Helicobacter pylori immune protection formulation and use thereof in preparation of immune cow milk

Abstract

The present invention relates to the field of biotechnology, and in particular, to a Helicobacter pylori immune protection formulation and use thereof in the preparation of immune cow milk. The present invention provides a Helicobacter pylori immune protection formulation comprising Helicobacter pylori, a natural antigen of Helicobacter pylori, and a recombinant antigen, immune cow milk prepared from the immune protection formulation, and use thereof in the preparation of a product for preventing and treating a Helicobacter pylori infection.

Description

Helicobacter pylori immune protection preparation and its application in preparing immune milk

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 20, 2023, with application number 202311765809.9 and invention name “Helicobacter pylori immune protection preparation and its application in the preparation of immune milk”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biotechnology, in particular to a Helicobacter pylori immune protection preparation and application thereof in preparing immune milk. Background Art

[0003] Helicobacter pylori (H. pylori) is a common pathogen of digestive tract diseases including gastritis and peptic ulcers, and is also associated with the development of gastric cancer. Therefore, H. pylori infection poses a serious threat to national health, and the prevention and treatment of related diseases consumes a large amount of social medical and health resources. Currently, for H. pylori infection, particularly virulent H. pylori infection, a multi-therapy treatment including antibiotics is usually used to eliminate the infection and treat related diseases. However, with the widespread development of antibiotic-based anti-H. pylori therapy, H. pylori resistance to antibiotics has become a clinical problem that cannot be ignored. The direct consequence of drug resistance is that the clearance rate of H. pylori by standard therapy is greatly reduced. Moreover, the antibiotics used for anti-H. pylori treatment will destroy the normal flora in the body during use, disrupting the normal microecological system that maintains the body's health, posing a significant threat and harm to the health of infected patients. Meanwhile, anti-Helicobacter pylori treatment options also include proton pump inhibitors and bismuth. Side effects of proton pump inhibitors include gastrointestinal adverse reactions, liver damage, allergic reactions, kidney damage, neuropsychiatric impairment, vision impairment, endocrine and reproductive system adverse reactions, and circulatory system adverse reactions. Side effects of bismuth include bismuth poisoning and bismuth encephalopathy for the nervous system; an ammoniacal taste in the mouth, diarrhea, bismuth in stool, and gray-black stool for the digestive system; renal impairment and renal failure for the urinary system; osteoarthritis for the musculoskeletal system; and the risk of drug-drug interactions. Current data show that the success rate of anti-Helicobacter pylori treatment has dropped from over 95% initially to 60-80%. Repeated antimicrobial treatment after treatment failure not only worsens H. pylori resistance but also disrupts the infected individual's normal microbiome and microbiome, seriously threatening patient health. Furthermore, repeated treatment inevitably increases medical expenses and burdens, diverting precious medical resources. Therefore, the current conventional anti-Helicobacter pylori treatment causes great damage to the body while clearing Helicobacter pylori infection. Doctors should carefully weigh the benefits and costs of the above treatment and treat the infected people under the premise that the harm to the patient can be controlled.

[0004] There are two coping strategies for the current difficulties in the treatment of Helicobacter pylori infection. One is to conduct in vitro drug sensitivity tests on Helicobacter pylori while maintaining the existing treatment plan. Sensitive antibiotics can be selected based on the results of in vitro antibiotic sensitivity tests, and the use of resistant antibiotics can be avoided, thereby guiding the design and formulation of clinical treatment plans, providing more precise treatment for Helicobacter pylori, greatly improving the success rate of treatment, and avoiding treatment failure. The other strategy is to choose non-antibiotic Helicobacter pylori elimination methods, especially using related products prepared based on immunological methods and principles to eliminate Helicobacter pylori infection.

