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WO1998044093A1 - Nouveau vecteur d'adn et vaccins renfermant ces nouveaux vecteurs d'adn recombinant comme principe actif - Google Patents

Nouveau vecteur d'adn et vaccins renfermant ces nouveaux vecteurs d'adn recombinant comme principe actif Download PDF

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WO1998044093A1
WO1998044093A1 PCT/JP1998/001358 JP9801358W WO9844093A1 WO 1998044093 A1 WO1998044093 A1 WO 1998044093A1 JP 9801358 W JP9801358 W JP 9801358W WO 9844093 A1 WO9844093 A1 WO 9844093A1
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dna vector
virus
dna
gene
vector
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Japanese (ja)
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Takanori Sato
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Zeon Corp
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Nippon Zeon Co Ltd
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Priority to AU65182/98A priority Critical patent/AU6518298A/en
Priority to JP54142298A priority patent/JP3924328B2/ja
Publication of WO1998044093A1 publication Critical patent/WO1998044093A1/fr
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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
    • 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
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a novel DNA vector, and more particularly, to a novel DNA vector useful as a vector having high expression in animal cells or a vaccine.
  • Conventional technology a novel DNA vector useful as a vector having high expression in animal cells or a vaccine.
  • Gene engineering is often aimed at introducing foreign genes into bacteria and cells for expression. In this case, it is not possible to separate and purify the DNA fragment containing the specific gene of interest and put it directly into bacteria or cells to increase it. DNA fragments cannot normally be replicated by themselves and may be degraded by various DNA degrading enzymes contained in cells.
  • a target foreign gene is covalently linked to a carrier that can be transferred to a host cell and that can multiply autonomously in the host cell (this carrier is generally called a vector). Let them act together.
  • Various vectors are known depending on the combination with the host cell. For example, when a microorganism such as Escherichia coli or yeast is used as a host, a DNA vector called a plasmid is generally used. There are many specific examples of this, such as PBR322 and pUC18, which are also described in detail in “Gene Engineering Experiments”, pp. 75-128 (Yasutaka Takagi, Kodansha Scientifiq).
  • viruses are used as vectors depending on the purpose and the host cell.
  • SV40 and poliovirus which have been used less recently because they are oncoviruses, but have been used since the late 1970s, or vaccinia virus, fowlpox virus, and baculo, which have been commonly used since the 1980s.
  • viral vectors such as viruses and retroviruses.
  • the disadvantages common to viral vectors that use animal cells as a host are: The point is that it takes time to produce the recombinant virus, and the expression level of the foreign gene is not always sufficient. Regarding the expression level, the viral vector has a much lower copy number (the ratio of the replicated vector per host cell) than the plasmid vector. The use of a strong promoter can improve the expression a little, but the expression level is large. The increase cannot be expected.
  • the present inventors have made intensive studies to develop a new vector using an animal cell as a host which has overcome the above-mentioned drawbacks, and as a result, a novel DNA vector which gives an animal cell an extremely higher copy number than a conventional virus vector. They found one and completed the present invention. Furthermore, they have found a method of using the recombinant novel DNA vector as a vaccine, and have completed the present invention.
  • the first embodiment of the present invention is a fowlpox virus-derived DNA vector of about 5 kb, which is replicated only in box virus-infected cells.
  • the DNA vector is characterized in that it contains an inverted terminal repeat sequence containing a sequence obtained by repeating the nucleotide sequence of SEQ ID NO: 1 twice or more. Further, the DNA vector is characterized by containing a regulatory gene and at least three or more protein coding regions.
  • a second aspect of the present invention is a recombinant DNA vector obtained by incorporating at least one or more foreign genes into the above DNA vector.
  • the at least one foreign gene is under the control of a foreign regulatory gene.
  • the integration site of the foreign gene is located in the untranslated region or the fourth codeic region.
  • the foreign gene is preferably a gene encoding an antigenic protein of a pathogen.
  • the gene encoding the antigenic protein of the pathogen may be a gene encoding the E protein of Japanese encephalitis virus, or an HN protein of the Newcastle disease virus.
  • Gene encoding protein, gene encoding F protein, gene encoding glycoprotein gB of Marek's disease virus, and infectious bursal disease virus It is preferably a gene selected from the group consisting of the genes encoding the structural protein VP2 of luz.
  • the regulatory gene may be a promoter of a vaccinia virus gene encoding a 7.5 kDa peptide or an 11K polypeptide or a variant thereof, a synthetic promoter having sequences of both an early promoter and a late promoter, and a base sequence described below. (SEQ ID NO: 5)
  • the promoter is selected from the group consisting of a promoter having a DNA sequence represented by
  • a third aspect of the present invention is a vaccine containing the above-mentioned recombinant DNA vector as an active ingredient.
  • the vaccine of the present invention preferably contains the above-mentioned recombinant DNA vector and an attenuated box virus.
  • a fourth aspect of the present invention is a method for transforming Escherichia coli with a plasmid containing a recombinant DNA vector containing a foreign gene, culturing the transformant to amplify the recombinant vector, And a step of introducing the vaccine into a box virus-infected cell to further amplify the vaccine.
  • the recombinant DNA vector contains a foreign gene and DNA represented by the nucleotide sequence of SEQ ID NO: 2.
  • a fifth aspect of the present invention is the use of the vaccine described above.
  • a sixth aspect of the present invention is a method for preventing and / or treating infectious diseases, comprising infecting a recombinant DNA vector of lOng lig with a poxvirus selected from the group consisting of orthopoxvirus and fowlpox virus.
  • a poxvirus selected from the group consisting of orthopoxvirus and fowlpox virus.
  • the above vaccine containing 1 ⁇ 10 3 to 1 ⁇ 10 5 pfu of attenuated poxvirus and 10 ng to 1 ⁇ g of the recombinant DNA vector is orally administered, intradermally administered, subcutaneously administered, intravenously administered, It is preferable to administer by an administration route selected from the group consisting of intramuscular administration and intraperitoneal administration.
  • a novel DNA vector that can be replicated only in a box virus-infected cell is provided, and (2) a recombinant DNA vector in which a foreign gene is inserted into the DNA vector is provided. And (3) the recombination A vaccine comprising a DNA vector as an active ingredient is provided.
  • FIG. 1 shows the construction of the vectors pNZ66, pNZ76, and pNZ76 used in the examples.
  • the 3.3 kb fragment of pMAOO1 cut with ⁇ anii I is ligated with pUC19 cut with BSIIM I to construct PNZ66. This is cut with T / '/ JcII, cut out from p-volume P-1 with pal I and £ cd l, and ligated with a DNA polymerase treated 7.5K promoter gene to construct PNZ76.
  • pNZ76 is further digested with dll, and PNZ87 is constructed by ligating the ⁇ a; rfil- / II fragment obtained from mplO-HN18RF.
  • Figure 2 shows the restriction map of the novel DNA vector and the positional relationship between the subcloned clones.
