JP2008061607A - Aggregated/immobilized antigen protein of cryptocaryon irritans, dna of the same and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling Cryptocaryon irritans that is a causing parasite of the white spot disease of marine fishes and preventing infection by the same, concretely, to provide an aggregated/immobilized antigen protein of the Cryptocaryon irritans, a DNA encoding the aggregated/immobilized antigen protein, an antibody against the aggregated/immobilized antigen of the parasite and a vaccine for preventing the infection by the parasite. <P>SOLUTION: The aggregated/immobilized antigen protein of the Cryptocaryon irritans, the DNA encoding the protein, the gene recombinant body obtained by expressing the DNA, the antibody against the aggregated/immobilized antigen of the parasite and the vaccine for preventing the infection by the parasite are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to proteins involved in parasite aggregation / immobilization, DNA encoding the same, and uses thereof. Specifically, it protects against aggregation / immobilized antigen protein of Cryptocaryon irritans, DNA encoding the aggregation / immobilized protein, antibodies against the aggregation / immobilized antigen of parasites and infection of parasites Regarding vaccines.

The marine white spot worm Cryptocaryon irritans , belonging to the protozoan Ciliate class, infests saltwater fish and causes white spot disease. As a result, the parasitic fish are weakened and die. In recent years, seawater white spotted insect parasitism on cultured fish has become a serious problem in the Japanese aquaculture industry. It has been reported that seawater white spotted insects parasitize flounder Paralichthys olivaceus , amberjack Seliola dumerili , yellowtail Seriola quinqueradiata , red sea bream Pugrus major , and tiger pufferfish . The life history of seawater white spotted worms is similar to that of freshwater white spotted worm Ichyhyophthitius multifiliis causing white spot disease of freshwater fish. It has a Seronto stage. Trohonto parasitizes the epidermis of the fish's epidermis and coral and grows to a size that is visible when nourished from the host tissue.

Tetrahymena thermophila , Paramecium aurelia, and freshwater white spotted worms, which also belong to the protozoan ciliate class, express aggregated antigens on their surface and produce antibodies that aggregate them in vitro when the protein is inoculated into rabbits. (Non-Patent Documents 1 and 2). In addition, it has been clarified that fish infected with freshwater white spotted insects recognize the antigen of freshwater white spotted caterpillar and produce antibodies that aggregate freshwater white spotted insects in vitro (Non-Patent Documents 3 and 4). ). Studies using immunological techniques have revealed that freshwater white spotted aggregating antigens are expressed on the surface of cilia (Non-patent Document 3). Although the role of this antigen in freshwater white spotworms has not been clarified, inoculation of catfish (channel catfish Ictalurus punctatus ) with this purified antigen can immunize freshwater white spotworms and prevent freshwater white spotworms from infesting In addition, the survival rate is improved in an attack test using freshwater white spotted insects (Non-patent Document 5). Similarly, it is known that fish immunized with Seronto can be resistant to subsequent infection in seawater white spotted insects (Non-patent Document 6).

BrunsPJ. Immobilization antigens of Tetrahymena pyriformis I. Assay and extraction. Exp Cell Res 1971; 65: 445-453. Jones IG. 1965. Immobilization antigen in heterozygous clones of Paramecium aurelia. Nature 1965; 207: 769. DickersonHW, Clark TG, Findly RC. 1989. Ichthyophthirius multufiliis hasmembrane-associated immobilization antigens. Journal of Protozool 1989; 36: 159-164. IglesiasR, Parama A, Aivarez MF, Leiro J, Ubeira FM, Sanmertin ML. Philasteridesdicentrarchi (Ciliophora: Scuticociliatide) express surface immobilizationantigens that probably induce protective immune responses in turbots.Parasitology 2002; 126: 125-134. WangX, Dickerson HW. Surface immobilization antigens of the parasitic ciliate Ichthyophthiriusmultufiliis elicits protective immunity in channel catfish (Ictaluruspunctatus). Clin Diagn Lab Immunol 2002; 9: 176-181. YoshinagaT, Nakazoe J. Acquired protection and production of immobilization antibodyagainst Cryptocaryon irritans (Ciliophora, Hymenostomatida) inmummichog (Fundulus heteroclitus). Fish Pathol 1997; 32: 229-230.

  An object of the present invention is to provide a method for controlling seawater white spot worms, which are causative parasites of white spot disease of marine fish, and for preventing infection. Specifically, seawater white spot worm aggregation / immobilization antigen protein, DNA encoding the aggregation / immobilization protein, antibody against parasite aggregation / immobilization antigen, and vaccine protecting against parasitic infection This is the issue.

  As a result of diligent research, the present inventors have found that trough puffers and rabbits immunized with a sea lion's serpent seronto produce an antibody that aggregates the sea scab's serotoon in vitro. The agglutinating antigens of seawater white spot worms were identified by immunological techniques for 114 extracted fractions, and the present invention was completed. The agglutinating / immobilizing antigen of this marine white spotted caterpillar is a major protein on the surface of seronto and trohonto and is recognized by rabbit and trough antiserum having aggregation / immobilizing activity.

The gist of the present invention is the protein (1) below.
(1) A protein represented by the following (a), (b), (c) or (d).
(A) Aggregated antigen protein of seawater white spot worm ( Cryptocaryon irritans ) represented by any amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 in the sequence listing.
(B) An isoform of a protein having the amino acid sequence of the aggregate antigen protein of Cryptocaryon irritans represented by any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, or 10 in the sequence listing.
(C) having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown by any of SEQ ID NOs: 2, 4, 6, 8, or 10 in the sequence listing; And the protein which has the biological activity of the aggregation antigen protein of seawater white spotted insects.
(D) having an amino acid sequence having 80% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8, or 10, and A protein having biological activity.

