WO1988006182A1 - Bovine leukemia virus vaccine prepared by using recombinant vaccinia virus - Google Patents

Abstract

A recombinant virus containing a gene of a surface antigen of a bovine leukemia virus incorporated in a vaccinia virus gene and capable of expressing a surface antigen of a bovine leukemia virus; a process for preparing the recombinant virus comprising the steps of (1) inserting a DNA fraction containing a hemagglutinin gene of vaccinia virus gene origin into a cloning vector to prepare a hemagglutinin gene-containing vector, (2) inserting a surface antigen gene of a bovine leukemia virus into the hemagglutinin gene in the hemagglutinin gene-containing vector and a promoter for the gene to prepare a vector containing the surface antigen gene of the bovine leukemia virus, and (3) incorporating the surface antigen gene of the bovine leukemia virus together with its promoter into the hemagglutinin gene region of the vaccinia virus gene by homologous recombination between the hemagglutinin gene region in the surface antigen gene-containing vector and the hemagglutinin gene region of the vaccinia virus gene; and a process for immunizing mammals against a bovine leukemia virus, which comprises inoculating mammals with the recombinant virus and then with a deactivated bovine leukemia virus or its part having antigenicity.

Description

 Description Leukemia virus vaccine using recombinant vaccine virus

 The present invention relates to a recombinant vaccinia virus having a surface antigen gene of E. coli leukemia virus, a method for producing the same, and a method for using the same. Background art

 ゥ Leukemia virus (BLV) is a virus that causes endemic ゥ leukemia (EBL), and some infected cattle become tumorous and follow a fatal course. In addition, even if asymptomatic, the blood of infected cattle remains infectious for a lifetime and becomes a source of infection. Up to now, EBL actin has not been developed, and development of an actin to prevent infection is desired.

 Vaccinia virus is a powerful virus that has been used as a vaccine for vaccinia vaccines.Vaccinia virus is nowadays used to treat various pathogens, especially pathogenic viruses. Various attempts have been made to produce a recombinant vaccinia virus by incorporating an antigen gene and use this recombinant vaccinia virus as a raw vaccine. However, development of a leukemia vaccine by such a method has not yet been performed.

The BLV gene has been identified and its entire structure has been determined in. Sagata et al., Proc. ^ fatl. Acad. Sci. 82 = 677-681 (1985)). However, no method has been disclosed for cutting out a gene (env gene) encoding the virus surface protein (BLV-env) of this gene and artificially expressing it.

 Accordingly, the present invention relates to a vaccinia virus gene in which the surface antigen gene of bacillary leukemia virus is integrated, a method of producing the vaccinia virus virus, and a method of using the same. It is intended to be provided.

 More specifically, the present invention relates to a recombinant vaccine capable of expressing a surface antigen of P. leukemia virus in which a surface antigen gene of P. leukemia virus is incorporated into a vaccinia virus gene. Serve Ruth.

 The present invention further relates to a method for producing the recombinant virus,

(1) Inserting a DNA fragment containing a hemagglutinin gene derived from a Vaccinia virus gene into a cloning vector to prepare a hemagglutinin gene-containing vector;

 (2) A surface antigen gene of ゥ leukemia virus by inserting a surface antigen gene of ゥ leukemia virus and a promoter for the gene into the hemagglutinin gene in the hemagglutinin gene-containing vector. Preparing a gene-containing vector; and

(3) By homologous recombination between the hemagglutinin gene region in the surface antigen gene building vector and the vaccinia virus gene (Γ> hemagglutinin gene region). The vaccinia virus remains Incorporating the surface antigen gene of Escherichia coli leukemia virus together with a promoter for the gene into the hemagglutinin gene region of the gene;

Provided is a method comprising steps.

 The present invention further provides a method of inoculating a mammal with the recombinant virus, and further inoculating an inactivated P. leukemia virus or a portion thereof having an antigenic property. Provided is a method for immunizing mammals other than humans against leukemia virus. BRIEF DESCRIPTION OF THE FIGURES

 Fig. 1 shows a method for preparing a plasmid PH U3 containing the hemagglutinin gene.

 FIG. 2 shows the structure of the hemagglutinin gene.

 FIG. 3 shows the nucleotide sequence of the 7.5 K protein promoter.

 FIG. 4 shows the nucleotide sequence of the DNA fragment in the chemical synthesis of the 7.5 K protein promoter.

 FIG. 5 shows a method for preparing a plasmid P9—7.5 Ps containing a 7.5 K protein promoter.

 FIG. 6 shows a method for preparing a plasmid pl3-Be having a BLV-env gene.

Figure 7 shows the plasmid P9-7.5 ps-Be containing the δΚ protein promoter and the BLV-env gene, and the 7.5 K protein promoter and the BLV-env in the hemagglutinin gene. The production of the plasmid pl3-HA-7.5Ps-Be into which the env gene was inserted is shown. Figure 8 shows the preparation of recombinant vaccinia virus by recombination with vaccinia virus L0-1 strain and plasmid P13——7.5 Bs-Be, and the screening method. Show.

 Fig. 9 is a drawing of 'Estan' plotting showing the expression of the BLV-en gene by recombinant vaccinia viruses No. 3 and No. 16.

 FIG. 10 shows Western Pro showing the production of antibodies in the serum of rabbits inoculated with inactivated BLV after inoculation with recombinant vaccinia virus No. 16 or L01. This is the result of the cutting.

 Figure 11 shows the temporal increase in antibodies in serum of rabbits inoculated with inactivated BLV after inoculation with recombinant vaccinia virus No. 16 Western blot. This is the result of the ringing.

 FIG. 2 shows a method for producing a pl8-AT pro536, which contains an ATI promoter.

 FIG. 13 shows a method for preparing a plasmid ρί2-9 containing an ATI promoter.

 FIG. 14 shows a method for preparing a plasmid in which an ATI promoter has been inserted into the hemagglutinin gene.

The first 5 FIG ATI promoter Maguruchini down gene into the and BLV- env genes plus Mi de is inserted _P 9 - HA - show a manufacturing method of AT I P- Be.

The first 6 Figure BLV- env gene and 7. [delta] kappa top flop port motor - shows a Be manufacturing method - the to舍有plus Mi de P2401- 7. 5 Ρ _Κ.

Fig. 17 shows the plasmid P13-HA- in which the 7.5K natural promoter and BL.V- _env gene have been inserted into the hemadaltin gene. This section describes how to make 7.5 PN-Be.

Fig. 18 shows the recombinant vaccine senior virus No. 16 (L01-7.5 Ps-Be) The recombinant vaccine senior virus No. 1 O-7.5 P _N- Be) _N and the combination The expression of the BLV-env gene by Xinnia virus No. 23 (m0-ATIP-Be) was compared with that of the negative control (mO) containing no B // enV gene and BLV-infected cells of sheep. FIG. 8 is a Western plotting diagram shown in comparison with a positive control which is FLK.