  • the restriction enzyme cleavage site of about 5 kb of DNA obtained from the fowlpox virus Sashimi strain and the position of each fragment subcloned into commercially available pUC18 plasmid are shown.
  • the subclones of the 0.6 kb, l. lkb, 711 of the coRI digestion fragment and the 2.4 kb fragment of the ⁇ al double digestion fragment are No. 17, No. 16, and No. 72-2, respectively.
  • No. 20 and No. 22 are the subclones of the 1.3 kb and 2.0 kb fragments that appeared after digestion with coRI after DNA polymerase I treatment.
  • No. 73-9 is a subclone containing a 1.53 ⁇ 4 fragment that appeared after double-cutting the .22 clone with 1 and 51.
  • the 0.8kb and 0.9kb subclones that appeared after double-cutting the clone of No. 20 with Si and ⁇ ; ⁇ 1 ⁇ and Stu ⁇ hi Bgl ⁇ I were No. 73-10 and No. 73-12, respectively. It is.
  • Figure 3 shows the construction method of pPADOl.
  • FIG. 4 shows a method for constructing a plasmid pPADin (4541-4759) P7.5: 1 acZ containing a recombinant DNA vector.
  • pPADOl was cut with coSlI / Spel to construct pPADdI (454-4922).
  • pPADOl was cut with o81I / 5 "peI, and pPA DrepL (454-4922) was constructed using a synthetic DNA linker.
  • PPADrepL (454-4922) was cut with // « II, and 4.
  • a 5 kb fragment was obtained.
  • P7.5: lacZ was constructed with a 3.3 kb fragment obtained by cutting 1 with //// 7dIII / 7; l.
  • FIG. 5 shows a method for constructing a plasmid pPADin (377) P7.5: HN containing a recombinant DNA vector.
  • PNZ87 Using a 1.9 kb fragment obtained by digesting with S1 nuclease and a fragment obtained by digesting pPADOl with ssHII and treating with S1 nuclease and BAP, pPADin (377) P7.5: HN Was built.
  • FIG. 6 shows the construction of pNZ1029 used to obtain fNZ1029 used in the examples.
  • the 7.3 kb fragment obtained by digesting the abipox virus with oRI is ligated with pUC18 digested with fcd and treated with alkaline phosphatase to construct pNZ133.
  • This is partially digested with oRV, and the; -3 ⁇ 4 ⁇ fragment cut out from pNZ76 is treated with DNA polymerase and ligated to construct PNZ1029.
  • FIG. 7 shows the construction of PNZ2237 used in the examples.
  • a 7.3 kb fragment obtained by treating Avibox virus with £ coRI and pUC18 treated with £ coRI and alkaline phosphatase were ligated to construct pNZ136, which was partially digested with Hind111 and £ coRV. The fragment is ligated with PVC18 to obtain PNZ136S. This is further processed to construct pNZl36SL, treated with HindIII and BadlI, and ligated with a fragment containing HN obtained from pNZ87 or the like to construct PNZ2237.
  • the DNA vector of the present invention (hereinafter, simply referred to as a DNA vector) is an approximately 5 kb DNA vector derived from fowlpox virus, but cannot replicate in box virus-uninfected cells, and does not replicate in box virus-infected cells. It is a concomitant DNA vector because it can
  • This DNA vector is a linear DNA, and its sequence contains the nucleotide sequence of SEQ ID NO: 1 twice or more.
  • the terminal structure of the vector of the present invention is such that the base sequence described in SEQ ID NO: 1 is defined as one unit, and the sequence includes this sequence two or more times, preferably three times. Then salt It is preferable that the base sequence is an inverted terminal repeat sequence (Inverted Terminal Repeat; hereinafter, it may be referred to as ITR).
  • ITR Inverted Terminal Repeat
  • the DNA vector of the present invention more preferably has the nucleotide sequence of SEQ ID NO: 2. That is, the adenine and thymidine content (AT content) of the present invention is high, and in addition to the above-mentioned inverted terminal repeat sequence, at least three protein coding regions (Open Reading Frame: hereinafter referred to as RF), promoters and other Those containing sequences are preferred.
  • RF Open Reading Frame
  • the AT content is preferably 60 to 65%.
  • the DNA vector of the present invention preferably has at least three or more ORFs, and preferably has four ORFs.If there are four ORFs, it may have the following nucleotide sequence. preferable.
  • 0RF1 is the 1,207-671st nucleotide sequence of SEQ ID NO: 2
  • 0RF2 is also the 1,257-7,776th nucleotide sequence
  • 0RF3 is the 1,806-6,397th nucleotide sequence
  • 0RF4 is the nucleotide sequence at positions 4,991 to 4,422.
  • the translation directions of 0RF1 and 0RF4 are opposite.
  • a promoter is preferably present before the translation initiation codon of the ORF in order to translate and express the ORF.
  • the promoter and other sequences include the vaccinia virus 7.5 kDa promoter, the vaccinia virus llkDa promoter, the synthetic promoter from Moss et al. (Moss et al., J. Mol. Biol., 215: 749-769 (1989)) and These variants are exemplified.
  • the vector of the present invention having the above structure has a high replication efficiency in a box virus-infected cell, and the replication efficiency is about 30 times or more the copy number of a natural box virus. preferable.
  • one or more bases in the base sequence are caused by a spontaneous mutation or the like. Mutations such as substitution / deletion / addition / insertion may be caused artificially by a known technique using a heterogenous substance or a restriction enzyme.
  • 0RF4 the nucleotide sequence at positions 4,991 to 4,422 of SEQ ID NO: 2
  • Various mutations such as deletion or integration of a foreign gene described below can be generated.
  • the function of the vector of the present invention is not impaired, so that similar mutations can be caused in these regions.
  • Examples of the mutation site in the untranslated region include a mutation at the 377th restriction enzyme II site of SEQ ID NO: 2.
  • Such a DNA vector is a livestock hygiene strain of fowlpox virus Sashimi (strain number: VA0101 / Japan Association of Biologicals for Animals) and a fowlpox virus Nishig ahara strain (strain number: VA0104, same) It can be obtained from fowlpox virus strains that are readily available, such as the Japan Association for Aging.
  • these preserved strains are infected with chicken embryo fibroblasts (CEF cells) according to a standard method, cultured at about 37 ° C for one week, and then the DNA present in the cytoplasm of the infected cells is recovered.
  • the fraction may be obtained by electrophoresis or the like, and a DNA fragment of about 5 kb other than the fowlpox virus genomic DNA confirmed thereby may be obtained.
  • the DNA vector of the present invention does not replicate when introduced into a box virus-uninfected cell, but replicates when introduced into a box virus-infected cell.
  • the DNA vector of the present invention is preferably a vector containing the DNA sequence represented by the nucleotide sequence of SEQ ID NO: 2.
  • box virus mentioned here examples include an orthobox virus represented by vaccinia virus and an avibox virus represented by fowlpox virus.