In addition, the gist of the present invention is DNA encoding the protein, DNAs (2) and (3) below, vectors containing these DNAs, and hosts incorporating the vectors.
(2) DNA encoding the protein according to (1) above.
(3) The DNA according to claim 2, which is a DNA represented by the following (a), (b), (c), or (d):
(A) DNA represented by any one of SEQ ID NOs: 1, 3, 5, 7, or 9 in the sequence listing.
(B) DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3, 5, 7, or 9 in the sequence listing.
(C) a base sequence having 80% or more homology with the base sequence represented by any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and DNA encoding a protein having biological activity.
(D) DNA that hybridizes under stringent conditions with the nucleotide sequence shown in any one of SEQ ID NOs: 1, 3, 5, 7, or 9 in the sequence listing.

The gist of the present invention is the following (4) to (7).
(4) A method for producing an agglutinated antigen protein of seawater white spot worms, which comprises culturing the host and collecting the agglutinated antigen protein of seawater white spotted worms from the culture.
(5) A gene recombinant protein obtained by the method of (4).
(6) An antibody that recognizes an agglutinating antigen protein of seawater white spotted insects as described in (1) or (5) above.
(6) A vaccine for protection against seawater white spot insect infection, comprising the protein according to (1) or (5) above.
(7) A DNA vaccine for protection against seawater white spot insect infection, comprising the DNA according to (2) or (3) above.

  INDUSTRIAL APPLICABILITY According to the present invention, an aggregated antigen protein of seawater white spot worms, a DNA encoding the same, an antibody that recognizes the antigen, and a vaccine containing the antigen are provided, and seawater white spot worms can be controlled and protected against infection. .

  Molecular biology, parasitology, immunology and biochemical techniques such as cDNA library preparation, gene cloning, screening and nucleotide sequence determination in the present invention are described in J. Sambrook, EFFritsch & T. Maniatis (1989). ): Molecular Cloning, alaboratory manual, second edition, Cold Spring Harbor Laboratory Press; Ed Harlow and David Lanc (1988): Antibodies, a laboratory manual, Cold Spring Harbor Laboratory Press; Veterinary Clinical Parasitology Editorial Board (1998) The methods described in literatures well known to those skilled in the art, such as veterinary clinical parasitology, Bunei-do, etc. may be used. DNA analysis can be performed using software such as GENETYX (Software Development).

  The protein of the present invention has an amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8, or 10, an isoform thereof, or a similar protein thereof, that is, SEQ ID NOs: 2, 4, 6, 8, Or having substantially the same properties as a protein having the amino acid sequence shown in any one of 10 and having one or several amino acids deleted, substituted, inserted or added in the amino acid sequence, Alternatively, it has a homology of 80% or more, preferably 90% or more, with the amino acid sequence of the seawater white spotworm aggregation / immobilized antigen. Here, the substantially homogeneous property means that it has the biological activity of agglutination / immobilized antigen protein of seawater white spot worms, and having biological activity means that the host that has ingested the protein has the biological activity. It means producing an antibody that aggregates / immobilizes seawater white spot worms.

  The DNA of the present invention includes all DNAs encoding a seawater white spot worm aggregation / immobilization antigen protein having any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, or 10. Further, in the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, or 10, the amino acid sequence has one or several amino acids deleted, substituted or added, and Also included are all DNAs that encode proteins having the biological activity of aggregated / immobilized antigen proteins.

  Furthermore, the DNA of the present invention is a DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, or 9 (DNA encoding a seawater white spot worm aggregation / immobilized antigen), or a sequence thereof. It contains DNA. Also included are DNAs having 80% or more homology with these DNAs, and all DNAs that hybridize under stringent conditions with DNAs that are complementary to these DNAs. Here, stringent conditions are high only when there is 80% or more homology, preferably 90% or more homology, between DNA sequences encoding the seawater white spot worm aggregation / immobilization antigen. It means the conditions under which hybridization occurs. Usually, the hybridization occurs at a temperature lower than the melting temperature of the complete hybrid by about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. Stringent conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), and the conditions described herein can be used.

The cDNA encoding the seawater white spot worm aggregation / immobilization antigen of the present invention can be obtained by the following method.
The seawater white spot worm Seronto can be collected from the cyst attached to the bottom of the aquarium where the seawater white spotted insects are bred, and the trohonto can be taken from the infected fish cage. MRNA (poly (A) RNA) is purified from Seronto using a commonly used method or a commercially available mRNA isolation kit. For example, after treating Seronto with guanidine reagent, phenol reagent, etc. to obtain total RNA, affinity column method using poly U-Sepharose etc. with oligo dT-cellulose or Sepharose 2B as carrier, or batch method poly (A) + mRNA can be obtained.

  Using the mRNA thus obtained as a template, synthesize single-stranded cDNA using reverse transcriptase, synthesize double-stranded DNA using DNA polymerase, and incorporate it into an appropriate vector. Then, transform E. coli etc. to prepare cDNA library. cDNA can be incorporated into a vector by a conventional method using an appropriate restriction enzyme and ligase. For example, there is a method in which the obtained cDNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of an appropriate vector DNA, and ligated to a vector. At this time, a cDNA library can be obtained using the vectors and host cells described below.

  The target DNA is selected from the cloned DNA library thus obtained. As a selection method, methods such as an immunoscreening method, a plaque hybridization method, and a colony hybridization method can be used. For example, the obtained clone group is expressed with an inducer such as isopropyl-1-thio-β-D-galactoside (IPTG), transferred to a nylon membrane or a cellulose membrane, and an antibody against the target protein or the Immunologically corresponding clones can be selected using serum containing antibodies. The nucleotide sequence of the target cDNA is determined from the thus prepared clone.

  The base sequence can be determined by, for example, the Maxam Gilbert method (Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sci. USA., 74, 560, 1977) or the dideoxy method (Messing, Jet al., Nucl. Acids Res. ., 9, 309, 1981) and the like. It is also possible to determine the sequence using an automatic base sequence analyzer that applies these principles.

  The obtained target DNA can be amplified by a gene amplification method such as polymerase chain reaction (PCR). PCR can be performed, for example, by the technique described in Protein Nucleic Acid Enzyme “Frontier of PCR Method: From Basic Technology to Application”, Vol. 4, No. 5, April 1996 issue, Kyoritsu Publishing. After cloning or amplifying the target DNA, the target DNA is recovered, incorporated into an appropriate expression vector that can be obtained, further transformed into an appropriate host cell, cultured and expressed in an appropriate medium, The target protein can be recovered and purified.

