JPH01125329A - Preparation of antigen component of bovine leukemia vaccine and bovine leukemia vaccine containing said antigen component - Google Patents

Abstract

PURPOSE:To obtain an antigen component of bovine leukemia vaccine sufficiently keeping the function as an antigen, by inactivating bovine leukemia virus (BLV) of crude protein containing BLV and subjecting to ultrafiltration. CONSTITUTION:A crude protein containing BLV is separated from a host cell infected with BLV or a cultured liquid of the cell. The BLV of the protein is inactivated with an inactivation agent (e.g. formalin or glutaraldehyde) and the obtained solution containing inactivated BLV is treated by ultrafiltration to remove a cell-originated component having a mol. wt. of <=10,000 and components such as inactivation agent and to obtain a concentrated fraction containing a protein useful as an antigen component of bovine leukemia vaccine. As an alternative method, the crude protein containing BLV is made to contact with a surfactant to effect the dissociation of the protein, separating a fraction containing core protein of BLV from the obtained mixture by ion exchange method and fractionating a fraction containing an envelope protein of BLV from the mixture free from said core protein by gel-filtration.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to an inactivated vaccine for protecting cattle from bovine leukemia virus (BLV), which is a type of retrovirus that causes bovine leukemia, and its use and production. Regarding the technology necessary for

[Prior art] Bovine leukemia virus (BLV) is known as a type of retrovirus that causes bovine leukemia, and BLV vaccine and BL
There is a strong demand for the practical application of antiserum or antiviral agents against V.

81. LV antigens, which are necessary for the development of vaccines, have been studied in sheep, goats, cows, etc., and in cells derived from these animals.

As for how to obtain BLV itself, for example, culture established cell lines such as FetaJ lamb k3dney cells (hereinafter abbreviated as FLK-BLV) persistently infected with BLV, which can be obtained from the Livestock Hygiene Laboratory of the Ministry of Agriculture, Forestry and Fisheries. However, a method for isolating BLV from cultured cells or culture supernatant is disclosed in JP-A-61-200471.

[Problems to be solved by the invention] With the increasing demand for anal V vaccines as mentioned above, immunity tests have been conducted on various animal models using BLV inactivated antigens, but There has been little research on this topic, and the current state of the art is that BLV vaccines have not been put into practical use using conventional techniques.

Moreover, inactivated B1, ① or BLV-derived proteins, which are necessary as antigenic components of BLV vaccines, can be used in a state where the function as an antigen is sufficiently retained and in a natural and raw state, such as FLK-BLV. A method for separating BLV-infected cells from cultured cells or culture supernatants has not yet been established.

For example, in the method disclosed in the above-mentioned Japanese Patent Application Laid-open No. 61-200471, BLV is separated using ultracentrifugal density gradient method using sucrose, etc., but this is costly and very complicated. However, this method is not practical because BLVs are damaged in the process of BLV separation, and a large amount of glycoproteins constituting the envelope is likely to be lost.

In addition, it is sufficient if the BLV host cell is a living cell, but FL
In the case of sheep cells such as K-BLV, when the sheep cells are violently destroyed, the sheep cell antigens are mixed with BLV antigens and fetal bovine serum to form a vaccine, and when cattle are immunized, the foreign antigens are completely absorbed. There is a risk that this may cause a stronger allergic reaction. Therefore, it was necessary to devise an operating method that does not include a process of treatment with surfactants or the like.

Furthermore, concentration methods such as ammonium sulfate salting out may denature and deactivate the BLV protein.

The present invention was accomplished with the aim of solving the technical problems necessary for the practical use of BLV vaccines and providing the technology necessary for the practical use of BLV vaccines.

[Means for Solving the Problems] The present invention provides BLV-infected cultured cells or culture supernatants thereof, which are useful as antigen components of BLV vaccines.
The present invention includes a method for separating and purifying crude proteins containing BLV, a BLV vaccine using the components obtained by the method, and a method for immunizing cattle with the vaccine.