  • avibox virus used in the present invention include fowlpox virus, ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nishigahara strain, Sashimi strain, and Examples include viruses such as the CEVA strain which is commercially available as a live fowlpox vaccine strain. These include the depository institutions such as the American Type Culture Collection. It is available as a commercial vaccine from Seki or from manufacturers such as Intervet In.
  • box virus infected cells Cells artificially infected with these box viruses or cells naturally infected with these viruses are called box virus infected cells.
  • aviboxvirus-susceptible infections include primary culture cells prepared from embryonated chicken eggs such as CEF cells and chorioallantoic membrane of embryonated chicken eggs.
  • Vaccinia virus-infected cells include cultured cell lines derived from animals such as Vero cells and RK13 cells, and epidermal cells and muscle cells of animal individuals instead of cultured cells.
  • the method of infecting these cells with the box virus may be a method of infecting cells with a normal virus. Specifically, for example, CEF cells are grown to subconfluence in a medium such as Eagle's MEM, D-MEM, and the like, and the medium is discarded and the box virus described above is m. Infect to be 1-10.
  • the DNA vector of the present invention has an extremely high replication efficiency in box virus-infected cells, and has the advantage that, when a foreign gene is inserted into the DNA vector, the foreign gene is also replicated very efficiently. More specifically, the replication efficiency is about 30 times or more the number of copies of the genome of the Avibox virus strain, and more than 10,000 copies per cell.
  • the DNA vector of the present invention is inserted into an appropriate vector in a conventional manner (Molecular Cloning; A Laboratory Manual (1982) to obtain a Cold Spring Harbor Laboratory recombinant vector. If one is used, it is possible to replicate the DNA vector (gene) even in a box virus-uninfected cell.
  • the vector into which the DNA vector of the present invention is incorporated is arbitrarily selected depending on the purpose. be able to.
  • examples of such vectors include plasmids such as pBR322, pBR325, pUC7, pUC8, pUC18, pUCBM-20, phages such as M13 phage, and cosmids such as pHC79.
  • a modified vector in which a gene such as a gene encoding _____________________________________________________________________________________________ either of which is incorporated in these vectors may be used.
  • These vectors may be treated with an appropriate restriction enzyme, and the DNA vector of the present invention may be incorporated according to a conventional method.
  • the recombinant vector into which the DNA vector of the present invention has been incorporated can replicate various Escherichia coli and other bacteria such as TG1 and JM103 as hosts.
  • the recombinant DNA vector of the present invention contains the DNA vector described in (1) above, a foreign gene, a promoter and other regulatory genes, and the like.
  • PNZ76 is constructed as described in Izukahei No. 1-157381 (US Pat. No. 5,286,639) and JP-A No. 1-168279 (US Pat. No. 5,387,519). (refer graph1) .
  • pMAOOKShirakawa et al., Gene, 28: 127- (1984)) is digested with a! I, extracted with phenol: chloroform, and recovered pMAOOl that has been cleaved by ethanol precipitation.
  • the 5'-terminal phosphate is removed by alkaline phosphatase treatment, and the DNA is again extracted with phenol: chloroform. Then, about 3.3 kb of the ⁇ -galactosidase gene (/ acZ) is recovered by ethanol precipitation.
  • PUC19 is digested with Sa! I, extracted with phenol: chloroform as described above, and recovered by ethanol precipitation.
  • the recovered fragment from PUC19 and 7acZ are ligated with ligase to obtain plasmid pNZ66.
  • M13-mplORF DNA manufactured by Amersham
  • ⁇ ; zill and ⁇ al extraction with phenol: cloth form is performed, and the cleaved M13-mpl0 RF DNA is recovered by ethanol precipitation.
  • an adapter consisting of the following nucleotide sequence (SEQ ID NO: 8) and consisting of a 40 bp oligodoxynucleotide partially having a single strand is chemically synthesized using Genet AIII (manufactured by Zeon Corporation).
  • This adapter includes site, site,: 1 site, A3 ⁇ 4oI site, and Badll site cleavage sites.
  • This adapter and the cleaved M13-mpl0 RF DNA are mixed, ligated with a ligase, and transduced into Escherichia coli, which is convenient according to a conventional method (Methods in Enzymology, vol. 101).
  • This transformant is cultured on 2 XYT agar medium containing 5-bromo-4-chloro-3-indolyl / 3 / 3-D-galactopyranoside and isopropyl-3-indolyl-) 3-D-galactoviranoside.
  • the phage RF DNA is recovered, digested with Bg! Ll, and phage mp10-mpa into which the adapter DNA is inserted is obtained by agarose electrophoresis.
  • Plasmid XLIII-10H containing the NDV HN gene (Virus Research, 7: 241-255 (1977)) was distributed by Assistant Professor Masao Kawakita of the University of Tokyo. Digest XLin-lOH with Irall, extract by agarose gel electrophoresis, and recover the cleaved mplO-mpa by ethanol precipitation.
  • the cleaved raplO-mpaRF DNA is mixed with ⁇ l all of the above HN gene DNA, the cohesive ends are blunt-ended with DNA polymerase, and the mixture is extracted with phenol: black form and then recovered by ethanol precipitation. .
  • the recovered DNA is ligated with ligase, transduced into Escherichia coli as described above, and grown on 2 XYT agar medium.
  • the phage RF DNA is recovered from the formed plaque, cut with ⁇ al and ftol, and mp10-HN180 containing about 1.8 kb of HN gene fragment is obtained by agarose gel electrophoresis. (2-3) Construction of pNZ87
  • PNZ76 is digested with Badi I, and an approximately 2.9 kb fragment containing no ⁇ -galactosidase gene is recovered by agarose gel electrophoresis.
  • the hybrid phage mpl 0-HN180 After that, a DNA fragment of about 1.8 kb of the HN gene is recovered by agarose gel electrophoresis. Both were ligated with ligase, transformed into E. coli, and plasmids were extracted according to the method of Birnboim and Dori (Nucleic Acid Research, 7: 1513- (1979)), and HN was extracted by agarose gel electrophoresis. The plasmid containing the gene, PNZ87, is obtained.
  • CEF Chicken embryo fibroblasts
  • No.16, No.17 and No.72-2 are obtained by obtaining fragments digested with Eco I and fragments digested with I and J ⁇ al and ligating them to pUC18XG (Fig. 1). ).
  • the DNA vector of the present invention was treated with DNA polymerase, and the fragment cut with coRI was ligated to PUC18XG treated with 3 ⁇ 4 / aI and £ coRI to obtain subclones No. 20 and No. 22 is obtained.
  • Subclones No.73-1, No.73-2 and No.73-3 are obtained by cutting No.22 with / «II II and ligating it to pUC18 treated with / ⁇ .
  • the recombinant vector of the present invention is constructed as follows (see FIGS. 3 to 5).
  • Subclone No. 20 was cut with BgJlI.
  • the subclones No.22 and No.72-2 are cut with and £ a U, and these fragments are ligated to obtain a subclone No.72-22.
  • pPADOl was obtained by ligating the fragment obtained by digesting No.72-22 with I and treating with BAP, and the ⁇ II fragment of subclone No.20 (Fig. 2).
