KR102200721B1 - Manufacturing method of immunoglobulin y for preventing or treating salmon rickettisia septicaemia - Google Patents

Abstract

The present invention relates to a method for producing an egg yolk antibody for prevention or treatment of salmon rickettsial septicaemia, wherein an antigen of Piscirickettsia Salmonis, which is the causative bacterium of salmon rickettsial septicaemia, is inoculated to an animal that is different in kind from chickens, so as to produce a heteroantibody, and then a composite obtained by coupling the antigen and the heteroantibody is inoculated to a laying hen, so as to produce an egg yolk antibody. According to the present invention, a method for producing an egg yolk antibody for prevention or treatment of salmon rickettsial septicaemia can mass-produce an egg yolk antibody by injecting an antigen-heteroantibody composite to laying hens. An egg yolk antibody produced according to the present invention has high specificity and binding properties with respect to the causative bacterium of salmon rickettsial septicaemia. Thus, the bacterium is effectively inhibited, and thus salmon rickettsial septicaemia can be prevented or treated.

Description

Manufacturing method of yolk antibody for preventing or treating salmon Rickettsia sepsis {MANUFACTURING METHOD OF IMMUNOGLOBULIN Y FOR PREVENTING OR TREATING SALMON RICKETTISIA SEPTICAEMIA}

The present invention relates to a method for preparing a yolk antibody for preventing or treating salmon Rickettsia sepsis, and more specifically, Piscirickettsia Salmonis antigen, which is the causative agent of salmon Rickettsia sepsis, is inoculated into chickens and heterogeneous animals After preparing the antibody, it relates to a yolk antibody for the prevention or treatment of salmon rickettsia sepsis by inoculating the egg yolk antibody with a complex in which the antigen and the heterologous antibody are combined.

Antibody is a globulin-based protein formed in lymph tissue by B lymphocytes or B cells, and is one of 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 an immune response, and the second is to remove the antigen by binding the antibody to the antigen. It is the role of attracting various substances and immune cells for doing so. For example, antibodies may bind to toxins to neutralize toxicity by changing a simple chemical composition of toxins, and by attaching to invading microorganisms, the motility of microorganisms is eliminated and invasion into somatic cells is prevented. In addition, antigens surrounded by antibodies cause a chemical chain reaction with complement, causing direct decomposition of invading microorganisms or attracting phagocytic cells. By applying the specificity and high affinity of the antigen-antibody reaction, and the diversity of antibodies capable of distinguishing tens of thousands of antigens, many types of antibody drugs, 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, antibodies are included in substances that not only humans but also various mammals and birds are made to protect themselves from infectious diseases or 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, which are slightly different in structure and function.

Conventionally, in order to produce antibodies against various pathogenic bacteria, a method of producing pathogenic bacteria-specific antibodies from serum obtained by immunizing mammals such as rabbits, cows, and goats has been widely used. However, this method has the disadvantage of having to collect antibodies from a small amount of serum from small mammals, and the amount of antibody production is very small. Therefore, as a method for easily producing antibodies of pathogenic bacteria, a method of collecting antibodies by immunizing the laying hens and transferring the generated antibodies to the eggs of the hens is in the spotlight. In particular, the specific antibody present in the egg yolk of the laying hen is called yolk immunoglobulin (Immunoglobulin Yolk, IgY) or the yolk antibody, which is produced by the transfer of immunoglobulin (IgG), a specific antibody present in the serum of the laying hen into the egg yolk As a result, it is accumulated in high concentration in the egg yolk. Since IgY can be easily separated, it can be used in various fields. In particular, the development of IgY, which has the effect of preventing and treating marine diseases, and its use as a feed additive is increasing.

A method for obtaining egg yolk antibodies from eggs of laying hens using pathogenic bacteria as an antigen, and a composition or food including the same are being actively developed, and the preparation of vaccines using antigen-antibody complexes or compositions for treating various diseases Development is also actively taking place. Specifically, Korean Patent Laid-Open Publication No. 2009-0088121 describes that the use of an antigen-antibody complex as an adjuvant for a vaccine can enhance the immune response by the antigen, and the paper Phase I trial of a murine antibody to In MUC1 in patients with metastatic cancer, it is known that when an antigen-heteroantibody complex is used to treat cancer cells, it can exhibit excellent effects in treating cancer cells by activating T cells (Ann Oncol. 2004 Dec;15(12)) :1825-33). However, a method using an antigen-heterologous antibody complex to produce a large amount of yolk antibodies has not been known.

