JP3062357B2 - Prevention vaccine for stomatosis of puffer fish - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass cultivation of a stomatosis virus of a pufferfish, useful for the preparation of a vaccine to prevent stomatosis of a pufferfish, and a vaccine to prevent stomatosis of a pufferfish. About.

[0002]

2. Description of the Related Art Albinism of a puffer fish is characterized by ulceration of the lip of the puffer fish, and is characterized by being aggressive to other fish. In particular, farmed tiger pufferfish is often affected during the summer when the water temperature rises, so the damage is large, and the establishment of effective preventive measures is required.

[0003] It is already known that the tinea pedis of the pufferfish is caused by virus infection, and the virus has also been isolated (Inoue et al., Fish Disease Research, 21).
(2), 129-130 (1985)). From this fact, it is expected that vaccination with an inactivated virus will be very effective in preventing desired albinism of Puffer fishes.
However, practical use of the vaccine requires a large amount of the oral leukovirus. Nevertheless, no effective method has been found for obtaining a large amount of the albinism virus of the puffer fish.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to establish a method for obtaining a large amount of stomatosis virus of a puffer fish, and to use the stomatosis virus of a puffer fish. To provide vaccines.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have inoculated and cultured the ovary-derived cells of the pufferfish, Pufferfish, with stomatosis virus of the pufferfish. Thus, the present inventors have found that the virus can be cultured in a large amount, and as a result, a vaccine for prevention of stomatosis of a Puffer fish can be prepared, thereby completing the present invention.

That is, according to the present invention, cells derived from the ovary of the Puffer fish are subcultured, and the cultured cells are inoculated with the stomatitis virus of the Puffer fish, and accumulated in the culture solution as the virus grows. A method for culturing tinea pedis virus of a Puffer fish, characterized by collecting the virus, and a preventive vaccine for tinea pedis of a Pufferfish, comprising an inactivated tuskera virus of a Pufferfish, as an active ingredient. To provide.

Hereinafter, the present invention will be described in detail. A. The culture method of the present invention is characterized by inoculating and subculturing the ovary-derived cells of the puffer fish, which are subcultured, with the stomatitis virus of the puffer fish. The type of ovary-derived cells used in the practice of the method of the present invention is not particularly limited as long as it is derived from the ovary of a Pufferfish that suffers from stomatosis. That is, ovary-derived cells of many types of puffer fish other than tiger puffer and kusafugu can be used. However, if the type of puffer fish intended to prepare a vaccine described later is clear, it is preferable to use the ovaries of this type of puffer fish from the viewpoint that more effective preparation of a prophylactic vaccine can be expected. In addition, in preparation of the ovary-derived cells, a tissue other than the cell part of the ovary of the blowfish is collected and digested with a protease. Such proteolytic enzymes include, for example, trypsin, dispase and the like. The reaction conditions for the enzyme can be appropriately set according to the type of the selected protease.

[0008] The suspension of ovary-derived cells obtained above is separately transferred to a culture vessel and cultured. Examples of the basal medium used for this culture include a medium such as an Eagle MGM medium and a Leibovitz L-15 medium. Usually, about 10% of fetal bovine serum is added to the basal medium for use in the main culture. The culturing is usually performed at a temperature of 20 to 25 ° C. in a closed state.

Next, the ovary-derived cells of the subcultured puffer fish prepared in this manner are inoculated with the oral leukomycosis virus of the puffer fish. That is, the cells were removed culture supernatant of a culture of the ovary cells is full sheet state becomes full culture surface, and then washed with a washing solution such as a phosphate buffer.
After the washing, inject the virus solution into the culture, and add
Let stand for about 60 to 120 minutes. During this standing,
It is preferred to occasionally gently agitate the culture.

[0010] In addition, the preparation of the oral leukomycosis virus of the puffer fishes can use a method that has already been established.
For example, as described above, “Inoue et al., Fish Disease Research, 21 (2), 129-
130 (1985) "can be used to prepare a desired albinism virus of the Pteratoid fish. Furthermore, it is necessary to culture the virus-infected ovary-derived cells of the puffer fish to propagate the virus.

