KR101414009B1 - Method for producing oral vaccine against fish viral nervous necrosis using transgenic plant and the plant thereof - Google Patents

Abstract

The present invention relates to a vaccine composition for preventing fish viral neurotoxicity comprising a transgenic plant or an extract thereof containing a gene encoding a coat protein derived from Nodaca virus, a vaccine composition for preventing fish viral neurotoxicity including the vaccine composition The present invention relates to a method for preventing fish viral neurotoxicity comprising oral administration of a feed additive and the vaccine composition or the feed additive to fish, and when the vaccine of the present invention is used in the fish culture industry, , Which is one of the species of fishes, can significantly contribute to productivity improvement by lowering the incidence of fatal diseases, and also growth of feed and disease control related industries can be expected.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing an oral vaccine for preventing viral < RTI ID = 0.0 > neuro < / RTI > necrosis using a transgenic plant,

The present invention relates to a method for producing an oral vaccine for the prevention of fish viral neurotoxicity using a transgenic plant and a plant, and more particularly, to a method for producing a plant viral endotoxin A method for producing a transformant plant comprising the steps of transforming a plant with the vector and expressing a nodavirus-derived coat protein, a method for producing a transformant plant produced by the method, a method for transforming the transformant plant , A vaccine composition for preventing fish viral neurotoxicity including a transgenic plant or an extract thereof containing a gene encoding a coat protein derived from Noda virus, a vaccine composition for preventing fish viral neurotoxicity including the vaccine composition Feed additives and And a method of preventing fish viral neurotoxicity comprising orally administering the vaccine composition or the feed additive to fish.

Recently, about 40 kinds of sea fish have been cultivated in Korea in order to protect and secure the sea fish resources. In addition, a large number of species of fry are being produced as seeds. Seedling production and breeding techniques for these fish have also been developed, but control of diseases caused by infection, especially virus infection, is increasingly difficult. For example, VNN (viral nervous necrosis) due to nodavirus infections in the louse or flounder, etc., has been reported as a virus infection with yellowtail ascites virus (YAV) .

Among them, viral neuro - necrosis caused by Nodavirus infection has been a threat to seed production and aquaculture since it has occurred in sea fish from all over the world since 1990 and is very lethal. In addition, there are more than 30 species of fish caught in the disease.

It is one of the best and most expensive seafood in the world. Especially, it is widely known as the most expensive and precious fish among the most famous fishes, which are caught only in the waters near Jeju Island. However, in Korea, commercial culture of fishes has not yet been established. This is due to the lack of overall understanding of fishes, but it can be said to be caused by viral nerve necrosis (VNN), which is one of the most fatal diseases in fishes. VNN is a disease caused by infection of sea urchin nodavirus, which has been causing serious damage to the seedling production plant since the late 1990s. It is caused by fish species such as domes, fauna, marine and flounder, When infected with this disease, it causes high mortality because of high mortality rate.

Recently, a survey of viral diseases in domestic farms revealed that the diseases caused by these diseases occurred in the lentils, domes, and flounder. Noda virus infection occurs mainly in fishes of 15-30 days after hatching, and infection by this virus has been confirmed in yellow clover, red mine, rockfish and lemur. Especially, it is reported that fatalities are very high in the initial stages of growth, and the fishes are characterized by swimmers swimming in the surface of the fish farms or spinning swimmers, and then falling on the bottom of the fish farms when they become weak. The disease often does not show any apparent lesions, but occasionally blackness of the body and abnormalities of the spine may be seen. Histologically, it is characterized by necrosis and collapse in the cells of the brain and retina, forming large vacuoles. It can be seen that small viral particles are concentrated at high density in the cytoplasm of such cells.

