KR102049976B1 - Antibody for shrimp’s early mortality syndrome and white spot syndrome virus, and applications thereof - Google Patents

Abstract

The present invention relates to an antibody against a shrimp early death syndrome and a white spot syndrome virus (WSSV), and an application thereof, and more particularly, to an antibody, which is isolated from an egg yolk of an egg laid by a layer chicken by inoculating an antigen against a shrimp early death syndrome and a WSSV to the layer chicken. According to the present invention, an antigen against a shrimp early death syndrome and a WSSV is inoculated to a layer chicken for immunization, and an antibody is isolated from an egg yolk of an egg obtained by an immunized layer chicken, and thus, is useful as a composition, a feed, or a feed additive for preventing shrimp death. In addition, the antibody of the present invention exhibits more excellent antibody titer than that of an antibody which uses vibrio genus and WSSV alone since formation of the antibodies of the vibrio genus and the WSSV which are a causative pathogen of a shrimp early death syndrome, causes a synergistic effect. Therefore, an effect for preventing or treating a shrimp early death syndrome and the WSSV is excellent, the stability is excellent since the antibody is produced from the layer chicken, and the antibody can be relatively easily produced.

Description

ANTIBODY FOR SHRIMP'S EARLY MORTALITY SYNDROME AND WHITE SPOT SYNDROME VIRUS, AND APPLICATIONS THEREOF

The present invention relates to antibodies against shrimp early mortality syndrome and white spot virus and its application, and more particularly, to inoculate antigens against shrimp early mortality syndrome and white spot virus, and to isolate the eggs from egg yolks laid. It relates to an antibody and its application technology.

Recently, as the interest in improving the diet for the prevention of geriatric diseases has increased rapidly around the world, the demand for seafood is continuously increasing and the production of fish farming is increasing rapidly. Therefore, aquaculture has become a major economic tool in many countries, and disease outbreaks in farmed fish have a great impact on economic development.

Among the farmed fish, vibrio and white spot viruses are known as the main causes of disease in shrimp. Vibrio genus is a Gram-negative bacillus, with one or more hair follicles on one end and two or more hairs depending on the species, and is a representative pathogen. Vibrio cholera ( Vibrio genus) causes cholera. colera ) The causes of shrimp Vibrio illness called Vibrio para itty Syracuse to Mora (Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi ) and Vibrio anguillarum , which are killed within 24 to 72 hours when shrimp are infected with these bacteria.

White spot syndrome virus (WSSV) has a tail-like attachment and has rod-shaped capsids and envelopes. When shrimp are infected with the white spot virus, white spots appear on the carapace, appendage and cuticle of the shrimp, and the hepatopancreas becomes red, and 3 to 5 days after infection It will die.

Therefore, yolk antibody (IgY) is emerging as one of the measures to prevent and treat shrimp diseases. Egg yolk antibodies are a form of passive immunization using the bird's immune system. The immune antibodies obtained by active chickens from mother chickens are transferred to egg yolk and the immune function is transmitted to the offspring. Egg yolk has already been proved by various studies to prevent and cure the disease, and when used in high concentrations, it is reported that it can be expected to have almost the same therapeutic effect as antibiotics. In addition, since it is derived from eggs, which are complete foods, it can be said to be a very useful substance without any safety concerns.

Republic of Korea Patent No. 10-1242821

An object of the present invention is to immunize the laying hens with antigens against shrimp early mortality syndrome and white spot syndrome, and the antibody isolated from egg yolk of the egg obtained from the immunized laying hens and the composition for preventing shrimp mortality using the same, feed or feed additives, To provide disease prevention and shrimp breeding methods.

The present invention comprises: (a) immunizing a laying hen by inoculating an laying hen with an antigen consisting of Vibrio genus and white spot syndrome virus (WSSV) recombinant protein, which are the causative agents of early shrimp mortality syndrome; (b) laying eggs from the immunized laying hens; And (c) separating the antibody from the egg yolk of the laid eggs, providing an antibody for preventing shrimp mortality.

