CN111349156A - Method for producing yolk antibody for preventing or treating digestive system diseases in calves, yolk antibody produced thereby, and use thereof - Google Patents
Method for producing yolk antibody for preventing or treating digestive system diseases in calves, yolk antibody produced thereby, and use thereof Download PDFInfo
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- CN111349156A CN111349156A CN201910091847.8A CN201910091847A CN111349156A CN 111349156 A CN111349156 A CN 111349156A CN 201910091847 A CN201910091847 A CN 201910091847A CN 111349156 A CN111349156 A CN 111349156A
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- Prior art keywords
- yolk antibody
- antibody
- digestive system
- stage
- calves
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Abstract
The present invention relates to a method for producing a yolk antibody for preventing or treating a calf digestive system disease by producing a foreign antibody using a calf digestive system disease pathogenic bacterium or virus antigen and then inoculating a complex in which the antigen and the foreign antibody are bound to a laying hen to produce the yolk antibody, and a yolk antibody produced thereby and use thereof.
Description
Technical Field
The present invention relates to a method for producing a yolk antibody for preventing or treating digestive system diseases in calves, a yolk antibody produced thereby, and uses thereof, and more particularly, to a method for producing a yolk antibody for preventing or treating digestive system diseases in calves, which comprises producing a heteroantibody (heteroantibody) using pathogenic bacteria or viral antigens of the digestive system diseases in calves, and then inoculating a complex in which the antigen and the heteroantibody are bound to a laying hen, a yolk antibody produced thereby, and uses thereof.
Background
Antibodies (antibodies) are globulin-based proteins that are based on B lymphocytes or B cells that form lymphoid tissues, and are important substances involved in specific immunological recognition. The antibody has two functions different from each other, the first is an action of specifically recognizing and binding to a specific site of an antigen causing an immune response to neutralize the antigen, and the second is an action of binding the antibody to the antigen, thereby exerting various substances for removing the antigen and an action of aggregating immune cells. For example, the antibody can bind to the toxin to change the chemical composition of the toxin alone to neutralize toxicity, and can attach to invading microorganisms to thereby inhibit the invasion of the microorganisms into body cells by eliminating their motility. In addition, the antigen surrounded by the antibody causes a chemical chain reaction with complement, causing direct decomposition of invading microorganisms or inducing phagocytes. Using the specificity and high affinity of such antigen-antibody reactions and the diversity of antibodies that can distinguish tens of millions of antigens, a variety of antibody pharmaceutical products including diagnostic and therapeutic agents, etc. have been developed (Jim e.
On the other hand, as a defense mechanism against diseases, antibodies are included in substances produced for protecting oneself or protecting offspring from infectious diseases, not only in humans, but also in various mammals and birds. Mammals, like most humans, produce IgG of about 150 Kd. In addition to producing IgG, mammals also produce IgA, IgD, IgE and IgM which differ slightly in their structure or function.
Conventionally, the following methods have been widely used for producing antibodies against various pathogenic bacteria: a mammal such as a rabbit, a cow, or a goat is immunized to obtain serum, and an antibody specific to a pathogenic bacterium is produced from the serum. However, such a method has a disadvantage that it is necessary to extract the antibody from a small amount of serum of a small mammal, and the amount of the produced antibody is extremely small. Therefore, as a method for easily producing antibodies against pathogenic bacteria, a method of transferring antibodies generated by immunizing a laying chicken to eggs of the laying chicken and extracting the antibodies has been attracting attention. In particular, a specific antibody present in the Yolk of a laying hen is called Immunoglobulin Yolk (IgY) or a Yolk antibody, which is an antibody produced by migration of Immunoglobulin (IgG) as a specific antibody present in the serum of a laying hen to the Yolk of an egg and is accumulated in the Yolk of an egg at a high concentration. Since IgY can be easily separated, it is increasingly used in various fields, particularly, development of IgY having a preventive and therapeutic effect on livestock diseases and effective use thereof as a feed additive.
A method for obtaining a yolk antibody from an egg of a laying chicken using a disease-causing bacterium as an antigen, and a composition, food, or the like containing the antibody have been actively developed, and the production of a vaccine using an antigen-antibody complex and the development of a composition for treating various diseases have been actively carried out. Specifically, Korean laid-open patent No. 2009-0088121 discloses that when an antigen-antibody complex is used as an adjuvant for a vaccine, the immune response to an antigen can be enhanced, and it is known from the paper Phase I tertiary of a human antibody to MUC1 in tissues with a metastacicanc that when an antigen-hetero antibody complex is used for the treatment of cancer cells, an excellent effect can be exhibited in treating cancer cells by activating T cells (Ann Oncol.2004 Dec; 15 (12): 1825-33). However, a method of using an antigen-antibody complex for the mass production of a yolk antibody has not been known so far.
On the other hand, a diarrheal disease, which is one of digestive diseases, is a disease that is caused by an abnormality in mutual movement of water in the contents of digestive tracts in the living body, can be fatal to calves and causes death, and even recovery causes growth failure and the like, and is known as a disease with a large economic loss.
The causes of diarrheal disease in calves can be distinguished from infectious and non-infectious, the non-infectious causes occurring due to improper feeding management and unclean barns, and the infectious causes occurring due to infection with various viruses, bacteria and parasites. When the symptoms of infectious diarrhea appear in calves, initial symptoms are present, and the calves rapidly develop within 3 to 5 days, and if symptomatic therapy is not performed, the calves die, so that rapid treatment is desired, and vaccination is important for preventing the cases.