[0005] There are two approaches to immune prevention and treatment of pathogenic microorganism infections: active immunization and passive immunization. Active immunization involves preparing a non-pathogenic inactivated form of the pathogen or key functional proteins as a vaccine. Through vaccination, the body is induced to produce antibodies and immune effector molecules against specific pathogens, thereby making the body immune to those pathogens. Passive immunization refers to the direct use of immune effector molecules, including antibodies, to help the body produce and enhance immunity to specific pathogens, thereby quickly eliminating pathogen infections. Similarly, the above two methods and routes can also be applied to the prevention and treatment of Helicobacter pylori infection. Immunological clearance methods for Helicobacter pylori infection and treatments for related diseases also include active immunization and passive immunization. One is a Helicobacter pylori vaccine based on active immunization, which uses Helicobacter pylori-related bacterial components to immunize the body, inducing the body to produce corresponding protective antibodies, thereby protecting the body from Helicobacter pylori infection. However, because Helicobacter pylori infection is a localized infection that colonizes the gastric mucosa, even if vaccination can induce the development of protective antibodies, with high titers of protective antibodies circulating in the gastric lumen, these antibodies are unlikely to accumulate in the gastric lumen where Helicobacter pylori colonizes and exert a protective effect. This is supported by the fact that, while most individuals infected with Helicobacter pylori have high titers of Helicobacter pylori antibodies in their serum, these antibodies do not exert a measurable protective effect against infection, much less clear existing infection. Numerous studies have shown that once Helicobacter pylori infects the human body, the infection cannot resolve on its own unless the individual receives systematic, standardized anti-H. pylori treatment.

[0006] Based on the current status of anti-Helicobacter pylori treatment, we urgently need to develop new therapeutic drugs and methods to deal with Hp infection. They should not only be safer and more effective, but also play an immune preventive role for the general susceptible population. At the same time, they should also have the greatest applicability and can be quickly and conveniently applied to all general populations. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a Helicobacter pylori immune protection preparation and its application in the preparation of immune milk. The present invention provides an effective, safe and easy-to-popularize Helicobacter pylori immune protection preparation, as well as its application in the preparation of immune milk. The prepared immune milk is safe and has a high Helicobacter pylori antibody titer.

[0008] The present invention provides an immunoprotective preparation for Helicobacter pylori, comprising at least one of Helicobacter pylori, a natural antigen, and a recombinant antigen of Helicobacter pylori, wherein:

[0009] The natural antigens include the whole bacterial protein of Helicobacter pylori;

[0010] The recombinant antigen includes at least one of cytotoxin-associated protein A, vacuolating toxin, urease and heat shock protein, and the urease includes urease A and / or urease B.

[0011] Compared with the existing technology, the present invention uses natural live Helicobacter pylori for immunization of dairy cows, and simultaneously uses natural antigens and recombinant antigens of Helicobacter pylori for comprehensive immunization of dairy cows. The three immunogens synergistically immunize, further improving the immune effect of the immune protection preparation, thereby achieving more excellent technical effects.

[0012] The present invention provides application of the immune protection preparation in preparing immune milk.

[0013] The present invention provides a method for preparing immune cow's milk, which comprises the steps of immunizing a dairy cow with the immune protection preparation to obtain the immune cow's milk.

[0014] In some embodiments, the immunization comprises:

[0015] feeding the live Helicobacter pylori bacteria to dairy cows; and / or

[0016] The natural antigen and / or recombinant antigen of Helicobacter pylori is injected and immunized into dairy cows.

[0017] In some specific embodiments, the feeding comprises using a mixture containing 5×10 10 Live CFU Helicobacter pylori bacteria are fed to dairy cows at a frequency of three times a day for 7 consecutive days.

[0018] In other specific embodiments, the method for preparing the natural antigen comprises: taking the Helicobacter pylori, lysing it with lysozyme, and ultrasonically disrupting it, collecting the lysis supernatant and precipitate, dissolving the precipitate with urea, and centrifuging it, dialyzing the supernatant after the centrifugation to obtain a precipitation supernatant, and mixing the lysis supernatant and the precipitation supernatant to obtain the natural antigen of the Helicobacter pylori;

[0019] The preparation method of the recombinant antigen comprises: constructing recombinant plasmids of cytotoxin-associated protein A, vacuolating toxin, urease and heat shock protein respectively, and obtaining the recombinant antigen after prokaryotic expression and purification.

[0020] In some embodiments, the urease A has:

[0021] (1) the amino acid sequence shown in SEQ ID NO: 11; or

[0022] (2) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0023] (3) an amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2);

[0024] The urease B has:

[0025] (4) the amino acid sequence shown in SEQ ID NO: 12; or

[0026] (5) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (4); or

[0027] (6) an amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5);

[0028] The vacuolating toxin has:

[0029] (7) the amino acid sequence shown in SEQ ID NO: 13; or

[0030] (8) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (7); or

[0031] (9) an amino acid sequence that is at least 80% identical to the amino acid sequence shown in (7) or (8);

[0032] The cytotoxin-associated protein A has:

[0033] (10), the amino acid sequence shown in SEQ ID NO: 14; or

[0034] (11) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (10); or

[0035] (12) an amino acid sequence that is at least 80% identical to the amino acid sequence shown in (10) or (11);

[0036] The heat shock protein has:

[0037] (13), the amino acid sequence shown in SEQ ID NO: 15; or

[0038] (14) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (13); or

[0039] (15) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (13) or (14).