  As the vector at this time, any vector can be used as long as it can be replicated in a host cell such as a plasmid, a phage, or a virus. Examples include E. coli plasmids such as pBR322, pBR325, pUC118, pUC119, pKC30, and pCFM536, Bacillus subtilis plasmids such as pUB110, yeast plasmids such as pG-1, YEp13, and YCp50, and phage DNA such as λgt110 and λZAPII. Examples of vectors for mammalian cells include viral DNA such as baculovirus, vaccinia virus, adenovirus, SV40 and its derivatives. The vector contains a replication origin, a selectable marker, and a promoter, and may contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like as necessary.

  Commercially available vectors can be used. For example, pHSG299 (manufactured by Takara Bio Inc.), pQE70, pQE60, pQE-9 (Qiagen), pBluescriptII, ptrc99a, pKK223- 3, pDR540, pRIT2T (GE Healthcare Bioscience), pT7blue, pET-11a (Novagen), etc.

  As an origin of replication, for E. coli vectors, for example, those derived from ColiE1, R factor, F factor, for yeast vectors, for example, 2 μm DNA, those derived from ARS1, for mammalian cells, for example, SV40, Those derived from adenovirus and bovine papilloma virus can be used. In addition, adenovirus or SV40 promoter as promoter, phage lambda PL promoter, phage lambda PL promoter, ADH, PHO5, GPD, PGK, AOX1 promoter for yeast, nuclear polyhedrosis virus-derived promoter for sputum cells, etc. Can be used.

  As selection markers, vectors for E. coli include kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, etc., yeast vectors include Leu2, Trp1, Ura3 gene, etc., and mammalian cells include neomycin resistance gene, thymidine kinase. Gene, dihydrofolate reductase gene and the like.

  DNA can be introduced into the vector by any method. The vector preferably contains a polylinker with various restriction sites therein or contains a single restriction site. A specific restriction site in the vector can be cleaved with a specific restriction enzyme, and DNA can be inserted into the cleavage site. An expression vector containing the DNA and regulatory sequences of the present invention can be used for transformation of an appropriate host cell to cause the host cell to express and produce the seawater white spot worm aggregation / immobilized antigen of the present invention.

  Hosts include bacterial cells such as Escherichia coli, Streptomyces, Bacillus subtilis, fungal cells such as Aspergillus spp., Yeast cells such as baker's yeast and methanol-utilizing yeast, insect cells such as Drosophila S2 and Spodoptera Sf9, CHO, COS And mammalian cells such as BHK, 3T3, and C127. Transformation can be performed by known methods such as calcium chloride, calcium phosphate, DEAE-dextran mediated transfection, electroporation and the like.

  The obtained recombinant protein can be separated and purified by various separation and purification methods. For example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used alone or in appropriate combination. In this case, when the expression product is expressed as a fusion protein with GST or the like, it can be purified by utilizing the properties of the protein or peptide fused with the target protein. For example, when expressed as a fusion protein with an amino acid sequence of 6 or more histidines, so-called histidine tag, the protein with histidine tag binds to the chelate column and can be purified using the chelate column. When expressed as a fusion protein with GST, GST has an affinity for glutathione and can therefore be efficiently purified by affinity chromatography using a column in which glutathione is bound to a carrier.

  Antibodies against seawater white spot worm aggregation / immobilized antigens can be prepared as follows. Using the agglutinated / immobilized antigen protein as an antigen, inoculate several times subcutaneously, intramuscularly, intraperitoneally, and intravenously in animals such as mice, guinea pigs, rabbits, goats and the like according to methods well known to those skilled in the art, Thereafter, blood is collected from the animal and serum is separated to produce an anti-roundworm-infected larva 16 kDa antigen antibody. At this time, an appropriate adjuvant can also be used. Monoclonal antibodies can also be produced by known methods. For example, a hybridoma obtained by cell fusion of spleen cells of mice immunized with agglutination / immobilized antigen of seawater white spotted worms and myeloma cells of mice is prepared, and the culture supernatant of the hybridoma or the hybridoma is administered intraperitoneally Can be prepared from ascites of isolated mice. The agglutination / immobilization antigen of seawater white spot worms used as an immunizing antigen may be a natural protein, a recombinant protein extracted from seawater white spotted worms, or a chemically synthesized one. Further, it may be a protein having the entire amino acid sequence, a peptide fragment having a partial structure of the protein, or a fusion protein with another protein. As the peptide fragment, a fragment obtained by degrading the protein with an appropriate proteolytic enzyme can be used, or a part of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9 is incorporated into an expression vector for expression. The product may be good. A fusion protein is produced by linking a part of the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, or 9 with a gene encoding another protein, incorporating it into an expression vector, and expressing it. Alternatively, the polypeptide fragment can be used after it is chemically bound to an appropriate carrier protein. The reactivity of the obtained antibody can be measured by methods well known to those skilled in the art, such as enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blotting.

  The seawater white spot worm aggregation / immobilized antigen can be used as a vaccine to prevent seawater white spot worm infection. The agglutination / immobilization antigen protein of seawater white spot worms used as a vaccine may be a natural protein, a recombinant protein extracted from sea water white spot worms, or a chemically synthesized one. Further, it may be a protein having the entire amino acid sequence, a peptide fragment having a partial structure of the protein, or a fusion protein with another protein. In the case of a fragment, it is necessary that the fragment contains an epitope (neutralizing epitope) recognized by an antibody (neutralizing antibody) that protects against infection with seawater white spot worms. As the peptide fragment, a fragment obtained by degrading the protein with an appropriate proteolytic enzyme can be used, or a part of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9 is incorporated into an expression vector for expression. The product may be good.

