JP2002125674A - Gene-open reading frame vaccine against infectious hematopoietic necrosis(ihn) virus, method for producing the same and method for administering the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new gene vaccine which exhibits an action for inducing a cell-mediated immunity in a living body against the infection of a virus by the transdermal or oral administration of the vaccine into a target fish. SOLUTION: This gene vaccine which has the action for inducing the cell- mediated immunity in the living body against the infection of the virus by the transdermal or oral administration of the vaccine into the target fish, characterized by containing as an active ingredient a DNA corresponding to the whole length-open reading frame of a gene or its partial DNA without integrating a gene DNA corresponding to a virus gene portion encoding the apoptosis- inducing protein of the fish infectious hematopoietic necrosis(IHN) virus with a gene-carrying factor such as a vector or a liposome. The method for producing the gene vaccine, and the method for administering the gene vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene reading frame vaccine against infectious hematopoietic necrosis (IHN) virus (Genevaccine of whole length-open readi).
ng frame), its production method and administration method, and more specifically, by administering to a target fish species,
The present invention relates to a novel gene vaccine having an effect of inducing cellular immunity in a living body against an infection with an IHN virus and exhibiting a high vaccine effect, and a method for producing and administering the same. The gene vaccine of the present invention uses the gene DNA corresponding to the viral gene portion encoding the apoptosis-inducing protein of the IHN virus as a vaccine component without incorporating it into a gene carrier such as a vector or a liposome. A new IH having the effect of inducing cellular immunity in the body against infection
An N virus vaccine has been realized and is useful as a vaccine against viral disease in fish.

[0002]

2. Description of the Related Art In general, the current status of virus vaccines is that, except for some polio vaccines, smallpox vaccines and rabies vaccines using live virus vaccines (improved infectious low-pathogenic viruses), AIDS No viral vaccine has been developed that is highly effective against human viral diseases, such as virus (HIV). And live vaccines always carry the danger that the virus will mutate and restore virulence. Also, there is no IHN virus vaccine against fish including live vaccines.
With the increasing global demand for fish as a source of animal protein, the development of vaccines for viral fish diseases that has hindered the production of fish has been considered an urgent task for a long time. Nothing is effective for IHN virus.

[0003] By the way, a so-called "DNA" which incorporates a specific gene DNA into a vector for various viruses, expresses the protein corresponding to the gene by administering to a living body, and expects an immune effect on the protein, is called "DNA".
Vaccines "have been studied around the world for more than a dozen years, but so far none have been put into practical use.
It can be said that it is under development. In addition, in this method, there is also a concern that the vector (a virus-like infectious genetic factor) may affect the living body.

[0004]

Under such circumstances, the present inventors, in view of the above-mentioned prior art, have proposed a new vaccine effective against the IHN virus which has no problems as seen in the above-mentioned conventional method. As a result of intensive studies with the aim of developing a gene, the intended purpose was achieved by using the full-length DNA reading frame of the gene DNA corresponding to the viral gene portion encoding the apoptosis-inducing protein of the pathogenic RNA virus as an active ingredient. They have found that this can be achieved and have completed the present invention. That is, the present invention provides a gene DN corresponding to a viral gene portion encoding an apoptosis-inducing protein of a pathogenic RNA virus.
An object of the present invention is to provide a gene vaccine containing the full-length reading frame DNA (cDNA) of A as an active ingredient, and a method for producing and administering the same. Another object of the present invention is to provide a novel gene vaccine which exerts an action of inducing cellular immunity in a living body against viral infection by transdermal or oral administration to a target fish species.

[0005]

The present invention for solving the above-mentioned problems comprises the following technical means. (1) A vaccine having an action of inducing cell-mediated immunity in a living body against virus infection by being administered to a target fish species, comprising an apoptosis-inducing protein of fish infectious hematopoietic necrosis (IHN) virus Without incorporating the gene DNA corresponding to the viral gene portion encoding the gene into a gene transfer factor such as a vector or liposome,
A gene vaccine comprising a DNA corresponding to the entire reading frame of the gene or a partial DNA thereof as an active ingredient. (2) The gene vaccine according to (1), wherein the M2 gene DNA, which is a gene encoding an apoptosis-inducing protein of the IHN virus, is used as an active ingredient. (3) The method for producing the gene vaccine according to the above (1), wherein the RNA virus gene portion encoding the apoptosis-inducing protein of the IHN virus is replaced with DNA
Production of a gene vaccine characterized in that a DNA corresponding to the entire reading frame of the gene or a partial DNA thereof is incorporated as an active ingredient without incorporating the gene DNA into a gene transfer factor such as a vector or a liposome. Method. (4) The method for administering the gene vaccine according to the above (1), wherein a gene DNA corresponding to a virus gene portion encoding an apoptosis-inducing protein of the IHN virus is incorporated into a gene transfer factor such as a vector or a liposome. A method for administering a gene vaccine, wherein a DNA corresponding to the full length of the reading frame of the gene or a partial DNA thereof is administered to the target fish species by dermal or oral administration.

