JP6709395B2 - Cold water vaccine and method for preventing cold water disease - Google Patents

Description

The present invention relates to a cold water disease vaccine and a method for preventing cold water disease.

Ayu is a representative freshwater fish in Japan and an important target fish for industry. In rivers and fish farms, cold water disease that causes the death of ayu is a big problem. Cold water disease is a bacterial disease caused by Flavobacterium cyclophilum , and exhibits symptoms such as perforation and non-hemolytic anemia. When ayu cold water disease occurs, production efficiency decreases due to a high mortality rate, and commercial value is significantly impaired due to trauma such as perforation. Ayu cold water disease control is an important issue in inland fisheries.

In general, administration of antibacterial agents is effective for bacterial diseases. As an antibacterial agent effective against Ayu cold water disease, sulfisosol has been approved as a veterinary drug and is used when cold water disease occurs (Patent Document 1). However, there are many cases in which death does not subside even when the above-mentioned therapeutic agent is administered, and it is difficult to completely cure an individual who already has external symptoms such as perforation without leaving a trace. In addition, a method has also been developed in which ayu breeding water is heated to remove the flavobacterium cyclophilum (Patent Documents 2 and 3). However, since it is impossible to keep several hundred tons or more of breeding water heated for a few days in most farms, the situation where the method can be applied is limited. It is difficult to say that the methods developed so far are perfect as measures against cold water disease.

From such a background, the demand from the field for the development of the cold water disease vaccine was extremely strong. Ayu is smaller than marine fish and is cultivated in the unit of hundreds of thousands to millions of fish in the farm, so it is not realistic to inject each one. Moreover, since the internal organs of ayu are also eaten, injecting the vaccine solution into the abdominal cavity is not preferable from the viewpoint of food hygiene, since there is a concern that an adjuvant may remain. Therefore, the Ayu cold water disease vaccine has been developed mainly in the immersion type. One of them is a formalin killed cell (FKC, hereinafter abbreviated as "FKC") vaccine inactivated by adding formalin to a culture medium containing Flavobacterium cyclophilum in a logarithmic growth phase. (Patent Document 4).

Regarding the FKC vaccine, the fisheries laboratories in various places have conducted effect tests, but although the injection method gives good results, the dipping method does not give good reproducible results. There is. In addition, a cold water disease vaccine in a dosage form that activates the immune system of ayu by combining rabbit-derived red blood cells has also been invented (Patent Document 5). However, none of the vaccines has been approved so far due to problems such as insufficient reproducibility obtained by the immersion method. Ayu belongs to the subfamily Ayu, but its characteristics, such as body shape and having fin fins, are similar to those of the Salmonidae.

Flavobacterium cyclophilum is the causative agent of cold water disease in salmonid fish. Bacteria that are pathogenic to salmonidae (salmon-type strains) and fungi that are pathogenic to ayu (ayu-type strains) are the same species, but the properties of the bacteria are different. There are also differences in the symptoms of cold water disease. Salmon-type cold water disease is mainly characterized by causing large-scale mortality in the fry stage, and it is rare in mature salmonid fish to cause large-scale mortality, but the product value is significantly reduced due to dissolution of caudal fins, etc. Therefore, countermeasures against cold water disease are also required at salmonid fish farms. Up to now, a vaccine effective against cold water disease in salmonids has not been developed, and there is a great demand for the vaccine.

JP-A-2000-191551 JP 2005-287303 A JP 2005-245318 JP 2004-210769A. JP 2004-352690 A

It is intended to provide a vaccine effective against cold water disease in sweetfish and salmonids.

In particular, it is an object of the present invention to develop a vaccine that can obtain a vaccine effect by the dipping method, which has not been able to obtain good reproducibility with conventional vaccines.

A large amount of collagenase, metalloprotease or an antigenic fragment thereof is obtained by culturing a strain belonging to Flavobacterium cyclophilum or by using a genetic engineering technique. It is used as a cold water disease toxoid vaccine by immersing sweetfish and salmonid fish in a solution containing collagenase, metalloprotease or an antigenic fragment thereof. In addition, a toxoid vaccine and a FKC vaccine of Flavobacterium cyclophilum or a solubilized Flavobacterium cyclophilum bacterial cell (Solubilized Flavobacterium psychlophilum: SFP, hereinafter referred to as “SFP”) vaccine may be used in combination or an appropriate adjuvant may be added. By doing so, the effect of the toxoid vaccine can be enhanced. Furthermore, the vaccine effect can be further enhanced by repeatedly treating these vaccines twice or more. In particular, since the SFP vaccine uses a smaller amount of formalin as compared with the conventional FKC vaccine, the toxicity to fish can be significantly reduced, and the time for immersing in the vaccine solution can be extended. Further, by prolonging the immersion time, the amount of antigen taken up can be improved, which in turn can contribute to the improvement of the vaccine effect. The present invention provides the following cold water disease vaccine and a method for preventing cold water disease.

