WO1998008936A1 - Recombinant canine herpesvirus - Google Patents
Recombinant canine herpesvirus Download PDFInfo
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- WO1998008936A1 WO1998008936A1 PCT/JP1997/000236 JP9700236W WO9808936A1 WO 1998008936 A1 WO1998008936 A1 WO 1998008936A1 JP 9700236 W JP9700236 W JP 9700236W WO 9808936 A1 WO9808936 A1 WO 9808936A1
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- 0 CCOC(CC1)(C1C12)C1(**)C2C(CC1)CC(*)C1*1CC1 Chemical compound CCOC(CC1)(C1C12)C1(**)C2C(CC1)CC(*)C1*1CC1 0.000 description 1
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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
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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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16741—Use of virus, viral particle or viral elements as a vector
- C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to the use of canine virus (CHV) as a vector virus. More specifically, a foreign nucleic acid sequence is located downstream of various promoters (including the promoter of CHV itself) that operate in eukaryotic cells. Alternatively, a method of producing a recombinant CHV that expresses a product derived from the inserted foreign nucleic acid sequence in infected cells by inserting one or more expression units in which a part thereof is linked into the CHV genome. The present invention relates to a recombinant CHV produced by this method and a method for inoculating an animal with the recombinant CHV to obtain the effect of the inserted foreign nucleic acid sequence.
- CHV canine virus
- Virology In the early days of virology, much research was done on its pathogenicity and the foundation of the current virology has been laid. Virology, coupled with research into pathogenicity elucidation, aims at pectinization of the virus, requires a long time of long-term passage using animals or tissue culture cells, is not so reliable, and is attenuated by methods. Have been detoxified. Later, when genetic engineering technology was introduced into virology, it became possible to elucidate the pathogenesis or vaccination of viruses at the molecular level, and vaccines using this technology are being actively developed.
- Recombinant viruses can be used, for example, by constructing a foreign nucleic acid sequence so as to express it in a cell, incorporating it into a vector virus, and infecting the recombinant virus in a test tube or a cell of an animal body.
- the effects of inoculation into an animal body include, for example, a vaccine effect when a gene encoding a protective antigen of various pathogenic microorganisms is introduced as a foreign nucleic acid sequence, and a gene coding for a bioactive substance.
- the therapeutic effect when introduced as a nucleic acid sequence is considered.
- the method of virus vectorization in other words, the method of producing a recombinant virus, it is possible to produce various types of recombinant viruses according to the purpose using the same method. Moreover, despite the wide range of effects that can be achieved, the method of producing recombinant virus that plays a role in many cases is similar to the method of producing wild-type virus, and many objectives can be achieved with a single production method. It is expected to contribute to the recovery, maintenance and promotion of the health of the target animals.
- viruses Although some viruses are being vectorized, the only known virus for canines is the adenovirus.
- Viruses belonging to the family Herpesviridae such as simple herpesvirus 1 (Malik et al., 1992, Virology, 190: 702-715), Aujeszky's disease virus (Thomsen et al., 1987, Gene, 57: 261-265), pertussis virus type 1 (Kit et al., 1992, Arch. Virol., 124: 1-20), cat herpes virus type 1 (Cole et al., 1990, J. Virol) , 64: 4930-4938) has already been developed as a vector virus for expressing foreign nucleic acid sequences in animals.
- the foreign nucleic acid sequence of interest is located in a non-essential gene region identified on the genome of the herpes virus, ie, a gene region that is not essential for the propagation of the virus.
- Gen one of the inventors of the present invention, conducted intensive studies and found that the nucleotide sequence of the thymidine kinase gene, which is considered to be one of the candidates for the foreign nucleic acid sequence insertion site in C CV Revealed. The structure of this region is being analyzed by Remond et al. (1995, Virus Res., 39: 341-354). Furthermore, Limbach et al. (1994, J. Gen.
- Virol., 75: 2029-2039 have determined the nucleotide sequences of the gB, gC, and gD genes, which are CHV structural proteins, and have described Remond et al. Gen. Virol., 77: 37-48) has been energetically determining the nucleotide sequence of a region called UL in the C ⁇ V genome.
- this CHV is used as a vector virus. There is currently no knowledge of whether it can be used.
- the inventors of the present invention aimed at developing a virus vector that is versatile for canines, aiming at vectorization of canine herpes virus (CHV), and completing the present invention. Reached.
- CHV canine herpes virus
- a first object of the present invention is to show that there are a plurality of sites for inserting a foreign nucleic acid sequence on a CHV genomic gene, that is, that CHV can be sufficiently used as a vector virus, It also aims to provide some ways to determine this position.
- the second purpose is to establish some methods for introducing a foreign nucleic acid sequence of interest into a determined site on the CHV genome, and to provide a method for producing this recombinant virus.
- a third object is to provide a method of constructing a foreign nucleic acid sequence to be inserted into a CHV genome for expressing a foreign nucleic acid sequence by infecting cells in a test tube or in vivo with recombinant CHV.
- the fourth object is to show that when the recombinant CHV thus produced is inoculated into an animal, the intended effect can be sufficiently enhanced, and the object of the present invention is to guarantee the practical application of the present invention.
- Another object of the present invention is to provide a recombinant CHV produced as described above. Disclosure of the invention
- the present invention provides, in order to achieve the above-mentioned object, a recombinant canine virus in which a nucleic acid sequence not present on the canine virus genome (hereinafter referred to as "foreign nucleic acid sequence") is inserted into its genome. is there.
- the site for inserting the foreign nucleic acid sequence is desirably a region that does not have a lethal effect on virus growth, but is not limited to this.
- the foreign nucleic acid sequence can be introduced into the CHV genome using, for example, a homologous recombination technique. Therefore, in the present invention, as a first step, an arbitrary DNA fragment of the CHV genome is cloned into plasmid DNA, and the transfer described later is performed. Used as vector.
- the genetic techniques used in cloning and subsequent steps have been compiled by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).
- a mutation is introduced at any position of the cloned DNA fragment. In this case, if a mutation is introduced in a manner that impairs the function of the gene located at that position, it can be determined whether or not the gene is a non-essential gene.
- the plasmid DNA into which this mutation has been introduced is transfected into CHV host cells, cultured for a certain period of time, and then infected with a wild-type virus.
- the cells are expected to produce CHV.
- the CHV genome DNA is transformed into a plasmid that has been mutated during its propagation. New generation of a recombinant virus genome by homologous recombination with DNA and DNA particles having the recombinant virus genome can be expected.
- the mutation-introduced site on the CHV genome can be used to identify the foreign nucleic acid sequence. It can be determined whether the site is an insertable site. By this method, there may be a plurality of sites where foreign nucleic acid sequence can be inserted.
- a similar technique can be applied or improved to achieve the desired foreign nucleic acid sequence. It is possible to construct a recombinant CHV that is constructed with a structure that expresses and that inserts it. The number of foreign nucleic acid sequences that can be inserted into the CHV genome does not need to be one, and a recombinant CHV in which a plurality of foreign nucleic acids are inserted can be produced depending on the purpose.
- CHV is pathogenic in puppies up to 2 weeks of age, but rarely in later dogs.
- Use of CHV with vector as a vector virus will effectively contribute to the recovery, maintenance and promotion of the health of dogs and other animals susceptible to recombinant CHV Can be given.
- FIG. 1 shows the cloning of a DNA fragment containing CHV and the thymidine kinase gene.
- the number following the name of the restriction enzyme indicates the position of cleavage by the enzyme in kb units, and the same applies to the following figures.
- FIG. 2 is a diagram showing cloning of a DNA fragment containing CHV, gC, and ORF2 genes.
- FIG. 3 shows the construction of a transfer vector derived from the thymidine kinase gene region.
- FIG. 4 is a diagram showing the construction of a transfer vector in which a beta-ga1 expression unit has been incorporated into the thymidine kinase gene transfer region.
- Figure 5 shows the construction of the rabies virus G protein expression unit.
- FIG. 6 shows the construction of a transfer vector in which a rabies virus G protein expression unit has been incorporated into the thymidine kinase gene region.
- FIG. 7 is a view showing the construction of a transfer vector in which a beta-ga1 expression unit and a madness virus G protein expression unit are incorporated into the thymidine kinase gene region.
- FIG. 8 is a diagram showing the closing of CDV, F protein gene and H protein gene.
- Figure 9 is a continuation of Figure 8.
- FIG. 10 is a diagram showing the construction of a transfer vector in which a beta-ga1 expression unit and CDV and F protein expression units have been incorporated into the thymidine kinase gene region.
- FIG. 11 shows the construction of a transfer vector in which a beta-a1 expression unit and CDV and H protein expression units have been incorporated into the thymidine kinase gene region.
- FIG. 12 shows that the thymidine kinase gene region has a beta-ga 1 expression unit and
- FIG. 2 is a diagram showing the construction of a transfer vector into which an expression unit of the E. coli disease virus thymidine kinase is incorporated.
- FIG. 13 is a diagram showing the construction of a transfer vector in which a CDV / F protein expression unit has been incorporated into the thymidine kinase gene region.
- FIG. 14 is a diagram showing the construction of a transfer vector in which a CDV / H protein expression unit has been incorporated into the thymidine kinase gene region.
- FIG. 15 shows construction of a transfer vector in which a beta—ga1 expression unit was incorporated into the 0 R F2 region.
- the basic techniques used are genetic engineering techniques and virology techniques.
- the former technology was described by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York), and the latter technology was developed by the National Institute of Health, National Institute of Health (1973). , Revised second edition, Maruzen Co., Ltd.).
- the first step in carrying out the present invention involves cloning the DNA fragment of the CHV genome.
- Any CHV strain can be used as the CHV used in the present invention, and examples thereof include strain D004 (ATCC VR-552) and strain F205 (ATCC VR-647). This process has already been described in Remond et al. C1995, Virus Res., 39: 341-354), Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039) or Reraond et al. (1996, J. Gen. Virol. , 77: 37-48) have been established and can be carried out according to their reported method.
- a synthetic DNA primer is arbitrarily prepared with reference to the nucleotide sequence known to date, and the desired fragment is amplified by polymerase-chain reaction (PCR) to obtain an appropriate plasmid DN. It can also be cloned on A.
- PCR polymerase-chain reaction
- Plasmid DNA which contains the DNA fragment of the CHV virus genome, can be used as a transfer vector for introducing a foreign nucleic acid sequence into the CHV genome by homologous recombination, and a source of a promoter that operates in eukaryotic cells. Also. As described above, the transfer vector is used for homologous recombination of a foreign nucleic acid sequence into a CHV gene. It is used when inserting into a nome, and the nucleic acid sequence before and after the insertion site on the CHV genome is positioned on both sides of the foreign nucleic acid sequence, and these sequences can cause homologous recombination with genomic DNA. These sequences must be of sufficient length to allow homologous recombination with the CHV genome.
- a mutation is introduced at any position of the CHV genomic DNA fragment on this transfer vector. Mutations can be introduced by methods such as deletion, insertion, or base substitution. Malik et al. (1992, Virology, 190: 702-715), Thorasen et al. (1987, Gene, 57: 261-265). ), Kit et al. (1992, Arch. Virol., 124: 1-20) or Cole et al. (1990, J. Virol., 64: 4930-4938).
- a / 3-galactosidase (beta-gal) expression unit having a promoter and a polyadenylation signal is prepared from plasmid pCHl10 (Pharmacia), and this is used to prepare the CHV genome on the transfer vector. Mutations can be introduced by inserting the fragment at any position.
- the term expression unit refers to a unit of a nucleic acid structure for autonomously expressing a foreign nucleic acid sequence in a eukaryotic cell, and specifically encodes the direction of the foreign nucleic acid sequence, for example, a polypeptide.
- the plasmid DNA into which the mutation has been introduced can be used together with the genomic DNA of CHV, for example, Malik et al. (1992, Virology, 190: 702-715), Thomsen et al. (1987, Gene, 57: 261-265), Kit et al. Virol., 124: 1-20), or according to the method of Cole et al. (1990, J.
- Virol., 64: 4930-4938 cotransfect into a host cell of CHV such as MDCK cell.
- CHV such as MDCK cell.
- cells may be transfected with the above-described plasmid into which a mutation has been introduced in advance, and then infected with CHV.
- these cells are cultured for several days, in addition to the parental virus in the culture supernatant and cells, a group that is likely to be regenerated by homologous recombination between the CHV genomic DNA and the mutated transfer vector DNA Production of a recombinant virus can be expected.
- the virus is recovered from this and virus clones are cloned by the Blackassay method.
- beta-gal expression unit 5-bromo-4--4-chloro-3-indolyl-yS-D-galactoside (by mixing or overlaying X-gal) in agar or methylcellulose medium, the appearance of recombinant virus can be easily confirmed as a blue black.
- the mutation when the mutation is introduced, it can be as high as 1 in 1,000 virus clones produced, and the use of beta-gal expression units and X-gal facilitates the appearance of recombinant CHV. You can check.
- the next step in the present invention is to insert the target nucleic acid sequence into that site.
- the target nucleic acid sequence is constructed, for example, with the structure of the expression unit described above.
- Promoters that operate in eukaryotic cells used at this time include, for example, the promoter of the immediate early gene of human cytomegalovirus, the promoter of the early gene of Simian Virus 40, or the promoter that naturally exists on the CHV genome.
- a nucleic acid sequence of interest for example, a sequence of an open reading frame (hereinafter abbreviated as “ORF”) encoding a rabies virus G protein is ligated downstream of this.
- the autonomous expression unit thus constructed is inserted into the foreign nucleic acid sequence insertion site on the transfer vector.
- a recombinant CHV in which a beta-gal expression unit has been inserted into the same foreign nucleic acid sequence insertion site in advance.
- homologous recombination is induced in cells, and then a clone of a recombinant virus can be selected by applying a method in which a foreign nucleic acid sequence insertion site has been searched.
- the recombinant CHV produced as described above is used as a host cell such as an MDCK cell (ATCC).
- the effect can be exerted by infecting CCL34).
- the inserted foreign nucleic acid sequence is a rabies virus G protein expression unit, it can be used for production of the protein using tissue culture.
- the effect of recombinant CHV can be directly obtained in animals by inoculating the recombinant virus directly into animals, for example, intranasally, subcutaneously, intradermally, intravenously, intramuscularly or orally. it can.
- inoculating an animal with a recombinant CHV into which a rabies virus G protein expression unit has been inserted has the effect of acquiring the ability to protect the recipient animal against rabies virus infection. This paves the way for vaccine production using recombinant CHV.
- CHV as a vector virus
- foreign nucleic acid sequences that may be introduced for the purpose of this use include, in addition to the above-mentioned rabies virus G protein gene, for example, the H- and F-protein genes of canine distemper virus, Inducible adenovirus type 1 or 2 hexon protein gene or fiber protein gene, parainfluenza virus F protein protein or HN protein gene, canine parvovirus VP 1 or Examples of the gene include a gene encoding a protective antigen of a pathogen to a host animal that is capable of infecting CHV, such as a dog, such as a VP2 protein gene.
- CHV when used as a vector virus, it can be used for therapeutic applications.
- a foreign nucleic acid sequence encoding a physiologically active substance such as lymphokine, interferon, or cytokine is used.
- a case where a child is introduced can be exemplified.
- MDCK cells suspended in Eagle's minimum essential medium supplemented with 5% fetal bovine serum (FBS) are seeded on a 6 cm diameter tissue culture plastic dish to form a monolayer cell sheet.
- FBS fetal bovine serum
- the CHV and YP11 strains with the University of Tokyo, Veterinary Microbiology Division II
- DFD-6 strains Neissei Laboratories
- This solution was recovered and extracted twice with an equal volume of a phenol-chloroform solution (1: 1) to recover an aqueous layer.
- a phenol-chloroform solution (1: 1)
- a filamentous DNA precipitates. Then, this is wound around a glass rod, collected, washed with a 70% ethanol solution, and dried. Dissolve in 0.1 ml of distilled water and store at 4 ° C until use. With this operation, 30 to 50 jug of DNA was recovered.
- Figure 1 shows the procedure for cloning the gene fragment. That is, the CHV genomic DNA 201 recovered in Example 1 above was treated with 50 units of the restriction enzyme Xbal overnight at 37, followed by agarose gel electrophoresis, and the agarose portion containing the 6.5 kilobase (kb) fragment was removed. The DNA was collected and collected using a Gene Clean Kit (BI0101). This was ligated to a plasmid pBluescript I1SKC +) that had been digested with Xbal and dephosphorylated with calf intestinal alkaline phosphatase (C1AP) using ligation kit (Takara Shuzo Co., Ltd.), and transformed into Escherichia coli DH5a.
- a Gene Clean Kit BI0101
- FIG. 2 shows the procedure for cloning gene fragments. Specifically, referring to the report of Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039), the following synthetic DN DN primer consisting of 20 residues:
- Example (1) was prepared, and the DNA extracted in Example (1) was used as a ⁇ type to perform a polymerase zethiane reaction (PCR).
- the reaction mixture contains 1 Ug of genomic DNA of the CHV YP11 strain in 50 ⁇ ⁇ , 2 M each of the above-mentioned synthetic DNA primer, 800 deoxynucleotides each, and lOXTaq polymerase attached to 51 enzymes.
- a 2.28 kb DNA fragment predicted from the nucleotide sequence was separated by agarose gel electrophoresis and recovered by Gene Clean Kit.
- This DNA fragment contains 0RF2, which encodes gC protein, in addition to 0RF2.
- the transformed E. coli was isolated by inoculating the LB agar medium containing 50 / g / ml of ampicillin from the transformed E. coli. A was recovered.
- the gene fragment was inserted into plasmid DNA in the direction opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples.
- the obtained 4.65 kbp plasmid was named p0RF2.
- Fig. 3 shows the procedure for constructing the transfer vector. That is, the pBS / CT baI6.5 obtained in Example (2) was digested with the restriction enzyme Xbal, the DNA ends were smoothed with the Klenow enzyme, and then the 6.50 kb fragment was purified by agarose gel electrophoresis and gene clean kit. Recovered. This was ligated with a 2.36 kb fragment of plasmid pUC19 digested with the restriction enzyme PvuII and treated with CIAP using a ligation kit, and introduced into a combined E. coli DH5 ⁇ , and 50 ⁇ g / ml of ampicillin was added.
- the transformed Escherichia coli was inoculated by inoculating the LB agar medium containing the plasmid, and recovered from Escherichia coli holding the plasmid DNA having the 6.50 kb fragment incorporated therein. Although the insertion direction of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was used in the following examples. The obtained 8.87 kb plasmid was designated as pUC (dl-P) CTKXbal6.5.
- This plasmid pUC (dl-P) CTKXbaI6.5 was digested with the restriction enzymes EcoRl and Hindlll, split into two fragments of 8.36 kb and 0.50 kb, separated by agarose gel electrophoresis, and 8.36 kbp by Gene Clean Kit. A fragment was obtained. Separately, pUC19 was digested with restriction enzymes EcoRI and Hindi II to prepare a 57 base pair (bp) fragment containing a multi-cloning site, and ligated to the above 8.36 kbp fragment using a ligation kit. This was introduced into the transformed E. coli 5 ⁇ by a transformation reaction, and the transformed E.
- coli was isolated by inoculation on an LB agar medium containing 50 g / nil of ampicillin. Retains integrated plasmid DNA This was recovered from Escherichia coli. The 8.41 kb plasmid obtained was named pCTKdlEH / MCS.
- Figure 4 shows the procedure for constructing the plasmid. That is, the plasmid pCHllO (Pharmacia) was digested with the restriction enzymes Tthllll and BamHI, treated with Kleno enzyme to blunt the ends of the DNA fragments, and the DNA fragment containing the 24-kbp beta-gal gene was digested. Recovered by agarose gel electrophoresis and Gene Clean Kit. This fragment contains the SV40 early promoter (prom) upstream of the ORF (beta-gal-ORF) encoding the beta-gal protein, and also downstream of the SV40 polyadenylation signal (poly (A)). ) Is collected as a beta-gal expression unit (Lac Z cassette).
- the ends are blunted with Klenow enzyme, and then subjected to CIAP treatment.
- the transfer vector and the 4.24 kbp beta-gal expression unit recovered above were ligated using a ligation kit, and introduced into a competent Escherichia coli DH5 cell by a transformation reaction.
- the transformed E. coli was isolated by inoculating the LB agar medium containing the E. coli, and recovered from the E. coli harboring the transfer vector DNA containing the beta-gal expression unit.