Recently, as interest in improving dietary habits for the prevention of adult diseases has increased rapidly around the world, the demand for aquatic products continues to increase and the production of fish farming is increasing rapidly. Therefore, aquaculture has become a major economic tool in many countries, and disease outbreaks of farmed fish have a great influence on economic development.

Recently, among farmed fish, the problem of salmon rickettisia septicaemia, which occurs in salmon, has emerged. Salmon rickettsia sepsis is a disease representing internal bleeding symptoms caused by infection of Piscirickettsia Salmonis.In general, salmon rickettsia sepsis is very difficult to prevent or treat without the use of antiseptics or pesticides. It is known to be difficult. In addition, even if anti-oxidants or pesticides are used to prevent or treat salmon rickettsia sepsis, the provision of salmon ingested anti-oxidants or pesticides as food may adversely affect those who consume them. There is a need to develop new treatments.

Meanwhile, in order to prevent or treat salmon rickettsia sepsis, attempts have been made to prevent or treat diseases such as salmon rickettsia sepsis by increasing the immunity of salmon by supplying the salmon with oral vaccines instead of antibiotics or pesticides in salmon farms. Is losing.

Accordingly, the present inventors have made extensive research efforts to overcome the problems of the prior art, and as a result of inoculating Piscirickettsia Salmonis antigen, which is the causative agent of salmon rickettsia sepsis, into chickens and heterogeneous animals, a heterologous antibody was prepared. Next, when the yolk antibody is prepared by inoculating the complex of the antigen and the heterologous antibody into a laying hen, it is possible to mass-produce and produce a yolk antibody with an increased immune response to the causative agent of salmon rickettsia sepsis. In the case of manufacturing a vaccine, it was confirmed that diseases such as salmon rickettsia sepsis can be prevented by preventing salmon disease, and the present invention was completed.

KR 10-2009-0088121 A

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

Therefore, the main object of the present invention is a salmon rickettsia sepsis-causing bacterium, Piscirickettsia Salmonis , which is capable of mass-producing and producing yolk antibodies with an increased immune response to salmon rickettsia sepsis prevention or treatment yolk It relates to a method for producing an antibody.

According to one aspect of the present invention, the present invention provides a first step of preparing an antigen using Piscirickettsia Salmonis , which is a causative agent of Salmon Rickettisia septicaemia, and the antigen of the first step. A second step of inoculating a chicken and a mouse or rabbit, which is a heterogeneous animal, to prepare a heterologous antibody, and a third step of preparing an antigen-heterologous antibody complex by combining the antigen of the first step with the heterologous antibody of the second step, A fourth step of inoculating a mixture of the first step antigen and the third step complex into the laying hens, and a fifth step of separating an egg yolk antibody against the Salmon Ricketts-related sepsis causative bacteria from the eggs of the fourth step. It provides a method for producing a yolk antibody for preventing or treating salmon Rickettsia sepsis comprising the step.

Yolk antibody (Immunoglobulin Yolk, IgY) is an antibody produced by the transfer of immunoglobulin (IgG), a specific antibody present in the serum of laying hens, to the egg yolk. It accumulates in a high concentration in the egg yolk and is easy to separate. It is being used in the field. As a result of research efforts to mass-produce and produce these yolk antibodies, antigen-antibody conjugates are used as adjuvants, and when using a heterologous antibody as an antibody, the immune response can be increased. When the yolk antibody is prepared using it, it was confirmed that it was possible to mass-produce and produce a yolk antibody with an increased immune response to the antigen, and the present invention was completed.

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

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

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 contains an adjuvant. The adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, lipofectin, lipotaxi, CaPO ₄ , 25 to 30% sucrose, DEAE dextran, poly Inorganic gels such as polybrene, saponin, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, surface active substances such as oil or hydrocarbon emulsions, Dinitrophenol may be used, but is not limited thereto. In the examples of the present invention, ISA70 incomplete adjuvant was used as an adjuvant.

