Additive containing anti-helicobacter pylori duck egg yolk antibody
By inoculating Helicobacter pylori as an antigen, it is immunized in ducks and purified the anti-Herrelicobacter pylori duck yolk antibody IgY in duck yolk, the drug resistance problem is solved and the existing research lacks the method of duck yolk antibody, achieving efficient and specific anti-Herrelicobacter pylori treatment effect.
Patent Information
- Application Number
- CN202510165257.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-14
- Publication Date
- 2025-05-13
AI Technical Summary
The existing anti-Herrelic pylori treatment methods have drug resistance problems, which leads to repeated onset of infection. In existing studies, duck yolk antibodies with Helicobacter pylori as the antigen and their preparation methods are lacking.
By inoculating Helicobacter pylori as an antigen, immunized in ducks, collecting and purifying anti-Herrelicobacter pylori duck yolk antibody IgY in duck yolk, an additive containing anti-Herrelicobacter pylori duck yolk antibody was prepared.
The anti-Herrelic pylori duck yolk antibody prepared by this method has the characteristics of high specificity and high titer, which can effectively prevent and treat diseases caused by Helicobacter pylori. The titer is as high as 1:1280,000 under the same immune dose and method, which is better than the anti-Herrelic pylori IgY of chickens.
Smart Images
Figure CN119971027A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an additive containing anti-Helicobacter pylori duck egg yolk antibodies. Background Art
[0002] Helicobacter pylori is Gram-negative, with arc-shaped, S-shaped or spiral-shaped bacteria, flagella, active movement, and no spores. The bacteria are not resistant to the external environment and are very sensitive to dryness and heat. Various commonly used disinfectants can easily kill it. The pathogenicity of Helicobacter pylori is related to factors such as the toxins and toxic enzymes it produces that damage the gastric mucosa and induce the body to produce inflammation and immune responses. Helicobacter pylori infection can induce inflammation and immune responses. Cell degeneration, necrosis and inflammatory cell infiltration can be seen in the infected gastric mucosa, and specific antibodies can be detected in the serum. Helicobacter pylori is associated with a variety of diseases such as gastritis, peptic ulcer, gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma (MALT lymphoma), non-steroidal anti-inflammatory drug-related gastric functional dyspepsia and gastroesophageal reflux disease.
[0003] More than 50% of the world's population is infected with Hp, and the incidence rate is closely related to the socioeconomic level, population density, public health conditions and water supply. Its main modes of transmission are "fecal-oral" transmission (fecal-oral) and "oral-oral" transmission (oral-oral). In the "fecal-oral" transmission route, Hp is shed with the renewal of the gastric mucosal epithelium, passes through the stomach, duodenum, small intestine and large intestine in turn, and is finally excreted from the feces. Eating food contaminated by feces may cause Hp infection. In the "oral-oral" transmission route, Hp is shed into the gastric juice, enters the oral cavity through gastroesophageal reflux, and then adheres to the oral cavity, especially on dental plaque, and is finally transmitted through saliva. At present, the commonly used anti-Hp drugs at home and abroad include clarithromycin, tetracycline, doxycycline, furazolidone, organic colloidal bismuth, and metronidazole (Mecura), etc. The course of treatment is generally two weeks. This treatment method often causes patients to have repeated attacks due to the problem of Helicobacter pylori resistance and the inability to completely eradicate Helicobacter pylori.
[0004] Yolk antibody (IgY) is produced by using egg-laying birds as bioreactors. It is low-cost and has obvious advantages over using rabbits, sheep, horses, cattle and other animals to produce antibodies. The immunoglobulins (Ig) of birds are also very different from those of mammals in immunological properties. The immune system of birds can recognize more immune response sites and more antigenic determinants, so the antibodies produced by the bird immune system to mammalian proteins or biological molecules have higher titers and affinity.
[0005] Yolk antibody is an antibody resource with great development potential, but in China, the production and application of chicken yolk antibody is mainly concentrated. Compared with chicken, the production cost of duck is lower. Studies have shown that an adult duck can lay 250 eggs a year, and 75-120 mg of antibody can be isolated from each egg. However, there are few studies on the production of duck yolk antibody by immunizing laying ducks, and there is no duck yolk antibody with Helicobacter pylori as antigen and its preparation method. Summary of the invention
[0006] The object of the present invention is to provide an additive containing anti-Helicobacter pylori duck egg yolk antibodies to solve the problems raised in the above background technology.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] An additive containing anti-Helicobacter pylori duck egg yolk antibodies, comprising:
[0009] Anti-Helicobacter pylori duck egg yolk antibody IgY;
[0010] Liquid duck egg yolk containing anti-Helicobacter pylori duck egg yolk antibody IgY;
[0011] Duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibody IgY.