In another aspect, the present invention, (a) shrimp early pathogen of Vibrio of mortality syndrome bacteria (Vibrio genus), white spot virus (white spot syndrome virus, WSSV) recombinant proteins and vibrio antigen consisting of the recombinant proteins of the antigenic determinant sites of bacterial outer membrane protein Immunizing the laying hens by inoculating the laying hens; (b) laying eggs from the immunized laying hens; And (c) separating the antibody from the egg yolk of the laid eggs, providing an antibody for preventing shrimp mortality.

The Vibrio bacterium is Vibrio parahamamoraiticus ( Vibrio parahaemolyticus ), Vibrio harveyi and Vibrio anguillarum .

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

The white spot virus recombinant protein may be VP28.

Recombinant proteins of the epitope region of the Vibrio bacteria outer membrane protein may be OmpW (Vibrio para-Haemolyticus outer membrane protein (W) and OmpK (Vibrio Havey outer membrane protein (Outer Membrane Protein K)).

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

In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Provided are antigens consisting of anguillarum ) and white spot syndrome virus (WSSV) recombinant protein inoculated into a laying hen, and there is provided an antibody for preventing shrimp mortality isolated from the egg yolk of the laying hen.

In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Provided is a composition for preventing or treating shrimp early mortality syndrome comprising inoculating an antigen consisting of anguillarum ) and white spot syndrome virus (WSSV) recombinant protein into a laying hen, and then separating the egg from the egg yolk of the laying hen. do.

In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Provided is a composition for the prevention or treatment of shrimp white spot syndrome, comprising an antibody isolated from egg yolk of eggs laid by the laying hen after inoculating an antigen consisting of anguillarum ) and white spot syndrome virus (WSSV) recombinant protein into the laying hen. do.

In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio An antigen consisting of anguillarum ) and a white spot syndrome virus (WSSV) recombinant protein is inoculated into a laying hen, and there is provided a shrimp feed or feed additive comprising an antibody isolated from egg yolk of the laying hen.

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

In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio After inoculating an egg consisting of anguillarum ) and a white spot syndrome virus (WSSV) recombinant protein into a laying hen, a composition for preventing or treating early mortality syndrome comprising a shrimp isolated from the egg yolk of the laying hen Provides a method for preventing shrimp disease in processing.

In addition, the present invention, Vibrio parahaemoriticus ( Vibrio parahaemolyticus), Vibrio Harvey (Vibrio harveyi), Vibrio Anguilla Anguilla room (Vibrio Shrimp which inoculate an egg consisting of anguillarum and white spot syndrome virus (WSSV) recombinant protein into a laying hen, and then feed it with a shrimp feed or feed additive containing an antibody isolated from the egg yolk of the laying hen. It provides a breeding method of.

According to the present invention, by inoculating immunizing eggs with antigens against shrimp early mortality syndrome and white spot syndrome, and separating the antibody from egg yolk of eggs obtained from the immunized laying hens, it can be usefully used as a composition for preventing shrimp mortality, feed or feed additives. have.

In addition, the antibody according to the present invention exhibits a synergistic (synergistic) effect of the composition of Vibrio bacteria and white spot virus antigens, which causes shrimp early mortality syndrome, and shows better antibody titers than antibodies using Vibrio or white spot virus alone. Accordingly, it is excellent in preventing or treating shrimp early mortality syndrome and white spot syndrome, and is excellent in stability because the antibody is produced from the laying hen, and the antibody can be produced relatively easily.

