KR102330545B1 - Manufacturing method of immunoglobulin y for preventing shrimp mortality - Google Patents

Abstract

The present invention prepares a heterologous antibody by inoculating a chicken and a heterologous animal with the causative agent of shrimp premature death syndrome or white spot virus antigen, and then inoculating the complex with the antigen and the heteroantibody to the laying hens to produce egg yolk antibody. It relates to a method for producing an anti-yolk antibody.
According to the present invention, the method for producing the yolk antibody for preventing shrimp mortality according to the present invention can manufacture and produce a large amount of the yolk antibody by injecting the antigen-heterologous antibody complex into the laying hens. Due to its high specificity and binding ability to the causative bacteria or white spot virus, it is possible to prevent the premature death of shrimp by the causative bacteria or white spot virus of shrimp premature death syndrome by effectively inhibiting the growth of the bacteria and virus.

Description

Manufacturing method of egg yolk antibody for preventing shrimp mortality

The present invention relates to a method for producing an egg yolk antibody for preventing premature shrimp death, and more specifically, a heterologous antibody is prepared by inoculating a causative agent of shrimp early death syndrome or a white spot virus antigen into an animal heterogeneous with a chicken, and then the antigen and the heteroantibody The present invention relates to a method for preparing an egg yolk antibody for preventing shrimp mortality by inoculating a complex bound to laying hens with an egg yolk antibody.

Antibody is a globulin-based protein formed in lymphoid tissue by B lymphocytes or B cells, and is one of the important substances involved in specific immune recognition. Antibodies have two different functions. The first is to neutralize the action of the antigen by specifically recognizing and binding to a specific site of the antigen that causes the immune response, and the second is to neutralize the action of the antigen by binding the antibody to the antigen. It is the role of attracting various substances and immune cells to be removed. For example, an antibody may bind to a toxin to neutralize the toxicity by changing the simple chemical composition of the toxin, or by attaching to the invading microorganism, remove the motility of the microorganism and prevent the invasion into body cells. In addition, the antigen surrounded by the antibody causes a chemical chain reaction with complement, causing direct degradation of the invading microorganism or attracting phagocytic cells. By applying the specificity and high affinity of these antigen-antibody reactions and the diversity of antibodies capable of discriminating tens of millions of antigens, many types of antibody medicines including diagnostic agents and therapeutic agents have emerged today (Jim E. Eyles et al., Vaccine, 25, pp73017306, 2007).

On the other hand, as a defense mechanism against disease, not only humans, but also various mammals and birds contain antibodies to substances produced to protect themselves from infectious diseases or to protect their offspring. Mammals produce about 150 Kd of IgG, similar to most humans. In addition to IgG, mammals produce IgA, IgD, IgE, and IgM with slightly different structures and functions.

Conventionally, a method of producing specific antibodies to pathogenic bacteria from serum obtained by immunizing mammals such as rabbits, cattle, and goats has been widely used to produce antibodies to various pathogenic bacteria. However, this method has a disadvantage in that the antibody must be collected from a small amount of serum from a small mammal, and the antibody production is also very small. Therefore, as a method for easily producing antibodies to pathogenic bacteria, a method of collecting antibodies by using the transfer of antibodies generated by immunizing laying hens to eggs of laying hens is currently in the spotlight. In particular, the specific antibody present in the yolk of laying hens is called yolk immunoglobulin (IgY) or yolk antibody, which is an antibody produced by the transfer of immunoglobulin (IgG), a specific antibody present in the serum of laying hens, to the yolk of the egg. As such, it accumulates in high concentration in the egg yolk of eggs. Since IgY can be easily separated, it can be used in various fields, and in particular, the development of IgY having preventive and therapeutic effects on marine diseases and its use as a feed additive is increasing.

Methods for obtaining yolk antibodies from eggs of laying hens using pathogenic bacteria as antigens, and compositions or foods containing the same are being actively developed. Preparation of vaccines using antigen-antibody complexes or compositions for treating various diseases. Development is also active. Specifically, Korean Patent Application Laid-Open No. 2009-0088121 discloses that antigen-antibody complexes can be used as adjuvants for vaccines to enhance immune responses by antigens, and the thesis Phase I trial of a murine antibody to In MUC1 in patients with metastatic cancer, it is known that when an antigen-xenoantibody complex is used to treat cancer cells, it can activate T cells and exhibit an excellent effect in treating cancer cells (Ann Oncol. 2004 Dec; 15(12)). :1825-33). However, a method using an antigen-heterologous antibody complex to mass-produce egg yolk antibody has not been known in the prior art.