A method for preparing a component containing inactivated BLV that is useful as an antigenic component of the BLV vaccine of the present invention is to prepare crude protein bovine leukemia virus containing bovine leukemia virus from a host cell infected with bovine leukemia virus or its culture solution. and a step of treating a solution containing crude protein containing the inactivated bovine leukemia virus with ultrafiltration to obtain a concentrated fraction containing a component useful as an antigen component for a bovine leukemia vaccine. It is characterized by

Host cells that can be used in this method include:
Examples of FL include sheep cells, goat cells, bovine cells, and Kolamori cells, and FLK is preferred because it is commonly used.

The medium used for culturing these host cells may be appropriately selected depending on the host cells to be cultured.

For example, in the case of FLK, RPM116 containing 5 to 20 parts of non-imaged fetal calf serum (hereinafter abbreviated as FC8)
40 (manufactured by Lasui), Dulbecco's M
EM (manufactured by Lasui) etc. can be used.

In general, infection of host cells with BLV and cultivation of BLV-infected host cells may be carried out according to conventional methods.

For example, in the case of culturing FLK-BLV, it is cultured in RPMI 1640 containing 5 to 20 t of non-imaged FC5 at 37° C. in a carbon dioxide atmosphere for 2 to 4 days. The cell concentration is, for example, 105 to 10'/ml.

After completion of the culture, cells are removed from the culture medium by low-speed centrifugation at, for example, about 2000 rpm to obtain a culture supernatant.

Next, BLV contained in the culture supernatant is inactivated.

For this inactivation treatment, treatment using an inactivating agent such as formalin, glutaraldehyde, paraformaldehyde, etc. can be used.

Formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) is added to the mixture so that the mixture is treated with gentle shaking at 4° C. for 24 to 72 hours.

Tween 100.80, Triton X-100
A feature of the present invention is that it does not include a step of strongly destroying cell components such as FLK-BLV with a surfactant such as. When the inactivation treatment is completed, the treated solution is immediately subjected to ultrafiltration without any other operations.

The operating conditions for this ultrafiltration are such that at least cell-derived components with a molecular weight of 10,000 or less and components such as the drugs used in the above inactivation treatment can be removed from the solution containing the inactivated former and BLV. The conditions are set to obtain a concentrated fraction containing inactivated BLV that can be used directly as an antigenic component of a vaccine.

As the filtration membrane used here, for example, Pelicon
Lab-cassette%Pore 5ize 10
,000 NMWL (@Hon Millibore, PT fil
ter), Amicon YMIO (manufactured by Amicon), and the like.

When performing ultrafiltration, for example, it takes 5 to 10 hours per 1 U of treatment solution to concentrate 50 to 90 times, and
A concentrated fraction from which S8, formalin, etc. are completely removed can be obtained.

This concentrated fraction can be freeze-dried to obtain a powder sample containing the inactivated compound, a solution of an appropriate concentration of this powder sample is prepared, and the solution is mixed with an adjuvant. It is possible to obtain a safe old vaccine without the risk of infection with old 4■.

The above example is an example using a BLV-infected cell culture supernatant, but the present invention is not limited to this. Instead of the culture supernatant, an antigen solution containing BLV, such as a rough, manually available BLV-containing antigen from Chemical Research Institute, can be used. It can be used as appropriate.

For the assay of the obtained powder sample, a standard solution of the sample (protein concentration: 1 mg/ml) is prepared, and it is injected with an anti-bovine leukemia virus envelope glycopeptide antigen monoclonal antibody (GPA-)]. , manufactured by Serotik)
When assayed using enzyme antibody immunoassay (ELISA method) using O, D, (4] Onm), it is judged as positive when O, D, (4] Onm) is 0.3 or more. This can be done by determining whether the ratio is 1:1
Preferably, a preparation that shows positivity up to serum dilutions of 6 or higher is desirable.

The concentration of the component obtained from the concentrated fraction is preferably, for example, 10 to 10,000 mg/ml in terms of protein concentration.