1 is a result of confirming the expression of recombinant protein VP28 of white spot syndrome virus (WSSV) through SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting results, Lane 1: IPTG induction ( before induction, Lane 2: after IPTG induction, M: protein marker.
2 is a schematic diagram of the pET-32a (+) OmpW plasmid.
3 is a schematic diagram of the pET-32a (+) OmpK plasmid.
Figure 4 shows the expression of recombinant protein OmpW (V1W) of the epitope region of the Vibrio para-Haemolyticus outer membrane protein by SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting Results: Lane 1: before induction of pET-32a (+) original vector_BL21 (DE3) with IPTG, Lane 2: after induction of pET-32a (+) original vector_BL21 (DE3) with 1.0 mM IPTG Lane 3: Before inducing OmpW + pET-32a (+) vector_BL21 (DE3) to IPTG, Lane 4: Inducing OmpW + pET-32a (+) vector_OmpW_BL21 (DE3) to 1.0 mM IPTG, M : Protein marker.
Figure 5 shows the expression of recombinant protein OmpK (VbK) of the epitope region of the Vibrio Habei outer membrane protein by SDS-PAGE and Western blotting, A: SDS-PAGE results, B: Western blotting results, Lane 1 : Before inducing pET-32a (+) vector_BL21 (DE3) with 1.0 mM IPTG, Lane 2: After inducing pET-32a (+) vector_OmpW_BL21 (DE3) with 1.0 mM IPTG, M: Protein marker )to be.
Figure 6 shows the results of confirming the yolk antibody titers generated for V1 (Vibrio parahaemoriaticus) after vaccination of each group. The horizontal axis shows the inoculation week (eg 1-1 is the first week), and the vertical axis shows the yolk antibody titer.
Figure 7 shows the results of confirming the yolk antibody titer generated for Vb (Vibrio Habei) after vaccination of each group.
Figure 8 is the result of confirming the yolk antibody titers generated for Va (Vibrio Anguilla Room) after vaccination of each group.
Figure 9 shows the result of confirming the yolk antibody titer generated for WSSV VP28 (white spot syndrome virus recombinant protein) after vaccination of each group.
10 shows the results of confirming IgY through SDS-PAGE and western blotting.
Figure 11 shows the binding affinity to the antigen through SDS-PAGE and Western blotting, 1: Vibrio anguillarum, 2: Vibrio harveyi, 3: Vibrio parahaemoriti Vibrio parahaemolyticus, 4: WSSV VP28, M: protein marker.
12 is a result of confirming the growth inhibitory effect on V1 (Vibrio parahaemoriaticus) after vaccination of each group. The horizontal axis represents time after inoculation and the vertical axis represents OD value.
Figure 13 shows the results of confirming the growth inhibitory effect on Vb (Vibrio Habei) after each group vaccination.
Figure 14 shows the results of confirming the growth inhibitory effect on Va (Vibrio Anguilla Room) after vaccination of each group.

Hereinafter, the present invention will be described in more detail with reference to Examples. The objects, features and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced herein are provided to sufficiently convey the spirit of the present invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

Experimental Example 1: Preparation of Anti-Shrimp Antibodies

1-1. Early mortality syndrome (early mortality syndrome, EMS) antigen production

Vibrio para to the antigen preparation to Mora ET kusu (Vibrio parahaemolyticus) KCTC 2471, Harvey V. (Vibrio harveyi) KCTC 12724 and Vibrio Anguilla Anguilla room (Vibrio anguillarum ) KCTC 2711BP was distributed and incubated for 20 hours in a shaking incubator at 37 ° C. at 120 rpm using tryptic soy broth (TSB) added with 3% NaCl. The culture broth was centrifuged at 6,000 rpm (10 min, 4 ° C), pellets were collected, washed twice with PBS, and 50 ml of suspension was prepared. A spectrophotometer was used to fix the absorbance value at 0.3 at OD 600 nm. 37% formaldehyde (formaldehyde) relative to the total volume was added and inactivated at room temperature for 24 hours to use as an antigen for immunity.