[0040] Furthermore, the reagent for injection immunization also includes complete Freund's adjuvant, and the reagent is obtained by mixing the complete Freund's adjuvant with the natural antigen, the recombinant antigen, or a mixture of the natural antigen and the recombinant antigen. The injection immunization includes taking the reagent and subcutaneously injecting it at the proximal end of the cow's limbs and both sides of the buttocks.

[0041] In some embodiments, the dairy cows are 16-month-old black and white cows, and the dairy cows are pre-immunized with drugs that inhibit gastric motility (such as scopolamine, etc.) and drugs that inhibit gastric acid secretion (such as omeprazole, etc.).

[0042] After a lot of preliminary research and experiments, it was proved that 16-month-old black and white cattle have good immune reactivity to natural and recombinant antigen proteins of Helicobacter pylori. After immunization, high titers of anti-Helicobacter pylori sIgA antibodies and IgG antibodies can be produced in the cow's milk. The use of cholinergic nerve inhibitors and proton pump inhibitors can weaken the gastric motility of immunized animals and inhibit the secretion of gastric acid, thereby facilitating the presence of oral live bacteria in the gastric cavity, improving immune efficiency, and then stimulating the immunized dairy cows to produce high titers of Helicobacter pylori-specific antibodies, thereby achieving better immune effects.

[0043] The invention provides immune milk prepared by the preparation method.

[0044] The present invention provides the use of the immune milk prepared by the preparation method in preparing products for preventing and / or treating Helicobacter pylori infection.

[0045] The present invention provides a product for preventing and / or treating Helicobacter pylori infection, including the immune milk prepared by the preparation method.

[0046] The present invention provides a method for preventing and / or treating Helicobacter pylori infection, comprising: administering the immune milk or the product.

[0047] The present invention provides an immunoprotective preparation for Helicobacter pylori comprising Helicobacter pylori, natural antigens of Helicobacter pylori, and recombinant antigens thereof, as well as immune milk prepared using the immunoprotective preparation and its use in preparing products for preventing and / or treating Helicobacter pylori infection. Compared with the prior art, the present invention has the following advantages:

[0048] 1. The final product of the present invention is presented in the form of a dairy product, which is a common food for people. It is more in line with common dietary habits in terms of taste, nutrition, and administration. In addition, the product of the present invention has the effect of resisting Helicobacter pylori infection, which makes it more easily accepted by people.

[0049] 2. Helicobacter pylori and related proteins only serve as immunogens to stimulate dairy cows to produce corresponding antibodies. They will not enter the blood directly, let alone the milk. Therefore, immune milk has no toxic side effects and no potential harm to health. Antibodies have no toxic side effects and will not produce drug resistance. They can be used for a long time. At the same time, cow milk as a carrier has a large output and is more economical and practical.

[0050] 3. The main functional factors are sIgA and IgG antibodies against Helicobacter pylori, which can exert very strong mucosal immune protection and bacterial neutralization effects. Therefore, it can not only be used to prevent Helicobacter pylori infection, but also can be used for treatment. It has a clearing effect on Helicobacter pylori that has colonized the gastric mucosa.

[0051] 4. Nine weeks after immunization, the antibody titer in the cow serum reached 1:25,000, and the antibody titer in the immune milk reached 1:8,000. After enhanced immunization, immune milk with anti-Helicobacter pylori biological activity can be obtained stably and continuously. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 shows the SDS-PAGE electrophoresis diagram and Western-Blot detection results of CagA protein, wherein, in the SDS-PAGE electrophoresis diagram, M: protein markers, lane 1: CagA protein, and in the Western-Blot detection results diagram, lane 1: CagA protein;

[0053] FIG2 shows the SDS-PAGE electrophoresis diagram and Western-Blot detection results of VacA protein, wherein, in the SDS-PAGE electrophoresis diagram, M: protein markers, lane 1: VacA protein, and in the Western-Blot detection results diagram, lane 1: VacA protein;

[0054] FIG3 shows the SDS-PAGE electrophoresis diagram and Western-Blot detection results of UreA protein, wherein, in the SDS-PAGE electrophoresis diagram, M: protein markers, lane 1: UreA protein, and in the Western-Blot detection results diagram, lane 1: UreA protein;