Examples of adjuvants to be mixed with the components obtained from the concentrated fraction include aluminum hydroxide [81 (Ko) 3], ePa
&Pa aluminum phosphate, salts obtained from aluminum hydroxide and acid such as aluminum hydroxide sulfate,
Aluminum phosphate (AlPO4), alum, alum potassium, calcium phosphate, bentonite, Freund's complete adjuvant, Freund's incomplete adjuvant, Keyholeson benthemocyanin (LH),
KLH-alum, pertussis vaccine, Cornebacterium pervum, thyroglobin, tetanus toxoid, cholera toxoid, Salmonella lipopolysaccharide sodium phosphate (L
PS) derivative, muramyl dipeptide, trehalose dimycolate, poly-L-(lysine:glutamic acid),
Beenut agglutinin, BS 8, wood-in-oil emulsion, oil-in-water emulsion, double emulsion, retinol, saponin [Kill (Q
ujl) A], protamine, sorbitol, sarcosine, glycerol, propylene glycol, carboxyvinyl polymer, dextran, diethylaminoethyl dextran, callous thymic factor (FTS), levamisole,
Tactocin, bestatin, imprinocin, 5tre
tococcus hemolyticus benicin phosphorus treated cells, 0K-432 agent, Vanil
, abridine [N,N-dioctoadecyl-N,N'-
bis(2-hydroxyethyl)propanediamine],
Tween 80, Intralipid
id), Arlacel A, peanut oil, Lipomal, liposome, etc., and one or more of these can be appropriately selected and used as desired.

In addition, when using aluminum hydroxide, 0.0. Old mg~1
It is possible to mix within the range of 00mg.

By immunizing cattle with the vaccine obtained as described above, antibodies against BUV can be induced.

As a vaccination method, a method appropriately selected from various vaccination methods may be used as necessary.

For example, the vaccine can be administered by intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, etc. However, if you want to delay absorption into the body, it is best to inject to a hard part of the body surface. Additionally, vaccination may be performed using a syringe gun if necessary.

The vaccination program, such as the amount of vaccine to be administered, the age of vaccination, and booster immunization, can be selected as appropriate.

To measure the antibody titer after inoculation, an Elisa method using a microplate adsorbed with a preparation obtained from the above-mentioned concentrated fraction, gp51, etc., which will be described later, can be effectively used.

In addition, in combination with the Elisa method, Western blotting using serum from immunized cows and BLV ferritin antibody method (immunoferritin) were used to detect BL.
It is recommended to test for the production of antibodies specific to the envelope protein, which is the infectious antigen of V.

In addition, BLV to cattle immunized by the above-mentioned vaccination.
The presence or absence of infection can be determined by syncytium assay (5yncy
This can be confirmed by the tium formation method).

The method for preparing the antigen component of the second BLV vaccine of the present invention is as follows:
A process of preparing a crude protein containing bovine leukemia virus from a host cell infected with bovine leukemia virus or its culture solution, a process of contacting the crude protein with a surfactant to convert the protein into M#, A process of separating a fraction containing the core protein of bovine leukemia virus from the mixture obtained in the Kakushun process by an ion exchange method, and a process of separating a fraction containing the envelope protein of bovine leukemia virus from the mixture from which the core protein has been removed by gel filtration. The method is characterized in that it includes a process of separating the components.

Note that in this method as well, crude BLV antigen or the like can be used instead of the culture supernatant as in the above method.

That is, by this method, gp5, one of the envelope proteins of bovine leukemia virus, and p24, one of the core proteins, can be separated and obtained, and these can be used to prepare BLV vaccine and to prepare BLV vaccine by Elisa method.
l. ■ It becomes possible to accurately test the antibody titer of vaccinated or BLV-infected cattle.

In addition, vaccines prepared by mixing either gp51 or p24 with an adjuvant have the advantage that it is possible to clearly distinguish whether a cow has been naturally infected with BLV or not, depending on the level of antibody production against BLV in the body. be.

The purification and separation method for gp51 and p24 will be described in detail below.

First, as in the case of obtaining the concentrated fraction above, a solution containing BLV, such as a culture supernatant, is treated with a surfactant to decompose the BLV contained in the solution into envelope protein and core protein.

For this decomposition treatment, one or more surfactants such as Nonidet P-40 and Triton X-100 can be used.

The amount of surfactant used or processing conditions can be selected as appropriate.
For example, when using Triton X-100, the final concentration is 0.1 to 5%, preferably about 1%, and the
This can be done by processing for about 10 minutes to 1 day and night.

Note that before and after this treatment with a surfactant, a step of concentrating a solution containing BLV by dialysis, ultrafiltration, a sugar concentration gradient method, etc. or a preliminary separation and purification step may be incorporated.

