KR100492492B1 - THE PRODUCTION METHOD FOR EGG CONTAINING ANTI-E.coli(K88) IgY, ANTI-E.coli(K99) IgY, ANTI-PEDV IgY AND ANTI-TEGV IgY AND THEREOF EGG - Google Patents

Abstract

The present invention relates to a method for producing an egg containing a complex special immune protein against E. coli (K88), E. coli (K99), pandemic diarrheal virus, infectious gastroenteritis virus, and more specifically to the egg produced by the above method. The present invention relates to a method for producing an egg containing special immune protein by antigenizing E. coli (K88), E. coli (K99), epidemic diarrheal disease virus and infectious gastroenteritis virus, and immunizing chicks, and an egg containing the special immune protein.

By adding the egg containing the special immune protein to the feed to feed the piglets to prevent diarrhea or gastroenteritis due to infectious, epidemic or digestive diseases, to reduce the mortality of piglets, and to improve the pig farming business of Korean farmers.

Description

Method for producing eggs containing complex special immune protein against Escherichia coli (K88), Escherichia coli (K99), pandemic diarrheal virus, infectious gastroenteritis virus and eggs produced by the above method {THE PRODUCTION METHOD FOR EGG CONTAINING ANTI-E.coli (K88) IgY, ANTI-E. Coli (K99) IgY, ANTI-PEDV IgY AND ANTI-TEGV IgY AND THEREOF EGG}

The present invention relates to a method for producing an egg containing a complex special immune protein against E. coli (K88), E. coli (K99), pandemic diarrheal virus, infectious gastroenteritis virus, and more specifically to the egg produced by the above method. The present invention relates to a method for producing an egg containing special immune protein by antigenizing E. coli (K88), E. coli (K99), epidemic diarrheal disease virus and infectious gastroenteritis virus, and immunizing chicks, and an egg containing the special immune protein.

In general, E. coli, Salmonella enteritidis, Salmonella typhimurium, Pandemic diarrheal virus, Infectious gastroenteritis virus, Isospora suis, Campylobacter, Rotavirus, etc. It is happening.

In particular, the onset of diarrhea in piglets caused by E. coli, epidemic diarrheal virus, and infectious gastroenteritis virus is causing enormous economic damage to a huge Korean farm.

Diarrhea in piglets caused by E. coli occurs at all ages in pigs, especially the most common occurrence in piglets. The incidence of diarrhea in piglets by E. coli occurs continuously throughout the year and shows strong susceptibility in the early and early weaning seasons. Escherichia coli invade the small intestine of pigs and causes disease by attaching to the barrier. At this time, adhesion factors (pili) that frequently cause diseases include K88, K99, 987p, and F41. Diarrhea occurs frequently after 1 to 2 weeks of lactation and weaning, and edema develops after weaning. Piglets that are not fed colostrum or piglets born to sows that do not receive antibodies to E. coli in colostrum are susceptible to E. coli.

The cause of the epidemic diarrheal virus (PEDV) is Coronaviridae Coronavirus (Coronaviridae Coronavirus), it is more likely to appear in the form of a mixed infection with infectious gastroenteritis virus than a single infection, especially mortality of piglets during mixed infection with infectious gastroenteritis virus Increases further. Swine pandemic diarrhea caused by the epidemic diarrheal virus (PEDV) is a gastrointestinal disease with vomiting and watery diarrhea. It occurs mainly in winter, occurs all year round, and has a slower spread rate than infectious gastroenteritis.

The infectious gastroenteritis virus occurs year-round, but occurs mainly in winter when the temperature is low. Acute diarrheal disease occurs in pigs of all ages, most often in piglets less than one week old. Diseases caused by infectious gastroenteritis virus occur all over the world, and in Korea, it has been commercialized since the 1950's and caused many economic damages.

Preventive measures to reduce pig mortality due to the above-mentioned Escherichia coli, epidemic diarrheal virus, and infectious gastroenteritis virus are to thoroughly control hygiene and disinfection of pig farms, and the second vaccination two to three weeks before the delivery of maternal pigs. Prevent diarrhea in piglets. However, this preventive practice continues to occur, and in some farms, in order to prevent piglets' diarrhea, feed feeding for sows is kept at a too low level during the lactation period, resulting in weaning weight loss and delayed post-weaning. Problems such as have appeared.

The present invention for solving the above-mentioned problems, E. coli (K88), E. coli (K99), epidemic diarrhea virus (PEDV), causing pancreatic diarrhea, infectious gastroenteritis virus (TGEV: Transmissible gastroenteritis virus) It is intended to provide an egg production method containing the complex special immune protein by antigen inoculation at the same time to the chicks and an egg containing the complex special immune protein produced by the method.