In the method for producing egg yolk antibody of the present invention, the mixing ratio of the mixture and the adjuvant may be 2 to 3: 7 to 9 weight ratio, and preferably the mixing ratio is 3 to 7 weight ratio. As the mixing ratio of the mixture increases, the function of the adjuvant as an emulsifier decreases, causing a problem that the water-soluble layer and the fat-soluble layer are separated when mixed with the antigen, so it is important to maintain the ratio.

In the method for producing an egg yolk antibody of the present invention, inoculation in the fourth step may be inoculated twice to 5 times in 0.1ml to 3ml, preferably 0.5 to 1ml in 3 times.

In the method for producing egg yolk antibodies of the present invention, the method for producing yolk antibodies is characterized in that the production yield of yolk antibodies 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 prepared rather than producing the yolk antibody by injecting only the antigen into the laying hen (Comparative Example). When the egg yolk antibody was prepared by injecting it into a laying hen, it was confirmed that the antibody content in the egg yolk was increased. These results show that, in view of the fact that it is difficult to significantly increase the total amount of antibody production, the increase in the production of yolk antibodies in the production method of the present invention imparts a synergistic effect as an adjuvant of antibody production. This suggests that the yolk antibody can be mass-produced and produced (see Experimental Example 3 and Table 4).

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-heterologous antibody complex was produced by injecting only the antigen into the laying hens (comparative example). In the case of the egg yolk antibody produced by injection into the laying hen (Example), it was confirmed that the binding power to the antigen was excellent. These results show that when the yolk antibody is produced according to the manufacturing method of the present invention, it is possible to increase the production of yolk antibody specific to a specific antigen. This suggests that the immune response to the causative bacteria antigen can be increased. In addition, the avidity suggests that IgY against the antigen was generated by recognizing the antigen-heterologous antibody complex injected into the laying hen as the first antigen, the causative agent of salmon rickettsia sepsis as an antigen (see Experimental Example 4 and Fig. 4).

The egg yolk antibody produced by the method for preparing a yolk antibody for preventing or treating salmon Rickettsia sepsis of the present invention can be used as a vaccine composition for preventing or treating salmon Rickettsia sepsis, and the vaccine is a pharmaceutically acceptable carrier , Adjuvants or excipients.

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 a solution, nasal solution, suspension, or emulsion. Examples of non-liquid solvents include vegetable oils such as propylene glycol, polyethylene glycol, olive oil and organic esters for injection such as ethyl oleate. The solution carrier may be water, an alcohol solution, an emulsion or a suspension such as physiological saline and a buffered culture solution.

Excipients and diluents for the vaccine of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, 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, polysorb Bait 60, methylparaben, propylparaben, and mineral oils.

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 a mode of administration including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion.

Parenteral administration of the vaccine of the present invention is prepared in a unit dosage form by mixing a pharmaceutically acceptable carrier, that is, a non-toxic to the receptor at the concentration and dosage used, and compatible with other ingredients under the desired purity. It is desirable 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 use, injection, or application. The above formulation can be prepared in the form of tablets, capsules, soft capsules, infusion solutions, granules, pills, or for injection (solution, suspension or emulsion), most preferably for injection.

The egg yolk antibody produced by the method for preparing a yolk antibody for preventing or treating salmon Rickettsia sepsis of the present invention may be used as a feed additive composition for preventing or treating salmon Rickettsia sepsis.

The feed additive of the present invention may be used as it is, or additionally, known carriers such as grains and by-products thereof allowed for livestock, stabilizers, etc. may be added, and if necessary, citric acid, humic acid, adipic acid, lactic acid, malic acid, etc. Organic acids, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate) and other phosphates, polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc. Natural antioxidants, antibiotics, antibacterial agents, and other additives may be added, and the shape may be in a suitable state such as powder, granules, pellets, and suspensions.

As described above, the method for preparing a yolk antibody for preventing or treating salmon Rickettsia sepsis according to the present invention can produce and produce a large amount of yolk antibody by injecting an antigen-heterologous antibody complex into the laying hen, and thus the prepared yolk antibody In this case, the specificity and binding to Piscirickettsia Salmonis , which is the causative agent of salmon rickettsia sepsis, is high, so that the growth of the bacterium is effectively inhibited, thereby preventing or treating salmon rickettsia sepsis.