- Fig. 5 and Fig. 6 The procedure for constructing the plasmid is shown in Fig. 5 and Fig. 6. That is, the plasmid pUC-ARAG disclosed in JP-A-3-139286 was renamed pUCRaGl.25 and used in this example. As shown in Fig. 5, this was cut with the restriction enzymes EcoRI and HindII and treated with Klenow enzyme to make the DNA ends blunt. After that, the 2.0 kb rabies virus G protein gene was subjected to agarose gel electrophoresis and gene Purified by clean kit did.
- Escherichia coli MC1061 / P3 was introduced by a transformation reaction, and the transformed Escherichia coli was isolated by inoculation on LB agar medium containing 7.5 g / ml tetracycline, and the rabies virus G protein gene was integrated. This was recovered from Escherichia coli retaining plasmid DNA.
- clones in which the G protein ⁇ gene is integrated in the same direction with respect to the human cytomegalovirus immediate-early promoter (CMV-prom) on PCM8 are plasmid DNA with the restriction enzymes Hindlll and EcoRl. Was selected as a clone in which a 5.95 kb fragment appeared when cleaved.
- the plasmid pCDM8-G prepared in this manner was digested with restriction enzymes Mlul and BamHI, treated with Klenow enzyme, and the DNA ends were blunt-ended.
- the G protein expression unit (G cassette) was purified using agarose electrophoresis and a gene clean kit.
- the transfer vector-pCTKdlEH / MCS prepared in Example (4) was digested with the restriction enzyme Xbal, followed by blunt-end blunting with Klenow enzyme and CIAP treatment, and the 3.42 kb rabies virus obtained above.
- the G protein expression unit was ligated using a ligation kit, introduced into a transformed E. coli DH5a by a transformation reaction, and inoculated on an LB agar medium containing 50 g, ml of ampicillin, and transformed. And the plasmid DNA was recovered from Escherichia coli holding the plasmid DNA into which the above 3.42 kb fragment had been incorporated.
- Figure 7 shows the procedure for constructing the plasmid. That is, pCTKdlEH / Lac Z prepared in Example (5) was digested with restriction enzyme Xbal, followed by blunt-ending with Klenow enzyme and CIAP treatment.
- the rabies virus G protein expression unit of 3.42 kb with blunt-ended DNA ends prepared in Example (6) was ligated using a ligation kit, and transformed into a competent Escherichia coli DH5a.
- the transformed E. coli was isolated by inoculating the LB agar medium containing 50 ⁇ g / ml of ampicillin, and the plasmid DNA was isolated from the E. coli harboring the 3.42 kbp fragment-incorporated plasmid DNA. Was recovered.
- the direction of insertion of the gene fragment into Plasmid DNA was the same as that shown in the figure due to ligation of blunt ends.
- the obtained 16.08 kb plasmid was designated as pCTKdlEH / Lac Z / RV-G.
- Vero cells are cultured in three roller bottles to form a monolayer cell sheet, which is then infected with Inu Distemper Virus (CDV) strain HK (Nissei Laboratories) and stored at 37 ° C. Cultured for days. After the culture, the culture supernatant was collected and centrifuged at 4000 rpm for 10 minutes to remove cell debris in the culture supernatant. This solution is centrifuged at 19,000 rpm for 2 hours using a Type 19 rotor (Beckman), and the precipitate is collected and suspended in 10 mM Tris-chloride buffer (TE buffer, pH 7.4) containing 5 ml of IIDM EDTA. I let it.
- CDV Inu Distemper Virus
- the RNA concentration was determined by measuring the absorbance at 280 nm.
- cDNA was synthesized from CDV genomic RNA as follows. That is, for 8 g of RNA, 51 of 10X reverse transcription reaction buffer (0.5 M Tris-HCl buffer, pH 8.3, 0.5 M KC 1.80 mM magnesium chloride, 0.1 M dithioleitol) ⁇ Random primer 1, 11 ribonuclease inhibitor (38 units / 1, Wako Pure Chemical Industries, Ltd.), to make a total amount of 431, leave at 90 ° C for 5 minutes, quench in ice water, In addition, 1 ⁇ 1 ribonuclease inhibitor, 51 deoxynucleotide mixture (each lOmM) and ⁇ ⁇ !
- Reverse transcriptase 29 units, 1 by Seikagaku Corporation
- the mixture was reacted at C for 90 minutes to synthesize cDNA.
- the random primer was removed using Ultrafree C3TK (Millipore) to obtain the final solution volume.
- MP2 5'-CAA (or G) CCATCAGTCCCACAG (or ⁇ ) GA-3 '
- FRP2 5'-GAGATATATGACCAGAATACT-3 '
- reaction mixture was added to 30/1 lOmM magnesium chloride, 5 mM dithiothreitol, 6 mM Dissolve in 70 mM Tris-HCl buffer containing ATP, pH 7.6, Ten units of T4 polynucleotide kinase were added and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment.
- the phosphorylated DNA fragment was digested with the restriction enzyme Sraa and ligated using a ligation kit to plasmid pUC19, which was dephosphorylated with alkaline phosphatase (BAP) derived from Pseudomonas aeruginosa.
- BAP alkaline phosphatase
- coli was introduced into XU-Blue by a transformation reaction and inoculated on LB agar medium containing 50 ⁇ g / ml of ampicillin to isolate transformed Escherichia coli. Plasmid DNA incorporating the above 2.59 kb fragment was isolated. Plasmid DNA was recovered from the retained E. coli. Although the gene fragment was inserted into the plasmid DNA in the direction opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 5.27 kb plasmid was designated as pUCl9-MP2-FRP2.
- FPfnl-2 5 '-CAACACAGCCAAGCCCCATG-3'
- coli MC1061 Introduced by transformation and containing 50 g'ml of ampicillin Transformed Escherichia coli was inoculated by inoculating a natural medium, and plasmid DNA was recovered from Escherichia coli holding the plasmid DNA in which the 2.03 kb fragment was incorporated. Clones were inserted in the opposite direction to the direction shown in the figure due to the ligation of blunt ends between gene fragments into plasmid DNA.In this example, clones in the direction shown in the figure were cloned. However, it was used in the following examples. The obtained 4.71 kbp plasmid was named pUC19-FPfn2-FRP2.
- the nucleotide sequence of the cloned DNA fragment was determined using a commercially available cycle sequencing kit (Perkin-Elma Japan) and the company's sequencing kit, and the previously reported CDV and F protein gene data (EMBL) Compared to the data library (Data of Accession Number L13194), it showed a homology of 90 mm or more, indicating that the target gene could be cloned.
- the plasmid pUC19-FPfn2-FRP2 was digested with the restriction enzymes BamHI and Sacl, the DNA ends were blunt-ended using Klenow enzyme, and the 2.04 kb DNA fragment was separated by agarose electrophoresis to generate a gene clean kit. Collected by the This was ligated to the plasmid pAcYMl (Matsuura et al., 1989, Virology, 173: 674-682), which had been cleaved with the restriction enzyme Smal and dephosphorylated by BAP treatment, using a ligature kit and made competent. Transformed E. coli was introduced into E.
- HRP5 5'-ATGCTGGAGATGGTTTAATTCAATC-3 '
- the lOXPfu polymerase buffer (attached to the polymerase) for the 2 ⁇ 1 cDNA synthesized in Example (8), 0.5 ⁇ 1 of the 10 // M synthetic primer , A mixture of 51 deoxynucleotides (2 mM each), 14 ⁇ 1 of distilled water and 0.5 / I of Pfu polymerase, overlayed with liquid paraffin, 94 ° C for 90 seconds, 56 ° C 30 30 cycles of PCR were performed at 72 ° C for 5 minutes per second, and the expected 1.93 kb DNA fragment was obtained as a PCR product.
- the reaction mixture was extracted with phenol: chloroform (1: 1) to remove the primers and deoxynucleotides with Ultrafree C3TK.
- the DNA fragment was dissolved in 70 mM Tris-HCl buffer, pH 7.6, added with 10 units of T4 polynucleotide kinase, and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment.
- the phosphorylated DNA fragment was ligated using a ligation kit to plasmid pUC19, which had been digested with the restriction enzyme Smal and dephosphorylated with BAP, and transformed into a competent E. coli XL1-Blue by a transformation reaction.
- the transformed E. coli was isolated by inoculating the LB agar medium containing 50 ⁇ g / ml of ampicillin, and the E.
- the nucleotide sequence of the cloned DNA fragment was determined using a commercially available cycle sequence kit (Perkin Elmer Japan, Inc.) and the company's sequencer, and the previously reported data for the CD V and H protein genes ( Compared to the EMBL data library (Data of Accession Number L13194), it showed 90% or more homology, indicating that the target gene could be cloned. (10) Introduction of beta-gal expression unit and canine distemper virus F protein expression unit into transfer vector pCTKdlEH / MCS
- Figure 10 shows the procedure for constructing the plasmid. That is, the plasmid pAcM-F prepared in Example (8) was digested with the restriction enzyme BamHI, the DNA ends were blunted using T4 DNA polymerase, and then agarose electrophoresis and gene clean kit were used. A 06 kb F protein gene fragment was recovered. Separately, in order to remove the rabies virus G protein gene from the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7), this was cut with the restriction enzyme Xbal, and then blunt-ended with Kleno monoenzyme.
- a clone having an EcoRI site present in the ORF of the F protein at a position of 92 kb was selected by analysis of a cleavage image with the restriction enzyme EcoRI.
- the F protein ORF is integrated in the same direction with respect to the human cytomegalovirus immediate-early promoter (CMV-prom), and the expression unit (F cassette as in the case of the G protein expression unit). ).
- CMV-prom human cytomegalovirus immediate-early promoter
- the obtained 16.09 kb plasmid was designated as pCTKdlEH / Lac Z / CDV-F.
- Figure 11 shows the procedure for constructing the plasmid.
- the plasmid created in the embodiment (9) After digestion with DNA and PvuII and blunting the 5 'end of the DNA using Klenow enzyme, a 2.5 kb H protein gene fragment was recovered using agarose electrophoresis and a gene clean kit. Separately from this, the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7) was used in the same manner as in Example (10). After digestion with the restriction enzyme Xbal, the ends were blunt-ended with Klenow enzyme and treated with CIAP, and the 2.5 kb H protein gene fragment recovered above was ligated using a ligation kit to obtain a recombinant E.
- Figure 12 shows the procedure for constructing the plasmid. That is, a plasmid pPTK obtained by cloning a Pst1-Kpnl fragment of the oeschis disease virus genome containing the 2.8 kb oeschis disease virus thymidine kinase gene (PTK) was obtained from Dr. S. Kit of the United States. Biochemical Virology, Baylor College of Medicine, Houston, Texas, USA). This is digested with EcoRI and Hindlll and the DNA ends are blunted using Klenow enzyme.Then, using agarose gel electrophoresis and Gene Clean Kit, the 2.79 kb Aujeszky's disease virus thymidine kinase gene is digested.
- PTK 2.8 kb oeschis disease virus thymidine kinase gene
- This fragment has the original promoter and polyadenylation signal of the thymidine kinase gene and operates as an expression unit.
- pCTKdlEH / Lac Z prepared in Example (5) was digested with the restriction enzyme BamHI, followed by blunt-end blunting with Kleno monoenzyme and dephosphorylation by CIAP treatment.
- a 79 kb thymidine kinase gene fragment was ligated using a ligation kit, introduced into a transformed Escherichia coli ⁇ ⁇ ⁇ 5 ⁇ by a transformation reaction, and inoculated on an LB agar medium containing 50 g / ral ampicillin. Isolate the formed E.
- coli and Blasmid DNA was recovered from E. coli harboring plasmid DNA in which the protein fragment was integrated.
- the direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but in this example, the clone in the direction shown in the figure was obtained.
- the obtained 15.42 kb plasmid was designated as pCTKdlEH / Lac Z / PTK.
- Fig. 13 shows the procedure for constructing a plasmid into which the F protein expression unit was inserted
- Fig. 14 shows the procedure for constructing a plasmid into which the H protein expression unit was inserted.
- the restriction enzyme Xba1 in order to remove the rabies virus G protein gene from pCTKdlEH / RV-G created in Example (6), it was cut with the restriction enzyme Xba1, followed by blunt-end with Cleno enzyme and CIAP.
- a treated 9.79 kb fragment was prepared, and this was combined with the 2.06 kb F protein gene fragment prepared in Example (10) or the 2.05 kb H protein gene fragment prepared in Example (11).
- coli DH5a by a transformation reaction, and inoculated on an LB agar medium containing 50 g / nil of ampicillin to separate transformed E. coli.
- the plasmid DNA was recovered from Escherichia coli harboring the plasmid DNA into which the protein gene fragment was integrated. In each case, clones were inserted in the opposite direction to the direction shown in the figure due to ligation of blunt ends with the insertion direction of the gene fragment into the plasmid DNA. Clones with sites were selected by analysis of cleavage images with the restriction enzyme EcoRI.
- the 11.85kbp plasmid incorporating the F protein gene was designated pCTKdlEH / CDV-F
- the 11.84kb plasmid incorporating the H protein gene was designated pCTKdlEH / CDV-H.
- Figure 15 shows the procedure for constructing the plasmid. That is, the plasmid p0RF2 produced in Example (3) was digested with the restriction enzyme Pvull, treated with CIAP, and prepared in Example (5). Expression unit The transformant was introduced into Escherichia coli DH5a which had been ligated using a kit and transformed into a concomitant strain, and the transformed E. coli was isolated by inoculating the LB agar medium containing 50 / g / ml ampicillin. Plasmid DNA was recovered from Escherichia coli holding plasmid DNA into which the kb fragment had been incorporated.
- the direction of insertion of the gene fragment into Plasmid DNA may be opposite to the direction shown in the figure due to ligation of blunt ends, but in this example, the clones in the direction shown in the figure are cloned. And used in the following examples.
- the resulting 8,89 kb plasmid was designated as p0RF2 / LacZ.
- a sheet of MDCK cells formed in a 75 cm 2 plastic culture flask is digested with a physiological buffered saline solution containing 0.25% tribsine and lmM EDTA, the cells are detached from the surface of the incubator, and a lidar containing 20 ml of 5% FBS And centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded to collect the cell precipitate.
- the cells were suspended in 20 ml of Eagle's minimum essential medium without FBS, centrifuged again at 1500 rpm for 5 minutes, and the centrifuged supernatant was discarded to collect the cell precipitate.
- the cell pellet was suspended in 5001 ET buffer (RPM1640 medium containing 10 mM D-glucose and 5 mM dithiothreitol) and transferred to a cuvette (BioRad) for electoral poration.
- 5001 ET buffer RPM1640 medium containing 10 mM D-glucose and 5 mM dithiothreitol
- BioRad cuvette
- 30 zl of the plasmid pCTKdlEH / Lac Z prepared in Example (5) and adjusted to lmg / ml, allowed to stand in ice water for 5 minutes, and then treated with Gene Pulser (Bio-Rad) at 960 ⁇ ⁇ ⁇ FD ( An electric pulse was applied under the conditions of microfarads) and 0.3 kV (kilovolt) / cm, and the DNA was again introduced into the cells by leaving still in ice water for 10 minutes.
- the cells were transferred to a plastic culture dish of 6 cm in diameter, to which 5 ml of the minimum essential culture medium of Eagle containing 10% FBS was added, and cultured in 37 carbon dioxide incubators for 6 to 12 hours. After the culture, the culture solution was removed, CHV and YP11 strains having an infectious titer equivalent to the number of cells were inoculated, and allowed to stand for 1 hour to adsorb the virus to the cells. After the adsorption, 2 ml of Eagle's minimum essential medium to which FBS was added was added dropwise, and the cells were cultured again in a carbon dioxide incubator at 37 ° C for 48 hours.
- the thymidine kinase gene In the offspring region, it is expected that homologous recombination with the plasmid DNA already introduced into the cells by the electroporation method will occur. The appearance of the virus is expected.
- the cells were freeze-thawed three times, and the infected cells were disrupted with an ultrasonic disrupter, centrifuged at 3,000 rpm for 5 minutes, and the supernatant was recovered as a virus solution and stored at -80 ° C until use. .
- the recombinant virus present in the virus solution recovered above was cloned by the following black assay. That is, the MDCK cell sheet formed on a plastic 6-well plate was inoculated with a 10-fold serially diluted virus solution, allowed to stand for 1 hour to adsorb the virus to the cells, and the inoculated virus solution was removed. Overlay 2 ml of Eagle's minimum essential medium containing 6 agarose and 2 FBS per well, and add 1 ml of Eagle's minimum essential medium containing 1 ml of FBS per well when the agarose solidifies, and add 37 ° C CO2 incubator. For 48 hours.
- the liquid layer was removed, and 1 ml of Eagle's minimum essential medium containing 600 g / inl of X-gal and 23 ⁇ 4FBS was added per well, followed by further culturing in a carbon dioxide incubator at 37 ° C for 12 hours. After this culture, the liquid layer was again removed, and a blue-colored plaque was recovered using a Pasteur pipet by enzymatic reaction using X-gal, a beta-gal gene product, as a substrate. The cells were suspended in Eagle's minimum essential medium containing 1% FBS, subjected to three freeze-thaw cycles and sonication, and again subjected to the same black assay to purify the virus clones.
- This example shows that the thymidine kinase gene region on the CHV genome is a site into which a foreign nucleic acid sequence can be inserted.
- the recombinant virus obtained in this example was named CHV (Y) dlTK / Lac Z.
- Example 15 The basic operation was performed in the same manner as in Example (15).
- the plasmid pCTKdlEH / RV-G produced in Example (6) was used as the plasmid to be introduced into MDCK cells by the electroporation method, and the plasmid-infected cells were infected.
- the recombinant virus CHV (Y) dlTK / LacZ produced in Example (15) was used as the virus.
- the homologous recombination between the beta-gal expression unit of CHV (Y) dlTK / Lac Z used as the parent virus and the rabies virus G protein expression unit on plasmid DNA was performed. We can expect to be replaced more.
- the recombinant CHV into which the rabies virus G protein expression unit is integrated has the phenotype of forming the parent virus blue black when black-assay using X-gal shown in Example (15) is performed.
- the phenotype changes from white to black.
- the recombinant virus forming a white plaque was cloned by BlackAssy, and the expression of the rabies virus G protein was confirmed by the fluorescent antibody method using the antirabies virus ⁇ sera serum against the recombinant virus-infected cells.
- anti-rabies virus egret serum was prepared by inoculating subcutaneous rabbits with purified rabies virus together with Freund's adjuvant.
- This example shows that the recombinant virus CHV (Y) dlTK / Lac Z enables a screening method based on the color of virus black and is effective in producing a new recombinant CHV of interest.
- the recombinant virus obtained in this example was named CHV (Y) dlTK / RV-G.
- Example 15 the basic operation was performed in the same manner as in Example (15).
- the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7) and the plasmid P CTKdlEH created in Example (10) were introduced into MDCK cells by the electroporation method.
- CHV, DFD-6 strain was used as a virus to infect cells into which this plasmid had been introduced.
- each virus protein expression unit canine distemper virus F protein
- canine distemper virus H protein canine distemper virus H protein
- virus plasmids that form blue black by introducing their respective plasmids were obtained. These virus clones, which were simultaneously introduced with their respective viral protein expression units, were found to be resistant to recombinant virus-infected cells. Fluorescent antibody method using rabies virus ⁇ heron serum or anti-innus temper virus ⁇ sera serum, or added to recombinant virus that incorporates a unit of expression of thymidine kinase of oeskinosis virus during virus propagation in tissue culture Confirmed by sensitivity to 5-1000-2'-DEOXYURI INE (IDU) at a concentration of 50 £ / 1111.
- IDU 5-1000-2'-DEOXYURI INE
- the anti-indistemper virus peregret serum was prepared by inoculating subcutaneous persimmons of purified canine distemper virus with Freund's adjuvant.
- This example shows that two or more independent expression units can be inserted into a single site of a foreign nucleic acid sequence insertion site, and provides, for example, a method for producing a multivalent vaccine.
- the recombinant viruses obtained in this example were the plasmids pCTKdlEH / Lac Z / RV-G, pCTKdlEH / Lac Z / CDV-F, pCTKdlEH / Lac Z / CDV-H, pCTKdlEH / Lac Z used for homologous recombination.