For example, a solution containing Bl, ■, O, ]96 Twee
n 80] OmM Tris-HCI, pt(7
, 2, and then concentrated by a sugar concentration gradient method using sucrose.

In addition, after treatment with a surfactant, ultrafiltration is performed using a filter with a molecular weight of 10,000 to 1,000,000, such as Labocassette (manufactured by Millipore) or Vericon (manufactured by Millipore), and the concentrated fraction is used for gp51 preparation. Also, both filtrates may be used for the preparation of p24.

The protein mixture obtained by surfactant treatment is
Next, it is treated by an ion exchange method using an anion exchanger.

The anion exchanger used here is DEAE-5e
phacel (manufactured by Pharmacia), DEAE-T
oyopearl (manufactured by Toso Corporation) or the like can be used, and the treatment may be performed multiple times as necessary.

The protein mixture obtained by surfactant treatment can be contacted with an anion exchanger to obtain both components that are not adsorbed to the ion exchanger. A standard product containing core protein p24 can be obtained by subjecting this non-adsorbed fraction to various special processes.

Purification of this non-adsorbed fraction includes a concentration step by ultrafiltration using Amicon YMIO, etc. excluding molecules with a molecular weight of 10,000 or less;
Uperose 6 or 12 (such as Affinity Chromatography, manufactured by Pharmacia) can be incorporated.

On the other hand, the protein mixture obtained by surfactant treatment is brought into contact with an anion exchanger, and both components containing gp51 are eluted from the components adsorbed on the anion exchanger, and a sample containing gp51 is obtained by further purification. Can be done.

GP from anion exchanger! ]] For the elution of both components,
A salt gradient method can be used.

For example, using a stepwise salt concentration gradient with a combination of 0.1M and 0.5M NaCl solutions, 0.5M NaCl
gp51 can be obtained in the fraction, and O − +0.
Using a linear salt concentration gradient of 5M Na1l, 0.2M
gp51 can be obtained in the ~0.3M fraction.

From the elution fraction containing gp51, gp! ] For purification of the standard containing 1, ultrafiltration using a filter such as Amicon YMIO, gel filtration using 5uperose 6 (manufactured by Pharmacia), TSK 3000SW (manufactured by Thor), Con A, etc. -5epharose (
(manufactured by Pharmacia) and α-methyl- as the eluent.
Affinity chromatography using mannosides, anti-g
p5] Monoclonal antibody binding-5epharose
One or more methods such as affinity chromatography can be incorporated.

gl) of the present invention! Representative examples of operations in the method for separating and purifying l1 and p24 are shown in FIGS. 1 to 3.

In addition, FIG. 2 is an example of the steps up to the treatment by the ion exchange method using an anion exchanger.

gp obtained as above! The preparation containing i 1 and the preparation containing p24 can be mixed with an adjuvant to obtain a BLV vaccine, similarly to the preparation obtained from the concentrated fraction described above.

Examples of adjuvants used here include those similar to those used in the specimen obtained from the concentrated fraction, and one or more of them can be used in combination. One part containing the specimen obtained from the fraction
It can be used in the same way as a vaccine.

The BLV vaccine of the present invention obtained by the method described above has BLV
activity has been lost, there is no risk of BLV infection, and there are some that maintain strong immunogenicity.

Further, even after boosting, allergic symptoms such as anaphylaxis are not caused.Furthermore, although FLK cell antigens and the like may be included, the induction of allergic symptoms caused by them is negligible, and no side effects are observed.

Therefore, the BLV vaccine of the present invention is a safe vaccine that is effective in preventing BLV infection.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example IF [,-BLV (established by Dr. Van der Maaten) was transferred to T-75falcon plastic
R containing 1096 FC5 in flask (50ml)
The cells were cultured in a PMI 1640 culture solution (manufactured by Lasui) at 37° C. in a carbon dioxide atmosphere for 2 to 4 days. The concentration of cells cultured in the Jinkinshakai culture was 106 cells/ml.

The obtained culture solution was centrifuged at 2000 rpm, and F
KL-BLV cultured cells were precipitated and the supernatant was collected.