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The present invention for achieving the above-described technical problem, E. coli (K88), E. coli (K99), epidemic diarrhea virus (PEDV), infectious gastroenteritis virus (TGEV) that causes piglets diarrhea It is characterized by a method of producing an egg containing the complex special immune protein by antigenization and inoculation at the same time by the chick, and the egg containing the complex special immune protein produced by the method.

Looking at the configuration of the present invention as follows.

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The present invention is inoculated once with 1 ml of a mixed bacterium emulsion emulsified with E. coli (K88) antigen, E. coli (K99) antigen, epidemic diarrheal virus (PEDV) antigen and infectious gastroenteritis virus (TGEV) antigen with aluminum hydroxide. Inoculate the mixed bacterial emulsifier emulsified with the emulsification adjuvant (ISA25) two times at a rate of 1 ml two times at two week intervals, and then the mixed bacterial emulsifier emulsified with the emulsification adjuvant (ISA25) at intervals of four months again. The present invention relates to a method of producing an egg containing a complex special immune protein by inoculating a total of five times, including two inoculations of 0.5 ml each.

More specifically, in the present invention, the antigen inoculated to chicks is E. coli (K88) antigen: Escherichia coli (K99) antigen: pancreatic diarrheal disease virus antigen: infectious gastroenteritis virus antigen: aluminum hydroxide ratio of 2.1: 0.7: 3.5: 0.7: 0.21 ml of 3.8 × 10 ⁸ / ml E. coli (K88) bacteriophage antigen, 0.07ml of 3.6 × 10 ⁸ / ml E. coli (K99) bacteriophage antigen, 3 × 10 ⁷ TCID ₅₀ / ml influenza diarrheal virus inactivating antigen 1 ml of a mixed bacterial emulsion, emulsified with 0.35 ml and 1.5 x 10 ⁷ TCID ₅₀ / ml 0.07 ml of infectious gastroenteritis virus inactivated antigen with 0.3 ml of aluminum hydroxide, was inoculated once in the chick's leg, and the same ratio of the same four The mixed bacterial emulsion prepared by emulsifying the antigen with the same amount of emulsifying aid (ISA25) was inoculated twice in 1 ml at 2 weeks intervals into the chick leg, and the mixed bacterial emulsion prepared by emulsifying with the emulsifying aid (ISA25) was grown. Lay the hens inoculated twice with 0.5 ml each at 4 month intervals. The present invention relates to a method for producing the egg containing a complex special immune proteins were inoculated five times.

In the present invention, 1 ml of a mixed bacteria emulsion emulsified with E. coli (K88) antigen, E. coli (K99) antigen, epidemic diarrheal virus (PEDV) antigen, and infectious gastroenteritis virus (TGEV) antigen with Freund's complete adjuvant 1 ml Inoculated twice, inoculated twice with 1 ml of the mixed bacterial emulsion emulsified with Freund's incomplete adjuvant from the second time, and the mixed bacterial emulsified solution emulsified with the Freund's incomplete adjuvant at intervals of 4 months at the growing laying hens. The present invention relates to a method of producing an egg containing a complex special immune protein by inoculating a total of five times, including two inoculations of 0.5 ml each.

More specifically, in the present invention, the antigen inoculated in chicks is E. coli (K88) antigen: Escherichia coli (K99) antigen: pancreatic diarrheal disease virus antigen: infectious gastroenteritis virus antigen: emulsification adjuvant ratio of 2.1: 0.7: 3.5: 0.7: 3 0.21 ml of 3.8 × 10 ⁸ / ml E. coli (K88) bacterium antigen and 0.07ml of 3.6 × 10 ⁸ / ml E. coli (K99) bacterium antigen and 3 × 10 ⁷ TCID ₅₀ / ml influenza diarrheal virus inactivating antigen 0.35 1 ml of mixed bacterial emulsion, emulsified with 0.3 ml of adjuvant complete Freund's and 0.07 ml of 1.5 × 10 ⁷ TCID ₅₀ / ml infectious gastroenteritis virus inactivated antigen, was inoculated once in the chick's leg, and from the second time A mixed bacterial emulsion prepared by emulsifying the same antigens of the same ratio with the same amount of adjuvant incomplete Freund's was inoculated twice at 1 ml intervals in 2 ml intervals on the chick leg, and the Freund's incomplete adjuvant ( oil painting by adjuvant incomplete Freund's It relates to a method for producing the egg containing a complex special immune proteins were inoculated five times, including two by 0.5㎖ a four month intervals once again inoculated in the growth of bacteria mixed emulsion prepared hens.