- / PTK corresponding to CHV (D) dlTK / Lac Z / RV-G, CHV (D) dlTK / Lac Z / CDV -F, CHV (D) dlT / Lac Z / CDV -H, or CHV ( D) It was named dlTK / Lac Z / PTK.
- Recombinant CHV in which rabies virus G protein expression unit, inudistemper virus F protein expression unit, or inudistemper virus F protein expression unit are incorporated into the thymidine kinase gene transfer region of the CHV genome.
- the basic operation was performed in the same manner as in Example (15), except that the recombinant virus CHV produced in Example (17) was used as a virus to be infected after introduction of plasmid DNA.
- dlTK / Lac Z / PTK was used. This virus expresses the thymidine kinase gene product of the oeschis disease virus and is sensitive to IDU as described in Example (17). Conversely, when this gene is deleted, it becomes resistant to this drug.
- the obtained virus solution was inoculated on an MDCK cell sheet on a plastic 12-well plate, adsorbed for 1 hour, and then the minimum essential medium of Eagle containing 50 jug / ml of IDU and 53 ⁇ 4FBS was applied to each well. Incubate in a carbon dioxide incubator at 37 ° C for 24 to 48 hours, freeze-thaw three times, crush the infected cells with an ultrasonic homogenizer, and centrifuge at 3,000 rpm for 5 minutes. Collected. This virus solution was again subjected to selective culture in the presence of IDU, and the resulting virus solution was subjected to plaque assay in the presence of X-gal.
- the recombinant virus expected to appear in this example is constructed so as to delete the beta-gal expression unit in addition to the Aujeszky's disease virus thyridine kinase expression unit, it appears as white black in Black Athesi. .
- the ratio of the recombinant virus forming white black was as high as one third of the total virus.
- the recombinant virus CHV (D) dlTK / Lac Z / PTK enables selection culture by IDU in addition to selection by plaque color, and provides a method for easily cloning a new recombinant virus. You.
- the resulting recombinant virus was used in response to the plasmids pCTKdlEH / RV-G, pCTKdlEH / CDV-F, or pCTKdlEH / CDV-H used for homologous recombination, CHV (D) dlTK / RV-G, CHV (D ) Named dlTK / CDV-F or CHV (D) dlTK / CDV-H.
- Example 15 the basic operation was performed in the same manner as in Example (15).
- the plasmid pORF2 / LacZ created in Example (14) was used as the plasmid to be introduced into MDCK cells by the electroporation method, and the virus that infects cells into which this plasmid was introduced was used. CHV. DFD-6 strains were used.
- CH (D) dlORF2 / Lac Z it is expected that the 0RF2 region on the CHV genome is also a site into which a foreign nucleic acid sequence can be inserted.
- Recombinant virus CHV (Y) dlTK / RV-G, CHV, YP11 strain produced in Example (16) or a commercially available inactivated rabies vaccine (Nisseiken Co., Ltd.) was used in two or three dogs per group. Then, the appearance of neutralizing antibodies against rabies virus and the antibody titers were observed.
- Recombinant CHV can to 2 X 10 3 ⁇ 75 TCID 5 () on both sides of the nasal cavity
- non-recombinant CHV is to 2 X 10 4 ⁇ 5 TCID ( ) on both sides of the nasal cavity, inactivated rabies vaccine according to its prescription It was inoculated subcutaneously.
- the use of genetic recombination technology for CHV provides a mass production system of useful proteins, a rational method for the production of veterinary vaccines and therapeutic agents, restoration of animal health, This has the effect of contributing to maintenance and promotion.
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Abstract
A recombinant canine herpesvirus usable as a vector virus carrying a nucleic acid sequence introduced thereinto, which sequence does not occur in a canine herpesvirus vector. A recombinant canine herpesvirus constructed by inserting a nucleic acid sequence which does not occur in canine herpesvirus genome into a region on the genome exerting no lethal effect on the growth of the virus.
Description
明細書 Specification
組換えィヌヘルぺスウィルス Recombinant canine virus
技術分野 Technical field
本発明はィヌヘルぺスウィルス (C H V) のベクターウィルスとしての利用に 関するものであり、 詳しくは、 真核細胞内で作動する各種プロモータ— (C H V 自身のプロモーターも含む) の下流に、 外来核酸配列あるいはその一部を連結さ せた発現単位を C H Vゲノム上に一つあるいは複数個挿入することにより、 挿入 された外来核酸配列に由来する産物を感染細胞内で発現する組換え C H Vを作出 する方法、 本法により作出した組換え C H V、 およびこの組換え C H Vを動物に 接種することにより、 挿入した外来核酸配列の効果を得る方法に関するものであ The present invention relates to the use of canine virus (CHV) as a vector virus. More specifically, a foreign nucleic acid sequence is located downstream of various promoters (including the promoter of CHV itself) that operate in eukaryotic cells. Alternatively, a method of producing a recombinant CHV that expresses a product derived from the inserted foreign nucleic acid sequence in infected cells by inserting one or more expression units in which a part thereof is linked into the CHV genome. The present invention relates to a recombinant CHV produced by this method and a method for inoculating an animal with the recombinant CHV to obtain the effect of the inserted foreign nucleic acid sequence.
背景技術 Background art
ウィルス学はその研究の初期にはその病原性に関して多くの研究がなされ現在 のウィルス学の基礎が築かれてきた。 病原性解明の研究と相まって、 ウィルス学 はウィルスのヮクチン化を目指し、 動物あるいは組織培養細胞を用いた長期継代 という長時間を要し且つ確実性のそれほど高くな t、方法で弱毒化ある t、は無毒化 してきた。 その後、 遺伝子工学の技術がウィルス学に導入されるとウィルスの病 原性あるいはワクチン化を分子レベルで解明できるようになり、 この技術を用い たワクチンの開発が盛んに進められている。 一方では、 この技術を用いて天然に 存在するウィルス (野生型ウィルス) のゲノム上に存在しない核酸配列 (外来核 酸配列) を揷入された組換えウィルスの作出すなわちウィルスのベクター化が盛 んに行われるようになつてきた。 組換えウィルスの用途としては、 外来核酸配列 を細胞内で発現するように構築してベクタ一ウィルスに組み込みこの組換えウイ ルスを試験管内あるいは動物体の細胞に感染させることにより、 例えば外来核酸 配列およびその産物の機能を解明するため、 遺伝子産物を生産させるため、 ある いは動物体に接種し接種された動物がベクターウイルスに導入された外来核酸配
列の効果を得るために用いられる。 動物体に接種したときの効果とは具体的には、 例えば各種病原微生物の感染防御抗原をコードする遺伝子を外来核酸配列として 導入した場合のワクチン効果、 生理活性物質をコ—ドした遺伝子を外来核酸配列 として導入した場合の治療効果などが考えられる。 ウィルスのベクタ一化、 換言 すれば組換えウィルスの作出法が一旦確立されるならば、 同様の手法を用いて、 目的に応じて多種類の組換えウィルスの作出が可能となる。 しかも、 目指す効果 は多岐に渡っているにも関わらずそれを担う組換えウィルス製造法は野生型ウイ ルスの製造法と同様である場合が多く、 多くの目的を一つの製造法で達成するこ とができ、 対象動物の健康の回復、 維持、 増進に資することが期待される。 In the early days of virology, much research was done on its pathogenicity and the foundation of the current virology has been laid. Virology, coupled with research into pathogenicity elucidation, aims at pectinization of the virus, requires a long time of long-term passage using animals or tissue culture cells, is not so reliable, and is attenuated by methods. Have been detoxified. Later, when genetic engineering technology was introduced into virology, it became possible to elucidate the pathogenesis or vaccination of viruses at the molecular level, and vaccines using this technology are being actively developed. On the other hand, the use of this technology to create a recombinant virus in which a nucleic acid sequence (foreign nucleic acid sequence) not present in the genome of a naturally-occurring virus (wild-type virus) has been introduced, that is, a viral vector has been actively used. It has come to be done. Recombinant viruses can be used, for example, by constructing a foreign nucleic acid sequence so as to express it in a cell, incorporating it into a vector virus, and infecting the recombinant virus in a test tube or a cell of an animal body. In order to elucidate the function of the product and to produce a gene product, or to inoculate the animal body and inoculate the animal into a foreign nucleic acid sequence introduced into a vector virus Used to get a column effect. Specifically, the effects of inoculation into an animal body include, for example, a vaccine effect when a gene encoding a protective antigen of various pathogenic microorganisms is introduced as a foreign nucleic acid sequence, and a gene coding for a bioactive substance. The therapeutic effect when introduced as a nucleic acid sequence is considered. Once the method of virus vectorization, in other words, the method of producing a recombinant virus, is established, it is possible to produce various types of recombinant viruses according to the purpose using the same method. Moreover, despite the wide range of effects that can be achieved, the method of producing recombinant virus that plays a role in many cases is similar to the method of producing wild-type virus, and many objectives can be achieved with a single production method. It is expected to contribute to the recovery, maintenance and promotion of the health of the target animals.
幾つかのウィルスでべクター化が進められているが、 ィヌ科の動物に対するべ クタ一ウィルスはアデノウイルスについて知られているのみである。 Although some viruses are being vectorized, the only known virus for canines is the adenovirus.
ヘルぺスウィルス科に属するウィルス、 例えば単純へルぺスウィルス 1型 (Malik et al. , 1992, Virology, 190: 702-715 ) 、 ォーエスキー病ウィルス (Thomsen et al. , 1987, Gene, 57 : 261-265 ) 、 ゥシヘルぺスウィルス 1型 (Kit et al. , 1992, Arch. Virol. , 124 : 1-20) 、 ネコへルぺスウィルス 1型 (Cole et al. , 1990, J. Virol. , 64: 4930-4938 ) などがすでに外来核酸配列を動物体内で発現さ せるためのベクタ一ウィルスとして開発されている。 ヘルぺスウィルス科のウイ ルスをベクターウィルスとして開発する場合、 目的とする外来核酸配列はヘルべ スウィルスゲノム上に同定された非必須遺伝子領域すなわちウィルスの増殖に必 須でない遣伝子領域に挿入することが多く、 本発明の発明者の一人である玄らは 鋭意研究を重ね、 C Η Vにおける外来核酸配列揷入部位の候補の一つになると考 えられるチミ ジンキナーゼ遺伝子の塩基配列を明らかにした。 又、 この領域は Remondら (1995, Virus Res., 39: 341-354 ) によってもその構造が解析されつつ ある。 さらに、 Limbach ら (1994, J. Gen. Virol. , 75: 2029-2039 ) は C H V構造 タンパク質である g B、 g C、 g D遺伝子の塩基配列を決定しており、 Remondら (1996, J. Gen. Virol. , 77: 37-48 ) は C Η Vゲノムの U Lといわれる領域の塩基 配列の決定を精力的に進めている。 しかし、 C H Vの遺伝子構造の解析は以上の ように近年精力的に進められているが、 この C H Vをベクターウィルスとして利
用できるか否かについての知見は現在のところ皆無である。 Viruses belonging to the family Herpesviridae, such as simple herpesvirus 1 (Malik et al., 1992, Virology, 190: 702-715), Aujeszky's disease virus (Thomsen et al., 1987, Gene, 57: 261-265), pertussis virus type 1 (Kit et al., 1992, Arch. Virol., 124: 1-20), cat herpes virus type 1 (Cole et al., 1990, J. Virol) , 64: 4930-4938) has already been developed as a vector virus for expressing foreign nucleic acid sequences in animals. When a virus of the family Herpesvirus is developed as a vector virus, the foreign nucleic acid sequence of interest is located in a non-essential gene region identified on the genome of the herpes virus, ie, a gene region that is not essential for the propagation of the virus. Gen, one of the inventors of the present invention, conducted intensive studies and found that the nucleotide sequence of the thymidine kinase gene, which is considered to be one of the candidates for the foreign nucleic acid sequence insertion site in C CV Revealed. The structure of this region is being analyzed by Remond et al. (1995, Virus Res., 39: 341-354). Furthermore, Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039) have determined the nucleotide sequences of the gB, gC, and gD genes, which are CHV structural proteins, and have described Remond et al. Gen. Virol., 77: 37-48) has been energetically determining the nucleotide sequence of a region called UL in the CΗV genome. However, although the analysis of the gene structure of CHV has been energetically advanced in recent years as described above, this CHV is used as a vector virus. There is currently no knowledge of whether it can be used.
本発明の発明者らはかかる状況に鑑み、 ィヌ科の動物に汎用性のあるウィルス ベクターの開発を目的とし、 ィヌヘルぺスウィルス (CHV) のベクター化をめ ざし、 本発明を完成させるに至った。 In view of such circumstances, the inventors of the present invention aimed at developing a virus vector that is versatile for canines, aiming at vectorization of canine herpes virus (CHV), and completing the present invention. Reached.
すなわち、 本発明の第一の目的は、 CHVゲノム遺伝子上に外来核酸配列を揷 入するための部位が複数個存在すること、 すなわち CHVがベクターウィルスと して十分利用可能であることを示し、 あわせてこの位置を決定する方法の幾つか を提供することにある。 That is, a first object of the present invention is to show that there are a plurality of sites for inserting a foreign nucleic acid sequence on a CHV genomic gene, that is, that CHV can be sufficiently used as a vector virus, It also aims to provide some ways to determine this position.
第二の目的は、 CHVゲノム上の決定された部位に目的の外来核酸配列を導入 する方法のいくつかを確立し、 この組換えウィルスの作成法を提供することにあ る。 The second purpose is to establish some methods for introducing a foreign nucleic acid sequence of interest into a determined site on the CHV genome, and to provide a method for producing this recombinant virus.
第三の目的は、 組換え CHVを試験管内あるいは生体内の細胞に感染させて外 来核酸配列を発現させるための CHVゲノム上に挿入する外来核酸配列の構築の 方法を提供することにある。 A third object is to provide a method of constructing a foreign nucleic acid sequence to be inserted into a CHV genome for expressing a foreign nucleic acid sequence by infecting cells in a test tube or in vivo with recombinant CHV.
第四の目的は、 このようにして作出した組換え CHVを動物に接種した場合に 目的とした効果を十分上げられることを示し、 本発明の実用化を保証することを 目的とする。 The fourth object is to show that when the recombinant CHV thus produced is inoculated into an animal, the intended effect can be sufficiently enhanced, and the object of the present invention is to guarantee the practical application of the present invention.
さらに本発明は以上のようにして作出した組換え CHVの提供を目的とする。 発明の開示 Further, another object of the present invention is to provide a recombinant CHV produced as described above. Disclosure of the invention
本発明は前記目的を達成するために、 ィヌヘルぺスウィルスゲノム上に存在し ない核酸配列 (以下 「外来核酸配列」 という) をそのゲノム上に挿入した組換え ィヌヘルぺスウィルスを提供するものである。 外来核酸配列を挿入する部位は、 ウィルス増殖に致死的な影響を与えない領域とすることが望ましいが、 これに限 定されるものではない。 The present invention provides, in order to achieve the above-mentioned object, a recombinant canine virus in which a nucleic acid sequence not present on the canine virus genome (hereinafter referred to as "foreign nucleic acid sequence") is inserted into its genome. is there. The site for inserting the foreign nucleic acid sequence is desirably a region that does not have a lethal effect on virus growth, but is not limited to this.
外来核酸配列は、 例えば相同組換えの手法を用いて CHVゲノム内に導入する ことができる。 そこで、 本発明ではその第一段階として C HVゲノムの任意の DN A断片をプラスミ ド DN A上にクローニングし、 後に述べるトランスファー
ベクターとして用いた。 クローニングおよびその後のステップで用いる遺伝子ェ 学的手法は Sambrookら (1989, Molecular Cloning : A laboratory manual , 2nd edition , Cold Spring Harbor Laboratory Press , New York) により集大成さ れている。 The foreign nucleic acid sequence can be introduced into the CHV genome using, for example, a homologous recombination technique. Therefore, in the present invention, as a first step, an arbitrary DNA fragment of the CHV genome is cloned into plasmid DNA, and the transfer described later is performed. Used as vector. The genetic techniques used in cloning and subsequent steps have been compiled by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).
次の段階ではクロ一ニングした DNA断片の任意の位置に変異を導入する。 こ の場合、 その位置に存在する遺伝子の機能を損なうような形で変異を導入すると その遺伝子が非必須遣伝子であるか否かを見極めることができる。 In the next step, a mutation is introduced at any position of the cloned DNA fragment. In this case, if a mutation is introduced in a manner that impairs the function of the gene located at that position, it can be determined whether or not the gene is a non-essential gene.
次にこの変異を導入されたプラスミ ド DNAを C HVの宿主細胞にトランスフ ヱク 卜し、 一定期間培養した後野生型ウィルスを感染させる。 このように DNA を導入され且つ野生型ウイルスの感染をうけた細胞を一定期間培養するとその紬 胞は CHVを產生すると予想され、 この過程で CHVゲノム DN Aはその増殖時 に変異を導入したプラスミ ド DN Aとの相同組換えによる組換えウィルスゲノム の新生およびこの組換えウィルスゲノムを有するウイルス粒子の新生が期待でき る。 このような状況下で産生されたウィルスの中にプラスミ ド DN A上で導入し た変異を取り込んだウィルスが存在するか否かを解析することにより CHVゲノ ム上の変異導入部位が外来核酸配列挿入可能部位であるか否かを見極めることが できる。 この方法により複数個あるかもしれな t、外来核酸配列挿入可能部位を順 次見つけ出していくことができる。 Next, the plasmid DNA into which this mutation has been introduced is transfected into CHV host cells, cultured for a certain period of time, and then infected with a wild-type virus. When cells transfected with the DNA and infected with the wild-type virus are cultured for a certain period of time, the cells are expected to produce CHV. In this process, the CHV genome DNA is transformed into a plasmid that has been mutated during its propagation. New generation of a recombinant virus genome by homologous recombination with DNA and DNA particles having the recombinant virus genome can be expected. By analyzing the presence or absence of a virus that incorporates the mutation introduced on plasmid DNA in the virus produced under such circumstances, the mutation-introduced site on the CHV genome can be used to identify the foreign nucleic acid sequence. It can be determined whether the site is an insertable site. By this method, there may be a plurality of sites where foreign nucleic acid sequence can be inserted.
C HVゲノム上の外来核酸配列揷入部位が判明したならば、 同様の技術を応用 あるいは改良して目的とする外来核酸配列を目的を達成するための核酸構造、 例 えば真核細胞內でポリベプチドを発現させるような構造で構築し、 これを挿入し た組換え CHVの作出が可能となる。 CHVゲノム上に挿入しうる外来核酸配列 の個数は一個である必要はなく、 目的に応じて複数個揷入した組換え CHVを作 出することができる。 Once the foreign nucleic acid sequence on the CHV genome has been identified, a similar technique can be applied or improved to achieve the desired foreign nucleic acid sequence. It is possible to construct a recombinant CHV that is constructed with a structure that expresses and that inserts it. The number of foreign nucleic acid sequences that can be inserted into the CHV genome does not need to be one, and a recombinant CHV in which a plurality of foreign nucleic acids are inserted can be produced depending on the purpose.
C HVは生後 2週齢までの子犬に対しては病原性を示すものの、 それ以降の犬 に対しては病原性を示すことは希であり、 もしこのようなほとんど病原性を持た ないという特徴を持つ CHVをベクターウィルスとして利用すればィヌをはじめ とする、 組換え CHVに感受性を持つ動物の健康の回復、 維持、 増進に有効に寄
与することができる。 図面の簡単な説明 CHV is pathogenic in puppies up to 2 weeks of age, but rarely in later dogs. Use of CHV with vector as a vector virus will effectively contribute to the recovery, maintenance and promotion of the health of dogs and other animals susceptible to recombinant CHV Can be given. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 C H V、 チミジンキナーゼ遣伝子を含む D N A断片のクローニング を示す図。 なお制限酵素名のあとに続く数字はその酵素で切断される位置を k b 単位で示し、 以下の図も同様である。 FIG. 1 shows the cloning of a DNA fragment containing CHV and the thymidine kinase gene. The number following the name of the restriction enzyme indicates the position of cleavage by the enzyme in kb units, and the same applies to the following figures.
第 2図は、 C H V、 g Cおよび O R F 2遺伝子を含む D N A断片のクローニン グを示す図。 FIG. 2 is a diagram showing cloning of a DNA fragment containing CHV, gC, and ORF2 genes.
