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WO1988006182A1 - Vaccin contre le virus de la leucemie bovine prepare en utilisant le virus recombinant de la vaccine - Google Patents

Vaccin contre le virus de la leucemie bovine prepare en utilisant le virus recombinant de la vaccine Download PDF

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Publication number
WO1988006182A1
WO1988006182A1 PCT/JP1988/000136 JP8800136W WO8806182A1 WO 1988006182 A1 WO1988006182 A1 WO 1988006182A1 JP 8800136 W JP8800136 W JP 8800136W WO 8806182 A1 WO8806182 A1 WO 8806182A1
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Prior art keywords
gene
virus
surface antigen
plasmid
leukemia virus
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English (en)
Japanese (ja)
Inventor
Kazue Oishi
Tadashi Maruyama
Masanobu Sugomoto
Yoshiyuki Sagata
Yoji Ikawa
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Tonen General Sekiyu KK
RIKEN
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Toa Nenryo Kogyyo KK
RIKEN
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • BLV ⁇ Leukemia virus
  • EBL ⁇ Leukemia virus
  • 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.
  • development of a leukemia vaccine by such a method has not yet been performed.
  • 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.
  • 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
  • a surface antigen gene of ⁇ leukemia virus by inserting a surface antigen gene of ⁇ leukemia virus and a promoter for the gene into the hemagglutinin gene in the hemagglutinin gene-containing vector. Preparing a gene-containing vector;
  • 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.
  • 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 ⁇ 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 ⁇ 2-9 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a vector can be any bacterial vector commonly used in genetic engineering, such as E. coli plasmids and mids.
  • 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.
  • pUC13 When pUC13 is used, it is digested with Hind1 ⁇ 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.
  • HA hemagglutinin
  • ⁇ 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).
  • the cleavage of this plasmid by the force of the plasmid, Pvu ⁇ and XhoI, and the partial digestion by the SmaI produce Bv '-A DNA fragment containing the onv gene can be obtained.
  • plasmid Pl3-Be can be obtained.
  • 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 ⁇ . 5 Ps by inserting positive Mi de pCU 9.
  • 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
  • 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.
  • 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
  • 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.
  • ATI A-type inclusion body
  • ATI promoter— cowpox virus
  • 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.
  • 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.
  • 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 ⁇ ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • the immunoress is collected from these samples and plated on the monolayer of RK13 cells to form plaques.
  • 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 (+).
  • 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.
  • 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.
  • each buffer has the following composition (1) Restriction enzyme buffer
  • Tr is' C i 66mM MgC H 2 5mM
  • 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 ⁇ ( ⁇ ⁇ 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.
  • plasmid pCl! 13 was digested with Hind ⁇ in a ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the plasmid pBR-322-BLV into which the BLV genomic gene has been inserted is digested with ⁇ ⁇ ⁇ 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.
  • the obtained Sma I — 2 Kbp DN fragment and the linearized plasmid CD13-Sma1 / BftP 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.
  • the plasmid was digested with Xbal in a high-salt buffer solution, and similarly blunt-ended with a polymerase enow fragment. .
  • 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.
  • 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.
  • Example 8 B'-env with recombinant xenia virus
  • the 9 candidate strains were subjected to ⁇ -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.
  • 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
  • lane 9 shows the results for BLV-infected cells in shed.
  • ⁇ 16 is also called L01 — 7.5 Ps — Be.
  • 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.
  • 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, ⁇ Sagi No.;! ⁇ 6 is collected until week 12 and ⁇ Sagi No.7 ⁇ 9 is sampled until week 10.
  • Octagony method Kerasato 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.
  • helper T cells 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).
  • 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. 3 ⁇ 4Example 10. Fabrication of ATI Promoter
  • 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.
  • 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 2 ⁇ ? By the alkaline method, and 10 was transfected into vaccinia virus-infected cells. did.
  • 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.
  • a restriction enzyme KpnI, SphI, PstI, or SacI.
  • any of the above plasmids can be used as an ATI promoter source.
  • Escherichia coli JM109-B23 containing the above-mentioned plasmid PB23 was designated as FERM P-8971 in 1986 by Escherichia coli JM109-B23.
  • 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.
  • 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.
  • 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.
  • 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 D ⁇ fragment containing the promoter was obtained.
  • 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.
  • 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.
  • the phage M13RF (replication type) DNA was
  • 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 Yalen Bio-technology Inc.), primer nucleotides were used.
  • 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 ⁇ -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. ⁇ 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 3 ⁇ 4C0 Z to be capital.
  • 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.
  • 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.
  • 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).
  • SDO241CLO6S'acrylamide gel was electrophoresed on SDO241CLO6S'acrylamide gel and further transferred onto a Dyuraphor paper (manufactured by Millipore).
  • Paokishida - was reacted labeled catcher anti Usagi I g antibody formic added at zero.
  • the recombinant vaccinia virus No. 29 strain also called m0-ATIP-Be strain expressed BLV-env in vitro.
  • Example 18 BLVenv by recombinant virus senior virus
  • inoculated virus The types and amounts of inoculated virus are as follows. Usagi ⁇ c I Angeles strain inoculation _ amount - recombinant ⁇ pulse No. 2 9 1 X 1 0 8 (PFU)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ —Be naming the e to ⁇ 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
  • 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.
  • Example 23 B env by recombinant virus senior virus
  • 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 ⁇ 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.
  • 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.
  • the present invention provides a new type of Escherichia coli leukemia virus, which is particularly useful in the fields of animal husbandry and drunkenness.

