JP2001161355A - Immortal cell line obtained from epinephelus coioides of family serranidae and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain (1) an immortal cell line prepared from fishes of the family Serranidae and to provide a method for establishing the cell line, to provide (2) a method for mass-producing and purifying an aquatic virus by using the immortal cell line obtained from the fishes of the family Serranidae, to obtain (3) an anti-nervous necrosis virus(NNV) antibody and to provide a method for producing the anti-NNV antibody and (4) to obtain a vaccine for the NNV and to provide a method for protecting the fishes from NNV infectious diseases. SOLUTION: This immortal cell line is obtained from the fishes of the family Serranidae and has sensitivity to viruses belonging to the family Birnaviridae such as Infectious Pncreatic Necrosis Virus(IPNV), the family Herpesviridae such as Eal Herpes Virus Formosa(EHVF), the family Reoviridae such as Hard- shell Clam Reovirus(HCRV) and the family Nodaviridae such as NNV.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immortal cell line (GF-1) obtained from the fin tissue of the grouper, Epinephelus coioides, and a method for establishing the GF-1 cell line. The GF-1 cell line is sensitive to several aquatic viruses. These several viruses are
Infectious Pancreatic Necros
is Virus, IPNV), Eal Herpes Virus Formosa (EHVF), and Nervous Necrosis Virus (NNV). The present invention also relates to a method for mass-producing and purifying aquatic virus using an immortal cell line obtained from a grouper fish such as the GF-1 cell line as a host. The invention further relates to an anti-NNV antibody, and the anti-NNV antibody.
The present invention relates to a method for producing an antibody. Finally, the invention relates to vaccines for NNV and methods for protecting fish from NNV infection.

[0002]

BACKGROUND OF THE INVENTION Nerve necrosis virus (NNV) is a pathogen found in many cultured marine fish and has caused massive deaths in these fish at the fry or juvenile stage.
NNV belongs to Nodaviridae. Fish nodule virus isolated from various fishes (eg SJNNV, B
FNNV, JFNNV, TPNNV, RGNNV, GNNV, etc.) are closely related to each other because the conserved regions of their coat protein genes are very similar. NNV, also known as fish encephalitis virus (FEV) and fish neuropathy nodule virus (PNN), has a particle size of 25
~ 34 nm globular virus. This virus is characterized by vacuolating neural tissue. Viral neuronecrosis (VNN) disease is caused by viral fish encephalitis, fish encephalomyelitis,
It is found in many countries under various names such as cardiomyopathy. NNV hosts include a wide variety of saltwater fish, including butterflies, perch, perch, turbot, grouper, pike, tiger pufferfish, pine kawa, flounder, salas squirrel, and spotted wolffish.

[0003] According to statistics in 1993, about 159 types of fish cell lines that have demonstrated the ability to grow fish viruses have been established (Fryer and Lannan, J. Tissue Culture Meth.
od (1994), 10: 57-94). These cell lines were mostly obtained from freshwater fish tissues, and only 34 cell lines were obtained from marine fish. Among these fish cell lines are, for example, RTG-2, CHSE-214, BF2, SBL, F
Some of them, such as HM and EPC, were measured for their susceptibility to fish nodule virus, but none showed a cytopathic effect (CPE) after inoculation of the virus.

[0004] In 1996, S obtained from the striped snakehead of the family Scrophulariaceae,
Using the SN-1 cell line, we successfully isolated a nodule virus from perch fish (Frerichs et al., J. General
Virology (1996) 77: 2067-2071). However, SNN
-1 cell line is permanently contaminated with type C retrovirus (Frerichs et al., J. General Virology (1991) 7
2: 2537-2539), making it unsuitable for the production of fish nodule virus.

[0005] Viral diseases cannot be treated with therapeutic reagents. The best ways to control viral disease include early detection and vaccine development. Either method requires a basic understanding of the biological, biochemical, and serological properties of the virus, including the ability of the industry to isolate the pure form of the virus, preferably in vitro. The ability to mass-produce with a cell culture system is required. Thus, the development of new cell lines sensitive to fish nodule virus is urgently needed to control the spread of viral disease in fish under infection with fish nodule virus. ing.

[0006] Groupers are important cultured fish in Taiwan. In recent years, many reports have been made on the establishment of cell lines obtained from grouper fishes. For example, Chen et al. Have reported that several cell lines have been established based on the fin and kidney tissues of the grouper grouper Epinephelus awoara ( Japan Scientific Society Press (Tokyo)).
(1988) 218-227). Lee also reported that he established a cell line based on eye pigment cells and brain tissue of the grouper Epinephelusamblycephalus (from a master's thesis in Zoology, National Taiwan University (1993)). However, Chen et al. Did not provide enough data to support the immortality of the established cell lines, and Lee also stated in his master's thesis that his cell lines were not immortal. I have. Furthermore, none of Chen et al. And Lee's established cell lines are susceptible to fish nodule virus.

[0007] Recently, there has been a succession of cases where grouper fishes die in large numbers mainly due to the nodule virus. At present, fish nodule virus has been found in grouper fish and can be isolated from moribund grouper fish showing symptoms of VNN disease (Chi et al., J. Fish Disease (1997)).
20: 185-193). Examination of tissues from grouper fish by electron microscopy revealed that grouper fish were susceptible to nodular and other viral infections (Chi, COA Fisheries
Series No.61, Reports on Fish Disease Research (1
997) 18: 59-69). Since some viruses have host specificity, cell lines obtained from groupers are
It is more suitable for use in research on certain viruses isolated from groupers.

[0008]

In the following, an immortal cell line obtained from the fin tissue of the grouper Chamilonha (Hamilton) will be introduced. The cell lines disclosed in the present invention are susceptible to various viruses, and are particularly susceptible to fish nodule viruses such as GNNV. Using the cell lines disclosed in the present invention, various aquatic viruses can be produced and purified in large quantities. The virus thus purified serves to produce antibodies and vaccines to protect fish from viral infections.

[0009]

According to a first aspect of the present invention, an immortal cell line obtained from a grouper fish is desirably an immortal cell line obtained from a fin tissue of an Epinephelus coioids. GF-1). GF-1 is a two-stage ribonucleic acid virus family (Birnaviridae)
(Eg, infectious pancreatic necrosis virus [IPNV], etc.), herpesviridae (eg, formosa virus, herpesvirus [EHVF], etc.), reoviridae (eg, hard clam reovirus [HCRV], etc.), and nodular virus (eg, It is susceptible to various viruses including but not limited to viruses belonging to grouper fish neuronecrosis virus [GNNV] and the like, and these viruses can be mass-produced.

[0010] In a first aspect, the present invention also provides a method for establishing an immortal cell line. This method involves the steps of (1) placing cells released from the fin tissue of the grouper Chairomaruta in a tissue culture flask, forming a cell monolayer, and establishing a primary cell culture, and (2) subculturing cells. Subculturing and maintaining the cell monolayer in a suitable medium, and (3) chromosome number distribution, plate efficiency, fetal bovine serum (FBS)
Monitoring the transformation of cells characterized by susceptibility to aquatic viruses, particularly fish nodule viruses such as GNNV.

