KR101127926B1 - Iridovirus antigenic peptide and vaccine comprising the same - Google Patents

Abstract

The present invention relates to a saltwater fish iridovirus antigenic peptide using an immunogenic site of the fish iridovirus, a pathogen causing fatal fatality in various saltwater fish, and a vaccine for preventing iridovirus using the same. Effective humoral immunity through the digestive system can prevent iridoviruses to increase the survival rate, and by mixing with the feed has the advantage of not giving stress to the fish at all due to inoculation.

Iridovirus, vaccine

Description

Iridovirus antigenic peptide and vaccine for preventing Iridovirus using the same

The present invention relates to a saltwater fish iridovirus antigenic peptide using a site having immunogenicity of a fish iridovirus, a pathogen causing fatal fatality in various saltwater fish, and a vaccine for preventing iridovirus using the same.

Iridovirus is a double-stranded DNA virus having a icosahedron shape and having a genome size of 170 to 200 kb. Iridoviruses are systemically infected with fish and are characterized by high morbidity and mortality. Fish iridoviruses have a wide regional distribution and host range, and usually develop at times of elevated water temperature from 18 to 20 ° C to 25 to 27 ° C.

Meat with iridovirus disease shows abnormal and lethargic swimming, with no special external symptoms, and cumulative mortality of 50-90% for two months. Histopathological findings show hypertrophic basophils in gills, kidneys, heart, liver and spleen. Iridoviruses are not only indigenous to imported fish, but also from natural to farmed fish and from larvae to adult fish. In the case of our country, from August to October 1998, various types of aquaculture have been identified since the cause of the mass mortality of the cultivated Olegnathus fasciatus on the south coast was found to be similar to red sea bream iridovirus (RSIV). Iridovirus infection occurs in fish. In the case of the flounder (Paralichthys olivaceus), the mass mortality of farmed flounder fry in 2003 also turned out to be an iridovirus similar to the sea bream iridovirus. The infection occurred at water temperatures between 23 and 26 ° C. and caused not only one-year-old but two- to three-year-old fish to death, causing enormous losses. Despite these outbreaks, there is no urgent vaccine or treatment for iridovirus, so urgent precautions are needed. Recent studies have shown that the capsid protein of fish iridovirus is genetically significantly different from lanavirus and lymphphocystivirus belonging to the same iridoviridae previously reported, but isolated from various fish species. Capsid proteins between iridoviruses have been found to be very genetically independent regardless of infected fish species, showing good conditions for serological diagnosis and development of preventive vaccines using recombinant proteins.

However, there is still only a diagnosis for the fish Irido virus, there is no effective preventive vaccine worldwide.

Therefore, the present inventors continued research on recombinant protein vaccines using immunogenic sites without the risk of iridovirus. As a result, the capsid protein, a structural protein in the gene of icosahedral cytoplasmic DNA virus, a fish idovirus, It was confirmed that this immunogenicity, using the same as the surprising vaccine effect of the recombinant protein of the virus also completed the present invention.

The present invention is to provide a vaccine for the prevention of iridovirus using a site having immunogenicity of the fish iridovirus, a pathogen causing fatal fatality in various sea fish, and an object of the present invention is a marine fish iridovirus antigen for using a yeast expression system To provide a sex peptide and recombinant nucleic acid encoding the same.

Still another object of the present invention is to provide a transformed microorganism expressing the saltwater fish iridovirus antigenic peptide and a method for producing the capsid protein of saltwater fish iridovirus by culturing the microorganism.

In order to achieve the above object, the present invention provides a saltwater fish iridovirus antigenic peptide and a recombinant nucleic acid encoding the same for using the yeast expression system.

The present invention also provides a transgenic microorganism expressing the saltwater fish iridovirus antigenic peptide and a method of culturing the microorganism to produce capsid proteins of saltwater fish iridovirus.

The saltwater fish iridovirus antigenic peptide according to the present invention is characterized in that the saltwater fish iridovirus antigenic peptide for using the yeast expression system, which is the immunogenic site of the capsid protein of the saltwater fish iridovirus It is characterized by being manufactured by optimizing (codon usage).

Iridovirus prevention vaccine containing the saltwater fish Iridovirus antigenic peptide according to the invention as an active ingredient is characterized in that the vaccine to induce effective humoral immunity through the digestive organs of the target fish.

