KR100364198B1 - An Improved Method for Inducing IgY to Helicobacter pylori and an Egg Prepared by Using the Same - Google Patents

Abstract

The present invention relates to a method for inducing improved IgY production for Helicobacter pylori and eggs produced by the same, and more particularly, to a method for inducing IgY production using a combination vaccine comprising a Helicobacter pylori vaccine and a typhoid vaccine. The present invention relates to an egg containing a large amount of IgY, wherein the method of the present invention not only produces a large amount of IgY against Helicobacter pylori, but also imparts immunity to typhoid, and the egg of the present invention prevents infection of Helicobacter pylori. And substantially assist in treatment.

Description

An Improved Method for Inducing IgY to Helicobacter pylori and an Egg Prepared by Using the Same}

The present invention relates to a method for inducing improved IgY production for Helicobacter pylori and eggs produced by the same, and more particularly, to a method for inducing IgY production using a combination vaccine comprising a Helicobacter pylori vaccine and a typhoid vaccine. To eggs containing large amounts of IgY.

Helicobacter pylori (Helicobacter pylori) was first separated, isolated from gastric biopsy specimens of a gram-negative bacillus, 1983 Australians gastritis (See: Warren, JR and Marshall, BJ , Lancet, 1: 1273-1275 (1983)) .

The Helicobacter pylori has several characteristics that can survive within the stomach. First, Helicobacter pylori in the stomach secretes enzymes called proteases and lipases to destroy the gastric mucus layer consisting of proteins, glycoproteins and lipids. Third, when Helicobacter pylori reaches the gastric mucosa, its own enzyme, urease, breaks down urea into ammonia and neutralizes the gastric acid in its surroundings, creating an environment in which it can live. In general, because the bacteria may be present in the stomach, it is easy to think that the optimal pH is acidic, but in reality, since the optimum condition is neutral within pH 7.0, the enzyme can be survived in the stomach by an enzyme called urease.

On the other hand, Helicobacter pylori is a causative agent of chronic active gastritis and gastric ulcer, especially duodenal ulcer, and has been reported to be closely related to the development of gastric cancer (Blaser, MJ, Gastroenterology , 102: 720-727 (1992)).

Until the discovery of Helicobacter pylori, the effect of treatment on gastrointestinal diseases was minimal, and the recurrence rate of peptic ulcer was very high. However, anti-helicobacter pylori therapy combined with antibiotics such as bismuth salt and omeprazole has shown a therapeutic effect of more than 80% of gastrointestinal diseases, and when helicobacter pylori is removed, the recurrence rate in duodenal ulcers is dramatically reduced. From the results (Peterson, WL, N. Engl. J. Med. , 324: 1043-1048 (1991)), the discovery of Helicobacter pylori is a new era in the treatment of gastrointestinal diseases.

The prevailing hypothesis is that Helicobacter pylori will be transmitted from the oral cavity to the oral or feces by the oral route, and epidemiological studies show that Helicobacter pylori infection is closely related to the economic level of society, which is not only low in the developed countries but also infected. While the predominant age group of the population is aging, the frequency of infection is increasing, and the probability of infection in childhood is high, and the frequency of infection is continuously maintained. In particular, Koreans report that 50% are infected when they are 5 years old, and about 90% are infected when they are 5 years old (see Kwangho Lee, et al . , Korean Journal of Microbiology , 25: 475-490 (1990)). ), The risk of Helicobacter pylori infection is very serious at the public health level.

Especially in Korea, the infection of Helicobacter pylori has been reported to have a tendency to be passed down to the household, so our dietary culture of Helicobacter Pylori infection, which is used by several people together, and the mother chews food and feeds the baby, It is assumed that there is a big association.

After Marshall and Baren separated Helicobacter pylori in 1994, the WHO International Agency for Research on Cancer (IARC) classified Helicobacter pylori as a highly pathogenic Type I carcinogen.

Helicobacter pylori is found in 92-100% of patients with gastric lymphoma, 90-95% of patients with duodenal ulcers, and 60-80% of patients with gastric ulcers.