1 is a view showing the antibody titer measurement result according to Experimental Example 1.
2 and 3 are diagrams showing the results of confirming the yolk antibody according to Experimental Example 2 (FIG. 2, SDSPAGE (12% gel staining); FIG. 3, Western-blot; 1, Natural-IgY protein (cat#.ab119138)) ; 2, P. salmonis -IgY; 3, P. salmonis + mAB complex-IgY)
4 is a view showing the result of confirming the antigen binding ability according to Experimental Example 4.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Preparation Example 1: Production of antigen

One) P. salmonis

To make TSA agar, put Red Sea Salt (RSS) in TSA, sterilize at 121℃ for 15 minutes, and then cool it to 50℃. After that, defibrinated sheep blood, L-cysteine, D-glucose FBS and ferric nitrate were added, and the pH was adjusted (6.8 ± 0.2). Spread P. salmonis on agar plate and incubate for 2 weeks at 20℃ incubator. After the collected bacteria were loosened with 1XPBS, 0.1% of formalin was added, left at room temperature for 1 day, and then stored in a refrigerator at 4°C. The prepared antigen was calculated to have an OD of 1.0 at 600mn using a spectrophotometer, and the concentration was adjusted.

Preparation Example 2: Preparation of mouse antibody

The antigen produced in Preparation Example 1 was inactivated to suppress toxicity and then refrigerated before use. In the case of a vaccine for producing a mouse antibody, a vaccine was prepared using a complete adjuvant. Each antigen-specific vaccine was prepared and antibodies were prepared according to a conventional mouse antibody production method. After production was completed, the mouse antibody was purified from the collected Serum using a protein-A column.

Preparation Example 3: Preparation of antigen-heteroantibody complex

In order to bind the antigen produced in Preparation Example 1 and the mouse antibody produced in Preparation Example 2, a test was conducted as follows. 100ug of the mouse antibody generated against the antigen was mixed with the inactivated mixed vaccine, and antigen-antibody binding was induced at 4℃ overnight. The sample after the binding was collected by centrifugation and used as a sample for inoculation of laying hens.

Preparation Example 4: Preparation of a vaccine for inoculation of laying hens

After mixing each antigen and mAb complex (antigen-heteroantibody complex) prepared in Preparation Example 1 with adjuvant at a weight ratio of 3:7 to prepare a mixed vaccine, a homogenizer was used to prepare a deadox vaccine, and then sterility test and inactivation were confirmed. After the test, a mixed vaccine was prepared for the production of specific egg yolk antibodies.

Example 1: Inoculation of vaccination for laying hens

Laying hens were inoculated with the prepared vaccine using a 25-week-old Hyine-brown breeding hen. Inoculation was performed at 1 ml each in the chest muscle, and inoculation was performed 3 times at 3 weeks intervals. Inoculation groups are shown in Table 1 below. In addition, the composition of the vaccine is shown in Table 2.

group Inoculation contents Vaccine composition Control No vaccination - Comparative example Preparation Example 1 (Vaccine containing SRS-inducing bacteria antigen) P. salmonis Example Preparation Example 1 + Preparation Example 4 (SRS-inducing bacteria antigen-heterologous antibody complex vaccine) P. salmonis +mAb complex (1:1)

group Vaccine composition Ratio (weight ratio) Control - Comparative example P. salmonis One Example P. salmonis +mAb complex ¹⁾ 1:1

¹⁾ : mAb complex = ( P. salmonis + mouse anti-SRS-IgG)

Experimental Example 1: Measurement of antibody titer

1) ELISA coating antigen production

The antigen produced in Preparation Example 1 was centrifuged at 8000 rpm for 50 minutes. After centrifugation, the supernatant was discarded, the pellet was resuspended with HEPES buffer, sonicated, and the supernatant lysed was centrifuged at 8000 rpm 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, N-Lauroly sarcosine (SIGMA, L-9150) was removed by centrifugation at 15,000rpm 4℃ for 50 minutes, suspended again with 50ml HEPES buffer, and centrifuged at 15,000rpm 4℃ for 50 minutes to collect OMP. The collected antigen was coated to 200 ng/ml after protein quantification by the BCA method and used for antibody titer measurement.