 Fig. 19 shows that after 6 weeks of inoculation of the rabbit with recombinant vaccinia virus No. 1 (itiO-7.5 PN-Be), the antibodies in the serum of the rabbit were compared with BLV virion as the antigen. The results of Western-plotting are shown.

 Fig. 20 shows that the antibodies in the rabbits, which were given a booster immunization 14 weeks after inoculation and 3 weeks after the rabbits mentioned in Fig. 19, were treated with BLV virions as antigens. Shows the results of the Estabent cutting. BEST MODE FOR CARRYING OUT THE INVENTION

As a method for inserting a foreign gene into the vaccinia virus gene, a sequence of a specific site in the vaccinia virus gene is linked to both ends of the foreign gene to be inserted, and this site is inserted into the vaccinia virus gene. A method based on homologous recombination with the corresponding sequence in the senior virus gene is generally used, and this method can be used in the present invention. Such a foreign gene insertion site allows virus insertion to be intrinsically unaffected by the insertion and allows for easy identification of the virus into which the foreign gene has been inserted. It must be such a part. In the present invention, it is preferable to use a site in the hemagglutinin gene as such a site. Hemagglutinin is not essential for the growth of vaccinia virus, and if the function of the hemagglutinin gene is lost due to the introduction of a foreign gene, such vaccinia virus is identified as a loss of hemagglutination function Because they can do it. This identification can be easily performed by staining viral plaques with nitrile erythrocytes.

 1. Cloning of the hemagglutinin (HA) gene (Figs. 1 and 2)

 The hemagglutinin (HA) gene is contained in the Sal I — Hind M fragment (approximately 1.8 kbp) located at the right end of the Hind IE A fragment of the vaccinia virus IHD-J strain gene. The entire DNA sequence of this fragment containing the hemagglutinin code sequence has been determined [H. Shida et al., Virology 150, 451-462 (1986) J. Therefore, it is convenient to use this fragment in the present invention.

This fragment is excised from A of vaccinia virus with the corresponding restriction enzymes, such as Hindi! And SalI, and cloned into an appropriate vector. Such a vector can be any bacterial vector commonly used in genetic engineering, such as E. coli plasmids and mids. For example, Escherichia coli plasmid PUC or PUC 9 can be used as such a vector. These plasmids, pUC13 and pC9, are commercially available as It is widely used in the world and is readily available.

 When pUC13 is used, it is digested with Hind1Π and SaII, and this large fragment is combined with the 1.8 kbp Hindi-SaII fragment containing the hemagglutinin gene. Ligation yields plasmid pHA13. By transforming this plasmid into, for example, E. coli JM103 strain, the desired hemagglutinin (HA) gene can be amplified.

 7

 2. Cloning of the leukemia surface antigen (BLV-env) gene (Fig. 6)

© Shi leukemia U genomic gene Lee pulse is (referred to as a plus Mi de _P BR 322-BLV herein) plus Mi de incorporated into plus Mi de pBR 322 has already been Construction [N Saga ta et al., Gene, 26, 1-10 (1983). In the present invention, as one mode, the cleavage of this plasmid by the force of the plasmid, PvuΠ and XhoI, and the partial digestion by the SmaI produce Bv '-A DNA fragment containing the onv gene can be obtained. Then, by inserting this into the Sinai site of commercially available plasmid pCU13, plasmid Pl3-Be can be obtained.

 3.7.5 Synthetic promoter of the K protein gene-containing plasmid 13-HA-7.5 Ps-Be

 Fabrication (Figs. 5 and 7)

(1) Cloning of promoter of 7.5 K protein gene In one embodiment of the present invention, 7.5 K protein is used as a promoter for expressing the -env gene. Genetic A promoter can be used. The base sequence of this promoter has been determined [Venktesan _: S. et al., Cell, 25, -805-813 (1981), which can be chemically synthesized. obtaining a plasmid p9- ¹ ί. 5 Ps by inserting positive Mi de pCU 9.

 (2) Linkage between BLV-env gene and Ί.5K protein gene promoter

 Next, the BLV-env gene in the plasmid pl3-Be is linked to the promoter of the L5K protein gene in the plasmid p9-7.5 Ps. DNA fragment containing BLV-env gene by digesting EcoRI and HindHI

 (About 2 kbp) and blunt the ends. On the other hand, after cutting the plasmid P9-7.5 Ps with XbaI, the ends are blunted.

 Next, this is ligated to obtain a plasmid P9-7.5Ps-Be in which the BLV-env gene is linked downstream of the 7.5K protein promoter (7.5K promoter).

 (3) 7.5 K promoter and B-isch env gene

 Of hemagglutinin into the gene

Next, the plasmid P9-7.5Ps-Be is digested with AccI, and the ends are blunted to obtain a DNA fragment having a 7.5K promoter and a BLV-env gene. On the other hand, after cutting the plasmid pHA13 with ccI, the ends were blunted and ligated, whereby the 7.5K promoter and BLV-env were inserted into the hemagglutinin gene. Plusmid P13— M-7.5 Ps—Be is obtained. 4. Promoter of A-type inclusion body (ATI) protein

 P9—HA—ATIP—Be

 Fabrication (Figs. 12 to 16)

 The ATI promoter and the BLV-env gene are included in the hemagglutinin gene using the promoter of the A-type inclusion body (ATI) protein gene of cowpox virus (ATI promoter—) as the promoter. You can get the inserted plasmid.

 (1) Cloning of ATI promoter "

 The plasmid pC6 containing the ATI promoter can be prepared from cowpox virus. The preparation of this plasmid is described later in Example 10. PC6 was cut with SacI and BamHI, the linearized plasmid was shortened with exonuclease, the ends were blunted, and then religated to remove the ATI structural gene. To obtain the plus P13C-6-2-32. This plasmid is quenched with Taq I and EcoR I to ATI. A 0.6 kbp DNA fragment containing an oral motor was obtained, and this was inserted into the HindIII site of pCU18 to obtain plasmid pl8-ATI-pro536. In this plasmid, ATG exists immediately downstream of the ATI promoter region, which is inconvenient when a foreign gene is introduced downstream of this promoter, so that the M13 RF phage is used. ATG is converted to ATA by introducing the mutation used to obtain the plasmid pA2-9.

(2) Insertion of ATI promoter into hemagglutinin gene The above plasmid PA2-9 is digested with EcoRI and HindEI, and the end is blunted to contain the ATI promoter. Fragment By inserting this into the Nru I site of the plasmid pHA13 containing the hemagglutinin gene described above, plasmid pHAA2-9 in which the ATI promoter was inserted into the hemagglutinin gene was obtained. obtain.