Accordingly, the present inventors have conducted extensive studies to overcome the above-mentioned problems of the prior art, and as a result, have found that a large amount of egg-yolk antibodies having an enhanced immune response against pathogenic bacteria or viral antigens of digestive system diseases of calves can be produced and produced when a heterogeneous antibody is produced using pathogenic bacteria or viral antigens of digestive system diseases of calves and then a complex in which the antigen and the heterogeneous antibody are bound is inoculated to a laying hen, and that a vaccine can be produced using the egg-yolk antibodies, thereby preventing digestive system diseases of calves, and have completed the present invention.
Documents of the prior art
Patent document
Patent document 1: KR 10-2009-0088121A
Non-patent document
Non-patent document 1: s.de Bono JS et al, Annals of oncology.2004 Dec; 15 (12): (1825-33)
Disclosure of Invention
Problems to be solved by the invention
Accordingly, a main object of the present invention is to provide a method for producing a yolk antibody for preventing or treating digestive system diseases in calves, which enables mass production and production of the yolk antibody, and a yolk antibody produced by the method and having an enhanced immune response to pathogenic bacteria or viral antigens of digestive system diseases in calves.
Another object of the present invention is to provide a method for producing the yolk antibody for preventing or treating a calf digestive system disease, and a vaccine composition for preventing or treating a calf digestive system disease or a feed additive composition for preventing or treating a calf digestive system disease, using the yolk antibody produced by the method.
Means for solving the problems
According to one embodiment of the present invention, there is provided a method for producing a yolk antibody for preventing or treating digestive system diseases in calves, the method comprising the steps of: stage 1, preparing antigen by using calf digestive system disease pathogenic bacteria or virus; a 2 stage of inoculating the antigen of the 1 stage to an animal xenogeneic to the chicken to produce a xenogeneic antibody; a 3 rd stage of binding the antigen of the 1 st stage to the foreign antibody of the 2 nd stage to produce an antigen-foreign antibody complex; a 4 stage of inoculating a mixture in which the antigen of the 1 stage and the complex of the 3 stage are mixed, to a laying chicken; and a 5 th stage of isolating yolk antibodies against pathogenic bacteria or viruses of digestive system diseases of calves from the eggs (egg) of the egg-laying chicken of the 4 th stage.
Since egg Yolk antibodies (IgY) are produced by transferring Immunoglobulin (IgG), which is a specific antibody present in the serum of a laying hen, to the egg Yolk, they are accumulated in the egg Yolk at a high concentration and easily separated, and thus can be used in various fields. As a result of intensive studies for mass production and production of such a yolk antibody, the present inventors have found that an immune response can be enhanced when an antigen-antibody conjugate is used as an adjuvant and a foreign antibody is used as an antibody, and, in view of this fact, a yolk antibody having an enhanced immune response against an antigen can be mass produced and produced when a complex in which an antigen and a foreign antibody are bound is used to produce a yolk antibody, and have completed the present invention.
The method for producing a yolk antibody of the present invention is characterized in that the pathogenic bacteria for digestive system diseases of calves at stage 1 may include any bacteria known to induce digestive system diseases of calves, preferably at least one pathogenic bacteria selected from the group consisting of escherichia coli (e.coli), Salmonella (Salmonella), Clostridium (Clostridium) and Mycobacterium paratuberculosis (Mycobacterium paratuberculosis), and more preferably escherichia coli (e.coli).
The method for producing a yolk antibody of the present invention is characterized in that the calf digestive system disease-causing virus of stage 1 may include any known virus that induces calf digestive system diseases, preferably at least one virus selected from Rotavirus (Rotavirus), CoronaVirus (CoronaVirus), Bovine viral diarrhea virus (Bovine viral diarrhea virus), Adenovirus (Adenovirus), parvovirus (Parbovirus), and Enterovirus (Enterovirus), and more preferably Rotavirus (Rotavirus).
In the method for producing a yolk antibody of the present invention, any mammal, preferably a mouse or a rabbit, which is conventionally used for producing antibodies can be used as the animal of the different species in the above stage 2.
In the method for producing a yolk antibody of the present invention, the ratio of the antigen to the complex in the 4 th stage is preferably 3: 0.5 to 1: 0.1. When the ratio of the complex is larger than this ratio, there is a problem in that an excessive amount of immunocytes causes recognition of the complex, and when the ratio is smaller than this ratio, it is difficult to exert a sufficient effect by application of the complex.
The method for producing a yolk antibody according to the present invention is characterized in that the mixture further contains an adjuvant (adjuvant) in the above-described stage 4. As the adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, liposome (lipofectin), lipotaxin, CaPO4, 25 to 30% sucrose, DEAE dextran, polybrene (polybrene), saponin, inorganic gel such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions (polyanions), peptides (peptides), surface active substances such as oil or hydrocarbon emulsion, dinitrophenol, and the like can be used, but not limited thereto. In the examples of the present invention, ISA70 incomplete adjuvant (incomplete adjuvant) was used as an adjuvant.
The method for producing a yolk antibody of the present invention is characterized in that the mixing ratio of the mixture to the adjuvant (adjuvant) may be 2 to 3: 7 to 9, preferably 3: 7. It is important to maintain the above ratio because the higher the mixing ratio of the mixture, the lower the function of the adjuvant as an emulsifier, the more the separation phenomenon of the water-soluble layer and the fat-soluble layer occurs when the adjuvant is mixed with the antigen.