第 3図は、 チミ ジンキナーゼ遺伝子領域に由来する トランスファ一ベクターの 構築を示す図。 FIG. 3 shows the construction of a transfer vector derived from the thymidine kinase gene region.
第 4図は、 チミ ジンキナーゼ遣伝子領域に b e t a - g a 1発現単位を組み込 んだトランスファーベクターの構築を示す図。 FIG. 4 is a diagram showing the construction of a transfer vector in which a beta-ga1 expression unit has been incorporated into the thymidine kinase gene transfer region.
第 5図は、 狂犬病ウィルス Gタンパク質発現単位の構築。 Figure 5 shows the construction of the rabies virus G protein expression unit.
第 6図は、 チミ ジンキナーゼ遺伝子領域に狂犬病ウィルス Gタンパク質発現単 位を組み込んだトランスファーべクタ一の構築を示す図。 FIG. 6 shows the construction of a transfer vector in which a rabies virus G protein expression unit has been incorporated into the thymidine kinase gene region.
第 7図は、 チミジンキナーゼ遣伝子領域に b e t a - g a 1発現単位および狂 大病ウィルス Gタンパク質発現単位を組み込んだトランスファーベクターの構築 を示す図。 FIG. 7 is a view showing the construction of a transfer vector in which a beta-ga1 expression unit and a madness virus G protein expression unit are incorporated into the thymidine kinase gene region.
第 8図は、 C D V、 Fタンパク質遺伝子および Hタンパク質遺伝子のクロ一二 ングを示す図。 FIG. 8 is a diagram showing the closing of CDV, F protein gene and H protein gene.
第 9図は、 第 8図の続き。 Figure 9 is a continuation of Figure 8.
第 1 0図は、 チミジンキナーゼ遺伝子領域に b e t a - g a 1発現単位および C D V、 Fタンパク質発現単位を組み込んだトランスファーベクターの構築を示 す図。 FIG. 10 is a diagram showing the construction of a transfer vector in which a beta-ga1 expression unit and CDV and F protein expression units have been incorporated into the thymidine kinase gene region.
第 1 1図は、 チミジンキナーゼ遺伝子領域に b e t a - a 1発現単位および C D V、 Hタンパク質発現単位を組み込んだトランスファーベクターの構築を示 す図。 FIG. 11 shows the construction of a transfer vector in which a beta-a1 expression unit and CDV and H protein expression units have been incorporated into the thymidine kinase gene region.
第 1 2図は、 チミジンキナーゼ遺伝子領域に b e t a - g a 1発現単位および
ォ一エスキー病ウィルスチミジンキナーゼ発現単位を組み込んだトランスファ一 ベクターの構築を示す図。 FIG. 12 shows that the thymidine kinase gene region has a beta-ga 1 expression unit and FIG. 2 is a diagram showing the construction of a transfer vector into which an expression unit of the E. coli disease virus thymidine kinase is incorporated.
第 1 3図は、 チミジンキナーゼ遺伝子領域に CD V、 Fタンパク質発現単位を 組み込んだトランスファーベクターの構築を示す図。 FIG. 13 is a diagram showing the construction of a transfer vector in which a CDV / F protein expression unit has been incorporated into the thymidine kinase gene region.
第 14図は、 チミジンキナーゼ遺伝子領域に CD V、 Hタンパク質発現単位を 組み込んだトランスファーベクターの構築を示す図。 FIG. 14 is a diagram showing the construction of a transfer vector in which a CDV / H protein expression unit has been incorporated into the thymidine kinase gene region.
第 1 5図は、 0 R F 2領域に b e t a— g a 1発現単位を組み込んだトランス ファ一べク夕一の構築。 発明を実施するための最良の形態 FIG. 15 shows construction of a transfer vector in which a beta—ga1 expression unit was incorporated into the 0 R F2 region. BEST MODE FOR CARRYING OUT THE INVENTION
本発明を完成させるに当たって、 用いられる基本的な手法は、 遺伝子工学的技 術とウィルス学の技術である。 前者の技術は Sambrookら (1989, Molecular Cloning : A laboratory manual , 2nd edition, Cold Spring Harbor Laboratory Press , New York) により、 また後者の技術は国立予防衛生研究所学友会 (1973、 ウイ ルス実験学総論、 改訂二版、 丸善株式会社) により集大成されている。 To complete the present invention, the basic techniques used are genetic engineering techniques and virology techniques. The former technology was described by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York), and the latter technology was developed by the National Institute of Health, National Institute of Health (1973). , Revised second edition, Maruzen Co., Ltd.).
本発明を遂行する第一段階では CHVゲノムの DN A断片のクローニングを行 う。 本発明で用いる C HVにはいかなる CHV株を用いることもでき、 例えば、 D 004株 (ATCC VR-552 ) あるいは F 205株 (ATCC VR-647 ) を挙げることがで きる。 この過程はすでに、 Remondら C1995, Virus Res. , 39: 341-354 ) 、 Limbach ら (1994, J. Gen. Virol. , 75: 2029-2039 ) あるいは Reraondら (1996, J. Gen. Virol. , 77: 37-48 ) が確立しており、 彼らの報告している方法により行うことが できる。 あるいは、 現在までに公知となっている塩基配列を参考にして任意に合 成 DNAプライマーを作成し、 ポリメラ一ゼ 'チヱ一ン反応 (PCR) により目 的の断片を増幅し適当なプラスミ ド DN A上にクローニングすることもできる。 The first step in carrying out the present invention involves cloning the DNA fragment of the CHV genome. Any CHV strain can be used as the CHV used in the present invention, and examples thereof include strain D004 (ATCC VR-552) and strain F205 (ATCC VR-647). This process has already been described in Remond et al. C1995, Virus Res., 39: 341-354), Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039) or Reraond et al. (1996, J. Gen. Virol. , 77: 37-48) have been established and can be carried out according to their reported method. Alternatively, a synthetic DNA primer is arbitrarily prepared with reference to the nucleotide sequence known to date, and the desired fragment is amplified by polymerase-chain reaction (PCR) to obtain an appropriate plasmid DN. It can also be cloned on A.
CHVウィルスゲノムの DNA断片を保持するプラスミ ド DNAは、 外来核酸 配列を相同組換えにより CHVゲノム上に導入するためのトランスファーべクタ一 として使用でき、 また真核細胞内で作動するプロモーターの供給源ともなる。 ト ランスファーべクターは上述のように外来核酸配列を相同組換えにより CHVゲ
ノムに揷入する場合に使用され、 C H Vゲノム上の挿入部位の前後の核酸配列を 外来核酸配列の両側に位置させこれらの配列によりゲノム D N Aとの相同組換え を起こさせることができる。 これらの配列の長さは C H Vゲノムとの相同組換え を生じさせ得るような適切な長さのものでなければならない。 Plasmid DNA, which contains the DNA fragment of the CHV virus genome, can be used as a transfer vector for introducing a foreign nucleic acid sequence into the CHV genome by homologous recombination, and a source of a promoter that operates in eukaryotic cells. Also. As described above, the transfer vector is used for homologous recombination of a foreign nucleic acid sequence into a CHV gene. It is used when inserting into a nome, and the nucleic acid sequence before and after the insertion site on the CHV genome is positioned on both sides of the foreign nucleic acid sequence, and these sequences can cause homologous recombination with genomic DNA. These sequences must be of sufficient length to allow homologous recombination with the CHV genome.
次の段階ではこのトランスファーベクター上の C H Vゲノム D N A断片の任意 の位置に変異を導入する。 変異の導入の仕方には欠失、 挿入、 あるいは塩基置換 などの方法を用いることができ、 Malik ら (1992, Virology, 190 : 702-715 ) 、 Thorasen ら (1987, Gene, 57: 261-265 ) 、 Kit ら (1992, Arch. Virol., 124 : 1-20) 、 あるいは Coleら (1990, J. Virol. , 64: 4930-4938 ) の示している方法に より実施することもできる。 具体的には例えばプラスミ ド pCHl lO (フアルマシア 社) よりプロモーターおよびポリアデニレーシヨンシグナルを持つ /3—ガラク 卜 シダーゼ (beta- gal) 発現単位を調製し、 これをトランスファ一ベクタ一上の C H Vゲノム断片の任意の位置に挿入することにより変異を導入できる。 発現単 位の語は外来核酸配列を真核細胞内で自律的に発現させるための核酸構造の単位 を指し、 具体的には外来核酸配列の方向、 例えばポリべプチドをコ—ドしている 方向と同一の方向性を持つて外来核酸配列の上流に真核細胞内で作動するプロモー 夕一を、 また下流にポリアデ二レーションシグナルを配した核酸配列の構造単位 を示す。 変異を導入したプラスミ ド D N Aは C H Vのゲノム D N Aとともに例え ば Malikら (1992, Virology, , 190 : 702-715 ) 、 Thomsen ら (1987, Gene, 57: 261-265 ) 、 Kit ら (1992, Arch. Virol. , 124 : 1-20) 、 あるいは Cole ら (1990, J. Virol. , 64: 4930-4938 ) の方法に従って C H Vの宿主細胞例えば MDCK細胞内にコトランスフヱク トする。 あるいはあらかじめ変異を導入した上記の プラスミ ドを細胞にトランスフエク トしておき C H Vを感染させてもよい。 この 細胞を数日間培養すると培養上清および細胞内に親ウィルスに加えて、 C H Vゲ ノム D N Aと変異を導入されたトランスファーべク夕一 D N Aとの間の相同組換 えにより新生すると考えられる組換えウィルスの産生が期待できる。 ここからゥ ィルスを回収しブラックァッセィ法によりウィルスクローンを分雜する。 これら のウィルスクローンをプラスミ ド D N A上で導入した変異に見合った方法、 例え
ば Malikら (1992, Virology, 190 : 702-715 ) 、 Thomsen ら (1987, Gene, 57 : 261-265 ) 、 Kit ら (1992, Arch. Virol. , 124 : 1-20) 、 あるいは Coleら (1990, J. Virol. , 64: 4930-4938 ) の方法で解析し、 変異が導入されたウィルス クローンすなわち組換えウィルスが存在するか否かを解析する。 具体的には例え ば上述の beta-gal発現単位を用いて変異を導入した場合にはブラックアツセィを実 施するときに 5- bromo- 4 - chloro- 3- indolyl- yS -D- galactoside(X-gal) を寒天ある いはメチルセルロース培地中に混合あるいは重層することにより、 青色のブラッ クとして容易に組換えウィルスの出現を確認できる。 変異が導入される確立はと きには産生されるウィルスクローン 1, 000個中 1個であることもある力く、 beta-gal 発現単位および X- galを用いれば組換え C H V出現を容易に確認できる。 このよう に低い確立でも一旦変異が導入できることが確認できれば、 その部位は外来核酸 配列揷入部位として同定されたことを意味し、 同様の方法で C H Vゲノム上に存 在する外来核酸配列挿入部位を複数個探し出すことができる。 In the next step, a mutation is introduced at any position of the CHV genomic DNA fragment on this transfer vector. Mutations can be introduced by methods such as deletion, insertion, or base substitution. Malik et al. (1992, Virology, 190: 702-715), Thorasen et al. (1987, Gene, 57: 261-265). ), Kit et al. (1992, Arch. Virol., 124: 1-20) or Cole et al. (1990, J. Virol., 64: 4930-4938). Specifically, for example, a / 3-galactosidase (beta-gal) expression unit having a promoter and a polyadenylation signal is prepared from plasmid pCHl10 (Pharmacia), and this is used to prepare the CHV genome on the transfer vector. Mutations can be introduced by inserting the fragment at any position. The term expression unit refers to a unit of a nucleic acid structure for autonomously expressing a foreign nucleic acid sequence in a eukaryotic cell, and specifically encodes the direction of the foreign nucleic acid sequence, for example, a polypeptide. A structural unit of a nucleic acid sequence having the same direction as that of a foreign nucleic acid sequence and operating in a eukaryotic cell upstream of a foreign nucleic acid sequence, and a polyadenylation signal arranged downstream thereof. The plasmid DNA into which the mutation has been introduced can be used together with the genomic DNA of CHV, for example, Malik et al. (1992, Virology, 190: 702-715), Thomsen et al. (1987, Gene, 57: 261-265), Kit et al. Virol., 124: 1-20), or according to the method of Cole et al. (1990, J. Virol., 64: 4930-4938), cotransfect into a host cell of CHV such as MDCK cell. Alternatively, cells may be transfected with the above-described plasmid into which a mutation has been introduced in advance, and then infected with CHV. When these cells are cultured for several days, in addition to the parental virus in the culture supernatant and cells, a group that is likely to be regenerated by homologous recombination between the CHV genomic DNA and the mutated transfer vector DNA Production of a recombinant virus can be expected. The virus is recovered from this and virus clones are cloned by the Blackassay method. Methods appropriate for the mutations introduced into these viral clones on plasmid DNA, such as For example, Malik et al. (1992, Virology, 190: 702-715), Thomsen et al. (1987, Gene, 57: 261-265), Kit et al. (1992, Arch. Virol., 124: 1-20), or Cole et al. 1990, J. Virol., 64: 4930-4938), and analyze whether or not a mutation-introduced virus clone, that is, a recombinant virus, is present. Specifically, for example, when a mutation is introduced using the above-mentioned beta-gal expression unit, 5-bromo-4--4-chloro-3-indolyl-yS-D-galactoside ( By mixing or overlaying X-gal) in agar or methylcellulose medium, the appearance of recombinant virus can be easily confirmed as a blue black. When the mutation is introduced, it can be as high as 1 in 1,000 virus clones produced, and the use of beta-gal expression units and X-gal facilitates the appearance of recombinant CHV. You can check. Once it is confirmed that a mutation can be introduced even with such a low probability, it means that the site has been identified as a foreign nucleic acid sequence insertion site, and the foreign nucleic acid sequence insertion site present on the CHV genome is identified in the same manner. You can find more than one.
C H Vゲノム上の外来核酸配列挿入部位が見いだされたならば、 本発明では次 のステップとして目的とする核酸配列をその部位に揷入する。 目的とする核酸配 列は例えば上に述べた発現単位の構造で構築する。 この時に用いる真核細胞内で 作動するプロモーターには例えばヒ トサイ トメガロウィルスの前初期遺伝子のプ 口モータ一、 Simian Virus 40 の初期遺伝子のプロモータ一あるいは天然に C H V ゲノム上に存在するプロモータ一などを用い、 この下流に目的の核酸配列、 例え ば狂犬病ウィルス Gタンパク質をコ一ドする読み取り枠 (Open reading frame, 以 下 「O R F」 と略記する) の配列を連結する。 このように構築した自律的な発現 単位をトランスファーベクター上の外来核酸配列揷入部位へ挿入する。 このよう に構築したトランスファ一ベクタ一と C H Vあるいは例えば組換えウィルスを選 択しゃすいようにするには、 あらかじめ同じ外来核酸配列挿入部位に beta-gal発現 単位を揷入した組換え C H Vを用い、 上記と同様に細胞内での相同組換えを誘起 し、 その後、 外来核酸配列挿入部位を探索した方法を応用して組換えウィルスの クローンを選択することができる。 Once a foreign nucleic acid sequence insertion site on the CHV genome is found, the next step in the present invention is to insert the target nucleic acid sequence into that site. The target nucleic acid sequence is constructed, for example, with the structure of the expression unit described above. Promoters that operate in eukaryotic cells used at this time include, for example, the promoter of the immediate early gene of human cytomegalovirus, the promoter of the early gene of Simian Virus 40, or the promoter that naturally exists on the CHV genome. A nucleic acid sequence of interest, for example, a sequence of an open reading frame (hereinafter abbreviated as “ORF”) encoding a rabies virus G protein is ligated downstream of this. The autonomous expression unit thus constructed is inserted into the foreign nucleic acid sequence insertion site on the transfer vector. In order to select the thus constructed transfer vector and CHV or, for example, a recombinant virus, use a recombinant CHV in which a beta-gal expression unit has been inserted into the same foreign nucleic acid sequence insertion site in advance. In the same manner as described above, homologous recombination is induced in cells, and then a clone of a recombinant virus can be selected by applying a method in which a foreign nucleic acid sequence insertion site has been searched.
以上のようにして作出した組換え C H Vはその宿主細胞例えば MDCK細胞 (ATCC
CCL34 ) に感染させることによりその効果を発揮させることができる。 例えば揷 入された外来核酸配列が狂犬病ウィルス Gタンパク質発現単位であるならばその タンパク質の組織培養を用いた産生に使用できる。 またさらには、 組換えウィル スを直接動物に接種、 例えば鼻腔内、 皮下、 皮内、 静脈内、 筋肉内あるいは経口 的に接種することにより、 動物体内で直接組換え C H Vの効果を得ることができ る。 例えば、 狂犬病ウィルス Gタンパク質発現単位を挿入した組換え C H Vを動 物に接種すれば、 被接種動物の狂犬病ウィルスに対する感染防御能の獲得という 効果が得られる。 これは組換え C H Vを用いたワクチン製造の道を開く ものであ る。 The recombinant CHV produced as described above is used as a host cell such as an MDCK cell (ATCC The effect can be exerted by infecting CCL34). For example, if the inserted foreign nucleic acid sequence is a rabies virus G protein expression unit, it can be used for production of the protein using tissue culture. Furthermore, the effect of recombinant CHV can be directly obtained in animals by inoculating the recombinant virus directly into animals, for example, intranasally, subcutaneously, intradermally, intravenously, intramuscularly or orally. it can. For example, inoculating an animal with a recombinant CHV into which a rabies virus G protein expression unit has been inserted has the effect of acquiring the ability to protect the recipient animal against rabies virus infection. This paves the way for vaccine production using recombinant CHV.
このように C H Vをベクターウィルスとして用いる場合の用途としては上に述 ベたべクタ一ワクチンが第一に考えられる。 またこの用途の目的で揷入が考えら れる外来核酸配列には上述の汪犬病ウィルス Gタンパク質遺伝子の他に、 例えば ィヌジステンパーウィルスの H夕ンパク質遣伝子および F夕ンパク質遺伝子、 ィ ヌアデノウイルス 1型あるいは 2型のへキソン夕ンパク質遺伝子あるいはフアイ バー夕ンパク質遺伝子、 パラインフルェンザウィルスの F夕ンパク質遺伝子ある いは H Nタンパク質遺伝子、 ィヌパルボウイルスの V P 1あるいは V P 2夕ンパ ク質遺伝子など、 C H Vの感染を許容する宿主動物例えばィヌに対する病原体の 感染防御抗原をコ一ドする遺伝子を挙げることができる。 As described above, the use of CHV as a vector virus is primarily considered to be the vector vaccine described above. In addition, foreign nucleic acid sequences that may be introduced for the purpose of this use include, in addition to the above-mentioned rabies virus G protein gene, for example, the H- and F-protein genes of canine distemper virus, Inducible adenovirus type 1 or 2 hexon protein gene or fiber protein gene, parainfluenza virus F protein protein or HN protein gene, canine parvovirus VP 1 or Examples of the gene include a gene encoding a protective antigen of a pathogen to a host animal that is capable of infecting CHV, such as a dog, such as a VP2 protein gene.
また、 C H Vをベクターウィルスとして用いる場合の用途として治療への応用 を挙げることができ、 この場合には外来核酸配列としてリ ンフォカイン、 インター フエロン、 あるいはサイ トカインのような生理活性物質をコードする遣伝子を揷 入する場合を例示できる。 実施例 In addition, when CHV is used as a vector virus, it can be used for therapeutic applications. In this case, a foreign nucleic acid sequence encoding a physiologically active substance such as lymphokine, interferon, or cytokine is used. A case where a child is introduced can be exemplified. Example
以下実施例を挙げて本発明をさらに具体的に説明する。 Hereinafter, the present invention will be described more specifically with reference to examples.