 (3) Insertion of B env gene into plasmid PH 2-9

 The plasmid pl3-Be was digested with EcoRI and Hind IE, and the end thereof was blunted to obtain a DNA fragment containing the BLV-env gene. This DNA fragment was used as an ATI promoter for the plasmid ρΗΑΑ · 2-9. The plasmid p9-M-ATIP-Be in which the ATI promoter and the BLV-env gene are inserted into the hemagglutinin gene is obtained by inserting the plasmid into the SmaI site downstream of the plasmid.

 5.7.5 Preparation of plasmid P13-HA-7.5P-Be containing natural promoter of K protein (Figs. 16 and II)

(1) 7.5 K of the natural promoter of the 7.5K protein

7: 5 K protein flop port motor one gene has already been described (Venktesan, S., etc., Cel 1, 25, 805-813 ( 1981) 3 0 to obtain a DNA fragment舍有the promoter according to the serial mounting By inserting this into pCU9, plasmid p2401 is obtained.

 (2) BLV-env gene and 7.5K protein natural promoter

 Concatenation with

The BLV-env gene in the plasmid P13-Be is linked to the natural promoter of the 7.5K protein gene in the plasmid p201. For this purpose, a DNA fragment (about 2 kb _P ) having the BLV-env: gene is obtained by cutting P13-Be with EcoRI and HindIII, and this end is blunted. On the other hand, plus midi p2401 The plasmid is digested with SmaI, ligated to the EcoR I-Hind EI fragment, and the plasmid P2401-7.5 PN in which the 7.5K natural promoter is linked to the BLV-env gene. — Get Be.

 (3) Insertion of 7.5K natural promoter — and BLV-env gene into hemagglutinin gene

Next, the plasmid P2401-7.5 PN-Be was cut with EcoR II and Hind ffl, and the end was smoothed to obtain a 7.5 K natural promoter. '-en Obtain a DNA fragment containing the V gene. On the other hand, after cutting the plasmid PHA13 with Accl, the ends were blunted and ligated to obtain a 7.5K promoter in the hemagglutinin gene. -And BLV-en V inserted plasmid pl3— HA—7.5 _PN —Be.

 6. Introduction of the BLV. Lenv gene into the vaccinia virus gene by recombination (Fig. 8) Finally, the recombinant plasmid Pl3- produced as described above.

HA-7.5Ps-Be, p9 - HA - AT IP - Be, and 13 - HA - 7. using 5 P _N -Be, because these BLV- env gene present in the positive Mi de of the gene Incorporation into the hemagglutinin gene region of the vaccinia virus gene together with the promoter. This recombination is carried out between the hemagglutinin gene region of the vaccinia virus gene and the hemagglutinin gene portion at both ends of the BLV-env gene in the recombination plasmid. This is performed by homologous recombination.

A variety of vaccine strains can be used as a vaccine for this purpose, a few examples of which are:

o

WR, 0-5, LC16m0, LC16m8 and the like can be mentioned. The LC16mO strain and the C16m8 strain were obtained from the L0 strain as attenuated strains with low trophopathic properties, and they have the following properties.

 Table 1 0 to C16m0 to C16m8 Non-proliferable temperature> 41 / C 414 (K5.C

(1TK cell)

 Black size Large <1

 2 Medium Φ

 (RK cells)

 Pot size Large Medium Small Various cells

 Proliferative

 GAM stalk

 Vero cell stalk -H +

 Cell

 CEF cells -H-5

 Proliferative

 ゥ Egret +

 monkey

 Mouse / /

 Intrusive

 Mouse

 Skin proliferation

 ゥ Egret +

0 hit

 Antibody production ability

 ゥ egret

Human recombination can be performed as follows. In other words, 5 kidney kidney cells are cultured to form a monolayer. Infects vaccinia viruses. Then, re-phosphate force Rushiu Ri by the beam method plus Mi de P13- HA- 7. 5 Ps - Be, P 9 one HA - ATIP - Be or pl3 - HA - 7. 5 PN - the Be door Lance effect.

 Next, the immunoress is collected from these samples and plated on the monolayer of RK13 cells to form plaques. Spread nitrile erythrocytes on this surface. The vaccinia virus in which the desired recombination has occurred does not express hemagglutinin [HA (-)] and thus does not adsorb erythrocytes, but the wild-type vaccinia virus is HA (+). Adsorbs erythrocytes. This was used as a marker to select candidate strains of recombinant vaccinia virus, and after cloning these viruses, the antiserum of a female afflicted with cystic leukemia was obtained. And a recombinant virus expressing the human leukemia surface antigen is selected by the enzyme antibody method.

 Next, the recombinant virus obtained as described above and the wild-type strain virus as a control were partially purified and adsorbed on a ditrocellulose membrane. By hybridization with the labeled surface antigen gene DNA, it is confirmed that the virus has the leukemia surface antigen gene incorporated therein.

In addition, when the recombinant virus of the present invention was inoculated into a egret and boosted with an inactivated leukemia virus, it is likely that the serum of the egret is an antibody against gp60. It is confirmed that the product is produced (Figs. 10 and 11). Also, in place of the inactivated virus, the part having antigenicity may be replaced. Can also be used.

 In the following examples, each buffer has the following composition (1) Restriction enzyme buffer

 (i) Medium salt concentration buffer solution (PH 7.5)

 NaC ϋ 50mM

 Tris-C ί lOmM

MgC I ₂ 10m «

 Dithiols ladle ImM

 (ii) High salt concentration buffer solution (PH 7.5)

 NaC ί lOOntM

 MgC ί z lOmM

 Dithiolate ImM

(iii) Sma I buffer solution ( _PH 8.0)

 C 20mM

 Tris · C ί lOmM

 Dithiols ladle ImM

 (iv) Nru I buffer (pH 8.0)

 Tris-HC ί lOmM

 MgC £ z TmM

 KC & 150m

 2 — Melcaptoethanol 7 mM

BSA 100 '^ / m twenty one

 o 5

(2) Ligation buffer solution (PH 7.6)

 Tr is' C i 66mM MgC H 2 5mM

 16

Dithiolate 5πιΜ

A T P ImM

(3) Nick transfusion buffer solution (PH 7.2)

 -1

 Five

 Tris-C ί 50mi1

10 MgS0 ₄ lOmM

 Dithiothreitol 0. ImM ゥ serum albumin 50

 (4) Exonuclease II buffer (PH 7.δ)

 Tris-HC i? 50mM

MgC ί z 5mM

D T T 5mM

B S A 50〃g mi

(5) SI nuclease buffer (pH 4.6)

 Sodium acetate 50mM Zinc acetate ImM

NaC & 250m

BSA 50 ^ / ms> Example 1. Production of plasmid PHA 13 [Hemaggluti

 Cloning of the nin (HA) gene] (Figs. 1 and 2)

The virus of the vaccinia virus WR strain was purified and suspended in 50 mM Tris-HC ^ (pH 7.4) (containing 1 mM EDTA and 0.5% sodium dodecyl sulfate). After adding proteinase K to 250-1000 Z ^ and incubating overnight at 37'C, extract three times with phenol: clonal form (1: 1) saturated with buffer solution. And settled the virus DMA with ethanol. This DNA was dissolved in 10 mil Tris-HC ί ( _Ρ Η 8.0) (containing 1 mM BDTA), digested with Hind HI, and a 50 kbp Hindm A fragment was analyzed by agarose gel electrophoresis. Isolated. This Hind IE A fragment was digested with Sal I in a high salt buffer, and the approximately 1.8 kbp Hind M-Sal I fragment present at the 3 ′ end of the Hind MA fragment was isolated by agarose electrophoresis. did.