As the size of domestic farms grows larger, the rate of incidence of infectious diseases is increasing due to high density and low water quality. Although antibiotics are used to prevent bacterial diseases in fish, viral diseases are not only horizontal but also cause vertical infection by fertilized eggs. In the high-temperature environment, especially in August-September, prevention of preventive measures is important because the virus incidence increases as the dissolved oxygen decreases. The continuous use of antibiotics to prevent infectious diseases of cultured fish causes resistant bacteria and causes problems such as environmental pollution. As a countermeasure against this, the developed countries are actively promoting the development of vaccines instead of restricting the use of antibiotics.

The most common types of fish vaccines are inactivated vaccines, live attenuated vaccines, DNA vaccines, and recombinant vaccines. Methods of administering vaccines include injection, dipping, and oral administration. Among them, the injection method is accurate in dose, requires less amount of vaccine, and is capable of adding immunity enhancer, which gives a certain immunity to fish, but has a disadvantage that it takes a lot of labor and is difficult to put into practical use. The immersion method includes a standard method for immersing fish in a container with a high concentration of vaccine for a short period of time and a low concentration method in which the vaccine is directly added at low concentration to a rearing tank. While the standard method requires a small amount of vaccine, but the treatment is cumbersome, the low concentration method is easy to work and uses a lot of vaccine. Injection and standard dipping methods are difficult to use for young fish because they are highly stressed during processing. Oral administration method is most convenient because it is used after mixing the vaccine with the feed. However, a large amount of vaccine should be administered for a long period of time, and the prevention effect is low. Therefore, it is more effective to develop a vaccine that compensates for the disadvantages of the oral vaccine, considering the production cost of the vaccine, the mass production system, and the safe storage and transportation system while reducing the stress of the fish.

Recently, a system for producing an exogenous protein using plants has been developed, and studies for producing an antibody and a vaccine for treatment of human or animal diseases have been actively conducted. Unlike animal cells or microbial expression systems, the exogenous protein produced by the plant expression system can be used for oral administration without separation and purification, which is economical and convenient.

Korean Patent Laid-Open Publication No. 2006-0131931 discloses a method for preventing and treating infection with Nodavirus, but the method for producing a vaccine for preventing fish viral neuropathy using transgenic plants as well as the plant according to the present invention has been disclosed Not at all.

Disclosure of the Invention The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a gene encoding a nodavirus capsid protein, , And the present inventors completed the present invention by confirming that the nodavirus antibody was produced in a mouse to which the transgenic plant transformed with the above-mentioned transgenic plant was transplanted.

In order to solve the above-described problems, the present invention provides a recombinant expression vector for plant-plant transformation in which a gene coding for Nodalovirus-derived coat protein is operatively inserted.

 In addition, the present invention provides a method for producing a transformant plant comprising transforming a plant with the vector and expressing a coat protein derived from Nodaca virus.

In addition, the present invention provides a plastid transgenic plant produced by the above method and seeds thereof.

In addition, the present invention provides a vaccine composition for preventing fish viral neurotoxicity comprising a transgenic plant comprising a gene coding for a coat protein derived from Nodaca virus or an extract thereof.

The present invention also provides a feed additive for preventing fish viral neurotoxicity comprising the vaccine composition.

The present invention also provides a method for preventing fish viral neurotoxicity comprising orally administering the vaccine composition or the feed additive to fish.

 If the vaccine developed through the present invention is used in the fish culture industry, it can greatly contribute to productivity improvement by lowering the incidence of fatal diseases in one of the highest-priced and highest-priced fishes in Korea at present. And disease control related industries can be expected to grow.