  The vaccine can be one or several adjuvants, such as Freund's complete or incomplete adjuvant, cholera toxin, heat-labile E. coli toxin, aluminum hydroxide, potassium alum, saponin or derivatives thereof, muramyl dipeptide, mineral oil or vegetable oil, NAGO , Novasome or nonionic block copolymers, DEAE dextran, and the like. Further, it may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be, for example, sterile water or a sterile physiological salt solution. In the case of more complex forms, the carrier can be, for example, a buffer.

The vaccine of the present invention can be administered by a normal active immunization method, and can be a systemic vaccine administered by injection or a mucosal-derived vaccine administered by oral administration or the like without injection. That is, single or repeated administration in a manner appropriate to the dosage form in a prophylactically effective amount (ie, an immunizing antigen capable of expressing an antigen that induces immunity in the animal against attack by sea scabs) Can be administered. The vaccine can be administered intramuscularly, intraperitoneally, intravenously, or orally. Also, the vaccine of the present invention can be effectively mixed with other antigen components of the same and / or other parasites.
The dose and frequency of administration of the vaccine may vary depending on the target fish. For example, the seawater white spot worm aggregation / immobilized antigen of the present invention is 0.1 to 100 mg, preferably 1 to 50 mg, per week to several weeks. Protective immunity can be induced by administration once to several times at a frequency of once.

The 32 kDa protein represented by any one of SEQ ID NOs: 2, 4, 6, 8, or 10 of the present invention is an aggregation / immobilization antigen of seawater white spot worms. It prevents the infection in the insect attack test. The molecular weight of the surface antigen reported for T. thermophila , P. aurelia and I. multifiliis , also belonging to the protozoan ciliate class, is about 40-250 kDa, and the 32 kDa protein of the present invention also has some function outside the cell. It is considered to be a GPI anchor protein having The 32 kDa protein is homologous to Paramecium tetraurelia 51A surface protein (GenBank: 159973), Paramecium primaurelia G surface protein (GenBank: 3452505), and Tetrahymena thermophila SerH3 immobilization antigen (GenBank: 102396) by homology search using BLAST search. The SMART program was found to have a motif similar to the surface antigen of the genus Paramecium with periodic cysteine residues. The 32 kDa protein of the present invention is the major protein on the surface of seronto and trohonto and was detected by sera with rabbit / troughfish agglutination / immobilization activity. Aggregation / immobilized antigen inoculation of freshwater white spot worms immunizes catfish, prevents subsequent freshwater white spotworm infection, and improves survival in challenge studies. Similarly, it can be said that the 32 kDa protein of the present invention can also be used as a vaccine for preventing infection by seawater white spot worms.
From the base sequence analysis of the 32 kDa protein, it was found that TAA and TAG codons were used as glutamine codons in seawater white spotted worms. The 32 kDa protein gene can be used for the production of recombinant proteins for vaccination of fish. In this case, the TAA codon in the 32 kDa protein-coding gene can be used as a freshwater white spot In this case, it is necessary to change to CAA or CAG, which is a normal codon for glutamine, by using a DNA point mutant preparation method such as assembly PCR.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

Aggregation / immobilization activity of rabbit and tiger puffer serum
1. Fish breeding Troughfish (about 150 g) hatched at our laboratory were bred in a 20-ton polycarbonate aquarium. The water tank was filtered with sand and seawater irradiated with ultraviolet rays was supplied at a flow rate of 1.2 L / min. A commercially available 3 mm pellet feed (manufactured by Nihon Suisan Co., Ltd.) was used as a bait, and an amount of 3% of the fish weight was fed twice a day. The seawater used was salinity 34 ppt, pH 8.1, chemical oxygen demand 1.0 mg / l throughout the year.
Before the test, 10 animals were collected arbitrarily, and their wrinkles and skin were observed under a microscope to confirm that the puffer fish used in the test was not infected with parasites.

2. Rearing of parasites Seawater white spotted caterpillars have been maintained at the Institute as a host in red sea bream raised in a 100-liter water tank at a water temperature of 25 ± 1 ° C. Since the cysts of seawater white spotted insects adhere to the bottom of the aquarium, glass slides were placed on the bottom of the aquarium and the slide glass with the cysts collected after 15 hours was collected. The Seronto was hatched after 5-8 days in a 300 mL beaker containing seawater maintained at 25 ° C., and used for the experiment within 12 hours after hatching. Trohonto dissects the wings of the infected fish under a microscope and collects them using a Pasteur pipette. The hatched seronto was concentrated using a cell strainer (diameter: 2.7 cm, mesh: 5 μm). Further, the seawater containing the seronto was transferred to a 1.5 mL tube and centrifuged at 2000 g / 5 minutes to remove the seawater. Seronto and trohonto were stored at -80 ° C until testing.

3. Fish immunization In order to observe the time course of antibody titer using the troughfish of Example 1, ultrasonically disrupted the seronto (0.5 mg / kg (fish weight)) obtained in Example 2 was FCA (Freund complete adjuvant). ) And inoculated intraperitoneally (11 individuals) of tiger puffer. As a negative control, BSA (bovine serum albumin) was similarly inoculated into trough fish (9 individuals). Two weeks after the first inoculation, the same amount was boosted by the same method. A small amount of blood was collected from the tail vein at 2, 6 and 10 weeks after the booster to obtain serum. Serum was stored at -80 ° C until testing.

4). Preparation of antiserum against rabbit immune serotopes was performed using female Japanese white rabbits. Seronto was mixed with FIA (Freund's incomplete adjuvant) and then inoculated 20 sites subcutaneously on the dorsal side. Seronto of 400 μg (about 1,000,000 individuals) was used for the first time, and 200 μg (about 500,000 individuals) was used for booster (total 3 times every 2 weeks). Blood was collected 8 weeks after the first immunization, coagulated at 4 ° C., and then centrifuged at 300 × g for 5 minutes to obtain serum. Serum was inactivated by incubation at 56 ° C. for 30 minutes and stored at −80 ° C. until used for testing.