Recently, as interest in improving diet for preventing adult diseases has rapidly increased around the world, the demand for aquatic products is continuously increasing and the production of fish culture is rapidly increasing. Therefore, aquaculture has become a major economic means in many countries, and disease outbreaks in farmed fish have a great impact on economic development.

Among farmed fish, Vibrio bacteria and white spot virus are known as major causes of disease in shrimp. Vibrio genus is a Gram-negative bacillus, which has one at one end and two or more cirri depending on the species, and is actively moving. Vibrio colera, which causes cholera, is a representative pathogen have. The causes of shrimp diseases include Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio anguillarum, called enteritis vibrio. It will die within 72 hours. In addition, the white spot syndrome virus (WSSV) has a tail-like attachment, and a rod-shaped capsid and envelope are present. When shrimp are infected with white spot virus, white spots appear on the carapace, appendage, and cuticle of the shrimp, the hepatopancreas becomes red, and 3 to 5 days after infection will die

Therefore, as described above, egg yolk antibody (IgY) is emerging as one of the measures to prevent premature death of shrimp due to disease. It is economically difficult to use to prevent it.

Accordingly, as a result of intensive research efforts to overcome the problems of the prior art, the present inventors prepared a heterologous antibody by inoculating a chicken and a heterogeneous animal with an antigen causative agent or white spot virus of shrimp early death syndrome, and then, the antigen and the heteroantibody When egg yolk antibody is produced by inoculating laying hens with a complex conjugated with If it does, it was confirmed that the mortality of the shrimp can be prevented by preventing the shrimp disease, and the present invention has been completed.

US 10-2009-0088121 A

J.S. de Bono JS et al., Annals of Oncology. 2004 Dec;15(12):1825-33

Accordingly, the main object of the present invention relates to a method for producing an egg yolk antibody for preventing shrimp mortality, which can mass-produce and produce an egg yolk antibody with an enhanced immune response to the causative agent of shrimp premature death syndrome or white spot virus.

According to one aspect of the present invention, the present invention provides a first step of preparing an antigen using the causative bacteria of shrimp early death syndrome and white spot syndrome virus (WSSV), and the antigen of the first step is heterologous to chicken. A second step of preparing a xenoantibody by inoculating a mouse or rabbit, an animal of A fourth step of inoculating laying hens with a mixture of the antigen and the complex of the third step, and a fifth step of isolating the yolk antibody to the causative agent or virus of shrimp premature death syndrome from the egg of the fourth step It relates to a method for producing an egg yolk antibody for preventing shrimp mortality, comprising the steps of, wherein the causative bacteria of the shrimp premature death syndrome are Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, and Vibrio parahaemolyticus E1 (vibrio parahaemolyticus E1) provides a method for producing an yolk antibody for preventing shrimp mortality, characterized in that at least one causative bacteria selected from the group consisting of.

Egg yolk antibody (Immunoglobulin Yolk, IgY) is an antibody produced by transferring immunoglobulin (IgG), a specific antibody present in the serum of laying hens, to the yolk of an egg. being used in the field. As a result of research efforts to mass-produce and produce such egg yolk antibodies, antigen-antibody conjugates are used as adjuvants, and a complex combining antigen and heteroantibodies was developed based on the fact that the immune response can be enhanced when a heterologous antibody is used as an antibody. When the yolk antibody is prepared using the yolk sac, it was confirmed that the yolk antibody with an increased immune response to the antigen can be mass-produced and produced, and thus the present invention has been completed.

In the method for producing an egg yolk antibody of the present invention, in the second step, the heterogeneous animal may be any mammal conventionally used to prepare the antibody, and preferably a mouse or rabbit.