In 1987, Cogran et al. Reported for the first time that the relapse rate of peptic ulcer was reduced due to the eradication of Helicobacter pylori (Coghlan, et al., Lancet , 2 (8568): 1109-1111 (1987)), and Graham et al. 1992 If ulcers were treated with gastric acid secretion inhibitors or antacids without disinfection of Helicobacter pylori, 80% of duodenal ulcers relapse within 20 weeks, and 70% of gastric ulcers relapse, but 10% of duodenal ulcers if helicobacter pylori was reinfected. Only 15% reported recurrence (Graham, et. Al., Am. J. Gastroenterol , 86 (9): 1158-1162 (1991)). In addition, gastric lymphomas have been cured after being disinfected with Helicobacter pylori.

Antibiotics are generally used to kill Helicobacter pylori, but since one type of antibiotic is generally not curable, a three-dose therapy that uses a combination of gastric acid secretion inhibitors and two antibiotics to suppress the progression of ulcers by gastric acid is recommended. It is widely used.

However, the three-agent regimen described above has the following disadvantages: First, Helicobacter pylori can achieve resistance to antibiotics used. In addition, colitis, diarrhea and black stools may occur due to the side effects of antibiotics, depending on the person may cause allergies to drugs, and the germicide rate is limited to about 90%.

Therefore, there is a need for a new bactericidal method that can supplement the disadvantages of conventional drug therapy for the bactericidal Helicobacter pylori.

The present inventors have made intensive research on a method that can replace the drug therapy against Helicobacter pylori according to the needs of the art described above, and as a result, helicobacter from eggs of laying hens inoculated with a combination vaccine including the Helicobacter pylori vaccine and Typhoid vaccine The present invention was completed by confirming that a large amount of IgY for pylori was generated.

Accordingly, it is an object of the present invention to provide a method of inducing improved IgY production for Helicobacter pylori.

Another object of the present invention is to provide an egg containing a large amount of IgY prepared according to the method of inducing IgY production.

Another object of the present invention is to provide a food comprising IgY isolated from the egg containing a large amount of IgY.

The invention features a method of inducing IgY production for Helicobacter pylori, comprising inoculating a laying hen with a combination vaccine comprising a Helicobacter pylori vaccine and a Typhoid vaccine.

In addition, the present invention is characterized by another egg containing a large amount of IgY for Helicobacter pylori prepared according to the method for inducing IgY production.

On the other hand, the present invention is another feature of the food containing IgY separated from eggs containing a large amount of IgY for the Helicobacter pylori.

The present invention is described in detail as follows:

The Helicobacter pylori vaccine used in the present invention is preferably an inactivated vaccine, but other vaccines such as attenuated vaccines, toxoids, synthetic vaccines and genetically engineered vaccines can also be used. The Helicobacter pylori inactivating vaccine includes helicobacter pylori as formalin, binary ethylenimine (hereinafter referred to as "BEI"), β-propiolactone (hereinafter referred to as "BPL"), thimerosal vaccines treated with inactivating agents such as thimerosal and the like.

The combination vaccine used in the present invention is preferably a Helicobacter pylori vaccine; Adjuvant selected from the group consisting of aluminum hydroxide gel and incomplete Freud adjuvant; And typhoid vaccine.

The combination vaccine employed in the invention is more preferably 1x10 ⁹ ~ 2x10 ⁹ Helicobacter pylori vaccine 10-20 dose containing the inactivated H. pylori in CFU / ㎖%; Adjuvant 65-75 wt%; And 10-20 dose% of a typhoid vaccine containing 0.5 × ^{10 11} to 1.5 × ^{10 11} CFU / mL inactivated typhoid strain.

According to a most preferred embodiment of the invention, the combined vaccine comprises 15% by volume of Helicobacter pylori vaccine containing 1.4 × 10 ⁹ CFU / ml of inactivated Helicobacter pylori; 70% by weight of incomplete Freud's adjuvant; And 15 dose% of a typhoid vaccine containing 1 × 10 ¹¹ CFU / ml of inactivated typhoid bacteria.

The combination vaccine is preferably inoculated into a laying hen 11 to 12 weeks old, because it is shown that the spawning decrease due to stress when inoculated in laying hens, in order to prevent spawning stress and to have antibodies to the first eggs Inoculate 4 weeks before spawning.