The method for producing a yolk antibody of the present invention is characterized in that the inoculation in the above-mentioned stage 4 can be performed 2 to 5 times in an amount of 0.1 to 3ml, preferably 3 times in an amount of 0.5 to 1 ml.
The method for producing a yolk antibody of the present invention is characterized in that the production yield of the yolk antibody is increased.
According to one embodiment of the present invention, the production yield of the yolk antibody according to the production method of the present invention was confirmed, and as a result, it was confirmed that: in the case where the egg-yolk antibody was produced by injecting the antigen-foreign antibody complex into a laying chicken, the antibody content in the egg yolk was increased as compared with the example (comparative example) in which the egg-yolk antibody was produced by injecting only the antigen into a laying chicken. Such results suggest that: in view of the fact that it is difficult to significantly increase the total production amount of antibodies, the production method of the present invention can produce and produce a large amount of yolk antibodies by imparting a synergistic effect to the antigen-foreign antibody complex as an adjuvant for antibody production with respect to the increase in the production of yolk antibodies (see example 3 and table 5).
Further, according to an embodiment of the present invention, the antigen binding force of the yolk antibody produced by the manufacturing method according to the present invention was confirmed, and as a result, it was confirmed that: the egg yolk antibody produced by injecting the antigen-xenoantibody complex into the egg-laying chicken (example) was superior in binding force to the antigen, compared to the egg yolk antibody produced by injecting only the antigen into the egg-laying chicken (comparative example). Such results suggest that: in the case where the yolk antibody was produced according to the production method of the present invention, the production of the yolk antibody specific to a specific antigen could be increased, and it was concluded that the superior binding force to the antigen could increase the immune response to the calf digestive system disease pathogenic bacteria or viral antigen. Furthermore, the above binding forces suggest: the antigen-xenoantibody complex injected into the laying hens was recognized as an initial antigen, that is, as an antigen, pathogenic bacteria or viruses of digestive system diseases of calves, and IgY against the antigen was generated (see example 6, table 8, and fig. 7).
According to another aspect of the present invention, there is provided a yolk antibody for preventing or treating digestive system diseases in calves, which is produced by the above method for producing a yolk antibody.
According to another embodiment of the present invention, there is provided a vaccine composition for preventing or treating digestive system diseases in calves, which comprises a yolk antibody as an active ingredient.
The vaccine of the present invention can be used together with a pharmaceutically acceptable carrier, adjuvant or excipient.
The carrier used in the vaccine of the present invention can use a physiologically balanced medium containing one or more stabilizers such as hydrolyzed protein, lactose, etc., and can also use 0.01 to 0.1M phosphate buffer or 0.8% physiological saline. The pharmaceutically acceptable carrier may be in the form of a solution, a non-solution, a suspension, or an emulsion. Examples of the non-solution solvent include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. As the soluble carrier, water, an alcohol solution, an emulsion, a physiological saline solution, or a suspension such as a buffer culture solution can be used.
The adjuvant used in the vaccine of the present invention can use complete Freund 'sadjivant, incomplete Freund's adjuvant, liposome (lipofectin), lipotaxix, CaPO4, 25 to 30% sucrose, DEAE dextran, polybrene (polybrene), saponin, inorganic gel such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions (polyanions), peptides (peptides), surface active substances such as oil or hydrocarbon emulsion, dinitrophenol, etc., without limitation.
Examples of the excipient and diluent used in the vaccine of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, cetyl alcohol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methyl hydroxybenzoate, propyl hydroxybenzoate, and mineral oil.
The vaccines of the present invention can be administered in the usual manner by oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intra-ocular or intradermal routes.
By parenteral administration is meant administration modes including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion. Non-oral administration of the vaccine of the invention is preferably: pharmaceutically acceptable carriers, i.e., carriers that are not toxic to the recipient at the concentrations and administration amounts used and that can be combined with other formulation ingredients, are mixed at a preferred purity and prepared in dosage forms of unit administration amounts.
The dosage form of the vaccine of the present invention can be applied to any dosage form, and the prepared dosage form can be used for oral administration, injection, or application. The above formulations can be prepared in the form of tablets, capsules, soft capsules, aqueous solutions, granules, pills or forms for injection (solutions, suspensions or emulsions), most preferably in the form of injections.
According to another embodiment of the present invention, there is provided a feed additive composition for preventing or treating digestive system diseases in calves, comprising a yolk antibody as an active ingredient.
The feed additive of the present invention may be used as it is or may be further added with known carriers, stabilizers and the like for grains and byproducts thereof which are acceptable for livestock, or organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), or polyphenols, catechin, α -tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, natural antioxidants such as chitosan, tannic acid and phytic acid, antibiotics, antibacterial agents and other additives, and may be added as needed, and the form thereof may be in a suitable state such as powder, granule, pill, suspension, and the like.
According to one embodiment of the present invention, it was confirmed that the egg yolk antibody produced by the production method of the present invention effectively inhibited the growth of bacteria as compared with the positive control group and the comparative example. Such results suggest that: the yolk antibody of the present invention can be easily used as a vaccine or feed additive for preventing or treating digestive system diseases in calves (see example 4 and fig. 5).