Next, a formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the culture supernatant (ift) to a final concentration of 0.1%, and the mixture was gently shaken at 4°C for 48 hours.

This formalin-treated culture supernatant was then transferred to Pe1li.
con Lab-cassette Pore 5iz
e It was subjected to ultrafiltration using 10,000 NMWL (manufactured by Millipore, PT filter).

Filtration took 5 to 10 hours per 11 formalin-treated culture supernatants and concentrated 50 to 90 times. Further, during the filtration operation, 200 ml of distilled water was added to the concentrated liquid, and the ultrafiltration was repeated several times.

The solution concentrated to about 20ml was freeze-dried to obtain a powder sample (BLV protein).

Example 2 The powder preparation obtained in Example 1 was dissolved in distilled water to prepare a solution with a protein concentration of 1 mg/ml, and this solution was mixed with an anti-bovine leukemia virus envelope glycopeptide antigen monoclonal antibody ( GP8-11 (manufactured by Serotik) was assayed by enzyme antibody immunoassay (Eljsa method), and O, o, (410 mm) of 0.3 or more was determined to have antigenic activity. Serum dilution showed positive.

Next, the standard powder was dissolved in distilled water to prepare a solution with a protein concentration of 300 mg/ml, and the same volume of Freund's complete adjuvant was added to obtain a BLV vaccine.

Example 3 Solution of powder sample obtained in Example 1 (protein concentration,
300n+g/ml) and the same volume of aluminum hydroxide gel solution (containing 300mg aluminum hydroxide) BLV vaccine was obtained.

Example 4 The BLV vaccine obtained in Example 2 was injected into two cows (Holstein, 21 months old) that were kept isolated from others and were proven not to be infected with BLV.
The antibody titer against V was assayed over time to examine vaccine efficacy. The results are shown in FIGS. 4 and 5.

In addition, for antibody titer assay, E shown in Reference Examples 1 to 3 below
A method such as the lisa method was used. In addition, FIG. 4 shows Reference Example 1 described below, in which a microplate to which a BLV protein preparation obtained from the concentrated fraction of Example 1 was adsorbed was used to assay the antibody titer.
FIG. 5 shows the results when using the Elisa method shown in Reference Example 2 described later using a microplate on which gp51 was adsorbed.

In addition, in combination with the Elisa method, Western blotting using serum from immunized cows, and BLV ferritin antibody method (immunoferritin) were used to detect BL.
The production of antibodies specific to gp51 and p24 (described below), which are infectious antigens of V, was examined.

As a result, at the time of first immunization, the protein amount of the above sample was 6.
When the mice were immunized with 00 mg, the antibody titer increased two weeks after immunization, and when they were boosted three weeks later with 300 mg without adjuvant, the antibody titer further increased two weeks later. The antibody titer persisted thereafter, and the results of Western immunotherapy, rotting, and ferritin antibody methods using the serum of immunized cows showed that the use of the above vaccine could reduce BLV
against BLV proteins, gp5] and p2 in particular
It was confirmed that antibodies against 4 were raised.

Furthermore, after conducting the following studies regarding the safety of the vaccine used, its safety was confirmed.

1 mg of the BLV vaccine used in this example was intraperitoneally injected into Wistar rats, and clinical findings and changes in body weight were observed for one week, but no abnormalities were found.

At the time of booster immunization, tests were conducted to check for the occurrence of allergic symptoms such as anaphylaxis, and no such symptoms were observed.

Furthermore, to determine whether the cows were infected with BLV due to vaccination, peripheral blood lymphocytes collected from nails at week 15 were tested using a syncytium assay (syncytium formation method), and no BLV infection was detected. 10 weeks, 2
Biochemistry and hematology clinical tests were conducted in one week, but no abnormalities were found in all items.

Furthermore, after vaccination, the cows were sacrificed in layers at about 24 pegs and pathological findings were performed, which revealed rheumatoid arthritis, edema, and edema due to abnormal immunity.
There was no evidence of cardiomyopathy or inflammation in the kidneys.

It should be noted that although the possibility of microscopic contamination of antigens derived from FLK cells into the vaccine cannot be denied, even if such contamination occurs, the above results indicate that the BLV protein antigen produced by the BLV protein antigen preparation method of the present invention cannot be excluded. It can be judged that the amount is extremely low and does not pose a safety problem.

