阅读说明：本技术 针对虾早期死亡综合症和白斑病毒的抗体及其应用 (Antibody against early death syndrome and white spot virus of shrimp and application thereof ) 是由 郑洪杰 权赫世 金秀娟 梁松益 于 2018-09-21 设计创作，主要内容包括：本发明涉及针对虾早期死亡综合症和白斑病毒的抗体及其应用,更详细地涉及对产卵鸡接种针对虾早期死亡综合症和白斑病毒的抗原,从产卵鸡产下的蛋的卵黄中分离的抗体及其应用技术。根据本发明,对产卵鸡接种针对虾早期死亡综合症和白斑综合症的抗原而使其免疫化,从由经免疫化的产卵鸡获得的蛋的卵黄中分离抗体,从而能够作为防虾死亡用组合物、饲料或饲料添加剂而有用地使用。本发明的抗体具有如下特征,作为虾早期死亡综合症的病原体菌的弧菌和白斑病毒抗原的组成引起协同(上升)效果,因此与单独使用弧菌、白斑病毒的抗体相比显现更优异的抗体效价,由此针对虾早期死亡综合症及白斑病毒的预防或治疗效果优异,而且安全性优越,易于生产抗体。(The present invention relates to an antibody against early death syndrome of shrimp and white spot virus and its use, and more particularly, to an antibody isolated from the yolk of an egg laid by a laying hen by inoculating the laying hen with an antigen against early death syndrome of shrimp and white spot virus and its use. According to the present invention, the antigen against early shrimp death syndrome and white spot syndrome is inoculated to and immunized against the laying hens, and the antibody is isolated from the yolk of the egg obtained from the immunized laying hens, thereby being usefully used as a composition for preventing shrimp death, feed or feed additive. The antibody of the present invention has a characteristic that the composition of vibrio and leukoderma virus antigens, which are pathogenic bacteria of early death syndrome of shrimp, causes a synergistic (increasing) effect, and thus exhibits an excellent antibody titer as compared with the use of antibodies against vibrio and leukoderma virus alone, thereby having an excellent preventive or therapeutic effect against early death syndrome of shrimp and leukoderma virus, and is excellent in safety and easy to produce an antibody.)

1. A method for producing an antibody for preventing death of shrimp, comprising the steps of:

(a) a stage of immunizing laying hens by inoculating the laying hens with an antigen composed of Vibrio genus, which is a pathogen of early death syndrome of shrimps, and white spot syndrome recombinant proteins, which are white spot syndrome recombinant proteins, wherein WSSV is an abbreviation for white spot syndrome,

(b) a stage of allowing the immunized laying chicken to lay eggs, and

(c) a stage of separating antibodies from the yolk of the egg.

2. A method for producing an antibody for preventing death of shrimp, comprising the steps of:

(a) a stage of immunizing a laying chicken by inoculating the laying chicken with an antigen composed of Vibrio gene, which is a pathogenic bacterium of early death syndrome of shrimp, recombinant protein of white spot syndrome, which is a white spot syndrome, and recombinant protein of an epitope part of outer membrane protein of Vibrio, wherein WSSV is an abbreviation,

(b) a stage of allowing the immunized laying chicken to lay eggs, and

(c) a stage of separating antibodies from the yolk of the egg.

3. The method for producing an antibody for preventing shrimp death according to claim 1 or 2,

the Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum.

4. The method for producing an antibody for preventing shrimp death according to claim 3,

the Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio anguillarum, Vibrio anguillarum are mixed in a ratio of 0.5-1.5: 0.3-1.0.

5. The method for producing an antibody for preventing shrimp death according to claim 1 or 2,

the white spot virus recombinant protein is VP 28.

6. The method for producing an antibody for preventing shrimp death according to claim 2,

the recombinant proteins of the antigenic determinant site of the vibrio Outer Membrane Protein are OmpW, namely vibrio parahaemolyticus Outer Membrane Protein (Outer Membrane Protein) W, and OmpK, namely vibrio harveyi Outer Membrane Protein (Outer Membrane Protein) K.

7. An antibody for preventing shrimp death is obtained by inoculating an antigen composed of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and white spot syndrome virus recombinant protein to a laying hen, and separating the antigen from the yolk of an egg laid by the laying hen, wherein the white spot syndrome virus is abbreviated as WSSV.