  • the pPADOl obtained in the above (4-1) was cut with co81I and Spel, treated with DNA polymerase to make the ends and ends, self-ligated to transform Escherichia coli, and selected to obtain pPADdl (454 to 4922). ) Can be obtained.
  • PPADOl obtained in (4-1) above was cut with ⁇ : o81I and ⁇ 9el to synthesize SEQ ID NO: 4.
  • Escherichia coli is transformed by ligating with an NA linker and, when selected, pPADrepl (454-4922) can be obtained.
  • the pNZ87 constructed in (2) above was cleaved with the moieties' ⁇ and 1 and treated with S1 nuclease to recover the fragment. Also, the pPADOl constructed in (3) above is cut with 5 ⁇ , treated with S1 nuclease and BAP, ligated with the fragment from pNZ87 recovered earlier, and transformed into E. coli. If selected, pPADin (377) p7.5: HN can be obtained (Fig. 5).
  • a foreign gene and a promoter which will be described later, can also be incorporated into the vector obtained as described above.
  • fNZ1029 is constructed as described in JP-A-168279 (FIG. 6).
  • PUC18 (Pharmacia) was digested with EcoRI and Hindlll, and extracted with phenol: chloroform, and PUC18 cleaved by ethanol precipitation was recovered. The 5'-terminal phosphoric acid is removed by alkaline phosphatase treatment, and the DNA is recovered again by extraction with phenol: chloroform and ethanol precipitation. The cleaved ⁇ 8 and the EcoRI fragment of the purified Aviboxvirus (strain) DNA were ligated with ligase, transformed into a competent Escherichia coli, and then treated with 5-bromo-4-chloro-3-. Incubate on LB agar medium containing indolyl 1 / 3-D-galactovyranoside, isopropyl 1 / 9-D-galactopyranoside and ampicillin.
  • the white colonies grown on the agar medium were cultured in LB liquid medium containing ampicillin,
  • the plasmid was extracted by the method of Birnboim and Doli (Nucleic Acid Research, 7: 1513- (1979)), digested with ⁇ 1 and // 3 ⁇ 41111, and then subjected to agarose gel electrophoresis to obtain the original Avibox virus DN.
  • a hybrid plasmid having a fragment of the same length as the CORI_7 /// K1III fragment of A is detected, and this is designated as pNZ133.
  • pNZ133 constructed in (5-1) was partially digested with oRV, extracted with phenol: cloth form, and recovered by ethanol precipitation.
  • the cleaved DNA of pNZ133 was mixed with the above fragment (ligated fragment of the 7.5K promoter gene and 3-galactosidase gene), the cohesive end was made blunt with DNA polymerase, and phenol: cloth form was used. After extraction, recover by ethanol precipitation.
  • the recovered DNA was ligated with ligase, transformed into E. coli, and grown on LB agar medium containing ampicillin.
  • Plasmid was collected from the grown E. coli by the method of Birnboim and Doley (Nucleic Acid Research, 7: 1513- (1979)). Then, a hybrid plasmid containing a mono-galactosidase gene fragment was selected by agarose gel electrophoresis and named PNZ1029.
  • PNZ1029 obtained as described above, a DNA fragment containing a 7.5K promoter gene and a / 3-galactosidase gene in avibox virus (Hatopox Nakano strain (also referred to as NP strain)) was prepared by a conventional method. To obtain fNZ1029 by homologous recombination.
  • PNZ2237 The construction of PNZ2237 is performed as follows according to the description in JP-A No. 157381 (FIG. 7).
  • PNZ87 is cut with ⁇ al and ////] dill, and 1 — /// ⁇ 11 fragment ( ⁇ ) is recovered. Similarly, ⁇ 87 is cleaved with 0al and rall, and the al— ⁇ rall fragment (B) is recovered. further, Cut PNZ87 with //// 7dIII and // rflll to obtain / ⁇ ⁇ ! ⁇ I — /// — rfl 11 fragment (C).
  • plasmid was extracted by the method of Birnboim and Dawley (described above), digested with coRI and // 7dIII, and then agarose gel electrophoresed. By electrophoresis, a hybrid plasmid having a fragment of the same length as the AcoRI-Hindi II fragment of the original avipox virus DNA was detected and designated as pNZ136.
  • the plasmid pNZ136S obtained in the above (6-2-2) is cut with a! I at / ⁇ , extracted with phenol: chloroform, and recovered by ethanol precipitation.
  • pNZ87, pNZ87-22 and pNZ87-37 were respectively cleaved with ⁇ ⁇ ; and a; rfll, subjected to agarose gel electrophoresis, and containing a vaccinia virus 7.5K promoter gene and HN gene DNA //// ⁇ — Recover a / rfll fragments.
  • PNZ2237 By ligating both with ligase and selecting as described above, PNZ2237 can be obtained.
  • the foreign gene to be incorporated into the recombinant DNA vector of the present invention is not particularly limited.
  • Preferred examples of such foreign genes include various enzyme proteins and antigenic proteins, structural genes encoding peptides, and regulatory genes that regulate gene expression.
  • examples of the enzyme protein include a gene encoding / 3-galactosidase and a gene encoding an enzyme protein such as human tissue plasminogen (tPA).
  • tPA human tissue plasminogen
  • antigen gene examples include those derived from mammalian cells, mammalian infectious pathogens, bird cells, bird infectious pathogens, fish cells, fish pathogens, and the like.
  • a gene encoding the E protein of Japanese encephalitis virus US Pat. No. 5,021,347 and a gene encoding the HN protein of Newcastle disease virus (Miller et al., J. Gen. Virol., ⁇ 2, 1917-). 1927 (1986)
  • F protein McGinnes et al., Virus Res., 5343-5356 (1986)
  • Marek's disease virus glycoprotein gB Rosss et al., J. Biol.
  • antigen genes Genes encoding antigens involved in the protection of infection, such as the gene encoding the structural protein VP2 of infectious bursal disease virus (Bay liss et al., J. Gen. Virol., 1303-1321 (1990)) Hereinafter, these genes are simply referred to as antigen genes).
  • the regulatory gene examples include a promoter of a vaccinia virus gene that encodes a 7.5 kDa peptide, a 11K polypeptide, and the like.
  • these promoters It may be a modified product in which a part is deleted.
  • synthetic promoters for example, synthetic promoters having both the early and late promoter sequences (J. Mol. Biol., 215, 749-769 (1989), ibid, 215, 771 781 (1989)) ) Or some of them are modified to the extent that promoter activity is not lost, deletions, modification or substitution of bases, for example, the base sequence is as follows (SEQ ID NO: 5)
  • Regulated genes that regulate gene expression used in gene recombination techniques such as
  • exogenous genes can be incorporated alone or, if necessary, in combination of two or more.
  • the insertion site of these genes is not particularly limited, as long as the DNA vector of the present invention does not lose its ability to replicate in a box virus-infected cell.
  • a region is preferably a non-translated region or a region of 0RF4, more preferably a 395th restriction enzyme site of SEQ ID NO: 2 or a 4,541 to 4,922th region.