On the other hand, the inoculation of the combined vaccine can be carried out by various methods generally used in the art, it is preferable to inject the muscle to the chest of the laying hens.

According to a preferred embodiment of the present invention, the method for inducing IgY production for the Helicobacter pylori of the present invention is 10-20% by volume of the Helicobacter pylori vaccine containing 1x10 ⁹ to 2x10 ⁹ CFU / ml of inactivated Helicobacter pylori; Adjuvant 65-75 wt%; And injecting a complex vaccine comprising 10-20 dose% of a typhoid vaccine containing 0.5 × ^{10 11} to 1.5 × ^{10 11} CFU / mL inactivated typhoid strain into the pectoral muscles of 11-12 week old laying hens.

The above-described method of inducing IgY production for Helicobacter pylori induces antibody production against Helicobacter pylori as increased titer. In addition, the method of the present invention also effectively induces antibody production against typhoid. Therefore, there is generally no need to separately inoculate the typhoid vaccine which is inoculated in laying hens in poultry farms, and immunity to typhoid can be simultaneously achieved through the practice of the present invention.

The advantage of the present invention is that, in order to prevent the disease occurring in chickens until the start of spawning after hatching, in general, various types of vaccines are inoculated according to the plan, and if only the additional Helicobacter pylori vaccine is additionally inoculated, Because it is difficult to complete the various types of vaccinations to be carried out during the spawning, the vaccine may be inoculated during spawning and delayed spawning may occur due to vaccine stress. Secondly, when the two types of vaccines are mixed as in the present invention, it is possible to reduce the cost of vaccine production and the labor cost incurred in vaccination.

The eggs of the present invention prepared by the method described above contain large amounts of IgY for Helicobacter pylori.

In view of our eating habits, if any family member is infected with Helicobacter pylori, the entire family using the same utensils may be infected. Not only is it not desirable for all families to take drugs to cure these infections. It is almost impossible. What's more, you can't take medicine to prevent the infection of Helicobacter pylori.

However, the egg containing a large amount of IgY for the Helicobacter pylori of the present invention is not only a problem of the conventional drug therapy described above, but also ingested as a food, and thus a very effective method for preventing or treating infection of Helicobacter pylori. Can be.

In order to effectively use the egg of the present invention for the infection or treatment of Helicobacter pylori, it is most recommended to ingest naturally without heating the yolk portion of the egg, but it is preferable to eat the cooked so that the yolk does not ripen.

Meanwhile, methods for isolating and purifying immunoglobulins are already known in the art, and many of these known methods can be applied to IgY, an immunoglobulin contained in eggs ( Short protocols in molecular biology , 2nd edition, 1992, 11). -40-41, John Wiley Sons, Inc. New York; A practical guide to monoclonal antibodies , 1991, 116, John Wiley Sons, Inc. New York; Practical immunology , 3rd edition, 1989, 282, Blackwell scientific pulication, oxford London Protein methods , 1991, 71-90, John Wiley Sons, Inc. New York; Antibodies , 1988, 298-299, Cold spring harbor Laboratory). Therefore, IgY for Helicobacter pylori can be easily separated from the egg yolk of the egg of the present invention using the above known method, and various foods containing the same can be prepared.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1 Preparation of Helicobacter Pylori Medium

First, the main components of the medium used in the present invention were prepared as follows:

Helicobacter Agar Base (pH 7.2)

28 g of Brucella broth; 10 g of noble agar; And distilled water was added to 2 g of activated carbon to 1 L, and prepared by autoclaving.

Helicobacter liquid medium base (pH 7.2)

28 g of Brucella broth was added to distilled water to 1 L, and prepared by autoclaving.

Helicobacter Additive (20X; pH 7.2)

28 g of Brucella broth; 20 g of β-cyclotextrin; And 0.8 g of TTC (triphenyltetrazolium chloride) was added to distilled water to 1 L, and prepared by filtration sterilization with a filter paper having a membrane pore size of 0.45 μm.