[0012] Preferably, the invention is used in the preparation of preparations for preventing and treating diseases caused by Helicobacter pylori.
[0013] Preferably, the preparation is a medicine, a health product or a food.
[0014] Preferably, the preparation method specifically comprises the following steps:
[0015] S1. Preparation of antigen: Helicobacter pylori was inoculated on blood agar plates. After 3-4 days of culture, the cells were collected and diluted to a concentration of 2×10 9 cfu / mL of bacterial solution, and ultrasonic treatment to obtain Helicobacter pylori bacterial solution;
[0016] S2, immunization of laying ducks: the Helicobacter pylori liquid of S1 was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in a volume ratio of 1:1, emulsified, and prepared into Freund's complete adjuvant emulsified vaccine and Freund's incomplete adjuvant emulsified vaccine, and the laying ducks were immunized for a total of five times. The first immunization was with Freund's complete adjuvant emulsified vaccine, with an immunization dose of 0.8-1.2 mL / duck, and injected subcutaneously at the back of the duck; on the 21st, 35th, and 56th days after the first immunization, the second, third, and fourth immunizations were changed to Freund's incomplete adjuvant emulsified vaccine, with an immunization dose of 0.3-0.7 mL / duck, and injected subcutaneously at the back of the duck; on the 77th day after the first immunization, the Helicobacter pylori liquid of S1 was used for the fifth immunization, with an immunization dose of 0.3-0.7 mL / duck, and injected intramuscularly at the base of the duck leg;
[0017] S3. Duck eggs and duck yolks were collected daily starting one week after the third vaccination, and the anti-Helicobacter pylori duck egg yolk antibody IgY was purified.
[0018] Preferably, in S1, the Helicobacter pylori strain is Helicobacter pylori ATCC 43504.
[0019] Preferably, in S1, the Helicobacter pylori strain is used after being subjected to ultrasonic treatment to form cell fragments.
[0020] Preferably, in S2, the laying ducks are healthy laying ducks of 20 weeks old.
[0021] Preferably, in S1, the ultrasonic treatment step is: diluting the mixture to a Helicobacter pylori concentration of 2×10 9 cfu / mL of bacterial solution was placed in an ice water bath and ultrasonicated at a power of 200 W for 20 s, with a rest period of 20 s, and repeated 15 to 25 times.
[0022] Preferably, in S3, the purification step is specifically divided into:
[0023] S31, adding 2 to 3 volumes of PBS buffer to the collected duck egg yolk, shaking, centrifuging, collecting the supernatant, adding PEG6000 to a final concentration of 8.5%, and stirring at room temperature;
[0024] S32, centrifuge the stirred liquid in S31 and discard the supernatant, add PBS to the precipitate to dissolve it, then add PEG6000 to a final concentration of 12%, stir at room temperature, centrifuge and discard the supernatant, redissolve the precipitate with PBS, add saturated ammonium sulfate to a final concentration of 47-50% in the solution, place it at 4°C, let it stand for more than 3 hours, centrifuge the mixed solution at 3000 rpm at room temperature, remove the supernatant, use PBS buffer to dissolve the precipitate, then add saturated ammonium sulfate dropwise to a final concentration of 33-35% in the solution, place it at 4°C, let it stand for more than 3 hours, repeat the above operation 1-2 times, and take the precipitate from the last centrifugation;
[0025] S33, dissolve the precipitate in S32 in 0.02% mol / L pH7 PBS buffer of the same volume as the original duck egg yolk, dialyze for 18 to 48 hours, replace the dialysate 2-3 times during the dialysis process, until the dialyzate external fluid is no longer yellow when measured by Naphthol reagent, then take out the IgY antibody liquid in the dialyzer, filter it through a filter membrane to obtain the purified anti-Helicobacter pylori duck egg yolk antibody IgY.