Hereinafter, the present invention will be described in detail.

The present invention removes amino acids 1 to 21 and 427 to 454 of the capsid protein of seawater fish iridovirus, amino acids 99, 153, 197, 203, 204, 306, 346 And the saltwater fish iridovirus antigenic peptide, wherein the CGC codon at site 395 is site-directed mutagenesis with an AGA codon.

More specifically, in order to prepare an antigenic peptide of the capsid gene of seawater fish iridovirus of about 1.21 kb, the base sequence 1 of the capsid gene of seawater fish Iridovirus after antigenic analysis The weak and hydrophobic portions of amino acids 63 to 63 (amino acids 1 to 21) and nucleotide sequences 1281 to the last 1362 (amino acids 427 to 454) were removed. The next step was to optimize eight arginine (R) codons that are not well understood in yeast with AGAs that are well translated in yeast. See FIG. 3. This is to prepare a vaccine for the purpose of adding the desired vaccine for iridovirus prevention to the feed which is the easiest way to inoculate and administer to fish using yeast. In addition, N-glycosylation, a phenomenon that does not appear in expression in prokaryotic cells such as Escherichia coli, occurs in yeast, thereby providing much enhanced immunity and antigenicity. This glycosylation site occurs at five sites (237th, 302th, 311th, 398th, 419th asparagine) in the antigenic peptide.

The saltwater fish iridovirus capsid protein of the present invention consists of the amino acid sequence of SEQ ID NO: 1, and the saltwater fish iridovirus antigenic peptide of the present invention is characterized in that the amino acid of SEQ ID NO: 2 consists of a sequence.

The present invention provides a recombinant nucleic acid encoding a marine fish iridovirus antigenic peptide and a recombinant nucleic acid, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 3 and a recombinant vector comprising the recombinant nucleic acid.

In addition, using a recombinant vector comprising the recombinant nucleic acid provides a transforming microorganism that expresses the saltwater fish Irivirus antigenic peptide, characterized in that the microorganism is Phichia pastoris ( Phichia pastoris ).

More specifically, the recombinant vector comprising a recombinant nucleic acid encoding the iridovirus antigenic peptide of the present invention used a pGAPZα A vector as a yeast expression vector, and expressed by SDS-PAGE and Western blot assay to confirm expression. It was confirmed. As a result, the protein expressed from the transformed yeast produced a band of about 55 kDa, slightly larger than the recombinant protein expressed in E. coli (about 46 kDa), which was affected by glycosylation in yeast. It was confirmed that the molecular weight of about kDa was increased. See FIG.

The present invention provides a method for producing a capsid protein of saltwater fish iridovirus by culturing a transformed microorganism expressing a saltwater fish iridovirus antigenic peptide, and containing the antigenic peptide in the capsid protein prepared by the above method as an active ingredient. Provides a vaccine for iridovirus prevention.

The vaccine is characterized in that oral or edible intake is possible.

More specifically, vaccination using the capsid recombinant protein of the saltwater fish iridovirus of the present invention confirmed the protective effect against iridovirus infection, and the survival rate in the test group using the recombinant capsid protein of the saltwater fish iridovirus expressed in yeast as a vaccine 91.7% of the control group compared to 100% mortality of the yeast-expressed Irivirus recombination capsid protein vaccine was confirmed that there is a clear protection against viral infection.

In addition, after oral administration of the recombinant capsid protein of seawater Irivirus expressed in yeast, it was confirmed that the anti-iridovirus gene was induced statistically significantly compared to the control group. It was confirmed that the rise of the immune response. This is also a result of showing that the recombinant capsid protein according to the present invention reacts with the antigenic capsid antibody, thereby having the same antigenicity as the antigen of the saltwater Irivirus capsid protein of the existing SEQ ID NO: 1. See FIG. 8.

Iridovirus prevention vaccine according to the present invention is effective to increase the survival rate by preventing the iridovirus by inducing effective humoral immunity through the digestive organs of the target fish, and also the stress caused by inoculation to fish by administering mixed with feed There is no advantage at all.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

[ Example  One] Saltwater fish Iridovirus Capsid  Gene Cloning

Phenol (Chlorophyllus olivaceus) from the spleen by the phenol / chloroform method DNA was extracted and dissolved in TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 7.5) to a concentration of 50 ng / ㎕, polymerase chain Used as a reaction template.