Item 1. A cold water disease vaccine containing an active ingredient of collagenase derived from Flavobacterium cyclophilum, metalloprotease or an antigenic fragment thereof.
Item 2. Item 2. The cold water disease vaccine according to Item 1, which is a cold water disease vaccine against sweetfish and the active ingredient is collagenase or an antigenic fragment thereof.
Item 3. Item 2. The cold water disease vaccine according to Item 1, which is a cold water disease vaccine against salmonid fish, wherein the active ingredient is a metalloprotease or an antigenic fragment thereof.
Item 4. A method for preventing cold water disease of sweetfish or salmonids, which comprises immersing the sweetfish or salmonids in a solution containing the cold water vaccine according to Item 1.
Item 5. Item 5. The sweetfish, salmonidae according to Item 4, wherein the flavobacterium cyclophilum formalin killed bacteria (FKC) vaccine or the flavobacterium cyclophilum solubilized (SFP) vaccine is further acted on ayu or salmonid fish. How to prevent cold water disease in fish.

According to the present invention, a high effect as a cold water disease vaccine can be obtained, and it becomes possible to make ayu and salmonid fish have acquired immunity against cold water disease bacteria by the dipping method.

A) Growth curve of collagenase-expressing Bacillus subtilis After inoculation into a medium, the cells were cultured at 37° C. for about 24 hours, and when the temperature was changed to 15° C., the collagenase expression amount became maximum. When the culture was continued at 37° C., O. D. _{Since the} growth stopped when the ₆₂₀ value was around 1.0, the collagenase is considered to have strong cytotoxicity against Bacillus subtilis. Therefore, it is expected that Bacillus subtilis continued to grow when the collagenase activity was reduced to 15°C. Samples (137, 161 and 216h) surrounded by an elliptical broken line and having a culture time were subjected to gelatin zymography in the following b). B) Confirmation of Collagenase Activity by Gelatin Zymography The Bacillus subtilis culture solution (cultured at 137, 161 and 216h) was subjected to gelatin zymography (containing 10% gel and 0.1% gelatin). The gel after electrophoresis was incubated at 24° C. for 1 h to decompose gelatin contained in the gel and visualized by CBB staining. Confirmation of specific antibody contained in serum of cold water disease-resistant ayu using ELISA method Cold water disease resistant ayu is an antibody that specifically recognizes and binds to collagenase produced by cold water disease bacteria in addition to cold water disease bacteria. Was produced. Cumulative mortality rate in toxoid vaccine test Vaccine test was conducted in two test tanks (25 fish per tank) in both test plots. The numerical values in the figure show the average mortality rates of the two aquariums. Vaccine plots had lower mortality rates than control plots (same treatment with vaccine-free aquifer). Cumulative mortality in the case of combined use of toxoid vaccine and FKC vaccine Vaccine test was carried out in two test tanks (20 fish per tank) in both test plots. The numerical value on the vertical axis (cumulative mortality rate) indicates the average mortality rate of the two water tanks. The combined ward had a significantly lower mortality rate than the control ward. Cumulative mortality when the toxoid vaccine and SFP vaccine are used together.

Two test tanks (25 fish per tank) were subjected to an attack test in both test plots. The change of the cumulative mortality in the challenge test performed 3 weeks (A) and 5 weeks (B) after the vaccine treatment is shown. The vaccine group had a lower mortality rate than the control group.