[0026] A yogurt containing anti-Helicobacter pylori duck egg yolk antibodies is prepared by using an additive containing anti-Helicobacter pylori duck egg yolk antibodies. 100 g of duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibody IgY is added to a fermented milk raw material which has been treated at 90°C for 5 minutes and then cooled to 65°C. The temperature is maintained at 65°C for 15 minutes, and then cooled, inoculated and fermented to obtain a yogurt with an anti-Helicobacter pylori duck egg yolk antibody IgY content of 2 mg / 100 g.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The anti-Helicobacter pylori egg yolk antibody prepared by the present invention is prepared from duck eggs laid by egg-laying ducks immunized with Helicobacter pylori, has the characteristics of high specificity and high titer, is used for preventing and treating diseases caused by Helicobacter pylori and immunological detection, and has good effects; and under the same immunization dose and immunization method, the titer of the anti-Helicobacter pylori duck IgY obtained by the prepared anti-Helicobacter pylori duck egg yolk antibody is as high as 1:1280000, which is higher than the titer of the anti-Helicobacter pylori IgY of chicken. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 It is a block diagram of the preparation method of the present invention. DETAILED DESCRIPTION
[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0031] See also Figure 1 As shown, an additive containing anti-Helicobacter pylori duck egg yolk antibodies comprises:
[0032] Anti-Helicobacter pylori duck egg yolk antibody IgY;
[0033] Liquid duck egg yolk containing anti-Helicobacter pylori duck egg yolk antibody IgY;
[0034] Duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibody IgY.
[0035] Application in the preparation of preparations for preventing and treating diseases caused by Helicobacter pylori.
[0036] The preparation is medicine, health product and food.
[0037] A method for preparing an additive containing anti-Helicobacter pylori duck egg yolk antibodies is used to prepare an additive containing anti-Helicobacter pylori duck egg yolk antibodies, and specifically comprises the following steps:
[0038] S1. Preparation of antigen: Helicobacter pylori was inoculated on blood agar plates. After 3-4 days of culture, the cells were collected and diluted to a concentration of 2×10 9 cfu / mL of bacterial solution, and ultrasonic treatment to obtain Helicobacter pylori bacterial solution;
[0039] S2, immunization of laying ducks: the Helicobacter pylori liquid of S1 was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in a volume ratio of 1:1, emulsified, and prepared into Freund's complete adjuvant emulsified vaccine and Freund's incomplete adjuvant emulsified vaccine, and the laying ducks were immunized for a total of five times. The first immunization was with Freund's complete adjuvant emulsified vaccine, with an immunization dose of 0.8-1.2 mL / duck, and injected subcutaneously at the back of the duck; on the 21st, 35th, and 56th days after the first immunization, the second, third, and fourth immunizations were changed to Freund's incomplete adjuvant emulsified vaccine, with an immunization dose of 0.3-0.7 mL / duck, and injected subcutaneously at the back of the duck; on the 77th day after the first immunization, the Helicobacter pylori liquid of S1 was used for the fifth immunization, with an immunization dose of 0.3-0.7 mL / duck, and injected intramuscularly at the base of the duck leg;
[0040] S3. Duck eggs and duck yolks were collected daily starting one week after the third vaccination, and the anti-Helicobacter pylori duck egg yolk antibody IgY was purified.
[0041] In S1, the Helicobacter pylori strain is Helicobacter pylori ATCC 43504. The Helicobacter pylori is inoculated on a blood agar plate and cultured at 37°C in a microaerobic environment. After culturing for 3 days, the colonies are washed with sterile PBS to collect the bacteria, and the A600 is measured to determine the cell concentration. According to the cell concentration, the bacterial solution is diluted with sterile PBS to obtain a cell concentration of 2×10 9 cfu / mL of ATCC 43504 Helicobacter pylori was then ultrasonicated at a power of 200 W for 20 seconds, with a rest period of 20 seconds, and repeated 20 times to break the cell wall and obtain a bacterial solution containing cell fragments, i.e., Helicobacter pylori bacterial solution, which was used as an antigen.
[0042] In S2, the laying ducks are healthy laying ducks of 20 weeks old.
[0043] In S1, the ultrasonic treatment step is: dilute to a Helicobacter pylori concentration of 2×10 9cfu / mL of bacterial solution was placed in an ice water bath and ultrasonicated at a power of 200 W for 20 s, with a rest period of 20 s, and repeated 15 to 25 times.