In the method for preparing the egg yolk antibody of the present invention, the mixing ratio of the antigen and the complex in the third step is preferably 3:0.5 to 1:0.1. When the ratio of the complex is more than this, there is a problem due to the recognition of excessive immune cells for the complex, and when it is included in less, it is difficult to exert a sufficient effect according to the complex application.

In the method for producing an egg yolk antibody of the present invention, the mixture in the fourth step is characterized in that it further comprises an adjuvant. The adjuvant includes complete Freund's adjuvant, incomplete Freund's adjuvant, lipofectin, lipotaxi, CaPO ₄ , 25-30% sucrose, DEAE dextran, poly Inorganic gels such as polybrene, saponins, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, surface active substances such as oil or hydrocarbon emulsions, Dinitrophenol and the like may be used, but the present invention is not limited thereto. In the example of the present invention, ISA70 incomplete adjuvant was used as an adjuvant.

In the method for preparing the egg yolk antibody of the present invention, the mixing ratio of the mixture and the adjuvant may be 2-3: 7-9, preferably, the mixing ratio is 3:7. As the mixing ratio of the mixture increases, the function of the adjuvant as an emulsifier decreases, which causes a problem of separation of the water-soluble layer and the fat-soluble layer upon mixing with the antigen, so it is important to maintain the above ratio.

In the method for preparing egg yolk antibody of the present invention, inoculation in the fourth step may be performed 2 to 5 times with 0.1 ml to 3 ml, preferably 3 times with 0.5 to 1 ml.

In the method for preparing the yolk antibody of the present invention, the method for preparing the yolk antibody is characterized in that the production yield of the yolk antibody is increased.

According to an experimental example of the present invention, as a result of confirming the production yield of the yolk antibody according to the production method of the present invention, the antigen and antigen-heterologous antibody complex were produced rather than preparing the yolk antibody by injecting only the antigen into the laying hens (Comparative Example). It was confirmed that the antibody content in the egg yolk was increased when the yolk antibody was prepared by injecting it into the laying hens. These results, given the fact that it is difficult to significantly increase the total antibody production, the increase in the production of the yolk antibody in the production method of the present invention gives a synergistic effect as the antigen-heterologous antibody complex as an adjuvant for antibody production. This suggests that the egg yolk antibody can be mass-produced and produced (see Experimental Example 3 and Table 5).

In addition, according to an experimental example of the present invention, as a result of confirming the antigen-binding ability of the yolk antibody produced according to the production method of the present invention, the antigen-heterogeneous antibody complex was obtained rather than the yolk antibody produced by injecting only the antigen into the laying hens (Comparative Example). In the case of the yolk antibody (Example) produced by injection into laying hens, it was confirmed that the antigen-binding ability was excellent. These results show that when the yolk antibody is prepared according to the preparation method of the present invention, the production of the yolk antibody specific to a specific antigen can be increased. It suggests that the immune response to the causative bacteria or white spot virus antigen can be enhanced. In addition, the binding force suggests that IgY for the antigen was generated by recognizing the antigen-xenoantibody complex injected into the laying hens as the initial antigen, that is, the causative agent of shrimp early death syndrome or white spot virus (Experimental Example 4, Fig. 5).

The egg yolk antibody prepared by the method for preparing the egg yolk antibody for preventing shrimp death of the present invention can be used as a vaccine composition for preventing or treating diseases caused by shrimp premature death syndrome or white spot syndrome virus, and the vaccine is pharmaceutically It may be used with an acceptable carrier, adjuvant or excipient.

As the carrier for the vaccine of the present invention, a physiologically balanced culture medium containing one or more stabilizers such as hydrated protein and lactose may be used, and 0.01 to 0.1 M phosphate buffer or 0.8% physiological saline may be used. . Additionally, the pharmaceutically acceptable carrier may be in the form of a solution or non-solution, suspension, or emulsion. Examples of non-solution solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The solution carrier may be water, an alcoholic solution, an emulsion, or a suspension such as physiological saline and buffered culture solution.

Excipients and diluents for the vaccine of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate bait 60, methylparaben, propylparaben and mineral oil.

The vaccine of the present invention can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes. Parenteral administration refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injections and infusions.