In the present specification, an antigenic fragment of collagenase or metalloprotease means a polypeptide capable of producing anti-collagenase antibody or anti-metalloprotease antibody in salmonid fish or ayu, and at least one epitope of collagenase or metalloprotease. Is included. In the present specification, the term "collagenase" may be used to mean "collagenase or an antigenic fragment thereof". Moreover, it may be described as “metalloprotease” in the sense of “metalloprotease or an antigenic fragment thereof”. Collagenase or an antigenic fragment thereof is particularly effective as a cold water vaccine for sweetfish, and metalloprotease or an antigenic fragment thereof is particularly effective as a salmonid vaccine. The metalloprotease preferably includes FPP1 and FPP2 (Secades et al., Appl. Environ. Microbiol., 67(2001) 2436-2444; Secades et al., FEMS. Microbiol. Lett., 226(2003). 273-279).

In the present specification, the collagenase derived from Flavobacterium cyclophilum may be referred to as “the collagenase”. The term "collagenase" includes both those obtained by culturing a microorganism belonging to Flavobacterium cyclophilum by the method described below and those obtained by genetic engineering techniques.

Microorganisms belonging to Flavobacterium cyclophilum can receive the strains deposited by the National Institute for Product Evaluation Technology (NITE). Examples of flavobacterium cyclophilum related to cold water disease of sweetfish include WA-1 strain (NBRC108951), WA-2 strain (NBRC109952), SG-1 strain (NBRC111643) strain, and cold water disease of salmonids. Examples of the Flavobacterium cyclophilum related to Escherichia coli include the WB-1 strain (NBRC108952). Cultivation of Flavobacterium cyclophilum can be carried out in MCY (containing 1 g of distilled water, 2 g of peptone, 0.5 g of yeast extract and 0.2 g of sodium acetate) in a solid or liquid medium. To this, 0.5 g of meat extract, 0.2 g of calcium chloride, and 10% (v/v) of fetal calf serum may be added per 1 L of the medium. In addition, a medium such as 1/2 CGY (containing bactocasitone 2.5 g, gelatin 1.5 g, yeast extract 0.5 g, 1 mM calcium chloride per 1 L) medium may be used.

When a collagenase or a metalloprotease is obtained from the culture solution of Flavobacterium cyclophilum, the collagenase or the metalloprotease is secreted into the medium and can be recovered from the culture medium by the following method. After passing the late logarithmic growth phase of Flavobacterium cyclophilum, the cells are removed by centrifugation (6,000 rpm×10 minutes) or the like for about 5 days, preferably about 48 hours. When calcium chloride or gelatin is contained in the medium, the expression levels of collagenase and metalloprotease are reduced, so it is desirable not to include these in the medium (Nakayama et al., Biosci. Biotech. Biochem. (2015) , Sep1:1-10, ePub ahead of print).

Collagenase in the culture medium can be purified and concentrated by the following method. The collagenase can be precipitated by adding 50% ammonium sulfate to the culture solution from which cells have been removed by centrifugation or the like, mixing the mixture at 4° C. for about 2 hours, and then centrifugation (20,000 g×30 minutes). On the other hand, it has been confirmed that the collagenase does not precipitate by centrifugation (6,000 g×15 minutes) when the ammonium sulfate concentration is 20%. Before performing 50% ammonium sulfate fractionation, if necessary, perform 20% ammonium sulfate fractionation to precipitate contaminating proteins other than collagenase, and recover the supernatant fragment containing collagenase to remove the contaminating proteins. You can Furthermore, the precipitated fragment after the 50% ammonium sulfate fractionation can be suspended in an appropriate buffer (50 mM Tris pH 8.0, 5 mM calcium chloride) and then purified by column chromatography. Also, the collagenase fragment can be concentrated by ultrafiltration (10 kDa cutoff).

A person skilled in the art can similarly purify and concentrate the metalloprotease by referring to the above-mentioned method for purifying and concentrating collagenase in the culture solution and known techniques.

One example of the collagenase derived from Flavobacterium cyclophilum used in the vaccine of the present invention is shown in SEQ ID NO: 1, and when used as a vaccine, a part of the N-terminal or C-terminal sequence in the antigenic fragment is It may be deleted. Specifically, the N-terminal and/or C-terminal sequence of the amino acid sequence of SEQ ID NO: 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 from each end. , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 deleted polypeptides (collagenase) can be used in vaccines. Therefore, an antigenic fragment in which 50 amino acids from the N terminus and 50 amino acids from the C terminus are deleted in total 100 amino acids can be used in the vaccine of the present invention. Further, as long as the epitope remains, at least one amino acid in the collagenase (non-terminal) may be substituted, added, or deleted.