5. Aggregation / immobilization assay The aggregation / immobilization assay is basically the same as Clark et al. (Clark TG, Dickerson HW, Findly RC. Immune response of channel catfish to ciliary antigens of Ichthyophthirius multifiliis . Dev Comp Immunol 1988; 12: 581-594) The method was followed. Seawater (990 μL) containing about 100 parasites was dispensed into a 24-well plate, serum (10 μL) diluted with seawater was added, and the mixture was incubated at 25 ° C. for 1 hour. Aggregation was observed under a microscope (BX50, Olympus Optical Co., Ltd.). After counting the aggregated individuals, 50 μL of formalin was added, and the total number of individuals contained in the wells was counted. Aggregation activity was calculated by (number of aggregated individuals) / (total number of individuals) × 100. Each serum was assayed in triplicate.

As a result, in rabbits and tiger puff sera immunized with Seronto, Seront became unable to swim and formed aggregates at the bottom (FIG. 1). In the immunized serum, aggregation was observed at 1/100 dilution, but in the non-immunized serum almost no aggregation was observed even at 1/10 dilution.
The antiseronto sera from tiger puffer and rabbits aggregated the seronto in vitro, as did the serotons on the surface, as in T. thermophila , P. aurelia , I. Multifiliis , P. dicentrarchi and N. gillerae. Is expressed. Since the cilia of the Seronto aggregated when observed with a microscope, it is considered that the agglutinate / immobilized antigen of seawater white spotted insects is expressed on the surface of the cilia as in the other ciliates described above.

Antibody titer in trough serum
1. Preparation of trough IgM Serum collected from immunized or uninfected tiger puffer was roughly fractionated by precipitation with ammonium sulfate, and the fraction containing IgM was dialyzed against 10 mM PBS. Next, it was subjected to gel filtration using Sephacryl S-300 HR (Amersham Pharmacia Bioscience). Gel filtration was performed at a flow rate of 0.216 mL / min, and the concentration of the eluted protein was monitored at an absorbance of 280 nm. The fraction containing IgM was subjected to SDS-PAGE, and bands considered to be heavy chains (about 75 kDa) and light chains (about 25 kDa) of IgM were cut out from the gel and recovered. Protein was eluted from the gel and used to immunize rabbits.

2. ELISA
The frozen Seronto was thawed and then suspended in PBS to a final concentration of 50 μg / mL. This Seronto suspension was added to a 96-well plate (Greiner Labortechnik) at 100 μL per well and incubated at room temperature for 2 hours. After blocking at room temperature for 2 hours with Block Ace, 1 / 100-diluted trough puff serum was added and incubated overnight at 4 ° C. After washing the plate three times with PBS-T, the rabbit anti-rafugu IgM serum diluted 1 / 1,000 and the alkaline phosphatase-conjugated goat anti-rabbit IgG antibody diluted 1 / 25,000 (made by Kirkegaard & Perry Laboratories) at room temperature, A 2 hour incubation was performed. Color development was performed using Alkaline-Phosphatase Substrate (manufactured by Bio-Rad), and the antibody titer was measured by measuring TECAN Rainbow Thermo (manufactured by Wako Pure Chemical Industries) with absorption at 405 nm. Each serum was tested in triplicate and the average value was used. The results are shown in FIG.

As a result, 2 weeks after the booster, obvious antibody production against Seronto was observed, which was significant compared to that in the control group (Student's t-test; P <0.001). Absorption at 405 nm showed a maximum value during 2-6 weeks.
FIG. 3 shows the correlation between the results of Example 1 and the antibody titer by ELISA. From this, it was found that there is a certain correlation between the aggregation / immobilization activity and the antibody titer against Seronto. This result indicates that Seronto surface antigen-specific antibodies are produced in rabbits and troughs, and that they contribute to the aggregation / immobilization activity.

Identification of aggregated / immobilized antigens
1. Extraction of membrane protein from seawater white spotworms Extraction of membrane protein from whitewater white spotted insects was carried out by phase separation using the previously reported Triton X-114 (Sigma-Aldrich) (Journal of Protozool 1989; 36: 159-164, J Biol Chem. 1981; 256: 1604-1607). Frozen seawater white spot worms were thawed and suspended in 100 μL, 10 mM Tris-HCl (pH 7.5) cooled in ice in a 1.5 mL tube. Next, an equal volume of extraction buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 2% [v / v] Triton X-114) was added and incubated on ice for 1 hour. The cytoskeletal protein was removed by centrifugation at 100,000 × g for 60 minutes at 4 ° C., and the resulting supernatant was removed from the separation buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.06% (v / v) Triton X-114). After incubation at 32 ° C. for 5 minutes, the Triton X-114 phase was separated by centrifugation at 300 × g for 3 minutes at room temperature. Again, the supernatant was recovered, Triton X-114 was added to a final concentration of 0.5% (v / v), and incubation was performed on ice for 1 hour. Again, after superimposing the supernatant in the previous separation buffer, incubate at 32 ° C for 5 minutes, and centrifuge at 300 xg for 3 minutes at room temperature to separate the Triton X-114 phase containing membrane protein did. The Triton X-114 phase was collected, 9 times the amount of ice-cooled acetone was added, and the mixture was incubated on ice for 1 hour, and then the protein was precipitated by centrifugation (16,000 g / 20 minutes). Acetone was removed after centrifugation, dried with a vacuum pump, suspended in 100 μL of 10 mM Tris-HCl (pH 7.5), and stored at −80 ° C. until use.

2. Purification of membrane protein The membrane protein of white spotworm was separated by anion exchange chromatography using a Mini Q PC 3.2 / 3 column (GE Healthcare Bioscience). Anion exchange chromatography uses 50 mM Tris-HCl buffer (pH 9.5) containing 0.5% NP 40 and 0.5% Triton X-100 at a flow rate of 0.2 ml / min, and a protein with a linear gradient of 0-0.5 M NaCl. Was eluted. The results are shown in FIG. The fraction containing C membrane protein was concentrated by ultrafiltration using an ultrafiltration spin column (manufactured by Vivascience) and subjected to acetone precipitation in order to remove the remaining surfactant. Fractionation was performed from 1 minute after injection to 7 minutes at 1 tube / min. The precipitate was suspended in PBS and stored at -80 ° C.