Helicobacter pylori medium containing the above ingredients was then prepared as follows:

I. Helicobacter liquid medium

The above Helicobacter liquid medium base 1,000 ml, Helicobacter additive 50 ml and fetal bovine serum (horse serum) or 50 ml was prepared by mixing.

Ⅱ. Helicobacter Agar Badge

1,000 ml of the Helicobacter agar medium base, 50 ml of Helicobacter additive, 50 ml of fetal bovine serum or horse serum and 50 ml of sheep blood (sheep blood) were prepared by mixing.

Example 2: Cultivation of Helicobacter pylori

Liquid culture

Helicobacter pylori KUH00-503 strain isolated from Koreans who had been placed in a 10% CO ₂ incubator overnight at 1000 ml of Helicobacter liquid medium prepared in Example 1 and then sold at Korea University College of Medicine anatomy, inoculated, 37 ℃, Incubated for 2 days in a 10% CO ₂ incubator.

Agar culture

The Helicobacter pylori liquid culture solution was soaked in a cotton swab, and then plated on the Helicobacter agar medium, and passaged at three-day intervals under the same conditions as the liquid culture.

Example 3: Preparation of Helicobacter Antigen for Vaccine

The Helicobacter pylori cultured in the agar medium of Example 2 was collected and suspended in 50 ml of 0.15 M PBS (pH 7.2) solution, and the concentration of the strain was 7 × 10 by adding PBS solution so that the absorbance at 630 nm was 0.27. Dilute to ⁷ CFU / mL.

Then, the diluted strain solution was centrifuged at 4,000 rpm for 20 minutes, then the supernatant was removed, and 20 times concentrated (1.4 × 10 ⁹ CFU / mL) by suspending the precipitate in 1/20 PBS solution of the initial volume. Strain solution was obtained.

Then, the strain solution was divided into equal amounts and inactivated with formalin and BEI, respectively, so that formalin had a final concentration of 0.1% and BEI with a final concentration of 0.01 M. Inactivation was performed at 37 ° C. for 2 days, and after inactivation, a part of the specimen was collected and subjected to the following inactivation confirmation test, and the rest was stored at 4 ° C., and when inactivation was confirmed, this was used as the antigen for vaccine in the present invention. It was.

As a result, it was confirmed that both strains treated with formalin and BEI were inactivated and could be used as a safe vaccine antigen in the present invention.

Inactivation confirmation experiment in this Example was carried out by inoculating the inactivation solution in the following seven medium, and cultured under the following conditions to observe the growth of bacteria and fungi:

In the case of the used Helicobacter agar medium, the cells were incubated for 7 days at 37 ° C. in a 10% CO ₂ incubator. In the case of _two 5% sheep blood agar medium (40 g of blood agar base and 950 ml of distilled water and 50 ml of blood) , And incubated for 7 days at 27 ℃ and 37 ℃, respectively. In the case of the two thioglycolate mediums used (29.8 g of thioglycolate medium and 1 L of distilled water), the cells were cultured at 27 ° C. and 37 ° C. for 7 days, respectively, and two tryptic soy liquid media (tryptic soy) were used. Even 30 g of broth and 1 L of distilled water were incubated at 27 ° C. and 37 ° C. for 7 days, respectively.

37 ℃ at the culture temperature is for observing the growth of general bacteria, 27 ℃ is the temperature adopted to observe the growth of fungi and low temperature bacteria. All of the above seven media in the inactivation confirmation test must be free of bacteria and fungi to be used as vaccine antigens.

Example 4 Preparation of Typhoid Antigen for Vaccine

First, 8 g of Tryptic Soy Broth was added to distilled water to make 1 liter, and sterilized by autoclaving to prepare a liquid medium (pH 7.0). Isolated from brown) and incubated for 24 hours in a 37 ℃ incubator.

In this way, 1,000 ml of cultured Typhoid culture medium was centrifuged at 4,000 rpm for 30 minutes, the supernatant was removed, and the precipitate was suspended in 50 ml of PBS. Subsequently, 1 ml of the sample is diluted to 10 ^-10 decimal steps, 1 ml each of tryptic soy agar medium (40 g of tryptic soy agar and 1 l of distilled water), and then incubated at 37 ° C for 2 days, followed by bacterial counting. Was measured.