Primer is a primer (5'-GAA GAATTC ATG TCT GCA ATC TCA GGT GCG-) containing restriction enzyme EcoR I site (underlined sequence) using GenBank registered rockbream iridovirus major capsid protein (MCP, AY849393) 3 ') and a reverse primer (5'-GGT GGTACC TTA CAG GAT AGG GAA GCC TGC-3') containing the Kpn I site (underlined base sequence) were prepared and used.

Polymerase chain reaction conditions were prepared by mixing 2 μl DNA, 1 μl of each primer (50 pM) prepared above, 5 μl of 10 mM dNTPs (2.5 mM each), and 1 μl of 5U Ex TaqTM DNA polymerase in 5 μl of 10 × reaction buffer. Then, sterile water was added to a total of 50 μl and amplified by polymerase chain reaction. The polymerase chain reaction was reacted under conditions of denaturation (95 ° C., 1 minute), annealing (60 ° C., 1 minute), extension (72 ° C., 2 minutes), and 30 cycles.

The specific band amplified by the polymerase chain reaction amplified in 1.0% agarose gel was confirmed the size of 1.21 kb, by separating only the specific band using a polymerase chain reaction product purification kit (PCR purification kit) After pure separation, the cells were cloned by inserting into the pGEM-T easy vector and transformed into E. coli JM109 and cultured.

The cultured transformant E. coli was extracted from the DNA plasmid cloned into the pGEM-T easy vector using the QIAprepTM Spin Mini Kit. In order to determine whether the amplified specific band was inserted into the extracted DNA plasmid, the restriction enzyme EcoR I + Kpn I was confirmed. The nucleotide sequence was analyzed using ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA) to confirm the nucleotide sequence of the inserted DNA, and compared with the nucleotide sequence of rockbream iridovirus MCP gene registered in GenBank. As a result, the iridovirus capsid gene having the amino acid sequence (SEQ ID NO: 1) of FIG. 2 could be cloned.

[ Example  2] Saltwater fish Iridovirus  Antigenicity Peptide  Produce

In order to prepare the antigenic peptide of the MCP gene of about 1.21 kb cloned in pGEM-T Easy in Example 1, from the base sequence No. 1 of the capsid gene of seawater iridovirus after analysis of antigenicity The antigenic and hydrophobic portions of 63 (amino acids 1 to 21) and base sequences 1281 to the last 1362 (amino acids 427 to 454) were removed.

As shown in FIG. 3, the removed peptides were optimized with AGA for eight arginine (R) codons that are poorly detoxified in yeast. For this purpose, a primer set having a corresponding base sequence to be changed was prepared. In other words, in order to convert the first arginine codon CGC into AGA, gag aac agc tac att AGA tgg tgt gat aat ttg was prepared, including 15 mer on each side of the CGC region, and caa att atc aca cca TCT aat as a reverse primer. Site-directed mutagenesis was performed with gta gct gtt ctc. Likewise, the remaining seven arginine codons were prepared by primers, respectively, as shown in Table 1 below.

The polymerase chain reaction used pfu DNA polymerase without 5-3 exonuclease function. After amplification, it was treated with restriction enzyme Dpn I to remove the amplified pGEM-T Easy vector, and then transformed into E. coli JM109 and arginine codon. The nucleotide sequence was analyzed to see if the phosphorus CGC was changed to AGA as desired.

As a result, as can be seen in Figures 5 and 6, the iridovirus antigenic peptide consists of a sequence of amino acids described in SEQ ID NO: 2, can produce a recombinant nucleic acid of SEQ ID NO: 3 encoding the iridovirus antigenic peptide there was.

Example 3 Expression of Capsid Proteins in Seawater Iridovirus

(1) Preparation of Vector for Expression of Recombinant Protein of Seawater Irivirus

The codon-optimized recombinant nucleic acid cloned into pGEM-T easy of Example 2 was treated with two restriction enzymes ( EcoR I + Kpn I), and both ends were made of sticky ends. PGAPZα A vector, a yeast expression vector, was treated with the same restriction enzyme ( Eco RI + Kpn I) to ligate a recombinant nucleic acid and yeast expression vector using T4 DNA ligase (Promega). One plasmid construct produced by the ligation was incubated in E. coli JM109, and the presence of recombinant nucleic acid and shift of open reading frame were reconfirmed by sequencing.