1-2. White spot virus recombinant protein production

In order to use VP28, a recombinant protein of white spot virus, as an antigen, LB (lysogeny broth) medium was prepared, sterilized, and prepared by adding ampicillin at a concentration of 50 ug / ml. One day ago, 1% of LB medium was prepared and inoculated with recombinant E. coli colonies expressing VP28 protein (GenBank: DQ007315.1) of white spot virus cultured by inoculation-staining on an LB agar plate. E. coli BL21 (DE3) was used to produce VP28 recombinant protein, which is the capsid protein of White Spot Syndrome Virus (WSSV). An expression vector was prepared by ligation of the pET30a (+) vector with the T7 promoter and VP28 gene, which was used for transformation of E. coli. Escherichia coli was plated on Luria-Bertani agar plate to which ampicillin (50 μg / ml) was added to secure transformed colonies, which were inoculated into 15 ml of LB liquid medium to which ampicillin was added. Shake incubation for 16 hours at ℃. 15 ml of the culture medium was passaged in 1.5 L of fresh LB liquid medium to which ampicillin was added, and optical density (O.D) was measured at a wavelength of 600 nm using a spectrophotometer. Gene expression was induced by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final 1.0 mM concentration when the optical density (O.D) was 0.6.

Cells were harvested by further incubation for 6 hours after IPTG addition, followed by centrifugation at 6,000 rpm for 30 minutes. Cells overexpressed by IPTG were resuspended in cell lysis buffer (50 mM Tris-HCI (pH 8.0), 1 mM EDTA, 100 mM NaCl) and 1 mg / ml of lysozyme and 10 ug. DNase of / ml was added and stirred for 1 hour in a shake incubator (200 rpm, 37 ℃). After 1 hour, the cells were crushed using a sonicator and centrifuged (6,000 rpm, 30 minutes) to collect the pellets. The process was repeated once using lysozyme. Pellets obtained as lysozyme were further crushed with a sonicator using urea buffer (100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 2 M Urea, 2% Triton X-100) Proteins were collected, urea was removed by dialysis using PBS (phosphate buffer saline), and the collected proteins were finally identified on SDS-PAGE. SDS-PAGE used a 10% SDS-PAGE gel.

Protein expression was further confirmed by western blotting. Western blotting was performed by a known method, and the first antibody used in the experiment was Anti-His-probe-IgG diluted 1: 1,000, and the second antibody was Anti-Mouse diluted 1: 5,000. -IgG-HRP was used.

As a result of confirming the protein expression, overexpression of the protein was observed at 12 kda, and the protein expression of the 6x-His attached to the recombinant protein target by Western blotting was confirmed. (FIG. 1).

1-3. Preparation of Recombinant Protein of Vibrio Outer Membrane

Among the outer membrane proteins for Vibrio para-Haamoraiticus and Vibrio Habei, the main antigens of early mortality syndrome, OmpW and OmpK, the recombinant proteins of epitope proteins, which are antigenic determinants, were YUAN Ye, WANG Xiuli, GUO Sheping, QIU Xuemei et al., Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus , Chinese Journal of Oceanology and Limnology, Vol. 29, No. 5, P. 952-957, 2011 and Li Ningqiu, Bai Junjie, Wu Shuqin, Fu Xiaozhe, Lao Haihua, Ye Xing, Shi Cunbin et al., An outer membrane protein, OmpK , is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper ( Epinephelus coioides ) , Fish & Shellfish Immunology, 25, 829-833, 2008.

In order to express OmpW and OmpK recombinant proteins, the cloned TA-Clone was reacted with restriction enzymes BamHI and XhoI to obtain only the inserted gene, and then combined with Novagen's pET32a (+) vector (# 69015-3CN). Recombinant clones were produced (FIG. 2, FIG. 3). The pET32a (+) vector has a T7 promoter gene at the N-terminus, which can overexpress the protein corresponding to the inserted external gene, and a his-tag whose six histidine residues are repeated after the T7 promoter gene. Present and can be used to detect recombinant proteins. The recombinant clones produced were transformed into BL21 (DE3) Competent Cells (# 69450-3CN, Novagen) to obtain a single colony. Single colonies were inoculated into 3 ml of LB broth and shaken at 37 ° C. for 16 hours. The culture was reinoculated into 50 ml of LB broth and shaken at 37 ° C. The absorbance (A600) was measured every hour using a spectrophotometer and cultured until it reached 0.5 to 0.6. In order to use the T7 promoter of the expression system, isopropyl-β-D-thio-galactopyranoside (IPTG; Sigma, USA) was added to a final concentration of 1 mM and incubated. Since 10 ml of the culture was obtained, only cells were obtained by centrifugation at 12000 rpm and 10 min. The obtained cells were resuspended with 1 ml of lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl), and then reacted at 25 ° C. for 30 minutes by adding 100 μg / ml lysozyme. . Then, the solution was dissolved using a sonicator. Next, the soluble part and the insoluble part were separated by centrifugation at 18000 rpm and 30 min, and the insoluble part was confirmed whether the protein was expressed through SDS-PAGE (FIGS. 4 and 5).