Effects of the invention
As described above, the method for producing a yolk antibody for preventing or treating a calf digestive system disease according to the present invention can produce and produce a large amount of yolk antibody by injecting an antigen-foreign antibody complex into a laying chicken, and the produced yolk antibody has high specificity and binding property to pathogenic bacteria or viruses of the calf digestive system disease, and can effectively inhibit the growth of the bacteria or viruses, thereby preventing or treating the calf digestive system disease.
Drawings
FIGS. 1 and 2 are graphs showing the results of measurement of antibody titers.
FIGS. 3 and 4 are each a graph showing the results of confirming the yolk antibody ((FIG. 3; SDS-PAGE (12% gel staining), FIG. 4; Western blot), 1. Natural chicken (Natural chicken) -yolk antibody protein (IgY protein) (cat # ab119138), 2. vaccination yolk antibody-example)
FIG. 5 is a graph showing the results of confirming inhibition of bacterial growth.
Fig. 6 is a graph showing the results of confirming the potency of the neutralizing antibody.
FIG. 7 is a graph showing the results of confirmation of antigen binding force.
Detailed Description
The present invention will be described in more detail below with reference to examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Production example 1: production of antigens
1)E.coli(09∶K35,K99,F41)KVCC-BA0000540-020800491
Aerobic culture was performed in a 37 ℃ incubator (incubator) using a Blood plate (Blood agar) medium. The colonies (Colony) were confirmed and inoculated into 1000ml of BHI broth (brain heart infusion broth), followed by shaking culture for 24 to 36 hours. After completion of the culture, the cells were inactivated by treatment with 0.3% Formalin (Formalin) at room temperature for 48 hours. The inactivated antigen was centrifuged at 6000rpm and recovered. The recovered antigen was dispersed in PBS (phosphate buffer, pH7.2), and then 0.1% Formalin (Formalin) was added thereto, and the mixture was left at room temperature for 1 day and then stored in a refrigerator at 4 ℃. The antigen was calculated and the concentration was adjusted so that the OD (optical density) of the produced antigen at 410nm by a spectrophotometer (spectrophotometer) became 1.0.
2) Rotavirus (KVCC-VR9200180, KVCC-VR9200179, KVCC-VR9200162)
Rota Virus (Rota Virus) was inoculated by culturing MA104(ATCC, CRL-2378.1) cells in a cell maintenance Medium (α -MEM, antibiotics (antibiotics), yeast (yeast), FBS 5%), washing (washing)3 times in a Medium supplemented with a non-serum Medium α -MEM (alpha-minimum Essential Medium) and trypsin (trypsin, 5. mu.g/ml) to form an 80% monolayer (monolayer), inoculating with a rotavirus diluent at a concentration of 100TCID50 (Tissue culture infectious dose 50, Tissue culture infection dose 50), incubating for 90 minutes with α -MEM containing 5% FBS (fetal bovine serum) for 48 hours, and then confirming the Cytopathic Effect (Cytopathic Effect) after 48 hours, recovering the Virus using 0.2M BEI (2-Hydrobromide) to inactivate the Virus (Bromide).
Rotavirus is distributed in KoreaAnd (5) a veterinary genetic resource bank (the accession number is KVCC-VR 9200179). The medium used for viral infection was prepared by adding 10ug/mL of trypsin (Gibco) to (-) alpha MEM medium. MA104(ATCC, CRL-2378.1) cells were prepared in a T175 flask (flash) so that the confluency (confluency) of the cells became 80 to 90%, and then washed 3 times with PBS. Rotaviruses 179, 162 were infected at 0.5m.o.i (multiplicity of infection) in infection medium for 1 hour. When the virus adsorption reaction time was over, the inoculum was removed and then washed 3 times with PBS. The maintenance medium was cultured in (-) alpha MEM medium supplemented with 2ug/mL of trypsin (Gibco) until the cytopathic effect was 80% or more. When it was confirmed (37 ℃, 5% CO)2) When cytopathic effect was observed, (-) alpha MEM supernatant was collected and centrifuged at 3000rpm for 10 minutes by a centrifuge (centrifuge). The cell pellet (pellet) was discarded, and the recovered supernatant was filtered (filtration) with a 0.22um syringe filter (system filter), followed by the use of the virus.
Production example 2: production of murine antibodies
The antigens produced in production example 1 were inactivated to suppress toxicity, and then stored in a refrigerator for use. In the case of a vaccine for murine antibody therapy, a complete adjuvant (complete adjuvant) was used to prepare the vaccine. Various vaccines corresponding to the respective antigens were produced, and antibodies were produced according to a usual method for producing murine antibodies. The murine antibody was purified from Serum (Serum) recovered after completion of production using a protein-A column (protein-Acolumn).
Production example 3: preparation of Calf antigen-binding samples (mAb complex)
In order to bind the antigen produced in production example 1 to the murine antibody produced in production example 2, the following test was performed. Mu.g of mouse antibody against each antigen was mixed with the inactivated mixed vaccine, and antigen-antibody binding was induced at 4 ℃ Overnight (Overnight). The bound sample was collected by centrifugation and used as a sample for inoculating a laying hen.
Production example 4: preparation of vaccine for inoculating egg-laying chicken
In order to produce a mixed calf Vaccine, each group of antigens and mAb complex (complex) prepared in production example 1 and an adjuvant (adjuvant) were mixed at a ratio of about 3: 7, and then inactivated Vaccine (killvaccine) was produced using a Homogenizer (homogenerizer), and then a mixed Vaccine for production of specific yolk antibody was prepared through sterility test and inactivation confirmation test.