( 1 ) C H VゲノムD N Aの抽出 (1) Extraction of C H V genome DNA
5¾のゥシ胎児血清 (FBS) を添加したィーグルの最小必須培地に懸濁した MDCK細胞 を直径 6cmの組織培養用プラスチックシャーレに播種し、 単層の細胞シ一卜が形成
されるまで炭酸ガス培養器內 (5¾炭酸ガス存在下、 37°C) で培養する。 細胞シート が形成された時点で、 培養液を除き、 シートを形成する細胞数以上の感染価を有 する CHV、 YP11株 (東京大学、 獣医微生物学教室所葳) あるいは DFD- 6株 (日 生研株式会社所蔵) を接種し、 1時間静置してウィルスを細胞に吸着させた。 吸 着後 2ml の^の FBSを添加したイーグルの最小必須培地を添加し、 再び、 炭酸ガス 培養器内で 22〜23時間培養した。 培養後、 培養液を除き MDCK細胞をリン酸緩衝食塩 液で一回洗浄し、 1ml の 1¾SDS、 0.1M NaCl 、 ImM EDTAを含む 0.1Mトリス一塩酸緩 衝液 (pH9) を添加し、 室温で 10分間静置した。 その後、 20mg/ml プロナ一ゼ溶液を 50^ 1添加し、 37てでー晚静置した。 この溶液を回収し、 等量のフヱノール · クロ ロフオルム溶液(1:1) で 2回抽出操作を行い、 水層を回収した。 回収した水溶液に 0.1ml の 3M NaCl 溶液と 2ml のエタノールを加えると、 糸状の DN Aが析出してく るのでこれをガラス棒に巻き付けて回収し、 70%エタノール溶液で洗浄して乾燥さ せ、 0.1ml の蒸留水にとかし、 使用時まで 4 °Cで保存した。 この操作で 30〜50ju g の DN Aが回収できた。 MDCK cells suspended in Eagle's minimum essential medium supplemented with 5% fetal bovine serum (FBS) are seeded on a 6 cm diameter tissue culture plastic dish to form a monolayer cell sheet. Incubate in a carbon dioxide incubator II (5 ° C, 37 ° C, in the presence of carbon dioxide) until complete At the time when the cell sheet is formed, the CHV and YP11 strains (with the University of Tokyo, Veterinary Microbiology Division II) or DFD-6 strains (Nissei Laboratories) that have an infectivity higher than the number of cells forming the sheet, excluding the culture solution The virus was adsorbed to the cells. After adsorption, Eagle's minimum essential medium supplemented with 2 ml of FBS was added, and the cells were cultured again in a carbon dioxide incubator for 22 to 23 hours. After the culture, remove the culture medium, wash the MDCK cells once with phosphate buffered saline, add 1 ml of 0.1 M Tris-monohydrochloride buffer (pH 9) containing 1¾SDS, 0.1 M NaCl and ImM EDTA, and add 10 ml at room temperature. Let stand for minutes. Thereafter, 50 ^ 1 of a 20 mg / ml protease solution was added, and the mixture was allowed to stand at 37 ° C. This solution was recovered and extracted twice with an equal volume of a phenol-chloroform solution (1: 1) to recover an aqueous layer. When 0.1 ml of a 3M NaCl solution and 2 ml of ethanol are added to the collected aqueous solution, a filamentous DNA precipitates.Then, this is wound around a glass rod, collected, washed with a 70% ethanol solution, and dried. Dissolve in 0.1 ml of distilled water and store at 4 ° C until use. With this operation, 30 to 50 jug of DNA was recovered.
(2) C HVゲノム DN Aからのチミジンキナーゼ遺伝子 (TK) を含む断片 のクロ一ニング (2) Cloning of fragment containing thymidine kinase gene (TK) from CHV genome DNA
第 1図に遣伝子断片のクローニング手順を示した。 すなわち、 上記の実施例 (1) により回収した CHVゲノム DNA20 1を 50単位の制限酵素 Xbalにより 37 で一晩処理した後ァガロース電気泳動を行い、 6.5キロベース(kb)断片を含むァ ガロース部分を回収し、 ジーンクリーンキッ ト (BI0101 社) でその中に含まれる DN Aを回収した。 これを、 Xbalで切断し子牛腸管由来アルカリフォスファターゼ (C1AP)を用いて脱リン酸化処理したプラスミ ド pBluescript I1SKC+) とライゲーシ ヨンキッ ト (宝酒造社) を用いて連結し、 コンビテン卜化した大腸菌 DH5aに形質 転換反応により導入した。 これを 50 g/mlのアンピシリンを含む LB寒天培地に接 種することにより トランスフオームした大腸菌を分離し、 アル力リ法により各ク ローンのプラスミ ド DN Aを調整した。 この中から上記 6.50kb断片が組み込まれた プラスミ ド D N Aを制限酵素 Xbalによる切断とァガロース電気泳動法による解析に より選び出した。 遺伝子断片のプラスミ ド DNAへの揷入の方向は図に示した方
^ ^ 向と逆の方向のクローンも得られたが、 本実施例では図に示した方向に挿入され たクローンをクロ一ニングし、 以下の実施例で用いた。 この組換えプラスミ ド上 にクローニングされた 6.50kb p断片が目的の遺伝子であるか否かについてはネコへ ルぺスウィルス C7301 株由来のチミジンキナーゼ遣伝子を含む HincII切断 1.5kb 断片 (Yokoyama et al. , 1995, J. Vet. Med. Sci. , 57 : 709-714 ) を用いた Maeda ら (1992, Arch. Virol. , 127 : 387-397 ) の方法によるハイブリダィゼ一シヨン 法により確認した。 得られた 9.46kbの組換えプラスミ ドは pBS/CTKXbaI6.5と命名し た。 Figure 1 shows the procedure for cloning the gene fragment. That is, the CHV genomic DNA 201 recovered in Example 1 above was treated with 50 units of the restriction enzyme Xbal overnight at 37, followed by agarose gel electrophoresis, and the agarose portion containing the 6.5 kilobase (kb) fragment was removed. The DNA was collected and collected using a Gene Clean Kit (BI0101). This was ligated to a plasmid pBluescript I1SKC +) that had been digested with Xbal and dephosphorylated with calf intestinal alkaline phosphatase (C1AP) using ligation kit (Takara Shuzo Co., Ltd.), and transformed into Escherichia coli DH5a. It was introduced by a transformation reaction. This was inoculated on an LB agar medium containing 50 g / ml of ampicillin to isolate transformed E. coli, and the plasmid DNA of each clone was prepared by the Algari method. Plasmid DNA into which the above 6.50 kb fragment was incorporated was selected from these by cleavage with the restriction enzyme Xbal and analysis by agarose electrophoresis. The direction of insertion of gene fragments into plasmid DNA is as shown in the figure. Although clones in the direction opposite to the ^ ^ direction were obtained, in this example, clones inserted in the directions shown in the figure were cloned and used in the following examples. Whether the 6.50 kbp fragment cloned on this recombinant plasmid was the target gene was determined by a HincII-cleaved 1.5 kb fragment containing the thymidine kinase gene derived from feline herpesvirus C7301 (Yokoyama et al. al., 1995, J. Vet. Med. Sci., 57: 709-714), and confirmed by the hybridization method according to the method of Maeda et al. (1992, Arch. Virol., 127: 387-397). The obtained 9.46 kb recombinant plasmid was designated as pBS / CTKXbaI6.5.
(3) CHVゲノム DNAからの g Cおよび ORF 2遺伝子のクローニング 第 2図に遺伝子断片のクロ—ニング手順を示した。 すなわち、 Limbach ら (1994, J. Gen. Virol. , 75: 2029-2039 ) の報告を参考にして 20残基よりなる下記 の合成 DN Αプライマー、 (3) Cloning of gC and ORF2 genes from CHV genomic DNA FIG. 2 shows the procedure for cloning gene fragments. Specifically, referring to the report of Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039), the following synthetic DN DN primer consisting of 20 residues:
5 ' - CGAGCCCTAATTATTGGTTT- 3 ' と 5 '-CGAGCCCTAATTATTGGTTT- 3' and
5 ' -TACAACTGTTTAATAAAGAC-3 ' 5 '-TACAACTGTTTAATAAAGAC-3'
を作成し、 上記実施例 (1) で抽出した DNAを铸型として、 ポリメラ一ゼチエー ン反応 (PCR) を行った。 反応液は 50 β\ 中に CHV YP11株のゲノム DNA 1 Ug 、 各 2 M の上記の合成 DNAプライマ一、 各 800 のデォキシヌクレオチ ド、 5 1 の酵素に添付された lOXTaqポリメラ一ゼ用バッファ一および 1.25単位の Taqポリメラーゼ (パーキン 'エルマ一ジャパン社) を混合し、 流動パラフィ ン を重層した後、 94°C1分、 50°C1分、 72 2分を 1サイクルとした PCRを 25サイク ル行つた。 塩基配列から予想される 2.28kbの D N A断片をァガ口―ス電気泳動法で 分離しジーンクリーンキッ 卜により回収した。 この DNA断片には 0RF2の他に g C タンパク質をコードする 0RFも含まれている。 この DNA断片を 50//1 の 10mM塩 化マグネシウム、 5πιΜ ジチオスレィ トール、 lmM A T Pを含む 70mM卜リス塩酸緩衝 液、 pH7.6 に溶かし、 20単位の T4ポリヌクレオチドキナーゼを加えて、 37°Cで 1時 間反応させ DNA断片の 5'末端をリン酸化した。 制限酵素 PvuII で消化し CIAPで処 理したプラスミ ド plIC19 の 2.36kb断片と上記のリン酸化した DNA断片をライゲー シヨンキッ トを用いて連結し、 コンビテン卜化した大腸菌 DH5aに形質転換反応に
より導入し、 50 /g/mlのアンピシリンを含む LB寒天培地に接種することにより ト ランスフォームした大腸菌を分離し、 上記 2.28kbp断片が組み込まれたプラスミ ド DN Aを保持した大腸菌よりプラスミ ド DN Aを回収した。 遺伝子断片のプラス ミ ド DN Aへの挿入の方向は平滑末端どうしのライゲーションのため図に示した 方向と逆の方向のクローンも得られたが、 本実施例では図に示した方向のクロー ンをクローニングし、 以下の実施例で用いた。 得られた 4.65kbp のプラスミ ドは p0RF2 と命名した。 Was prepared, and the DNA extracted in Example (1) was used as a 铸 type to perform a polymerase zethiane reaction (PCR). The reaction mixture contains 1 Ug of genomic DNA of the CHV YP11 strain in 50 β \, 2 M each of the above-mentioned synthetic DNA primer, 800 deoxynucleotides each, and lOXTaq polymerase attached to 51 enzymes. After mixing buffer and 1.25 units of Taq polymerase (Perkin 'Elma Japan Co., Ltd.) and overlaying liquid paraffin, 25 cycles of PCR were performed at 94 ° C for 1 minute, 50 ° C for 1 minute, and 722 minutes for 1 cycle. I went. A 2.28 kb DNA fragment predicted from the nucleotide sequence was separated by agarose gel electrophoresis and recovered by Gene Clean Kit. This DNA fragment contains 0RF2, which encodes gC protein, in addition to 0RF2. Dissolve this DNA fragment in 70 mM Tris-HCl buffer, pH7.6 containing 50 // 1 of 10 mM magnesium chloride, 5πι ス レ dithiothreitol, and lmM ATP, add 20 units of T4 polynucleotide kinase, and heat at 37 ° C. The reaction was performed for 1 hour to phosphorylate the 5 'end of the DNA fragment. The 2.36 kb fragment of plasmid plIC19, digested with the restriction enzyme PvuII and treated with CIAP, was ligated with the above phosphorylated DNA fragment using a ligation kit, and used to transform E. coli DH5a, which had been transformed into a combination. The transformed E. coli was isolated by inoculating the LB agar medium containing 50 / g / ml of ampicillin from the transformed E. coli. A was recovered. Although the gene fragment was inserted into plasmid DNA in the direction opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 4.65 kbp plasmid was named p0RF2.
(4) プラスミ ド pBSZCTKXbaI6.5からの トランスファーべクターの構築 (4) Construction of transfer vector from plasmid pBSZCTKXbaI6.5
第 3図にトランスファ一ベクターの構築手順を示した。 すなわち、 実施例 (2) で得られた pBS/CT baI6.5を制限酵素 Xbalで消化しクレノー酵素で D N A末端を平 滑化した後、 ァガロース電気泳動法とジーンクリーンキッ 卜により 6.50kb断片を回 収した。 これを、 制限酵素 PvuII で消化し CIAPで処理したプラスミ ド pUC19 の 2. 36kb断片とライゲーションキッ 卜を用いて連結し、 コンビテン卜化した大腸菌 DH5 αに導入し、 50〃g/mlのアンピシリンを含む LB寒天培地に接種することにより ト ランスフォームした大腸菌を分離し、 上記 6.50kb断片が組み込まれたプラスミ ド DNAを保持した大腸菌よりこれを回収した。 遣伝子断片のプラスミ ド DNAへ の挿入の方向は平滑末端どうしのライゲーションのため図に示した方向と逆の方 向のクローンも得られたが、 本実施例では図に示した方向のクローンをクロ一二 ングし、 以下の実施例で用いた。 得られた 8.87kbのプラスミ ドは pUC(dl- P) CTKXbal6.5 と命名した。 Fig. 3 shows the procedure for constructing the transfer vector. That is, the pBS / CT baI6.5 obtained in Example (2) was digested with the restriction enzyme Xbal, the DNA ends were smoothed with the Klenow enzyme, and then the 6.50 kb fragment was purified by agarose gel electrophoresis and gene clean kit. Recovered. This was ligated with a 2.36 kb fragment of plasmid pUC19 digested with the restriction enzyme PvuII and treated with CIAP using a ligation kit, and introduced into a combined E. coli DH5α, and 50 μg / ml of ampicillin was added. The transformed Escherichia coli was inoculated by inoculating the LB agar medium containing the plasmid, and recovered from Escherichia coli holding the plasmid DNA having the 6.50 kb fragment incorporated therein. Although the insertion direction of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was used in the following examples. The obtained 8.87 kb plasmid was designated as pUC (dl-P) CTKXbal6.5.
このプラスミ ド pUC(dl - P)CTKXbaI6.5 を制限酵素 EcoRl と Hindlll で消化して 8. 36kbと 0.50kbの 2つの断片に分割し、 ァガロース電気泳動法で分離しジーンクリ一 ンキッ トにより 8.36kbp 断片を得た。 これとは別に pUC19 を制限酵素 EcoRI と Hindi II で消化し、 マルチクロ一ニングサイ トを含む 57ベースペア(bp)の断片を調 製し、 上の 8.36kbp断片とライゲーションキッ トを用いて連結した。 これをコンビ テント化した大腸菌 5αに形質転換反応により導入し、 50 g/nilのアンピシリ ン を含む L B寒天培地に接種することにより トランスフォームした大腸菌を分離し、 8.36kb断片にマルチクローニングサイ 卜の組み込まれたプラスミ ド DNAを保持し
た大腸菌よりこれを回収した。 得られた 8. 41kbのプラスミ ドは pCTKdlEH/MCSと命名 した。 This plasmid pUC (dl-P) CTKXbaI6.5 was digested with the restriction enzymes EcoRl and Hindlll, split into two fragments of 8.36 kb and 0.50 kb, separated by agarose gel electrophoresis, and 8.36 kbp by Gene Clean Kit. A fragment was obtained. Separately, pUC19 was digested with restriction enzymes EcoRI and Hindi II to prepare a 57 base pair (bp) fragment containing a multi-cloning site, and ligated to the above 8.36 kbp fragment using a ligation kit. This was introduced into the transformed E. coli 5α by a transformation reaction, and the transformed E. coli was isolated by inoculation on an LB agar medium containing 50 g / nil of ampicillin. Retains integrated plasmid DNA This was recovered from Escherichia coli. The 8.41 kb plasmid obtained was named pCTKdlEH / MCS.
( 5 ) 卜ランスファーべクタ一 pCTKdlEH/MCSへの —ガラク トシダーゼ (beta -gal) 発現単位の挿入 (5) Insertion of a galactosidase (beta-gal) expression unit into the transfer vector pCTKdlEH / MCS
プラスミ ドの構築手順を第 4図に示した。 すなわち、 プラスミ ド pCHl lO (フアル マシア社) を制限酵素 Tthlll l と BamHI で消化しクレノ一酵素で処理して D N A断 片の末端を平滑化し、 4. 24kbp の beta-gal遺伝子を含む D N A断片をァガロース電 気泳動法とジーンクリーンキッ トにより回収した。 この断片は beta - galタンパク質 をコードしている ORF (beta-gal-ORF) の上流に SV40の初期プロモータ一 (prom) を持ち、 同じく下流には SV40のポリアデニレーシヨ ンシグナル(poly(A) ) を持つ beta-gal発現単位 (Lac Z cassette) として回収される。 さらに、 実施例 (4 ) で 作出したトランスファ一べクタ一 ( 11(11£}1/»^3を制限酵素^11£111 1 で切断後クレ ノー酵素で末端を平滑化し、 CIAP処理を施す。 このトランスファ一ベクターと上で 回収した 4. 24kbp の beta- gal発現単位をライゲーシヨンキッ トを用いて連結し、 コ ンピテント化した大腸菌 DH5なに形質転換反応により導入し、 のアンピシ リンを含む L B寒天培地に接種することにより トランスフォームした大腸菌を分 離し、 beta- gal発現単位の組み込まれたトランスファーベクター D N Aを保持した 大腸菌よりこれを回収した。 遺伝子断片のプラスミ ド D N Aへの挿入の方向は平 滑末端どうしのライゲーションのため図に示した方向と逆の方向のクローンも得 られたが、 本実施例では図に示した方向のクローンをクローニングし、 以下の実 施例で用いた。 得られた 12. 66kbpのプラスミ ドは pCTKdlEH/Lac Zと命名した。 Figure 4 shows the procedure for constructing the plasmid. That is, the plasmid pCHllO (Pharmacia) was digested with the restriction enzymes Tthllll and BamHI, treated with Kleno enzyme to blunt the ends of the DNA fragments, and the DNA fragment containing the 24-kbp beta-gal gene was digested. Recovered by agarose gel electrophoresis and Gene Clean Kit. This fragment contains the SV40 early promoter (prom) upstream of the ORF (beta-gal-ORF) encoding the beta-gal protein, and also downstream of the SV40 polyadenylation signal (poly (A)). ) Is collected as a beta-gal expression unit (Lac Z cassette). Furthermore, after cutting the transfer vector (11 (11 £) 1 / »^ 3 with the restriction enzyme ^ 11 £ 111 1) created in Example (4), the ends are blunted with Klenow enzyme, and then subjected to CIAP treatment. The transfer vector and the 4.24 kbp beta-gal expression unit recovered above were ligated using a ligation kit, and introduced into a competent Escherichia coli DH5 cell by a transformation reaction. The transformed E. coli was isolated by inoculating the LB agar medium containing the E. coli, and recovered from the E. coli harboring the transfer vector DNA containing the beta-gal expression unit. As a result, clones in the direction opposite to the direction shown in the figure were also obtained due to ligation between smooth ends, but in this example, clones in the direction shown in the figure were cloned, and Was used in the real 施例. Plasmid obtained 12. 66Kbp was named pCTKdlEH / Lac Z.