For parenteral administration of the vaccine of the present invention, a unit dose formulation is prepared by mixing a pharmaceutically acceptable carrier, i.e., nontoxic to the recipient at the concentration and dosage used, and compatible with other formulation ingredients, under the desired purity. It is preferable to do

In addition, the formulation of the vaccine of the present invention can be applied in any formulation, and the prepared formulation can be used for oral, injection, and application. The formulation can be prepared in the form of tablets, capsules, soft capsules, infusions, granules, pills or injections (solutions, suspensions or emulsions), and it is most preferable to prepare for injection.

The egg yolk antibody prepared by the method for producing the egg yolk antibody for preventing shrimp death of the present invention can be used as a feed additive composition for preventing or treating diseases caused by the shrimp premature death syndrome or white spot syndrome virus.

The feed additive of the present invention may be used as it is, or additionally known carriers such as grains and by-products allowed for livestock, stabilizers, etc. may be added, and, if necessary, citric acid, humic acid, adipic acid, lactic acid, malic acid, etc. Phosphates such as organic acids, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc. of natural antioxidants, antibiotics, antibacterial agents, and other additives may be added, and the shape may be in a suitable state such as powder, granule, pellet, suspension, and the like.

As described above, the method for producing an yolk antibody for preventing shrimp mortality according to the present invention can manufacture and produce a large amount of yolk antibody by injecting an antigen-heterologous antibody complex into laying hens. Due to the high specificity and binding properties to the causative bacteria or white spot virus of the syndrome, it is possible to prevent the premature death of shrimp by the causative bacteria or white spot virus of shrimp early death syndrome by effectively inhibiting the growth of the bacteria and virus.

1 and 2 are diagrams showing the antibody titer measurement results according to Experimental Example 1.
3 and 4 are diagrams showing the results of confirming the egg yolk antibody according to Experimental Example 2 (FIG. 3, SDSPAGE (12% gel staining); FIG. 4, Western-blot; 1, Natural-IgY protein (cat#.ab119138)) ; 2, Vibrio EMS-IgY + WSSV-IgY; 3, Vibrio EMS + WSSV + mAB complex-IgY)
5 is a view showing the results of confirming the antigen binding force according to Experimental Example 4.
6 and 7 are diagrams showing the results of confirming the survival rate of shrimp according to Experimental Example 5 (Fig. 6; survival rate after infection with V. parahaemolyticus; Fig. 7, survival rate after infection with WSSV).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Preparation Example 1: Production of antigen

1) Vibrio EMS (V1, Vb, Va, VPE1)

Each of the four EMS species (vibrio parahaemolyticus (V1), vibrio harveyi (Vb), vibrio anguillarum (Va), and Vibrio parahaemolyticus E1 (VPE1)) was aerobically cultured in an incubator at 37°C using TSA agar medium. . After confirming the colony, each TSA broth was inoculated in 1.5L and cultured for 20 hours. Each cultured EMS 4 strains were collected by centrifugation at 6,000 rpm, dissolved in PBS, and then 0.1% of formalin was added, left at room temperature for 1 day, and then stored at 4°C refrigerated. The concentration of the prepared antigen was adjusted by calculating the antigen to be O.D 1.0 at 600mn using a spectrophotometer.

2) white spot syndrome virus (WSSV)

Infected white spot virus (WSSV) shrimp were used to produce antigens. Thaw WSSV-infected dead shrimp frozen at -80°C. The muscle tissue of the infected shrimp is homogenized using a homogenizer by adding 10 times the volume of PBS at 4°C. After homogenization, the cells were collected by centrifugation at 4° C. and 12,000 rpm for 10 minutes and filtered through a 0.45 μm membrane. The filtered supernatant is inactivated with 0.2M BEI (2-Bromoethylamine Hydrobromide).

Preparation Example 2: Preparation of mouse antibody

Each antigen produced in Preparation Example 1 was inactivated to suppress toxicity, and then stored in a refrigerator before use. In the case of a vaccine for mouse antibody production, a vaccine was prepared using a complete adjuvant. Antibodies were prepared according to a conventional mouse antibody production method by preparing a vaccine for each antigen. Mouse antibody was purified using protein-A column from serum collected after production was completed.