Pichia was transformed into YPD liquid medium [1% (w / v) yeast extract, 2% (w / v) peptone, and 1% (v / v) in order to transform the plasmid construct identified above into Phichia pastoris , a yeast for protein expression. ) After growth in glycerol] and YPD solid medium competent cells were prepared and transformed using Pichia EasyComp ^™ Kit. In order to find only transformed Phichia , transformed yeast expressing recombinant protein of seawater Irivirus was isolated by adding antibiotic Zeocin at a concentration of 100 ㎍ / ml.

(2) Confirmation of Expression of Irido Virus Capsid Protein

Selected and isolated transgenic yeasts were expressed in 100 ml of YPD medium and stirred at 160 rpm for 28 hours at 28 ° C. while the culture cell concentration was about 0.2 at OD ₆₀₀ (1% yeast extract, 2% peptone, 4 × 10 ⁻⁵ % Biotin) was incubated for 2 to 4 days after inoculation to induce protein expression. In order to confirm the expression, the expression was confirmed by SDS-PAGE and Western blot assay. MCP protein expressed in E. coli was used as a control.

As can be seen from the results of FIG. 7, SDS-PAGE confirmed whether the protein was expressed from the transformed yeast, and a band of about 55 kDa slightly larger than the recombinant protein (about 46 kDa) expressed in E. coli was generated. . It was confirmed that the molecular weight of about 9 kDa was increased due to glycosylation in yeast.

[ Example  4] Protective effect against iridovirus infection

(1) Oral administration experiment

Defense experiments were conducted to determine whether vaccination with the capsid recombinant protein of the saltwater fish Irivirus of the present invention has a protective effect against Irido virus infection. That is, after preparing 60 experimental objects in each group divided into two groups for 2 months old flounder, the control group was prepared by dipping the EP feed for fry in distilled water, and then taking out and drying again. The test group was prepared by soaking 1 liter of yeast culture solution of the recombinant protein of seawater iridovirus of the present invention for 1 kg of fry EP feed and wet again and dried again. Feed was fed about 50 g per 100 horses five times a day and for two weeks.

The above is shown in more detail in Table 2 below.

In addition, the two-week rest period was applied, and then, for another two weeks, the feed containing and not containing the recombinant capsid protein of the seawater iridovirus expressed in yeast was fed.

The effect of oral vaccine was confirmed by examining the survival rate against iridovirus attacks. That is, the virus was taken from tissues of individuals naturally infected with the virus and used for infection. The virus population from infected tissue was calculated as 1 × 10 ⁵ copy / ml by quantitative polymerase chain reaction.

As a result, only 5 animals died (8.3% mortality) in the test group using the recombinant capsid protein of seawater Irivirus expressed in yeast, and the remaining 55 (91.7% survival) survived, whereas the control group survived 60. All died (100% mortality). This showed that the iridovirus recombinant capsid protein vaccine expressed in yeast has a definite defense against iridovirus infection.

(2) the antibody is measured

The antibodies of the test group and the control group were collected from a small amount of blood from the vein on the 5th day of the end of feed feeding to measure the serum was used after separation. That is, in order to determine the presence of anti-iridovirus MCP antibody (IgM) in the separated serum, 100 μl of the recombinant protein antigen expressed in E. coli at a concentration of 1 ug / ml was coated on a 96-well ELISA plate, followed by primary Diluted serum of experimental fish 1: 100 as an antibody, 1: 100 dilution of anti-flounder IgM monoclonal antibody as a secondary antibody, and anti-mouse IgG HRP-conjugated as a tertiary antibody The antibody was measured.

As a result of oral administration of the recombinant capsid protein of seawater fish Irivirus expressed in yeast, as shown in FIG. 8, it was confirmed that the anti-iridovirus gene was induced statistically significantly compared to the control group. In addition, it was confirmed that the antibody titer is raised in the blood and the immune response is induced. This is also a result of showing that the recombinant capsid protein according to the present invention reacts with the antigenic capsid antibody, thereby having the same antigenicity as the antigen of the saltwater Irivirus capsid protein of the existing SEQ ID NO: 1.