The N-terminus of collagenase means both the N-terminus where the signal peptide is cleaved and the N-terminus containing the signal peptide. The amino acid at positions 1 to 75 shown in SEQ ID NO: 1 is a signal peptide, and this peptide may be cleaved at any length. Further, 50 amino acids at positions 76 to 125 may be deleted at any number. You may lose.

Similarly, for metalloproteases, the N-terminal and/or C-terminal sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 11, 12, 13, 14, from the respective ends. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, Polypeptides with 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 deletions can be used in vaccines.

The collagenase gene and metalloprotease gene used for producing collagenase and metalloprotease in bacteria such as Bacillus subtilis and Escherichia coli are genes encoding flavobacterium cyclophilum-derived collagenase or metalloprotease. Preferred collagenase genes are (a) a DNA encoding the amino acid sequence represented by SEQ ID NO: 1 or an antigenic fragment thereof, (b) a DNA represented by SEQ ID NO: 2, (c) the above (a) or (b). It is a complementary strand.

One embodiment of the collagenase gene and the metalloprotease gene of the present invention can be obtained by cloning a natural gene derived from the genomic DNA of a strain isolated from ayu and salmonid fish among Flavobacterium cyclophilum. The procedure that the present inventors actually performed on ayu is as follows. Genomic DNA was extracted from cultured cells of Flavobacterium cyclophilum isolated from sweetfish. Primers capable of amplifying a DNA fragment containing the collagenase gene set (F: tttcgctgca ggatcc TATACTTTGCCAGTAGTATTTC (SEQ ID NO: 3), underlined the recognition sequence for the restriction enzyme BamHI; R: ccggggtacc gaattc TTATTTTTTAATTATTTTTTTAG (SEQ ID NO: 4), underlined restriction enzymes EcoRI A DNA fragment containing the collagenase gene was obtained by PCR reaction using the recognition sequence of When the DNA fragment is used, the collagenase can be produced in a large amount by a gene recombination technique. That is, a host cell can be transformed by incorporating the collagenase gene of the present invention into an appropriate vector. The collagenase gene of the present invention can be expressed in each host cell by introducing an appropriate promoter or a sequence involved in expression into these vectors. As a host cell, Escherichia coli, Bacillus subtilis, or Brevibacillus chosinensis can be used. , PGEM4 and the like, pUC110, pE194, pC194 and the like, which can be replicated in Bacillus subtilis, and pNCMO2, pNY326 and the like, which can be replicated in Brevibacillus choshinensis.

The collagenase can be produced by culturing a host cell transformed with the vector containing the collagenase gene of the present invention and collecting the collagenase from the culture. Culturing of host cells can be performed by liquid culture in a medium containing an appropriate carbon source, nitrogen source and a trace amount of metal element (Lecroisey et al., FEBS Lett., 59 (1975) 167-172). The obtained culture is recovered, and, for example, the culture supernatant or the solubilized bacterial cell supernatant is subjected to ammonium sulfate precipitation (50% saturation), and subjected to various column chromatography (for example, DEAE cellulose column chromatography, gelatin sepharose column chromatography). Chromatography, Sephadex G-100 column chromatography, or chelate affinity chromatography) to give the desired collagenase. An antigenic fragment of collagenase can be similarly obtained.

A solution containing collagenase obtained by the above method is appropriately diluted with ground water or the like to prepare a solution, which can be used as a cold water disease vaccine solution. In the case of a collagenase solution expressed using Brevibacillus choshinensis, it is desirable to dilute it about 1 to 20 times, and an appropriate adjuvant may be added. Antigen presentation is performed by immersing ayu, which has been confirmed not to be infected with Flavobacterium cyclophilum, in this solution. It is better to immerse the sweetfish in the vaccine solution for 5 minutes or more while performing aeration using air stone or the like, and to extend the soaking time as long as the sweetfish is alive.

The vaccine effect can be improved by using the FKC vaccine or the SFP vaccine of Flavobacterium cyclophilum in combination with the above vaccine using collagenase or before the above vaccine. Further, by carrying out these vaccine treatments twice or more with an interval of about 2 weeks, the vaccine effect can be further improved by the booster effect.