Furthermore, observations up to 23 weeks after vaccination showed that no side effects were observed due to vaccination.

Example 5 Inactivated BLV (manufactured by Kako Research Institute) at 1 OmM
) soot buffer, pH 7,2 (0,1't; T
(containing 80) at 4° C. for 1 to 2 days and nights.

After dialysis, take out the dialysis fluid and add it to a final concentration of 1.
% of Triton X and dissolve it at 4°C.
It was left for 15 minutes.

Next, it was filtered with a 0.45) u+ membrane (manufactured by Millipore), and the filtrate was equilibrated with the above dialysis buffer.
- Toyopearl 650S () - manufactured by Thor Co., Ltd.) column (1 x 10C [11)]. The effluent fraction (passage fraction) obtained here was used in the p24 separation and purification method of Example 6.

Furthermore, after thoroughly washing the column with the above dialysis buffer, the protein adsorbed on the column was eluted using a linear gradient with a sodium chloride concentration of 0 to 0.5M.

This elution operation was performed at 0.6 ml/min, and 2 ml fractions were collected.

The absorbance at 280 nm of each portion was measured, and the elution pattern of the antigen was further analyzed by the Elisa method shown in Reference Example 4 below.

As a result, it was confirmed that the target gp51 was contained in the 0.2 M to 0.3 M Na11 fraction.

Fractions containing gp51 were collected and transferred to Amicon YMIO.
Menpuran (molecular weight cutoff, 10,000, manufactured by Amicon)
It was concentrated by ultrafiltration using .

The solution with a volume of about 1 ml was added to 0.1M phosphate buffer, pH 7.4 (containing 0.3M sodium chloride).
Dialysis was performed for one day and one night.

After completion of dialysis, the dialyzed fluid was transferred to a 5uperose 6 column (IX30cm,
(manufactured by Pharmacia).

Furthermore, the column was treated with the same buffer to obtain an eluate. The elution rate at that time was 0.1 ml/min, and the elution rate was 0.5 m
1 fractions were collected.

When each fraction obtained was analyzed by the Elisa method shown in Reference Example 4 below, it was found that gp was found at the void volume.
51 activity was observed. Therefore, both fractions in which gp51 activity was observed were collected and used as a fraction containing gp51.

Example 6 DEAE-Toyopearl 650 obtained in Example 5
The effluent fraction from S was concentrated by ultrafiltration using Amicon YMIO (molecular weight 10,000 cutoff, manufactured by Amicon).

The solution with a volume of about 1 ml was added to 0.1M phosphate buffer, pH 7.4 (containing 0.3M sodium chloride).
Dialyzed against.

Next, after the end of dialysis, the dialyzed fluid was transferred to a 5uperose B column (1x3
0 cm, manufactured by Pharmacia).

The column was treated with the same buffer to obtain an eluate. The elution rate at that time was 0.1 ml/min, and fractions of 0.5 ml were collected.

As a result, Tri eluted near the total volume.
ton X 100 were removed.

In addition, each of the obtained fractions was analyzed using Elis
It was analyzed by method a, and both fractions in which p24 activity was observed were collected and used as the p24-containing fraction.

Example 7 The gp51 vaccine obtained in Example 5 was mixed with 10 mg of aluminum hydroxide to obtain a gp51 vaccine.

Example 8 1 mg of both p24-containing portions obtained in Example 6 and 1 mg of aluminum hydroxide were mixed to obtain a p24 vaccine.

Example 9 10 mg/ml of gp51 obtained in Example 5 and 1 mg/ml of serum thymus factor (FTS) were each given Iip.
The mixture was added to an osome and mixed to obtain a gp51 vaccine.

Reference Example I Eli by attaching BLV protein to a plate
Measurement of the immune antibody titer by the sa method was carried out as follows.

First, the BLV protein obtained in Example 1 was prepared from 20 to 60
BLV protein at a density of 1.16 g/ml was collected by centrifugation at 80,000 x g using the sucrose gradient method up to *. This BLV protein was placed in a 96-hole Nunk.
400 to 800 ng/well in microplate manufactured by
1, then 3% glutaraldehyde solution was added to the wells, treated at 4°C for 15 minutes, and BLV
Proteins were immobilized.