  As a result, the purified membrane protein was not bound to the Mini Q column in the 50 mM Tris-HCl (pH 9.5) buffer containing 0.5% NP 40 and 0.5% Triton X-100, and was found in the flow-through fraction ( (Figure 5). On the other hand, the narrow protein was bound to the column and was found in the gradient buffer.

3. SDS-PAGE, immunoblotting and N-terminal amino acid sequence analysis
SDS-PAGE was performed using a 4-20% polyacrylamide gradient gel (10 μg / lane) and stained with Coomassie Brilliant Blue (CBB). The molecular weight of the protein was estimated by comparing with a molecular weight marker manufactured by Bio-Rad. Proteins separated by SDS-PAGE are Pall Fluoro Trans W Membranes (manufactured by Nippon Genetics) using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) at 2 mA / cm2 for 1 hour. Transcribed to. After the transfer, blocking was performed with Block Ace (Dainippon Pharmaceutical Co., Ltd.) containing 0.1% (v / v) Tween 20. Next, incubation was performed at room temperature for 1 hour in trough puffer (1/100 (v / v)) and rabbit serum (1 / 1,000 (v / v)) in which the membrane was diluted. Trafugu antibodies bound to the proteins on the membrane were 1 / 1,000 diluted rabbit anti-Tragugu IgM serum and 1 / 25,000 diluted alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Kirkegaard & Perry Laboratories) at room temperature 1 After incubation for a period of time, color was detected with nitroblue tetrazolium (NBT) / 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Roche Diagnostics) and detected. The rabbit antibody bound to the protein on the membrane was detected by coloring with NBT / BCIP after incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody at room temperature for 1 hour.
The protein band of about 32 kDa on the membrane was further subjected to N-terminal amino acid sequence analysis using PPSQ-21 protein sequencer (manufactured by Shimadzu Corporation). For immunoblot analysis, 10 ng of Seronto's major membrane protein (rabbit serum) and 5 μg (rafugu serum) were transferred onto a PDVF membrane by electroblotting.

As a result, it was considered that components of the Seronto membrane extracted by phase separation using Triton X-114 are involved in the aggregating activity. When the entire Seronto is electrophoresed (FIG. 6, lane 1), various proteins are observed, but when the Seronto membrane extract fraction is electrophoresed (FIG. 6, lane 2 (under non-reducing conditions) and lane 3 ( It can be seen that a protein of about 32 kDa is the major protein under reduced conditions)). When analyzed by immunoblot, it was strongly detected by rabbits and trough sera immunized with Seronto in a non-reducing state (FIG. 7). On the other hand, in the reduced state, this rabbit serum was hardly detected, and this trough puff serum was not detected at all.
The result of N-terminal amino acid sequence analysis of the approximately 32 kDa protein is shown in SEQ ID NO: 11 in the Sequence Listing.

Mixed Indirect immunofluorescence staining rabbit anti Seronto serum 10μl of 40μl of purified membrane proteins (10 mg / ml) solution of Seronto, or BSA solution (10 mg / ml) (negative control) were incubated for 3 hours at room temperature. Prior to use, the precipitate was removed by centrifugation at 10,000 g / 10 min.
In addition, Seronto's joint immunofluorescence staining was performed to examine the localization of the purified membrane protein. Seronto was fixed in breeding water by adding formalin to a final concentration of 1%, and collected using a 5 μm mesh filter. The formalin-fixed Seronto was washed several times with distilled water, spotted on a glass slide and air-dried at room temperature. After blocking with Block Ace to prevent non-specific antibody binding, the above absorbed serum is diluted with PBS-T so that the final serum concentration is 1/200, and incubated with Seronto for 2 hours at room temperature. It was. After washing with PBS-T three times, incubation with Alexa-conjugated goat anti-goat IgG antibody diluted 400-fold with PBS-T was performed at room temperature for 2 hours. After washing 3 times with PBS-T, it was embedded with Prolong Antifade Kit (Molecular Probes) and observed with a confocal laser microscope (FLUOVIEW FV300, Olympus Optical).

  When immunoblot using the above-mentioned absorbed serum was performed, purified membrane protein was detected in the antiserum absorbed with BSA, but purified membrane protein was not detected in the antiserum absorbed with purified membrane protein. It was judged that the absorption of the antiserum by the protein was successful (FIG. 8). When immobilization assay was performed using these sera, aggregation of white spot worms was observed in the antiserum absorbed with BSA, but almost no aggregation was observed in the antiserum absorbed with the purified membrane protein. In addition, in order to investigate the localization of the purified membrane protein, indirect immunofluorescence staining of Seronto was performed using these antisera, but the antisera absorbed with BSA strongly stained the ciliate of white spotworms. The antiserum absorbed with the membrane protein hardly stained, and as a result, it was considered that the purified membrane protein was expressed on the cilia surface (FIG. 9).

Isolation of approximately 32 kDa protein gene Extraction of total RNA from the Seront was performed using the RNeasy Midi kit (Qiagen), and 5 '/ 3' rapid amplification of cDNA ends (RACE) kit (Roche Diagnostics). CDNA synthesis was performed using A degenerate primer was synthesized from the N-terminal amino acid sequence analysis result of the 32 kDa protein (SEQ ID NO: 12 in the sequence listing), and an amplified fragment was obtained by 3′-RACE method. Amplification (PCR) was performed at 94 ° C./30 seconds after incubation at 94 ° C. for 15 minutes in a 50 μL reaction solution containing 25 μL HotStarTaq Master Mix (Qiagen), 10 pM degenerate primer and anchor primer, and 1 μL cDNA. A cycle of 55 ° C./1 minute and 72 ° C./2 minutes 30 seconds was performed 30 times, and finally, incubation at 72 ° C. for 10 minutes was performed. Subsequently, for 5′-RACE, cDNA was synthesized using 5′-Full RACE Core Set (Takara Bio Inc.) and 5 ′ phosphorylated oligonucleotide (SEQ ID NO: 13 in the Sequence Listing). Further, 25 μL HotStarTaq Master Mix (manufactured by Qiagen), 10 pM primer set (SEQ ID NO: 14 and 15), and 50 μL reaction solution containing 1 μL cDNA were incubated at 94 ° C. for 15 minutes. Thereafter, a cycle of 94 ° C./30 seconds, 58 ° C./1 minute, and 72 ° C./2 minutes was performed 30 times, and finally, incubation was performed at 72 ° C. for 10 minutes. Furthermore, primers were designed from unique sequences on the 5 ′ and 3 ′ sides, and the resulting nucleotide sequences were confirmed. The sequence was analyzed using a DNA sequencer (Model 3100, manufactured by Applied Biosystems), and 5 clones were analyzed to avoid PCR errors. As a result, five types of cDNA were obtained. The DNA and amino acid sequences are shown in SEQ ID NO: 1, 3, 5, 7, or 9 (DNA sequence) and SEQ ID NO: 2, 4, 6, 8, or 10 (amino acid sequence), respectively. Each amplified DNA fragment was subcloned into pHSG299 (Takara Bio Inc.) and transformed with JM109 competent cells (Takara Bio Inc.).