The above description of the collagenase vaccine for ayu and the method for producing the same can be applied to a metalloprotease vaccine for salmonid fish. Since the type of metalloprotease produced by salmon-type cold water fungus is considered to function as a toxin in the same manner as collagen for ayu, the present invention uses a metalloprotease instead of collagenase in a cold water disease vaccine for salmonid fish. Can be carried out.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[Extraction of genomic DNA from Flavobacterium cyclophilum]
Flavobacterium cyclophilum WA-1 strain (NBRC108951) isolated from ayu that died from cold water disease was treated with MCY agar medium (2 g of peptone per 1 L, 0.5 g of yeast extract, 0.2 g of sodium acetate, 0.2 g of calcium chloride). 2 g, agar 15 g) and static culture at 18° C. for about 3 days gave slightly transparent yellow colonies. When the colony is further inoculated into an MCY liquid medium and swirling culture (120 rpm) at 18° C. for about 3 days, turbidity occurs. Therefore, the culture solution is subjected to centrifugation (6,000 rpm×15 minutes) to remove bacterial cells. Allowed to settle. Total genomic DNA was isolated from the cells by a conventional method. Examples of the method for isolating microbial DNA include the Saito Miura method (Saito et al., Biochem. Biophys. Acta, 72 (1963) 619-629).

[Cloning of collagenase gene]
Primer set (F: ttcgctgca ggatcc TATACTTTGCCATAGTAGTTTTC (SEQ ID NO: 3), underlined portion is recognition sequence of restriction enzyme BamHI; R: ccgggggtacc gaattc TTATTTTTTTATTTTTTTTE, sequence number of restriction enzyme ( B )). Using the genomic DNA of the WA-1 strain extracted in Example 1 as a template, PCR amplification was performed using DNA polymerase (KOD Dash, TOYOBO) (heat denaturation: 96° C.×30 seconds, annealing: 50° C.×15). Second, extension: 72°C x 3 minutes). By subjecting the PCR product to 1% agarose gel electrophoresis, it was confirmed that a PCR product of about 3.0 kb was amplified.

Next, the PCR product and the pNY326 vector for cloning are digested with restriction enzymes BamHI and EcoRI (37° C.×1 hour), and the digested product is subjected to 1% agarose gel electrophoresis to contain the ORF of the collagenase gene. The DNA fragment and the linearized pNY326 vector were excised and purified. 2 μL of the solution containing the DNA fragment containing the purified collagenase gene ORF was mixed with 1 μL of the linearized pNY326 vector, 3 μL of ligase (Mighty Mix, Takara) was added, and the mixture was incubated at 16° C. for 20 hours. The reaction product was introduced into a competent cell of Brevibacillus choshinensis according to the manual, and the transformed bacterial solution was applied to 2SY (hereinafter abbreviated as "2SYNm") agar medium containing neomycin and 37°C x 40. Incubated for hours. Due to the cytotoxicity of the cloned Flavobacterium cyclophilum-derived collagenase gene, the number of colonies produced was extremely small and was within 10 to 10 per plate. The colony was picked up with a sterilized platinum loop or the like, inoculated into 2SYNm medium, and cultured at 24° C. for 40 hours. After culturing, the bacterial solution was collected by centrifugation (6,000 rpm×15 minutes), and plasmid DNA was extracted from the bacterial cells using a plasmid extraction kit (Nippon Gene). Using the extracted plasmid as a template, the nucleotide sequence was determined using the dideoxy method (F. Sanger et al., Proc. Nat. Acad. Sci. USA, 74 (1977) 5963-5967) and the correct position was determined. It was confirmed that the mature collagenase gene derived from Flavobacterium cyclophilum was linked to.

[Selection of collagenase expressing strain]
Using the plasmid for which the insert was confirmed in Example 2 above, the plasmid was introduced into the competent cells of Brevibacillus choshinensis and the cells again, and 10 colonies were obtained by the same method. Each of these colonies was inoculated into a test tube containing a separate 2SYNm liquid medium, cultured at 24°C for 5 days, and the culture solution was subjected to centrifugation (6,000 rpm × 15 minutes) for culturing. A supernatant was obtained. The culture supernatant was subjected to gelatin zymography, and an expression strain with a high collagenase expression level was selected using the activity as an index. For gelatin zymography, a 10% polyacrylamide gel containing 0.1% gelatin was prepared, and a sample obtained by mixing an equal amount of the culture supernatant with 2× loading buffer was electrophoresed. The gel after electrophoresis was washed twice with a washing buffer (50 mM Tris pH 8.0, 0.1% Tween 20), and incubated in an incubation buffer (50 mM Tris pH 8.0, 0.1% Tween 20, 5 mM calcium chloride) at 24°C. It was carried out by culturing for 20 hours. As a result, gelatin around collagenase is decomposed, and the gel is subjected to CBB staining, so that the approximate amount of collagenase can be estimated by the area of the clear zone which is not stained. For clones with high expression levels of collagenase, 20% glycerol (final concentration) was added to a part of the liquid culture and frozen and stored at -80°C.