In a second aspect of the present invention, there is provided a method of growing a virus using an immortal cell line obtained from a grouper fish, preferably a GF-1 cell line. This method
(1) inoculating the cell line with the virus, and (2) culturing the cell line in a nutrient medium suitable for virus growth and replication. Viruses that are susceptible to and can replicate in immortal cell lines include viruses belonging to the two-stage ribonucleic acid virus family (Birnaviridae), the helpswirl family, the reoviridae family, and the nodal virus family. In particular, IPNV of the two-stage ribonucleic acid virus family, EHVF of the herpesviridae family, HCRV of the reoviridae family, and GNNV of the nodal virus family.

The second aspect of the present invention also provides a method for mass-producing and purifying a virus using an immortal cell line of a grouper fish and detecting the virus in the cell line. The method for mass production of virus is (1)
Inoculating the grouper fish cell line with the virus, (2)
Culturing the cell line in a nutrient medium suitable for virus growth and replication, and (3) recovering the virus from the cell line.

[0013] The method for purifying the virus is described in (1)
Inoculating the grouper fish cell line with the virus, (2)
Culturing the cell line in a nutrient medium suitable for virus growth and replication until a cytopathic effect (CPE) appears,
(3) recovering the virus from the cell line; and (4) purifying the virus using density gradient centrifugation. Preferably, the density gradient is a CsCl density gradient.
However, other density gradients from which sufficient virus can be recovered are also within the scope of the invention.

A method for detecting a virus in an immortal cell line of a grouper fish includes observing the development of a cytopathic effect (CPE) in the cell line under a microscope. The virus is
It can be further confirmed using electron microscopy.
This method comprises the steps of (1) fixing the cell line to glutaraldehyde and osmium tetroxide, (2) cutting the fixed cells into ultrathin sections, and (3) ultra-thinning the fixed cell lines by electron microscopy. Detecting the virus particles in the thin section.

The following four methods can be provided as special detection methods for detecting NNV in immortal cell lines of grouper fishes after CPE is observed in the cell lines.
The first method uses the polymerase chain reaction (PCR), (1) extracting viral RNA from a cell line,
(2) Virus RN was obtained by PCR using a reverse primer (SEQ ID NO: 1) and a forward primer (SEQ ID NO: 2).
Amplifying A. The second method is a method using western blotting (western immunoblot).
(1) extracting viral proteins from cell lines, (2) electrophoresing the viral proteins in an SDS polyacrylamide gel, and (3) running a polyacrylamide gel by Western blot using anti-NNV serum. Including the step of analyzing. The third method is to detect NNV protein using enzyme-linked immunosorbent assay (ELISA). The fourth method uses immunofluorescence staining to detect NNV contained in cells by binding a goat anti-mouse antibody labeled with fluorescein isothiocyanate (FITC) to mouse anti-NNV serum. is there.

In a third aspect, the present invention provides an anti-NNV antibody and a method for producing the anti-NNV antibody. Preferably, the anti-NNV antibody is a monoclonal antibody. Anti-NNV
Antibodies are prepared by administering an effective amount of NNV to a suitable animal, preferably a mouse or rabbit, to stimulate the animal's immune response. NNV used to prepare anti-NNV antibodies was recovered from a grouper fish cell line and
Desirably purified by sCl density gradient centrifugation (the buoyant density in CsCl of NNV is about 1.34 g / m
l).

A fourth aspect of the present invention provides NNV vaccines and methods for protecting fish from NNV infection. NNV vaccines contain an immunogenically effective amount of killed NNV. The vaccine further comprises a material selected from the group consisting of adjuvants, plasticizers, pharmaceutical excipients, diluents, carriers, binders, lubricants, glidants, anesthetic compounds, and mixtures thereof. Including. This vaccine is
It can be administered orally or by injection. As a method for vaccination of fish, oral administration is preferred. For oral administration of the vaccine, an enteric coating that does not dissolve in the fish stomach can be added to the vaccine. NNV used to prepare anti-NNV antibodies
Is preferably recovered from a grouper fish cell line and further purified by CsCl density gradient centrifugation (the suspension density of NNV in CsCl is about 1.34 g / ml). NNV
Is preferably inactivated. A method for protecting fish from NNV infection comprises administering to the fish an effective amount of an NNV vaccine.

The cell line according to the present invention is characterized in that it is (1) an immortal cell line obtained from a grouper fish which is susceptible to aquatic virus.

In the cell line according to the present invention,
(2) The immortal cell line according to the above (1), wherein the grouper fish is a black-spotted grouper (Epinephelus coioides).

In the cell line according to the present invention,
(3) The immortal cell line according to (2) above, wherein the ATCC accession number is PTA-859.

In the cell line according to the present invention,
(4) The immortal cell line according to (3), wherein the cell line is a cell line obtained from a fin tissue of a grouper fish.

In the cell line according to the present invention,
(5) The immortal cell line according to the above (4), wherein the cell line is a cell line that is sensitive to a certain virus and mass-produces the virus.

In the cell line according to the present invention,
(6) The immortal cell line according to the above (5), wherein the virus is selected from the group consisting of a two-stage ribonucleic acid virus family (Birnaviridae), a herpesviridae family, a reoviridae family, and a nodal virus family. It is a cell line.

In the cell line according to the present invention,
(7) The immortal cell line according to the above (6), wherein the two-stage ribonucleic acid virus family (Birnaviridae) is an infectious pancreatic necrosis virus (IPNV).

In the cell line according to the present invention,
(8) The immortal cell line according to the above (6), wherein the herpesviridae is a formosa virus herpesvirus (EHVF).

Further, in the cell line according to the present invention, (9) the immortal cell line according to (6) above, wherein the reoviridae is a hard clam reovirus (HCRV). It is characterized by.

In the cell line according to the present invention,
(10) The immortal cell line according to the above (6), wherein the nodal virus family comprises a nerve necrosis virus (NNV), a fish encephalitis virus (FEV), a fish neuropathy nodule virus (PNN),
Grouper Fish Nerve Necrosis Virus (GNNV), Trevally Nerve Necrosis Virus (SJNNV), Troutfish Nerve Necrosis Virus (TPN)
NV), Matsukawa nerve necrosis virus (BFNNV), and pheasant grouper nerve necrosis virus (RGNNV).

In the method according to the present invention, (1)
1) A method for establishing an immortal cell line, comprising placing cells isolated from the fin tissue of the grouper Chairomaruhata in a tissue culture flask to form a cell monolayer and establishing a primary cell culture; Subculturing and maintaining the cell monolayer in a medium suitable for subculturing cells, and monitoring transformation of the subcultured cells, characterized by susceptibility to aquatic virus. The method is characterized by including the method.

In the method according to the present invention, (1)
2) A method for growing a virus, the method comprising:
(1) inoculating the established cultured cells containing the immortal cell line according to the virus, and culturing the cultured cells in a nutrient medium suitable for the growth and replication of the virus. The method is characterized by including the method.

