body to secrete antitoxin antibodies, and the most basic receptor GM1 exists on the surface of most mammalian cells, so the CTB can be used as a good adjuvant. CTB can 1) assist in enhancing the presentation of DC and other APC to antigen, and promote antigen to cross mucosal barrier; 2) inducing the organism to secrete specific antibodies; 3) stimulating the expression of receptors and the secretion of cytokines on the surface of T cells, B cells and APC. Many scholars at home and abroad successfully construct expression vectors of CTB and antigen or antigenic determinant fusion protein and express the CTB and the antigen or antigenic determinant fusion protein in host bacteria, so that the method for enhancing the immunogenicity of specific antigens through the fusion protein is a feasible way. In the invention, the CTB sequence is added to the HpaA antigen, so that the titer of the yolk antibody against the HpaA is obviously enhanced.

The pentavalent complex antigen of urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA which are researched and designed improves the expression quantity and purity of the antigen and reduces the preparation cost of the antigen by specific antigen compounding, sequence optimization and selection of an antigen region; further, the induced yolk antibody can better identify and neutralize extracellular protein of helicobacter pylori, effectively prevent the helicobacter pylori from being colonized in vivo, thereby efficiently eliminating the helicobacter, and the obtained yolk antibody has broad spectrum.

Example 1

The embodiment provides a Hp pentavalent complex antigen, which consists of five antigen proteins, namely urease (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA; the amino acid sequences of the five antigen proteins are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 after being optimized; wherein, the sequence optimization comprises the optimization according to the escherichia coli code preference and the helicobacter pylori sequence conservation, and the polypeptide sequence CTB is added at the N end or the C end of the HpaA antigen protein, and the CTB sequence is shown as SEQ ID No. 11; protein expression tags are added to the N-terminal or C-terminal of each antigenic protein.

The preparation method comprises the following steps:

expression of S1, Hp Complex antigen

Carrying out sequence optimization on nucleotides corresponding to five antigen proteins of urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, and then cloning the five antigen proteins into a prokaryotic expression vector to construct an expression plasmid; transforming the expression plasmid into competent bacteria BL21, Rosetta or OrigamiB, inducing for 4h by IPTG at 37 ℃, centrifugally collecting bacteria, cracking the bacteria by adopting ultrasonic disruption, and centrifugally collecting bacterial cracking supernatant;

wherein, the competent bacteria BL21, Rosetta or OrigamiB can be obtained by purchasing;

purification of S2 Hp Complex antigen

Purifying the antigen by adopting a nickel ion affinity chromatography column: cracking the bacteria expressing the antigen to obtain supernatant, filtering, adding the sample to a nickel ion affinity column, washing with a buffer solution to a baseline state, eluting with an eluent, and collecting an elution peak; dialyzing and concentrating the eluted protein, and freeze-drying and storing;

SDS-PAGE detection of S3, Hp composite antigen

(1) Mixing the prepared five Hp antigen protein samples with 5 × loading buffer according to the volume ratio of 1:4, performing denaturation by metal bath at 95 ℃ for 5min, and storing in a refrigerator at-80 ℃ for later use;

(2) Preparing glue: preparing separation gel and concentrated gel by referring to the specification of an SDS-PAGE gel kit;

(3) sampling: cleaning a glass plate, detecting leakage, filling glue, transferring the glue-making plate into an electrophoresis tank after the concentrated glue is solidified, taking down a comb after the electrophoresis tank is filled with electrophoresis buffer, adding denatured protein samples into sample adding holes of gel, paying attention to ensure that the total protein content of each hole is consistent, and adding a marker into another hole;

(4) electrophoresis: adding a proper amount of electrophoresis buffer solution into an electrophoresis box, assembling an electrophoresis apparatus, carrying out electrophoresis at a constant voltage of 80V, and stopping electrophoresis when a sample runs to the bottom of a gel;

(5) coomassie brilliant blue staining: placing the SDS-PAGE gel into Coomassie brilliant blue staining for 1 h;

(6) and (3) decoloring: decolorizing the dyed SDS-PAGE gel with a decolorizing solution until protein bands are clear;

(7) the test results are shown in fig. 1, and the purity of the five antigens is more than 85%.