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Abstract

Virus recombinant contenant un gène d'un antigène superficiel du virus de la leucémie bovine incorporé dans un gène du virus de la vaccine et pouvant exprimer un antigène superficiel du virus de la leucémie bovine; procédé de préparation du virus recombinant, consistant (1) à introduire une fraction d'ADN contenant un gène d'hémagglutinine provenant du gène du virus de la vaccine dans un vecteur de clonage pour préparer un vecteur contenant le gène d'hémagglutinine, (2) à introduire un gène d'antigène superficiel du virus de la leucémie bovine dans le gène d'hémagglutinine présent dans le vecteur contenant le gène d'hémagglutinine et un promoteur du gène pour préparer un vecteur contenant le gène de l'antigène superficiel du virus de la leucémie bovine, et (3) à incorporer le gène de l'antigène superficiel du virus de la leucémie bovine en combinaison avec son promoteur dans la région du gène d'hémagglutinine du gène du virus de la leucémie bovine par la recombinaison homologue entre la région du gène d'hémagglutinine dans le vecteur contenant le gène de l'antigène superficiel et la région du gène d'hémagglutinine du gène du virus de la vaccine. Est également décrit un procédé d'immunisation de mammifères contre le virus de la leucémie bovine, consistant à inoculer aux mammifères le virus recombinant et ensuite un virus désactivé de la leucémie bovine ou une partie de ce dernier présentant des propriétés d'antigénicité.
PCT/JP1988/000136 1987-02-10 1988-02-10 Vaccin contre le virus de la leucemie bovine prepare en utilisant le virus recombinant de la vaccine Ceased WO1988006182A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2648712A1 (fr) * 1989-06-23 1990-12-28 Rhone Merieux Vaccin contre le virus de la leucemie bovine
US5521083A (en) * 1994-05-13 1996-05-28 The Research Foundation Of State University Of New York Et Al. Large granular lymphocyte leukemia associated virus
WO2007014977A1 (fr) * 2005-07-30 2007-02-08 Consejo Superior De Investigaciones Científicas Vecteurs recombines bases sur le virus ankara modifie (mva) utilises en tant que vaccin contre la leishmaniasis

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58129971A (ja) * 1981-12-24 1983-08-03 ヘルス・リサ−チ・インコ−ポレ−テツド 変異化ワクチニアウイルス並びにその製造及び使用方法
DD240029A1 (de) * 1985-08-02 1986-10-15 Akad Wissenschaften Ddr Verfahren zur herstellung eines blv-kodierten huellproteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58129971A (ja) * 1981-12-24 1983-08-03 ヘルス・リサ−チ・インコ−ポレ−テツド 変異化ワクチニアウイルス並びにその製造及び使用方法
DD240029A1 (de) * 1985-08-02 1986-10-15 Akad Wissenschaften Ddr Verfahren zur herstellung eines blv-kodierten huellproteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol. 82, No. 3, (1985), N. SAGATA et al., "Complete Nucleotide Sequence of the Genome of Bovine Leukemia Virus; Its Evolutionary Relationship to other Retroviruses", p. 677-681. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2648712A1 (fr) * 1989-06-23 1990-12-28 Rhone Merieux Vaccin contre le virus de la leucemie bovine
WO1991000105A1 (fr) * 1989-06-23 1991-01-10 Rhone Merieux Vaccin contre le virus de la leucemie bovine
US5521083A (en) * 1994-05-13 1996-05-28 The Research Foundation Of State University Of New York Et Al. Large granular lymphocyte leukemia associated virus
WO2007014977A1 (fr) * 2005-07-30 2007-02-08 Consejo Superior De Investigaciones Científicas Vecteurs recombines bases sur le virus ankara modifie (mva) utilises en tant que vaccin contre la leishmaniasis
ES2313807A1 (es) * 2005-07-30 2009-03-01 Consejo Superior De Investigaciones Cientificas Vectores recombinantes basados en el virus vaccinia modificado de ankara (mva) como vacunas contra la leishmaniasis.
ES2313807B1 (es) * 2005-07-30 2009-12-23 Consejo Superior De Investigaciones Cientificas Vectores recombinantes basados en el virus vaccinia modificado de ankara (mva) como vacunas contra la leishmaniasis.

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