2) Measurement of antibody titer using ELISA

The titer of specific egg yolk antibody in yolk was measured by the Indirect ELISA method. First, the antigen is diluted in a coding buffer, coated on a 96 well polystyrene plate at 100ul per well, and over night at 4℃ (or left at 37℃ 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, followed by washing in the same manner as described above. Dilute negative control, positive control and sample by 2X, add 100µl per well, and stand at 37°C for 1 hour. After 1 hour, washing 3 times, diluting the secondary antibody (anti-chicken: Sigma, U.S.A) in an appropriate amount in PBS, dispensing 100ul into each well, and reacting at 37°C for 1 hour. After that, wash 3 times, dispense 100ul of substrate (OPD, sigma) into each well, react at room temperature for about 10 minutes, dispense 50ul of Stop solution to stop the reaction, and absorb the absorbance of each well with ELISA reader at 450nm wavelength. Was measured and expressed as an ELISA value. The measured results were multiplied by the negative value and substituted into the measured value of the processed egg yolk, and the titer of the analyzed data is shown in Table 3 and FIG. 1 below.

Inoculation order SRS Control Comparative example Example parking Primary One 200 800 1,600 2 200 800 1,600 3 400 800 3,200 Secondary One 200 800 3,200 2 400 1600 12,800 3 200 400 3,400 3rd One 400 800 12,800 2 200 800 25,600 3 400 800 25,600 4 200 1600 25,600 5 200 1600 25,600 6 200 1600 25,600 7 200 1600 25,600

As a result, as can be seen in Table 3 above, in the case of the embodiment in which the heterologous antibody was combined with the antigen and inoculated into the laying hen with the additional antigen, the titer of the antibody was formed very high compared to the comparative example in the first to third inoculation. Confirmed.

Experimental Example 2: Confirmation of egg yolk antibody

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

Specifically, eggs showing the highest antibody titer in the yolk produced by each group (control group, comparative examples and examples) were collected at weeks 2 to 3, and IgY was isolated from the yolk. After separating the yolk and the egg white, only the yolk is collected, and then purified water (D.W) is diluted in the collected yolk and stirred for 30 minutes. D.W and the agitated sample are frozen and thawed to separate lipids, and a water-soluble protein sample is collected. After adding ammonium sulfate to the collected sample, the egg yolk antibody is precipitated by refrigerating at 4℃. A sample precipitated for about 18 hours in refrigeration was collected and collected by centrifugation at 6,000 rpm at 4° C., and a sample resuspended in PBS on the collected antibody sample was dialyzed against PBS to obtain a final sample. The obtained sample was quantified with a BCA protein quantification kit, and the purity of separation was confirmed on a 12% SDS-PAGE gel, and the results are shown in FIGS. 2 and 3.

As a result, as can be seen in FIG. 2, the test result was isolated without significant difference from the commercially available purified egg yolk antibody, and as a result of confirming through the Western-blot test, all antibody proteins were found to be suitable, so the pure separation of the antibody seems to be well performed.

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

The egg yolk antibody quantitative test was conducted using HPLC. The IgY standard for quantification was Abcam's normal chicken IgY (Abcam Cat No ad119138). The standard solution, normal IgY, was taken and centrifuged at 12,000 rpm for 20 seconds to use the supernatant. A standard curve is made 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 0.0625mg/ml. After diluting the yolk solution to a concentration of 0.2mg/ml, thaw for 5 minutes using a shaker. The solution was centrifuged at 3,000 rpm for 5 minutes, the supernatant was taken and filtered through a 0.45 um syringe filter, which was measured by HPLC. Measure the standard solution by concentration (1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL) on HPLC, and obtain a linear expression drawn by drawing a linear graph according to the concentration of the standard solution. The value of the test solution measured by HPLC was introduced and calculated, and the results are shown in Table 4 below.

IgY (mg/ml) weeks Control Comparative example Example 1-1 11.50 15.25 25.12 1-2 12.20 14.55 25.24 1-3 11.60 13.95 25.52 2-1 10.50 12.85 25.80 2-2 11.50 15.25 25.21 2-3 12.20 14.55 26.65 3-1 12.10 14.85 26.40 3-2 11.20 14.95 32.54 3-3 11.40 13.75 28.21 3-4 11.50 15.25 26.61 3-5 11.00 13.35 25.83 3-6 11.20 14.95 25.80 3-7 12.10 15.85 25.65 Mean 11.54 14.75 26.51

As a result, as can be seen in Table 4, it was found that the content of the yolk antibody was increased compared to the group (control) in which the vaccination group was not inoculated, and the antibody content in the egg yolk was increased in the example compared to the comparative example. appear.