Experimental examples for obtaining the amount of antigen expression:

the expression level of the truncated and expressed BabA antigen is higher than that of the full-length BabA antigen, prokaryotic expression plasmids expressing the full length BabA and the BabA-OPM region are respectively transformed into BL21 escherichia coli, after IPTG induction expression, bacteria are subjected to ultrasonic lysis, protein is purified through affinity chromatography (the specific method is the same as the examples 1 and 2), the protein concentration of the stored protein is measured by a BCA method, and the result shows that the expression level of the plasmid expressing the full length BabA is 0.42mg/L, the expression level of the plasmid expressing the BabA-OPM is 1.34mg/L, and the expression level of the BabA antigen soluble protein expressed by truncation is 3.2 times of that of the full-length antigen.

Comparative examples of the yolk antibody inhibiting effect on helicobacter pylori of the pentavalent complex antigen prepared in this example and the tetravalent complex antigen studied (see chinese patent application CN 111793137A):

MGC-803 cells in logarithmic growth phase are digested with pancreatin, and the cell concentration is regulated to 2 × 10⁴Adding cells/ml into 6-well plate with sterile cover glass, culturing at 37 deg.C for 24 hr, after cell adherence, adding culture medium containing pentavalent anti-helicobacter pylori and tetravalent anti-helicobacter pylori yolk antibody 1ml into experimental group, respectively, the yolk antibody concentration is 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, common yolk antibody 20 μ g/ml is added into control group, and continuously adding helicobacter pylori strain A (HpaA high expression, BabA low expression) or helicobacter pylori strain B (HpaA low expression, BabA high expression) 4 × 10⁶1ml of each cfu/ml (4 wells per concentration), placing the cfu/ml into a 37 ℃ cell incubator for further culture for 2 hours, washing the cfu/ml with PBS for 3 times, taking out a cover glass, airing, fixing the cfu/ml with methanol for 15 minutes, washing the cfu/ml with PBS for one time, then dyeing the cfu/ml with a methylene blue method, observing the number of bacteria of the helicobacter pylori adsorbed on each cell by an oil-mirror observation technology, counting 15 cells per group, and calculating the average number of bacteria adhered to each cell.

Referring to FIG. 2, the test results showed that the pentavalent anti-H.pylori yolk antibody had a significantly better inhibitory effect on both H.pylori A and H.pylori B tested than the tetravalent anti-H.pylori yolk antibody. It can be seen that the inhibition of H.pylori A and H.pylori B by the pentavalent H.pylori resistant egg yolk antibody of the invention is significantly improved.

Example 2

This example provides a yolk antibody against helicobacter pylori, which was obtained by inducing hen immunization with the pentavalent complex antigen prepared in example 1, and further obtaining a yolk antibody against five Hp antigens. The preparation method comprises the following specific steps:

(1) emulsifying the five Hp combined antigens by adopting an adjuvant; the adjuvant is Freund's complete adjuvant;

(2) immunizing egg-laying hens: healthy laying hens aged 10-40 weeks are selected for isolated feeding, subcutaneous injection is carried out, 2-6 injection points are selected for injection of each hen, the antigen amount for immunization is 80-200 mug/chicken, preferably 80-200 mug/chicken, 100 mug/chicken is adopted in the embodiment, and after the first immunization, the immunization is strengthened once every two weeks;

(4) separating and purifying eggs containing yolk antibodies for resisting five Hp antigens to obtain the Hp-resistant yolk antibodies, which comprises the following steps: cleaning and sterilizing eggs containing anti-Hp yolk antibodies, and opening the openings to allow egg white to flow out; turning and rolling the yolk on common filter paper for several times, removing egg white, and breaking yolk membrane to collect yolk; adding distilled water 5 times the volume of the yolk solution, diluting, stirring, freezing at-40 deg.C for 24 hr, taking out, slowly thawing at 16 deg.C, and filtering with 400 mesh sterile filter membrane. Filtering the supernatant with ceramic membranes of 8 μ M, 1 μ M and 0.2 μ M, collecting yolk antibody solution, measuring absorbance at 280nm, and calculating yolk antibody content; the purity of the obtained yolk antibody is identified by SDS-PAGE electrophoresis.