[0011] The conditions of cell culture intended for such growth are as follows:
The cultivation can be performed under substantially the same conditions as those for the subculture of ovary-derived cells described above. However, the amount of fetal bovine serum added in the culture medium is usually about 5%, which is smaller than the amount added in the subculture of the ovary-derived cells. Preferably, the cultivation of the virus-infected cells is continued until the virus is grown in a large amount as much as possible and the infected cells do not die. Specifically, a point in time when the microscopic observation shows that it is an extreme stage of denaturation of the cells that exhibit fibroblast-like cells out of the infected cells and are spheroidized and detached, which are induced in synchronization with the growth of the virus. The culture solution is separated into a cell fraction and a culture supernatant by a generally known separation method such as centrifugation, and the culture supernatant is treated as a virus suspension. B. Preparation of the vaccine of the present invention can be carried out by inactivating the oral leukomycosis virus of the puffer fish.

[0012] The target albinism virus of the Puffer fish
The cultivation method is not particularly limited, but since a large amount of tinea wilt virus of the puffer fish is required for practical use of the vaccine, the bacterium was propagated by the method of multiplying the tinea wilt virus of the puffer fish of the present invention described above. It is particularly preferable and practical to use the virus. Virus inactivation means
A commonly known method used for preparing a normal inactivated virus vaccine can be employed. For example,
Means such as formalin treatment and β-propiolactone treatment can be employed. When performing formalin treatment, 0.1 to 0.3%, preferably about 0.1% of formalin is added to the virus solution. In the case of performing β-propiolactone treatment, 0.2-0.4% β-propiolactone is added to the virus solution.

[0013] The vaccine of the present invention can be composed of only the inactivated virus. However, if necessary, other components such as an adjuvant can be mixed and used. In addition, the dosage form does not matter whether injection or immersion. Usually before the hot summer season (5
接種 to June) at two to three week intervals.
Such inoculation is performed in the first or second year, or in the first and second years, when the puffer eggs have hatched.

[0014]

The present invention will be specifically described below with reference to examples. [Example 1] Culture of Trafugu albinism virus 200 to 500 g of female ovaries of Trafugu are aseptically collected, penicillin (100 IU / ml), streptomycin (100 µg / m
l) Washed 2-3 times with a phosphate buffer to which kanamycin (100 μg / ml) was added. Subsequently, after removing the coating covering the tissue, the ovary was cut into 1 mm square pieces with ophthalmic scissors.

5% to 10 times 0.05% trypsin-0.53m
The minced tissue was suspended in MEDTA-4Na, and the tissue was digested while stirring with a stirrer. After treating the digest at room temperature for 3 hours, the solution is filtered through a metal mesh, and the filtrate is subjected to 1000 rpm.
And centrifuged for 10 minutes in a cell culture medium (a defined amount of Leibovitz L-15 medium as a basal medium, and 10 ml of fetal bovine serum).
%, Penicillin 100 IU / ml, streptomycin 100μ
g / ml, kanamycin 100 μg / ml) to obtain a cell suspension.

This cell suspension is dispensed into a culture flask.
The cells were cultured at 20 ° C. The cultured cells thus obtained were detached with trypsin when the cells became full on the culture surface, centrifuged, and the resulting precipitate was suspended in a culture solution for cell culture and dispensed into a culture flask. Teens. Next, the culture solution for cell culture of the above-described subcultured cells of the tiger ovary was removed, and washed twice with a phosphate buffer. The washed subcultured cells were inoculated with the virus isolated from tinea pedis infection. The isolation of the virus, which is a prerequisite for inoculation, is described in Inoue et al., Fish Disease Research, 21 (2), 12
9-130 (1985) ".

A virus solution was mixed at a ratio of 1 ml to a subcultured cell cultured in a flask (manufactured by Falcon) having a culture bottom of 25 cm ² for cell culture until it reached a full sheet state. After 90 minutes, the virus solution was removed with a Pasteur pipette, the virus-inoculated cells were washed once with a phosphate buffer, and then a culture medium for cell maintenance (a defined amount of Leibovitz L-15 as a basal medium) was used. A medium, 5% fetal calf serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, 100 μg / ml kanamycin) were added, and the virus was cultured at 20 ° C. At the extreme stage of the cell degeneration, the culture supernatant was collected with a pipette, while identifying the degeneration of the cell that occurred in synchronization with the growth of the virus by observation with a light microscope. The collected culture solution was centrifuged at 3000 rpm for 20 minutes, and the supernatant was filtered with a 0.22 μm membrane filter to obtain a virus suspension. This step was repeated twice to obtain a large amount of the virus suspension. [Example 2] Preparation of vaccine of the present invention 10% formalin was added to the virus suspension obtained in Example 1 above with stirring so that the final concentration was 0.1%, and this was used as a stock solution of vaccine.