On the other hand, plasmid pCl! 13 was digested with HindΠΙ in a Φ salt buffer and then with Sal I in a high-concentration buffer to linearize it. The above-mentioned HindII-SalI fragment separated by electrophoresis and the linearized plasmid were ligated with T4 DNA ligase in a Ligation buffer, and Escherichia coli 103 was transformed using this reaction mixture. did. A plasmid was extracted from each clone by a rapid extraction method, and a plasmid having the desired composition was selected by restriction enzyme analysis and named PHA13. 荬 Example 2.7.5 Synthesis of white promoter

 (Fig. 4)

 Vaccinia wineless has its own RNA polymerase and therefore has its own promoter sequence. The 7.5 kd protein promoter, encoded by the early gene of vaccinia virus, is powerful and, when incorporated into a recombinant virus, efficiently expresses downstream structural genes. I know that. The nucleotide sequence of this promoter region has already been determined.

 7

 3 As shown in the figure.

 There are no sequences that exactly correspond to the eukaryotic TATA box or the prokaryotic privno box, but there are sequences rich in TA as shown in the figure above. With reference to these, DNA was synthesized so as to cover two TATA-like sequences and the transcription start site of RNA. To simplify later experiments, several restriction enzyme sites were added, and EcoRI linkers were added at the ends.

 Since the synthetic region was long, it was divided into two regions, each of which was combined with the complementary DNA, and four were synthesized, and named VAC1, -2, and 34, respectively. These are shown in Figure 4.

 These single DNA fragments were synthesized by a phosphor-amidite method using a DNA synthesizer (Applied Biosystems), purified, and then ligated.

Example 3 Preparation of P9-7.5 Ps (Fig. 5)

The obtained synthetic promoter was ligated with pCli 9 cut with EcoRI and linearized in a ligation buffer, and E. coli 103 was used with this reaction mixture. Transformation. The resulting clone was confirmed to be a plasmid having the correct structure by analysis with various restriction enzymes, and was named P9-7.5 Ps. The direction of insertion was found to be the same as the direction of the DNA of the lacZ gene of PCU 9. The nucleotide sequence of the region excluding EcoRI at both ends was determined by the dideoxy method.

Example 4. Production of a plasmid P13—Be (Fig. 6)

 The plasmid pBR-322-BLV into which the BLV genomic gene has been inserted is digested with Ρνιι 中 で in a medium salt buffer, and after ethanol precipitation, it is further added to a high salt buffer. These XhoI-PvuII fragments digested with XhoI were separated by agarose gel electrophoresis, and a DNA fragment of about 2.1 Kbp was isolated and purified.

 Next, this was completely digested with Sma'I in a Smal buffer solution first, and after confirming that the expected band appeared, a partial digestion pilot experiment was performed. Pvu E · Xho I-2. For 1 kbp DNA 1, change Sma I to 0.1, 0.075, 0.05 = 0.025, 0.09 in 5 steps, 3? And digested for 30 minutes. As a result, 0.075 was found to be appropriate, so partial digestion was performed at this concentration. The resulting SmaI fragment was separated by agarose electrophoresis, and a DNA fragment of about 2 Kbp was isolated and purified.

The obtained Sma I — 2 Kbp DN fragment and the linearized plasmid CD13-Sma1 / BftP (Pharmacia Nodio Technology 27- 4975-01) were ligated in a ligation buffer. Ligation was performed using T4 DNA ligase, and Escherichia coli 107 was transformed using the reaction mixture. The obtained clone was analyzed by various restriction enzymes. As a result, a plasmid having the correct configuration was obtained and named P13-Be. The insertion direction was the same as the DNA direction of the lacZ gene of PCU13.

Example 5. Production of plasmid P9—7.5 Ps-Be

 (Fig.）)

 After increasing the amount of the above plasmid P13-Be in a large amount, this was digested with EcoRI and 11 indII in a buffer solution of medium salt concentration, separated by agarose gel electrophoresis, and purified. In addition, the protruding 3 'end of DNA was filled with a polymerase k1 enow fragment in a nick'translation buffer solution to make the end blunt.

 After increasing the amount of the plasmid P9-7.5 Ps in a large amount, the plasmid was digested with Xbal in a high-salt buffer solution, and similarly blunt-ended with a polymerase enow fragment. .

 Both were ligated with T4 DNA ligase in Ligation buffer, and E. coli JM107 was transformed using the reaction mixture. The obtained clone was examined by restriction enzyme for the composition and insertion direction of the plasmid, and the orientation of the 7.5K protein promoter and the direction of the BLV-env were consistent. The one was named p9'7.5 Ps-Be. Further, this was confirmed by examining the internal region of B or env as a probe with a colony and an hybridization.

Example 6 Preparation of P13—HA—7.5 Ps-Be

 (Fig.）)

After large amount增and to the plus Mi de P9- 7. 5 Ps- Be, 7. 5 K protein promoter - was cut out a region including the a BLV-env _c That is, it was digested with Accl in a medium salt concentration buffer solution, separated by agarose electrophoresis, and purified. In addition, blunt ends were made with polymerase Klenow fragment in nick transfusion buffer.

 In addition, plasmid PHA13 containing the hemagglutinin gene was digested with Accl in a buffer solution of a medium salt concentration, and similarly blunt-ended with a Volamelase Klenow fragment.

 The two were ligated in a ligation buffer using T4 DNA ligase, and E. coli 107 was transformed using the reaction mixture.

 The Escherichia coli JM107-plSBe containing the plasmid P13—HA—7.5 Ps—Be obtained in this way was obtained from the microorganism of the National Institute of Advanced Industrial Science and Technology as FERM BP-1286. Deposited internationally with the National Institute of Advanced Industrial Science and Technology, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture, Japan, on February 10, 1987, based on the Budapest Treaty.