Then, formalin was added to 0.05% of the remaining Typhoid suspension and inactivated at 37 ° C for 2 days. After inactivation is completed, a part of the sample is collected and the inactivation confirmation test is carried out in the same manner as in Example 3, and the remainder is stored at 4 ° C., and when inactivation is confirmed, the inactivation bag is diluted to 1 × ^{10 11} CFU / mL. Used as a credit antigen.

Example 5 Vaccination

Preparation of the vaccine

Helicobacter antigen solution for inactivated vaccine prepared in Example 3, aluminum hydroxide gel as an adjuvant and PBS were mixed in a ratio of 15:20:65 to prepare a gel vaccine.

Helicobacter antigen solution for inactivated vaccine prepared in Example 3, Montanide ISA70 (including non metabolic mineral oil and mannide oleate, Sepiax, France) and PBS at a ratio of 15:70:15 The oil vaccine was prepared by mixing.

A typhoid vaccine was prepared by mixing the antigen solution for the inactivated vaccine prepared in Example 3, the typhoid antigen solution for vaccine prepared in Example 4, and Montanide ISA70 in an adjuvant ratio of 15:15:70. .

An oil vaccine was prepared by mixing the antigen solution for the inactivated vaccine prepared in Example 4, ISA70 and PBS in an adjuvant ratio of 15:70:15.

Vaccination

The prepared vaccines were inoculated with 0.5 ml of pectoral muscles of five 12-week-old laying hens (highline brown) per treatment group as shown in Table 1 below. At this time, the number of the final inoculation strains were 1 × 10 ⁸ CFU for Helicobacter pylori, 1 × 10 ¹⁰ CFU for Typhoid bacteria.

division Deactivator Adjuvant Antigen Control Uninoculated Treatment group A formalin Aluminum Hydroxide Gel Helicobacter Treatment group B formalin oil Helicobacter Treatment Group C BEI oil Helicobacter Treatment Group D formalin oil Helicobacter + Typhoid Treatment group E formalin oil typhus

Experimental Example 1 Measurement of IgY Titer for Helicobacter Pylori

IgY for Helicobacter pylori generated by Helicobacter pylori antigen inoculation performed in Example 5 was measured by ELISA method as follows:

First, the strain cultured in agar medium in Example 2 was suspended in 30 ml of 20 mM sodium phosphate buffer solution, and then ultrasonically pulverized three times at 30 second intervals. Subsequently, the mixture was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was filtered through a filter paper having a membrane pore size of 0.45 µm to obtain 30 ml of the filtrate, which was used as an antigen for ELISA.

The protein concentration of the ELISA antigen thus obtained was found to be 2.7 mg / ml as measured by the BCA method. Then, 1 mL of the ELISA antigen was diluted in 26 mL of coating buffer (pH 9,5), the protein concentration was adjusted to 1.0 mg / mL, and then 0.1 mL of each well of the ELISA plate was dispensed at 4 ° C. Coated overnight at.

Then, five eggs per group were collected from the laying hens 6 weeks after the vaccination performed in Example 5, and egg yolk was separated from the egg whites. The isolated yolk was then diluted 1,000-fold in PBS and inactivated for 30 minutes at 56 ° C.

After the antigen coating was finished, the antigen solution of the plate was removed and washed three times with PBS, 0.1 ml each of the 1,000-fold diluted egg yolk was dispensed in a plate well and reacted at 37 ° C for 30 minutes. After the reaction was completed, washed three times with PBS and the antibody to chicken immunoglobulin to which the horseradish peroxidase is bound 0.1 ml of each well of the plate was reacted for 30 minutes at 37 ℃.