Experimental example for identifying the immunological Effect of the antibody

1. ELISA titer of prepared Hp-resistant yolk antibody

(1) Dissolving the lyophilized yolk antibody with sterile water to 1 mg/ml;

(2) diluting UreB (2. mu.g/ml), CagA (0.5. mu.g/ml), VacA (0.5. mu.g/ml), HpaA (1. mu.g/ml) and BabA (0.5. mu.g/ml) with a carbonate buffer solution or PBS buffer solution of pH9.6, adding the diluted antigens to the wells of an ELISA plate (100. mu.l/well), applying a sealing membrane, and standing overnight at 4 ℃;

(3) the coating solution was spun off and washed 3 times with TBST containing 0.05% Tween 20;

(4) add 300. mu.l TBS blocking solution containing 3% BSA per well, incubate 1h at 37 ℃ and wash 3 times with TBST;

(5) diluting yolk antibody (1mg/ml) by 1 ten thousand times with PBS, diluting by multiple times, adding into enzyme-linked immunosorbent assay plates coated with different antigens, incubating for 2h at 37 ℃, and washing for 5 times with TBST;

(6) adding 100 μ l/well of horse radish peroxidase-labeled (HRP) anti-chicken IgY antibody diluted with TBS 1:5000, incubating at 37 deg.C for 1h, and using TBST system for 5 times;

(7) adding 100 mul/hole TMB color development solution, incubating at room temperature for 5-15 min, stopping color development reaction by 2N sulfuric acid, reading an OD value of 450nm by an enzyme-labeling instrument, wherein the OD value is 1.6 times greater than that of a negative control and is positive, and the maximum dilution multiple of a positive value hole is the titer of an antibody;

(8) The result shows that the titer of the yolk antibody (1mg/ml) for resisting helicobacter pylori, which is prepared by immunizing laying hens with the composite antigen provided by the invention, is as follows: the ELISA titer of the anti-UreB is 1:64 ten thousand; the titer of the ELISA for resisting CagA is 1:64 ten thousand; the titer of the VacA-resistant ELISA is 1:32 ten thousand; the titer of ELISA for resisting HpaA is 1:64 ten thousand; the ELISA titer against BabA was 1:32 ten thousand. Therefore, the yolk antibody obtained by the quinquevalent composite antigen immune layer chicken has higher titer.

2. CTB fusion protein for enhancing immune effect of HpaA

Immunizing laying hens with HpaA and HpaA-CTB, collecting egg yolk antibodies prepared from egg yolks, and adjusting the concentration of the egg yolk antibodies to be 1 mg/ml; the HpaA antigen was diluted to 1. mu.g/ml with a carbonate buffer solution of pH9.6, added to an ELISA plate at 100. mu.l/well, and incubated overnight at 4 ℃ in the test procedure of reference example 5, which revealed that the titer of ELISA against egg yolk antibody HpA after HpaA immunization was 1:16 ten thousand and the titer of ELISA against egg yolk antibody HpA after HpaA-CTB immunization was 1:64 ten thousand.

3. The affinity of the IgY (IgY-OPM) generated by the immunization of the BabA OPM region and the antigen is obviously superior to that of the IgY (IgY-BabA) generated by the full-length immunization of the BabA

Antibody affinity was detected using BLITZ:

yolk antibody was diluted to 25ug/ml with buffer (1 × PBS containing 0.02% Tween, 0.1% BSA) to 200ul per tube; BabA-OPM and BabA antigens were diluted to 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 0, 20ul each, respectively, and added to the probe. The probe was placed in PBS for 10min activation. The antibody was incubated at room temperature for 1 h. The machine was operated, 200ul of buffer was loaded into 500ul of brown EP tubes, and placed on the machine for detection.

Data analysis was performed and probes were recovered.