Fig. 1 shows the nucleotide sequence (top) of the original gene encoding the capsid protein of Nodavirus and the nucleotide sequence (bottom) of the gene with chloroplast condon-optimization thereof. In the bottom sequence, the sequence shown in red is a codon-optimized sequence.
Fig. 2 shows an Overlap extension PCR method performed to analyze a gene encoding a capsid protein of Nodavirus.
Figure 3 is a vector used in the present invention. (Top) nicotine-free tobacco chromosome transfection vector, vector for expression of Nodavirus envelope protein utilizing nicotine-free tobacco chromosome (bottom).
FIG. 4 shows the results of PCR of the regenerated tobacco plastid transformants. WT; Wild type, C; Plasmid vector control (TIA_Rclp :: synNoda).
FIG. 5 shows the results of RT-PCR of the tobacco plastid transformants.
FIG. 6 shows the results of Western blot analysis of the expression of the Nodavirus envelope protein in the transgenic tobacco pigments. M; Size marker, C; Nodavirus protein extracted from Escherichia coli, WT; Wild type, kD; Kilodalton.
Figure 7 shows the immunization method and mouse population for each group for mouse immunization.
FIG. 8 shows the results of measurement of mucosal immunoglobulin IgA in a sample collected from a small intestine after a mouse immunization experiment. A; Group only, B; One priming group, C; One priming group and one boosting group. Control at A, B and C; Negative control (general feed grade), 10%; 10%, 20% of the transgenic plant content; The transgenic plant content represents 20% of the feed.

In order to accomplish the object of the present invention, there is provided a recombinant expression vector for plant transformation of a plant, wherein a gene coding for a coat protein derived from Nodaca virus is operatively inserted.

The recombinant expression vector for plant plastid transformation according to an embodiment of the present invention may be a vector pTIARclp :: synNoda shown at the bottom of FIG. 3, but is not limited thereto.

The gene coding for the envelope protein derived from Nodaca virus may be a gene encoding the original novirus envelope protein having the nucleotide sequence of SEQ ID NO: 1, but preferably a chloroplast codon optimization having the nucleotide sequence of SEQ ID NO: 2 (chloroplast condon-optimization) gene.

In addition, variants of the gene sequence encoding the Nodavirus-derived coat protein are included within the scope of the present invention. The mutant is a base sequence having a functional characteristic similar to that of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, although the nucleotide sequence thereof is changed. Specifically, the gene sequence has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% sequence homology with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 May contain a base sequence.

"% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The recombinant plant expression vector of the present invention can be used as a transient expression vector capable of transient expression in a plant into which the foreign gene is introduced and a plant expression vector capable of permanently expressing the foreign gene in the introduced plant.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples thereof include, but are not limited to, aadA (spectinomycin resistance gene), betainaldehyde gene, and gfp (green fluorescent protein) gene.

In the plant expression vector according to an embodiment of the present invention, the terminator may be a psbA terminator, an rrnB1 / B2 terminator or the like, but is not limited thereto.

In order to accomplish still another object of the present invention, the present invention provides a method for producing a transformant plant comprising transforming a plant with the vector to express Nodaca virus-derived coat protein.

The recombinant plant expression vector of the present invention can transform any plant regardless of dicotyledonous or monocotyledonous plants. In the present invention, however, the transformant is transformed into a tobacco plant, particularly nicotine-free tobacco. This is because it is desirable to use a nicotine-free cigarette to provide the transgenic tobacco in feed form.

According to one embodiment of the present invention, the plant is selected from the group consisting of Arabidopsis thaliana, tomato, eggplant, cigarette, red pepper, chinese broad bean, lettuce, bellflower, spinach, modern celery, parsley, parsley, gab, watermelon, melon, Root crops such as strawberry, soybean, mung bean, kidney bean, or pea or radish crops such as radish, turnip, cabbage, cabbage, carrot, burdock, sweet potato, maize, lotus root, potato and ginger; Medicinal plants such as ginseng, 칡, and udon; Forage crops such as corn and soybeans; Grain crops such as rice; But are not limited to, plants that can be used as roots, including oilseed rape, and the like.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), infiltration of plants, or Agrobacterium tumefaciens by transformation of mature pollen or microparticles of various plant elements (DNA or RNA-coated) Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention is particle bombardment.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

"Plant tissue" refers to cells of differentiated or undifferentiated plants, such as, but not limited to, various types of cells used in fruit, stem, leaf, pollen, seed, protoplasts, shoots and callus tissue. The plant tissue may be in planta or may be in an organ culture, tissue culture or cell culture.