[0006]

Next, the present invention will be described in more detail. In the present invention, fish disease infectious hematopoietic necrosis (IH)
N) The gene of the virus is used. The IHN virus is an RNA virus having a single-stranded RNA as a gene. As a method for preparing gene DNA from this virus, nucleic acid is extracted from cultured rainbow trout cells infected with the IHN virus, primers for the gene are designed, and RT is used.
(Reverse transcription) -PCR shows that the double-stranded D
A method for synthesizing NA is exemplified. In the present invention, preferably, the M2 gene RN of the IHN virus is
An M2 gene DNA (cDNA) corresponding to A is synthesized. Further, using this gene DNA, for example, by transforming Escherichia coli according to a known method, an M2 protein can be synthesized. This M2
As a method of administering gene DNA and M2 protein to fish, for example, a method of immersing a target fish species in an aqueous solution to which these are added, a method of breeding the target fish species in water containing them, and an appropriate method equivalent thereto Is exemplified.

In the present invention, as shown in the examples described below, M2 gene DNA is administered to juvenile rainbow trout,
As a result of inoculation of the IHN virus and examination of the subsequent onset, it was confirmed that administration of the M2 gene DNA resulted in a vaccine effect for reducing the onset and death. This vaccine effect was maintained for at least three months after administration of the M2 gene DNA. In addition, it was confirmed that the administration of the M2 protein had a smaller vaccine effect than that of the M2 gene DNA. M2 gene D in rainbow trout fry
As a result of administering NA and M2 protein and examining the M2 antibody titer (antibody titer of anti-M2 serum), a higher antibody titer was obtained in the case of administering the M2 protein as compared to the case of administering the M2 gene DNA. Humoral immune response) was detected. On the other hand, as a result of examining the relationship between the administration of the M2 gene DNA and the cellular immune response, the administration of the M2 gene DNA showed a strong killer activity (cellular immune response) that destroys IHN virus-infected cells in lymphocytes of rainbow trout fry. Was done.

From the above results, the following matters can be pointed out. (1) Among the genes of the infectious fish disease IHN virus (RNA virus), M2 gene DNA has a vaccine activity so high that it cannot be compared with N gene or G gene DNA. (2) M2 DNA exerts its effectiveness as a vaccine, for example, by immersing an individual in a dilute M2 DNA solution using M2 gene full-length reading frame DNA.
Since the in vivo translocation of components by immersion administration is presumed to be due to the body epidermal layer, gills, and the route of swallowing, this vaccine effect is due to the effect of entering the living body through a mixed route of transdermal / transginal / oral administration. it is conceivable that. (3) Therefore, the M2 DNA open reading frame full-length gene vaccine enables simple, short-time, large-scale treatment of individuals. (4) For example, administration of M2 DNA as an antigen has a greater vaccine effect than administration of M2 protein as an antigen, and the effect is exhibited two days after administration. Therefore, this effect is caused by M2 DNA itself. It is thought to be done. In general, it takes at least three days or more after antigen administration until the first immune activity appears, regardless of human or fish. (5) The full-length M2 DNA reading frame vaccine exerts its effect by eliciting a cellular immune response to IHN virus infection rather than the effect of an anti-M2 protein antibody resulting from a humoral immune response to the living body. Demonstrate strongly. That is, the vaccine effect of M2 DNA is considered to be due to induction of cellular immunity.

The gene vaccine of the present invention does not use gene transfer factors such as vectors and liposomes as components and uses the full-length DNA of the gene itself as a vaccine, and has no components other than the target gene. Therefore, it is a feature and advantage that there is no danger that some kind of mutation caused by a gene carrier, which has been a concern, is caused and the virulence is restored. Furthermore, transdermal and oral administration allow simple and large-scale treatment of individuals. This reading frame full length gene vaccine, after administration,
The gene vaccine exhibits its effect in a short time, and its vaccine effect lasts for at least three months or more. Therefore, the gene vaccine has a high preventive effect for preventing fish disease. Originally, the gene (M2 DNA) is known to cause disease by inducing apoptosis (programmed death of host cells) in lymphocytes, which are major cell components of the host immune response, by expressing the M2 protein in the host. (Hatayama / Suzuki / Sakai, aquaculture, 445, p.86-
87, 1999). Therefore, this M2 gene full-length vaccine has a specific mechanism of action that allows the host (living organism to which the vaccine is administered) to avoid apoptosis and exert its vaccine effect. Vaccine ".