Example 7 Transfer of BLV-env Gene to Vaccinia Virus

 Installation (Fig. 8)

ゥ The heron kidney cell line RK13 was seeded on a 6 cm diameter dish and cultured at 5% C0z to form a monolayer.- Next, the cultured cells were The L.-1 strain of Vaccinia virus was infected at a dose of 0.1 moi and left for 1 hour. Next, the plasmid pl3-HA-7.5P3-Be prepared in Example 6 was subjected to the calcium phosphate method (Weir, JP, et al. (1982) Proc. Natl. Acad. Sci. USA). , I £ _: 1210-1214). From this culture, the virus is frozen and thawed to remove the medium. And the virus was recovered.

 RK13 cells were cultured at 37 ° C. for 24 hours in a MEM medium of chicks housed in a 6 era-diameter share to form a monolayer. Next, the virus recovered as described above was sown on this layer, and incubated for 2 days to form plaque. Next, the erythrocytes of the nitrile were spread on this layer, and the plaque [HA (-)) which did not turn red was picked up.

After cloning the HA virus, it is possible to determine whether these recombinant vaccinia virus strains have the BLV-env gene of interest. Dot blotting was conducted. The above-mentioned recombinant vaccinia virus candidate strains (20 strains) and 0-1 strains (targeted) and BLV-enV gene DNA radiolabeled with ³² P, adsorbed to the 2-trocel membrane, and Was hybridized. As a result, it was found that 9 strains had the target BLV-env gene.

Example 8: B'-env with recombinant xenia virus

 Confirmation of gene expression

 The 9 candidate strains were subjected to ヱ -stand blotting to determine whether BLVenv was actually expressed in the cultured cells.

RK 13 cells were spread on a 6 era-diameter plate to form a monolayer, and the recombinant vaccinia virus candidate strain was infected at the moi 1 (multiplicity of infection) amount. After culturing for 4 hours, the cells were collected. 1/20 of these was electrophoresed on an SDS / acrylamide gel and further purified by nitrosenorrose. Transfection at once Was. Next, the cells were treated with a rabbit antibody against gp60, followed by addition of a peroxidase-labeled goat anti-Peagle Ig antibody to react. As a result, it was found that two strains, recombinant vaccinia virus No. 3 and recombinant vaccinia virus No. 16, expressed ΒΙ'-env in vitro. The results are shown in FIG. In this area, lanes 1 to 3 are recombinant vaccinia winnowles No. 3, lanes 4 to (recombinant vaccinia winnowles No. 1S, lane 7 is vaccinia virus L0— One strain (negative control), lane 8 shows the results for BLV-infected cells in commerce, and lane 9 shows the results for BLV-infected cells in shed. Να 16 is also called L01 — 7.5 Ps — Be.

Example 9. BLV-env with recombinant vaccinia virus

 In vivo expression of genes

 In Example 8, the in vivo expression of the recombinant vaccinia viruses ffe3 and No. 16 in which the expression of the BLV-env gene was confirmed in the mouth of the in vivo mouth was determined by the L01 virus strain ( It was confirmed in Egret in comparison with the negative control.

The type and amount of inoculated virus are as follows.

Heron o. Virus strain contact 1 Control virus L 0 1 5 1 0 (PFU) Control virus L 0 1 5 X 1 0 ⁷

3 Recombination virus No. 1 6 5 I 0 ⁷ 4 Recombination virus No. 1 6 5 1 0 ⁷ 5 Recombination virus No. 3 5 1 0 ⁷ 6 Recombination virus No. 3 5 1 0 ⁷

Recombination virus No. 1 6 2 1 0 ⁸

8 Recombinant virus No. 1 6 2 X 1 0 ⁸ 9 Control virus L 〇 1 2 1 0 ⁸

Viruses were intradermally inoculated in three places with a total volume of 0.5. One week later, the redness of the inoculation site was measured. As a result, red spots of about 10 wt each were formed, and it was determined that the inoculation was successful. Every week after inoculation, ゥ Sagi No.;! ~ 6 is collected until week 12 and ゥ Sagi No.7 ~ 9 is sampled until week 10. Octagony method (Kitasato Institute: Bovine Leukemia Diagnosis The antibody titer was measured by kit), but no antibody was detected. The same was true for Western brosorting using FLK cells or BLV virions as antigens.

As described above, although humoral immunity was not induced by the recombinant vaccinia virus, there is a good possibility that helper T cells have been activated. In order to confirm this, Against c I pulse No. 1 6 and L 0 1 strain (control) 2 x 1 0 ⁸ PFU of amounts Usagi said and No. 8 were each rather inoculation No. 9, © I Luz inoculated 1 0 After a week, BLV antigen (obtained by incubating the virus suspension of bovine leukemia virus-sustained fetal sheep kidney cells HK-11 and inactivating the virus suspension) was added to about 200 (the antigen concentration was changed to env antigen). Inoculation was followed by booster immunization, blood was collected every other week after this antigen inoculation, and measurement was performed by Western plotting. As antigen for Western 'plotting, virion of E. coli leukemia virus was used. Egret serum was diluted 50-fold and used for measurement. The results at 1 week and 3 weeks after the boost are shown in FIG. As is clear from this figure, the serum of the egret inoculated with the recombinant vaccinia winnowles No. 16 contains a product that appears to be an antibody to gp60, but the serum from the heron inoculated with L01 was present. The corresponding substance was not detected in the serum.As a result, the heron inoculated with the recombinant vaccinia virus alone and the heron inoculated with the non-recombinant vaccinia virus and then with the BLV antigen were inoculated. No substance was detected in the serum that reacts with the virulence of the leukemia virus, but was added after inoculation with the recombinant xenia virus to the inactivated leukemia virus. A substance that was considered to be an antibody to gp60 was detected in the serum of the immunized egret.

FIG. 1 shows the time course of antibodies to gp60 up to 3 weeks after the boost. As is clear from this figure, the anti-gp60 antibody was not detected at the time of booster immunization, and thereafter increased temporarily. ¾Example 10. Fabrication of ATI Promoter

5 cowpox c I Angeles CPR0 6 share (Niigata Univ.,巿撟by Yasuo assistant professor Ri available) were infected with moi 0. 2 to 1 0 ⁸ Vero cells (A full re-mosquito Mi drill monkey kidney-derived cells) Two days later, the virus was collected when cytopathicity became significant. This virus was purified by sucrose density gradient centrifugation to obtain about 3 mm of virus.

 3% of the resulting purified virus was added to 0.5% sodium raurylsulfate (SDS) and 1% sodium (πΜethylenamine tetraacetate (EDTA)).

 Two

 Digested with 1Ζ [^ proteinase 5 in the presence of 晚 3 7. For deproteinization, extraction was performed three times with phenolic black form and three times with ethyl ether. One to ten amounts of sodium triacetate and two times the amount of isopropanol were added and stirred, and the aggregated DΝ was rolled up with a glass rod. DNA was dissolved in TE [10 mM Tris-HCl (Tris-HC) pH 8.0; 1 mM EDTAj. The yield of DNA was 180.