In addition, the present invention, the egg containing the complex special immune protein produced by the above method was added 2 kg of egg yolk from which a yolk film was removed from a predetermined container, and stirred by adding an equivalent amount of alkaline ionized water (pH 10) 2 kg and stirring for 24 hours. After standing at (5-10 ° C), yolk: 18 times (72 kg) of alkaline ionized water (1: 1) is mixed and left for 48 hours to extract water-soluble protein, and then the supernatant is ultra filtered. The present invention relates to a method for producing a complex special immune protein which is concentrated and lyophilized by Hollow fiber membrane separation method.

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Hereinafter, the configuration of the present invention will be described in more detail based on embodiments and drawings or diagrams. However, the scope of the present invention is not limited to the claims of the present invention by the embodiments or the drawings.

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Example 1

In Example 1, E. coli (K88), Escherichia coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) were antigenicized and inoculated into chicks to separate the complex special immune protein from the chick-produced eggs. It relates to a piglet feed containing the complex special immune protein.

1. Preparation of Enteropathogenic E. coli Antigen

A) Escherichia coli

E. coli used as antigen in this test was used serotypes K88 and K99 according to the adhesion factor, and the serotypes K88 and K99 were used by the gene bank (KCTC). The selected colonies grew into pink colonies on MacConkey agar, and the specific colonies of E. coli (K88) and E. coli (K99), which grew on metallic green by smearing on EMB medium (EMB agar), were developed. Confirmed.

B) E. coli (K88), E. coli (K99) antigen production

The E. coli (K88) and E. coli (K99) strains were inoculated in Trypticase soy agr and incubated for 18 hours at 37 ° C. in a colony (colony) 5ml trypticase Soy Broth ( Trypticase soy broth). After 2 hours, inoculated in a large amount of tryticase soy broth and incubated for 48 hours at 37 ℃. The cultured bacterial solution was mixed with formaldehyde (0.5%) of the total amount and inactivated at room temperature for 24 hours. The inactivated cells were centrifuged at 4,000 rpm for 20 minutes to harvest the cells and washed three times with sterile physiological saline (pH 7.2). The harvested cells were used by cryopreservation at -70 ℃.

2. Preparation of Pandemic Diarrheal Virus (PEDV) Antigen

1) propagation of viruses

① Cell line

Cell lines used as host cells for the propagation of the virus used vero cells derived from monkey kidney cells.

② Medium used

Α-Minimum essential Medium (MEM, Gibco BRL) was used as a cell maintenance medium, and 5% of calf serum was added to the medium, and yeast extract per 1 L of the medium. ) 0.02%, tryptose phosphate broth (Typtose phosphate broth) was used to be 0.3%.

③ Amplification of pandemic diarrheal virus

Vero cells were dispensed into the cell culture flask at 1 × 10 ⁵ cells / ml, and when the Vero cells were filled to about 70% of the flask in a single layer, a non-serum-containing medium containing 10 μg / ml trypsin was used. After washing, the epidemic diarrhea virus solution was inoculated. The cells were sensitized at 37 ° C. for 90 minutes and added to the used medium containing 2 μg / ml trypsin, and then observed for cell degeneration at 37 ° C. incubator. After 2 days, the cell degeneration was frozen. Stored at ° C.

④ inactivation of the pandemic diarrheal virus

Inactivation of the pandemic diarrheal virus was performed using 2-Bromoethylamide (BEI). BEI with a final concentration of 0.004M was added to the epidemic diarrheal virus solution and inactivated by stirring at 37 ° C for 5 hours. The stirring reaction was stopped with 0.004M sodium thiosulfate solution (2M).

⑤ Preparation of Pandemic Diarrheal Disease Virus Antigens for ELISA

Epidemic diarrhea virus amplified in Vero cells was centrifuged for 90 minutes at 25,000 rpm. After centrifugation, the precipitated virus was suspended in 2 ml of buffer, left at 4 ° C. for 16 hours, and resuspended. The suspended liquid was sonicated three times for 1 minute at 18 Hz and used as an ELISA antigen.

3. Preparation of Infectious Gastritis Virus (TGEV) Antigen

1) propagation of viruses

① Cell line

The cell line used as a host cell for propagation of infectious gastroenteritis virus was used as a swine testis cell (ST cell).

② Medium used

As a cell maintenance medium, α-Minimum Essential Medium (MEM, Gibco BRL) was used, and 5% fetal bovine serum was added to the medium.

③ Amplification of infectious gastroenteritis virus

Infectious gastroenteritis virus after dispensing the porcine testis cells (ST cell) into the cell culture flask at 1 × 10 ⁵ cell / ml and washing the porcine testis cells three times with a non-serum-containing medium when the pig testis cells were filled to about 70% of the flask as a monolayer. The solution was inoculated. Sensitize at 37 ° C. for 90 min, wash once and add use medium containing 0.5 μg / ml trypsin. Cell degeneration was observed in a 37 ° C. thermostat. After 2 days, the cell degeneration was frozen and thawed and stored at -70 ° C. three times.