Preparation Example 3: Preparation of antigen-heteroantibody complex

In order to bind the antigen produced in Preparation Example 1 to the mouse antibody produced in Preparation Example 2, the test was performed as follows. 100 ug of mouse antibody generated for each antigen was mixed in the inactivated mixed vaccine, and antigen-antibody binding was induced overnight at 4°C. After the binding was completed, the sample was collected by centrifugation and used as a sample for inoculation of laying hens.

Preparation Example 4: Preparation of vaccine for laying hens inoculation

To prepare a mixed vaccine, each antigen prepared in Preparation Example 1 and the mAb complex (antigen-heteroantibody complex) prepared in Example 2 were mixed with an adjuvant at a ratio of 3:7, and then a dead poison vaccine was prepared using a homogenizer, followed by aseptic sterility. A mixed vaccine for the production of specific egg yolk antibodies was prepared through tests and inactivation confirmation tests.

Example 1: Inoculation of a vaccine for inoculation of laying hens

Laying hens were inoculated with the vaccine prepared in Preparation Example 4 using 25-week-old Hyine-brown-type laying hens. Inoculation was carried out by inoculating 1 ml each in the pectoral muscle, and inoculation was performed three times at 3-week intervals. The inoculation group is shown in Table 1 below. In addition, the composition of the vaccine is shown in Table 2.

group Inoculation details Vaccine configuration control not vaccinated - comparative example Preparation Example 1 (Shrimp Early Death Inducing Antigen Mixed Vaccine) Vibrio EMS (V1, Vb, Va VPE1) + WSSV Example Preparation Example 1 + Preparation Example 4 (antigen-xenoantibody complex vaccine inducing early prawn death) Vibrio EMS (V1, Vb, Va VPE1) + WSSV + mAB complex

group Vaccine configuration ratio control - comparative example Vibrio EMS (V1, Vb, Va VPE1) + WSSV 1:1:1:1:1 Example Vibrio EMS (V1, Vb, Va VPE1) + WSSV + mAB complex ¹⁾ 1:1:1:1:1:1

¹⁾ : mAb complex = (4 types of EMS: V1, Vb, Va, VPE1+ WSSV+ mouse anti-EMS-IgG)

Experimental Example 1: Measurement of antibody titer

1) ELISA coating antigen preparation

The antigen produced in Preparation Example 1 was centrifuged at 8,000 rpm for 50 minutes. After centrifugation, the supernatant was discarded, the pellet was resuspended with HEPES buffer, and the supernatant lysed by sonication was centrifuged at 8,000 rpm at 4° C. for 30 minutes to harvest the supernatant. 1% N-Lauroly sarcosine (SIGMA, L-9150) was added to the harvested supernatant to a final concentration of 0.01%, and then treated at room temperature for 10 minutes. After treatment, centrifugation was performed at 15,000 rpm at 4 ° C for 50 minutes to remove N-Lauroly sarcosine (SIGMA, L-9150), resuspended in 50 ml of HEPES buffer, and centrifuged at 15,000 rpm 4 ° C for 50 minutes to collect OMP. The collected antigen was coated to 200ng/ml after protein quantification by BCA method and used for antibody titer measurement.

2) Measurement of antibody titer using ELISA

The titer of specific egg yolk antibody in egg yolk was measured using the Indirect ELISA method. First, the antigen is diluted in a coating buffer, coated on a 96-well polystyrene plate by 100ul per well, and allowed to over night at 4°C (or leave at 37°C for 1 hour). After washing with PBS-T (phosphate buffer saline, 0.05% Tween 20, pH 7.4), blocking was performed at 37° C. for 1 hour with PBS buffer containing BSA and washed in the same manner as above. Dilute the negative control group, the positive control group, and the sample by 2X, put 100 μl per well, and let stand at 37° C. for 1 hour. After 1 hour, wash 3 times, dilute an appropriate amount of secondary antibody (anti-chicken: Sigma, U.S.A) in PBS, dispense 100ul into each well, and then react at 37°C for 1 hour. After that, wash 3 times and dispense 100ul of substrate (OPD, sigma) into each well, then react at room temperature for about 10 minutes, stop the reaction by dispensing 50ul of Stop solution, and absorb the absorbance of each well with an ELISA reader at a wavelength of 450nm was measured and expressed as an ELISA value. The titers of the analyzed data are shown in Tables 3 and 4 and FIGS. 1 and 2 below by substituting the measured value for the measured value of the processed egg yolk by multiplying the negative value in the measured result.