 牛 0 cowpox virus DNA with Hind ID or Sa} I

10 mH Tris-HC H ( _PH 7.5), 60 NaC £, 7 mM MgC ί ₂ or 10 mM Tris-HC £ (pH 7.5), 150 mM NaC £, 7 mM MgC ί 3 7 in _z . C at cut 120 minutes, _P UC 13 off cut with pCU 18 plus Mi de DNA or Sa 1 I cut respectively Hind ffl. Mixed with lasmid DNA, 66 mM Tris-HC (pH 7.5), δ mil MgC £ 2,5 dithiothreitol, with T4 ligase in 1 ATP 16 For 16 hours. Escherichia coli strain JM103 or Jil109 was transformed with the above recombinant plasmid to obtain a genomic DNA library of cowpox virus. Recombination Rasmid contains a genomic DNA fragment of 1 kb to 25 kb.

 Plasmid containing bovine spastic virus DNA was transfected into transfected vaccinia virus cells (CV-1 cells) to express the A-type inclusion body gene Was confirmed by Western blotting using an anti-A-type inclusion body antibody, and DNA harboring the A-type inclusion body gene was identified.

First to 3 X 1 0 ⁵ pieces of CV _ 1 cells (monkey. Kidney-derived cells) were infected with vaccinia Shiniau Lee Angeles WR strain at the mo i 5 0 placed between one o'clock. A plasmid containing bovine spastic virus DNA was extracted from the above-described culture solution of E. coli 2πι? By the alkaline method, and 10 was transfected into vaccinia virus-infected cells. did.

After 30 minutes at room temperature, 3 ml of a medium containing 5% fetal bovine serum was added, the medium was incubated at 37 for 5 hours, the medium was changed once, and then overnight at 37'C. After sonication, the cell suspension was electrophoresed on a SDS-polyacrylamide gel and subjected to Western blotting using an anti-A type A inclusion body antibody. That is, the proteins on the gel were electrically transferred to a bitrocellulose filter. Nitrocellulose filter was treated with 10 Tris-HCJ2 (H7.5), 0.15 M NaCί.5% bovine serum albumin, and then the anti-human IgG antibody and 10 mM Tris _{-HC & (P h 7. 5)} , was allowed to react at 1 hour 3 7 at 0.15M NaC ^ medium. Further, an anti-Egret IgG antibody conjugated with peroxidase was reacted at 37 in the same solution as above. Finally, 0.01% hydrogen peroxide solution, 0.5 to 4%, including black naphthol 10 Tris-HC ί (ρΗΤ.ο) The cells were stained with a 0.15 M NaC solution to confirm the expression of the type A inclusion body gene. As a result, it was found that the Sai22-kb fragment contained the A-type inclusion body gene. This fragment is excised from the plasmid containing the fragment in the library with SalI, and the fragment is further digested with a restriction enzyme, KpnI, SphI, PstI, or SacI. By cutting and ligating to pUC18 or plJC19 plasmid cut with the corresponding restriction enzyme, recombinant plasmid ρΒ6, PB20, ΡΒ23, pC3, PC6, etc. are obtained. Plastic

 Two

 When the same procedure was performed for the peptide 7, the Kpn I-Sph I 9.1 kbp fragment (F. rasmid pB23) and the Sac I-Sal I 8.9 Kbp fragment (Plasmid PC 3) Shows that the full length of the A-type inclusion body gene is contained, and that the Sac I-Sal I 6.2 Kbp fragment (plasmid pC6) contains a part of the A-type inclusion body gene. did.

 Therefore, in the present invention, any of the above plasmids can be used as an ATI promoter source.

 In addition, Escherichia coli JM109-B23 containing the above-mentioned plasmid PB23 was designated as FERM P-8971 in 1986 by Escherichia coli JM109-B23. On March 19, the Institute for Microbiological Research, National Institute of Advanced Industrial Science and Technology, was deposited at Tsukuba East 1-chome 1-3, Ibaraki Prefecture, Japan, and was transferred to Souken Kenjo No. 1459 (FERM BP-1459). It was transferred to an international deposit under the Budapest Treaty on September 1, 1987.

Example 11. Preparation of a plasmid P13 C — G — 2 — 32

 (Fig. 12)

The plasmid pC 6 prepared in Example 10 was buffered with a low salt concentration. Digested with Sac I in the solution. Subsequently, it was digested with BamH [in a high salt buffer. Furthermore, after phenol extraction and ethanol precipitation, exonuclease II was used to move from the BamHI site to the ATI promoter region in exonuclease m buffer. About 3.3 Kbp. In addition, S1 nuclease was allowed to act in S1 nuclease buffer, and the protruding single protein was digested to blunt ends. Next, both ends were religated and cyclized using T4 DNA ligase, and Escherichia coli JM109 was transformed using the reaction mixture. A plasmid was extracted from the obtained clones by the alcoholic method and analyzed by various restriction enzymes to obtain a desired plasmid, which was then added to the plasmid pl3C-6-2-132. And

Example 12. Plus P18-ATI-pr. Preparation of 536 (Fig. 12) A cloning site for incorporating a foreign gene was introduced downstream of the ATI promoter as follows.

 : Blasmid P13C-6-2-32 was digested with Taq I and EcoR I in a high salt buffer, and then blunt-ended by treating with KI enow polymerase. A DΝ fragment containing the promoter was obtained. On the other hand, plasmid pUC18 was digested with HincH in a high salt buffer. Both were ligated with T4 DNA ligase, and E. coli JM109 was transformed using the reaction mixture.

The obtained clone was analyzed by various restriction enzymes to obtain a clone in which the direction of the ATΓ promoter was directed from the Hindffl site to the EcoRI site, and this was added to plasmid pl8-ATI. -pro536 Example 13. Preparation of PA2-9 (Figure 13)

 ATG exists immediately downstream of the ATI promoter region integrated in pUC18. This ATG is the start codon of the ATI protein gene, but its presence is inconvenient when a foreign gene is incorporated therein. Because of this, this Ding. An operation to convert to is performed.

 The plasmid pl8-ATI-pro536 was digested with Hind.DI in a medium salt buffer and further digested with EcoRI in a high salt buffer. On the other hand, the phage M13RF (replication type) DNA was

Digested with E [and EcoR I. Both were ligated with T4 DNA ligase, and the reaction mixture was transfected into E. coli JM109. After DNA was extracted by the alkaline method, it was analyzed with various restriction enzymes to confirm that the ATI gene had been incorporated into -13-RF. This was designated as .113-ATI.

 A phage (single-stranded DNA) was recovered from the culture supernatant of E. coli in which M13-ATI was transfected. The recovered single DNA was purified by ethanol precipitation. Next, using primers called Origonucleotide Site-Directed Mutagenesis in! 113-3 (manufactured by Anglian Bio-technology Inc.), primer nucleotides were used.