④ Inactivation of infectious gastroenteritis virus

Inactivation of infectious gastroenteritis virus was performed using 2-Bromoethylamide (BEI). Infectious gastroenteritis virus solution was added with BEI having a final concentration of 0.004M and stirred at 37 ° C. for 5 hours to inactivate it. The stirring reaction was stopped with 0.004M sodium sulphate solution (2M).

⑤ Preparation of infectious gastroenteritis virus antigen for ELISA

Epidemic diarrhea virus amplified in porcine testicular cells (ST cell) was centrifuged at high speed for 2 minutes at 25,000 rpm. After centrifugation, the precipitated virus was suspended in 2 ml of buffer, left at 4 ° C. for 16 hours, and resuspended. The suspended liquid was sonicated three times for 1 minute at 18 Hz and used as an ELISA antigen.

4. Inoculate the combined four antigens into chicks and boost the laying hens

A) The antigens inoculated into 12-week-old chicks were E. coli (K88) antigens: E. coli (K99) antigens: pancreatic diarrheal virus antigens: infectious gastroenteritis virus antigens: aluminum hydroxide ratio of 2.1: 0.7: 3.5: 0.7: 3. 0.21 ml of 3.8 × 10 ⁸ / ml E. coli (K88) bacteriostatic antigen and 0.07ml of 3.6 × 10 ⁸ / ml E. coli (K99) bacteriostatic antigen and 3 × 10 ⁷ TCID ₅₀ / ml 0.35 ml of pandemic diarrheal virus inactivating antigen 1.5 × 10 ⁷ TCID ₅₀ / ㎖ infectious gastroenteritis virus inactivation supplements the first doses, and used for boost from the second emulsion by mixing fungus 1㎖ emulsifying the antigen 0.07㎖ with aluminum hydroxide 0.3㎖ the chick leg is known emulsion The mixed bacterial emulsifier emulsified with the adjuvant (ISA25) was inoculated twice at 2 weeks intervals in 1 ml portions of the chicks' legs, and emulsified with the emulsification adjuvant (ISA25) prepared at the same ratio at 4 month intervals in a 28-week-old laying hen. Inoculate the mixed bacterial emulsion twice with 0.5 ml each The antigens were simultaneously vaccinated a total of five times. As a result, one egg produced from the laying hens inoculated with the mixed bacterial emulsification solution had anti-E. Coli (K88) special immune protein and anti-E. Coli (K99) special immune protein, anti-pandemic diarrheal virus special immune protein and anti-infectious gastritis. Eggs that simultaneously shared the virus special immune protein were produced.

B) The antigens inoculated into 12-week-old chicks are E. coli (K88) antigens: E. coli (K99) antigens: pancreatic diarrheal virus antigens: infectious gastroenteritis virus antigens: emulsifiers: 2.1: 0.7: 3.5: 0.7: 3 0.21 ml of 3.8 × 10 ⁸ / ml E. coli (K88) bacteriostatic antigen and 0.07ml of 3.6 × 10 ⁸ / ml E. coli (K99) bacteriostatic antigen and 3 × 10 ⁷ TCID ₅₀ / ml 0.35 ml of pandemic diarrheal virus inactivating antigen to 1.5 × 10 ⁷ TCID ₅₀ / ㎖ infectious gastroenteritis virus inactivation boosting the antigen 0.07㎖ Freund's complete adjuvant (complete Freund's adjuvant) vaccinated once the bacteria were mixed with the emulsion 1㎖ emulsion 0.3㎖ the chick leg, and from the second time The adjuvant used was inoculated twice with 2 ml of 1 ml each of the mixed bacterium emulsion emulsified with known adjuvant incomplete Freund's at 2 weeks intervals, and at 28 weeks old laying hens at the same rate. Prepared Freund's incomplete adjuvant (adjuv 0.5 ml each of the mixed bacterial emulsions emulsified with ant incomplete Freund's) was inoculated five times in total for the four antigens. As a result, anti-E. Coli (K88) special immune protein and anti-E. Coli (K99) special immune protein and anti- pandemic diarrheal virus special immune protein and anti-infectious gastritis virus Eggs that simultaneously shared the immunoprotein were produced.

5. Egg yolk separation, egg yolk powder extraction, special immune protein separation and titer measurement

A) Separation of egg yolk

Eggs produced by the above method were broken and egg whites were removed, and fresh egg yolks were separated and stored in a designated container.

B) egg yolk powder

The separated fresh yolk was lyophilized to prepare yolk powder and stored in a cold place.