In order to amplify the gene of the recombinant protein OmpW (Outer membrane protein W) at the epitope region of the Vibrio para-Haemolyticus outer membrane protein, the primer (see strain zj2003; GenBank accession No: DQ425109.1) was referred to. primers) were synthesized, and showed a 99% homology when compared with the recombinant clones made from them. Therefore, the BL21 (DE3) strain transformed with the recombinant gene pET-32a OmpW was incubated for 4 hours by treatment with 1.0 mM IPTG, which was confirmed to be suitable for OmpW recombinant protein production (FIG. 4).

In addition, primers were prepared by referring to OmpK (GenBank accession No .: AY332563.1) registered in GenBank to amplify the gene of the recombinant protein OmpK (Outer membrane protein K) at the epitope region of the Vibrio Havey outer membrane protein. By amplification to confirm the base sequence. Compared with the nucleotide sequence referenced by GenBank, the sequence homology was 98%. When recombinant BL21 (DE3) strain was transformed using recombinant clone pET-32a OmpK, 4 hours after treatment with 1.0 mM IPTG As a result of incubation, it was confirmed that OmpK recombinant protein can be efficiently produced (FIG. 5).

1-4. Manufacture of Vaccine for Laying Hens for Production of Egg Yolk Antibody (IgY)

Vaccine containers, rubber caps, PBS (1 ×), sieves, forceps, syringes, 50 ml tubes, 20 ul pipettes, 20 ul tips, plastic beakers (500 mL, 1000 mL) were prepared for laying hens. The vaccine mixed with the antigen prepared according to 1-1 to Experimental Example 1-3 was prepared as follows.

Each adjuvant oil was added to a water bath warmed at 37 ± 1 ° C. and warmed for 15 to 20 minutes. The homogenizer was sterilized by adding 0.1% of 37% formaldehyde solution to sterile distilled water, removing water, and then dried by spraying 70% ethanol. Antigens prepared according to refrigerated Experimental Example 1-1 to Experimental Example 1-3 were taken out at room temperature, and mixed antigens were prepared according to the configuration of Table 1 below. The mixed antigen was filtered aseptically on a sieve sprayed with 70% ethanol. At antigen mixing, sterile PBS was added when the final volume of antigen was insufficient. The mixed antigen and the adjuvant oil were mixed, and a homogenizer was turned for 25 minutes to prepare a vaccine for laying hens. In the antigen composition included in the vaccine of Table 1, the vaccine consisting of only the white spot virus recombinant protein is Comparative Example 1, the vaccine consisting only of Vibrio bacteria, the causative agent of shrimp early mortality syndrome, is compared with Comparative Example 2, Vibrio bacteria mixed in the same ratio. The vaccine consisting of a mixed antigen of the white spot virus recombinant protein was prepared in Example 1, in a ratio of 1: 1: 0.5: 1: 0.5: 0.5 of the epitope region of the Vibrio bacteria, the white spot virus recombinant protein and the Vibrio bacteria outer membrane protein. A vaccine consisting of a mixed antigen of recombinant proteins OmpW (V1W) and OmpK (VbK) was designated as Example 2.