Site-directed mutagenesis was performed using [5'-AATAAATAGAGGTCACGAACC-3 '). Nucleotide changes were confirmed by the dideoxy method, and the plasmid containing the desired mutation was named pA2-9. Example 14. Preparation of plasmid PHAA2-9 (Fig. 14)

Digestion of plasmid pA2-9 with Hind HI in medium salt buffer Subsequently, it was digested with EcoRI in a high salt buffer. This was further treated with Klenow polymerase to blunt the ends to obtain EcoRI-Hindii [[fragment.

 On the other hand, the fragment having blunt-ended ends from PA2-9 was ligated to the NruI site of P13HA obtained in Example 1, and Escherichia coli was transformed using the reaction mixture. Plasmid was extracted from the obtained clones, and analyzed by restriction enzymes to obtain a plasmid in which the ATI promoter was inserted in the reverse direction into the hemagglutinin gene, which was named pH A2-9. did.

Example 15. Preparation of plasmid p9——ATIP—Be (FIG. 15) Brassmid P13—Be obtained in Example 4 was digested with Hind H [in a medium salt concentration buffer, and further digested. And digested with EcoRI in high salt buffer. The resulting gene fragment was blunt-ended by Klenow polymerase treatment. On the other hand, the plasmid obtained in Example 14 was digested with Sma! In Smal buffer. Both were ligated with T4 ligase, and E. coli was transformed with the reaction mixture. Plasmid was extracted from the resulting clones and analyzed with various restriction enzymes to confirm that the Benv gene had been incorporated downstream of the ATI promoter in the same direction as the ATI promoter. This plasmid was named p9—HA—ATIP—Be. Eschcerichia col i Τβ-P9—HA—ATIP—Be, which has this plasmid, was established on August 8, 1988, by the Institute of Microbiological and Industrial Technology, Institute of Industrial Science and Technology, 1-3-1 Higashi, Tsuguba City, Ibaraki Prefecture. FERH BP-1 was deposited internationally in Japan under the Budapest Treaty. 荬 Example 16. Incorporation of BLV-em 'mosquitoes into vaccinia viruses

The Usagi kidney cell line RK13 plated on sheet catcher Moltrasio diameter 6 era, to form a mono-les over Layer chromatography was incubated at 3 7, 5 ¾C0 _Z to be capital. Next, the cultured cells were infected with vaccinia virus L0-1 strain as a vector at an amount of mo i 0.1 and left for 1 hour. Next, plasmid p9-HA-ATIP-Be prepared in Example 15 was combined with the calcium phosphate method (Weir, JP, ^ (1982) Proc. Natl. Cad. Sci. USA, 7_9, 1210-1214), the virus was released from the culture by freezing and thawing, and the virus was recovered.

 RK13 cells were stored in a 6-inch diameter dish.

 The cells were cultured in MEM medium at 37 at 24 hours to form a monolayer. Next, on this layer, the collected wirelesss as described above were sowed and incubated for 2 days to form plaques. Next, we spread erythrocytes of the nitrile onto this layer and picked up plaques [HA (-)] that did not turn red.

 In this way, 32 candidate virus strains were obtained.

Example 17. Confirmation of BLV-enV-gene expression by recombinant vaccinia virus

 For the above-mentioned 32 candidate strains, ゥ Estan 'plotting was carried out after cloning in order to examine whether BLVenv was actually expressed in the cultured cells.

RK13 cells are spread on a 6 cm diameter plate to form a monolayer, and the recombinant vaccinia virus candidate strain is infected with the amount of mo i 1 The cells were cultured for 24 hours, and the cells were collected. One-twentieth of these were electrophoresed on SDO241CLO6S'acrylamide gel and further transferred onto a Dyuraphor paper (manufactured by Millipore). Next, after treatment with antibodies to g _P 60, Paokishida - was reacted labeled catcher anti Usagi I g antibody formic added at zero. As a result, it was found that the recombinant vaccinia virus No. 29 strain (also called m0-ATIP-Be strain) expressed BLV-env in vitro.

 Three

 The results are shown in FIG. Two

Example 18. BLVenv by recombinant virus senior virus

 In vivo expression of genes

 For the recombinant vaccinia virus ΐ¾29 in which expression of the BLVenv gene was confirmed in the in vivo mouth in Example 17, the in vivo expression was evaluated using the m0 virus strain (negative control). ) Was confirmed in Egret.

The types and amounts of inoculated virus are as follows. Usagi Να c I Angeles strain inoculation _ amount - recombinant © pulse ^{No. 2 9 1 X 1 0 8} (PFU)

1 X 1 0 ⁸

1 1 0 ⁸ Control virus m 0 1 X 1 0 ⁸

1 x 1 0 ⁸ 1 1 0 ⁸ Viruses were intradermally inoculated in two places with a total volume of 0.5. One week later, when the redness of the inoculation site was measured, about 10 red spots were formed in each case, and it was judged that the inoculation was successful.

 Example 19: Preparation of P2401

 According to the method of Venktesan, S. et al., Cel 1, 25, 80.5-813 (1981), a 0.26 kbp Rsal-Hinc II fragment containing the promoter of the 7.5 K protein was obtained, and Plasmid P2401 was obtained by insertion into the Hinc II site.

 Three

Example 3

 20. Preparation of P2401-7.5 P N-Be

 (Fig. 16)

 Plasmid P2401, into which the 7.5 Kd promoter had been inserted, was digested with Sma I in Sma I buffer. After inactivation of the enzyme by phenol extraction, ethanol precipitation was performed.

 In addition, the plasmid pl3-Be obtained in Example 4 was digested with EcoRI and HindHI in a medium salt concentration buffer. The EcoRI-Hind ffl fragment was separated by agarose electrophoresis, and a DNA fragment of about 2.0 Kbp was isolated and purified. In addition, the protruding 3 'end of DNA was filled in with a polymerase K] enow fragment to make it a blunt end.

The plasmid p2401 digested with SmaI and a 2 Kb fragment containing the BLV-env gene were ligated using T4 DN ligase, and E. coli TB-1 was ligated using the reaction mixture. Transformation. The obtained clone was analyzed by various restriction enzymes, and it was confirmed that the BL V-en V gene was inserted in the same direction as the 7.5 Kd promoter. — 7.5 _PN — Be named Be. Example 21. Preparation of plasmid P13—HA—7.5 PH-Be

 (Fig. 17)

The plasmid P2401-7.5P _N- Be obtained in Example 20 was digested with HindIII and EcoRI in a medium salt buffer to obtain a BLVenv gene fragment linked to a 7.5Kd promoter. Was. The approximately 2.2 Kb _P fragment was isolated and purified by agarose gel electrophoresis. The 3 ′ end of the DNA fragment protruded by the polymerase Klenow fragment was blunt-ended.