[0055] FIG4 shows the SDS-PAGE electrophoresis diagram and Western-Blot detection results of UreB protein, wherein, in the SDS-PAGE electrophoresis diagram, M: protein markers, lane 1: UreB protein, and in the Western-Blot detection results diagram, lane 1: UreB protein;

[0056] FIG5 shows the SDS-PAGE electrophoresis diagram and Western-Blot detection result diagram of groEL protein, wherein, in the SDS-PAGE electrophoresis diagram, M: protein markers, lane 1: groEL protein, and in the Western-Blot detection result diagram, lane 1: groEL protein. DETAILED DESCRIPTION

[0057] The present invention provides a Helicobacter pylori immune protection preparation and its application in the preparation of immune milk. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] The purpose of the present invention is to prevent and treat Helicobacter pylori infection through immunological methods (passive immunization). The implementation of the present invention is divided into two technical implementation routes. One is to boost immunization of specific breeds of dairy cows with Helicobacter pylori-related immunogens to induce the cows to produce specific antibodies against Helicobacter pylori, thereby producing high titers of anti-Helicobacter pylori sIgA antibodies and IgG antibodies in the secreted milk. sIgA can play a mucosal immune role, and IgG antibodies can play a neutralizing and protective role. The above-mentioned immune milk can be orally ingested and bind to uncolonized and colonized Helicobacter pylori. On the one hand, it can block the functional groups on the Helicobacter pylori outer membrane that are used for adhesion and colonization, thereby inhibiting the successful colonization of the bacteria on the gastric mucosa. On the other hand, the anti-Hp antibodies in the immune milk bind to Hp and activate the complement system and other immune-active cells to eliminate Helicobacter pylori. Another technical route for preparing immune milk is to prepare monoclonal antibodies against different virulence proteins and other functional proteins of Helicobacter pylori through genetic engineering. These specific antibodies are added to milk to prepare functional immune milk that can eliminate Helicobacter pylori infection.

[0059] Immune milk rich in anti-Helicobacter pylori sIgA and IgG antibodies is prepared by active immunization of specific breeds of dairy cows: Helicobacter pylori whole-cell lysis antigens are prepared, and at the same time, important virulence factors of Helicobacter pylori, including urease, vacuolating toxin (VacA), cytotoxin-associated protein A (CagA), heat shock protein (groEL) and other Helicobacter pylori-related proteins are artificially synthesized. Specific breeds of dairy cows are actively immunized in combination with corresponding immune adjuvants. After 4-6 weeks of immunization, the corresponding antibodies against Helicobacter pylori in the cow serum and milk are detected, especially the secretory IgA antibodies in the milk, and the titer and function of the above antibodies are evaluated. Finally, anti-Helicobacter pylori immune milk with protective antibodies against Helicobacter pylori is obtained for the prevention and treatment of Helicobacter pylori infection.

[0060] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0061] Example 1

[0062] 1. Preparation of Helicobacter pylori immune antigens

[0063] 1.1 Preparation of natural lytic antigens of Helicobacter pylori whole cells

[0064] 1.1.1 Collection of Helicobacter pylori strains

[0065] Clinically derived Helicobacter pylori strains were collected from patients with chronic gastritis, peptic ulcer, and gastric cancer. Electronic gastroscopy was performed on the patients, and biopsy samples were taken from patients with clear gastric tissue pathological changes. Helicobacter pylori was isolated and cultured using Helicobacter pylori isolation and selection medium. The culture was carried out at 37°C for 3-5 days under microaerobic conditions (5% O2, 10% CO2, 85% N2) to obtain pure culture of Helicobacter pylori. A total of 100 Helicobacter pylori strains were collected and frozen for future use.

[0066] The above-mentioned strains were tested for virulence factors such as CagA, VacA, and urease UreA / UreB. The specific method was to sample the above-mentioned bacteria and perform SDS-PAGE electrophoresis. Then, Western-Blot detection was performed using anti-CagA, VacA, and urease UreA / UreB corresponding antisera to confirm that the relevant Helicobacter pylori strains produced the above-mentioned virulence factors. The confirmed Helicobacter pylori strains were frozen for future use.