( 6 ) トランスファーベクタ一 pCTKdlEH/MCSへの狂大病ウィルス Gタンパク質発 現単位の挿入 (6) Insertion of mad virus G protein expression unit into transfer vector pCTKdlEH / MCS
プラスミ ドの構築手順を第 5図および第 6図に示した。 すなわち、 特開平 3— 1 3 9 2 8 6号公報に示されているプラスミ ド pUC- ARaG を pUCRaGl. 25と改名し本 例で用いた。 第 5図に示すようにこれを制限酵素 EcoRI と Hindl l l で切断しクレ ノー酵素による処理を施して D N A末端を平滑化した後、 2. 04kbの狂犬病ウィルス Gタンパク質遺伝子をァガロース電気泳動法とジーンクリーンキッ 卜により精製
した。 これを制限酵素 Xbalで切断後、 クレノー酵素による処理を施して D N A末端 を平滑化し CIAP処理したプラスミ ド pCDM8Gnvitorogen 社) の 3, 90kb断片とライ ゲ一シヨンキッ トを用いて連結し、 コンビテント化した大腸菌 MC1061/P3 に形質転 換反応により導入し、 7. 5 g/mlのテトラサイクリンを含む L B寒天培地に接種す ることにより トランスフォームした大腸菌を分離し、 狂犬病ウィルス Gタンパク 質遺伝子の組み込まれたプラスミ ド D N Aを保持した大腸菌よりこれを回収した。 図に示したように PCM8上のヒ トサイ トメガロウィルス前初期プロモーター (CMV -prom) に対して同一の方向に Gタンパク質遗伝子が組み込まれたクローンは制限 酵素 Hindl l l と EcoRl でプラスミ ド D N Aを切断したときに 5. 95kbの断片が出現す るクローンとして選択した。 更に第 6図に示すようにこのようにして作成したプ ラスミ ド pCDM8 - G を制限酵素 Mlulと BamHI で切断しクレノー酵素による処理を施し て D N A末端を平滑化した後、 3. 42kbの狂犬病ウィルス Gタンパク質発現単位 (G cassette) をァガロース電気泳動法とジーンクリーンキッ 卜を用いて精製した。 これとは別に実施例 (4 ) で作成したトランスファ一ベクタ一 pCTKdlEH/MCSを制限 酵素 Xbalで切断後クレノー酵素による末端平滑化と CIAP処理を施し、 これに上記で 得た 3. 42kbの狂犬病ウィルス Gタンパク質発現単位をライゲ一ションキッ トを用い て連結させ、 コンビテント化した大腸菌 DH5 aに形質転換反応により導入し、 50 g, mlのァンピシリンを含む L B寒天培地に接種することにより トランスフォーム した大腸菌を分離し、 上記 3. 42kb断片が組み込まれたプラスミ ド D N Aを保持した 大腸菌よりプラスミ ド D N Aを回収した。 遺伝子断片のプラスミ ド D N Aへの揷 入の方向は平滑末端どうしのライゲ一ションのため図に示した方向と逆の方向の クローンも得られたが、 本実施例では図に示した方向のクローンをクロ一ニング し、 以下の実施例で用いた。 得られた 11. 84 kbのプラスミ ドは pCTKdlEH/RV-G と命 した。 The procedure for constructing the plasmid is shown in Fig. 5 and Fig. 6. That is, the plasmid pUC-ARAG disclosed in JP-A-3-139286 was renamed pUCRaGl.25 and used in this example. As shown in Fig. 5, this was cut with the restriction enzymes EcoRI and HindII and treated with Klenow enzyme to make the DNA ends blunt. After that, the 2.0 kb rabies virus G protein gene was subjected to agarose gel electrophoresis and gene Purified by clean kit did. This was digested with the restriction enzyme Xbal, treated with Klenow enzyme to make the DNA end blunted, and ligated with a 3,90 kb fragment of plasmid (pCDM8Gnvitorogen), which had been treated with CIAP, using a ligation kit to form a combination. Escherichia coli MC1061 / P3 was introduced by a transformation reaction, and the transformed Escherichia coli was isolated by inoculation on LB agar medium containing 7.5 g / ml tetracycline, and the rabies virus G protein gene was integrated. This was recovered from Escherichia coli retaining plasmid DNA. As shown in the figure, clones in which the G protein 遗 gene is integrated in the same direction with respect to the human cytomegalovirus immediate-early promoter (CMV-prom) on PCM8 are plasmid DNA with the restriction enzymes Hindlll and EcoRl. Was selected as a clone in which a 5.95 kb fragment appeared when cleaved. As shown in Fig. 6, the plasmid pCDM8-G prepared in this manner was digested with restriction enzymes Mlul and BamHI, treated with Klenow enzyme, and the DNA ends were blunt-ended. The G protein expression unit (G cassette) was purified using agarose electrophoresis and a gene clean kit. Separately, the transfer vector-pCTKdlEH / MCS prepared in Example (4) was digested with the restriction enzyme Xbal, followed by blunt-end blunting with Klenow enzyme and CIAP treatment, and the 3.42 kb rabies virus obtained above. The G protein expression unit was ligated using a ligation kit, introduced into a transformed E. coli DH5a by a transformation reaction, and inoculated on an LB agar medium containing 50 g, ml of ampicillin, and transformed. And the plasmid DNA was recovered from Escherichia coli holding the plasmid DNA into which the above 3.42 kb fragment had been incorporated. Although the direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The resulting 11.84 kb plasmid was designated pCTKdlEH / RV-G.
( 7 ) トランスファーベクタ一 pCTKdlEH/MCSへの beta- gal発現単位および狂犬病 ウィルス Gタンパク暂発現単位の挿入 (7) Insertion of beta-gal expression unit and rabies virus G protein II expression unit into transfer vector pCTKdlEH / MCS
プラスミ ドの構築手順を第 7図に示した。 すなわち、 実施例 (5 ) で作成した pCTKdlEH/Lac Zを制限酵素 Xbalで切断後クレノー酵素による末端平滑化と CI AP処理
を施し、 これに実施例 (6) で調製した 3.42kbの DN A末端を平滑化した狂犬病ゥ ィルス Gタンパク質発現単位をライゲーションキッ 卜を用いて連結させ、 コンビ テント化した大腸菌 DH5aに形質転換反応により導入し、 50^g/mlのアンピシリン を含む LB寒天培地に接種することにより トランスフォームした大腸菌を分離し、 上記 3.42kbp 断片が組み込まれたプラスミ ド DN Aを保持した大腸菌よりプラスミ ド DN Aを回収した。 遣伝子断片のプラスミ ド DN Aへの挿入の方向は平滑末端 どう しのライゲーションのため図に示した方向と逆の方向のクローンも得られた 力 本実施例では図に示した方向のクローンをクローニングし、 以下の実施例で 用いた。 得られた 16.08 kb のプラスミ ドは pCTKdlEH/Lac Z/RV-G と命名した。 Figure 7 shows the procedure for constructing the plasmid. That is, pCTKdlEH / Lac Z prepared in Example (5) was digested with restriction enzyme Xbal, followed by blunt-ending with Klenow enzyme and CIAP treatment. The rabies virus G protein expression unit of 3.42 kb with blunt-ended DNA ends prepared in Example (6) was ligated using a ligation kit, and transformed into a competent Escherichia coli DH5a. The transformed E. coli was isolated by inoculating the LB agar medium containing 50 ^ g / ml of ampicillin, and the plasmid DNA was isolated from the E. coli harboring the 3.42 kbp fragment-incorporated plasmid DNA. Was recovered. The direction of insertion of the gene fragment into Plasmid DNA was the same as that shown in the figure due to ligation of blunt ends. Was cloned and used in the following Examples. The obtained 16.08 kb plasmid was designated as pCTKdlEH / Lac Z / RV-G.
(8) ィヌジステンパーウィルス F夕ンパク質遺伝子のクローニング (8) Cloning of canine distemper virus F protein protein gene
ローラ一ボトル 3本に Vero細胞を培養して単層の細胞シ一トを形成させ、 これに ィヌジステンパーウィルス(CDV)HK株 (日生研株式会社所蔵) を感染させ、 37°Cで 4日間培養した。 培養後、 培養上清を回収し、 4000回転で 10分間遠心し、 培養上清 中の細胞の破片を取り除いた。 この液をタイプ 19ロータ一 (ベックマン社) を用い て 19000 回転で 2時間遠心し、 沈殿を回収して 5mlの IIDM EDTAを含む 10mMトリス塩 酸緩衝液 (TE緩衝液、 pH7.4 ) に浮遊させた。 これをさらに 3000回転で 10分間遠 心し、 その上清を 60%(«'/W)シユークロース溶液と 24¾(W/W)シュ一クロース溶液の上 に重層し、 SW28ロータ一 (ベックマン社) を用いて 25000 回転で 90分遠心し、 60 ¾(I/W)シユークロース溶液と 24¾(W/W)シユークロース溶液の境界に沈降した成分を ウィルス分画として回収した。 この回収した成分をさらに TE緩衝液で 3倍以上 に希釈し、 再度 SW28ロータ一を用いて 25000 回転で 90分遠心し、 沈殿を精製ウィル スとして回収した。 沈殿は 2ml の TE緩衝液に浮遊させ、 一部を用いて口一リー法 によりそのタンパク質濃度を測定し、 残りの部分は使用時まで - 80°Cで保存した。 Vero cells are cultured in three roller bottles to form a monolayer cell sheet, which is then infected with Inu Distemper Virus (CDV) strain HK (Nissei Laboratories) and stored at 37 ° C. Cultured for days. After the culture, the culture supernatant was collected and centrifuged at 4000 rpm for 10 minutes to remove cell debris in the culture supernatant. This solution is centrifuged at 19,000 rpm for 2 hours using a Type 19 rotor (Beckman), and the precipitate is collected and suspended in 10 mM Tris-chloride buffer (TE buffer, pH 7.4) containing 5 ml of IIDM EDTA. I let it. This was further centrifuged at 3000 rpm for 10 minutes, and the supernatant was overlaid on a 60% («'/ W) sucrose solution and a 24% (W / W) sucrose solution, and the SW28 rotor (Beckman) was used. The mixture was centrifuged at 25,000 rpm for 90 minutes, and the component precipitated at the boundary between the 60¾ (I / W) sucrose solution and the 24¾ (W / W) sucrose solution was recovered as a virus fraction. The recovered components were further diluted 3 times or more with TE buffer, and centrifuged again at 25,000 rpm for 90 minutes using a SW28 rotor to collect the precipitate as a purified virus. The precipitate was suspended in 2 ml of TE buffer, and the protein concentration was measured by the oral method using a part of the precipitate, and the remaining part was stored at -80 ° C until use.
C D Vゲノム RN Aを回収するために、 タンパク質量として 1.5mgを含む 250 (il の精製ウィルス液に、 1.5^1 の 1Mトリス塩酸緩衝液 (pH8 ) 6 il の 0.5M EDTA 溶液、 3μ1 の lOmg/ml プロティナーゼ K溶液および 37.5 « 1 の 10% SDS 溶液を加え、 37 で 30分間反応させた。 これをフヱノール: クロロフオルム溶液(1: 1) で 2回抽出操作を行い、 水層の部分を回収してこれに 30 1 の 3M酢酸ナ卜リ ゥ
厶溶液 (pH5.2 ) と lml のエタノールを加え、 - 80°Cで 20分間静置した後、 15000 回転で 10分間遠心して沈殿を回収し、 100^1 の TE緩衝液 (pH8 ) に溶かした。To recover the CDV genomic RNA, 250 mg (1.5 μl of 1 M Tris-HCl buffer (pH 8), 6 μl of 0.5 M EDTA, 3 μl of lOmg / ml Proteinase K solution and 37.5 «1 in 10% SDS solution were added and reacted for 30 minutes at 37. This was extracted twice with a phenol: chloroform solution (1: 1), and the aqueous layer was recovered. 30 1 3M sodium acetate Solution (pH5.2) and lml of ethanol were added. The mixture was allowed to stand at -80 ° C for 20 minutes, centrifuged at 15,000 rpm for 10 minutes to collect the precipitate, and dissolved in 100 ^ 1 TE buffer (pH8). Was.
RN Aの濃度は 280nmの吸光度を測定することにより求めた。 The RNA concentration was determined by measuring the absorbance at 280 nm.
C D Vの Fタンパク質遣伝子のクローニング手順を第 8図および第 9図に示し た。 すなわち、 CDVゲノム RNAより c DNAを以下のようにして合成した。 すなわち、 8 g の RNAに対して、 5 1 の 10X逆転写反応用緩衝液 (0.5Mトリ ス塩酸緩衝液、 pH8.3、 0.5M KC1. 80mM塩化マグネシウム、 0.1Mジチオスレィ ト一 ル) 、 の ΙΟμΜ ランダムプライマ一、 1 1 のリボヌクレアーゼ阻害剤 (38 unit/ 1 、 和光純薬工業株式会社) 、 を加えて総量 43 1 とし、 90°Cで 5分間 静置した後、 氷水中で急冷し、 さらに、 1^1 のリボヌクレア一ゼ阻害剤、 5 1 のデォキシヌクレオチド混合液 (各 lOmM) 、 \μ! の逆転写酵素 (29 unit, 1 、 生化学工業株式会社) を加え、 42°Cで 90分間反応させ c DNAを合成した。 反 応終了後、 ウルトラフリー C3TK (ミ リポア社) を用いてランダムプライマ一を除去 し、 最終溶液量 とした。 The cloning procedure of the CDV F protein gene is shown in FIGS. 8 and 9. That is, cDNA was synthesized from CDV genomic RNA as follows. That is, for 8 g of RNA, 51 of 10X reverse transcription reaction buffer (0.5 M Tris-HCl buffer, pH 8.3, 0.5 M KC 1.80 mM magnesium chloride, 0.1 M dithioleitol) ΙΟμΜ Random primer 1, 11 ribonuclease inhibitor (38 units / 1, Wako Pure Chemical Industries, Ltd.), to make a total amount of 431, leave at 90 ° C for 5 minutes, quench in ice water, In addition, 1 ^ 1 ribonuclease inhibitor, 51 deoxynucleotide mixture (each lOmM) and \ μ! Reverse transcriptase (29 units, 1 by Seikagaku Corporation) The mixture was reacted at C for 90 minutes to synthesize cDNA. After the reaction, the random primer was removed using Ultrafree C3TK (Millipore) to obtain the final solution volume.
C D Vの Fタンパク質遺伝子を PCR法により合成するために、 EMBLデータライブ ラリ一の塩基配列データベースより CDVの全塩基配列を登録している Accession Number L13194 のデータを入手し、 これを参考にして 2本の合成プライマー、 To synthesize the CDV F protein gene by PCR, we obtained the Accession Number L13194 data, which contains the entire CDV nucleotide sequence, from the nucleotide sequence database of the EMBL Data Library. Synthetic primers,
MP2: 5'-CAA (または G) CCATCAGTCCCACAG (または Λ) GA-3' と MP2: 5'-CAA (or G) CCATCAGTCCCACAG (or Λ) GA-3 '
FRP2: 5 ' -GAGATATATGACCAGAATACT-3 ' FRP2: 5'-GAGATATATGACCAGAATACT-3 '
を合成した。 上記で合成した 2^1 の c DNAに対して の lOXPfuポリメラ一 ゼ用バッファ (ポリメラーゼに添付のもの) 、 各 0·5〃1 の 10/ M 合成プライ マー、 5 1 のデォキシヌクレオチド混合液 (各 2mM) 、 14// 1 の蒸留水、 0.5 μ ΐ の Pfu ポリメラーゼ ( 2.5 unit///l ) を加え、 流動パラフィ ンを重層した 後、 94°C90秒、 56°C30秒、 72 5分を 1サイクルとした PCRを 30サイクル行い、 予 想される 2.59kbの DNA断片を PCR産物として得た。 この反応液をフヱノール: ク ロロフオルム(1:1) で抽出操作を施し、 ウルトラフリー C3TKでプライマ一およびデ ォキシヌクレオチドを除去した後、 これを 30/ 1 の lOmM塩化マグネシウム、 5mM ジチオスレィ トール、 6mM A TPを含む 70mMトリス塩酸緩衝液、 pH7.6 に溶かし、
10単位の T4ポリヌクレオチドカイネースを加えて、 37°Cで 1時間反応させ DN A断 片の 5'末端をリン酸化した。 リン酸化した DNA断片は、 制限酵素 Sraa】で切断し紬 菌由来アルカリ性フォスファタ一ゼ (BAP) で脱リン酸化したプラスミ ド pUC19 と、 ライゲーシヨンキッ トを用いて連結し、 コンビテント化した大腸菌 XU- Blueに形質 転換反応により導入し、 50^g/mlのアンピシリンを含む L B寒天培地に接種するこ とにより トランスフォームした大腸菌を分離し、 上記 2.59kb断片が組み込まれたプ ラスミ ド DNAを保持した大腸菌よりプラスミ ド DNAを回収した。 遺伝子断片 のプラスミ ド DN Aへの挿入の方向は平滑末端どうしのライゲ一ションのため図 に示した方向と逆の方向のクローンも得られたが、 本実施例では図に示した方向 のクローンをクローニングし、 以下の実施例で用いた。 得られた 5.27kbのプラスミ ドは pUCl 9-MP2-FRP2と名付けた。 Was synthesized. LOXPfu polymerase buffer (attached to polymerase) for 2 ^ 1 cDNA synthesized above, 0.5 01 of 10 / M synthetic primer and 51 deoxynucleotides mixed Solution (2 mM each), 14 // 1 distilled water, 0.5 μΐ Pfu polymerase (2.5 unit /// l), and overlay with liquid paraffin. 30 cycles of PCR were performed with 5 minutes as one cycle, and the expected 2.59 kb DNA fragment was obtained as a PCR product. The reaction mixture was extracted with phenol: chloroform (1: 1) to remove the primers and deoxynucleotides with Ultrafree C3TK. Then, the reaction mixture was added to 30/1 lOmM magnesium chloride, 5 mM dithiothreitol, 6 mM Dissolve in 70 mM Tris-HCl buffer containing ATP, pH 7.6, Ten units of T4 polynucleotide kinase were added and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment. The phosphorylated DNA fragment was digested with the restriction enzyme Sraa and ligated using a ligation kit to plasmid pUC19, which was dephosphorylated with alkaline phosphatase (BAP) derived from Pseudomonas aeruginosa. Transformed E. coli was introduced into XU-Blue by a transformation reaction and inoculated on LB agar medium containing 50 ^ g / ml of ampicillin to isolate transformed Escherichia coli. Plasmid DNA incorporating the above 2.59 kb fragment was isolated. Plasmid DNA was recovered from the retained E. coli. Although the gene fragment was inserted into the plasmid DNA in the direction opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 5.27 kb plasmid was designated as pUCl9-MP2-FRP2.
さらに、 第 9図に示すように Fタンパク質をコードする 0RF領域 (F- 0RF) のみを リクローニングするために合成プライマー Furthermore, as shown in Fig. 9, synthetic primers were used to reclone only the 0RF region encoding the F protein (F-0RF).
FPfnl-2: 5 ' - CAACACAGCCAAGCCCCATG - 3 ' FPfnl-2: 5 '-CAACACAGCCAAGCCCCATG-3'
を合成し、 5//1 のプラスミ ド pUC19- MP2-FRP2 (\ng/ μΐ ) に対して 2 1 の lOXPfuポリメラーゼ用バッファ (ポリメラーゼに添付のもの) 、 各 0.5 1 の 10 M 合成プライマ一 FPfn卜 2 および FRP2、 ΙβΙ のデォキシヌクレオチド混合液Of 5 // 1 plasmid pUC19-MP2-FRP2 (\ ng / μΐ) to 21 lOXPfu polymerase buffer (attached to the polymerase), 0.51 each of 10 M synthetic primer-FPfn Mixture of DNA 2 and FRP2, ΙβΙ
(各 lOmM) 、 10.5 1 の蒸留水、 0,5 1 の Pfu ポリメラーゼを加え、 流動バラフ ィ ンを重層した後、 94°C60秒、 56°C30秒、 72°C 3分を 1サイクルとした PCRを 20 サイクル行い、 予想される 2.03kbの DNA断片を PCR産物として得た。 この反応液 をフヱノール: クロロフオルム(1:1) で抽出操作を施し、 ウルトラフリー C3TKでプ ライマ一およびデォキシヌクレオチドを除去した後、 これを 30 1 の lOmM塩化マグ ネシゥム、 5mM ジチオスレィ トール、 6mM A T Pを含む 70mMト リ ス塩酸緩衝液(Each lOmM), 10.51 distilled water and 0.51 Pfu polymerase were added, and the flow buffer was overlaid.Then, 94 ° C for 60 seconds, 56 ° C for 30 seconds, and 72 ° C for 3 minutes were defined as one cycle. The PCR was performed for 20 cycles, and the expected 2.03 kb DNA fragment was obtained as a PCR product. The reaction mixture was extracted with phenol: chloroform (1: 1) to remove the primers and deoxynucleotides with Ultrafree C3TK. 70 mM Tris-HCl buffer containing ATP
(pH7.6 ) に溶かし、 10単位の T4ポリヌクレオチドカイネースを加えて、 37°Cで 1 時間反応させ DN A断片の 5'末端をリン酸化した。 リン酸化した DN A断片は、 制 限酵素 Smalで切断し細菌由来アルカリ性フォスファターゼ (BAP) で脱リン酸化した プラスミ ド PUC19 と、 ライゲーシヨンキッ トを用いて連結し、 コンビテント化した 大腸菌 MC1061に形質転換反応により導入し、 50 g 'mlのアンピシリ ンを含む LB寒
天培地に接種することにより トランスフォームした大腸菌を分離し、 上記 2.03kb 断片が組み込まれたプラスミ ド DNAを保持した大腸菌よりプラスミ ド DNAを 回収した。 遺伝子断片のプラスミ ド DNAへの挿入の方向は平滑末端どうしのラ ィゲーションのため図に示した方向と逆の方向のクローンも得られたが、 本実施 例では図に示した方向のクローンをクローニングし、 以下の実施例で用いた。 得 られた 4.71kbp のプラスミ ドは pUC19- FPfn卜 2- FRP2と名付けた。 (pH 7.6), 10 units of T4 polynucleotide kinase were added, and the mixture was reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment. The phosphorylated DNA fragment was ligated using a ligation kit to plasmid PUC19, which had been cleaved with the restriction enzyme Smal and dephosphorylated with bacterial alkaline phosphatase (BAP), and ligated using a ligation kit to form E. coli MC1061 Introduced by transformation and containing 50 g'ml of ampicillin Transformed Escherichia coli was inoculated by inoculating a natural medium, and plasmid DNA was recovered from Escherichia coli holding the plasmid DNA in which the 2.03 kb fragment was incorporated. Clones were inserted in the opposite direction to the direction shown in the figure due to the ligation of blunt ends between gene fragments into plasmid DNA.In this example, clones in the direction shown in the figure were cloned. However, it was used in the following examples. The obtained 4.71 kbp plasmid was named pUC19-FPfn2-FRP2.