Group Antigen Composition in Vaccines Comparative Example 1 WSSV VP28 Comparative Example 2 V1, Vb, Va, VPE1 (1: 1: 1: 1) Example 1 V1, Vb, Va + WSSV VP28 (1: 1: 1: 1: 1) Example 2 V1, Vb, Va + WSSV VP28 + V1W + VbK (1: 1: 0.5: 1: 0.5: 0.5)

V1: Vibrio parahaemolyticus , Vb: Vibrio harveyi , Va: Vibrio anguillarum , WSSV: White Spot Syndrome Virus, V1W: Vibrio parahaemolyticus Outer Membrane Protein's recombinant protein (OmpW), VbK: Vibrio harveyi Outer Membrane Protein's recombinant protein (OmpK)

1-5. Laying Hens

The vaccine prepared according to Experimental Example 1-4 was inoculated into the chest muscle by 1 ml in a 22-week-old Hyline-brown family laying hen.

1-6. Egg yolk sample collection

The yolk sample began with the collection of eggs laid by the immunized laying hens daily. Collected eggs were collected by date and collected at 7-day intervals. In order to collect the potency sample for each parking lot, five egg yolks were randomly collected to separate egg yolks, and sterile DW (deionized water) equivalent to 10 times the weight was mixed and stored in a 50 ml tube for 24 hours. It was. After 24 hours, the mixture was dissolved in a hot water at 37 ° C., centrifuged at 3,500 rpm at 4 ° C. for 30 minutes, and the supernatant was taken and stored as a sample for potency analysis.

Experimental Example 2: Measurement of yolk antibody titer

2-1. OMP separation of EMS antigens

After cultivation of Vibrio bacteria, the antigen of early mortality syndrome (EMS), transfer the bacterial culture to a centrifuge tube, centrifuge at 9,000 rpm for 30 minutes at 4 ° C, discard the supernatant, wash the centrifuge tube twice with PBS, and wash 10 mM HEPES. The cells were suspended in 500 ml of buffer (GibcoBRL, USA), centrifuged again and washed once. After centrifugation, the supernatant was discarded and the pellet was suspended in 300 ml of 10 mM HEPES buffer, and then 30 ml aliquots were sonicated to dissolve the antigen. The dissolved supernatant was centrifuged at 4 ° C. at 8,000 rpm for 30 minutes to separate the supernatant, and the pellet was discarded. 1% N-Lauroly sarcosine (SIGMA USA) was added to the separated supernatant, and then allowed to stand at room temperature for 10 minutes and centrifuged at 15,000 rpm for 50 minutes at 4 ° C. The pellet was resuspended in 50 ml of 10 mM HEPES buffer and centrifuged at 15,000 rpm for 50 minutes to discard the supernatant and remove the detergent. After centrifugation the supernatant was discarded and the pellet (OMP) was suspended in 25 ml of 10 mM HEPES buffer. The pellet was suspended and sonicated, filtered to 0.45 um, and formalin and thimerosal were added. Proteins were quantified and tested by ELISA using standard serum to confirm the antigenicity of the OMP antigen.

2-2. Confirmation of Yolk Antibody Titer by ELISA (enzyme-linked immunosorbent assay)

The titer of specific yolk antibody in egg yolk was measured by applying the Indirect ELISA method. First, dilute in a carbonate coating buffer at a predetermined concentration for each antigen, and coat 100 ul of each well on a 96 well polystyrene plate to overnight at 4 ° C., It was left at 37 ° C. for 1 hour. After washing with PBS-T (phosphate buffer saline, 0.05% Tween 20, pH 7.4), blocking with PBS buffer containing 2% BSA (bovine serum albumin) at 37 ° C. for 1 hour. Washed. Negative control, positive control and egg yolk samples were diluted twice, and 100 μl per well was added and allowed to stand at 37 ° C. for 1 hour. After 1 hour, washed three times, and the secondary antibody (anti-chicken: Sigma, USA) diluted in an appropriate amount in PBS and 100 ㎕ divided into each well and reacted for 1 hour at 37 ℃. After washing three times, TMB (3,3 ', 5,5'-Tetramethylbenzidine) solution was dispensed into each well for 100 ul with a substrate and reacted for about 10 minutes at room temperature. The reaction was stopped by dispensing 100 ul of 1.6 NH ₂ SO _{4, and} the absorbance of each well was measured with an ELISA reader at a wavelength of 450 nm. The average value of the negative sample was doubled in the measured result, and the titer of the analyzed data was represented by substituting the measured value of the processed yolk.