In the method according to the present invention, (1)
3) The method for growing a virus according to the above (12), wherein the virus is a two-stage ribonucleic acid virus family (Bi
rnaviridae), herpesviridae, reoviridae,
And a nodal virus family.

In the method according to the present invention, (1)
4) The method for growing the virus according to the above (13), wherein the two-stage ribonucleic acid virus (Birnaviridae) is used.
Is an infectious pancreatic necrosis virus (IPNV).

In the method according to the present invention, (1)
5) A method for growing the virus according to the above (13), wherein the herpesviridae is the formosa virus herpesvirus (EHVF).

In the method according to the present invention, (1)
6) A method for growing the virus according to the above (13), wherein the reoviridae is a hard clam reovirus (HCRV).

In the method according to the present invention, (1)
7) The method for growing a virus according to the above (13), wherein the nodular virus family comprises a nerve necrosis virus (NN
V), fish encephalitis virus (EFV), fish neuropathy nodule virus (PNN), grouper fish necrosis virus (GNNV),
Shimaji Nerve Necrosis Virus (SJNNV), Trafugu Nerve Necrosis Virus (TPNNV), Matsukawa Nerve Necrosis Virus (BFN)
NV), and a fish nodule virus selected from the group consisting of pheasant grouper neuronal necrosis virus (RGNNV).

In the method according to the present invention, (1)
8) A method for mass-producing a virus, the method comprising:
(1) inoculating an established cell culture containing the immortal cell line according to (1) with the virus, and in a nutrient medium suitable for the growth and replication of the virus, a cytopathic effect (CP
Culturing the cultured cells until E) appears; and recovering the virus from the cell line.

In the method according to the present invention, (1)
9) A method for purifying a virus, the method comprising:
Inoculating established cultures containing said immortal cell line with said virus, and growing said cultures in a nutrient medium suitable for the growth and replication of said virus until a cytopathic effect (CPE) appears. A method comprising culturing, recovering the virus from the cell line, and purifying the virus using a density gradient centrifugation method.

In the method according to the present invention,
(20) The method for purifying a virus according to the above (19), wherein the density gradient centrifugation is a CsCl density gradient centrifugation.

Further, in the method according to the present invention, (2)
1) The method for purifying a virus according to the above (19), wherein the virus is NNV, and the NNV is contained in CsCl.
The method has a buoyant density of 1.34 g / ml.

In the method according to the present invention, (2)
2) A method for detecting a virus in an immortal cell line, wherein the development of the cytopathic effect (CPE) in an established cultured cell containing the immortal cell line according to the above (1) is examined by a microscope. The method includes a step of observing the image.

In the method according to the present invention, (2)
3) A method for detecting a virus in an immortal cell line according to the above (22), wherein the cell line is fixed to glutaraldehyde and osmium tetroxide; and Detecting virus particles in an ultrathin section of the cell line.

In the method according to the present invention, (2)
4) A method for detecting NNV in an immortal cell line, wherein the development of the cytopathic effect (CPE) in established cultured cells containing the cell line according to (1) above is observed with a microscope. And extracting viral RNA from the cultured cells, by a PCR method using a reverse primer having a DNA sequence of SEQ ID NO: 1 and a forward primer having a DNA sequence of SEQ ID NO: 2, And amplifying the viral RNA.

In the method according to the present invention, (2)
5) A method for detecting NNV in an immortal cell line, wherein the development of cytopathic effect (CPE) in established cultured cells containing the cell line described in (1) above is observed with a microscope. Extracting the viral proteins from the cultured cells; electrophoresing the viral proteins in an SDS polyacrylamide gel;
Analyzing the polyacrylamide gel by Western blotting using serum.

In the method according to the present invention, (2)
6) A method for detecting NNV in an immortal cell line, wherein the development of cytopathic effect (CPE) in established cultured cells containing the cell line according to (1) above is observed with a microscope. And extracting NNV protein from the cell line, and detecting NNV using ELISA.

In the method according to the present invention, (2)
7) A method for detecting NNV in an immortal cell line, wherein the cell line according to (1) above is fixed in formalin, and a mouse anti-NNV antibody serum is added to the cell line. And staining the cell line with a goat anti-mouse antibody labeled with fluorescein isothiocyanate (FITC).

In the antibody according to the present invention, (2)
8) An anti-NNV antibody, comprising an anti-NNV serum produced by administering an effective amount of NNV purified by the method described in (21) to an appropriate animal and stimulating an immune response of the animal. It is an antibody.

In the antibody according to the present invention, (2)
9) The anti-NNV according to the above (28), which is a monoclonal antibody
It is an antibody.

In the method according to the present invention, (3)
0) A method for preparing an anti-NNV antibody, the method comprising:
1) Injecting an effective amount of NNV purified by the method described in 1) into a suitable animal, stimulating an immune response of the animal, and collecting and purifying serum from the animal. Features.

In the vaccine according to the present invention,
(31) A vaccine for protecting fish from NNV infection, which is a vaccine containing an immunogenically effective amount of killed NNV purified by the method described in (21) above. And

In the vaccine according to the present invention,
(32) The vaccine according to the above (31), wherein the adjuvant, a plasticizer, a pharmaceutical excipient, a carrier, a diluent, a binder,
It is characterized in that the vaccine further contains one or more materials selected from the group consisting of lubricants, glidants, and narcotic compounds.

In the vaccine according to the present invention,
(33) The vaccine according to the above (31), which is orally administered.

In the vaccine according to the present invention,
(34) The vaccine according to the above (33), wherein the orally administered vaccine further contains an enteric coating,
The enteric coating is a vaccine that does not dissolve in the fish stomach.

In the vaccine according to the present invention,
(35) The vaccine according to the above (31), which is provided by injection.

In the method according to the present invention, (3)
6) A method for protecting fish from NNV infection, comprising providing the vaccine produced by the method described in (31) above to fish.

[0054]

DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of the present invention, there is provided an immortal cell line obtained from a fin tissue of a grouper fish, preferably a grouper Chairomaruhata, more preferably a grouper Chairomaruhata. A biological sample of an immortal cell line obtained from the fin tissue of the grouper
l sample), ie, the GF-1 cell line, is subject to the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, VA 20110-2209)
Has been deposited. The accession number assigned to this cell line is
PTA-859.

This embodiment also provides a method for establishing an immortal cell line. The following examples describe experimental designs and results for the establishment of immortal cell lines, but do not limit the invention.

[0056]

[Embodiment 1 ] Establishment of GF-1 cell line GF-1 cell line was established and maintained by the following procedure. (1) Primary culture For the establishment of the primary culture, grouper fishes (Chairomarha: Epinephelus coioides, Hamilton) weighing 1 kg were used. Fish were soaked in 5% chlorex for 5 minutes and wiped with 70% alcohol. Dissect the fins from the body and use the washing solution (4
Washed three times with 00 IU (international units) / ml of penicillin, 400 μg / ml of streptomycin and 10 μg / ml of Hungisone. The washed fin tissue is cut into small pieces with scissors, and a 0.25% trypsin solution (0.25%
% Trypsin and 0.2% EDTA in phosphate buffered saline [P
BS]). The tissue fragments in the trypsin solution were slowly stirred at 4 ° C. using a magnetic stirrer. After 30 minutes, the cells released from the tissue fragments are
Collected by centrifugation. Next, the collected cells were added to a complete culture solution (L15, 20% fetal bovine serum [FB-S],
(IU / ml penicillin, 100 μg / ml streptomycin, and 2.5 μg / ml fungisone), and then transferred to a 25 cm ² tissue culture flask and cultured at 28 ° C. .