  • the method for producing a recombinant DNA vector containing the novel DNA vector of the present invention is not particularly limited.
  • a recombinant plasmid is prepared by incorporating the DNA vector of the present invention into a plasmid, and incorporating a foreign gene into an insertable site in the DNA vector sequence according to a conventional method.
  • This recombinant plasmid is introduced into an appropriate cell such as E. coli, and the recombinant plasmid is propagated and purified.
  • a recombinant DNA vector containing a foreign gene from the recombinant plasmid with an appropriate restriction enzyme, for example, 1 is used.
  • an appropriate restriction enzyme for example, 1
  • the method for introducing the recombinant DNA vector and the box virus into cells is not particularly limited.
  • Escherichia coli is transformed and grown in a recombinant vector in which the recombinant DNA vector obtained as described above is incorporated into an appropriate vector, and the recombinant vector is prepared in a large amount.
  • the recombinant vector may be directly or cut out from the recombinant vector by a conventional method into a cell previously infected with a box virus by an electroporation method, a lipofection method, etc., and introduced.
  • the method of infecting the cell with the box virus is not particularly limited, and may be performed by a known method such as contacting the virus with the cell at a desired m.o.i.
  • a recombinant DNA vector containing an antigen gene as a foreign gene is introduced into cells infected with a live box virus strain of a box virus, and transferred from the cell. Produced by repeating purification.
  • the antigen gene to be inserted is not particularly limited as described in (2) above.
  • the box virus to be used is not particularly limited as long as it is a strain recognized as a live vaccine strain.
  • a fowlpox live vaccine strain of avibox virus which is still widely used, is a preferred example.
  • the selection method at the time of subculture / purification is not particularly limited.However, whether or not the foreign gene shows a posi signal by the hybridization method using the foreign gene inserted into the recombinant DNA vector as a probe, or whether the foreign gene It is preferable to use an antibody against the encoded antigen to confirm whether the antigen is expressed. Then, let the box virus plaques form and recombine in all the plaques It is more preferable to repeat the purification work of subculturing and selecting the selected plaques until the presence of the foreign gene inserted into the DNA vector or the expression of the antigen can be confirmed.
  • the vaccine solution of the present invention can be obtained by using the preparation solution containing the recombinant DNA vector and the box virus obtained as described above in the same manner as in a method for preparing a normal box virus live vaccine.
  • This preparation method is a general method and is not particularly limited.
  • cells capable of proliferating the recombinant DNA vector of the present invention are simultaneously infected with a box virus, and the cells are cultured.
  • the cells are collected, disrupted, and then centrifuged to obtain a high-titer recombinant DNA vector.
  • a centrifugal supernatant containing box virus and a precipitate containing box virus and a precipitate.
  • This centrifuged supernatant which is essentially free of host cells and no or debris thereof, but contains the cell culture medium, the recombinant DNA vector, and the box virus, can be used as the vaccine of the present invention.
  • an attenuated box virus may be appropriately added to a preparation containing the recombinant DNA vector and box virus prepared as described above, and used as a vaccine.
  • the attenuated box virus is not particularly limited, but an avibox virus, an orthobox virus and the like can be preferably used.
  • Avipoxvirus include fowlpox virus: ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nisshi gahara strain, Sashimi strain And viruses such as the CEVA strain which is commercially available as a fowlpox vaccine strain.
  • vaccinia viruses examples include Lister strain LC16mO strain, WR strain, and New York 'Board' ob 'health strain. These viruses can be attenuated in a variety of ways as needed, but the use of foul-box virus (FPV) is particularly preferred for increasing vaccine efficacy.
  • FMV foul-box virus
  • the supernatant can be stored as it is, or, if necessary, diluted or concentrated and frozen or lyophilized. When freeze-dried, it may be reconstituted with pharmacologically acceptable saline or distilled water before use.
  • the vaccine of the present invention can be prepared by any method as long as the above-described recombinant DNA vector is expressed in an animal body and biosynthesizes an antigen protein encoded by a foreign gene. It may be administered by such a method.
  • a vaccination can be made by scratching the skin and inoculating the target animal subcutaneously with a needle or other device. It is also possible to suspend the vaccine in the drinking water of the animal to be vaccinated or to orally administer the vaccine by mixing it with the solids of the feed.
  • vaccines can be inhaled by aerosol or spray, intravenous inoculation, intramuscular inoculation, intraperitoneal inoculation, and the like.
  • the amount of box virus is usually 10 3 to 10 6 pfu per bird (plaque forming unit) It is desirable that the amount of the recombinant DNA vector be 10 ng to 1 tg.
  • I Li is preferably a Li 10 4 pfu per 1 birds.
  • dilute with physiologically acceptable liquid such as saline and inoculate about 0.1 mL per bird.
  • the vaccine of the present invention can be stored and used under the same conditions as ordinary poxvirus live vaccines. For example, if the vaccine of the present invention is lyophilized, it can be stored, handled, and transported at room temperature (about 20 to 22 ° C.) for a long time. In addition, the vaccine of the present invention can be stored by freezing the suspension at ⁇ 20 ° C. to ⁇ 70 ° C. Example
  • fowlpox virus Vial One strain of fowlpox virus (Cul tures for Animal Hygiene) managed by the Japan Association of Veterinary Biologies (Cultures for Animal Hygiene); fowlpox A virus strain (strain number VA0101)) was transformed into chicken embryo fibroblasts cultured to subconfluence in a 1.5 cm diameter culture dish. 1 infected. After infection, and cultured for 1 week in 37 ° C in C0 2 incubator primary, disk Le - Remove the cells from the dish par, i collect peel cells, 000 x g, and centrifuged at 4 ° C 5 minutes.
  • PBS physiological equilibration solution
  • lysis buffer 1.25% Triton-X100, 250 mM 2-mercapto 0.8 mL of a PBS solution containing ethanol and 50 mM EDTA was added. After stirring with a vortex mixer, the mixture was left at room temperature for 5 minutes and centrifuged at 2,000 Xg for 5 minutes to remove cell debris.
  • the supernatant was transferred to two Eppendorf tubes and centrifuged at 10,000 X g for 20 minutes at 4 ° C to collect a precipitate containing fowlpox virus.
  • the separated aqueous layer was transferred to another Eppendorf tube by centrifugation at 20,000 Xg at 4 ° C for 2 minutes. To this, 16 L of 5M NaCl was added, and 1 mL of 100% ethanol cooled at -20 ° C was further added, and the mixture was left at -20 ° C for 30 minutes. Subsequently, DNA was precipitated by centrifugation at 4 ° C. and 20.000 ⁇ g for 10 minutes. The precipitate was washed with 70% ethanol and dried.
  • This DNA was dissolved in an appropriate amount of a 10 mM Tris-HCl (pH 8.0) solution containing ImM EDTA (hereinafter referred to as a TE solution) and used.