Example 1: preparation of mixed vaccine for oviposition chicken and calf
The egg-laying chicken inoculation was carried out by inoculating the produced vaccine to 25-week-old egg-laying chickens of the Hyine-brown series. The inoculation was carried out in the following manner: the pectoral muscle was inoculated with 1ml each time, 3 times at 3-week intervals. The inoculation groups are shown in table 1 below.
TABLE 1
Specifically, the vaccination group was composed as follows to produce a vaccine. In comparative examples, antigens inactivated against viruses and bacteria causing digestive diseases in calves (production example 1) were prepared in equal amounts. In examples, mabs that bind to antigens in comparative examples were produced, and complexes that bind to antigens and antibodies (production example 4) were prepared by adding them to vaccines, and the compositions thereof were as shown in table 2 below.
TABLE 2
| Group of | Vaccine compositions | Ratio of |
| Control group | - | |
| Comparative example | Rotavirus 162+ rotavirus 179+ escherichia coli | 1∶1∶1 |
| Examples | Rotavirus 162+ rotavirus 179+ escherichia coli + mAb-complex1) | 1∶1∶0.5 |
1): mAb complex (complex) (rotavirus (RotaV) + murine anti-rotavirus immunoglobulin G (mousingi-RotaV-IgG)) + (e.coli) + murine anti-e.coli immunoglobulin G (mousingi-e.coli-IgG))
Example 1: antibody titer determination
1) ELISA coated antigen production
The antigen produced in production example 1 was centrifuged at 8000rpm for 50 minutes. After centrifugation, the supernatant was discarded, the pellet (pellet) was resuspended in HEPES buffer (buffer) (suspension), and then dissolved by ultrasonication (Lysis), and the dissolved supernatant was centrifuged at 8000rpm at 4 ℃ for 30 minutes to obtain a supernatant. To the resulting supernatant, 1% N-lauroylsarcosine (SIGMA, L-9150) was added so that the final concentration became 0.01%, and then treated at room temperature for 10 minutes. After the treatment, centrifugation was carried out at 15000rpm at 4 ℃ for 50 minutes to remove N-lauroylsarcosine (SIGMA, L-9150), and the resulting solution was resuspended in 50ml of HEPES buffer (buffer) (suspension) and centrifuged at 15000rpm at 4 ℃ for 50 minutes to recover OMP. The recovered antigen was quantified by BCA method, and then coated at 200ng/ml for antibody titer measurement.
2) Antibody titer determination by ELISA
The titer of specific yolk antibodies in yolk was determined by Indirect enzyme-linked immunosorbent assay (Indirect ELISAmethod). The antigen was first diluted in coating buffer and coated in 96-well polystyrene plates (wells) where 100. mu.l was coated per well, followed by overnight at 4 ℃ (or 1 hour at 37 ℃). After washing with PBS-T (phosphate buffer saline, 0.05% Tween 20, pH7.4), blocking (blocking) was performed with BSA-containing PBS buffer at 37 ℃ for 1 hour, and washing was performed as described above. The negative control group, the positive control group and the sample were diluted 2X, 100. mu.l was added to each well, and the mixture was allowed to stand at 37 ℃ for 1 hour. After 1 hour, 3 washes were performed, and 2 washes of the antibody (anti-chicken: Sigma, U.S. A) were diluted in PBS in appropriate amounts, dispensed in 100. mu.l per well, and then reacted at 37 ℃ for 1 hour. Then, 3 times of washing were performed, 100. mu.l of a substrate (substrate, OPD, sigma) was dispensed to each well, and then reacted at room temperature for about 10 minutes, 50. mu.l of a Stop solution (Stop solution) was dispensed to terminate the reaction, and absorbance at 450nm was measured by ELISA reader to each well and expressed as ELISA value (value). The positive/negative values (P/Nvalue) of each sample were calculated by performing inverse calculation on the dilution factor of OD value 2 times or more as compared with the OD value of the negative sample (negative control), and the results thereof are shown in tables 3 and 4 below and fig. 1 and 2.
TABLE 3
TABLE 4
As a result, as can be confirmed in tables 3 and 4 and fig. 1 and 2, in the case of the examples in which the foreign antibody was bound to the antigen and the laying hens were inoculated with the additional antigen, the titer of the antibody formed was significantly higher in 1 and 2 inoculations than in the comparative examples, and particularly in the case of the virus antigen, the titer was continuously high as a whole, and thus the antibody formation effect against the virus antigen was increased.
Example 2: identification of yolk antibody
After separating the antibody from the inoculated yolk using Ammonium sulfate (Ammonium sulfate), experiments were performed to confirm the separation purity.
Specifically, 2-3 weeks of eggs showing the highest antibody titer were collected from the egg yolks produced in each group (control group, comparative example, and example), and IgY was isolated from the egg yolks. The yolk and egg white were separated, and only the yolk was collected, and then the recovered yolk was diluted with purified water (D.W) and stirred for 30 minutes. The sample stirred with D.W was repeatedly frozen and thawed to separate lipids and recover a water-soluble protein sample. Ammonium sulfate (Ammonium sulfate) was added to the collected sample, and the sample was stored at 4 ℃ under refrigeration to precipitate the yolk antibody. The samples precipitated in the cold storage for about 18 hours were recovered, centrifuged at 6000rpm at 4 ℃ and recovered, and then the samples obtained by resuspending the recovered antibody samples with PBS were dialyzed with PBS to obtain final samples. The obtained sample was quantified with BCA protein quantification kit (kit), and the separation purity was confirmed on 12% SDS-PAGE gel (gel), and the results are shown in fig. 3 and 4.