[0010] The vaccine of the present invention comprises, as an active ingredient, DNA corresponding to the entire length of the open reading frame of the M2 gene, and a partial DNA which is a part thereof and has the same activity can be used as the active ingredient. Here, the partial DNA is a part of the DNA having the full length of the reading frame, and has a partial sequence having the same activity, and a part of the sequence having the same or similar activity as the DNA having the full length of the reading frame. Is defined as a derivative obtained by performing substitution, deletion, addition, and insertion by ordinary means. The gene vaccine of the present invention comprises
It is applicable as a vaccine against fish virus diseases and is not restricted to fish species. As a method for administering the gene vaccine of the present invention, a method of percutaneously or orally administering the gene vaccine to a target fish species is preferable.Specifically, the target fish species is added to an aqueous solution to which the gene vaccine of the present invention is added. Examples include a method of immersion, a method of breeding a target fish species in water containing the gene vaccine, and an appropriate method equivalent thereto. The gene vaccine of the present invention is preferably applied as a diluted M2 gene DNA solution by mixing the above M2 gene DNA with the above aqueous solution or water so as to have a concentration of, for example, 0.1 to 1.0 μg / ml. . Further, the gene vaccine of the present invention basically suffices to contain the above-mentioned M2 gene DNA as a component, but if necessary, the above-mentioned M2 gene D
For example, a suitable material such as a pharmaceutically acceptable carrier, a stabilizer, a bulking agent, an auxiliary substance, and other compounding ingredients may be compounded or combined with NA to form a product in an appropriate form by a normal preparation means. Is also possible as appropriate.

[0011]

EXAMPLES Hereinafter, the present invention will be specifically described based on examples of the present invention, but the present invention is not limited to the following examples. Example 1 (1) Infectious fish disease infectious hematopoietic necrosis (IHN) virus IHN virus is RNA having a single RNA strand as a gene.
In this example, the virus was isolated from adult rainbow trout fish that died of IHN, cultured, and treated with IHN anti-rabbit serum for IHN.
A virus strain confirmed for N virus was used. For cultivation of the virus, rainbow trout cultured cells (RTG-2 cells) were used.
The cell culture broth disrupted by virus growth was centrifuged to remove cell debris, and stored at -80 ° C as a virus solution. The virus concentration was 1 × 10 ⁶ (pfu) / ml.

(2) Preparation of gene DNA Cultured rainbow trout cells (RTG) infected with IHN virus
-2) extracting nucleic acid from the cells of (2), M2 of IHN virus;
Primers were designed for each gene, N gene, and G gene. The primers designed for RT-PCR replication of the IHN virus gene are shown in Table 1. Then
Each gene double-stranded DNA was obtained by RT (reverse transcription) -PCR. The details are shown in Tables 2-6. Furthermore,
If necessary, use the obtained DNA as a template for the G gene D
NA, M2 gene DNA and N gene DNA were prepared in large quantities by PCR (FIG. 1).

[0013]

[Table 1]

[0014]

[Table 2]

[0015]

[Table 3]

[0016]

[Table 4]

[0017]

[Table 5]

[0018]

[Table 6]

(3) Recombinant M2 protein The M2 RNA gene was reverse transcribed into M2 DNA, and the M2 DNA was ligated to the multiple cloning site of Escherichia coli plasmid vector pCR-2.1 (ligation) to transform Escherichia coli (Top10F '). Convert to pCR
-2.1 / M2 is expressed in large amounts, and the restriction enzyme Eco R
1 and the vector pGEX-6P-1 / incorporating glutathione S-transferase (GST).
GST was ligated with M2 DNA, and E. coli (B
L-21) to express the GST-M2 fusion protein. This Escherichia coli is lysed, bound to an affinity chromatography column of glulathione Sepharose 4B, and a specific protease (human rhinovirus 1) that specifically cleaves GST and M2 proteins.
42C protease) to obtain a recombinant M2 protein.

(4) M2 antibody A rabbit was immunized twice with 1 mg of the recombinant M2 protein, and the serum was collected to obtain an anti-M2 rabbit antibody.