  • a 10 mM Tris-HCl (pH 8.0) solution containing ImM EDTA hereinafter referred to as a TE solution
  • the above DNA was dissolved in a TE solution, and 0.6 g of the DNA was subjected to electrophoresis using a 0.8% agarose gel as a carrier, and electrophoresed at 100 V for 2 hours.
  • the intensity of UV absorption (260 ⁇ ) of this 5 kbp DNA band on agarose gel was about half that of fowlpox virus genome DN ⁇ (about 300 kb). Therefore, despite the size of the genome being about 60: l (300kbp: 5kbp), the fact that the intensity of UV absorption is about half means that the number of copies of this 5kb DNA per cell is large. The copy number was estimated to be about 30 times that of fowlpox virus.
  • the number of copies of this 5 kb DNA was 10,000 or more per cell, calculated from the amount of L //; 2din-cleaved DNA, the amount of sample DNA, and the number of cells from which the sample was prepared. Met.
  • the plasmid vector used for subcloning was a plasmid pUCXG obtained by introducing one site into the 5 5 restriction site (hereinafter, the restriction site is called a site) of commercially available pUC18 plasmid using synthetic DNA. Using.
  • the DNA solution (DNA concentration: 10 to 100 g / mL) prepared by the procedure of Example 1 was cleaved with 1 to 10 units / mL of restriction enzymes £ coRI, £ gJli, and J3 ⁇ 4al alone or in combination of two types.
  • the double digestion of Bg I and? Al resulted in three fragments of 1.0 kb, 2.4 kb and 1.7 kb.
  • Example 2 To subclone the region containing the ends, the 5 kb DNA (10 g) found in Example 1 was treated with DNA polymerase I (10 units / mL), cut with £ coRI, and cut into 1.3 kb DNA. A fragment and a 2.0 kb fragment were obtained. These two fragments, respectively Subcloning into pUC18XG plasmid double-cut with al and £ coRI yielded a plasmid into which the target DNA fragment had been inserted. These clones were named No. 20 and No. 22 (see Fig. 2).
  • the .22 clone was cloned into 7 /// 2 (0.9410 that appeared by cutting at 1111), 0.5 kb, and 0.5 kb fragments from clones No. 73-1, No. 73-2, and No. 73-3, respectively. These were subcloned into PUC18 cut with.
  • the 1.5 kb fragment that appeared when the No. 22 clone was double-cut with 1 and 2 was named No. 73-9, and was subcloned into PUC18XG double-cut with 3 ⁇ 4al and ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 4.
  • nucleotide sequence of each subclone prepared in Example 2 was decoded using a nucleotide sequence analysis kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit) and an ABI fully automatic sequencer.
  • Plasmid containing each subclone was used as a template, and P7 and P8 oligonucleotides commercially available from Toyobo Co., Ltd. were used as primers.
  • the base sequence was similarly decoded by using a synthetic nucleotide created based on the sequence at the read position as a new primer.
  • the sequences of these primers are shown in SEQ ID NOs: 6 and 7.
  • the DNA had the following characteristics.
  • the total length is 5,243 bp and the AT content is as high as 63.5 ° / 0 .
  • a 217 bp inverted terminal repeat sequence is present at both ends, and the 30 bp repeated sequence represented by SEQ ID NO: 1 in these ITRs is repeated three times in tandem with no single base space.
  • a consensus sequence TAAA T of the late transcription promoter of vaccinia virus exists upstream of the translation initiation codon of ⁇ RF.
  • pPADOl plasmid containing the 5,243 bp full-length DNA was obtained by the procedure shown in FIG.
  • the plasmid was constructed and named pPADOl. That is, No. 72-2 and No. 22 were double-cut with ⁇ 3 ⁇ 4al and a / dll respectively to recover DNA fragments of 5.lkb and 1.8kb, ligated, and ligated. 72-22 was built. .1 kb fragment obtained by cutting 20 with ⁇ 1 1 and 6.9 kb fragment obtained by cutting No. 72-22 with 711 and BAP (Bacterial Alkaline Phosphatase; manufactured by Takara Shuzo) The fragments were ligated to construct pPADOl.
  • E. coli TG1 was transformed with PPDA01 according to a conventional method.
  • Escherichia coli transformed with this pPADOl can be selected for its resistance to ampicillin, and can be easily prepared from Escherichia coli in the same manner as a commonly used plasmid such as PUC18. Furthermore, a linear DNA fragment of 5,243 bp can be cut out only by cutting pPADOl with the restriction enzyme ay / Ml.
  • Escherichia coli transformed with pPADOl was cultured in LB medium containing ampicillin for 16 hours at 37 ° C for 16 hours, and pPADOl plasmid was prepared therefrom according to a conventional method. After cutting 5 g of this pPADOl brassmid with ⁇ , self-fry with T4 DNA ligase. A gated DNA was prepared in advance.
  • the DNA vector of the present invention was not detected in CEF cells infected with the CEVA strain (approved by the US Department of Agriculture), one of the live fowlpox vaccine strains.
  • the DNA fragmented from pPADOl and self-ligated was introduced by electroporation.
  • MEM medium containing serum (5% CS) to stop trypsin digestion, and remove 50 mL of cells. Collected in a falcon tube. '1, 000 X g at centrifuged for 5 minutes, then discarding the centrifugal supernatant, Saline G buffer (8.0g NaCl, 0.4g KC1, 0.395g Na 2 HP0 4 ⁇ 12 ⁇ 2 0, 0.2g KH 2 P0 4, 0. lg MgCl 2 '6H 2 0, 0. lg CaCl 2 -6H 2 0, 1, the cells were washed with lg of glucose / L).
  • the cells were suspended once again in 0.8 mL of Saline G, and 0.7 mL of the suspension was transferred to an election port cuvette (manufactured by Bio-Rad). To this, a previously prepared self-ligated DNA was added, and electrophoresis was performed under the conditions of 1.2 kV and 25 zF. Thereafter, the cells were allowed to stand at room temperature for 10 minutes, transferred to a 25 cm 2 flask, added with 5 mL of MEM medium, and cultured in a CO 2 incubator at 37 ° C. for 6 days.
  • the culture was frozen and thawed with the flask. After three freeze-thaw cycles, the culture was transferred to a Falcon tube and centrifuged at 5,000 Xg for 5 minutes at 4 ° C to remove the cell debris and collect the supernatant. This solution contains the fowlpox virus CEVA strain and the replicated DNA vector.
  • pPADO1 was double digested with the restriction enzymes £ co811 and Spel and blunt-ended with DNA polymerase I (10 units / mL) in reaction buffer. Thereafter, Escherichia coli TG1 was transformed by cyclization with T4 DNA ligase, and the end of the 7.5 kb plasmid fragment was cultured in an LB medium containing ampicillin.
  • This plasmid is a plasmid containing a deletion of the DNA region from SEQ ID NO: 2 to positions 4,541 to 4,922, and was named pPADdl (4541-4922).
  • pPADO 1 was double-cleaved with the restriction enzymes fcoS 11 and Spel, and a 7.5 kb plasmid fragment was recovered.