As a result, as can be confirmed in fig. 3 and 4, the isolation was not significantly different from the commercial purified yolk antibody, and it was found that the antibody was isolated in good purity by confirming all the antibody proteins by Western blot (Western blot) test.
Example 3: determination of antibody content based on egg-laying chicken inoculation
Quantitative assay of yolk antibody assay was performed by HPLC. As the standard substance for the quantification, there was used normal chicken IgY (Abcam Cat No ad119138) available from Abcam Co. The normal IgY was used as a standard solution, centrifuged at 12000rpm for 20 seconds, and the supernatant was used. The centrifugally separated standard solution was diluted with distilled water to 5 concentrations to prepare a standard curve. The standard solution was diluted at 1, 0.5, 0.25, 0.125, 0.0625 mg/ml. The egg yolk solution was diluted to a concentration of 0.2mg/ml and then dissolved for 5 minutes by a shaker. The solution was centrifuged at 3000rpm for 5 minutes, and the supernatant was collected, filtered through a 0.45um syringe filter (syringe filter), and the filtrate was measured by HPLC. The standard solutions were measured by HPLC at concentrations (1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL), linear expressions were prepared from the concentrations of the standard solutions, and values of the test solutions measured by HPLC were introduced and calculated, and the results are shown in Table 5 below.
TABLE 5
**：VS G1，p＜0.01
As a result, as can be confirmed in table 5, the vaccinated group showed an increase in the content of yolk antibodies compared to the group without vaccination, and the examples showed an increase in the content of yolk antibodies compared to the comparative examples.
Example 4: confirmation test for bacterial growth inhibition
Coli (E.coli) used as an antigenic bacterium was subjected to growth inhibition test in LB Broth at 1 × 107Prepared for inoculation at cfu/ml concentration, the isolated yolk antibodies were treated uniformly at the same concentration, i.e. at a concentration of about 10 mg/ml. The test period was measured at 1-hour intervals until about 12 hours after inoculation, and then at 2-hour intervals, and the results are shown in fig. 5.
As a result, as can be confirmed in fig. 5, in the case of e.coli, the yolk antibodies of all groups did not differ greatly from all positive control groups until the initial 4 hours. In the case of E.coli, there was a slightly decreased interval compared with the positive control group, but no large difference was observed. In the case of the examples, the increase was gradual by inhibition up to 5 hours of the test, and after 10 hours, the increase was large. The yolk antibody of the example was shown to be more effective in inhibiting the growth of the antigen bacteria than the comparative example.
Example 5: neutralizing antibody assay
In order to confirm virus neutralization potency of the yolk antibody produced against rotavirus used as an antigen, an experiment was performed using srb (sulforhodamine B colorimetric assay). As a method for measuring the proliferation and survival of cells, the following methods can be used: the SRB was used to stain proteins that are commonly produced in the cells, and the viability of the cells of the test group and the control group was confirmed by optical density (o.d) values. The absorbance represents the amount of total protein present in each well (well) and is proportional to the number of viable cells remaining in that well. The percentage value of the average value of the optical density at 560nm in the test group to the average value of the optical density at 560nm in the control group was calculated. This percentage corresponds to the value of the cell survival rate of the test group compared with the control group.
Specifically, in 96-well plates (well plates) at 2 × 105cell/ml 100. mu.l MA104 cells were dispensed separately in 5% CO2Cultured in an incubator (incubator) for 24 hours. The virus and IgY were diluted as follows, mixed, and reacted at 4 ℃ for 10 minutes. The 96-well plate inoculated (seeded) with each cell was washed 3 times with PBS (washing). 100. mu.l of each well of the washed 96-well plate was added. Incubation (incubation) for 2 days, provided time for infection. It was confirmed that (37 ℃, 5% CO)2) After CPE (cytopathic effect), the 96-well plates were washed 3 times with PBS (1 ×). Mu.l of acetone solution (70%) was added to a 96-well plate, and the mixture was reacted at-20 ℃ for 10 to 30 minutes to fix the cells. The acetone solution (70%) was prepared for use at-20 ℃. To completely remove the acetone, the 96-well plate was uncapped and placed in a dry box (dry oven) for 10 minutes. Add 75. mu.l of SRB solution to each well. The 96-well plate to which the SRB solution was added was left for 4 hours at room temperature in the lacker. After the SRB solution was removed, the SRB solution was prevented from remaining by washing 5 times with 1% acetic acid (acetic acid). Whether washed or not was read by microscopic observation. The SRB solution is prevented from remaining on the wall surface and peripheral portions of the 96-well. To completely remove 1% acetic acid, the reaction mixture was left in the dry box for 10 minutes. The stained 96-well plate was observed with a microscope. Add 20mM tris (Tris) solution to each well (Tris>ph8.5), after mixing with the SRB solution, the optical density values were read at 560nm using a spectrophotometer (spectrophotometer). The results were confirmed by an optical micrograph of cells stained with SRB, and the cell viability was confirmed by the optical density value, and the results are shown in tables 6 and 7 and fig. 6.