In order to accomplish still another object of the present invention, there is provided a transgenic plant and seed produced by the above method.

In order to accomplish still another object of the present invention, the present invention provides a vaccine composition for preventing fish viral neurotoxicity comprising a transgenic plant, a fragment thereof or an extract thereof, which comprises a gene encoding a coat protein derived from Nodaca virus to provide.

In the vaccine composition according to one embodiment of the present invention, the gene may preferably have the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the vaccine composition according to one embodiment of the present invention, the fish includes all kinds of fish to be cultured, and preferably includes fishes such as domes, gnat, flounder, hornbill, red sea bream, , And more preferably, fish, but the present invention is not limited thereto.

As used herein, "vaccine" is a composition used to administer to a fish comprising at least one immunoprotective antigenic substance.

The extract may be in the form of a liquid, or may be in the form of a powder obtained by lyophilizing a plant or a section thereof. The vaccine composition according to the present invention can be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are commonly used in pharmaceuticals. Preferably, it is preferable to formulate the composition for oral administration. For example, it is preferable to formulate a solid preparation for oral administration such as tablets, pills, powders, granules and capsules, or a liquid preparation such as a suspension, a solution, an emulsion or a syrup. CM-chitin can be used as an adjuvant when formulating into a solid preparation. In the case of formulation with a liquid formulation, various excipients such as wetting agent, sweetening agent, fragrance, preservative, etc. may be mixed with the vaccine composition according to the present invention in addition to water or liquid paraffin.

In order to accomplish still another object of the present invention, there is provided a feed additive for preventing fish viral neurotoxicity comprising the vaccine composition.

The vaccine composition according to the present invention may be added to a feed to be ingested into a fish, thereby inducing the formation of an antibody against novirus of fish viral neurotoxicity in a fish fed the feed to induce an immune effect. Wherein the vaccine composition may be added to the feed in powder form or in liquid form.

In order to accomplish still another object of the present invention, the present invention provides a method for preventing fish viral neurotoxicity comprising orally administering the vaccine composition or the feed additive to fish.

Preferably, the transgenic plants according to the present invention may be lyophilized, pulverized, mixed with feed and starch, and fed to fish in the form of pills. When the transgenic plant according to the present invention is mixed with a feed as a feed additive and fed to the fish, the effect of continuously administering the vaccine to the fish can be shown.

It is believed that plants containing envelope proteins derived from Nodaca virus can be administered to subjects, i.e., fish, in a variety of ways depending on needs and circumstances. &Quot; Administration " is defined as the introduction of a substance into the body of a fish and includes oral, nasal, rectal, vaginal, and extra-intestinal routes. The compositions of the present invention may be administered individually via any route of administration, including subcutaneous (SQ), intramuscular (IM), intravenous (IV), mucosal, nasal or oral, or in conjunction with other therapeutic agents. Particularly preferably, it is an oral route.

The specific dose of the antigenic composition according to the invention will depend on various factors, including the species, age, overall condition of the fish to which the composition is administered, and the mode of administration of the composition. The effective amount of the composition according to the present invention can be readily determined using routine experimentation. In vitro and in vivo models can be used to confirm the dosage.

The efficacy of the vaccine according to the invention exemplifies the fact that administration of the vaccine prevents or alleviates the symptoms of the disease to be treated or caused by the intended pathogens by at least 5%, preferably 10-20%, more preferably 25-100% .