1 is an amplification picture of the capsid gene of seawater fish iridovirus using a polymerase chain reaction,

Figure 2 shows the amino acid sequence of the capsid protein (SEQ ID NO: 1) of the sea fish Iridovirus (iridovirus),

FIG. 3 is a diagram showing the locations of amino acid sites and site-directed mutagenesis removed in the capsid protein of seawater fish iridovirus,

Figure 4 shows the results confirming the site-specific mutagenesis of seawater fish Iridovirus antigenic peptides according to the present invention.

Figure 5 shows the amino acid sequence (SEQ ID NO: 2) of the saltwater fish iridovirus antigenic peptide according to the present invention,

Figure 6 shows the nucleotide sequence (SEQ ID NO: 3) of the recombinant nucleic acid coating the marine fish iridovirus antigenic peptide according to the present invention,

7 is a result confirming the expression of the seawater fish iridovirus antigenic peptides according to the present invention from the transformed yeast,

(M: size marker, 1: Escherichia coli MCP protein, 2: Saltwater fish iridovirus antigenic peptide, 3: Salted fish iridovirus capsid protein)

8 is a diagram measuring the antibody value induced after antigen recognition of the recombinant capsid protein of seawater fish iridovirus according to the present invention.

(1: control, 2: test group, P <0.001)