C) Isolation of special immune proteins

20 g of egg yolk from which egg yolk was removed from a predetermined container was added to the egg produced by the above a) or b), and 20 ml of the same amount of alkaline ionized water (pH 10) was added thereto, followed by stirring. Left to stand, egg yolk: Alkaline water (1: 1) and 18 times (720 ml) of alkaline water are mixed and left for 48 hours. The water-soluble protein is extracted and the supernatant is filtered through an ultra-filtration system. Hollow fiber) was concentrated by membrane separation and lyophilized to prepare a special immune protein powder.

D) titer measurement

The content of special immune protein in the water-soluble protein obtained by the above method was measured as follows.

In addition, the measurement of the content of special immune protein was performed by sandwich ELISA (ELISA) method. The absorbance (OD) values of Escherichia coli (K88) and Escherichia coli (K99) were diluted in a buffer solution so as to be 0.05 at 660 nm, and epidemic diarrheal virus and infectious gastroenteritis virus were diluted 100-fold using ELISA antigen. It was. The diluted causal agent was coated on a microplate and left overnight. After washing the microplate, the filtered water-soluble protein was added and the reaction was washed, and rabbit anti-cheek IgG Ab-HRP diluted to 1 / 10,000 was added. TMB was used as a substrate for HRP, and absorbance was measured at 450 nm using 2N-sulfuric acid (2N-H ₂ SO ₄₎ as a reaction stopper. This result is shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG.

As shown in Figure 1, when E. coli (K88), E. coli (K99), epidemic diarrheal disease virus, infectious gastroenteritis virus is inoculated using Freund's emulsification adjuvant and emulsification adjuvant (ISA25), the bacteria and viruses are emulsification adjuvant The titer was higher when inoculated with Freund's emulsification aid than (ISA25), followed by E. coli (K88), E. coli (K99), epidemic diarrheal virus, and infectious gastroenteritis virus.

As shown in Figures 2 and 6, E. coli (K88) showed the highest titer at the time of inoculation using Freund's emulsification aid at the time of complex inoculation rather than single inoculation, the titer was maintained even after 40 days of inoculation .

As shown in Figures 3 and 7, E. coli (K99) showed the highest titer at the time of inoculation using Freund's emulsifying adjuvant at the time of multiple inoculation rather than a single inoculation, the titer was maintained even after 40 days of inoculation .

As shown in Figures 4 and 8, the epidemic diarrheal virus virus was the highest titer at the time of inoculation using Freund emulsification aid at the time of multiple vaccinations, even after 60 days of inoculation schedule without much difference with the maximum titer Was maintained.

As shown in FIG. 5 and FIG. 9, the infectious gastroenteritis virus showed the highest titer in the case of single inoculation, but maintained high titer even after 60 days of inoculation at the time of inoculation with Freund's emulsifying adjuvant.

6. Preparation of pig feed containing four special immune proteins

A) Water Soluble Protein Extraction

Water-soluble protein extraction was carried out in the following manner. 35 g of the yolk from which the membrane was removed was placed in a 250 ml bottle, and 35 ml of alkaline ionized water was added and stirred. It is allowed to stand at low temperature (5 ~ 10 ℃) for 24 hours, mixed with 18 times (1260 ml) of alkaline ionized water (1260 ml) of egg yolk: alkaline water (1: 1), left for 48 hours, extracted and ultra-filtered. Ultra filteration) was concentrated by Hollow fiber membrane separation and lyophilized.

B) Preparation of piglets containing extracted water-soluble protein

As in the previous section, after inoculation with the combined antigen mixed with E. coli (K88) antigen, E. coli (K99) antigen, epidemic diarrheal disease virus antigen, and infectious gastroenteritis virus antigen, the egg yolk was separated from the produced egg and then extracted with water-soluble protein (crude). IgY) powder was added and used as pig feed (Table 1, Table 2).

C) Preparation of piglets containing egg yolk powder

As shown in the previous section, egg yolks were isolated from eggs produced by inoculation of a mixed antigen mixed with E. coli (K88), E. coli (K99), epidemic diarrheal virus, and infectious gastroenteritis, followed by lyophilization and complex special immunity. Egg yolk powder containing protein was added and used as a pig feed (Table 1, Table 2).