(5) Rainbow trout juvenile Rainbow trout juveniles, which have been subjected to artificial insemination, eye development, hatching, and capsular absorption, are further bred to 0.6-1.2 g with an artificial compound feed, during which time they are used as gene vaccine test fish. Tested.
Thirty fry were put in each 10 l water tank, and groundwater at 9 ° C. was supplied to the water tank at 0.1 l / min. Air was supplied into the water tank through the disperser. For 1 to 1.5 weeks before the start of the test, fry were reared in an aquarium in advance for acclimation, and it was confirmed that there were no infectious diseases.

(6) Immersion (vaccination) of DNA and protein M2 DNA and N in 200 ml of breeding water in a small container
DNA and G DNA were added to each at a concentration of 0.2 μg / ml.
After immersion for 15 minutes with no tails, the animals were returned to the breeding aquarium.
M2 protein was prepared at 1 μg / ml, and rainbow trout fry were immersed under the same conditions. DNA and protein solutions were used once.

(7) Immersion infection of virus 30 small fish in 200 ml of breeding water (for one aquarium)
Fry, and thawed virus solution 0.2 ml (10
³ pfu / ml), and after immersion for 15 minutes, the fry was returned to the 10 l water tank. The virus solution was used once.

(8) Measurement of Antibody Titer of Anti-M2 Serum The antibody titer (humoral immune response) of anti-M2 serum was measured by the enzyme antibody method. After binding the M2 protein to the microplate and washing with a phosphate buffer, serum obtained from rainbow trout fry was serially diluted two-fold and added. After washing, anti-M2 was further added.
Protein rabbit IgG antibody was added as the primary antibody.
After washing, an enzyme-labeled (hydroperoxidase) anti-rabbit IgG goat antibody was bound to the remaining primary antibody. Unbound antibody was washed away, the enzyme substrate was added, and color development was measured with a microplate reader. The antibody titer was determined using normal serum (no antibody) as a control.

(9) Measurement of killer activity (cellular immune response) Lymphocyte fraction was collected from blood of rainbow trout treated with heparin by Percoll density gradient centrifugation, washed with tissue culture medium, and suspended in lymphocytes. A liquid was prepared and stored at 4 ° C.
Lymphocyte fluid was added to rainbow trout gonad-derived cultured cells (RTG-2 cells) previously cultured in a flask, and the mixture was added to a 15 ° C.
For 5 hours. ED from bottom of flask
The cells were detached with TA-trypsin, and the number of surviving cells was counted with a hemocytometer under a phase-contrast microscope. Killer lymphocytes are M2
RTG-2 cells infected with IHN virus and passed for 3 days were used as target cells for lymphocytes and lymphocyte killer activity obtained from rainbow trout treated with DNA immersion.

(10) Results 1) Vaccine effect of full length DNA reading frame of IHN virus gene G gene DNA, M2 gene DNA and N gene DNA were immersed and administered to rainbow trout fry, and one day later, IHN virus was immersed and inoculated. The effect of a vaccine to reduce the disease-causing mortality was investigated. The result is shown in FIG. The G gene and N gene DNA have almost no vaccine effect,
A high vaccine effect was observed for the two-gene DNA. IHN virus was detected in all dead fish.

2) Vaccine Effect of Recombinant M2 Protein The recombinant M2 protein is immersed in rainbow trout fry,
The vaccine was infected with the IHN virus and tested for its vaccine efficacy. The result is shown in FIG. It was observed that the onset of mortality was delayed by several days, and the cumulative mortality was reduced by more than ten percent. However, despite the high concentration of immersion compared to M2 DNA, the vaccine effect was small.

3) Persistence of M2 full-length DNA vaccine The rainbow trout fry is soaked with M2 DNA (0.2 μg / ml), and after one month, two months and three months, the IHN virus is soaked and inoculated (10 ³ pfu / ml). ), Followed by one month of death. The result is shown in FIG.
Shown in When M2 DNA was treated one month and two months before, the final cumulative mortality was as low as 2 out of 30 (7%) and extremely effective, and the cumulative mortality was 4 out of 30 (13 months) even before 3 months.
%), And the vaccine effect lasted for at least 3 months.

4) Serum M2 antibody titer (humoral immune response) M2 DNA (0.2 μg / ml) and rainbow trout
After immersion administration of M2 protein (1.0 μg / ml),
Measure M2 antibody titer in rainbow trout serum for one month at daily intervals
Was. As a result, a high antibody titer was detected in M2 protein immersion.
And the antibody titer continued to rise for 30 days (2^Five / 10
Day 2,⁹ / 20 days, 2^Ten/ 30 days). On the other hand, M2 DN
The A-immersion has a significantly lower resistance than the M2 protein
Price (2^Three / 10 days, 2^Five / 20 days, 2⁶ / 30 days)
Indicated.