After the reaction was completed, washed three times with PBS and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid: ABTS) substrate solution was added 0.1 ml per well of the plate and reacted for 5 minutes at room temperature After stopping the reaction with 3 M sulfuric acid, the absorbance was measured with an ELISA reader at 405 nm, and the results are shown in Table 2 below:

division Absorbance (Mean ± Standard Deviation) P / N ratio ^* Control 0.005 ± 0.014 Treatment group A 0.036 ± 0.006 0.719 Treatment group B 0.210 ± 0.066 4.221 Treatment Group C 0.172 ± 0.055 3.462 Treatment Group D 0.243 ± 0.036 4.889 ^* Absorbance of treated group divided by absorbance of control [less than 1.5: negative (no difference); 1.5 and above 2.0: false positive (may vary); Positive over 2.0 (with differences)]

As can be seen in Table 2 above, there is a high antibody titer against Helicobacter pylori in Treatment Group D administered a combination vaccine comprising Helicobacter pylori vaccine and Typhoid vaccine.

Experimental Example 2: Investigation of antibody titers against poultry typhoid

Antibody titers in blood against Typhoid bacteria produced by Typhoid antigen inoculation performed in Example 5 were performed by aggregation reaction as follows:

First, the vaccine antigen produced in Example 5 was diluted 10-fold with PBS and used as an antibody titer test. Subsequently, blood was collected from 5 chickens in each group from the laying hens 6 weeks after the vaccination performed in Example 5 to separate serum. After each serum was inactivated at 56 ° C. for 3 minutes, dilute the binary step to 2 ^-12 with PBS using a U plate, add the same amount of antigen for aggregation reaction, and leave it at room temperature for 1 hour, then observe whether the antigen was aggregated. The antibody titers were measured, and the results are shown in Table 3 below.

division Antibody Titers (log ² base) Average Treatment Group D 6, 8, 9, 7, 9 7.8 Treatment group E 6, 7, 7, 6, 6 6.4

As can be seen in Table 3, it can be seen that there is almost no difference in the titus antibody titers of the treatment group E inoculated with poultry typhoid bacteria alone and the treatment group D treated with the combination vaccine of the present invention.

As described in detail above, the present invention provides a method of inducing improved IgY production for Helicobacter pylori. The present invention also provides an egg containing a large amount of IgY prepared according to the method of inducing IgY production. On the other hand, the present invention provides a food comprising IgY isolated from the egg containing a large amount of IgY. The improved method of inducing IgY production of the present invention not only allows the production of large amounts of IgY for Helicobacter pylori, but also confers immunity to typhoid. In addition, since the eggs of the present invention are consumed by the general consumer as food, it is possible to substantially assist in the prevention and treatment of infection of Helicobacter pylori.

Claims (8)

A method for inducing IgY production for Helicobacter pylori, comprising: 10-20% dose of Helicobacter pylori vaccine containing 1x10 ⁹ -2x10 ⁹ CFU / mL of inactivated Helicobacter pylori; And inoculating a 11-12 week old laying hen with a multiple vaccine comprising 10-20 doses of a typhoid vaccine containing 0.5 × ^{10 11} −1.5 × ^{10 11} CFU / ml of an inactivated typhoid strain, for improved Helicobacter pylori. Induction method of IgY production. 2. The IgY production of improved Helicobacter pylori according to claim 1, wherein the inactivation of Helicobacter pylori is made by an inactivating agent selected from the group consisting of formalin, binary ethylenimine, β-propiolactone and thimerosal. Method of induction. 2. The induction of IgY production for improved Helicobacter pylori according to claim 1, wherein the combined vaccine further comprises 65-75% by weight of an adjuvant selected from the group consisting of aluminum hydroxide gel and incomplete Freud adjuvant. Way. delete The method of claim 1, wherein said inoculation is performed by intramuscular injection to the chest of a laying hen. A method for inducing IgY production for Helicobacter pylori, comprising: 10-20% dose of Helicobacter pylori vaccine containing 1x10 ⁹ -2x10 ⁹ CFU / mL of inactivated Helicobacter pylori; Adjuvant 65-75 wt%; And intramuscularly injecting a complex vaccine comprising 10-20 dose percent of a typhoid vaccine containing 0.5x10 ¹¹ -1.5x10 ¹¹ CFU / ml of an inactivated typhoid strain into the chest of an 11-12 week old laying hen. Derivation of IgY production for pylori. An egg containing a large amount of IgY for Helicobacter pylori prepared according to any one of claims 1, 2, 3, 5 and 6. Food containing IgY isolated from the egg yolk of the said egg of Claim 7.