8. A composition for preventing or treating early shrimp death syndrome, comprising an antibody isolated from the yolk of an egg laid by a laying hen after inoculating the laying hen with an antigen composed of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and white spot virus recombinant protein, wherein WSSV is an abbreviation for white spot syndrome virus.

9. A composition for preventing or treating white spot syndrome in shrimp, comprising an antibody isolated from the yolk of an egg laid by a laying hen after inoculating the laying hen with an antigen composed of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and white spot syndrome virus recombinant protein, wherein WSSV is an abbreviation for white spot syndrome virus.

10. A shrimp feed or feed additive comprising an antibody obtained by inoculating an antigen consisting of Vibrio parahaemolyticus, Vibrio harzii, Vibrio anguillarum and white spot syndrome virus recombinant protein to a laying hen and separating the antigen from the yolk of an egg laid by the laying hen, wherein the WSSV is an abbreviation for white spot syndrome virus.

11. The shrimp feed or feed additive of claim 10, wherein,

the shrimp feed or feed additive is used for preventing or treating early death syndrome and shrimp white spot syndrome of shrimps.

12. A disease prevention method for shrimps by treating shrimps with a composition for early death syndrome prevention or treatment comprising an antibody obtained by inoculating an antigen consisting of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and white spot virus recombinant protein to a laying hen and isolating the antigen from the yolk of an egg laid by the laying hen, wherein WSSV is an abbreviation of white spot syndrome virus.

13. A method for feeding shrimp, wherein a shrimp feed or feed additive containing an antibody, which is obtained by inoculating an antigen consisting of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and white spot virus recombinant protein, to a laying hen and separating the antibody from the egg yolk of the egg laid by the laying hen, is supplied to the shrimp, wherein WSSV is an abbreviation for white spot syndrome virus.

Technical Field

The present invention relates to an antibody against early death syndrome of shrimp and white spot virus and its use, and more particularly, to an antibody isolated from the yolk of an egg laid by a laying hen by inoculating the laying hen with an antigen against early death syndrome of shrimp and white spot virus and its use.

Background

In recent years, with the worldwide interest in improving dietary life for the prevention of adult diseases has proliferated, the demand for aquatic products has continuously increased and the yield of fish farming is proliferating. Therefore, it is the actual situation that the aquaculture industry is becoming the main economic means in many countries, and the occurrence of diseases in cultured fish seriously affects the economic development.

In farmed fish, vibrio and white spot virus are known as the main causes of diseases induced by shrimp. Vibrio (Vibrio) is a gram-negative bacterium having 1 flagella (polar hairs) at one end and 2 or more flagella (polar hairs) depending on the species and actively moves, and Vibrio cholerae (Vibrio colera) which causes cholera is a representative pathogenic bacterium. The causes of shrimp diseases include Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio harveyi (Vibrio harveyi) and Vibrio anguillarum (Vibrio anguillarum), which are called Vibrio enteritis, and shrimp die within 24 to 72 hours when infected with these bacteria.

White Spot Syndrome Virus (WSSV) has a form of an attachment similar to a tail, a shell (capsid) and an envelope (envelop) in the form of a rod (rod). When a shrimp is infected with white spot virus, white spots appear on the shell (carapace), outer limb (apendage) and epidermis (cuticle) of the shrimp, and the liver and pancreas (hepatopancreas) appear red, and the shrimp dies after 3 to 5 days from infection.

Therefore, egg yolk antibody (IgY) has been proposed as one of the means for preventing and treating shrimp diseases. Yolk antibody is a means of passive immunization utilizing the immune system of birds, and immune antibodies obtained by the hen through active immunization are transferred to yolk to transfer immune functions to the offspring. Various studies have demonstrated that the yolk antibody has prophylactic and therapeutic effects on diseases, and it has also been reported that the yolk antibody can be expected to have almost the same therapeutic effect as an antibiotic when used at a high concentration. Further, since it is derived from eggs as a complete food (complete food), it can be said that it is a very useful substance without any concern for safety.

Prior patent literature

Patent document

Korean registered patent No. 10-1242821.