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. Noda virus Antigen protein  Codon optimization for plant expression optimization of genes

In order to maximize the expression of foreign proteins in plants, the Nodavirus capsid gene was used in a graphical codon usage analyzer program and http: // www. Using the database of kazusa.or.jp/codon, the nucleotide sequence was optimized to match the codon of the tobacco chromosome (Fig. 1). Codon optimized Nodavirus capsid The genes were synthesized by applying an overlap extension PCR technique (Fig. 2). Thirteen primers with a length of about 60 bp and thirteen primers in the reverse direction overlapping the forward primer by about 20 bp were prepared. Initial denaturation for 5 minutes at 95 ° C using 8 primer combinations (F1 to F7, R2 to R8); 30 cycles of denaturation at 95 DEG C for 20 seconds, annealing at 51 DEG C for 15 seconds, and polymerization at 72 DEG C for 30 seconds; Then, PCR was performed by an additional 5 minute polymerization at 95 ° C to prepare a block 1 having a length of about 340 bp (1st step). After preparing Blocks 2 and 3 under the same conditions, the three PCR reaction products were gel-purified and mixed at the same ratio to prepare a template. 2 μl of template, 1 μl of each of F1 and R26 primers of 10 pmole concentration, 2 μl of 2.5 mM dNTPs, 0.3 μl of pyrobest DNA polymerase (Takara) and 2.5 μl of 10 × PB buffer were added to make a total clone of 1,042 bp, Lt; RTI ID = 0.0 > 95 C < / RTI > for 5 minutes; 30 cycles of 30 sec denaturation at 95 캜, 30 sec annealing at 50 캜 and 1 min 30 sec polymerization at 72 캜; Subsequently, secondary PCR was performed under the condition of additional 5-minute polymerization at 95 캜 (2nd step). The final amplified product was gel-purified and cloned into pGEM T-Easy vector to analyze and confirm the base sequence.

Example  2. Plant Plasmid Transformation Vector Production

The codon-optimized Nodavirus capsid gene was introduced into TIA :: RclpGAH, a transgenic basic vector containing the rclp promoter designed to prevent secondary recombination between genes during plant plastid transformation (Fig. 3, top). First, to remove the mGFP region from the basic vector TIA :: RclpGAH, DNA was digested with restriction enzyme Pst I and treated with Klenow enzyme. The treated reaction product was digested with Sal I restriction enzyme, and then a vector was prepared by purifying phenol / chloroform and ethanol. The Nodavirus capsid gene in the pGEM T-Easy vector was treated with EcoR V and Sal I restriction enzyme and then isolated using a gel purification kit (Bioneer). The isolated Nodavirus capsid gene was subcloned into a TIA :: RclpGAH vector ( Sal I / Pst I *) site with mGFP removed using DNA Ligation kit-Mighty mix (Takara) and introduced into E. coli To prepare TIARclp :: synNoda (FIG. 3, bottom).

Example  3. Noda virus Antigen protein  Nicotine in the gene free ( nicotine - free ) Introduction into tobacco pigments

Tobacco that expresses the antigen protein is provided directly as feed, so a nicotine-free tobacco ( Nicotiana tabacum cv.MD609, professor Yang Moon-shik of Chonbuk National University) was used. Tobacco leaves grown on MS medium without antibiotics for 5 weeks or more after germination were slaughtered on Rmop medium and transformed with Bio-Rad PDS-1000. The transformed plasmid vector was coated with 0.6 탆 gold particles using CaCl ₂ and spermidine. The vacuum in the aseptic chamber was 28 in. Hg, the pressure was 1100 psi, and the distance was 9 cm. The bombarding was applied to the tobacco leaf sprouted on the medium. The shocked tobacco leaves were cultured on the same medium for 2 days, cut into 5 mm x 5 mm and cultured for 6-7 weeks in a selective medium supplemented with Rmop for spectinomycin to induce resistant shoots Respectively. The shoots induced in the spectinomycin resistant medium were cut into 3 mm x 3 mm size and then regenerated in the same selection medium to induce resistance shoots again. Regeneration was repeated in the selection medium in the same manner as above to increase the homoplasmy of the introduced gene.