クローニングされた DN A断片について市販のサイクルシークェンスキッ 卜 (パーキン · エルマ一 · ジャパン社) および同社のシークェンサ一を用いて塩基 配列を決定し、 すでに報告されている CDV、 Fタンパク質遺伝子のデータ (EMBL データライブラリ一の Accession Number L13194 のデータ) と比較したところ 90 ¾以上のホモロジ一を示したことから目的の遺伝子がクローニングできたと判断し た。 The nucleotide sequence of the cloned DNA fragment was determined using a commercially available cycle sequencing kit (Perkin-Elma Japan) and the company's sequencing kit, and the previously reported CDV and F protein gene data (EMBL) Compared to the data library (Data of Accession Number L13194), it showed a homology of 90 mm or more, indicating that the target gene could be cloned.
さらにブラスミ ド pUC19- FPfn卜 2 - FRP2を制限酵素 BamHI と Saclで切断しクレノー 酵素を用いて DN A末端を平滑化した後、 2.04kbの DNA断片をァガロース電気泳 動法により分離しジーンクリーンキッ トにより回収した。 これを、 制限酵素 Smal で切断し BAP処理により脱リン酸化したプラスミ ド pAcYMl (Matsuura et al. , 1989, Virology, 173 : 674-682 ) にライゲ一シヨンキッ トを用いて連結し、 コン ピテント化した大腸菌 MC1061に形質転換反応により導入し、 50 g/mlのァンピシリ ンを含む L B寒天培地に接種することにより トランスフォームした大腸菌を分離 し、 上記 2, 04kb断片が組み込まれたプラスミ ド DN Aを保持した大腸菌よりプラス ミ ド DNAを回収した。 遣伝子断片のプラスミ ド DNAへの揷入の方向は平滑末 端どうしのライゲーションのため図に示した方向と逆の方向のクローンも得られ たが、 本実施例では図に示した方向のクローンをクロ一ニングし、 以下の実施例 で用いた。 得られた 11.24kb のプラスミ ドは pAcHK- F と命名した。 Furthermore, the plasmid pUC19-FPfn2-FRP2 was digested with the restriction enzymes BamHI and Sacl, the DNA ends were blunt-ended using Klenow enzyme, and the 2.04 kb DNA fragment was separated by agarose electrophoresis to generate a gene clean kit. Collected by the This was ligated to the plasmid pAcYMl (Matsuura et al., 1989, Virology, 173: 674-682), which had been cleaved with the restriction enzyme Smal and dephosphorylated by BAP treatment, using a ligature kit and made competent. Transformed E. coli was introduced into E. coli MC1061 by a transformation reaction and inoculated on LB agar medium containing 50 g / ml ampicillin.The transformed E. coli was separated, and the plasmid DNA containing the 2,04 kb fragment was retained. Plasmid DNA was recovered from the resulting E. coli. Although the direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained. Clones were cloned and used in the examples below. The obtained 11.24 kb plasmid was designated as pAcHK-F.
(9) ィヌジステンパーウィルス H夕ンパク質遺伝子のクローニング (9) Cloning of canine distemper virus H protein protein gene
CDVの Hタンパク質遺伝子のクロ一ニング手順を第 8図に示した。 すなわち、 CDVの Hタンパク質遺伝子を PCR法により合成するために、 EMBLデータライブラ リーの塩基配列データベースより CD Vの全塩基配列を登録している Accession
Number L13194 のデータを参考にして 2本の合成プライマー、 The procedure for cloning the H protein gene of CDV is shown in FIG. In other words, in order to synthesize the CDV H protein gene by PCR, the entire nucleotide sequence of CDV is registered from the nucleotide sequence database of the EMBL data library. Two synthetic primers, referring to the data of Number L13194,
HPfnl : 5'-(^:八0 丁461(じ八じ4八丁6(:1>(;-3'と HPfnl: 5 '-(^: 80-chome 461 (ji-hachi 4-8-chome 6 (: 1 > (;-3'
HRP5: 5 ' -ATGCTGGAGATGGTTTAATTCAATC-3 ' HRP5: 5'-ATGCTGGAGATGGTTTAATTCAATC-3 '
を合成し、 実施例 (8) で合成した 2〃1 の c DNAに対して の lOXPfuポリ メラ一ゼ用バッファ (ポリメラーゼに添付のもの) 、 各 0.5〃1 の 10//M 合成プラ イマ一、 5 1 のデォキシヌクレオチド混合液 (各 2mM) 、 14^1 の蒸留水、 0. 5 /I の Pfu ポリメラーゼを加え、 流動パラフィ ンを重層した後、 94°C90秒、 56°C 30秒、 72°C 5分を 1サイクルとした PCRを 30サイクル行い、 予想される 1.93kbの DNA断片を PCR産物として得た。 この反応液をフヱノール: クロロフオルム(1: 1) で抽出操作を施し、 ウルトラフリー C3TKでプライマーおよびデォキシヌクレオ チドを除去した後、 これを 30// 1 の 10mM塩化マグネシウム、 5mM ジチオスレィ トー ル、 6mM A T Pを含む 70mMトリス塩酸緩衝液、 pH7.6 に溶かし、 10単位の T4ポリヌ クレオチドカイネースを加えて、 37°Cで 1時間反応させ DN A断片の 5'末端をリン 酸化した。 リ ン酸化した DNA断片は、 制限酵素 Smalで切断し BAP で脱リン酸化し たプラスミ ド pUC19 と、 ライゲーシヨンキッ トを用いて連結し、 コンビテント化し た大腸菌 XL1 - Blueに形質転換反応により導入し、 50^g/mlのアンピシリ ンを含む L B寒天培地に接種することにより トランスフォームした大腸菌を分離し、 上記 1.93kb断片 (H-ORF ) が組み込まれたプラスミ ド DN Aを保持した大腸菌よりブラ スミ ド DNAを回収した。 遺伝子断片のプラスミ ド DNAへの挿入の方向は平滑 末端どうしのライゲ一ションのため図に示した方向と逆の方向のクローンも得ら れたが、 本実施例では図に示した方向のクローンをクローニングし、 以下の実施 例で用いた。 得られた 4.62kbのプラスミ ドは pUC19- HPfn HRP5と命名した。 The lOXPfu polymerase buffer (attached to the polymerase) for the 2〃1 cDNA synthesized in Example (8), 0.5〃1 of the 10 // M synthetic primer , A mixture of 51 deoxynucleotides (2 mM each), 14 ^ 1 of distilled water and 0.5 / I of Pfu polymerase, overlayed with liquid paraffin, 94 ° C for 90 seconds, 56 ° C 30 30 cycles of PCR were performed at 72 ° C for 5 minutes per second, and the expected 1.93 kb DNA fragment was obtained as a PCR product. The reaction mixture was extracted with phenol: chloroform (1: 1) to remove the primers and deoxynucleotides with Ultrafree C3TK. The DNA fragment was dissolved in 70 mM Tris-HCl buffer, pH 7.6, added with 10 units of T4 polynucleotide kinase, and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment. The phosphorylated DNA fragment was ligated using a ligation kit to plasmid pUC19, which had been digested with the restriction enzyme Smal and dephosphorylated with BAP, and transformed into a competent E. coli XL1-Blue by a transformation reaction. The transformed E. coli was isolated by inoculating the LB agar medium containing 50 ^ g / ml of ampicillin, and the E. coli harboring the plasmid DNA incorporating the 1.93 kb fragment (H-ORF) was introduced. More plasmid DNA was recovered. Although the direction of insertion of the gene fragment into plasmid DNA was opposite to that shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 4.62 kb plasmid was designated as pUC19-HPfn HRP5.
クローニングされた DN A断片について市販のサイクルシークェンスキッ ト (パーキン ·エルマ一■ ジャパン社) および同社のシークェンサ一を用いて塩基 配列を決定し、 すでに報告されている CD V、 Hタンパク質遺伝子のデータ (EMBL データライブラリーの Accession Number L13194 のデータ) と比較したところ 90 %以上のホモロジ一を示したことから目的の遺伝子がクローニングできたと判断し た。
( 1 0 ) トランスファーベクター pCTKdlEH/MCSへの beta - gal発現単位およびィヌ ジステンパーウィルス Fタンパク質発現単位の揷入 The nucleotide sequence of the cloned DNA fragment was determined using a commercially available cycle sequence kit (Perkin Elmer Japan, Inc.) and the company's sequencer, and the previously reported data for the CD V and H protein genes ( Compared to the EMBL data library (Data of Accession Number L13194), it showed 90% or more homology, indicating that the target gene could be cloned. (10) Introduction of beta-gal expression unit and canine distemper virus F protein expression unit into transfer vector pCTKdlEH / MCS
プラスミ ドの構築手順を第 1 0図に示した。 すなわち、 実施例 (8 ) で作出し たプラスミ ド pAcM- F を制限酵素 BamHI で切断し T4DNAポリメラーゼを用いて D N A末端を平滑化した後、 ァガロース電気泳動法とジーンクリーンキッ トを用 いて 2. 06kbの Fタンパク質遺伝子断片を回収した。 これとは別に、 実施例 (7 ) で 作出したプラスミ ド pCTKdlEH/Lac Z/RV-G から狂犬病ウィルス Gタンパク質遺伝子 を除去する目的でこれを制限酵素 Xbalで切断後クレノ一酵素による末端平滑化と C1AP処理を施し、 これと上で回収した 2. 06kbの Fタンパク質遺伝子断片をライゲ一 シヨンキッ 卜を用いて連結し、 コンビテント化した大腸菌 DH5ひに形質転換反応に より導入し、 50 g/mlのアンピシリンを含む L B寒天培地に接種することにより 卜 ランスフォームした大腸菌を分離し、 Fタンパク質遺伝子断片が組み込まれたプ ラス ミ ド D N Aを保持した大腸菌よりプラスミ ド D N Aを回収した。 遺伝子断片 のプラスミ ド D N Aへの挿入の方向は平滑末端どうしのライゲ一ションのため図 に示した方向と逆の方向のクローンも得られたが、 図に示したようにプラスミ ド 上の 3. 92kbの位置に Fタンパク質 O R F内に存在する EcoRI 部位のくるクローンを 制限酵素 EcoRI による切断像の解析により選択した。 この構築により、 Fタンパク 質 O R Fはヒ トサイ トメガロウィルス前初期プロモー夕一 (CMV- prom) に対して同 一の方向に組み込まれ Gタ ンパク質発現単位の場合と同様に発現単位 (F cassette) として機能する。 得られた 16. 09kbのプラスミ ドは pCTKdlEH/Lac Z/CDV - Fと命名した。 Figure 10 shows the procedure for constructing the plasmid. That is, the plasmid pAcM-F prepared in Example (8) was digested with the restriction enzyme BamHI, the DNA ends were blunted using T4 DNA polymerase, and then agarose electrophoresis and gene clean kit were used. A 06 kb F protein gene fragment was recovered. Separately, in order to remove the rabies virus G protein gene from the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7), this was cut with the restriction enzyme Xbal, and then blunt-ended with Kleno monoenzyme. C1AP treatment was performed, and the 2.06 kb F protein gene fragment recovered above was ligated using a ligation kit, and introduced into a competent Escherichia coli DH5 cell by a transformation reaction to give 50 g / ml. The transformed Escherichia coli was isolated by inoculating the LB agar medium containing ampicillin, and the plasmid DNA was recovered from Escherichia coli carrying the plasmid DNA in which the F protein gene fragment was incorporated. The direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but as shown in the figure, 3. A clone having an EcoRI site present in the ORF of the F protein at a position of 92 kb was selected by analysis of a cleavage image with the restriction enzyme EcoRI. By this construction, the F protein ORF is integrated in the same direction with respect to the human cytomegalovirus immediate-early promoter (CMV-prom), and the expression unit (F cassette as in the case of the G protein expression unit). ). The obtained 16.09 kb plasmid was designated as pCTKdlEH / Lac Z / CDV-F.
( 1 1 ) トランスファーベクタ一 pCTKdlEH/MCSへの beta- gal発現単位およびィヌ ジステンパーウィルス Hタンパク質発現単位の挿入 (11) Insertion of beta-gal expression unit and canine distemper virus H protein expression unit into transfer vector-pCTKdlEH / MCS
プラスミ ドの構築手順を第 1 1図に示した。 すなわち、 実施例 (9 ) で作出し たプラスミ
と PvuI I で切断しクレノー酵素を 用いて D N Aの 5 '末端を平滑化した後、 ァガロース電気泳動法とジーンクリーンキ ッ トを用いて 2. 05kbの Hタンパク質遺伝子断片を回収した。 これとは別に、 実施例 ( 7 ) で作出したプラスミ ド pCTKdlEH/Lac Z/RV-G から実施例 (1 0 ) と同様にこ
れを制限酵素 Xbalで切断後クレノー酵素による末端平滑化と CIAP処理を施し、 これ と上で回収した 2. 05kbの Hタンパク質遺伝子断片をライゲーションキッ 卜を用いて 連結し、 コンビテン ト化した大腸菌 DH5 aに形質転換反応により導入し、 mlのアンピシリンを含む L B寒天培地に接種することにより トランスフォームした 大腸菌を分離し、 Hタンパク質遺伝子断片が組み込まれたプラスミ ド D N Aを保 持した大腸菌よりプラスミ ド D N Aを回収した。 遺伝子断片のプラスミ ド D N A への挿入の方向は平滑末端どうしのライゲーションのため図に示した方向と逆の 方向のクローンも得られたが、 図に示したようにプラスミ ド上の 3. 72kbと 4. 16kb の位置に Hタンパク質 O R F内に存在する EcoRI 部位のくるクローンを制限酵素 EcoRI による切断像の解析により選択し、 プラスミ ド上に Hタンパク質発現単位 (11 cassette) を構築した。 得られた 16. 08kbのプラスミ ドは pCTKdlEH/Lac Z/CDV Hと命名した。 Figure 11 shows the procedure for constructing the plasmid. In other words, the plasmid created in the embodiment (9) After digestion with DNA and PvuII and blunting the 5 'end of the DNA using Klenow enzyme, a 2.5 kb H protein gene fragment was recovered using agarose electrophoresis and a gene clean kit. Separately from this, the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7) was used in the same manner as in Example (10). After digestion with the restriction enzyme Xbal, the ends were blunt-ended with Klenow enzyme and treated with CIAP, and the 2.5 kb H protein gene fragment recovered above was ligated using a ligation kit to obtain a recombinant E. coli DH5. a) Transformed Escherichia coli was isolated by inoculating a LB agar medium containing ampicillin into the LB agar medium containing the H protein gene fragment. Was recovered. The direction of insertion of the gene fragment into plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but a clone of 3.72 kb on the plasmid was obtained as shown in the figure. 4. A clone having an EcoRI site present in the H protein ORF at the position of 16 kb was selected by analyzing the cleavage image with the restriction enzyme EcoRI, and an H protein expression unit (11 cassette) was constructed on the plasmid. The obtained 16.08 kb plasmid was designated as pCTKdlEH / LacZ / CDVH.
( 1 2 ) トランスファーベクター pCTKdlEH/MCSへの beta- gal発現単位およびォ一 エスキー病ウィルスチミ ジンキナーゼ発現単位の挿入 (12) Insertion of beta-gal expression unit and oeskey disease virus thymidine kinase expression unit into transfer vector pCTKdlEH / MCS
プラスミ ドの構築手順を第 1 2図に示した。 すなわち、 2. 8kbのォ一エスキー病 ウィルスチミ ジンキナーゼ遣伝子(PTK) を含むォ一エスキ一病ウィルスゲノムの Pst l-Kpnl 断片をクローニングしたプラスミ ド pPTKは米国 S. Kit 博士 (Division of Biochemical Virology, Baylor College of Medicine, Houston , Texas, USA ) より分与をうけた。 これを、 制限酵素 EcoRI と Hindl l l で切断しクレノー酵素 を用いて D N A末端を平滑化した後、 ァガロース電気泳動法とジーンクリ一ンキ ッ トを用いて 2. 79kbのォーエスキー病ウィルスチミジンキナーゼ遣伝子断片を回収 した。 この断片にはチミジンキナーゼ遺伝子本来のプロモータ一およびポリアデ 二レーシヨ ンシグナルを有し、 発現単位として作動する。 これとは別に、 実施例 ( 5 ) で作成した pCTKdlEH/Lac Zを制限酵素 BamHI で切断後クレノ一酵素による末 端平滑化と CIAP処理による脱リン酸化をを施し、 これと上で回収した 2. 79kbのチミ ジンキナーゼ遺伝子断片をライゲーションキッ トを用いて連結し、 コンビテン 卜 化した大腸菌 Μ5 αに形質転換反応により導入し、 50 g/ralのアンピシリンを含む L B寒天培地に接種することにより トランスフォームした大腸菌を分離し、 F夕
ンパク質遣伝子断片が組み込まれたプラスミ ド DN Aを保持した大腸菌よりブラ スミ ド DNAを回収した。 遣伝子断片のプラスミ ド DNAへの挿入の方向は平滑 末端どうしのライゲーションのため図に示した方向と逆の方向のクローンも得ら れたが、 本実施例では図に示した方向のクローンをクロ一ニングし、 以下の実施 例で用いた。 得られた 15.42kbのプラスミ ドは pCTKdlEH/Lac Z/PTKと命名した。 Figure 12 shows the procedure for constructing the plasmid. That is, a plasmid pPTK obtained by cloning a Pst1-Kpnl fragment of the oeschis disease virus genome containing the 2.8 kb oeschis disease virus thymidine kinase gene (PTK) was obtained from Dr. S. Kit of the United States. Biochemical Virology, Baylor College of Medicine, Houston, Texas, USA). This is digested with EcoRI and Hindlll and the DNA ends are blunted using Klenow enzyme.Then, using agarose gel electrophoresis and Gene Clean Kit, the 2.79 kb Aujeszky's disease virus thymidine kinase gene is digested. The fragments were recovered. This fragment has the original promoter and polyadenylation signal of the thymidine kinase gene and operates as an expression unit. Separately, pCTKdlEH / Lac Z prepared in Example (5) was digested with the restriction enzyme BamHI, followed by blunt-end blunting with Kleno monoenzyme and dephosphorylation by CIAP treatment. A 79 kb thymidine kinase gene fragment was ligated using a ligation kit, introduced into a transformed Escherichia coli に よ り 5α by a transformation reaction, and inoculated on an LB agar medium containing 50 g / ral ampicillin. Isolate the formed E. coli and Blasmid DNA was recovered from E. coli harboring plasmid DNA in which the protein fragment was integrated. The direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but in this example, the clone in the direction shown in the figure was obtained. Was cloned and used in the following Examples. The obtained 15.42 kb plasmid was designated as pCTKdlEH / Lac Z / PTK.