As a result, as shown in FIGS. 6 to 9, IgY titers of three Vibrio strains and the white spot syndrome virus recombinant protein (WSSV VP28) were confirmed for each group. In Comparative Example 1, antibody titers were not formed for other antigens except for the WSSV VP28 antigen. In Comparative Example 2, except for WSSV VP28 antigen, the titer gradually increased by parking for each antigen, and the titer after the third inoculation increased to 6,400. In the case of Example 1, the titer was increased stably according to the inoculation parking for the target antigen, but the threshold value of the titer increase was not higher than 6,400 on average. In the case of Example 2, the titer was increased by parking, and the average titer was increased to 12,800 or more for all antigens, and the period of maintaining the elevated titer was also shown to be longer than other groups. The results show that the titer of the antigen according to Example 2 is superior to that of Comparative Example 1 and Comparative Example 2 containing Vibrio bacteria antigen and white spot virus recombinant protein antigen alone.

Experimental Example 3: Isolation of Egg Yolk Antibodies Treated with Ammonium Sulfate

In order to purely separate the produced specific yolk antibody protein, separation and purification were performed using ammonium sulfate treatment. Eggs of high titer were collected, egg whites and strips were removed using an egg yolk separator, egg yolk was removed with Whatman No. 1 filter paper, and only egg yolks were separated in a beaker. The amount of separated egg yolk was measured, and the primary distilled water was added to 4 times the amount by weight, stirred for 1 hour, and then adjusted to pH 5 using 1 M HCl. This yolk solution was stored for one day in a -70 ° C cryogenic refrigerator. The yolk solution was taken out at room temperature, dissolved for about one day, and centrifuged (9,000 rpm, 4 ° C.) for 10 minutes to obtain a supernatant, which was prepared by filtration on filter paper using a vacuum pump. The volume of the filtered supernatant was measured, and ammonium sulfate was added little by little under ice conditions to sufficiently dissolve. The ammonium sulfate treated solution was left overnight at 4 ° C., then centrifuged (9,000 rpm, 4 ° C.) for 30 minutes to collect pellets and resuspended in 1 × PBS buffer to collect samples. The supernatant was measured by volume and again added ammonium sulphate to collect the pellet and the supernatant and confirmed the purity of the isolated purified yolk antibody through SDS-PAGE and Western blotting. SDS-PAGE and western blotting were performed under the same conditions as in Experimental Example 1-2.

As a result, the heavy chain (70kda) and light chain (21kda) portions of the IgY antibody are well represented and confirmed by Western blotting, it was confirmed that the yolk antibody protein (Fig. 10).

Experimental Example 4: Confirming the binding affinity to the antigen

To confirm the binding affinity of the produced egg yolk antibody to the antigen, the antigen was suspended in PBS in a certain amount, 5 × sample dye was added, followed by electrophoresis, and transfer to the membrane. After blocking with 5% skim milk, it was washed with PBS-T and treated with IgY antibody at a concentration of 2 ug / ml at 4 ° C. and washed with PBS-T. After diluting with 1: 20,000 with Anti-Chicken-IgG-HRP, the results were confirmed using a horseradish peroxidase (HRP) substrate.

As a result, it was confirmed that the yolk antibody binds to the specific portion of each antigen using the yolk antibody (Fig. 11).

Experimental Example 5: Confirmation of growth inhibition for EMS antigen

When yolk antibodies are treated with EMS antigens, the same treatment with 1 × 10 ⁵ cfu / ml antigen bacteria to 10 mg / ml yolk antibodies is performed to determine whether they exhibit growth inhibition effects. Absorbance was measured at OD 600 nm until 9, 12, 24, 36 hours, and the effect of the composition of the antigen was confirmed. The experiment was conducted by designating that the control group was nothing treated with EMS antigen.