(2) Subculture and maintenance If a confluent cell monolayer is formed in the primary culture, 0.1%
The surface of the flask was treated with a trypsin solution (containing 0.1% trypsin and 0.2% EDTA in PBS) to release cells. The released cells were then transferred to two new flasks containing fresh L15 medium supplemented with 20% FBS. Cells were subcultured at a split ratio of 1: 2. GF-1
Until the first teenage of cells, mix old and new cultures
Maintained at 50% each. The concentration of FBS contained in the L15 medium is
GF-1 cells are maintained at 10% from the 11th to the 70th generation, and after the 70th generation
Reduced to 5%. GF-1 cells were subcultured at 9-day intervals during the first 20 generations, at 5 day intervals during the next 21-70 generations, and at 3 day intervals from the 71st generation onward. It was subcultured.

(3) Measurement of Mycoplasma Contamination in the GF-1 Cell Line The GF-1 cell line was grown for 3 metastases in L15-10% FBS containing no antibiotic, and bacteria, fungi and mycoplasma were removed. The presence or absence was measured. For mycoplasma measurement, use the Mycoplasma Contamination Kit (Flow Laboratories,
USA) was used.

(4) Measurement of viability of GF-1 cell line The viability of GF-1 cell line was measured by the following procedure. First, GF
-1 cells are removed from the flask, and the cells are separated from the culture solution by centrifugation, and then 10% dimethyl sulfoxide (DM
The cells were resuspended in a freezing medium consisting of (SO) and 90% FBS. 5x
Ampoule containing 10 ⁶ cells / ml / ampoule (NUN
C, Denmark) first at -20 ° C for 1 hour, then at -70 ° C
, And transferred to liquid nitrogen (-176 ° C).
One month and one year later, the ampules were thawed in a 30 ° C. water bath.
Cells are separated from the freezing medium by centrifugation, and L15-10%
Resuspended in FBS. Surviving cells were determined by trypan blue staining. Cell numbers were counted using a hemocytometer. For further observation,
Thawed cells were re-inoculated into 25 cm ² flasks.

(5) Distribution of Chromosome Number The distribution of the number of chromosomes in GF-1 cells in their 50s and 80s was examined using semi-fusing cells in the growth phase. First, 0.1μg
After pretreatment of the cells for 5 hours at 28 ° C. using a / ml Colcemid (Gibco, Grand Island, NY), the cells were released with a 0.1% trypsin solution. Subsequently, after centrifugation at 1000 g for 10 minutes, a hypotonic solution (8% distilled water and 1
% PBS) for 30 minutes. Further, a few drops of Carnoy's fixative (containing 1% glacial acetic acid and 3% 100% methanol) were added to partially fix the cells, and 4 ° C, 800g
For 10 minutes. The supernatant was discarded and the cells were fixed for 20 minutes in fresh and cold Carnoy's fixative. The suspension of fixed cells was dropped onto a 76 × 26 mm slide. After air-drying the slides, 0.4% Giemsa stain (Sigma, St.
Louis, Mo., USA) for 30 minutes. Olympus V
Chromosome numbers were observed and counted using anox microscopy.

(6) Plate Efficiency Plate efficiency of GF-1 cells in the 50th to 80th generations was estimated. Cells were first seeded in 25 cm ² flasks at a density of 100 cells / flask. After 15 days of culture, the culture was removed and cell colonies were fixed with 70% ethanol and stained with 0.4% Giemsa stain. Colony in each flask is
s Counted using an IM inverted microscope. For comparison,
Carp fin (CF), black porgy spleen (B
PS-1), tilapia ovary (TO-2), and eel kidney (EK) cell lines were plated and cultured in the same manner as the GF-1 cell line.

(7) Effects of FBS concentration and temperature on growth of GF-1 cells The effects of FBS concentration on growth of GF-1 cells were determined in the 50th to 80th generations. Two replicates were performed for each FBS concentration. At regular time intervals, two flasks corresponding to each FBS concentration were removed and the average number of cells was counted.

To determine the effect of temperature on the growth of 80th generation GF-1 cells, cell cultures in 25 cm ² flasks containing L15-10% FBS were grown at 18 ° C., 28 ° C., and 35 ° C. Cultured. At regular time intervals, two flasks corresponding to each temperature were removed and the average number of GF-1 cells was counted.

Results: Approximately two weeks after primary and subculture implantation of GF-1 cells, a monolayer of cells formed in the primary culture. Fibroblast-like cells and epithelial cells coexisted in the cell group (see FIG. 1). Since 1995, subcultures of GF-1 cells have been 160
It has been successful for more than a generation and continues to be a permanent cell line.

The subculture of GF-1 cells was performed using L15-
The test was performed every 9 days in 20% FBS, every 5 days in L15-10% FBS for 21-70 generations, and every 3 days in L15-5% FBS from the 71st generation. Contact suppression was observed in GF-1 cells until the first 50 generations, which gradually decreased between the 51st and 80th generations.

The viability of GF-1 cells in the 80th generation was 73% after one year and one month. When reinoculated in L15-5% FBS at 28 ° C., the cells grew vigorously.

Chromosome number The number of chromosomes in GF-1 cells in the 50th generation is distributed between 7 and 44,
The peak was 32 (see FIG. 2A). GF in the 80s
-1 The number of chromosomes in cells was 17-42 when 100 cells were examined.
And a bimodal curve with peaks at 32 and 36 (see FIG. 2B). In cells arrested in metaphase, both micro and macro chromosomes were observed.

Plate Efficiency The plate efficiency of GF-1 cells inoculated at a density of 100 cells / flask increased from 21% in the 50th passage to 80% in the 80th passage. On the other hand, the plating efficiencies of the CF, BPS-1, TO-2, and EK cell lines, also inoculated at a density of 100 cells / flask, were 22%, 13%, 48%, and 63%, respectively.
Increased plating efficiency suggests that GF-1 cells had undergone a transformation during the 50-80 generation.

[0069] The FBS concentration and temperature affect the growth of GF-1 cells.
Boss Effect Figure 3, FBS concentration is a graph showing the effect on the growth of GF-1 cells in the 50s and the 80s. In both the 50th and 80th generations, GF-1 cell growth corresponds to the concentration of FBS, with higher FBS concentrations resulting in faster cell growth. However, comparing the growth rates of GF-1 cells in the 50th and 80th generations, the GF-1 cells in the 80th generation showed
It was found that it showed greater growth potential than GF-1 cells in their 50s. This was especially noticeable at FBS concentrations of 2%, 5%, and 10%. For example, 10% FBS
On the fourth day of cell culture containing, the concentration of 50th generation GF-1 cells is 3.5 × 10 ⁶ cells / 25 cm ² flask, while the concentration of the 80th generation GF-1 cells is 5.0 × 10 ⁶ cells / 25 cm ² flask. This result suggests that the need for FBS in cell growth was reduced at passage 80, indicating that cell transformation occurred between passages 50-80.