[0067] 1.1.2 Preparation of natural antigens of Helicobacter pylori strains

[0068] Helicobacter pylori strains were revived using isolation culture medium. After 3 days of microaerobic culture, 3 mL of sterile saline was used to collect bacteria from the surface of each culture plate. 1 mL of lysis buffer containing lysozyme was added and the bacteria were lysed using a cell sonicator in an ice bath (power setting of 300-350 W, 10 seconds continuous, 10 seconds rest). After sonication, the bacterial suspension was shaken to mix thoroughly and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant and precipitate were collected separately for later use.

[0069] Dissolve the above-mentioned precipitate with 3 mL of 3 M urea and stir thoroughly at 4°C overnight (>12 hours). Centrifuge at 12,000 g for 30 minutes at 4°C. Remove the supernatant and dialyze it to reduce the urea concentration to 0.5 M. Then store the solution at -80°C until ready for use. Combine the two supernatants (lysis supernatant + urea-dissolved precipitate supernatant) and adjust the protein concentration in the supernatant to 10-20 mg / mL. Store at -80°C until ready for use.

[0070] 1.2 Preparation of Helicobacter pylori-specific virulence-related proteins

[0071] 1.2.1 Cloning of Helicobacter pylori virulence-related protein genes

[0072] 1.2.1.1 Cloning of the Helicobacter pylori urease gene

[0073] Helicobacter pylori urease is typically composed of large and small subunits. We amplified the urease A / B encoding genes using primers targeting the urease A and urease B subunits, respectively. We then expressed urease A and urease B in Escherichia coli, such as pTrcHis2A\B\C, as prokaryotic expression hosts. After obtaining highly expressed products, we purified urease A and urease B to obtain purified recombinant proteins. Following the prokaryotic expression protocol for UreA / UreB, we also performed prokaryotic expression and protein preparation for the cagA, vacA, and groEL genes.

[0074] The primers for amplifying the UreA, UreB, cagA, vacA and groEL genes are shown in Table 1 below.

[0075] Table 1

[0076] The amino acid sequence of UreA is as follows:

[0077] The amino acid sequence of UreB is as follows:

[0078] The amino acid sequence of VacA is as follows:

[0079] The amino acid sequence of CagA is as follows:

[0080] The amino acid sequence of groEL is as follows:

[0081] 1.2.2 Prokaryotic expression of Helicobacter pylori virulence proteins

[0082] A clinical strain of Helicobacter pylori was used for lysis and extraction of genomic nucleic acid, and then the gene fragment was synthesized using the PCR method with the above-mentioned gene primers. The above-mentioned gene fragment was introduced into pTrcHis2c by double digestion with restriction endonucleases to construct a recombinant plasmid, which was introduced into Escherichia coli for expression, purification and identification.

[0083] The specific operation process is as follows:

[0084] 1.2.2.1 Expression and purification of Helicobacter pylori urease gene in prokaryotic cells

[0085] ①Collect fresh culture of Helicobacter pylori from clinical sources and extract genomic nucleic acid using a bacterial genomic DNA extraction kit;

[0086] ②Using bacterial genomic nucleic acid as a template, amplify the urease genes ureA and ureB, add restriction enzyme sites on both sides of the gene, and obtain the above-mentioned gene and plasmid fragments by purifying the PCR product and plasmid pTrcHis2c with double enzyme digestion;

[0087] ③ Use ligase to construct the recombinant plasmid of ureA and ureB and plasmid pTrcHis2C and identify it;

[0088] ④ Introduce the above recombinant plasmid into BL21 or Rosset to express urease;

[0089] ⑤Pick the E. coli carrying the expression plasmid and culture it in SOB medium. When the OD600 of the cultured bacteria reaches 0.4-0.6, add IPTG to a final concentration of 1 mM to induce expression.

[0090] ⑥ Induce expression on a 37°C constant temperature shaker for 3 hours, and harvest the bacteria by low-temperature centrifugation;

[0091] ⑦After the expression of UreA and UreB proteins, protein identification and confirmation were performed using Western-BLOT;

[0092] ⑧After confirming the expression of the above protein, affinity chromatography purification is performed using a nickel particle column, and elution is performed using imidazole elution buffer of different concentrations to collect the target protein, and then the protein is verified and confirmed.

[0093] 1.2.2.2 Expression of Helicobacter pylori cagA / vacA / groEL genes and preparation of recombinant proteins According to the above methods and procedures, sufficient amounts of corresponding CagA, VacA and groEL proteins for dairy cow immunization were finally obtained.

[0094] The SDS-PAGE electrophoresis patterns and Western-Blot detection results of CagA, VacA, groEL and urease UreA / UreB in the above experiments are shown in Figures 1 to 5.