Diarrhea Prevention Pig Feed Formulation Example Raw material Compounding ratio A Compounding ratio B Compounding ratio C corn 62.5 64.00 64.00 Soybean meal 24.8 22.25 17.90 Fishmeal 3.00 3.50 3.50 Limestone 0.90 0.90 0.90 Raise calcium 1.50 1.40 4.00 Processed salt 0.40 0.40 1.40 molasses 1.00 2.00 0.40 Confectionery 2.00 2.00 2.00 Nutrients 3.70 3.50 2.00 * Yolk powder containing complex special immune protein 0.20 - 3.50 Complex Special Immune Protein Powder - 0.05 - ME (㎉ / ㎏) 3,250 3,300 3,300 calcium 1.12 1.10 1.20 sign 0.68 0.60 0.60 * Combination special immune protein is extracted from eggs produced by inoculating four antigens

Diarrhea Prevention Pig Feed Formulation Example Raw material Compounding ratio A Compounding ratio B Compounding ratio C corn 36.8 36.8 36.6 Soybean meal 21.8 20.05 18.6 Fishmeal 2.0 2.0 2.0 Limestone 0.5 0.5 0.5 Processed salt 0.2 0.2 0.2 Powder Uji 9.6 9.5 9.6 glucose 11.0 11.0 11.0 Soybean oil 15.0 15.0 15.0 MCP 1.5 1.5 1.5 Nutrients 1.6 1.6 1.6 Yolk Powder Containing Complex Special Immune Protein 0.2 2.0 2.0 Complex Special Immune Protein Powder - 0.05 - ME (㎉ / ㎏) 3,300 3,280 3,300 calcium 1.00 1.00 1.00 sign 0.67 0.67 0.67 Complex Immune Protein Extracted from Eggs Produced by Combining Four Antigens

Example 2

In Example 2, E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) were antigenically challenged to different chicks, and the individual eggs produced by the different chicks The present invention relates to a piglet feed containing a mixed composition in which each special immune protein is separated and a predetermined ratio of each special immune protein is mixed.

1. Production of E. coli (K88), E. coli (K99) antigens

Antigen preparation of Escherichia coli (K88) and Escherichia coli (K99) used in the present invention is as shown in Example 1.

2. Antigen Preparation of Pandemic Didemic Diarrhea Virus (PEDV)

The preparation of a pancine epidemic diarrhea virus (PEDV) antigen is also as shown in Example 1.

3. Preparation of Transmissible Gastroenteritis Virus Antigen

Transmissible gastroenteritis virus antigen preparation is also as shown in Example 1.

3. Inoculate the four antigens individually into chicks and boost the laying hens

A) The number of cells inoculated into 12-week-old chicks was 10 ⁸ / ml, and the ratio of E. coli (K88) antigen: emulsification aid was 1: 1, and the ratio of E. coli (K99) antigen: emulsification aid was 1: 1. The ratio of epidemic diarrheal virus antigen: emulsification adjuvant shall be 1: 1, and the ratio of infectious gastroenteritis virus antigen: emulsification adjuvant shall be 1: 1,0.5ml of 2.0 × 10 ⁸ / ml E. coli antigen antigen and aluminum hydroxide 0.5 ml of emulsified, 2.0 × 10 ⁸ / ml E. coli (K99) bacterium antigen 0.5ml and 0.5ml of aluminum hydroxide were emulsified, and 2 × 10 ⁷ TCID ₅₀ / ml epidemic diarrheal virus inactivating antigen and 0.5ml of aluminum hydroxide Emulsified, each individual bacterial emulsion prepared by emulsifying 2 × 10 ⁷ TCID ₅₀ / ml 0.5 ml of infectious gastroenteritis virus inactivating antigen and 0.5 ml of aluminum hydroxide was inoculated once with 1 ml of different chicken legs, and the second time Each of the four antigens from the emulsification adjuvant (ISA25) The emulsified individual emulsifier was inoculated twice in 2 ml intervals in 1 ml each of the different chicks, and 0.5 ml of the individual emulsified liquor emulsified in the emulsification aid (ISA25) at 4 month intervals in the growing laying hens. A total of 5 inoculations were included.

B) The number of cells inoculated into 12-week-old chicks was 10 ⁸ / ml, and the ratio of E. coli (K88) antigen: emulsifier was 1: 1, and the ratio of E. coli (K99) antigen: emulsifier was 1: 1. The ratio of epidemic diarrheal virus antigen: emulsification adjuvant is 1: 1, and the ratio of infectious gastroenteritis virus antigen: emulsification adjuvant is 1: 1, 0.5 × 2.0 × 10 ⁸ / ml E. coli antigen 0.5ml and aluminum hydroxide 0.5ml Emulsified 2.0 × 10 ⁸ / ml E. coli (K99) bacterium antigen and 0.5ml of aluminum hydroxide, emulsified 2 × 10 ⁷ TCID ₅₀ / ml epidemic diarrheal virus inactivating antigen and 0.5ml of aluminum hydroxide , 1 × 1 ml of each individual bacterial emulsion prepared by emulsifying 2 × 10 ⁷ TCID ₅₀ / ml 0.5 ml of infectious gastroenteritis virus inactivating antigen and 0.5 ml of Freund's complete adjuvant. Vaccination, and from the second time Freund's incomplete adjuvant (adjuv After inoculating the individual bacterial emulsion using ant incomplete Freund's twice at 2 weeks intervals at 1 ml at the same rate as the individual bacterial emulsion, 0.5 µl of the individual bacterial emulsion prepared at the same rate was applied to the different growing laying hens at 4 month intervals. A total of five inoculations were included, including two inoculations of 2 ml each.