FIG. 4 is a graph showing the effect of temperature on the growth of 80th generation GF-1 cells. This result indicates that GF-1 cells grow well at 28 ° C. and 35 ° C.
However, the growth rate of GF-1 cells cultured at 35 ° C.
Start decreasing on day four. This suggests that maintaining the culture temperature at 35 ° C. has a long-term effect on cell growth. GF-1 cells did not grow well at 18 ° C.

According to a second aspect of the present invention, there is provided a method for producing and purifying an aquatic virus in GF-1 cells and detecting the virus in an immortal cell line. Hereinafter, the experimental design of this embodiment will be described.

Embodiment 2 FIG . Virus using GF-1 cell line
And a virus in the cell line.
To infect GF-1 cells in the measurement 80th generation susceptibility to aquatic viruses methods (1) GF-1 cells to detect, infectious pancreatic necrosis virus (IPNV: AB, SP, VR299 , and EVE of Virus strain), hard clam reovirus (HCRV), formosa virus help virus (EHVF), and nerve necrosis virus (NNV: GNNV isolate). The susceptibility to GNNV was also tested using a BGF-1 cell line obtained from the fin of a green grouper (Epinephelus awoara).

Each monolayer of GF-1 cells was inoculated with 0.5 ml of various aquatic viruses at a titer of 10 ³ TCID ₅₀ /0.1 ml. After 30 minutes of adsorption, the cells in each flask were washed three times with PBS, followed by the addition of 5 ml of L15-2% FBS to each flask. The two flasks were then separately cultured at 20 ° C and 28 ° C, respectively. Six days after virus infection, the supernatant of the cultured cells was collected and the virus titer was measured.

(2) Propagation and Purification of Aquatic Virus in GF-1 Cell Line The virus isolate was isolated from GF-1 at an MOI of 0.01 (multiplicity of infection).
The cell line was inoculated. When CPE occurred, the GF-1 cells were scraped, placed in a culture solution, and centrifuged at 10,000 × g for 30 minutes to precipitate cell debris (first precipitate). Transfer the supernatant to a bottle,
Polyethylene glycol (PEG, molecular weight 20,000) and
NaCl was added to adjust the final concentrations to 5% and 2.2%, respectively. Next, the supernatant was stirred at 4 ° C. for 4-6 hours, and the virus particles were precipitated by centrifugation at 10,000 × g for 1 hour (second
Precipitate). Remove the first and second precipitates with a small amount
TNE buffer (0.1 M Tris, 0.1 M NaCl, 1 mM EDTA, pH 7.
The suspension was resuspended in 3), and an equal volume of Freon-113 was added. Shake the mixture vigorously for 5 minutes and wash the resulting emulsion with 3,
The mixture was centrifuged at 000 × g for 10 minutes to separate the freon from the aqueous phase. Recover the aqueous phase and pre-formed
Place on a 10-40% (w / w) CsCl gradient layer at 160,000xg
Centrifuged for 20 hours. Collect virus streak, 10ml
And then centrifuged at 150,000 g for 1 hour to precipitate again. The resulting precipitate was resuspended in a small amount of TE buffer (0.1 M Tris, 1 mM EDTA, pH 7.3).

(3) Detection of aquatic virus in GF-1 cell line In general, when a virus infects a cell line that is susceptible to the virus, CPE is observed in cultured cells within 2 days after infection. You. The occurrence of CPE is evidence that the virus has successfully transmitted and propagated within the cell line. The presence or absence of a viral infection in the cell line can be further confirmed using electron microscopy. That is, the cells infected with the virus are first fixed with 2.5% glutaraldehyde dissolved in 0.1 M phosphate buffer (pH 7.4), and then fixed with 1% osmium tetroxide, and then fixed. To make an ultrathin section, uranyl acetate-
Those stained with lead citrate were examined with a Hitachi H-600A electron microscope. Virus particles should be observed as homogeneous spherical particles in the cytoplasm.

There are three other methods for detecting NNV in the GF-1 cell line. (A) Method of detecting NNV in GF-1 cells by amplification by polymerase chain reaction (PCR) PCR amplification was used to confirm whether GF-1 cells could propagate NNV. In this method, Rneasy (registered trademark) mini kit is used from the supernatant of NNV-infected cells after CPE has occurred.
(QIAGEN) must be used to extract viral RNA. 2 units of MMLV reverse transcriptase (Promega) for reverse transcription
And 0.4 units of RNsin (Promega), 0.25 mM dNTPs,
0.5 μM reverse primer R3 (5′-CGAGTCAACACGGGTGAA
GA-3 ′) (SEQ ID NO: 1) and 40 μl of 2.5 × PC
R buffer (25 mM Tris HCl, pH 8.8, 3.75 mM MgCl ₂ , 12
Extracted viral RNA was cultured in 5 mM KCl and 0.25% Triton X-100) at 42 ° C. for 30 minutes. Following cDNA synthesis, 40 μl of the cDNA mixture was mixed with diethyl pyrocarbonate (D
Was treated with EPC) H ₂ O (0.025 units of DNA polymerase [Bi
ometra], 0.1 mM dNTP, and 0.5 μM forward primer F2 [5′-CGTGTCAGTCATGTGTCGCT-3 ′] [SEQ ID NO:
2), and dilute 2.5-fold with an automatic thermal cycler (To
uchDown (R) thermalcycler, Hybaid company)
Cultured in The target region of the primer set (F2, R3) is T4 (400 bp). PCR products corresponding to T2 and T4 were obtained by amplification from nucleic acids of GF-1 cells infected with NNV.

(B) Method of Detecting NNV in GF-1 Cells by Western Blot In order to specifically detect NNV protein, Western blot was used. A virus sample was prepared according to the following procedure. First, NNV was inoculated into GF-1 cells, and the temperature was 20 to 32 ° C.
And cultured. After 5 days of culture, cells infected with NNV
The mixture was centrifuged at 00 g for 10 minutes to precipitate. After cell sediment was electrophoresed on a 10% SDS polyacrylamide gel, proteins were transferred to immobilon-P transfer membrane (Mil
lipore) and soaked in 3% skim milk-Tris buffered saline (TBS) for 1 hour. Subsequently, this membrane is anti-NN
After culturing for 1 hour at room temperature with the antiserum of V, washing with TBS, further reacting for 1 hour with a goat antibody system labeled with peroxidase, 6 mg of 4-chloronaphthol dissolved in 20 ml of methanol and 100 ml of TBS Was stained with a substrate containing 60 μl of H ₂ O ₂ dissolved in E. coli.