[Preparation of vaccine solution containing collagenase]
From the glycerol stock of the clone in which good collagenase expression was confirmed in Example 3 above, 1 L of 2SYNm liquid medium was inoculated and cultivated at 37° C. for 24 hours in a rotating culture. Then, the temperature was lowered to 15° C., and the cells were further cultured for 8 days (FIG. 1). It has been confirmed that if the culture is continued at 37°C, the growth of the host bacterium may be stopped, probably because of the cytotoxicity of the collagenase derived from Flavobacterium cyclophilum. The culture broth was subjected to centrifugation (6,000 rpm×15 minutes) to obtain a culture supernatant. The supernatant was frozen at -20°C and stored until the time of vaccine treatment.

[Collecting serum from cold-water disease-resistant over-ayu]
Preparation of sweetfish serum having anti-cold water disease antibody: Blood was collected from a tail of an ayu suffering from cold water disease and resistant to infection using a 1 mL syringe. The collected blood was allowed to stand overnight at 4° C. and centrifuged (1,500×g, 10 minutes) to collect serum. Serum was stored at −20° C. until the ELISA test was carried out, and when the ELISA test was carried out, a product dissolved by leaving it at room temperature was used.

[Detection of specific antibody contained in ayu serum]
Coating wells with antigen: cold water fungus used as an antigen in the ELISA test was coated with a coating buffer (in 1 L of distilled water, Na ₂ CO ₃ : 1.59 g, NaHCO ₃ : 2.93 g, NaN ₃ : 0.2 g, pH with HCl. (Adjusted to 9.6) (cells 10 mg/mL). The collagenase solution obtained in Example 4 was diluted 10 times with the coating buffer. Also, PBS buffer was used as a control. 50 μL of each of these solutions was placed in a 96-well microplate for ELISA (Maxisorp, Nunc) and allowed to stand at 25° C. for 1 hour, and the plate surface was coated with each antigen.

Wash-off of uncoated antigen: After discarding the antigen solution, wash buffer (Tris: 2.4 g, NaCl: 8.0 g, KCl: 0.2 g, NaN ₃ : 0.2 g, Tween 20 in 1 L of distilled water). : 0.5 mL, adjusting pH to 7.4 with HCl) was added to each well in an amount of 200 μL and discarded. This operation was repeated 4 times.
Blocking: 200% of 1% Block Ace (manufactured by DS Pharma Biomedical) solution was added to each well and allowed to stand at 25°C for 1 hour.
Washing of wells: After discarding the blocking solution, wash buffer (in 1 L of distilled water, Tris: 2.4 g, NaCl: 8.0 g, KCl: 0.2 g, NaN ₃ : 0.2 g, Tween 20: 0.5 mL, 200 μL each of which was adjusted to pH 7.4 with HCl) was added to each well and discarded, which was repeated 4 times.
Primary antibody reaction: The serum collected in Example 5 was diluted 10-fold with a washing buffer, 50 μL of each was added to each well, and the mixture was allowed to stand at 25° C. for 1 hour.
Washing of primary antibody: After discarding the primary antibody solution, the above-mentioned washing buffer (200 μL) was added to each well, and the wells were discarded four times.
Secondary antibody reaction: A secondary antibody (Anti-Ayu IgM, Mouse, mono, AQD) was diluted 2,000-fold with the above-mentioned washing buffer, 50 μL was added to each well, and the mixture was allowed to stand at 25° C. for 1 hour. did.
Washing of secondary antibody: After discarding the secondary antibody solution, 200 μL of the above washing buffer was added to each well, and the operation of discarding was repeated 4 times.
Tertiary antibody reaction: Tertiary antibody (Anti-Mouse IgG, Horse, poly, AP-2000, Vector Co.) was diluted 2000 times with the above-mentioned washing buffer, and 50 μL of each was added to each well, and 25° C.×1 hour I let it stand.
Washing of the tertiary antibody: After discarding the tertiary antibody solution, 200 μL of the above washing buffer was added to each well, and the discarding operation was repeated 4 times.
Substrate decomposition reaction: Substrate (p-nitrophenylphosphate hexahydrate) 10 mg, substrate solution (diethanolamine: 97 mL, magnesium chloride hexahydrate: 0.1 g, NaN ₃ : 0.2 g in distilled water 800 mL) It was dissolved and adjusted to pH 9.5 by adding hydrochloric acid, and then dissolved in 10 mL of which the volume was adjusted to 1 L with distilled water. 100 μL of the substrate solution was added to each well and left at 37° C. for 30 minutes.
Reaction termination: When sufficient color development was observed in the above substrate decomposition reaction, 50 μL of a reaction termination solution (2N NaOH) was added to each well.
Absorbance measurement: Absorbance (Abs. 405 nm) of each well was measured using a microplate reader (NJ-2300, Nunc).