Disclosure of Invention

Problems to be solved by the invention

The object of the present invention is to provide: an antibody isolated from the yolk of an egg obtained from an immunized laying hen after immunizing the laying hen with an antigen against early death syndrome and white spot syndrome of shrimp, and a composition for preventing shrimp death, feed or feed additive, a method for preventing diseases, and a method for feeding shrimp using the same.

Means for solving the problems

The present invention provides a method for producing an antibody for preventing death of shrimp, comprising the steps of: (a) a stage of immunizing a laying chicken by inoculating the laying chicken with an antigen composed of Vibrio (Vibrio gene) and white spot virus (WSSV) recombinant proteins, which are pathogenic bacteria of early death syndrome of shrimp; (b) a stage of allowing the immunized laying chicken to lay eggs; and (c) a stage of separating the antibodies from the yolk of the egg.

The present invention also provides a method for producing an antibody for preventing death of shrimp, comprising the steps of: (a) a step of immunizing a laying chicken by inoculating the antigen composed of Vibrio (Vibrio genus), white spot virus (WSSV) recombinant proteins, which are pathogenic bacteria of early death syndrome of shrimp, and recombinant proteins at an epitope site of outer membrane proteins of Vibrio, thereby immunizing the chicken; (b) a stage of allowing the immunized laying chicken to lay eggs; and (c) a stage of separating the antibodies from the yolk of the egg.

The Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum can be selected from Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum.

The Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum may be mixed in a ratio of 0.5-1.5: 0.3-1.0, preferably in a ratio of 0.7-1.3: 0.4-0.7, more preferably in a ratio of 1.0:1.0: 0.5.

The above-mentioned white spot virus recombinant protein may be VP 28.

The recombinant Protein of the epitope portion of the Vibrio Outer Membrane Protein may be OmpW (Outer Membrane Protein) W) or OmpK (Outer Membrane Protein) K).

In the present invention, OmpW and OmpK can be represented as V1W and VbK, respectively.

The present invention also provides an antibody for preventing shrimp death, which is obtained by inoculating an antigen comprising a Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio harveyi (Vibrio harveyi), Vibrio anguillarum (Vibrio anguillarum) and a white spot virus (WSSV) recombinant protein to a laying hen and isolating the antigen from the yolk of an egg laid by the laying hen.

The present invention also provides a composition for preventing or treating early shrimp death syndrome, which comprises an antibody obtained by inoculating an antigen comprising a recombinant protein of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and Vibrio anguillarum, and white spot virus (WSSV) to a laying hen and isolating the antigen from the yolk of an egg laid by the laying hen.

The present invention also provides a composition for preventing or treating white spot syndrome in shrimp, comprising an antibody obtained by inoculating an antigen comprising a recombinant protein of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and Vibrio anguillarum, and white spot virus (WSSV) to a laying hen and isolating the antigen from the yolk of an egg laid by the laying hen.

The present invention also provides a shrimp feed or feed additive comprising an antibody obtained by inoculating an antigen comprising a recombinant protein of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum and white spot virus (WSSV) to a laying hen and separating the antigen from the yolk of an egg laid by the laying hen.

The above shrimp feed or feed additive can be used for preventing or treating early death syndrome and shrimp white spot syndrome.

Further, the present invention provides a method for preventing diseases in shrimp, which comprises treating shrimp with a composition for preventing or treating early death syndrome in shrimp, the composition comprising an antibody obtained by inoculating a laying hen with an antigen comprising a recombinant protein of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, and white spot virus (WSSV), and separating the antigen from the yolk of an egg laid by the laying hen.

The present invention also provides a method for feeding shrimp, which comprises administering a shrimp feed or a feed additive comprising an antibody obtained by inoculating a oviposition chicken with an antigen comprising a recombinant protein of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, and white spot virus (WSSV), and isolating the antibody from the yolk of an egg laid by the oviposition chicken.

Effects of the invention

According to the present invention, the antigen against early shrimp death syndrome and white spot syndrome is inoculated to and immunized against the laying hens, and the antibody is isolated from the yolk of the egg obtained from the immunized laying hens, thereby being usefully used as a composition for preventing shrimp death, feed or feed additive.