(C) Method for Detecting NNV in GF-1 Cells Using Enzyme-Linked Immunosorbent Assay (ELISA) ELISA is performed using an immunoreactive substance (antigen or antibody) labeled with an enzyme and an immunosorbent ( An antigen or antibody bound to a solid) is a type of immunological method for identifying antibodies or antigens of a particular serum or tissue. ELIS
The test method of A is performed in the following procedure. First, an effective amount of the purified NNV protein was spread on a microtiter plate and allowed to adsorb at 4 ° C. overnight. Subsequently, 3% bovine serum albumin (BSA) was added to the plate (used as a blocking agent) and incubated at 37 ° C. for 1 hour. The plate was then washed three times with buffer. Next, the diluted rabbit anti-N
NV serum was added to the plate and incubated at 37 ° C. for 1 hour. Subsequently, goat anti-rabbit serum IgG labeled with horseradish peroxidase was added, and the mixture was cultured at 37 ° C. for 1 hour, and then 3,3 ′, 5,5′-tetramethylbenzidine was added for coloring. The color reaction was stopped with H ₂ SO ₄ of 1N. The optical density of the inner wall of the microtiter plate was measured using an ELISA reader (Dynatech MR 5000).
Measured at 450 nm.

(D) Detection of NNV in GF-1 cells using immunofluorescence staining method To detect the virus grown in GF-1 cells, 10% after 12 hours from virus infection The cultured cells were fixed with formalin. Fix the cultured cells with 0.2% Trito
Treated with nX-100 and washed with PBST (phosphate buffer containing 0.05% Tween 20). Cultured cells treated with Triton were further washed with 3% skim milk as a blocking agent and reacted with mouse anti-NNV serum. The antibody-treated cultured cells were finally stained with a goat anti-mouse antibody labeled with fluorescein isothiocyanate (FITC).

Results: Table 1 shows that IPNV (AB, S
It summarizes the results of measuring virus susceptibility to P, VR299, and EVE virus strains), HCRV, EHVF, and NNV (GNNNV isolate). The measurement was performed after the cells were inoculated with CPE after virus inoculation.
D ₅₀ / ml).

[0081]

[Table 1] Virus susceptibility of 80th generation GF-1 cells ND: No titer was measured. +: A cytopathic effect (CPE) was observed.

As shown in Table 1, in the case of the IPNV virus strain and HCRV, CP was only obtained when the cells were cultured at 20 ° C.
E occurred. In the case of EHVF, CPE occurred at both 20 ° C and 28 ° C. However, in the case of GNNV, CPE occurred when cultured at 28 ° C. The yield of the virus in the 80s GF-1 cells (when cultured EHVF at 20 ℃) 10 ^7.0 ~10 ^{11
. 0} (when HCRV is cultured at 20 ° C.), which is an extremely high figure.

In the case of aquatic viruses such as GNNV, CP
E typically begins to appear three days after infection. First, round granular refraction cells began to form in the cell culture (see FIG. 5). Eventually, more cells became round and swollen, and the swollen cells grew in size, eventually separating from the cell culture and floating in culture. Most of the detached cells were completely disrupted. Cultures from cell cultures indicate that CPE can transmit to other GF-1 cells. In this experiment, the BGF-1 cell line (Aohata: Epine
phelus awoara fin) (obtained from fins) was also measured. As a result, CPE after virus infection
Was not observed.

In the case of aquatic viruses such as GNNV, they can typically be observed with an electron microscope. GF in electron microscope
In the cytoplasm of -1 cells, a large number of spherical to icosahedral, non-hulled, homogeneous particles with a particle size of 20-25 nm can be observed (see FIG. 7). Some of the virus particles are contained in inclusion bodies, and the rest are contained in the cytoplasm (see FIG. 7). The isolated virus particles are CsC
l Can be further purified by density gradient centrifugation. Taking GNNV as an example, the purified virus was an icosahedral virion without hulls (mature virus particles) with a particle size of 20-25 nm. GNNV buoyant density in CsCl is
It was 1.34 g / cm ³ .

In addition to the method for detecting the occurrence of CPE in GF-1 cells, the following four methods can also be used to determine whether aquatic virus is present in GF-1 cells and to determine whether the virus is present in the cells. Can be confirmed whether or not it is capable of breeding. That is, there are four methods: (1) PCR, (2) Western blot, (3) ELISA, and (4) immunofluorescence staining.

Taking GNNV as an example, the PCR method can be achieved by selecting a pair of primers, ie, R3 (SEQ ID NO: 1) and F2 (SEQ ID NO: 2) and performing PCR amplification. Target region T4 is present in fish nodule virus. Therefore, the PCR method using F2 and R3 is applicable not only to GNNV but also to fish nodule virus. As a result of PCR, G
NNV was found to be replicated in GF-1 cells and released into the supernatant of cultured cells (FIG. 6).

According to the Western blotting method using mouse anti-GNNV serum, it was found that a viral protein was present in cells of GNNV infection cultured at 20 to 32 ° C. This means that when the culture is maintained at 20-32 ° C, the viral mRNA is successfully translated into a viral polypeptide in the host cell.

The ELISA and immunofluorescent staining methods are also
Infectious GF-1 cells were detected to show a positive reaction with anti-GNNV serum. This means that GNNV can grow in GF-1 cells.

According to a third aspect of the present invention, there is provided an anti-NNV antibody and a method for producing the antibody. The experimental design of this embodiment is described below.

Embodiment 3 FIG . Production of Anti-NNV Antibody (1) Production of Anti-NNV Antibody A polyclonal antibody is produced by, for example, sequentially injecting a purified NNV immunogen into a suitable animal such as a rabbit, rat, or mouse based on a conventional method. be able to. For example, rabbits can be boosted intravenously, subcutaneously, or intraperitoneally with a suitable amount of the NNV immunogen at 2 or 3 week intervals. The contents to be injected are
If necessary, it may contain an appropriate amount of Freund's complete adjuvant or Freund's incomplete adjuvant.

For production of monoclonal antibodies, immunized mice are preferred. Three or four days after the final boost, the splenocytes of the mice are separated, and myeloma cells such as
It can be fused with SP2 / 0-Ag14 myeloma cells (ATCC CRL 1581). This is based on the conventional method described by Mishell and Shiigi (Selected Mthods in Cellular Immunolo
gy, WH Freeman & Company, 1980). Splenocytes and myeloma cells can be used in a ratio of 1: 1 to 1: 4. To promote cell fusion,
Fusion promoters such as polyethylene glycol (PEG) 4000
May be used. Culture media suitable for use in the cell fusion stage is RPMI 1640 (Gibco BRL, Life Technologies,
Inc.), and this culture typically contains 10-15% (v / v) fetal bovine serum (FBS).

The fused cells are composed of hypoxanthine, thymidine,
And RPMI1640-15% FBS supplemented with aminopterin. After culturing for 7 to 10 days, a positive hybridoma clone capable of producing an antibody specific to NNV can be selected by ELISA using the supernatant of the culture solution. Further selection of positive clones can be achieved by conventional methods such as, for example, limiting dilution, plaque, spot, agglutination efficiency assays, and autoradiographic immunoassays.