[0095] Example 2

[0096] In order to evaluate the immunogenicity of the above-mentioned Helicobacter pylori natural antigens and recombinant antigens, we designed animal experiments to evaluate the immune efficacy of the above-mentioned Helicobacter pylori-related antigens.

[0097] Evaluation Methods: 6-8 week old female BALB-C mice were immunized with either native or recombinant antigens. The antigens used for immunization included Helicobacter pylori lysate supernatant, Helicobacter pylori precipitate urea-dissolved supernatant, recombinant proteins CagA, VacA, UreaA / UreaB, and GeoEL recombinant protein. These components were mixed with complete Freund's adjuvant to prepare the immunogen, which was injected intramuscularly (200 μL) for a total of 100 μg. Immunization was performed, followed by weekly booster immunizations for two consecutive weeks. Serum was collected at week 4, and the titer of Helicobacter pylori-specific antibodies was determined by ELISA. The results are shown in Tables 2 and 3.

[0098] Table 2

[0099] Table 2 shows the serum antibody titers produced by animals immunized with different immunogens alone and in combination. The results show that higher antibody titers can be obtained by immunizing animals with two antigen components in combination.

[0100] Table 3

[0101] Table 3 shows that higher antibody titers can be obtained by co-immunizing animals with natural antigens (bacterial lysis supernatant + precipitated urea supernatant) and recombinant antigens (UreA / UreB, CagA, VacA and GroEL).

[0102] The results showed that immunizing animals with both natural and recombinant H. pylori antigens resulted in the highest antibody titers. Therefore, we subsequently used both natural and recombinant antigens in our dairy cow immunizations to obtain the highest titers of corresponding antibodies.

[0103] Example 3

[0104] Immunity of dairy cows

[0105] 1. Selection of dairy cow breeds

[0106] Select 16-month-old black and white cattle, tested to ensure they are free of foot-and-mouth disease, anthrax, and tuberculosis. Weigh 300-350 kg.

[0107] 2 Pre-immunization treatment

[0108] Gastric motility inhibitors and antacids

[0109] Before antigen immunization, the cows were treated with scopolamine and omeprazole (1 g scopolamine, twice daily) for one week.

[0110] 3. Choice of immunization regimen:

[0111] 3.1 Immunity with oral live bacteria

[0112] Oral feeding of Helicobacter pylori whole cell suspension in experimental dairy cows

[0113] Helicobacter pylori was revived on the culture medium, then passaged and cultured at 37°C for 3 days. 50 mL × 10 9 CFU / mL, the above culture solution containing fresh live bacteria was added to the feed of immunized dairy cows that had been fasting for 1 day, three times a day, for 7 consecutive days; blood samples and milk samples were collected 2 weeks, 3 weeks, 6 weeks and 9 weeks after the end of immunization to detect Helicobacter pylori-specific antibodies, and the results are shown in Tables 4 and 5.

[0114] 3.2 Immunization with Helicobacter pylori natural antigens

[0115] Take the Helicobacter pylori lysis supernatant (natural immunogen, including natural bacterial lysis supernatant and precipitated urea supernatant) and add an equal amount of Freund's complete adjuvant to fully emulsify the Helicobacter pylori antigen lysis supernatant. The specific method includes:

[0116] i. Suspension method: Take 15 mL of complete Freund's adjuvant and add 15 mL of Helicobacter pylori supernatant. Add the adjuvant first, then suspend the adjuvant in a suspension vessel while slowly adding the Helicobacter pylori supernatant dropwise to thoroughly mix the antigen and adjuvant until a viscous emulsion is formed.

[0117] ii. Syringe mixing: Add 15 mL of complete Freund's adjuvant to 15 mL of Helicobacter pylori supernatant and draw the mixture into two 150 mL syringes. Connect the two syringes with a thin rubber tube and expel all air. First, push the antigen into the adjuvant, then push the syringes alternately until a viscous emulsion is formed that is difficult to push.

[0118] Each dairy cow was immunized with 30 mL of the emulsified antigen. 5 mL of the emulsified antigen was injected subcutaneously into the proximal end of each limb, and then 5 mL of the emulsified antigen was injected into each side of the cow's buttocks.

[0119] Three and six weeks later, booster immunizations were performed according to the aforementioned method. Weekly, 10 mL of blood and 500 mL of milk were collected from the immunized cows for testing for anti-Helicobacter pylori antibody titers. Blood samples were tested for IgG antibodies at 2, 3, 6, and 9 weeks after immunization, and milk samples were tested for sIgA and IgG antibodies. The results are shown in Tables 4 and 5.