Example  4. Generation of antigen genes in nicotine-free tobacco chromosome genome T0  And T1  Generation analysis data)

① On the starter medium  Of the selected tobacco plastid transformants PCR  analysis

Genomic DNA was extracted from each plant to confirm that the regenerated plant in the antibiotic-supplemented medium was transformed with the target gene, and vector-specific primers trnIF (5'-ATCTCTCGAGCACAGGTTTA-3 '; SEQ ID NO: 3) and trnAR 5'-TTCTTGACAGCCCATCTTT-3 '; SEQ ID NO: 4). Because of the large PCR product size, Ex Taq (Takara) was used and initial denaturation at 95 ° C for 5 minutes; 30 cycles of denaturation at 95 DEG C for 30 seconds, annealing at 58 DEG C for 30 seconds, and polymerization at 72 DEG C for 5 minutes; PCR was then carried out under conditions of an additional 5 minute polymerization at 95 < 0 > C. A DNA band of 1.6 kb was detected in the wild type in which the gene was not introduced, whereas a DNA band of 4.5 kb in which the vector such as the promoter and the target gene was introduced in the T0 and T1 generation transformants was detected (FIG. 4).

② Genome DNA PCR Of a tobacco chromosome transformant RT - PCR  analysis

Total RNA was extracted from each plant and cDNA was synthesized using reverse transcriptase in order to confirm the target gene transcript (expression at the RNA level) in the plant in which gene introduction was confirmed by genomic DNA PCR. PCR was performed with Nodavirus gene-specific primers. As a result, it was found that lines with relatively high expression levels (# 1-5, # 3-4, # 3-6, # 4-3, # 5-5, ) Can be selected. As shown in Fig. 5, the transcript of the nodavirus gene was found to be at least 2-3 times larger than that of the actin gene primer that always expresses using the same amount of cDNA (Fig. 5).

③ RT - PCR Of a tobacco chromosome transformant Western Blat  analysis

The plants expressing the transcript of the Nodavirus antigen protein were selected by RT-PCR and Western blotting was performed to confirm whether the target gene expressed at the protein level. Total protein was extracted from the plant leaves with protein extraction buffer, loaded onto SDS-PAGE gel, and then immersed in 0.2 μM immuno-blot ^TM Transferred to a PVDF membrane (Bio-Rad) and blocked with 5% defatted milk powder. As the primary antibody, the antibody of Nodavirus antigen protein was diluted 1: 5000, reacted at room temperature overnight, diluted with a goat anti-rabbit HRP-labeled secondary antibody at a concentration of 1: 10000, and reacted at room temperature for 6 hours . HRP conjugated secondary antibody was reacted with a chemiluminescent substrate, supersignal westpico (Thermo scientific), and the signal was confirmed by chemidoc ^TM XRS + (Bio-Rad). As a result, as shown in FIG. 6, it was confirmed that nodavirus protein of about 43 kD was detected in the tobacco plastid transformant and not detected in the wild type (WT) (FIG. 6).

Example  5. Oral administration of mice

① Immune response experiment by mouse oral administration

Six-week-old female ICR mice were prepared and divided into four groups. Group A is a group in which only plant recombinant recombinant antigen compound feed is fed, Group B is a recombinant antigen expressed in E. coli in a single injection, followed by antigen priming in mouse, followed by addition of plant recombinant recombinant antigen only feed , Group C was used to perform single-step boosting injection after antigen-priming in mice by injecting a recombinant antigen expressed in E. coli once, and grouping plant-expressing recombinant antigen-containing feed only into a group . Group D, fed only normal diet, was used as a negative control. Feeding was carried out by subdividing into two groups so that the contents of transgenic plants were 10% and 20% in each group (Fig. 7).