After removing the formalin from the stock solution of this vaccine, the subcultured cells of the tiger pufferfish shown in Example 1 were inoculated at the ratio shown in Example 1. As a result, it was confirmed that no cell denaturation was caused. . [Test Example 1] Test for confirming the ability of the vaccine of the present invention to induce antibody production After removing formalin from the stock solution of the vaccine prepared in Example 2 above, use the stock solution and the culture solution for cell maintenance.
A 10-fold dilution and a 100-fold dilution were prepared. 50 for test fish
g and 300 g of pufferfish were used. One test group consists of 25 fish,
Four groups at 50 g and four groups at 300 g (each including one control group) were inoculated subcutaneously in the back at a dose of 0.1 ml each with the vaccine of the above-mentioned dilution ratio.

Blood was collected every week after the inoculation, and the amount of antibody titer against oral leukoplakia virus in the blood of Trafugu was measured by a neutralization test by serum dilution. The results are shown in FIG. This figure reveals that the antibody titer in the blood reaches the peak at 3 weeks each, and that the inoculation of the vaccine of the present invention induces the production of antibodies against oral leukovirus in test fish. became.

The same results were obtained for the undiluted solution of the above vaccine with an adjuvant added. The results of inoculating the vaccine of the present invention into 50 g of a puffer fish by the dipping method were examined. That is, with one test group consisting of 10 fishes, 50 g of the puffer fish of the group were immersed in 10 L of seawater and a vaccine solution obtained by adding 1 L of the stock vaccine solution for 10 minutes.

As described above, blood is collected every week,
The antibody titer against stomatosis virus in blood was measured. As a result, as in the case of subcutaneous inoculation, the antibody titer reached its peak three weeks after inoculation, and it was revealed that production of antibodies against oral leukovirus in the test fish was also induced by the immersion method. [Test Example 2] Test of the protective ability of the vaccine of the present invention against infection The test fish were prepared by diluting the concentration of the vaccine 100 times from the undiluted solution using 300 g of tiger puffer, each group consisting of 25 fish. Subcutaneous subcutaneous inoculation of 0.1 ml was performed. The control group was subcutaneously inoculated with 0.1 ml of physiological saline in the back, with 25 groups as one group.

Two weeks after inoculation, 1 × 10 ^3.7 TCID ₅₀ / m
All fish were inoculated subcutaneously in the back at a titer of l with 0.1 ml of the solution. The results of this test are shown in FIG. According to this figure, the response of each group after the vaccination was as high as 80% in the vaccine-administered group, whereas the control group was 20% in the control group.
%, Clearly indicating a difference between the administration group and the non-administration group.

Thus, it has been clarified that inoculation of the vaccine of the present invention provides an effective infection-protecting effect against albinism in puffer fish.

[0024]

Industrial Applicability According to the present invention, a method for obtaining a large amount of stomatosis virus of a pufferfish is established, and a vaccine using the stomatosis virus of a pufferfish is provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of inoculation of the vaccine of the present invention on the amount of oral leukoplakia virus antibodies in the blood of the puffer fish.

FIG. 2 is a diagram showing the results of a test on the ability to prevent infection of stomatosis virus by inoculation of the vaccine of the present invention.

Continued on the front page (72) Inventor Michio Nonaka 16-2 Wadai, Tsukuba-shi, Ibaraki Prefecture Central Research Institute of Ocean Fisheries Co., Ltd. (72) Inventor Seiya Ide 3-3-12 Kita-Kashiwa, Kashiwa-shi, Chiba Pref. No. (72) Inventor Nobutoshi Maehara 4-16-9 Minami Koshigaya, Koshigaya City, Saitama Prefecture (72) Inventor Katuro, 2-1-25 Oma, Kounosu City, Saitama Prefecture (72) Inventor Yujiro Fujisaki 1 Kichijoji Minamicho, Musashino City, Tokyo No. -6-18-904 (56) Reference Fish Disease Research, Vol. 21, No. 2, p. 129-130 (1986) Fish Disease Research, Vol. 16, No. 3, p. 129-137 (1981) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 39/00 C12N 7/00 JICST file (JOIS)

Claims (1)

(57) [Claims] The present invention provides a method for inactivating the virus obtained by inoculating a passage cell derived from the ovary of a puffer fish, an oral leukosis virus of a puffer fish, and collecting virus particles from the culture solution. A vaccine for the prevention of stomatosis of pufferfishes, comprising the pufferfish tinea virus as an active ingredient.