As a result of the above a), b) anti-E. Coli (K88) special immune protein, anti-E. Coli (K99) special immune protein, anti- pandemic diarrheal virus special immune protein, Eggs containing individual anti-infective gastroenteritis virus special immune proteins were produced.

4. Egg yolk separation, egg yolk powder extraction, special immune protein isolation and titer measurement

Separation and measurement of special immune proteins used in egg yolk separation, egg yolk powder extraction, and test analysis are the same as in Example 1.

5. Preparation of piglets containing anti-mixed bacteria special immune protein

As described in the previous section, each of the special immune protein powder (crude IgY) or egg yolk dry powder extracted from eggs produced after inoculation with E. coli (K88), E. coli (K99), pandemic diarrheal virus, and infectious gastroenteritis virus is 1: 1: Select any one of the ratios of 1: 1, 2: 1: 1: 1, 3: 1: 1: 1, 4: 1: 1: 1, 5: 1: 1: 1, and mix them as feed additives. It was used, the preparation ratio of piglet feed is as shown below.

Diarrhea Prevention Pig Feed Formulation Example Raw material Compounding ratio A Compounding ratio B Compounding ratio C corn 62.5 64.0 64.0 Soybean meal 24.8 22.25 17.9 Fishmeal 3.0 3.5 3.5 Limestone 0.9 0.9 0.9 Calcium phosphate 1.5 1.4 4.0 Processed salt 0.4 0.4 1.4 molasses 1.0 2.0 0.4 Confectionery 2.0 2.0 2.0 Nutrients 3.7 3.5 2.0 * Yolk powder containing anti-mixed bacteria special immune protein 0.2 - 3.5 * Anti-Mixed Special Immune Protein - 0.05 - ME (㎉ / ㎏) 3,250 3,300 3,300 calcium 1.12 1.1 1.2 sign 0.68 0.6 0.6 Mixed bacteria are extracted from eggs produced by inoculating four antigens

The present invention is not limited to the technical spirit described in the specific embodiments, diagrams or drawings described above, and those skilled in the art without departing from the gist of the invention claimed in the claims. As long as anyone can carry out various modifications, such changes are within the scope of the claims.

As described above, yolk powder and special immunity possessing a special immune protein for E. coli (K88), E. coli (99), pandemic diarrheal virus, infectious gastroenteritis virus that cause diarrhea of piglets extracted from the eggs produced by the present invention Feed containing protein can prevent diarrhea in piglets from infectious, pandemic or digestive diseases.

In addition, the present invention can prevent mortality due to lack of antibodies in the body when piglets are infected with infectious or epidemic diarrhea after birth or shortly after weaning.

In addition, the present invention can avoid the use of expensive antibiotics or vaccines by adding special immune proteins to the feed, improve the quality of the feed and improve the pig farming business of Korean farmers.

Figure 1 shows the change in titer of each special immune protein when co-inoculated with E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV).

Figure 2 shows the titer of anti-E. Coli (K88) special immune protein when inoculated with E. coli (K88) alone and when co-inoculated with E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) It is a change.

Figure 3 shows the titer of anti-E. Coli (K99) special immune protein when inoculated with E. coli (K99) alone and when co-inoculated with E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) It is a change.

4 shows anti-pandemic diarrheal virus (PEDV) specific when inoculated with pandemic diarrhea virus (PEDV) alone and when co-inoculated with E. coli (K88), E. coli (K99), pandemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) It shows a change in the titer of the immunoprotein.

FIG. 5 shows anti-infective gastroenteritis virus (TGEV) specific when inoculated with infectious gastroenteritis virus (TGEV) alone and when co-inoculated with E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) It shows a change in the titer of the immunoprotein.

Figure 6 shows the average of anti-E. Coli (K88) special immune protein when E. coli (K88) alone inoculation and E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) Potency change is shown.

Figure 7 shows the average of anti-E. Coli (K99) special immune protein when inoculated with E. coli (K99) and E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) Potency change is shown.

FIG. 8 shows anti- pandemic diarrheal virus special immune protein upon inoculation of epidemic diarrhea virus (PEDV) alone and when co-inoculated with E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV). Mean titer change is shown.

9 shows anti-infective gastroenteritis virus (TGEV) specific when inoculating infectious gastroenteritis virus (TGEV) alone and inoculated with E. coli (K88), E. coli (K99), epidemic diarrheal virus (PEDV), infectious gastroenteritis virus (TGEV) The average titer change of immunoprotein is shown.