<110> Industry Foundation of Chonnam National University <120> Iridovirus antigenic peptide and vaccine comprising the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 453 <212> PRT <213> Paralichthys olivaceus <220> <221> PEPTIDE (222) (1) .. (453) <223> iridovirus capsid protein <400> 1 Met Ser Ala Ile Ser Gly Ala Asn Val Thr Ser Gly Phe Ile Asp Ile   1 5 10 15 Ser Ala Phe Asp Ala Met Glu Thr His Leu Tyr Gly Gly Asp Asn Ala              20 25 30 Val Thr Tyr Phe Ala Arg Glu Thr Val Arg Ser Ser Trp Tyr Ser Lys          35 40 45 Pro Pro Val Thr Leu Ser Lys Gln Thr Gly His Ala Asn Phe Gly Gln      50 55 60 Glu Phe Ser Val Thr Val Ala Arg Gly Gly Asp Tyr Leu Ile Asn Val  65 70 75 80 Trp Leu Arg Val Lys Ile Pro Ser Ile Thr Ser Ser Lys Glu Asn Ser                  85 90 95 Tyr Ile Arg Trp Cys Asp Asn Leu Met His Asn Leu Val Glu Glu Val             100 105 110 Ser Val Ser Phe Asn Asp Leu Val Ala Gln Thr Leu Thr Ser Glu Phe         115 120 125 Leu Asp Phe Trp Asn Ala Cys Met Met Pro Gly Ser Lys Gln Ser Gly     130 135 140 Tyr Asn Lys Met Ile Gly Met Arg Ser Asp Leu Val Gly Gly Ile Thr 145 150 155 160 Asn Gly Gln Thr Met Pro Ala Ala Tyr Leu Asn Leu Pro Ile Pro Leu                 165 170 175 Phe Phe Thr Arg Asp Thr Gly Leu Ala Leu Pro Thr Val Ser Leu Pro             180 185 190 Tyr Asn Glu Val Arg Ile His Phe Lys Leu Arg Arg Trp Glu Asp Leu         195 200 205 Leu Ile Ser Gln Ser Thr Gln Ala Asp Met Ala Ile Ser Thr Val Thr     210 215 220 Leu Ala Asn Ile Gly Asn Val Ala Pro Ala Leu Thr Asn Val Ser Val 225 230 235 240 Met Gly Thr Tyr Ala Val Leu Thr Ser Glu Glu Arg Glu Val Val Ala                 245 250 255 Gln Ser Ser Arg Ser Met Leu Ile Glu Gln Cys Gln Val Ala Pro Arg             260 265 270 Val Pro Val Thr Pro Val Asp Asn Ser Leu Val His Leu Asp Leu Arg         275 280 285 Phe Ser His Pro Val Lys Ala Leu Phe Phe Ala Val Lys Asn Val Thr     290 295 300 His Arg Asn Val Gln Ser Asn Tyr Thr Ala Ala Ser Pro Val Tyr Val 305 310 315 320 Asn Asn Lys Val Asn Leu Pro Leu Leu Ala Thr Asn Pro Leu Ser Glu                 325 330 335 Val Ser Leu Ile Tyr Glu Asn Thr Pro Arg Leu His Gln Met Gly Val             340 345 350 Asp Cys Phe Thr Ser Val Asp Pro Tyr Tyr Phe Ala Pro Ser Met Pro         355 360 365 Glu Met Asp Gly Val Met Thr Tyr Cys Tyr Thr Leu Asp Met Gly Asn     370 375 380 Ile Asn Pro Met Gly Ser Thr Asn Tyr Gly Arg Leu Ser Asn Val Thr 385 390 395 400 Leu Ser Cys Lys Val Ser Asp Asn Ala Lys Thr Thr Ala Ala Gly Gly                 405 410 415 Gly Gly Asn Gly Thr Gly Tyr Thr Val Ala Gln Lys Phe Glu Leu Val             420 425 430 Val Ile Ala Val Asn His Asn Ile Met Lys Ile Ala Asp Gly Ala Ala         435 440 445 Gly Phe Pro Ile Leu     450 <210> 2 <211> 406 <212> PRT <213> Paralichthys olivaceus <220> <221> PEPTIDE (222) (1) .. (406) <223> iridovirus antigen peptide <400> 2 Met Glu Thr His Leu Tyr Gly Gly Asp Asn Ala Val Thr Tyr Phe Ala   1 5 10 15 Arg Glu Thr Val Arg Ser Ser Trp Tyr Ser Lys Pro Pro Val Thr Leu              20 25 30 Ser Lys Gln Thr Gly His Ala Asn Phe Gly Gln Glu Phe Ser Val Thr          35 40 45 Val Ala Arg Gly Gly Asp Tyr Leu Ile Asn Val Trp Leu Arg Val Lys      50 55 60 Ile Pro Ser Ile Thr Ser Ser Lys Glu Asn Ser Tyr Ile Arg Trp Cys  65 70 75 80 Asp Asn Leu Met His Asn Leu Val Glu Glu Val Ser Val Ser Phe Asn                  85 90 95 Asp Leu Val Ala Gln Thr Leu Thr Ser Glu Phe Leu Asp Phe Trp Asn             100 105 110 Ala Cys Met Met Pro Gly Ser Lys Gln Ser Gly Tyr Asn Lys Met Ile         115 120 125 Gly Met Arg Ser Asp Leu Val Gly Gly Ile Thr Asn Gly Gln Thr Met     130 135 140 Pro Ala Ala Tyr Leu Asn Leu Pro Ile Pro Leu Phe Phe Thr Arg Asp 145 150 155 160 Thr Gly Leu Ala Leu Pro Thr Val Ser Leu Pro Tyr Asn Glu Val Arg                 165 170 175 Ile His Phe Lys Leu Arg Arg Trp Glu Asp Leu Leu Ile Ser Gln Ser             180 185 190 Thr Gln Ala Asp Met Ala Ile Ser Thr Val Thr Leu Ala Asn Ile Gly         195 200 205 Asn Val Ala Pro Ala Leu Thr Asn Val