(2) Purification of Antibody The antibody can be purified by a conventional immunoglobulin purification procedure such as ammonium sulfate precipitation, gel electrophoresis, dialysis, affinity chromatography, and ultrafiltration. In addition, ion exchange, size exclusion (size exc
lusion) Hydroxyapatite or hydrophobic interaction chromatography can be used alone or in combination. Light and heavy chains can be obtained using gel electrophoresis or isoelectric focusing, and other techniques known in the art.

According to a fourth aspect of the present invention, there is provided a vaccine for NNV and a method for protecting fish from NNV infection. The experimental design of this embodiment is described below.

Embodiment 4 FIG . Production of NNV Vaccines The vaccines according to the invention for preparing vaccines using dead NNV are to be administered as killed vaccines and include any killing methods now known or later developed. A preferred method is a heat treatment method. The heat treatment method can be achieved by heating the purified NNV to a temperature sufficient to inactivate the virus (eg, 70 ° C.) for a sufficient time (eg, 24 hours). Once the heating phase is complete, inactivated NNV is removed for intraperitoneal or intramuscular vaccination using Freund's incomplete adjuvant (FI) using a mixer for several minutes.
A) Emulsify within. Then, the vaccine is injected into fish (first injection). 30-45 days after the first injection, fish can be booster injected (boost injection). The booster injection generally uses about half the amount of the vaccine used in the first injection. Ten days after the first booster injection, a second booster injection can be given. At each time point, a fish serum sample can be taken for titer determination.

For enteral administration, enteric coatings containing non-toxic polymeric materials can be added to the vaccine for oral administration. The enteric coating material is preferably a material that does not dissolve at the pH of the stomach, but can dissolve when it passes through the stomach and reaches the pyloric cecum and the intestinal tract. For example, preferred materials include cellulose acetate phthalate, hydroxypropylmethyl phthalate, carboxymethylethyl cellulose, hydroxypropylmethyl acetate succinate cellulose, cellulose trimellitate, polyvinyl acetate phthalate, EUDAGRIT L-30D and 1100-55, EUDAGRI
TL 12.5 and L 100, EUDRAGIT E, RL, RS, and NE
And the like. Additional materials can be used in combination with the enteric coating. For example, plasticizers (polyethylene glycol 200, 400, 1000, 4000, 6000,
Propylene glycol, PVPK-90, glycerin or glycerol, diethyl phthalate, oleic acid, isopropyl myristate, liquid paraffin or mineral oil, triacetin, glycerol monostearate, dibutyl sebacate, triethyl citrate, tributyl citrate, acetylated monoglyceride, dibutyl Phthalate,
Acetyl tributyl citrate, castor oil, glycerol tributyrate, etc., disintegrants (sodium starch glycolate, etc.), adjuvants (immunostimulants [eg, β-glucan], etc.), binders (starch, polyvinylpyrrolidone, polyvinyl alcohol, etc.) ), Diluents (such as lactose), lubricants (such as magnesium stearate) and the like can all be used with enteric coatings. The vaccine can be administered orally to fish for a total of 30 days on an alternate day basis. The efficacy of the vaccine was monitored using ELISA.

Oral administration of the vaccine is a generally preferred method for vaccination of fish. This is because there is no limit to the size of fish that can be handled, and the stress associated with immersion and intraperitoneal injection on fish can be reduced. Oral administration can also stimulate lymphatic tissue associated with the gastrointestinal tract more strongly than with intraperitoneal injection.

The present invention has been described with reference to certain preferred embodiments. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples contained herein be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0099]

According to the present invention, (1) an immortal cell line obtained from a grouper fish and a method for establishing the cell line, and (2) an immortal cell line obtained from a grouper fish are used. A method for mass producing and purifying aquatic virus,
(3) Anti-NNV antibodies and methods for producing the anti-NNV antibodies, and (4) vaccines for NNV and methods for protecting fish from NNV infection are provided.

[Sequence Listing] Sequence Listing <110> CHI, Shau-Chi <120> Immortalized cell line obtained from grouper Chairomaruha and its application <130> 32350-151224 <140> N / A <141> N / A <150 > TW 88 120 590 <151> 1999-11-25 <160> 2 <170> Microsoft Word <210> 1 <211> 20 <212> DNA <213> Artificial Seqence <220> <223> reverse primer <400> 1 cgagtcaaca cgggtgaaga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 cgtgtcagtca atgtgtcgct 20

[Brief description of the drawings]

FIG. 1 is a view showing the morphology of GF-1 cells observed with an inverted microscope. (A) a semi-fusible cell monolayer in which fibroblastic and epithelial cells coexist, and (B) GF-1 cells
A confluent monolayer of confluent cells at age 0, with fibrillar cells being the dominant cells. In the figure, the arrowheads indicate fibrous cells, and similarly, the arrows indicate epithelial cells. One scale is 10 μm.

FIG. 2 is a graph showing the distribution of chromosome numbers of GF-1 cells in (A) 50th generation and (B) 80th generation.

FIG. 3 is a graph showing the effect of fetal bovine serum (FBS) on the growth rate of GF-1 cells in (A) 50th generation and (B) 80th generation.

FIG. 4 is a graph showing the effect of temperature on the growth rate of GF-1 cells in the 80th generation.

FIG. 5: GF-1 cells in the 80th generation were (A) IPNV AB virus strain, (B) IPNVSP virus strain, (C) IPNV VR299 virus strain, (D) IPNV EVE virus strain, (E) HCRV,
(F) Fish nodule virus GNNV isolate, and (G) HEVF
(H) Comparison of cytopathic effect (CPE) produced after infection with GF-1 cells not infected with (H).

FIG. 6 is a diagram showing agarose gel electrophoresis of a product obtained by RT-PCR amplification using a pair of primers (SEQ ID NO: 1 and SEQ ID NO: 2) specific to a target region T4 of fish nodule virus SJNNV. . Lane 1 is the PCR product of GF-1 cells infected with GNNV, and lane 2 is the PCR product of uninfected GF-1 cells. M is a pGEM marker.

FIG. 7 is an electron micrograph of GF-1 cells infected with GNNV. Inclusion bodies (indicated by arrowheads) and a number of unencapsulated virus particles are shown in the cytoplasm. I for inclusion bodies filled with virus particles, M for mitochondria, N
Denotes cell nuclei, respectively. Arrowheads indicate virus particles. One division is 1 μm.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 7/02 C12P 21/08 C12P 21/08 C12Q 1/68 A C12Q 1/68 1/70 1/70 (C12N 7/00 // (C12N 7/00 C12R 1:93) C12R 1:93) C12N 5/00 EF term (reference) 4B063 QA01 QA18 QA19 QQ02 QQ10 QQ52 QQ79 QQ96 QR08 QR32 QR55 QR62 QS08 QS12 QS16 QS25 QS36 QX01 4B064 AG27 CC03 CC24 CD21 CE02 CE07 CE13 DA04 DA11 DA13 4B065 AA90X AA95X BA06 BD15 CA23 CA24 CA25 CA43 CA45 CA46 4C085 AA03 BA51 CC08 DD08 DD22 DD33 DD37 DD41 DD43 EE07 FF03 GG06 GG08 4H075 AA11A11A11A11