Claims (15)

A 12-week-old chick was inoculated once with 1 ml of a mixed bacterium emulsified solution of E. coli (K88) antigen, E. coli (K99) antigen, epidemic diarrheal virus antigen, and infectious gastroenteritis virus antigen emulsified with aluminum hydroxide at a ratio. From the second time, the mixed bacterial emulsion prepared by emulsifying the four antigens with an emulsification aid (ISA25) was inoculated twice in 1 ml at intervals of 2 weeks on the chick's legs. After the 28-week-old laying hens, the mixed bacterial emulsion using the emulsification aid (ISA25) was inoculated five times, including two inoculations of 0.5 ml each at the same ratio as the mixed bacterial emulsion. A method for producing an egg having a complex special immune protein, characterized in that it simultaneously shares a special immune protein for one of the four antigens. The method of claim 1, The antigen inoculated to the 12-week-old chick is E. coli (K88) antigen: E. coli (K99) antigen: pancreatic diarrheal virus antigen: infectious gastroenteritis virus antigen: aluminum hydroxide ratio of 2.1: 0.7: 3.5: 0.7: 3, 3.8 × 10 ⁸ / mL Escherichia coli (K88) bacteriostatic antigen 0.21 ml, 3.6 × 10 ⁸ / mL Escherichia coli (K99) Escherichia coli antigen 0.07 ml, 3 × 10 ⁷ TCID ₅₀ / ml Epidemic diarrheal virus inactivating antigen 0.35 ml, 1.5 × 10 ⁷ TCID ₅₀ / ml Infectious gastroenteritis virus inactivated antigen (0.07 ml) 1 ml of mixed bacterial emulsion emulsified with 0.3 ml of aluminum hydroxide was inoculated once in the chick's legs, From the second time, the mixed bacterial emulsion prepared by emulsifying the same antigens of the same ratio with the same amount of emulsification aid (ISA25) was inoculated twice in 1 ml at 2 weeks intervals into the chick leg, The mixed bacterial emulsion prepared by emulsifying with the emulsification aid (ISA25) was produced by inoculating a total of five antigens, including two inoculations of 0.5 ml each at four-month intervals after 28 weeks of growing laying hens. Egg production method comprising a complex special immune protein, characterized in that sharing the special immune protein at the same time. A 12-week-old chick was inoculated with 1 ml of E. coli (K88) antigen, E. coli (K99) antigen, epidemic diarrheal virus antigen, and infectious gastroenteritis virus antigen (1 ml) mixed with Freund's complete adjuvant at a rate. , From the second time, the mixed bacterial emulsion prepared by emulsifying the four antigens with Freund's incomplete adjuvant was inoculated twice in 1 ml at intervals of 2 weeks on the chick leg, After the 28-week-old laying hens, the mixed bacterial emulsion using the emulsification aid (ISA25) was inoculated five times, including two inoculations of 0.5 ml each at the same ratio as the mixed bacterial emulsion. A method for producing an egg having a complex special immune protein, characterized in that it simultaneously shares a special immune protein for one of the four antigens. The method of claim 3, 3.8 × so that the antigens inoculated into the 12-week-old chick are E. coli (K88) antigens: E. coli (K99) antigens: pancreatic diarrheal disease virus antigens: infectious gastroenteritis virus antigens: emulsification adjuvant ratio of 2.1: 0.7: 3.5: 0.7: 3 10 ⁸ / ml Escherichia coli (K88) 0.21ml and 3.6 × 10 ⁸ / ml Escherichia coli (K99) bacterium antigen 0.07ml and 3 × 10 ⁷ TCID ₅₀ / ml Epidemic diarrheal virus inactivating antigen 0.35ml and 1.5 × 10 ⁷ Inoculate the chicks' legs once with 1 ml of a mixed bacterial emulsion emulsified with 0.07 ml of TCID ₅₀ / ml infectious gastroenteritis virus inactivated antigen with 0.3 ml of Freund's complete adjuvant. From the second time, the mixed bacterial emulsion prepared by emulsifying the same antigens of the same ratio with the same amount of Freund's incomplete adjuvant was inoculated twice into the chick's legs twice at 1 week intervals. Specialized to the four antigens produced by the method of inoculating a total of five antigens, including two inoculations of 0.5ml each at 4 months intervals from the growing laying hens 28 weeks after the emulsified with the Freund's incomplete adjuvant Egg production method comprising a complex special immune protein, characterized in that sharing the immunoprotein at the same time. Eggs containing the complex special immune protein produced by the method of any one of claims 1 to 4. delete delete delete delete delete delete delete delete delete delete