The detection result shows that the yolk antibody obtained by using the BabA-OPM immunizationThe affinity of the body to BabA is KD is 2.3 × 10^-8The affinity of the yolk antibody obtained by full-length immunization with the BabA to the BabA is KD ═ 5.2 × 10^-7. Therefore, the affinity of the IgY (IgY-OPM) generated by the BabA OPM region immunization and the antigen is obviously better than that of the IgY (IgY-BabA) generated by the BabA full-length immunization and the BabA.

4. Protective effect of prepared anti-Hp egg yolk antibody on mice

The reference literature establishes a mouse Hp infection model, and experiments are divided into a model group, a common IgY control group and an anti-Hp IgY group prepared by immunization of five antigens.

The experimental procedure was as follows:

40 BALB/c mice were equally divided into 5 groups (blank group, infected group, common yolk antibody group (i.e., yolk antibody extracted from non-specific immunized egg), tetravalent yolk antibody group, and pentavalent yolk antibody group), fasted for 24h, the blank group was gavaged with 0.2ml Brookfield broth, and the other groups were gavaged with 3% NaHCO₃0.1ml of yolk antibody group, 0.2ml of yolk antibody with the concentration of 1mg/ml, and after 15 minutes, the groups except the blank group have the concentration of 0.2ml of yolk antibody with the concentration of 1 multiplied by 10 ⁹The same gavage is performed on the CFU/ml pylorus screw strain B bacterial liquid 1 time every two days, and 3 times are total. Mice were sacrificed 8 weeks after the last gavage. Taking the stomach under aseptic condition, placing on sterilized filter paper, removing forestomach, cutting along the stomach bay, washing off residual food in the stomach by using sterile physiological saline, dividing the stomach into two parts in the longitudinal row, wherein one part is used for bacterial fast urease experiment, and the other part is used for bacterial culture. Mice positive to the fast urease test and to the bacterial culture are judged to be positive to the helicobacter pylori infection. Urokinase assay: the mouse gastric mucosa suspension is added into a rapid urease determination kit, and the reagent turns positive from yellow red within 30 minutes. Helicobacter pylori culture experiment: placing urease-positive stomach tissue on helicobacter pylori selective culture medium, repeatedly smearing mucosa surface downwards, and streaking at 5% O₂、10％CO₂、85％N₂Culturing at 37 ℃ for 3-4 days. Calculation of infection Rate: the infection rate was (infection positive mice/8) × 100%, and the results of the experiment were statistically analyzed by Fisher's exact probability method, and the results are shown in table 1 below.

TABLE 1

The result shows that the protective effect of the pentavalent egg yolk antibody on the helicobacter pylori strain B is superior to that of the tetravalent egg yolk antibody and is obviously superior to that of the common egg yolk antibody.

5. In vivo treatment safety and effect evaluation of prepared anti-Hp egg yolk antibody

Immunizing laying hens with the composite antigen, collecting eggs, preparing egg yolk antibodies, adjusting the concentration of the egg yolk antibodies to 1mg/ml, detecting that the titer of anti-UreB is more than 1:64 ten thousand, the titer of anti-CagA is more than 1:64 ten thousand, the titer of anti-VacA is more than 1:32 ten thousand, the titer of anti-HpaA is more than 1:64 ten thousand and the titer of anti-helicobacter pylori is more than 1:32 ten thousand by ELISA, collecting the eggs, preparing the egg yolk antibodies of anti-helicobacter pylori, processing the egg yolk antibodies into oral capsules (250 mg/capsule, namely each oral capsule contains 250mg of the antibodies) by adopting the prior art, recruiting 39 helicobacter pylori positive subjects relapsed after being treated by a standard quadruple therapy scheme, wherein 16 subjects have different and different degrees of clinical symptoms comprising stomachache, gastrospasm, acid resistance and the like, after signing consent, continuously taking the egg yolk antibodies of anti-helicobacter pylori for 2 weeks by each subject, respectively 1 time in the morning and evening, 1 capsule is orally taken within half an hour after each meal. DOB values detected by C13 were measured before and 1 week and 2 weeks after administration. The subjects were evaluated for clinical symptoms and adverse events at the treatment end. The DOB values detected by C13 of the test subjects before and after the anti-helicobacter pylori yolk antibody treatment are subjected to statistical analysis by using a paired T test, Graphad8.0.1 software is used for drawing, and the P value is less than 0.05, so that the test subjects are judged to have statistical difference.