5) Killer activity (cellular immune response) Lymphocytes were isolated from rainbow trout fry one week after immersion administration of M2 DNA into rainbow trout fry, and cultured cells three days after IHN virus infection (before cytodegeneration was observed) (RTG-2
Cells) and killer activity was measured by the number of surviving cells. Table 7 shows the results. M2 DNA treated rainbow trout lymphocytes were found to show strong killer activity (cellular immune response) to destroy IHN virus infected cells.

[0031]

[Table 7]

[0032]

As described above in detail, the present invention relates to a gene reading frame vaccine against infectious hematopoietic necrosis (IHN) virus, a method for producing and administering the same, and according to the present invention, 1) IHN It is possible to provide a novel gene vaccine having an action of inducing cellular immunity in a living body against virus infection. 2) The gene vaccine of the present invention exhibits its vaccine effect in a short time after administration. In addition, the effect is maintained, 3) the vaccine effect is exerted by inducing so-called cellular immunity in the host, and 4) the vaccine is effective as a vaccine against viral diseases of fish.

[0033]

[Sequence List] SEQUENCE LISTING <110> Director of Hokkaido Fish Hatchery <120> Gene vaccine of whole length-open reading frame against IHN, method of production and administration there <130> 009910P <141> 2000-10-20 <160 > 8 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <400> 1 caaacgagag catgtctatt t 21

<210> 2 <211> 21 <212> DNA <213> Artificial Sequence <400> 2 ccgatggatg ggtgagtgag a 21

<210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 cggatcacga acgatgacaa g 21

<210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 tcagtggaat gcgtcggagt c 21

<210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 tttcgtgctg gagtatttgg a 21

<210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 ggttctttgc ggcttggttg a 21

<210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 accgcaactc gcagagacc 19

<210> 8 <211> 21 <212> DNA <213> Artificial Sequence <400> 8 gtctggtggg gaggaagtga a 21

[Brief description of the drawings]

FIG. 1 shows the prepared M2 gene DNA and N gene DN.
The electrophoresis photograph of A and G gene DNA is shown.

FIG. 2 shows a vaccine effect of IHN virus gene DNA on IHN virus-infected rainbow trout fry.

FIG. 3 shows the vaccine effect of recombinant M2 protein on rainbow trout fry infected with IHN virus.

FIG. 4. M2 against rainbow trout fry infected with IHN virus
The result of having examined the persistence of the vaccine effect of gene DNA is shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:93) C12R 1:93) (72) Inventor Kunio Suzuki 238 Hamanaka-cho, Yoichi-cho, Yoichi-gun, Hokkaido North Sea F-term (Reference) 4B024 AA10 BA32 BA80 CA04 CA07 CA20 DA02 DA06 EA04 GA11 HA03 4C084 AA13 NA14 ZB332 ZC612 4C085 AA03 BA51 CC05 DD22 DD62 GG08

Claims (4)

[Claims] Claims 1. A vaccine having an effect of inducing cellular immunity in a living body against virus infection by administering to a target fish species, comprising a fish disease infectious hematopoietic necrosis (I)
HN) The DNA corresponding to the entire reading frame of the gene or its partial DNA can be effectively used without incorporating the gene DNA corresponding to the viral gene portion encoding the apoptosis-inducing protein of the virus into a gene carrier such as a vector or a liposome. A gene vaccine characterized by containing as a component. 2. The gene vaccine according to claim 1, wherein M2 gene DNA, which is a gene encoding an apoptosis-inducing protein of IHN virus, is used as an active ingredient. 3. The method for producing a genetic vaccine according to claim 1, wherein the RNA virus gene portion encoding the apoptosis-inducing protein of the IHN virus comprises:
DNA corresponding to the entire reading frame of the gene or a partial DN thereof without reverse transcription into DNA and incorporating the gene DNA into a gene transfer factor such as a vector or liposome.
A method for producing a gene vaccine, comprising mixing A as an active ingredient. 4. The method for administering the gene vaccine according to claim 1, wherein a gene DNA corresponding to a virus gene portion encoding an apoptosis-inducing protein of an IHN virus is converted into a gene transfer factor such as a vector or a liposome. A method for administering a gene vaccine, which comprises administering, without integration, DNA corresponding to the entire reading frame of the gene or a partial DNA thereof to a target fish species by transdermal or oral administration.