As a result of the experiment, in the case of Comparative Example 1, there was no antigen for EMS, and thus the growth curve appeared in the same manner as the control. In Comparative Example 2, each of the antigens was cultured for 3 to 9 hours after incubation. Although the growth of the antigen was slightly reduced, the growth curve appeared in the same pattern as the control. In the case of Example 1, it was shown to have the effect of inhibiting the growth of antigen bacteria up to 3-9 hours. In Example 2, the growth curve between 3-9 hours for all antigens showed a low growth curve with a lower OD value than all other experimental groups, and the OD value up to the last 36 hours was also lower than that of the other groups. As shown in the above antibody titer experiments, it is shown that high antibody titers are maintained to improve the binding ability to the antigen (FIGS. 12 to 14).

Experimental Example 6: Confirmation of the effect of yolk antibody on white spot virus

To confirm the effect of yolk antibody on white spot virus (WSSV), challenge experiments were conducted. Forty days old white larvae larvae were fed IgY for 14 days in filtered seawater (salt 30 ‰) at 25 ° C. WSSV was infected by the oral route of shrimp. In preliminary experiments, shrimp mortality rates ranged from 50-70% on day 4, so to control this, one shrimp infected with WSSV was fed about 10% of body weight per group. Accumulated mortality was recorded daily over 96 hours, and the results were expressed as percent mortality. In addition, the experimental group was assigned to the normal control group, shrimp group infected with WSSV not infected with WSSV, and the experimental group was designated as a positive control group.

As a result, in the case of Comparative Example 1, the sole antigen composition test group against WSSV, the mortality was reduced by about 11% compared to the positive control. For Comparative Example 2 without the antigen against WSSV, mortality was found to be 68.75%. In Examples 1 and 2, which are complex antigens, mortality was reduced by 15 to 28.75% compared to the positive control. Among the antigen groups, Example 2 showed the lowest mortality in all experimental groups with a mortality of 41.25%, confirming that the antigen combination of Example 2 was most suitable as a vaccine for preventing shrimp mortality (Table 2).

IgY Effect on WSSV (Aggression) Experimental group Number of experiments dosage 24h
After our population 48h
After our population 72h
After our population 96h
After our population Total mortality Mortality (%) normal
Control One - 0 0 0 One One 1.25% 2 0 0 0 0 0 positivity
Control One 19 g 3 One 13 14 31 70.00% 2 0 2 13 10 25 Comparative Example 1 One 19 g One 0 9 12 22 58.75% 2 One 3 10 11 25 Comparative Example 2 One 19 g 2 One 11 14 28 68.75% 2 One 3 12 11 27 Example 1 One 19 g One 4 5 11 21 55% 2 One 3 7 12 23 Example 2 One 19 g One 2 8 8 19 41.25% 2 0 3 5 6 14

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

delete (a) Mixtures of Vibrio genus, white spot syndrome virus (WSSV) recombinant protein, and Vibrio bacterial outer membrane protein, which are the causative agents of early mortality of shrimp, in a ratio of 2.5: 1: 1 Inoculating the laying hens with the antigen to immunize the hens;
(b) laying eggs from the immunized laying hens; And
(C) relates to a method for producing egg yolk antibody for preventing shrimp mortality, comprising the step of separating the antibody from the egg yolk of the laid egg,
Vibrio bacteria in step (a) are Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum, with a ratio of 0.7-1.3: 0.7-1.3: 0.4-0.7 Mixed, the white spot virus recombinant protein in step (a) is VP28, and in step (a) the recombinant protein of the epitope site of Vibrio fungal envelope protein is derived from Vibrio parahaemoriticus derived envelope membrane OmpW and Vibrio Havey The outer membrane protein OmpK, characterized in that the egg yolk antibody for preventing shrimp mortality.
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