[0120] 3.3 Immunization with Helicobacter pylori recombinant antigens (UreA / UreB, CagA, VacA and GeoEL)

[0121] Take the Helicobacter pylori recombinant antigen and add an equal amount of Freund's complete adjuvant to fully emulsify the Helicobacter pylori antigen lysis supernatant. The specific method includes:

[0122] i. Suspension method: Take 15 mL of complete Freund's adjuvant and add 15 mL of Helicobacter pylori supernatant. Add the adjuvant first, then suspend the adjuvant in a suspension vessel while slowly adding the Helicobacter pylori supernatant dropwise to thoroughly mix the antigen and adjuvant until a viscous emulsion is formed.

[0123] ii. Syringe mixing: Add 15 mL of complete Freund's adjuvant to 15 mL of Helicobacter pylori supernatant and draw the mixture into two 150 mL syringes. Connect the two syringes with a thin rubber tube and expel all air. First, push the antigen into the adjuvant, then push the syringes alternately until a viscous emulsion is formed that is difficult to push.

[0124] Each dairy cow was immunized with 30 mL of the emulsified antigen. 5 mL of the emulsified antigen was injected subcutaneously into the proximal end of each limb, and then 5 mL of the emulsified antigen was injected into each side of the cow's buttocks.

[0125] Three and six weeks later, booster immunizations were performed according to the aforementioned method. Weekly, 10 mL of blood and 500 mL of milk were collected from the immunized cows for testing for anti-Helicobacter pylori antibody titers. Blood samples were tested for IgG antibodies at 2, 3, 6, and 9 weeks after immunization, and milk samples were tested for sIgA and IgG antibodies. The results are shown in Tables 4 and 5.

[0126] 3.4 Oral vaccination of dairy cows with whole-bacteria immunization and Helicobacter pylori natural antigens (natural bacterial lysis supernatant and precipitated urea supernatant)

[0127] According to the above scheme, while the dairy cows are being immunized with live bacteria orally, they are also immunized with Helicobacter pylori natural antigens. After the booster immunization with natural antigens is completed, serum and milk samples are collected to detect the antibody titers and levels of different antibodies in the samples.

[0128] 3.5 Oral vaccination of dairy cows with whole bacteria + immunization with recombinant Helicobacter pylori antigens (UreA / UreB, CagA, VacA and GeoEL)

[0129] According to the above scheme, while the dairy cows were immunized with live bacteria orally, they were also immunized with Helicobacter pylori recombinant antigens. After the completion of the booster immunization with the recombinant antigen, serum and milk samples were collected from the dairy cows 2 weeks, 3 weeks, 6 weeks and 9 weeks after the end of the immunization. The antibody titers and levels of different antibodies in the samples were tested. The results are shown in Tables 4 and 5.

[0130] 3.6 Oral vaccination of dairy cows with whole-bacteria immunization and combined immunization with natural and recombinant Helicobacter pylori antigens

[0131] While feeding the above-mentioned live bacteria, the immune cows were immunized with a mixture of Helicobacter pylori natural antigens and recombinant antigens. The immunogen was prepared by mixing equal amounts of the aforementioned Helicobacter pylori natural antigens (natural antigen supernatant + urea supernatant) and Helicobacter pylori recombinant antigens (UreA / UreB, CagA, VacA and GroEL). The immunogen was then mixed with complete Freund's adjuvant according to the above method to prepare an immune suspension. The specific method is as follows:

[0132] The mixed immunogen and Freund's complete adjuvant are mixed in equal amounts to prepare an immune emulsion suspension to fully emulsify the Helicobacter pylori antigen. The specific method includes:

[0133] i. Suspension method: Take 15 mL of complete Freund's adjuvant and add 15 mL of Helicobacter pylori supernatant. Add the adjuvant first, then suspend the adjuvant in a suspension vessel while slowly adding the Helicobacter pylori supernatant dropwise to thoroughly mix the antigen and adjuvant until a viscous emulsion is formed.

[0134] ii. Syringe mixing: Add 15 mL of complete Freund's adjuvant to 15 mL of Helicobacter pylori supernatant and draw the mixture into two 150 mL syringes. Connect the two syringes with a thin rubber tube and expel all air. First, push the antigen into the adjuvant, then push the syringes alternately until a viscous emulsion is formed that is difficult to push.