Experimental Example 4: Antigen binding ability test

In order to confirm the antigen specificity of the salmon of the vaccine produced by Adbiotech, it was analyzed using a flow cytometer. P. salmonis was used as the antigen, and the primary antibody was salmon IgY produced by the company, and the secondary antibody was combined with ANnti-chicken IgG-FITC to perform flow cytometry, and the results are shown in FIG. 4.

As a result, as can be seen in FIG. 4, cells showing a fluorescent signal of 60.6% were observed in the comparative example, and cells sending a high fluorescent signal of 74.6% were observed in the Example. Therefore, it can be confirmed that the egg yolk antibody having high specificity was generated in the examples compared to the comparative examples.

Experimental Example 5: Confirmation of the growth inhibition of the causative agent of salmon rickettsia sepsis

In order to confirm the efficacy of IgY prepared in Example 1 against P. salmonis , the SRS bacteria, a test was conducted to determine whether or not the growth of bacteria was inhibited. In the test, the inhibitory effect was confirmed by reacting P. salmonis with the IgY step-diluted sample for an hour and then checking the copy number of P. salmonis through RT-PCR, and the results are shown in Table 5 below.

Sample Copy number average in 0.5ml ^* Inhibition% Non infected cells control 0 - Infected cells without IgY 1.6x10 ⁷ - Infected cells +1:125 dilution 1.7x10 ² 99.99% Infected cells +1:250 dilution 6.2x10 ² 99.99% Infected cells +1:500 dilution 1.4x10 ² 99.99% Infected cells +1:1000 dilution 1x10 ³ 99.99% Infected cells +1:2000 dilution 0.5x10 ³ 99.99% Infected cells +1:4000 dilution 1x10 ³ 99.99% Infected cells +1:8000 dilution 1.3x10 ⁶ 91.88% Infected cells +1:16000 dilution 1.4x10 ⁷ 12.5%

* the results are an average of 4 replicates

As a result of the test, as can be seen in Table 5, IgY was diluted from 1:125-fold to 1:16,000-fold by binary dilution, and the effect of inhibiting 99.99% compared to the control was shown up to 1:4000 dilution. : It showed the effect of inhibiting 90% growth compared to the control at the concentration up to 8000 times dilution. These results show that produced IgY effectively inhibits P. salmonis .

Claims (6)

Salmon Rickettisia sepsis (Salmon Rickettisia septicaemia) using the causative bacteria Piscirickettsia Salmonis ( Piscirickettsia Salmonis ) a first step of preparing an antigen;
A second step of inoculating the antigen of the first step into a mouse or rabbit, which is a chicken and a heterogeneous animal, to prepare a heterologous antibody;
A third step of preparing an antigen-heteroantibody complex by combining the antigen of the first step with the heterologous antibody of the second step;
A fourth step of inoculating a laying hen with a mixture of the antigen of the first step and the complex of the third step; And
A fifth step of separating an egg yolk antibody against the causative agent of salmon rickettsia sepsis from the laying egg (egg) of the fourth step; Salmon rickettsiae sepsis prevention or treatment method for producing yolk antibodies comprising a.
The method of claim 1,
In the third step, the mixture ratio of the antigen and the complex is 3:0.5 ~ 1:0.1 weight ratio of salmon rickettsia sepsis prevention or treatment yolk antibody production method, characterized in that.
The method of claim 1,
In the fourth step, the mixture further comprises an adjuvant. Method for producing a yolk antibody for preventing or treating salmon rickettsia sepsis.
The method of claim 3,
The mixing ratio of the mixture and the adjuvant is 2-3: 7-9 weight ratio of salmon rickettsia sepsis prevention or treatment yolk antibody production method, characterized in that.
The method of claim 1,
Inoculation in the fourth step is a method for producing yolk antibody for preventing or treating salmon Rickettsia sepsis, characterized in that the inoculation is 2 to 5 times at 0.1ml to 3ml.
The method of claim 1,
The method for producing yolk antibodies is a method for producing yolk antibodies for preventing or treating salmon Rickettsia sepsis, characterized in that increasing the production yield of yolk antibodies.

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