 On the other hand, 4 Brasmid pHA13 obtained in Example 1 was linearized by digestion with Acc I in a medium salt buffer, and the enzyme was inactivated by phenol extraction. I went.

 Next, the 2.2 Kb fragment containing the 7.5 Kb promoter and the BLVenv gene was combined with the linearized plasmid pHA13.

Ligation was carried out using T4 Mk ligase, and the reaction mixture was used to transform E. coli TB-1.

The resulting clone was analyzed by various restriction enzymes to confirm the introduction of the 5 Kd promoter—the BUenv gene. The obtained plasmid was identified as P13—HA—7.5 P κ—Be naming the _e to舍有this plus Mi de Escherichia col i TB - pl3 - HA - 7. 5 P N -Be is, on February 8, 1988, Agency of industrial Science and technology Fermentation research Institute, if get Ibaraki Prefecture Deposited internationally under the Budapest Treaty on 1-chome i-chome i-3 as FERM BP-1703.

Example 22. Delivery of Vaccinia virus to & _-ENV gene

 Integration

ま い Sprinkle the heron kidney cell line RK13 onto a 6-inch The cells were cultured under 5% CO2 to form a monolayer. Next, the cultured cells were infected with vaccinia virus L0-1 strain as a vector at a moi of 0.1 and left for 1 hour. Next, the plasmid P13-HA-7.5 PN-Be prepared in Example 21 was subjected to the calcium phosphate method (Weir, JP, et al. (1982) Proc. Natl. Acad. Sci. USA, 79 , 1210-1214). From this culture, the virus was released into the medium by freezing and thawing, and the virus was recovered.

 RK13 cells were cultured for 24 hours at 37 ° C. for 24 hours in a CID medium containing 6 cm diameter dishes to form a monolayer. Next, on this layer, the virus collected as described above was sown and incubated for 2 days to form plaque. Next, the erythrocytes of the nitrile were spread on this layer, and the plaque [HA (-)] that did not turn red was picked up.

 As a result, 23 candidate virus strains were obtained.

Example 23. B env by recombinant virus senior virus

 Confirmation of gene expression

 To examine whether the above 23 candidate strains actually expressed BLVenv in the cultured cells, Western-blotting was performed after cloning.

RK13 cells are spread on a 6 cm diameter dish to form a monolayer, and infected with recombinant vaccinia virus strain at an amount of moi 1 and cultured for 24 hours. Was recovered. These 120 volumes were electrophoresed on an SDS / acrylamide gel, and further transferred onto a Durapore paper (Millipore). next After treatment with an antibody against gp60, a peroxidase-labeled goat anti-Peagle Ig antibody was added and reacted. As a result, this results Kumi換Wa click Shiniau I pulse No. 1 strain (m O-7. that also referred to as 5 P _N -B e) it was found that by expressing the BLVe nv with I Nbi preparative port It is shown in Figure 18.

Example 24 Recombinant Vaccinia Virus B

 In vivo expression of genes

 With respect to recombinant vaccinia virus No. 1 in which expression of the BL Venv gene was confirmed in vitro in Example 23, expression in vivo was confirmed in Persian heron.

The type and amount of inoculated virus are as follows. © heron o. © I Angeles stock inoculation amount of recombinant c I Luz ^{No. 1 1 1 0 8 (P} FU)

The virus was intradermally inoculated in two places with a total volume of 0.5 m. One week later, the redness of the inoculation site was measured, and the redness of each urine was about 10 urn, indicating that the inoculation was successful.

Every week after the inoculation, blood was collected from the ear vein of the egret, and a 50-fold dilution of the serum was used for Western blotting using BLV virion as an antigen. The results for the serum at week 6 are shown in FIG. gp60 specific band It was found in the sera of three rabbits. A clearer band could be obtained by increasing the dilution factor by a factor of 5 or 10. A similar experiment was performed with sera at 14 weeks, but with the same results.

C I Luz vaccination after 1 week 4 to 0.4 percent - propionitrile La click tons in about 1 1 0 ^seven Kumi換Wa click senior cormorant I Angeles infected cells that have been inactivated was injected intramuscularly. The results of the Western blotting 3 weeks after the booster immunization are shown in FIG. 20, where the band specific to gp60 is intensified.

 Three

You can see that it is. [F, Industrial applicability

 INDUSTRIAL APPLICABILITY The present invention provides a new type of Escherichia coli leukemia virus, which is particularly useful in the fields of animal husbandry and drunkenness.

Claims

The scope of the claims 1. A recombinant virus capable of expressing a surface antigen of P. leukemia virus, wherein a surface antigen gene of P. leukemia virus is incorporated into the vaccinia virus gene.  2. The recombinant virus according to claim 1, wherein the surface antigen of Escherichia coli leukemia virus is integrated into the hemagglutinin gene region of the vaccinia virus gene.  3. Upstream of the surface antigen gene of the ゥ leukemia virus 7.5 K protein promoter — and type A inclusion body of cowpox virus The recombinant virus according to claim 1, which has a promoter selected from the group consisting of (ATI) gene promoter.  4. The method according to claim 1, wherein all or most of the gene other than the gene for the surface antigen of P. leukemia virus and the control sequence for the gene is derived from the gene of V. cinerea. Recombined virus listed.  5. A method for producing a recombinant virus capable of expressing a surface antigen of a leukemia virus, wherein the surface antigen gene of a leukemia virus is incorporated into a vaccinia virus gene. ,  (1) a hemagglutinin gene-containing vector derived from the vaccinia virus gene is inserted into a cloning vector to prepare a hemagglutinin gene-containing vector; (2) Hemagglutinin in the hemagglutinin gene-containing vector Inserting a surface leukemia virus surface antigen gene and a promoter for the gene into the Tinin gene to prepare a vector containing the leukemia virus surface antigen gene; and  (3) The vaccinia virus gene is obtained by homologous recombination between the hemagglutinin gene region in the vector containing the surface antigen gene and the hemagglutinin gene region of the vaccinia virus gene. Integrating the surface antigen gene of Escherichia coli leukemia virus into a hemagglutinin gene region together with a promoter for the gene; A method comprising steps.  6. A recombinant virus capable of expressing a surface antigen of leukemia virus is integrated into a vaccinia virus gene, and the recombinant virus is capable of expressing a surface antigen of leukemia virus in mammals. A method of immunizing a mammal against E. leukemia virus, which comprises inoculating the mammal.  7. A recombinant virus capable of expressing a surface antigen of leukemia virus, which has a leukemia virus surface antigen gene incorporated into the vaccinia virus gene, is a mammal. Immunizing a mammal against P. leukemia virus, characterized by inoculating the P. leukemia virus or an inactivated P. leukemia virus or a portion thereof having antigenicity. Method.