Ser Val Met Gly Thr Tyr Ala     210 215 220 Val Leu Thr Ser Glu Glu Arg Glu Val Val Ala Gln Ser Ser Arg Ser 225 230 235 240 Met Leu Ile Glu Gln Cys Gln Val Ala Pro Arg Val Pro Val Thr Pro                 245 250 255 Val Asp Asn Ser Leu Val His Leu Asp Leu Arg Phe Ser His Pro Val             260 265 270 Lys Ala Leu Phe Phe Ala Val Lys Asn Val Thr His Arg Asn Val Gln         275 280 285 Ser Asn Tyr Thr Ala Ala Ser Pro Val Tyr Val Asn Asn Lys Val Asn     290 295 300 Leu Pro Leu Leu Ala Thr Asn Pro Leu Ser Glu Val Ser Leu Ile Tyr 305 310 315 320 Glu Asn Thr Pro Arg Leu His Gln Met Gly Val Asp Cys Phe Thr Ser                 325 330 335 Val Asp Pro Tyr Tyr Phe Ala Pro Ser Met Pro Glu Met Asp Gly Val             340 345 350 Met Thr Tyr Cys Tyr Thr Leu Asp Met Gly Asn Ile Asn Pro Met Gly         355 360 365 Ser Thr Asn Tyr Gly Arg Leu Ser Asn Val Thr Leu Ser Cys Lys Val     370 375 380 Ser Asp Asn Ala Lys Thr Thr Ala Ala Gly Gly Gly Gly Asn Gly Thr 385 390 395 400 Gly Tyr Thr Val Ala Gln                 405 <210> 3 <211> 1218 <212> DNA <213> Paralichthys olivaceus <220> <221> transit_peptide (222) (1) .. (1217) <223> iridovirus antigen peptide sequence <400> 3 atggagaccc acttgtatgg cggcgacaat gccgtgacct actttgcccg tgagaccgtg 60 cgtagttcct ggtacagcaa gccgcccgtc accctatcaa aacagactgg ccatgctaat 120 ttcggccagg agtttagtgt gactgtggca aggggtggcg actacctcat taatgtgtgg 180 ctgcgtgtta agatcccctc catcacgtcc agcaaggaga acagctacat tagatggtgt 240 gataatttga tgcacaatct agttgaggag gtgtcggtgt catttaacga cctggtggca 300 cagaccctga ccagcgagtt ccttgacttt tggaacgcct gcatgatgcc tggcagcaaa 360 caatctggct acaacaagat gattggcatg agaagcgacc tggtgggcgg tatcaccaac 420 ggtcagacta tgcccgccgc ctaccttaat ttgcccattc ccctgttctt tacccgtgac 480 acaggccttg cattgcctac tgtgtctctg ccgtacaatg aggtgagaat ccacttcaag 540 ctgagaagat gggaggacct gctcatcagc cagagcaccc aggccgacat ggccatatcg 600 actgtcaccc tggctaacat tggcaatgta gcacccgcac tgaccaacgt gtccgtgatg 660 ggcacctacg ctgtactgac aagtgaggag cgtgaggttg tggcccagtc tagccgtagc 720 atgctcattg agcagtgtca ggtggcgcct cgtgtgcctg tcacacccgt agacaattcc 780 ttggtgcatc tcgacctgag gttcagtcac cctgtgaagg ccttgttctt tgcagtcaag 840 aatgtcactc acagaaacgt gcaaagcaat tacaccgcgg ccagccccgt gtatgtcaac 900 aacaaggtga atctgccttt gctggccacc aatcccctgt ccgaggtgtc gctcatttac 960 gagaacaccc ctagactcca ccagatggga gtagactgct tcacatctgt cgacccctac 1020 tactttgcgc ccagcatgcc tgagatggat ggtgttatga cctactgtta tacgctggac 1080 atgggcaata tcaaccctat gggctcgacc aactacggca gactgtccaa cgtcaccctg 1140 tcatgtaagg tgtcggacaa tgccaagacc accgcggcgg gcggtggagg caacggcacc 1200 ggctacacgg tcgcccaa 1218  

Claims (11)

Amino acids 1 to 21 and 427 to 454 of the capsid protein of the seawater fish iridovirus consisting of the amino acid sequence of SEQ ID NO: 1 are removed, and amino acids 99, 153, 197, 203 and 204 Sea fish irido consisting of the amino acid sequence set forth in SEQ ID NO: 2, wherein the CGC or CGG codons (203 and 346) at sites 306, 346 and 395 are site-directed mutagenesis with an AGA codon Viral antigenic peptides. delete delete Recombinant nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 3 encoding the marine fish iridovirus antigenic peptide according to claim 1. delete Recombinant vector comprising a recombinant nucleic acid according to claim 4. A transformed microorganism expressing the antigenic peptide according to claim 1. The method of claim 7, wherein The microorganism is a transformed microorganism, characterized in that the Phichia pastoris ( Phichia pastoris ). A method of producing a capsid protein of saltwater fish idovirus by culturing the microorganism according to claim 7. Iridovirus prevention vaccine containing the antigenic peptide according to claim 1 as an active ingredient. The method of claim 10, The vaccine is a vaccine for the prevention of iridovirus, characterized in that oral or edible ingestion is possible.

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