  • the DNA sequence represented by SEQ ID NO: 3 was paired with a DNA fragment (SEQ ID NO: 4) forming a pair.
  • SEQ ID NO: 4 was synthesized using T4DNA ligase and transformed into Escherichia coli.
  • a plasmid was constructed in which the DNA region at positions 4,54 1 to 4,922 of SEQ ID NO: 2 of the obtained transformant was replaced with a synthetic adapter-1.
  • This synthetic adapter has the base sequences of A3 ⁇ 4el and Bln Sal Xhod sites, which facilitate the introduction of foreign genes into these locations.
  • This plasmid was named pP ADrepL (4541-4922).
  • This transformant was treated with 0.03% of 5-bromo-4-cucto-3-indolyl- ⁇ -D-galactopyranoside, 0.03 mM of isopropyl- / 3-D-galactopyranoside, 40 g / mL.
  • the cells were cultured on LB agar medium containing ampicillin for 15 hours at 37 ° C.
  • white colonies were picked and cultured in LB liquid medium containing 40 g / mL ampicillin for 15 hours at 37 ° C.
  • Transformants were recovered, plasmid was extracted according to the method of Birnboim and Dolly (Nucleic Acid Research, 7: 1513-, (1979)), and double digested with oRI and // ⁇ .
  • the ⁇ -galactosidase gene (about 3.3 Kbp) was recovered from this double digested fragment by 0.6% agarose electrophoresis.
  • 0.3 pUC19 was digested with ⁇ , extracted with phenol-chloroform (1: 1), and recovered by ethanol precipitation.
  • the recovered PUC19 and the / 3-galactosidase gene were ligated with ligase to prepare recombinant plasmid PNZ66.
  • the pNZ76 obtained as in (3-1) above was cut with ⁇ /; 7 II and Approximately 3kbp DNA fragment, which is linked to the 7.5K promoter gene of E. coli virus and the gene encoding / 3 galactosidase of E. coli immediately after, was excised and subjected to 0.8% agarose gel electrophoresis (100V, 40 minutes). Recovered from agarose gel. In addition, a 3.3 kb DNA fragment that appeared by cutting pPADOl with //// ⁇ I and a 4.5 kb DNA fragment that appeared by cutting pPADrepL (454-4922) with ZI were also recovered.
  • the three recovered fragments were ligated with a T4 DNA ligase to transform E. coli TG1. From the ampicillin-resistant transformants that emerged by the transformation, a DNA fragment of about 3 kb containing the lacZ gene was inserted into the DNA region at positions 4,541 to 4,759 in SEQ ID NO: 2. Those having a plasmid of 10.8 kb were selected.
  • This plasmid was named pPADin (4544544759) P7.5: lacZ.
  • nucleotide sequence of 4,541 to 4,922 in SEQ ID NO: 32 was changed in exactly the same manner except that pPADrepL (4541-4922) constructed in (2) of Example 5 was used instead of pPADOl in Example 4. It was examined whether the recombinant DNA vector substituted with the bp multicloning site could not be introduced into cells.
  • Vaccinia virus 7.5K promoter is located in the nucleotide sequence region of 4,541 to 4,759 of SEQ ID NO: 2.
  • pPADin (4541-4759) P7.5 Escherichia coli TG1 transformed with lacZ was cultured in MEM at 37 ° C for 40 mL, and about 50 g of plasmid pPADin (4541-4759) P7.5: lacZ was obtained from the cells. It was prepared.
  • CEF cells (1 ⁇ 10 7 cells) were collected by trypsinization, and suspended in 0.7 mL of Saline G as shown in Example 4.
  • the culture solution was transferred to a 15 mL Falcon tube, and a preparation solution containing FPV from which cell debris had been removed and the recombinant DNA vector was collected by centrifugation at 5,000 X for 5 minutes.
  • This solution was serially diluted four times in 10-fold MEM medium, and 1 mL of the solution was added to another CEF cell cultured at 37 ° C in a 9 cm diameter dish so that it became subconfluent. After discarding the supernatant later, a MEM medium containing 0.8% agar was overlaid and cultured for about 6 days.
  • Buruogaru Halogenated indolyl- ⁇ -D-galactoside; GIBC0 BRL trade name and Bluo-ga further overlayed with 0.8% agar medium supplemented with, the 37 ° C C0 2 ink Yube - in ter After 3 hours, a number of blue plaques were found in the dish after three hours, indicating that -galactosidase was expressed.
  • Example 6 The blue plaque obtained at the end of Example 6 was extracted with a pasteur pit, Suspend in 1 mL of MEM medium, serially dilute 10- to 1,000-fold, and add ME to a 9 cm-diameter dish.
  • the cells were added to CEF cells (1 ⁇ 10 7 ) cultured in M medium, and after 1 hour, the supernatant was discarded. Thereafter, 0.8% agar medium was overlaid and cultured at 37 ° C for about 6 days.
  • This suspension was added to CEF cells (1 ⁇ 10 6 ) cultured in MEM medium in a 1.5 cm dish. One hour later, the supernatant was discarded, and 20 mL of fresh MEM medium was added, followed by culturing for 1 week .
  • Example 4 a preparation containing the fowlpox virus and the recombinant new DNA vector was recovered from the cultured cells by freeze-thawing. Then, the amount of fowlpox virus contained in this solution was measured.
  • infected cells were collected from one dish, washed with PBS, and suspended in 0.5 mL of PBS. Later, the sample was frozen at -20 ° C.
  • This reaction solution was incubated at 28 ° C and dissolved in an enzyme reaction solution to a concentration of 4 mg / mL.
  • Substrate 0NPG (0-nitrophenyl / 3-D-galactopyranoside (Sigma; product number N1 127)) the enzymatic reaction was initiated by adding 0.2 mL, stop the enzymatic reaction in 1M K 2 C0 3 at appropriate time, the absorbance was measured at 420 nm.
  • fNZ1029 is disclosed in Japanese Unexamined Patent Publication No. 168279 (US This is a recombinant fowlpox virus into which the / 3-galactosidase gene is inserted as disclosed in National Patent No. 5,387,519 (Fig. 6).
  • 3-galactosidase (product number G6512) commercially available from Sigma was used as an enzyme standard.
  • Nishigahara chicken pox virus strain (strain number VA0104) was obtained from the Cultures for Animal Hygiene strain managed by the Japanese Association of Veterinary Biologics. did.
  • a 5 kb DNA was again obtained from infected cells by the same operation using this strain in place of the Sashimi strain of Example 1. This DNA was hybridized by Southern hybridization using a new DNA vector obtained from Sashimi strain as a probe. I did
  • PNZ76 was digested with 5aH and electrophoresed on a 0.8% agarose gel to recover an approximately 2.9 kb fragment that did not contain the / 3-galactosidase gene.
  • hybrid phage mplO-HN180 was double-digested with ⁇ 711 and electrophoresed on a 0.8% agarose gel to recover an approximately 1.8 kb NDV HN gene fragment.