The results of the test are shown in fig. 3, and show that none of the 39 subjects had adverse effects after treatment, and among them, the DOB value detected by C13 of 34 subjects was reduced, and the effectiveness was 88.2%. Among them 8 subjects (20.5%) had a DOB value below 4 after treatment, achieving the clinical effect of helicobacter pylori clearance.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.

<110> Yulikang (Jiangsu) biomedical Co., Ltd

<120> helicobacter pylori composite antigen, antibody, preparation method and application

<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

Thr Gly 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 Thr Phe 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 Thr Thr 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 Phe Asn 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

Gly Asn 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 Gly Cys 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 Gly Thr 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> PRT

<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. The helicobacter pylori complex antigen is characterized by consisting of five antigen proteins, namely urease (UreB), vacuole cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and truncated adhesin BabA; the amino acid sequences of the five antigen proteins are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5.

2. The helicobacter pylori composite antigen as claimed in claim 1, wherein five antigen proteins, including urease (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are expressed by optimizing their corresponding nucleotide sequences, and the optimized nucleotide sequences are shown as SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10.

3. The method for producing a helicobacter pylori complex antigen according to claim 1 or 2, comprising the steps of:

4. The method for preparing a helicobacter pylori composite antigen as claimed in claim 3, wherein in step (1), the corresponding nucleotides of five antigen proteins, urease (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA), adhesin HpaA and adhesin BabA, are subjected to sequence optimization and then cloned into a prokaryotic expression vector to construct an expression plasmid.

5. The method for preparing the helicobacter pylori composite antigen as claimed in claim 4, wherein the sequence optimization comprises adding a polypeptide sequence CTB to the N-terminal or C-terminal of the antigen protein, wherein the CTB sequence is shown as SEQ ID No. 11; and adding a protein expression tag at the N-terminal or the C-terminal of the antigen protein; and selecting a hydrophilic region and a conservative region of the antigen protein.

6. A yolk antibody against helicobacter pylori, characterized in that it is produced by immunizing a hen with the complex antigen of helicobacter pylori according to claim 1 or 2, and then laying an egg.

7. The method for preparing yolk antibody against helicobacter pylori according to claim 6, comprising the steps of:

(2) immunizing egg-laying hens: selecting healthy laying hens of 10-40 weeks old for isolated feeding, performing subcutaneous injection, selecting 2-6 injection points for each hen injection, immunizing with antigen amount of 80-200 mug/chicken, and after primary immunization, enhancing the immunization once every two weeks;

(4) and separating and purifying the eggs containing the yolk antibodies for resisting the five Hp antigens to obtain the Hp-resistant yolk antibodies.

8. The method for preparing a yolk antibody against helicobacter pylori according to claim 7, wherein the step (4) is specifically as follows: cleaning and sterilizing egg with anti-Hp yolk antibody, and opening to allow egg white to flow out; turning and rolling the yolk on common filter paper for several times, removing egg white, and breaking yolk membrane to collect yolk; adding distilled water with the volume 5 times of that of the egg yolk solution for dilution, fully stirring, freezing at-40 ℃ for 24 hours, taking out, slowly melting at 16 ℃, filtering by using a 400-mesh sterile filter membrane while melting, sequentially filtering filtered supernatants by using ceramic membranes of 8 mu M, 1 mu M and 0.2 mu M, collecting egg yolk antibody solution, measuring the absorbance at 280nm, and calculating the content of the egg yolk antibody; the purity of the obtained yolk antibody is identified by SDS-PAGE electrophoresis.

9. The use of the yolk antibody against helicobacter pylori according to claim 6 in the manufacture of food, food additives, pharmaceuticals and health foods.