In addition, the antibody of the present invention has a characteristic that the composition of vibrio and leukoderma virus antigens, which are pathogenic bacteria of early death syndrome of shrimp, causes a synergistic (increasing) effect, and thus exhibits an excellent antibody titer (potential) as compared with the use of antibodies of vibrio and leukoderma virus alone, thereby having an excellent effect of preventing or treating early death syndrome of shrimp and leukoderma virus, and moreover, the antibody is produced from a laying chicken, and thus is excellent in safety, and the antibody can be produced relatively easily.

Drawings

FIG. 1 shows the results of SDS-PAGE and Western blotting for confirming the expression of VP28, a recombinant protein of White Spot Syndrome Virus (WSSV), and the results of SDS-PAGE and Western blotting for detecting the expression of protein, before induction (induction) of Lane1 and after induction (induction) of Lane2 and protein labeling (marker) for detecting the expression of protein.

FIG. 2 is a schematic diagram of the plasmid pET-32a (+) OmpW.

FIG. 3 is a schematic diagram of pET-32a (+) OmpK plasmid.

FIG. 4 shows the results of confirming the expression of the recombinant protein OmpW (V1W) at the epitope site of the outer membrane protein of Vibrio parahaemolyticus by SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting results, Lane1: before induction (induction) of pET-32a (+) original vector _ BL21(DE3) with IPTG, Lane2: after induction (induction) of pET-32a (+) original vector _ BL21(DE3) with 1.0mM IPTG, Lane3: after induction of OmpW + pET-32a (+) vector _ BL21(DE3) with IPTG, Lane4: after induction of OmpW + pET-32a (+) vector _ OmpW _ BL21(DE3) with 1.0mM G, M: protein marker (marker).

FIG. 5 shows the results of SDS-PAGE and Western blotting for confirming the expression of the recombinant protein OmpK (VbK) at the epitope site of the outer membrane protein of Vibrio harveyi, wherein A represents the result of SDS-PAGE and B represents the result of Western blotting, and M represents the protein marker (marker) after Lane1, pET-32a (+) vector _ BL21(DE3) was induced with 1.0mM IPTG and Lane2, pET-32a (+) vector _ OmpW _ BL21(DE3) was induced with 1.0mM IPTG.

FIG. 6 shows the results of confirming the yolk antibody titer produced against V1 (Vibrio parahaemolyticus) after the respective groups of vaccination. The horizontal axis represents the number of inoculation cycles (for example, 1-1 is 1 cycle and 1 week), and the vertical axis represents the yolk antibody titer.

FIG. 7 shows the results of confirming the yolk antibody titer produced against Vb (Vibrio harveyi) after each group of vaccination.

Fig. 8 shows the results of confirming the yolk antibody titer produced against Va (vibrio anguillarum) after each group vaccination.

Fig. 9 shows the results of confirming the yolk antibody titer produced against WSSV VP28 (white spot syndrome virus recombinant protein) after each group vaccination.

FIG. 10 shows the results of confirmation of IgY by SDS-PAGE and Western blotting.

FIG. 11 shows the results of confirming the binding affinity for antigen by SDS-PAGE and Western blotting, wherein the antigen was labeled with Vibrio anguillarum (Vibrio anguillarum), Vibrio harveyi (Vibrio harveyi), Vibrio parahaemolyticus (Vibrio parahaemolyticus) 4 WSSV VP28, and M protein.

FIG. 12 shows the results of confirming the growth inhibitory effect against V1 (Vibrio parahaemolyticus) after the respective groups of vaccination. The horizontal axis represents time after inoculation, and the vertical axis represents o.d. value.

FIG. 13 shows the results of confirming the growth inhibitory effect against Vb (Vibrio harveyi) after each group of vaccination.

Fig. 14 shows the results of confirming the growth inhibitory effect against Va (vibrio anguillarum) after each group vaccination.

Detailed Description

The present invention will be described in more detail below with reference to examples. The objects, features and advantages of the present invention can be easily understood by the following examples. The present invention is not limited to the embodiments described herein, and can be embodied in other forms. The embodiments described herein are provided to fully convey the concept of the present invention to those skilled in the art to which the present invention pertains. The following examples are therefore not intended to limit the invention.