Next, it was washed several times with PBS-Tween 20 solution.

Before adding the serum for which the antibody titer is to be measured, wash the plate with 1% BS and then add the diluted bovine serum (Reference Example 2gl)! l] is attached to the plate.
Measurement of the immune antibody titer by the method was carried out as follows.

gl) obtained in Example 5)! ] 50 for both sides including
It was diluted with mM carbonate buffer, pH 9.6, to give a solution with a protein concentration of 1 μg/m I.

50 μl of this diluted solution was added to each well of a 96-well microplate for Elisa method (manufactured by Flow), and incubated at 4°C.
After leaving for 18 hours, Dulbecco PBS (-) (0,
Each well was washed with 0.05% Tween 8020 (hereinafter abbreviated as PT).

Next, 100 μl of PT containing 1% ovalbumin (OVA, Sigma) was added to each well for 6 hours at room temperature.
After standing for a while, each well was further washed with PT.

Antiserum obtained from a cow immunized with the vaccine was administered at 0.1 tOV.
It was diluted 1:100 with PT containing A and used as a sample for analysis.

After adding 50 μm of this analytical sample to the wells of the plate prepared as described above, it was left at 4° C. for 18 hours and washed with PT.

Then 1:1 with PT containing 0.1% OVA
50 μl of rabbit anti-bovine IgG (H+L)-peroxidase diluted to
It was left to stand for a while and washed with PT.

After washing with PT, the wells were treated with 0.025%;
- Azinobis (50 mM citric acid buffer containing 3-ethylbenzthiazoline sulfonic acid diammonium salt and 0.15% hydrogen peroxide, pH 4.0)
0 μm was added to each well and allowed to react for 15 minutes at room temperature.
Immediately measure the absorbance at 414 nm using Immunoreader NJ-2.
000 (manufactured by Nihon Intermed Co., Ltd.) to determine the antibody titer.゛Reference Example 3 Both parts containing p24 obtained in Example 6 were diluted with 50111M carbonate, pH 9.6, and the protein concentration was 10.
The antibody titer was detected in the same manner as in Reference Example 2 except that the solution was used as a 0 ng/ml solution.

Reference Example 4 BL by Elisa method in each fractionation operation in the above examples
Detection of V protein, BLV core protein, and envelope protein was performed as follows.

5 μm of both fractions obtained at each fractionation step was
Add to each well of a 6-well microplate (70-at) and leave to stand at 4°C for 18 hours, then add Dalvey Co. PBS (-) (0,054k Tween 8020
(hereinafter abbreviated as PT)) was used to wash each well.

Next, 100 μm of PT containing 1% OVA was added to each well, left at room temperature for 6 hours, and each well was further washed with PT.

Here, bovine anti-old, ■ polyclonal antibody (manufactured by Noji Research Institute)
or mouse anti-BLV gp69 monoclonal antibody (
After adding 50 .mu.m of PTFE (manufactured by Serotik) to the wells of the plate prepared as described above, it was left at 4.degree. C. for 18 hours and washed with PT.

Next, PTTE1:1 containing O,1% OVA
50 μl of rabbit anti-bovine IgG (H+L)-peroxidase or anti-mouse IgG (Fc)-peroxidase diluted to 0.000 was added to the wells, left for 2 hours at room temperature, and washed with PT.

After washing with PT, 0.025! Nino 2,2'
-Azinobis (3-ethylbenzthiazolinesulfonate diammonium salt and 0.15!L; 100μm of 50mM citric acid buffer containing hydrogen peroxide, pH 4.0) was added to each well and reacted for 15 minutes at room temperature. Immediately measure the absorbance at 414 nm using Immunoreader NJ
-2000 (manufactured by Nihon Intermed Co., Ltd.).

[Effects of the Invention] The present invention provides a vaccine useful for preventing bovine leukemia, which is highly safe and has high durability of immunity.

Among these, the anal V vaccine of the present invention is obtained by directly mixing proteins separated and purified in raw (intact form) with various adjuvants without using particularly complicated methods, and is reliable. It has a vaccine effect, making it easy to provide an excellent preventive measure against bovine leukemia infection.