② In mice given orally IgA  Measure

Oral immunity (mucosal immunity) differs greatly from the systemic immune system in that immune is induced by administering the antigen directly to the mucosal surface, the site where the infection begins. For this reason, it is important to understand the mucosal immunity mechanism first. Unlike the IgG or IgM antibodies that are induced when the antigen is orally administered, IgA antibodies are mainly produced locally in the mucosal immunity. IgA is an immortal antibody that plays an important role in mucosal immunity and captures many pathogenic substances to stop its activity. It is present not only in the intestinal mucosa but also in tears, saliva, colostrum, intestinal fluid, vaginal fluid, prostate fluid, respiratory epithelium, etc., and also in the blood. Because it is resistant to degradation by enzymes, it does not lose its function even in a harsh environment such as digestive or respiratory machines, thus fighting against bacterial infections. IgA is the body's primary line of defense against bacteria, food residues, fungi, parasites and viruses present in the intestinal tract. Therefore, IgA was analyzed and immunity was confirmed by oral administration.

On the 7th day after the final immunization via feeding, a small intestine sample of the mouse was collected to measure antibody titer. The small intestine was sampled about 3 cm, and the feces in the intestinal tract was removed. 0.5 ml of PBS was added to the small intestine sample. The tissue was ground with a tissue grinder and centrifuged to obtain supernatant. To measure the IgA concentration in the supernatant, 1 μg / well / 90 μl of the recombinant antigen expressed and purified in E. coli was coated on a 96-well plate and blocked with PBS supplemented with 1% defatted milk powder. PBST PBS + 0.05% Tween-20, pH 7.4). The supernatant of the sample to be measured was used as a primary antibody and allowed to react at 37 ° C for 1 hour. After the reaction, the secondary antibody conjugated with goat anti-mouse IgA HRP was diluted 1: 500 with 3 times of washing with PBST, and reacted at 37 ° C for 1 hour. After the reaction, the cells were washed three times with PBST. Non-primed murine samples of mice were used as negative control. Detection of bound secondary antibody was performed using substrate solution (100 mM citric acid buffer, pH 4.0, ABTS stock solution, H ₂ O ₂ ). After the reaction with the substrate solution in the dark room was carried out for 5 minutes, the OD value was measured using an ELISA reader at an absorbance of 405 nm. As shown in FIG. 8, IgA in the sample collected from the small intestine showed a statistically significant increase ( P <0.05) in antibody titer in the group administered with the feed containing 20% of transgenic plants in each experimental group Respectively. In addition, it was confirmed that most of the antibodies were generated in the C group in which the feedstuffs containing the transgenic plants were fed after primary immunization and boosting (FIG. 8).