Claims (36)

[Claims] An immortal cell line obtained from a grouper fish that is susceptible to aquatic virus. 2. The immortal cell line according to claim 1, wherein the grouper fish is Epinephelus coioid.
es). 3. The immortal cell line of claim 2, wherein the cell line is AT.
A cell line whose CC accession number is PTA-859. 4. The immortal cell line according to claim 3, wherein the cell line is obtained from a fin tissue of a grouper fish. 5. The immortal cell line according to claim 4, wherein the cell line is susceptible to a virus and mass-produces the virus. 6. The immortal cell line according to claim 5, wherein the virus is a two-stage ribonucleic acid virus (Birnavirida).
e), a cell line selected from the group consisting of Herpesviridae, Reoviridae, and Nodularviridae. 7. The immortal cell line according to claim 6, wherein the two-stage ribonucleic acid virus (Birnaviridae) is an infectious pancreatic necrosis virus (IPNV). 8. The immortal cell line according to claim 6, wherein the herpesviridae is the formosa virus herpesvirus (EHVF). 9. The immortal cell line of claim 6, wherein the reoviridae is a hard clam reovirus (HCRV). 10. The immortal cell line of claim 6, wherein
The nodular virus family includes neural necrosis virus (NNV), fish encephalitis virus (FEV), fish neuropathy nodule virus (P
NN), grouper fish necrosis virus (GNNV), horse mackerel nerve necrosis virus (SJNNV), tiger puffer nerve necrosis virus (TPNNV), Matsukawa nerve necrosis virus (BFNNV), and pheasant grouper neuronecrosis virus (RGNNV) A cell line that is a fish nodule virus. 11. A method for establishing an immortal cell line, comprising placing cells isolated from the fin tissue of the grouper Chairomaruhata in a tissue culture flask to form a cell monolayer and establishing a primary cell culture. Subculturing and maintaining the cell monolayer in a medium suitable for subculturing cells; and monitoring the transformation of the subcultured cells, characterized by susceptibility to aquatic virus. And a method comprising steps. 12. A method for growing a virus, the method comprising: inoculating an established cultured cell containing the immortal cell line according to claim 1 with the virus; In a suitable nutrient medium,
Culturing the cultured cells. 13. The method for growing a virus according to claim 12, wherein said virus is a two-stage ribonucleic acid virus family (Birnavirid).
ae), a method selected from the group consisting of Herpesviridae, Reoviridae, and Nodularviridae. 14. The method for growing a virus according to claim 13, wherein the double-stage ribonucleic acid virus (Birnaviridae) is infectious pancreatic necrosis virus (IPNV). 15. The method for growing a virus according to claim 13, wherein the herpesviridae is a Formosa virus herpesvirus (EHVF). 16. The method for growing a virus according to claim 13, wherein the reoviridae is a hard clam reovirus (HCRV).
The way that is. 17. The method for growing a virus according to claim 13, wherein said nodular virus family comprises neural necrosis virus (NNV), fish encephalitis virus (EFV), fish neuropathy nodule virus (P).
NN), grouper fish necrosis virus (GNNV), horse mackerel nerve necrosis virus (SJNNV), tiger puffer nerve necrosis virus (TPNNV), Matsukawa nerve necrosis virus (BFNNV), and pheasant grouper neuronecrosis virus (RGNNV) How to be a fish nodule virus. 18. A method for the mass production of a virus, comprising inoculating an established cell culture containing the immortal cell line of claim 1 with the virus; In a suitable nutrient medium,
Culturing the cultured cells until a cytopathic effect (CPE) appears; and recovering the virus from the cell line. 19. A method for purifying a virus, comprising inoculating the established cultured cells containing the immortal cell line according to claim 1 with the virus; and suitable for the growth and replication of the virus. In a nutrient medium,
Culturing the cultured cells until a cytopathic effect (CPE) appears; collecting the virus from the cell line; and purifying the virus using density gradient centrifugation. 20. The method for purifying a virus according to claim 19, wherein the density gradient centrifugation is a CsCl density gradient centrifugation. 21. The method for purifying a virus according to claim 19, wherein the virus is NNV, and the NNV is about 1.34 g in CsCl.
A method with a buoyant density of / ml. 22. A method for detecting a virus in an immortal cell line, comprising the step of developing a cytopathic effect (CPE) in an established cultured cell containing the immortal cell line according to claim 1. And observing with a microscope. 23. The method for detecting a virus in an immortal cell line according to claim 22, wherein the cell line is fixed on glutaraldehyde and osmium tetroxide; Detecting virus particles in an ultrathin section of the isolated cell line. 24. A method for detecting NNV in an immortal cell line, wherein the development of cytopathic effect (CPE) in established cultured cells containing the cell line according to claim 1 is carried out using a microscope. , A step of extracting viral RNA from the cultured cells, a PCR method using a reverse primer having a DNA sequence of SEQ ID NO: 1 and a forward primer having a DNA sequence of SEQ ID NO: 2. And amplifying the viral RNA. 25. A method for detecting NNV in an immortal cell line, the method comprising microscopically examining the development of the cytopathic effect (CPE) in established cultured cells containing the cell line according to claim 1. Observing the virus protein from the cultured cells; electrophoresing the viral protein in an SDS polyacrylamide gel; and Western blotting using anti-NNV serum. Analyzing the acrylamide gel. 26. A method for detecting NNV in an immortal cell line, wherein the development of cytopathic effect (CPE) in established cultured cells containing the cell line according to claim 1 is carried out using a microscope. A method comprising: detecting NNV protein from the cell line; and detecting NNV using ELISA. 27. A method for detecting NNV in an immortal cell line, comprising: fixing the cell line according to claim 1 to formalin; and adding a mouse anti-NNV antibody serum to the cell line. Fluorescein isothiocyanate (FIT)
Staining with a goat anti-mouse antibody labeled in C). 28. An anti-NNV serum, which is an anti-NNV antibody produced by administering an effective amount of NNV purified by the method according to claim 21 to a suitable animal and stimulating an immune response of the animal. Antibodies. 29. The anti-NNV antibody according to claim 28, which is a monoclonal antibody. 30. A method for preparing an anti-NNV antibody, comprising: injecting an effective amount of NNV purified by the method according to claim 21 into a suitable animal to stimulate an immune response of the animal. Recovering and purifying serum from said animal. 31. A vaccine for protecting fish from NNV infection, comprising an immunogenically effective amount of killed NNV purified by the method of claim 21. 32. The vaccine of claim 31, comprising an adjuvant, a plasticizer, a pharmaceutical excipient, a carrier, a diluent, a binder, a lubricant, a glidant, and a narcotic compound. A vaccine further comprising one or more materials selected from the group. 33. The vaccine of claim 31, which is administered orally. 34. The vaccine of claim 33, wherein the orally administered vaccine further comprises an enteric coating, wherein the enteric coating does not dissolve in the fish stomach. 35. The vaccine of claim 31, wherein the vaccine is provided by injection. 36. A method for protecting fish from NNV infection, comprising providing a vaccine produced by the method of claim 31 to fish.