[0135] After the 1:1 mixture of antigen and adjuvant is prepared, it should be refrigerated at 2-8°C for subsequent immunization injections:

[0136] Blood and milk samples were collected from cows 2, 3, 6, and 9 weeks after immunization to detect the corresponding specific antibodies. The results of the serum antibody level (IgG) of the cows are shown in Table 4, and the results of the milk antibody level (IgG+sIgA) of the cows are shown in Table 5.

[0137] Table 4

[0138] Table 5

[0139] These results show that single and combined immunization with different antigens resulted in different titers of specific antibodies in serum and milk samples. Oral administration combined with immunization with natural and recombinant antigens produced the highest H. pylori antibody titers in dairy cows. Therefore, our final H. pylori immunization regimen for dairy cows involved a combined oral administration with immunization with natural and recombinant antigens.

[0140] 4. Enhance immunity and collect immune milk

[0141] After the cows complete the above-mentioned combined immunization (week 9), immune milk can be collected and sequential booster immunizations are performed once every three months using Helicobacter pylori natural antigens and recombinant antigens, respectively, to ensure the production of high titer sIgA antibodies and IgG antibodies in the milk.

[0142] Table 6. sIgA antibody levels in milk after immunization

[0143] Sequential booster immunization method for dairy cows:

[0144] Natural and recombinant H. pylori antigens were prepared as described above. Booster immunizations were performed three months after the start of milk collection. First, 50 mL of natural antigen plus adjuvant was used for booster immunization according to the aforementioned method. Three months later, 50 mL of recombinant H. pylori antigen plus adjuvant was used for booster immunization. This sequential booster immunization process ensured high titers of sIgA and IgG antibodies in the cow's milk.

[0145] Table 7. Determination of sIgA and IgG antibody titers in pregnant dairy cows after sequential booster immunization

[0146] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An immunoprotective preparation for Helicobacter pylori, characterized in that: The invention comprises at least one of Helicobacter pylori, natural antigen and recombinant antigen of Helicobacter pylori, wherein: The natural antigens include whole bacterial proteins of Helicobacter pylori; The recombinant antigen comprises at least one of cytotoxin-associated protein A, vacuolating toxin, urease and heat shock protein, and the urease comprises urease A and / or urease B.

2. Use of the immunoprotective preparation according to claim 1 in the preparation of immune milk.

3. A method for preparing immune milk, characterized in that: The immune protection preparation according to claim 1 is used to immunize dairy cows to obtain the immune milk.

4. The preparation method according to claim 3, characterized in that: The immunization includes: Feeding the live bacteria of Helicobacter pylori to dairy cows; and / or The natural antigen and / or recombinant antigen of Helicobacter pylori is taken to perform injection immunization on dairy cows.

5. The preparation method according to claim 4, characterized in that: The feeding comprises a feed containing 5×10 10 The live bacteria of CFU Helicobacter pylori are fed to dairy cows three times a day for 7 consecutive days.

6. The preparation method according to claim 4 or 5, characterized in that: The method for preparing the natural antigen comprises: taking the Helicobacter pylori, lysing it with lysozyme, and then ultrasonically disrupting it, collecting the lysate supernatant and precipitate, dissolving the precipitate with urea, and centrifuging it, dialyzing the supernatant after the centrifugation to obtain the precipitate supernatant, and mixing the lysate supernatant and the precipitate supernatant to obtain the natural antigen of the Helicobacter pylori; The preparation method of the recombinant antigen comprises: constructing recombinant plasmids of cytotoxin-associated protein A, vacuolating toxin, urease and heat shock protein respectively, and obtaining the recombinant antigen after prokaryotic expression and purification.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The reagent for injection immunization also includes complete Freund's adjuvant. The reagent is obtained by mixing the complete Freund's adjuvant with the natural antigen, the recombinant antigen or a mixture of the natural antigen and the recombinant antigen. The injection immunization includes taking the reagent and injecting it subcutaneously at the proximal end of the cow's limbs and both sides of the buttocks.

8. The immune milk obtained by the preparation method according to any one of claims 3 to 7.

9. Use of the immune milk prepared by the preparation method according to any one of claims 3 to 7 in preparing products for preventing and / or treating Helicobacter pylori infection.

10. A product for preventing and / or treating Helicobacter pylori infection, characterized in that The invention comprises immune milk prepared by the preparation method according to any one of claims 3 to 7.

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