<110> GenDocs, Inc. <120> Method for producing oral vaccine against fish viral nervous          necrosis using transgenic plant and the plant thereof <130> PN12275 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 1017 <212> DNA <213> Nodavirus <400> 1 atggtacgca aaggtgagaa gaaattggca aaacccgcga ccaccaaggc cgcgaatccg 60 caaccccgcc gacgtgctaa caatcgtcgg cgtagtaatc gcactgacgc acctgtgtct 120 aaggcctcga ctgtaactgg atttggacgt gggaccaatg acgtccatct ctcaggtatg 180 tcgagaatct cccaggccgt cctcccagcc gggacaggaa cagacggata cgttgttgtt 240 gacgcaacca tcgtccccga cctcctgcca cgactgggac acgctgctag aatcttccag 300 cgatacgctg ttgaaacact ggagtttgaa attcagccaa tgtgccccgc aaacacgggc 360 ggtggttacg ttgctggctt cctgcctgat ccaactgaca acgatcacac cttcgacgcg 420 cttcaagcaa ctcgtggtgc agtcgttgcc aaatggtggg aaagcagaac agtccgacct 480 cagtacaccc gcacgctcct ctggacctcg tcgggaaagg agcagcgtct cacgtcacct 540 ggtcggctga tactcctgtg tgtcggcaac aacactgatg tggtcaacgt gtcagtgctg 600 tgtcgctgga gtgttcgact gagcgttcca tctcttgaga cacctgaaga gaccaccgct 660 cccatcatga cacaaggttc cctgtacaac gattcccttt ccacaaatga cttcaagtcc 720 atcctcctag gatccacacc actggacatt gcccctgatg gagcagtctt ccagctggac 780 cgtccgctgt ccattgacta cagccttgga actggagatg ttgaccgtgc tgtttattgg 840 cacctcaaga agtttgctgg aaatgctggc acacctgcag gctggtttcg ctggggcatc 900 tgggacaact tcaacaagac gttcgcagat ggcgttgcct actactctga tgagcagcct 960 cgtcaaatcc tgctgcctgt tggcactgtc tgcactaggg ttgactcgga aaactaa 1017 <210> 2 <211> 1017 <212> DNA <213> Artificial Sequence <220> <223> Nodavirus coat protein chloroplast codon-optimized sequence <400> 2 atggtacgaa aaggtgagaa gaaattggca aaacctgcaa caacaaaggc tgcaaatcct 60 caaccacgac gacgtgctaa caatcgtaga cgttctaatc gaactgacgc acctgtatct 120 aaggcttcta ctgtaactgg atttggacgt ggaacaaatg acgttcatct ttcaggtatg 180 tctagaatct ctcaggccgt ccttccagct ggaacaggaa cagacggata cgttgttgtt 240 gacgcaacaa tcgttcccga ccttttgcca cgattgggac acgctgctag aatcttccag 300 cgatacgctg ttgaaacatt ggagtttgaa attcagccaa tgtgccctgc aaacactgga 360 ggtggttacg ttgctggatt cttgcctgat ccaactgaca acgatcacac tttcgacgca 420 cttcaagcaa ctcgtggtgc agttgttgct aaatggtggg aaagtagaac agttcgacct 480 cagtacacac gaacgcttct ttggacatct tctggaaagg agcagcgtct tacttctcct 540 ggtcgattga tacttttgtg tgttggaaac aacactgatg tagttaacgt atcagtattg 600 tgtcgatgga gtgttcgatt gagtgttcca tctcttgaga cacctgaaga gacaacagct 660 cctatcatga cacaaggttc tttgtacaac gattctcttt ctacaaatga cttcaagtct 720 atccttttag gatctacacc attggacatt gctcctgatg gagcagtttt ccagttggac 780 cgtccgttgt ctattgacta cagtcttgga actggagatg ttgaccgtgc tgtttattgg 840 caccttaaga agtttgctgg aaatgctgga acacctgcag gatggtttcg atggggaatc 900 tgggacaact tcaacaagac tttcgcagat ggtgttgctt actactctga tgagcagcct 960 cgtcaaatct tgttgcctgt tggaactgtt tgcactagag ttgactctga aaactaa 1017 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tRNA primer <400> 3 atctctcgag cacaggttta 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> trnAR primer <400> 4 ttcttgacag cccatcttt 19

Claims (13)

A recombinant expression vector comprising a nucleotide sequence of SEQ ID NO: 2, wherein a gene coding for a coat protein derived from nodavirus is operatively inserted. delete A method for producing a transgenic plant comprising transforming a plant with the vector of claim 1 and expressing a nodal virus-derived coat protein. 4. The method according to claim 3, wherein the plant is a dicotyledonous plant. 5. The method of claim 4, wherein the dicotyledonous plant is a nicotine-free tobacco plant. A transgenic plant produced by the method of claim 3. A seed of the transgenic plant of claim 6. 1. A vaccine composition for preventing fish viral neurotoxicity comprising a transgenic plant comprising a nucleotide sequence of SEQ ID NO: 2 and a gene encoding a nodavirus-derived coat protein, or an extract thereof. delete 9. The vaccine composition according to claim 8, wherein the fish is a freshwater fish. A feed additive for preventing fish viral neurotoxicity comprising the vaccine composition of claim 8. 12. The feed additive of claim 11, wherein the fish is a freshwater fish. A method for preventing fish viral neurotoxicity comprising orally administering the vaccine composition of claim 8 or the feed additive of claim 11 to fish.

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