[Culture of cold water fungus SG-1 strain]
Flavobacterium cyclophilum virulent strain SG-1 strain (hereinafter abbreviated as "SG-1 strain") is used in 1/2 CGY medium (2.5 g of bactocasitone per 1 L, gelatin 1.5 g, yeast extract). 0.5g, containing 1mM calcium chloride) and incubating at 18°C for about 2 days while culturing until the late logarithmic growth phase. This bacterial solution was used as an attacking bacterial solution in a vaccine test or as a material for producing a vaccine.

[Preparation of FKC vaccine]
A final concentration of 0.1% formalin was added to the culture solution of the SG-1 strain cultivated in Example 6 above, and the mixture was incubated at 24° C. for 1 hour while swirling to produce an FKC vaccine. The strain used for the vaccine does not have to be the SG-1 strain, and may be a flavobacterium cyclophilum strain having pathogenicity isolated from ayu.

[Toxoid vaccine treatment using collagenase solution]
Ayu artificial seedlings produced at the Kanagawa Prefectural Fisheries Technology Center were immersed in the toxoid vaccine solution prepared in Example 4 for 5 minutes for vaccine treatment. The ayu to be treated at one time had a weight of about 1/10 of the weight of the vaccine liquid. Two weeks after the vaccination, the flavobacterium cyclophilum was artificially infected by immersing the ayu in the culture solution of the SG-1 strain cultured by the method of Example 6 for 30 minutes. After that, the ayu were bred in an aquarium that was flushed with groundwater, and the cumulative mortality rate for 2 weeks after artificial infection was examined (Fig. 3). As a result, the vaccine efficacy rate of the toxoid vaccine alone was about 60%. The formula for calculating the vaccine efficacy rate is as follows.
Vaccine effectiveness rate (%) = (1-vaccine mortality rate / vaccine non-treatment mortality rate) x 100

[Vaccine treatment using a combination of collagenase solution and FKC vaccine]
Natural ayu from Lake Biwa collected in Lake Biwa, Shiga Prefecture, are often found to carry flavobacterium cyclophilum during the collection stage. A population in which the Flavobacterium cyclophilum had been removed beforehand was prepared by heating to 28° C. for 3 days (3 g size or less), and this was used in the vaccine test.

First, according to the method of Example 8 above, sweetfish was dipped in a collagenase solution for vaccine treatment. Subsequently, it was immersed in the FKC vaccine prepared in Example 7 for 5 minutes. After the vaccine treatment, the sweetfish was bred in an aquarium flushed with groundwater, and two weeks after the first vaccine treatment, the second vaccine treatment was performed again by the same method.

Three weeks after the second vaccine treatment, a cold water disease artificial infection was carried out by intraperitoneally injecting the bacterial solution (=10 ⁶ CFU) of the SG-1 strain cultured by the method of Example 6 above. Then, the animals were reared in an aquarium that was flushed with groundwater, and the cumulative number of deaths during the three weeks after the attack was counted. As a result, when the toxoid vaccine and the FKC vaccine were used in combination, and the vaccine was administered twice in total, the vaccine efficacy rate was about 86% (Fig. 4). The vaccine efficacy rate was calculated using the same formula as in Example 8 above.