TABLE 6
| Total mean Virus (%) | Cell population average (%) |
| 32.9 | 100 |
TABLE 7
| mg/ml | 4.0 | 2.0 | 1.0 | 0.5 | 0.25 | 0.125 | 0.063 | 0.031 | 0.016 | 0.008 | 0.004 | 0.002 |
| |
100 | 100 | 100 | 65.1 | 45.8 | 41.1 | 30.7 | 35.4 | 48.7 | 34.2 | 30.4 | 40.4 |
| Comparative example | 100 | 100 | 100 | 83.6 | 81.3 | 49.7 | 38.7 | 36.7 | 44.4 | 39.8 | 38.5 | 41.5 |
| Examples | 100 | 100 | 100 | 92.5 | 86.8 | 73.8 | 50.2 | 39.1 | 40.5 | 26.1 | 37.0 | 36.0 |
As a result, when the yolk antibodies isolated from each group were diluted in binary from the concentration of 4mg/ml, the neutralizing antibody potency was confirmed as follows: the control group showed the result at 0.5mg/ml, and the comparative example showed 16 and the example showed 32. In the case of examples, the neutralizing antibody titer was 4 times higher than that of comparative examples.
Example 6: antigen binding force determination test-calf
The binding force to the antigen and the production ratio of the antigen-specific yolk antibody were confirmed for the separated antibody using a Flow cytometer (Flow cytometer). Coli (e.coli) used as an antigen was cultured and then inactivated with 0.1% formalin. The recovered antigen was subjected to the following treatments: the absorbance was measured at an Optical Density (OD) of 600nm and the dilution was 1.0. The isolated yolk antibody was prepared from a concentration of 50mg/ml to 500mg/ml, and then mixed with the antigen and bound to the antigen at 4 ℃ for 18 hours.
The sample bound to the antigen was centrifuged at 3000rpm for 10 minutes, and the supernatant was discarded, and the unbound antibody was discarded. As a secondary antibody, Anti-chicken-IgG-PE antibody was diluted at 1: 2000 and washed twice with PBS, and the results were confirmed in a Flow cytometer (Flow cytometer) in Table 8 and FIG. 7(1. E.coli; 2.E.coli + G1-IgY + Anti-chicken-IgG-PE; 3.E.coli + G2-IgY + Anti-chicken-IgG-PE; 4.E.coli + G3-IgY + Anti-chicken-IgG-PE).
TABLE 8
| Group of | Affinity (%) |
| Control group | 12.3％ |
| Comparative example | 46.7％ |
| Examples | 79.5％ |
As a result, as can be confirmed in table 8 and fig. 7, the ratio of the antibodies bound to the antigenic bacteria was 12.3% in the control group, 46.7% in the comparative example, and 79.5% in the example. Even the normal IgY that was not sensitized to the antigen of the control group showed a binding force of 12.3%, indicating that the antibody possessed by the parent of escherichia coli is substantially present to some extent. Therefore, the following steps are carried out: in the case of comparative example and example, the binding force was further increased by 3 vaccinations with antigen, compared to the control group. As can be seen from this test, the bonding force was further increased in the examples as compared with the comparative examples.
Example 7 clinical trial
1) Preparation of animals
10 calves were numbered and randomly divided into 5 experimental groups and 5 control groups. For calf, 1 times a day in the morning/afternoon in 1 week 2L of milk substitute, pellet feed, and drinking water are supplied alternately and freely. After 1 week, 2-2.5L of milk substitute is supplied every morning, 250g of pellet feed for calf containing more than 18% protein is supplied after milk substitute is supplied, 250g of pellet feed for calf is supplied every afternoon, and drinking water is alternately and freely supplied every day.
2) Calf maternal transitional antibody test
10 calves were bled from the tail before colostrum, and serum was isolated for examination of bovine rotavirus-specific IgG and E.coli antibodies. Specifically, all calves were bled 5ml from the jugular vein before feeding colostrum, left for 30 minutes, centrifuged at 3000r/min for 15 minutes, serum was separated, and specific Ig-G and E.coli antibodies against rotavirus were examined.
3) Product use and challenge vaccination
The test group calves were administered once at 20 g/head a vaccine (Ig-Guard Paste C) containing IgY produced according to the invention within 6 hours after birth and once again 24 hours later with the same amount. The vaccine was mixed with the formula at a dose of 10 g/head daily from 3 days of age until the end of the trial, and was given once in the morning and afternoon. The control group was not given vaccine. Blood is collected from calves aged 4 days in the experimental group and the control group, serum is separated, and the bovine rotavirus specific IgG and the Escherichia coli antibody are measured. After blood collection, the calves of the experimental group were orally administered with rotavirus and escherichia coli to infect them.
4) Examination of viral (rotavirus) IgG in serum
The virus IgG examination was performed in serum isolated according to the use of rotavirus ELISA antibody examination kit. The percentage (%) of inhibition of each test sample and positive serum was calculated based on the absorbance value given by a microplate reader (enzyme-labeled instrument), and the results are shown in Table 9.
TABLE 9
Remarks that the sample inhibition ratio%
And (4) analyzing results:
the inhibition rate is less than 20 percent, and the positive degree is 0
The inhibition rate is more than or equal to 20 percent and less than 40 percent, and the positive degree is +
The inhibition rate is more than or equal to 40 percent and less than 60 percent, and the positive degree is +
The inhibition rate is more than or equal to 60 percent and less than 80 percent, and the positive degree is +++
The inhibition rate is more than or equal to 80 percent, and the positive degree is ++++
As a result, as can be confirmed in table 9 above, in the case where the calf was inoculated with the vaccine containing the IgY produced by the production method of the present invention, the inhibition rate of rotavirus was confirmed to be 80% at maximum. These results suggest that IgG against rotavirus is produced in a large amount when a vaccine containing IgY produced by the production method of the present invention is administered.