  • pPADOl was cleaved with ⁇ sfill, the cleaved end was blunted with SI nuclease, then the end was dephosphorylated with BAP, and phenol-treated. This treated pADOl was ligated with the previously recovered fragment of about 1.9 kb to transform E. coli TG1.
  • plasmid having a total length of about 9.8 kb, in which a DNA fragment of about 1.9 kb containing the HN gene was inserted at position 377 of SEQ ID NO: 2 was selected. It was named pPADin (377) P7.5: HN.
  • the plasmid pPAD in (377) P7.5 prepared in Example 9 was cultured at 37 ° C in 40 mL of an LB medium containing ampicillin at 37 ° C, and pPADin (377) P7.5: HN Was prepared. After cutting 5 ⁇ g of the plasmid with aI, DNA self-ligated with T4 DNA ligase was prepared in advance.
  • the self-ligated DNA was dissolved in 50 L of HBS buffer (2.0 mM HEPES, 150 mM NaCl (pH 7.4)) after ethanol precipitation.
  • solution A a solution obtained by diluting 10 L of the reagent DOSPER solution with 40 L of HBS buffer was used as solution B.
  • solution A and solution B were mixed gently and allowed to stand at room temperature for 15 minutes. 100 L of this mixture was slightly dropped onto infected cells immersed in 0.5 mL of MEM medium culture solution. After 1 hour at 37 ° C, add 1.5 mL of culture solution and Cultured for days.
  • the infected cells were collected together with the culture solution, and freeze-thawing was repeated three times, and then the supernatant was recovered by removing the cell debris by centrifugation at 5,000 xg for 5 minutes.
  • This solution contains the fowlpox virus CEVA strain and the recombinant DNA vector.
  • This solution was diluted 6 times 10 times, each concentration in the liquid lml were infected with CEF cells cultured in a 9 cm diameter dishes and overlaid with MEM medium lOmL C0 2 Inkyube containing 0.8% agar - C. in ter The cells were cultured at 37 ° C. About 6 days later, when plaques due to fowlpox virus were clearly visible, 24 plaque viruses were removed using pasteur pipets, and the viruses were infected with fresh CEF cells cultured on 24-well plates. I let it.
  • the baked membrane was immersed in a pre-hybridization washing solution (5X SSC, 0.5% SDS, lmM EDTA) and washed at 42 ° C for 15 minutes.
  • a pre-hybridization washing solution (5X SSC, 0.5% SDS, lmM EDTA)
  • hybrida I See Chillon was allowed 1 hour (6X SSC, 0.5% SD S , 3% Sukimumiru h) and transferred to 68 ° C during the subsequent, HN gene radioisotopes monument - in 32 P -dCTP The solution was transferred to a hybridization solution to which a labeled DNA probe had been added, and left at 68 ° C. for 3 hours.
  • the membrane was washed twice with 2 ⁇ SSC solution, once with 1 ⁇ SSC solution, once with 0.2 ⁇ SSC solution, air-dried, and subjected to autoradiography using X-ray film.
  • the MEM medium containing 10 mL was overlaid and cultured at 37 ° C. in a 5% CO 2 incubator.
  • plaques Once plaques have formed, remove the virus from the 24 blacks with Pasteur pits as before using Pasteur pits and remove the CEF cells from the 24-well plate. The vesicles were each infected. The same operation as above was repeated until all the spots of all the wells reacted with the DNA probe.
  • a vaccine solution was prepared in the same manner as in the preparation of a fowlpox vaccine by culturing it in CEF cells.
  • the vaccine solution thus prepared contained about 20 times as many recombinant DNA vectors as fowlpox virus vaccine strain DNA.
  • Vaccinated solution prepared in Example 10 was diluted at a titer at 10 6 pfu appropriately by Uni becomes / ml PB S fowlpox virus, to the right wing membrane SPF chicken O.OlmL seven day old did.
  • ND-HI antibody Hemagglutination-inhibiting antibody for Newcastle disease
  • test serum was serially diluted 2-fold to 25; L, and 4 hemagglutinating units of NDV antigen (25 zL) were added thereto, mixed and allowed to stand for 10 minutes to sensitize. Thereafter, 50 L of a 0.5% chicken erythrocyte solution was added, and the mixture was allowed to stand at room temperature for 45 minutes, and red blood cell aggregation was observed.
  • the ND-HI antibody titer was represented by the highest dilution of serum in which hemagglutination inhibition was observed.
  • a novel DNA vector having a copy number several tens times that of a viral vector in animal cells, and a method for highly expressing a foreign gene using this vector are obtained. It can be used as a vector for use or as a vaccine.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA

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Abstract

L'invention concerne un vecteur d'ADN d'environ 5Kb provenant du virus de la variole aviaire, des vecteurs d'ADN recombinant renfermant ce vecteur d'ADN et au moins un gène étranger soumis à la régulation d'un gène régulateur, et des vaccins renfermant ces vecteurs comme principe actif. Le vecteur d'ADN d'environ 5 Kb précité provenant du virus de la variole aviaire est un vecteur concomitant qui ne se développe pas dans les cellules infectées par un autre virus que le virus Pox, mais uniquement dans les cellules infectées par le virus Pox. Etant donné qu'il possède un grand nombre de répliques par cellule, ce vecteur d'ADN permet d'obtenir des vaccins présentant des effets préventifs excellents.
PCT/JP1998/001358 1997-03-28 1998-03-26 Nouveau vecteur d'adn et vaccins renfermant ces nouveaux vecteurs d'adn recombinant comme principe actif Ceased WO1998044093A1 (fr)

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AU65182/98A AU6518298A (en) 1997-03-28 1998-03-26 Novel dna vector and vaccines containing novel recombinant dna vectors as the active ingredient
JP54142298A JP3924328B2 (ja) 1997-03-28 1998-03-26 新規ｄｎａベクター、及び組み換え新規ｄｎａベクターを有効成分とするワクチン

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JP9/94875 1997-03-28
JP9487597 1997-03-28

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WO1998044093A1 true WO1998044093A1 (fr) 1998-10-08

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PCT/JP1998/001358 Ceased WO1998044093A1 (fr) 1997-03-28 1998-03-26 Nouveau vecteur d'adn et vaccins renfermant ces nouveaux vecteurs d'adn recombinant comme principe actif

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JP (1) JP3924328B2 (fr)
AU (1) AU6518298A (fr)
WO (1) WO1998044093A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05192165A (ja) * 1991-08-26 1993-08-03 Immuno Ag 組換え鶏痘ウイルス
WO1996040880A1 (fr) * 1995-06-07 1996-12-19 Syntro Corporation Virus de variole aviaire recombines et leurs utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05192165A (ja) * 1991-08-26 1993-08-03 Immuno Ag 組換え鶏痘ウイルス
WO1996040880A1 (fr) * 1995-06-07 1996-12-19 Syntro Corporation Virus de variole aviaire recombines et leurs utilisations

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AU6518298A (en) 1998-10-22

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