[Preparation of Flavobacterium Cyclophilum Cell Solubilized Vaccine Solution]
By the method of Example 6 above, 250 mL of a culture solution of SG-1 strain was prepared. Cultured cells were obtained by subjecting the culture solution to centrifugation (6,000 rpm×15 minutes). The cells were frozen and stored at -20°C until the day before the vaccine treatment. On the day before the vaccine treatment, the stored cells were thawed at room temperature and pipetted with 5 mL of 0.5% SDS solution to dissolve the cells. 5 μL of Benzonase Nuclease (Sigma-Aldrich) was added to this solution, and the mixture was incubated at 24° C. for 20 hours. The flavobacterium cyclophilum-derived genomic DNA eluted by the action of the DNA-degrading enzyme was reduced in molecular weight, and the resulting viscosity was reduced to form a homogeneous solution. Further, in order to completely sterilize the Flavobacterium cyclophilum, formalin was added to a final concentration of 0.1%, and the mixture was incubated at 24°C for 1 hour. This solution was used as an SFP vaccine stock solution and diluted to 2.5 L with ground water when used as a vaccine. Since the SFP vaccine forms innumerable bubbles by aeration even after dilution, an appropriate amount of antifoaming agent KM-72 (Shin-Etsu Silicone) for food addition was added.

[Combination of toxoid vaccine and SFP vaccine using collagenase solution]
A natural ayu collected from the coast of Wakayama prefecture was heated at 28° C. for 3 days to prepare a population from which flavobacterium cyclophilum had been removed in advance, which was used for a vaccine test.

First, by the same method as in Example 8 above, ayu was immersed in a collagenase solution for 30 minutes for vaccine treatment. Subsequently, the SFP vaccine prepared in Example 10 was immersed for 30 minutes. After the vaccine treatment, the sweetfish was bred in an aquarium flushed with groundwater, and two weeks after the first vaccine treatment, the second vaccine treatment was performed again by the same method.

Three weeks after the second vaccine treatment, the bacterial solution of the SG-1 strain cultured by the method of Example 7 (=2.5×10 ⁶ CFU) was intraperitoneally injected to induce cold water disease human infection. Carried out. Then, the animals were reared in an aquarium that was flushed with groundwater, and the cumulative number of deaths during the two weeks after the attack was counted. As a result, when the toxoid vaccine and the SFP vaccine were used in combination and they were performed twice in total, the vaccine efficacy rate was about 50% (FIG. 5A). Further, 5 weeks after the second vaccine treatment, the same cold water disease artificial infection was carried out. As a result, the vaccine efficacy rate was about 66% (FIG. 5B). The vaccine efficacy rate was calculated using the same formula as in Example 8 above.

[Concentration of collagenase solution]
The culture supernatant of Brevibacillus choshinensis containing cold water fungus collagenase obtained in Example 4 was concentrated up to 10 times using an ultrafiltration membrane (Vivaspin 20-10k cutoff, GE Healthcare). The samples before and after concentration were subjected to the gelatin zymography described in Example 3 above to confirm the degree of concentration.

When an ultrafiltration membrane with a 30 k cutoff was used, a part of the low-molecular collagenase was permeated, and an active band was confirmed in the flow-through fragment, although slightly. No active band was observed in the flow-through fragment when using a 10 k cut-off ultrafiltration membrane. When the collagenase is used as a vaccine, even a low-molecular-weight collagenase protein may be recognized as an epitope by the ayu immune system. Therefore, it is desirable to recover all protein components derived from collagenase. Therefore, when the collagenase is concentrated, it is efficient to concentrate it using an ultrafiltration membrane with a 10k cutoff.

On the other hand, the collagenase can be concentrated by ammonium sulfate fractionation. It has been confirmed that the collagenase hardly precipitates at an ammonium sulfate concentration of 20% and precipitates at an ammonium sulfate concentration of 50% or more.

Claims (2)

Characterized by immersing sweetfish in a solution containing collagenase derived from flavobacterium cyclophilum or an antigenic fragment thereof and a formalin killed bacteria (FKC) vaccine not treated with SDS of flavobacterium cyclophilum , Ayu Cold Water Disease Prevention Method. The ayu is immersed in a solution containing collagenase derived from flavobacterium cyclophilum or an antigenic fragment thereof and a formalin killed bacteria (FKC) vaccine that has not been treated with SDS of flavobacterium cyclophilum two or more times. The method for preventing ayu cold water disease according to claim 1, which is repeated.