number of inoculations Vibrio EMS control comparative example Example parking Primary One 200 800 3,200 2 200 800 3,200 3 400 1,600 3,200 Secondary One 200 800 6,400 2 200 800 6,400 3 200 1,600 12,800 tertiary One 400 1,600 12,800 2 200 1,600 25,600 3 400 12,800 25,600 4 200 12,800 25,600 5 200 12,800 12,800 6 200 6,400 12,800

number of inoculations WSSV control comparative example Example parking Primary One 200 800 1,600 2 200 800 1,600 3 400 1,600 3,200 Secondary One 200 800 3,400 2 200 1,600 12,800 3 200 1,600 12,800 tertiary One 400 1,600 12,800 2 200 1,600 51,200 3 400 12,800 51,200 4 200 12,800 51,200 5 200 12,800 25,600 6 200 6,400 25,600

As a result, as can be seen in Tables 3 and 4 above, in the case of the example in which the heterologous antibody was combined with the antigen and inoculated to the laying hens together with the additional antigen, the antibody titer was very high in the first to third inoculations compared to the comparative example. formation was confirmed.

Experimental Example 2: Identification of egg yolk antibody

After the antibody was isolated from the inoculated egg yolk using ammonium sulfate, a test was performed to confirm the separation purity.

Specifically, eggs at 2 to 3 weeks of age showing the highest antibody titer in egg yolk produced by each group (control group, comparative example, and example) were collected and IgY was isolated from the yolk. Separate the yolk from the white, collect only the yolk, dilute the collected yolk with purified water (D.W) and stir for 30 minutes. The sample stirred with D.W is frozen and thawed repeatedly to separate lipids, and a water-soluble protein sample is collected. Ammonium sulfate is added to the collected samples and stored in a refrigerator at 4°C to precipitate the egg yolk antibody. After collecting the sample precipitated in refrigeration for about 18 hours, centrifugation at 4° C. 6,000 rpm and centrifugation, the collected antibody sample is resuspended in PBS, and the sample is dialyzed against PBS to obtain a final sample. The obtained sample was quantified with a BCA protein quantification kit, and separation purity was checked on 12% SDS-PAGE gel, and the results are shown in FIGS. 3 and 4 .

As a result, as can be seen in FIGS. 3 and 4 , the test results showed that the purified egg yolk antibody was separated without significant difference from the commercially available purified egg yolk antibody. see.

Experimental Example 3: Measurement of antibody content according to inoculation of laying hens

The yolk antibody quantitative test was conducted using HPLC. IgY standard material for quantification was Abcam's normal chicken IgY (Abcam Cat No ad 119138). The standard solution, normal IgY, was taken and centrifuged at 12,000 rpm for 20 seconds to use the supernatant. A standard curve is prepared by diluting the centrifuged standard stock solution to 5 concentrations with distilled water. The standard solution was diluted to 1, 0.5, 0.25 0.125, and 0.0625 mg/ml. Dilute the egg yolk solution to a concentration of 0.2 mg/ml and thaw it for 5 minutes using a shaker. The solution was centrifuged at 3,000 rpm for 5 minutes, the supernatant was collected and filtered through a 0.45um syringe filter, and the result was measured by HPLC. Measure the standard solution by concentration (1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL) by HPLC and draw a linear graph according to the concentration of the standard solution to obtain a linear expression The values of the test solutions measured by HPLC were introduced and calculated, and the results are shown in Table 5 below.