[0044] In S3, the purification step is specifically divided into:
[0045] S31, breaking the duck egg shell, carefully separating the duck yolk with an egg yolk separator, and ensuring that the duck yolk membrane is not broken. Roll the duck yolk on ordinary filter paper several times, absorb the egg white as much as possible, break the duck yolk membrane and collect the duck yolk, add 2 to 3 times the volume of PBS buffer and shake, centrifuge, collect the supernatant, add PEG6000 to a final concentration of 8.5%, and stir at room temperature;
[0046] S32, centrifuge the stirred liquid in S31 and discard the supernatant, add PBS to the precipitate to dissolve it, then add PEG6000 to a final concentration of 12%, stir at room temperature, centrifuge and discard the supernatant, redissolve the precipitate with PBS, add saturated ammonium sulfate to a final concentration of 47-50% in the solution, place it at 4°C, let it stand for more than 3 hours, centrifuge the mixed solution at 3000 rpm at room temperature, remove the supernatant, use PBS buffer to dissolve the precipitate, then add saturated ammonium sulfate dropwise to a final concentration of 33-35% in the solution, place it at 4°C, let it stand for more than 3 hours, repeat the above operation 1-2 times, and take the precipitate from the last centrifugation;
[0047] S33, dissolve the precipitate in S32 in 0.02% mol / L pH7 PBS buffer of the same volume as the original duck egg yolk, dialyze for 18 to 48 hours, replace the dialysate 2-3 times during the dialysis process, until the dialyzate external fluid is free of yellow when measured by Naphthol reagent, then take out the IgY antibody solution in the dialyzer, filter it through a filter membrane to obtain purified anti-Helicobacter pylori duck egg yolk antibody IgY, dilute it 20 to 100 times with PBS, measure the absorbance at a wavelength of 280nm, and calculate the IgY content (IgY concentration = 1.45×A280-0.74×A260, unit: mg / ml).
[0048] Collect the immune duck eggs obtained by immunization, break the duck egg shells, and carefully separate the duck yolk with an egg yolk separator to ensure that the duck yolk membrane is not broken. Roll the duck yolk on ordinary filter paper several times to remove the egg white as much as possible, and break the duck yolk membrane to collect the duck yolk. Spread the duck yolk liquid on a sterile tin foil tray so that its thickness does not exceed 0.5 cm, put it in a -80℃ refrigerator to freeze for 24 hours, then take out the tin foil tray, put it in a freeze dryer for freeze drying, freeze drying at 0.150mbar for 48 hours, and grind it into freeze-dried powder.
[0049] During treatment, the obtained duck egg yolk freeze-dried powder was stored in a refrigerator at 4°C, and one pack of duck egg yolk freeze-dried powder (7g / pack) was taken every day for a total of 30 days as a course of treatment. The powder was taken two hours after dinner, and fasting was required after taking it. Cold water or room temperature water was taken, and alcohol was prohibited during the period of taking it. The results of the C-13 or C-14 breath test (DOB value or DMP value, respectively) showed that after one month of treatment, the detection result of Helicobacter pylori turned from positive to negative.
[0050] A yogurt containing anti-Helicobacter pylori duck egg yolk antibodies is prepared by using an additive containing anti-Helicobacter pylori duck egg yolk antibodies. 100 g of duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibody IgY is added to a fermented milk raw material which has been treated at 90 DEG C for 5 minutes and then cooled to 65 DEG C. The temperature is maintained at 65 DEG C for 15 minutes, and then cooling, inoculation and fermentation operations are performed to obtain a yogurt with an anti-Helicobacter pylori duck egg yolk antibody IgY content of 2 mg / 100 g.
[0051] A powder containing anti-Helicobacter pylori duck egg yolk antibodies is prepared by mixing 5-10 parts by weight of astragalus powder, 3-5 parts by weight of dandelion powder, 1-3 parts by weight of pinellia tuber, and 40-50 parts by weight of duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibodies, uniformly stirring to form antibody powder, and then mixing the antibody powder with liquid in a mass ratio of 1:5-7 for oral administration.
[0052] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. An additive containing anti-Helicobacter pylori duck egg yolk antibodies, characterized in that: include: Anti-Helicobacter pylori duck egg yolk antibody IgY; Liquid duck egg yolk containing anti-Helicobacter pylori duck egg yolk antibody IgY; Duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibody IgY.
2. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 1, characterized in that: Application in the preparation of preparations for preventing and treating diseases caused by Helicobacter pylori.
3. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 2, characterized in that: The preparation is medicine, health product and food.
4. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 1, characterized in that: The preparation method specifically comprises the following steps: S1. Preparation of antigen: Helicobacter pylori was inoculated on blood agar plates. After 3-4 days of culture, the cells were collected and diluted to a concentration of 2×10 9 cfu / mL of bacterial solution, and ultrasonic treatment to obtain Helicobacter pylori bacterial solution; S2, immunization of laying ducks: the Helicobacter pylori liquid of S1 was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in a volume ratio of 1:1, emulsified, and prepared into Freund's complete adjuvant emulsified vaccine and Freund's incomplete adjuvant emulsified vaccine, and the laying ducks were immunized for a total of five times. The first immunization was with Freund's complete adjuvant emulsified vaccine, with an immunization dose of 0.8-1.2 mL / duck, and injected subcutaneously at the back of the duck; on the 21st, 35th, and 56th days after the first immunization, the second, third, and fourth immunizations were changed to Freund's incomplete adjuvant emulsified vaccine, with an immunization dose of 0.3-0.7 mL / duck, and injected subcutaneously at the back of the duck; on the 77th day after the first immunization, the Helicobacter pylori liquid of S1 was used for the fifth immunization, with an immunization dose of 0.3-0.7 mL / duck, and injected intramuscularly at the base of the duck leg; S3. Duck eggs and duck yolks were collected daily starting one week after the third vaccination, and the anti-Helicobacter pylori duck egg yolk antibody IgY was purified.
5. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 4, characterized in that: In the S1, the Helicobacter pylori strain is Helicobacter pylori ATCC 43504.
6. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 4, characterized in that: In S1, the Helicobacter pylori strain was used after being subjected to ultrasonic treatment to form cell fragments.
7. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 5, characterized in that: In S2, the laying ducks are healthy laying ducks of 20 weeks old.
8. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 7, characterized in that: In S1, the ultrasonic treatment step is: dilute to a Helicobacter pylori concentration of 2×10 9 cfu / mL of bacterial solution was placed in an ice water bath and ultrasonicated at a power of 200 W for 20 s, with a rest period of 20 s, and repeated 15 to 25 times.
9. The additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 7, characterized in that: In S3, the purification step is specifically divided into: S31, adding 2 to 3 volumes of PBS buffer to the collected duck egg yolk, shaking, centrifuging, collecting the supernatant, adding PEG6000 to a final concentration of 8.5%, and stirring at room temperature; S32, centrifuge the stirred liquid in S31 and discard the supernatant, add PBS to the precipitate to dissolve it, then add PEG6000 to a final concentration of 12%, stir at room temperature, centrifuge and discard the supernatant, redissolve the precipitate with PBS, add saturated ammonium sulfate to a final concentration of 47-50% in the solution, place it at 4°C, let it stand for more than 3 hours, centrifuge the mixed solution at 3000 rpm at room temperature, remove the supernatant, use PBS buffer to dissolve the precipitate, then add saturated ammonium sulfate dropwise to a final concentration of 33-35% in the solution, place it at 4°C, let it stand for more than 3 hours, repeat the above operation 1-2 times, and take the precipitate from the last centrifugation; S33, dissolve the precipitate in S32 in 0.02% mol / L pH7 PBS buffer of the same volume as the original duck egg yolk, dialyze for 18 to 48 hours, replace the dialysate 2-3 times during the dialysis process, until the dialyzate external fluid is no longer yellow when measured by Naphthol reagent, then take out the IgY antibody liquid in the dialyzer, filter it through a filter membrane to obtain the purified anti-Helicobacter pylori duck egg yolk antibody IgY.
10. A yogurt containing anti-Helicobacter pylori duck egg yolk antibodies, using the additive containing anti-Helicobacter pylori duck egg yolk antibodies according to claim 1, characterized in that: 100 g of duck egg yolk powder containing anti-Helicobacter pylori duck egg yolk antibody IgY was added to the fermented milk raw material that was treated at 90°C for 5 minutes and then cooled to 65°C. The temperature was maintained at 65°C for 15 minutes, followed by cooling, inoculation and fermentation operations to obtain yogurt with an anti-Helicobacter pylori duck egg yolk antibody IgY content of 2 mg / 100 g.