5) Examination of bacterial (E.coli) IgG in serum
The antibody titer of the serum for measurement was recorded based on the degree of agglutination after mixing the 2-fold diluted serum with the already prepared agglutinative antigen solution. The results are shown in table 10.
Remarking: when the serum antibody titer is less than or equal to 1, the determination is negative, and when the serum antibody titer is greater than 1, the determination is positive.
As a result, as can be confirmed in table 10 above, it was confirmed that the serum antibody titer was greater than 1 when the calf was inoculated with the vaccine containing the IgY produced by the production method of the present invention. These results suggest that a large amount of IgG can be produced in escherichia coli when a vaccine containing IgY produced by the production method of the present invention is administered.
Claims (10)
1. A method for preparing yolk antibody for preventing or treating digestive system diseases of calf comprises the following steps:
stage 1, preparing antigen by using calf digestive system disease pathogenic bacteria or virus;
a 2 stage of inoculating the antigen of the 1 stage to an animal xenogeneic to a chicken to produce a xenogeneic antibody;
a 3 stage of binding the antigen of the 1 stage to the foreign antibody of the 2 stage to produce an antigen-foreign antibody complex;
a 4 stage of inoculating a mixture in which the antigen of the 1 stage and the complex of the 3 stage are mixed, to a laying chicken; and
and a 5 th stage of isolating yolk antibodies against pathogenic bacteria or viruses of digestive system diseases of calves from eggs, of the laying hens of the 4 th stage.
2. The method for producing a yolk antibody for the prevention or treatment of digestive system diseases in calves according to claim 1, wherein the yolk antibody is a yolk antibody,
the pathogenic bacteria of digestive system disease of calf at stage 1 are more than one pathogenic bacteria selected from Escherichia coli (E.coli), Salmonella (Salmonella), Clostridium (Clostridium) and Mycobacterium paratuberculosis (Mycobacterium paratu berculosis).
3. The method for producing a yolk antibody for the prevention or treatment of digestive system diseases in calves according to claim 1, wherein the yolk antibody is a yolk antibody,
the calf digestive system disease pathogenic Virus of stage 1 is more than one Virus selected from Rotavirus namely Rotavirus, coronavirus namely Corona Virus, Bovine viral diarrhea Virus namely Bovine viral dialrea Virus, Adenovirus namely Adenovirus, parvovirus namely Parbovirus and Enterovirus namely Enterovirus.
4. The method for producing a yolk antibody for the prevention or treatment of digestive system diseases in calves according to claim 1, wherein the yolk antibody is a yolk antibody,
in stage 2, the xenogeneic animal is a mouse or rabbit.
5. The method for producing a yolk antibody for the prevention or treatment of digestive system diseases in calves according to claim 1, wherein the yolk antibody is a yolk antibody,
in stage 4, the mixture further comprises an adjuvant, i.e., adjuvant.
6. The method for producing a yolk antibody for the prevention or treatment of digestive system diseases in calves according to claim 5, wherein the yolk antibody is a yolk antibody,
the mixing ratio of the mixture to an adjuvant, namely adjuvant, is 2-3: 7-9.
7. The method for producing a yolk antibody for the prevention or treatment of digestive system diseases in calves according to claim 1, wherein the yolk antibody is a yolk antibody,
in the 4 th stage, the inoculation is carried out 2 to 5 times at 0.1 to 3 ml.
8. A yolk antibody for preventing or treating digestive system diseases in calves, which is produced by the production method according to claim 1.
9. A vaccine composition for preventing or treating digestive system diseases in calves, comprising the egg yolk antibody according to claim 8 as an active ingredient.
10. A feed additive for preventing or treating digestive system diseases in calves, comprising the yolk antibody according to claim 8 as an active ingredient.
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| KR1020180167140A KR102047784B1 (en) | 2018-12-21 | 2018-12-21 | Manufacturing method of Immunoglobulin Y for preventing or treating calf digestive diseases, and Immunoglobulin Y thereby and the use thereof |
| KR10-2018-0167140 | 2018-12-21 |
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| CN118000298A (en) * | 2024-03-04 | 2024-05-10 | 江苏雅博动物健康科技有限责任公司 | Premixed feed composition for calves and preparation method and application thereof |
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| KR102260009B1 (en) * | 2019-11-18 | 2021-06-03 | (주)애드바이오텍 | Manufacturing method of immunoglobulin y for preventing or treating obesity through repression of cholesterol absorption |
| KR102200721B1 (en) * | 2019-11-18 | 2021-01-13 | (주)애드바이오텍 | Manufacturing method of immunoglobulin y for preventing or treating salmon rickettisia septicaemia |
| KR102219100B1 (en) * | 2019-11-18 | 2021-02-24 | (주)애드바이오텍 | Manufacturing method of immunoglobulin y against helicobacter pylori |
| KR20240086721A (en) * | 2022-11-22 | 2024-06-19 | (주)애드바이오텍 | Manufacturing method of single domain vhh antibody for preventing or treating calf digestive diseases |
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