[Brief explanation of the drawing]

Figures 1 to 3 are diagrams showing an outline of a representative example of a method for separating and purifying gp51 and P24, and Figures 4 and 5 show changes in antibody titers of two cows in Example 4. It is a graph. Patent applicant: Mitsui Toatsu Chemical Co., Ltd. Mitsui Pharmaceutical Industries, Ltd. Japan Holstein Breeding Service Co., Ltd.

Claims (1)

[Claims] 1) A process for inactivating crude protein bovine leukemia virus containing bovine leukemia virus from a host cell infected with bovine leukemia virus or a culture solution thereof, and comprising the inactivated bovine leukemia virus. 1. A method for preparing an antigen component for a bovine leukemia vaccine, comprising the step of treating a solution containing crude protein with ultrafiltration to obtain a concentrated fraction containing a protein useful as an antigen component for a bovine leukemia vaccine. 2) The preparation method according to claim 1, wherein the host cell is Fetallamb Kidney cell (FLK). 3) A process of contacting crude protein containing bovine leukemia virus from a host cell infected with bovine leukemia virus or its culture solution with a surfactant to dissociate the protein, and ion exchange from the mixture obtained in the protein dissociation process. a step of separating a fraction containing the core protein of the bovine leukemia virus by a method, and a step of fractionating a fraction containing the envelope protein of the bovine leukemia virus by gel filtration from the mixture from which the core protein has been removed. A method for preparing an antigenic component of a bovine leukemia vaccine, characterized by: 4) The preparation method according to claim 3, wherein the fraction containing the envelope protein of the bovine leukemia virus is further processed by ion exchange chromatography using an anion exchanger. 5) After the treatment with the ion exchange chromatography,
The method for preparing an antigen component of a bovine leukemia vaccine according to claim 4, which further comprises gel filtration to obtain the envelope protein of bovine leukemia virus in the filtrate. 6) The preparation method according to any one of claims 3 to 5, wherein the host cell is a Fetallamb Kidney cell (FLK). 7) After inactivating the bovine leukemia virus in the crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or its culture solution, a solution of the crude protein containing the inactivated bovine leukemia virus is prepared. A bovine leukemia vaccine characterized by containing a concentrated fraction obtained by ultrafiltration as an antigen component. 8) The bovine leukemia vaccine according to claim 7, which further comprises an adjuvant. 9) The bovine leukemia vaccine according to claim 7 or 8, wherein the host cells are Fetallamb Kidney cells (FLK). 10) A crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or its culture solution is brought into contact with a surfactant to dissociate the protein, and the resulting dissociated product mixture is selectively isolated using an ion exchange method. A bovine leukemia vaccine characterized by containing as an antigen component the envelope protein of bovine leukemia virus isolated in . 11) The bovine leukemia vaccine according to claim 10, which further comprises an adjuvant. 12) The host cell is Fetallamb kidney
The bovine leukemia vaccine according to claim 10 or 11, which is a cell (FLK). 13) A crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or its culture solution is brought into contact with a surfactant to dissociate the protein, and the resulting dissociated product mixture is selectively isolated using an ion exchange method. A bovine leukemia vaccine characterized by containing as an antigen component the core protein of bovine leukemia virus isolated in 14) The bovine leukemia vaccine according to claim 13, further comprising an adjuvant. 15) The host cell is Fetallamb kidney
The bovine leukemia vaccine according to claim 13 or 14, which is a cell (FLK). 16) After inactivating the bovine leukemia virus in the crude protein containing bovine leukemia virus prepared from a host cell infected with bovine leukemia virus or its culture solution, a solution of the crude protein containing the inactivated bovine leukemia virus is prepared. A solution obtained as a concentrated fraction obtained by ultrafiltration and having a protein concentration of 1 mg/ml was mixed with an anti-bovine leukemia virus envelope glycopeptide antigen monoclonal antibody (
Measured by enzyme antibody immunoassay using GPA-11),
O. D. (410nm) of 0.3 or more is determined to be positive, the component in the fraction showing a positive reaction with a titer of 1:16 or more after dilution is defined as a protein concentration of 10 to 10,000.
An immunization method characterized by inoculating cattle together with an adjuvant at a concentration of 0 mg/ml.