As a result, the protein of the present invention encoded 309 amino acid residues. In the 32 kDa protein gene, the TAA and TAG codons were presumed to be glutamine codons as in the genera Tetrahymena and Paramecium . Moreover, ACT and ACA tended to be used as the threonine codon in this gene as in the Paramecium genus. Furthermore, in the deduced amino acid sequence of this gene, a highly hydrophobic region on the C-terminal side peculiar to the GPI anchor protein was found by the Kyte-hydropathy plot (FIG. 10). Analysis using the big-PI Predictor program revealed that the amino acid to which the C-terminal peptide chain was cleaved and GPI was added was the 315th serine. On the other hand, an analysis using the SMART program revealed that it has a motif similar to the cysteine repeat structure unique to the surface antigen of the genus Paramecium (FIG. 11).

Immunoblotting and RT-PCR analysis of trohonto In order to examine whether the 32 kDa protein was expressed on the surface of trophoton as well as Seronto, analysis by immunoblotting was performed. Extraction of total RNA from Seronto and Trophon was performed in the same manner as in Example 1, and cDNA synthesis was performed using ProSTAR First-Strand RT-PCR kit (Stratagene). Reverse transcription was performed in 35 PCR cycles in the same reaction system as in Example 4 using primers specific to 5 ′ and 3 ′ (SEQ ID NOs: 16 and 17 in the sequence listing). The amplification product (984 bp) and molecular weight marker were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. In order to confirm whether the target gene was amplified by PCR, the amplified DNA fragment was subcloned and subjected to sequence analysis.

  As a result, about 32 kDa protein was found as a major protein on SDS-PAGE in the Triton X-114 extract of Trohont, and this protein could be detected by immunoblotting with rabbit anti-Seronto serum ( FIG. 12). As a result of RT-PCR, a band was observed at the expected position (984 bp) in both the seronto and the trophone (FIG. 13). As a result of sequence analysis, this PCR product was subcloned and confirmed to have a partial sequence of the desired 32 kDa protein gene.

Recombinant protein expression by E. coli The cDNA obtained from white spot insects used a stop codon (TAA) as a glutamine codon, and also used many rare codons. A synthetic gene encoding a protein excluding the signal sequence was prepared. 5 μL 10 × PCR buffer (Stratagene), 4 μL 250 mM dNTPs, 0.5 U Pfu DNA polymerase (Stratagene) and 100 bp or 80 bp 10 pM DNA oligo (Sigma Genosys, SEQ ID NO: 18 to 35) The reaction of 94 ° C./30 seconds, 63 ° C./1 minute, and 72 ° C./4 minutes was performed 10 cycles in the 50 μL reaction system. Furthermore, using this reaction product as a template, the reaction was carried out for 30 cycles of 94 ° C./30 seconds, 63 ° C./1 minute, and 72 ° C./3 minutes using primers (SEQ ID NOs: 36 and 37). The obtained DNA fragment was subcloned into pHSG299 (manufactured by Takara Bio Inc.) to confirm the sequence. The sequence of the obtained DNA fragment is shown in SEQ ID NO: 38 in the sequence listing. This plasmid was further digested with Bam HI and Eco RI, and the resulting DNA fragment was subcloned into pCold II (Takara Bio Inc.) to obtain an expression vector. Furthermore, the expression host E. coli BL21 (DE3) pLysS was transformed with this expression vector. For expression of the recombinant protein, the transformant was cultured with shaking in LB medium at 37 ° C, and when the OD600 reached 0.4, the culture solution was cooled at 15 ° C for 1 hour. IPTG was added to the culture solution to a final concentration of 1 mM, and shaking culture was performed at 15 ° C. for 24 hours. After incubation, E. coli was recovered by centrifugation, solubilized with 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 8 M urea, and 20 mM imidazole, and used for His-Trap HP (GE Healthcare Bioscience). After washing the column with 10 column volumes of 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 8 M urea, 20 mM imidazole, the column with 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 8 M urea, 500 mM imidazole The protein bound to was eluted to obtain the recombinant protein of the present invention.

According to the present invention, an agglutination / immobilization antigen protein of seawater white spot worms, a DNA encoding the aggregation / immobilization protein, an antibody against the aggregation / immobilization antigen of a parasite, and a vaccine that protects against infection of the parasite are provided. Can do.