IgY (mg/ml) weeks control comparative example Example 1-1 11.40 14.29 15.54 1-2 12.20 14.10 15.60 1-3 11.60 13.85 15.85 2-1 10.50 14.55 16.05 2-2 11.50 14.82 16.30 2-3 12.20 14.56 15.78 3-1 12.10 14.55 15.86 3-2 10.20 14.75 16.02 3-3 11.50 14.33 15.78 3-4 11.50 14.65 15.82 3-5 11.90 13.99 15.84 3-6 11.00 14.28 15.80 3-7 11.50 14.44 15.81 Mean 11.50 13.99 15.81

As a result, as can be seen in Table 5 above, it was found that the content of the yolk antibody was increased in the vaccinated group compared to the non-vaccinated group (control group), and the Example showed that the antibody content in the egg yolk was increased compared to the comparative example. appear.

Experimental Example 4: Test for confirming antigen binding affinity

In order to confirm the antigen specificity of shrimp IgY (SH-IgY) produced by Adbiotech, that is, IgY prepared in Example 1, it was analyzed using a flow cytometer. Vibrio harveyi, which is one of the causes of early mortality syndrome, was used as an antigen. Fluorescence staining was performed with Goat Anti-chicken IgY-PE (#ab46852, abcam). As a control group, Normal Chicken IgY isolated from non-immunized chickens was used for comparison, and the results are shown in FIG. 5 .

As a result, as can be seen in FIG. 5 , cells exhibiting a fluorescence signal of 18.3% were observed in the control group, and cells exhibiting a high fluorescence signal of 85.88% in the test group treated with Vibrio EMS and mAb complex (Example) were observed. became Therefore, it was confirmed that the specific egg yolk antibody was produced 4 times higher than that of the control group.

Experimental Example 5: Confirmation of bacteria causing shrimp death syndrome and virus resistance

Thirty white-legged shrimp weighing 3-5 g were housed in each tank, and the control group was fed a general feed for 30 days, and the treated group was fed a feed containing 0.5% specific egg yolk antibody. Vibrio parahaemolyticus or WSSV was artificially infected with white-legged shrimp raised on the 30th day to confirm the survival rate of the shrimp, and the results are shown in FIGS. 6 and 7 .

As a result, as can be seen in FIG. 6 , the control group fed the normal feed 7 days after infection with V. parahaemolyticus showed a survival rate of 40%, and the survival rate of the treatment group fed with a feed containing 0.5% egg yolk antibody was 75% As a result, the survival rate was 1.88 times higher than that of the control group.

In addition, as can be seen in FIG. 7 , as a result of breeding for one week after infection with WSSV, the control group died within 5 days. On the other hand, the survival rate of the treatment group fed with the specific egg yolk antibody was 41%, confirming the effect of suppressing virus disease in shrimp.

Claims (6)

A first step of preparing an antigen using the causative agent of shrimp early death syndrome or white spot syndrome virus (WSSV);
a second step of preparing a heterologous antibody by inoculating the antigen of the first step into a mouse or rabbit, which is a heterogeneous animal from chicken, which is an animal that produces egg yolk antibody;
a third step of preparing an antigen-xenoantibody complex by binding the antigen of the first step with the heteroantibody of the second step;
a fourth step of inoculating laying hens with a mixture of the antigen of the first step and the complex of the third step; and
a fifth step of isolating the yolk antibody to the causative bacteria or virus of shrimp premature death syndrome from the laying hen eggs (egg) of the fourth step; It relates to a method for producing an egg yolk antibody for preventing shrimp mortality, comprising:
The causative agent of the shrimp premature death syndrome is a group consisting of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, and Vibrio parahaemolyticus E1 A method for producing an egg yolk antibody for preventing shrimp death, characterized in that it is one or more causative bacteria selected from
According to claim 1,
A method for producing an egg yolk antibody for preventing shrimp death, characterized in that the mixing ratio of antigen and complex in the third step is 3:0.5 to 1:0.1.
According to claim 1,
The method for producing egg yolk antibody for preventing shrimp death, characterized in that the mixture in the fourth step further comprises an adjuvant.
4. The method of claim 3,
The mixing ratio of the mixture and the adjuvant is 2-3: 7-9.
According to claim 1,
In the fourth step, the inoculation is 0.1ml to 3ml 2 to 5 times, the method for producing an egg yolk antibody for preventing shrimp death, characterized in that the inoculation.
According to claim 1,
The method for producing yolk antibody is a method for producing an yolk antibody for preventing shrimp mortality, characterized in that it increases the yield of yolk antibody production.

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