FIG. 4 represents a photograph of a seron aggregated in the aggregation / immobilization assay of Example 1 by the addition of serum from rabbits or trough immunized with 1/100 diluted seronto. (A and B are rabbit sera, C is added with trough puffer serum) The figure which showed the time-dependent change of the antibody titer with respect to the seroto of the serum of the trough puffer immunized with the seronto in Example 2 is represented. (● is control using trough puff serum ingested by Seronto, ○ is control using trough puff serum ingested bovine serum albumin. Each point indicates the mean value ± standard error of absorbance at 405 nm when each serum was added. (* Means that there is a significant difference of P <0.001 in Student'st-test compared to the control group) The figure which shows the correlation of the antibody titer with respect to the aggregation / immobilization activity of Example 1 and the seronto of Example 2 is represented. (The correlation between the agglutination / immobilization activity of 1: 100 diluted serum and the ELISA activity of serum is shown. ○ indicates the serum of fish before immunization, Δ indicates data after 2 weeks of immunization, and ◇ indicates data after 6 weeks of immunization. The dotted line is the regression line (Y = 0.0016X + 0.0675, R2 = 0.6796). Fig. 4 represents an elution profile of anion exchange chromatography in the purification of membrane protein of the present invention. The dotted line indicates the NaCl gradient curve, and the thick line on the upper left indicates the fractionated area. The result of SDS-PAGE of the fraction in this invention membrane protein purification is represented. The upper number indicates the fraction number, 2 is the fraction of 2-3 minutes after injection, 3 is the fraction of 3-4 minutes, 4 is the fraction of 4-5 minutes, 5 is 5-6 minutes Fraction, 6 indicates a 6-7 minute fraction, respectively. The results of SDS-PAGE of the whole Seronto (lane 1) and the Seronto membrane extraction fraction (lane 2) under non-reducing conditions and the SDS-PAGE (lane 3) of the Seronto membrane extraction fraction under reducing conditions are shown, respectively. FIG. 4 shows a diagram showing immunoblot analysis with rabbits immunized with Seronto in the non-reducing state and with trough puffer serum. (Lane 1; rabbit anti-seronto serum, lane 2; BSA and trough antiserum as negative control, lane 3; trough puff anti-seronto serum. The numbers on the left indicate molecular weight markers. The 32 kDa antigen is indicated by the arrow. .) The result of the immunoblot of the absorption by the purified membrane protein of the present invention of the anti-Seronto serum is shown. Lane 1 shows the antiserum absorbed with BSA (positive subject), and lane 2 shows the antiserum absorbed with membrane protein. The result of indirect immunofluorescence staining of Seronto using antiserum is represented. 1 and 2 are antiserum absorbed with BSA, 3 and 4 are the results using antiserum absorbed with purified membrane protein, 1 and 3 are fluorescence observation images, 2 and 4 are transmission images of 1 and 3, respectively. To express. The figure which shows the Kyte-hydropathy plot (hydrophobicity parameter | index) of protein of this invention is represented. The figure which arranged the amino acid sequence of this invention protein and the surface antigen of Paramecium tetraurelia 51A (51A PAR) side by side is shown. (* Indicates a part that is the same amino acid. The box is surrounded by a repetitive structural motif of cysteine.) Represents a trohonto immunoblot. The results of SDS-PAGE (lane 1) of the major membrane protein of trohonto under non-reduction and immunoblotting with rabbit anti-seronto serum (lane 2) are shown. The numerical value on the left indicates a molecular weight marker. The 32 kDa antigen is indicated by the arrow. The result of RT-PCR analysis of Trohont is shown. Lane 1 is Seronto and Lane 2 is Trohont.

SEQ ID NO: 11
N-terminal amino acid sequence SEQ ID NO: 12
Degenerate primer SEQ ID NO: 13
Primer SEQ ID NO: 14
Primer SEQ ID NO: 15
Primer SEQ ID NO: 16
Primer SEQ ID NO: 17
Primer SEQ ID NO: 18
Primer SEQ ID NO: 19
Primer SEQ ID NO: 20
Primer SEQ ID NO: 21
Primer SEQ ID NO: 22
Primer SEQ ID NO: 23
Primer SEQ ID NO: 24
Primer SEQ ID NO: 25
Primer SEQ ID NO: 26
Primer SEQ ID NO: 27
Primer SEQ ID NO: 28
Primer SEQ ID NO: 29
Primer SEQ ID NO: 30
Primer SEQ ID NO: 31
Primer SEQ ID NO: 32
Primer SEQ ID NO: 33
Primer SEQ ID NO: 34
Primer SEQ ID NO: 35
Primer SEQ ID NO: 36
Primer SEQ ID NO: 37
Primer SEQ ID NO: 38
Synthetic cDNA

Claims (11)

The protein shown in the following (a), (b), (c) or (d).
(A) Aggregated antigen protein of seawater white spot worm ( Cryptocaryon irritans ) represented by any amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 in the sequence listing.
(B) An isoform of a protein having the amino acid sequence of the aggregate antigen protein of Cryptocaryon irritans represented by any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, or 10 in the sequence listing.
(C) having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown by any of SEQ ID NOs: 2, 4, 6, 8, or 10 in the sequence listing; And the protein which has the biological activity of the aggregation antigen protein of seawater white spotted insects.
(D) having an amino acid sequence having 80% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8, or 10, and A protein having biological activity. DNA encoding the protein according to claim 1. The DNA according to claim 2, which is the DNA represented by the following (a), (b), (c), or (d).
(A) DNA represented by any one of SEQ ID NOs: 1, 3, 5, 7, or 9 in the sequence listing.
(B) DNA comprising the base sequence represented by any one of SEQ ID NOs: 1, 3, 5, 7, or 9 in the sequence listing.
(C) a base sequence having 80% or more homology with the base sequence represented by any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and DNA encoding a protein having biological activity.
(D) DNA that hybridizes under stringent conditions with the nucleotide sequence shown in any one of SEQ ID NOs: 1, 3, 5, 7, or 9 in the sequence listing. A vector comprising the DNA of claim 2 or 3. A host into which the vector of claim 4 has been incorporated. A method for producing an agglutinated antigen protein of seawater white spot worms, which comprises culturing the host of claim 5 and collecting the agglutinated antigen protein of seawater white spot worms from the culture. A genetically modified protein obtained by the method according to claim 6. The antibody which recognizes the aggregation antigen protein of the seawater white spot worm of Claim 1 or 7. The antibody of claim 8, which is a monoclonal antibody. A vaccine for protection against seawater white spot insect infection, comprising the protein according to claim 1 or 9. A DNA vaccine for protection against seawater white spot insect infection, comprising the DNA according to claim 2 or 3.