(13) トランスファーべクタ一 pCTKdlEH/MCSへのィヌジステンパーウィルス F 夕ンパク質発現単位あるいはィヌジステンパーウィルス Hタンパク質発現単位の (13) Transfer vector expression unit of canine distemper virus F protein or expression unit of canine distemper virus H protein to pCTKdlEH / MCS
Fタンパク質発現単位を挿入したプラスミ ドの構築手順を第 1 3図に、 また H タンパク質発現単位を揷入したプラスミ ドの構築手順を第 1 4図に示した。 すな わち、 実施例 (6) で作出した pCTKdlEH/RV-Gから狂犬病ウィルス Gタンパク質遺 伝子を除去する目的でこれを制限酵素 Xba 1で切断後ク レノ一酵素による末端平滑化 と CIAP処理を施した 9.79kb断片を調製し、 これと実施例 (10) で調製した 2.06kb の Fタンパク質遗伝子断片、 あるいは実施例 (1 1) で調製した 2.05kbの Hタンパ ク質遺伝子断片をそれぞれライゲーションキッ 卜を用いて連結し、 コンビテン ト 化した大腸菌 DH5aに形質転換反応により導入し、 50 g/nilのアンピシリンを含む LB寒天培地に接種することにより トランスフォームした大腸菌を分離し、 F夕 ンパク質遺伝子断片が組み込まれたプラスミ ド D N Aを保持した大腸菌よりブラ スミ ド DN Aを回収した。 いずれの場合でも遺伝子断片のプラスミ ド DNAへの 挿入の方向は平滑末端どうしのライゲ一ションのため図に示した方向と逆の方向 のクローンも得られたが、 それぞれ図に示した位置に EcoRI 部位の認められるク ローンを制限酵素 EcoRI による切断像の解析により選択した。 Fタンパク質遺伝子 を組み込んだ 11.85kbpのプラスミ ドは pCTKdlEH/CDV- F、 Hタンパク質遺伝子を組 み込んだ 11.84kbのプラスミ ドは pCTKdlEH/CDV- Hと命名した。 Fig. 13 shows the procedure for constructing a plasmid into which the F protein expression unit was inserted, and Fig. 14 shows the procedure for constructing a plasmid into which the H protein expression unit was inserted. In other words, in order to remove the rabies virus G protein gene from pCTKdlEH / RV-G created in Example (6), it was cut with the restriction enzyme Xba1, followed by blunt-end with Cleno enzyme and CIAP. A treated 9.79 kb fragment was prepared, and this was combined with the 2.06 kb F protein gene fragment prepared in Example (10) or the 2.05 kb H protein gene fragment prepared in Example (11). Were ligated using a ligation kit, introduced into the transformed E. coli DH5a by a transformation reaction, and inoculated on an LB agar medium containing 50 g / nil of ampicillin to separate transformed E. coli. The plasmid DNA was recovered from Escherichia coli harboring the plasmid DNA into which the protein gene fragment was integrated. In each case, clones were inserted in the opposite direction to the direction shown in the figure due to ligation of blunt ends with the insertion direction of the gene fragment into the plasmid DNA. Clones with sites were selected by analysis of cleavage images with the restriction enzyme EcoRI. The 11.85kbp plasmid incorporating the F protein gene was designated pCTKdlEH / CDV-F, and the 11.84kb plasmid incorporating the H protein gene was designated pCTKdlEH / CDV-H.
(14) トランスファーベクター p0RF2 への beta- gal発現単位の揷入 (14) Insert beta-gal expression unit into transfer vector p0RF2
プラスミ ドの構築手順を第 1 5図に示した。 すなわち、 実施例 (3) で作出し たプラスミ ド p0RF2 を制限酵素 Pvull で切断し CIAPで処理しておき、 これに実施例 ( 5 ) で調製し D N A末端を平滑化した 4.24kbの beta-gal発現単位 ライゲ—ショ
ンキッ トを用いて連結し、 コンビテント化した大腸菌 DH5aに形質転換反応により 導入し、 50/ g/mlのアンピシリ ンを含む LB寒天培地に接種することにより トラン スフオームした大腸菌を分離し、 上記 4.24kb断片が組み込まれたプラスミ ド DNA を保持した大腸菌よりプラスミ ド DN Aを回収した。 遺伝子断片のプラスミ ド DN Aへの挿入の方向は平滑末端どうしのライゲーションのため図に示した方向 と逆の方向のクローンも得られるが、 本実施例では図に示した方向のクローンを クロ一ニングし、 以下の実施例で用いた。 得られた 8,89kbのプラスミ ドは p0RF2/ Lac Z と命名した。 Figure 15 shows the procedure for constructing the plasmid. That is, the plasmid p0RF2 produced in Example (3) was digested with the restriction enzyme Pvull, treated with CIAP, and prepared in Example (5). Expression unit The transformant was introduced into Escherichia coli DH5a which had been ligated using a kit and transformed into a concomitant strain, and the transformed E. coli was isolated by inoculating the LB agar medium containing 50 / g / ml ampicillin. Plasmid DNA was recovered from Escherichia coli holding plasmid DNA into which the kb fragment had been incorporated. The direction of insertion of the gene fragment into Plasmid DNA may be opposite to the direction shown in the figure due to ligation of blunt ends, but in this example, the clones in the direction shown in the figure are cloned. And used in the following examples. The resulting 8,89 kb plasmid was designated as p0RF2 / LacZ.
(1 5) beta- gal発現単位を CHVゲノムのチミジンキナーゼ遺伝子領域に組み 込んだ組換え C HVの作出 (15) Creation of recombinant CHV incorporating beta-gal expression unit into thymidine kinase gene region of CHV genome
75cm2 のプラスチック製培養用フラスコに形成された MDCK細胞のシートを 0.25 % トリブシンと lmM EDTA を含む生理緩衝食塩水溶液で消化して細胞を培養器表面 から剥離し 20mlの 5¾ FBSを含むィ一ダルの最小必須培地に分散させ 1500回転で 5分 間遠心し、 遠心上清を捨て細胞の沈殿を回収した。 この細胞を 20mlの FBS を含まな いイーグルの最小必須培地に浮遊させ、 再度 1500回転で 5分間遠心し、 遠心上清を 捨て細胞の沈殿を回収した。 細胞の沈殿は 500 1の E.T.バッファー (10mM D- グルコース、 5mMジチオスレィ トールを含む RPMI1640培地) に浮遊させエレク ト口 ポレーシヨン用のキュべッ ト (バイオラッ ド社) に移した。 これに実施例 (5) で作出し lmg/mlに調製したプラスミ ド pCTKdlEH/Lac Zを 30 zl 加え、 氷水中で 5分 間静置した後、 ジーンパルサー (バイオラッ ド社) で 960〃FD (マイクロファラッ ド) 、 0.3kV (キロボルト) /cmの条件で電気パルスをかけ、 再び、 氷水中に 10 分間静置することにより細胞内に DN Aを導入した。 この細胞を直径 6cmのプラス チック製培養用シャーレに移し、 これに 5mlの 10% FBS を含む Eagle の最小必須培 地を添加して 37 の炭酸ガス培養器內で 6〜12時間培養した。 培養後培養液を除 き、 細胞数と等量の感染価を有する CHV, YP11株を接種し、 1時間静置してゥ ィルスを細胞に吸着させた。 吸着後、 の FBS を添加したイーグルの最小必須培地 を 2ml滴下し、 再び、 37°Cの炭酸ガス培養器内で 48時間培養した。 この培養の間の CHV親ウィルスのゲノムの複製過程で、 このゲノム上のチミジンキナーゼ遺伝
子の領域ではエレク トロポレーション法によりすでに細胞に導入されているブラ スミ ド D N Aとの間での相同組換えが起こることが期待でき、 このために新生す る組換え C H Vゲノムを有する組換えウィルスの出現が期待される。 この培養後、 凍結融解を 3回行いさらに超音波破砕機で感染細胞を破砕して 3000回転で 5分間遠 心し、 上清をウィルス液として回収して使用時まで - 80°Cに保存した。 A sheet of MDCK cells formed in a 75 cm 2 plastic culture flask is digested with a physiological buffered saline solution containing 0.25% tribsine and lmM EDTA, the cells are detached from the surface of the incubator, and a lidar containing 20 ml of 5% FBS And centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded to collect the cell precipitate. The cells were suspended in 20 ml of Eagle's minimum essential medium without FBS, centrifuged again at 1500 rpm for 5 minutes, and the centrifuged supernatant was discarded to collect the cell precipitate. The cell pellet was suspended in 5001 ET buffer (RPM1640 medium containing 10 mM D-glucose and 5 mM dithiothreitol) and transferred to a cuvette (BioRad) for electoral poration. To this was added 30 zl of the plasmid pCTKdlEH / Lac Z prepared in Example (5) and adjusted to lmg / ml, allowed to stand in ice water for 5 minutes, and then treated with Gene Pulser (Bio-Rad) at 960 バ イ オ FD ( An electric pulse was applied under the conditions of microfarads) and 0.3 kV (kilovolt) / cm, and the DNA was again introduced into the cells by leaving still in ice water for 10 minutes. The cells were transferred to a plastic culture dish of 6 cm in diameter, to which 5 ml of the minimum essential culture medium of Eagle containing 10% FBS was added, and cultured in 37 carbon dioxide incubators for 6 to 12 hours. After the culture, the culture solution was removed, CHV and YP11 strains having an infectious titer equivalent to the number of cells were inoculated, and allowed to stand for 1 hour to adsorb the virus to the cells. After the adsorption, 2 ml of Eagle's minimum essential medium to which FBS was added was added dropwise, and the cells were cultured again in a carbon dioxide incubator at 37 ° C for 48 hours. During the culture of the CHV parent virus genome during this culture, the thymidine kinase gene In the offspring region, it is expected that homologous recombination with the plasmid DNA already introduced into the cells by the electroporation method will occur. The appearance of the virus is expected. After this culture, the cells were freeze-thawed three times, and the infected cells were disrupted with an ultrasonic disrupter, centrifuged at 3,000 rpm for 5 minutes, and the supernatant was recovered as a virus solution and stored at -80 ° C until use. .
上で回収したウィルス液中に存在する組換えウィルスは以下のブラックアツセ ィによりクローニングした。 すなわち、 プラスチック製 6穴プレートに形成され た MDCK細胞シー卜に 10倍階段希釈したウィルス液を接種し、 1時間静置してウィル スを細胞に吸着させ、 接種ウィルス液を除去した後、 0. 6¾ ァガロースと 2¾ FBS を含むイーグルの最小必須培地をゥヱル当たり 2ml重層し、 ァガロースが固化し た時点でゥヱル当たり 1mlの FBSを含むイーグルの最小必須培地を加えて 37°Cの 炭酸ガス培養器内で 48時間培養した。 培養後、 液層の部分を除去し、 600 g/inl の X- gal と 2¾FBS を含むイーグルの最小必須培地をゥヱル当たり 1ml加えてさらに 37°Cの炭酸ガス培養器内で 12時間培養した。 この培養の後再び液層の部分を除去 し、 beta-gal遣伝子産物の X- gal を基質とした酵素反応により青色を呈したプラッ クをパスツールピぺッ 卜を用いて回収して 1mlの 1%FBS を含むイーグルの最小必須 培地に浮遊させ、 3回の凍結融解と超音波処理を施し、 さらに上記と同様のブラ ックアツセィを再度行い、 ウィルスクローンを純化した。 本実施例は C H Vゲノ ム上のチミジンキナーゼ遺伝子領域が外来核酸配列挿入可能部位であることを示 している。 本実施例で得られた組換えウィルスは CHV(Y)dlTK/Lac Zと命名した。 The recombinant virus present in the virus solution recovered above was cloned by the following black assay. That is, the MDCK cell sheet formed on a plastic 6-well plate was inoculated with a 10-fold serially diluted virus solution, allowed to stand for 1 hour to adsorb the virus to the cells, and the inoculated virus solution was removed. Overlay 2 ml of Eagle's minimum essential medium containing 6 agarose and 2 FBS per well, and add 1 ml of Eagle's minimum essential medium containing 1 ml of FBS per well when the agarose solidifies, and add 37 ° C CO2 incubator. For 48 hours. After the culture, the liquid layer was removed, and 1 ml of Eagle's minimum essential medium containing 600 g / inl of X-gal and 2¾FBS was added per well, followed by further culturing in a carbon dioxide incubator at 37 ° C for 12 hours. After this culture, the liquid layer was again removed, and a blue-colored plaque was recovered using a Pasteur pipet by enzymatic reaction using X-gal, a beta-gal gene product, as a substrate. The cells were suspended in Eagle's minimum essential medium containing 1% FBS, subjected to three freeze-thaw cycles and sonication, and again subjected to the same black assay to purify the virus clones. This example shows that the thymidine kinase gene region on the CHV genome is a site into which a foreign nucleic acid sequence can be inserted. The recombinant virus obtained in this example was named CHV (Y) dlTK / Lac Z.
( 1 6 ) 狂犬病ウィルス Gタンパク質発現単位を C H Vゲノムのチミジンキナー ゼ遗伝子領域に組み込んだ組換え C H Vの作出 (16) Production of recombinant CHV incorporating rabies virus G protein expression unit into thymidine kinase gene region of CHV genome
基本的な操作は実施例 (1 5 ) と同様に行った。 本実施例ではエレク トロポレー シヨン法によって MDCK細胞内に導入するプラスミ ドには実施例 (6 ) で作出したプ ラスミ ド pCTKdlEH/RV- G を使用し、 このプラスミ ドを導入された細胞に感染させる ウィルスとして実施例 (1 5 ) で作出した組換えウィルス CHV(Y)dlTK/Lac Zを使用 した。 この場合には親ウィルスとして使用した CHV(Y)dlTK/Lac Zの beta-gal発現単 位とプラスミ ド D N A上の狂犬病ウィルス Gタンパク質発現単位が相同組換えに
より入れ替わることが期待できる。 このため、 狂犬病ウィルス Gタンパク質発現 単位が組み込まれた組換え C H Vは、 実施例 (1 5 ) に示した X- gal を用いたブラ ックアツセィを行うと親ウィルスの青色のブラックを形成するという表現型から 白色のブラックを形成するという表現型に変異する。 実際、 本実施例では白色の プラックを形成する組換えウィルスをブラックアツセィによりクローニングし、 この組換えウィルス感染細胞に対する抗狂犬病ウィルスゥサギ血清を用いた蛍光 抗体法により狂犬病ウィルス Gタンパク質の発現が確認された。 なお、 抗狂犬病 ウィルスゥサギ血清は、 精製狂犬病ウィルスをフレゥン ドのアジュバン トと共に ゥサギの皮下に接種することにより作成した。 本実施例は組換えウィルス CHV (Y) dlTK/Lac Zがウィルスブラックの色による選別法を可能にし、 目的とする新たな組 換え C H Vを作出する上で有効であることを示している。 本実施例で得られた組 換えウィルスを CHV(Y)dlTK/RV- G と命名した。 The basic operation was performed in the same manner as in Example (15). In this example, the plasmid pCTKdlEH / RV-G produced in Example (6) was used as the plasmid to be introduced into MDCK cells by the electroporation method, and the plasmid-infected cells were infected. The recombinant virus CHV (Y) dlTK / LacZ produced in Example (15) was used as the virus. In this case, the homologous recombination between the beta-gal expression unit of CHV (Y) dlTK / Lac Z used as the parent virus and the rabies virus G protein expression unit on plasmid DNA was performed. We can expect to be replaced more. Therefore, the recombinant CHV into which the rabies virus G protein expression unit is integrated has the phenotype of forming the parent virus blue black when black-assay using X-gal shown in Example (15) is performed. The phenotype changes from white to black. In fact, in this example, the recombinant virus forming a white plaque was cloned by BlackAssy, and the expression of the rabies virus G protein was confirmed by the fluorescent antibody method using the antirabies virus ゥ sera serum against the recombinant virus-infected cells. Was. In addition, anti-rabies virus egret serum was prepared by inoculating subcutaneous rabbits with purified rabies virus together with Freund's adjuvant. This example shows that the recombinant virus CHV (Y) dlTK / Lac Z enables a screening method based on the color of virus black and is effective in producing a new recombinant CHV of interest. The recombinant virus obtained in this example was named CHV (Y) dlTK / RV-G.
( 1 7 ) beta - gal発現単位と汪犬病ウィルス Gタンパク質発現単位、 beta- gal 発現単位とィヌジステンパーウィルス Fタンパク質発現単位、 beta - gal発現単位と ィヌジステンパーウィルス H夕ンパク質発現単位、 あるいは beta-gal発現単位と ォーエスキー病ウィルスチミジンキナーゼ発現単位を C H Vゲノムのチミジンキ ナーゼ遺伝子領域に組み込んだ組換え C H Vの作出 (17) Beta-gal expression unit and the canine disease virus G protein expression unit, beta-gal expression unit and the Inu distemper virus F protein expression unit, beta-gal expression unit and the Inu distemper virus H protein expression unit Or Recombinant CHV in which the beta-gal expression unit and the Aujeszky's disease virus thymidine kinase expression unit are integrated into the thymidine kinase gene region of the CHV genome
本実施例においても基本的な操作は実施例 (1 5 ) と同様に行った。 本実施例 ではエレク トロポレーシヨン法によって MDCK細胞内に導入するプラスミ ドには実施 例 (7 ) で作出したプラスミ ド pCTKdlEH/Lac Z/RV-G 、 実施例 (10) で作出したプ ラスミ ド PCTKdlEH/Lac Z/CDV- F、 実施例 (11) で作出したプラスミ ド pCTKdlEH/Lac Z 'CDV- H、 あるいは実施例 (12) で作出したプラスミ ド pCTKdlEH/Lac Ζ, 'ΡΤΚを使 用し、 このプラスミ ドを導入された細胞に感染させるウィルスとして C H V、 D F D - 6株を使用した。 本実施例では、 親ウィルスである C H V、 D F D— 6 株のチミジンキナーゼ遺伝子とそれぞれのプラスミ ドの 2価の発現単位、 すなわ ち beta- gal発現単位および各ウィルスタンパク質発現単位 (狂犬病ウィルス Gタン パク質、 ィヌジステンパーウィルス Fタンパク質、 ィヌジステンパーウィルス H タンパク質、 あるいはォーエスキー病ウィルスチミジンキナーゼ発現単位) の間
で相同組換えを起こし、 2つの発現単位を組み込まれた組換えウィルスの出現が 期待される。 このようにして新生してくる組換えウィルスは beta- gal遺伝子産物に より、 X- gal を用いたブラックアツセィで青色のブラックを形成する。 実際、 それ ぞれのプラスミ ドを導入して青色のブラックを形成するウィルスクローンが得ら れ、 これらは、 同時にそれぞれのウィルスタンパク質発現単位を導入されている ことが、 組換えウィルス感染細胞に対する抗狂犬病ウィルスゥサギ血清あるいは 抗ィヌジステンパーウィルスゥサギ血清を用いた蛍光抗体法、 あるいはォ一エス キ一病ウィルスチミジンキナーゼ発現単位を組み込んだ組換えウィルスの場合に は組織培養でのゥィルス増殖時に添加した50 £/1111の濃度の5- 1000-2 ' - DEOXYURI INE(IDU) に対する感受性により確認された。 なお、 抗ィヌジステンパー ウィルスゥサギ血清は精製ィヌジステンパーウィルスをフレゥン ドのアジュバン トと共にゥサギの皮下に接種することにより作成した。 本実施例は一力所の外来 核酸配列挿入部位に 2個以上の独立した発現単位を挿入することができることを 示しており、 例えば多価ワクチンの製造法を提供するものである。 本実施例で得 られた組換えウィルスは相同組換えに使用したプラスミ ド pCTKdlEH/Lac Z/RV-G、 pCTKdlEH/Lac Z/CDV- F、 pCTKdlEH/Lac Z/CDV - H、 pCTKdlEH/Lac Z/PTKに対応して CHV (D)dlTK/Lac Z/RV-G、 CHV(D)dlTK/Lac Z/CDV - F、 CHV(D)dlT /Lac Z/CDV - H、 ある いは CHV(D)dlTK/Lac Z/PTKと命名した。 In this example, the basic operation was performed in the same manner as in Example (15). In this example, the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7) and the plasmid P CTKdlEH created in Example (10) were introduced into MDCK cells by the electroporation method. / Lac Z / CDV-F, the plasmid pCTKdlEH / Lac Z 'CDV-H created in Example (11) or the plasmid pCTKdlEH / Lac Ζ,' ΡΤΚ created in Example (12). CHV, DFD-6 strain was used as a virus to infect cells into which this plasmid had been introduced. In this example, the parent virus CHV, the thymidine kinase gene of the DFD-6 strain and the bivalent expression unit of each plasmid, ie, beta-gal expression unit and each virus protein expression unit (rabies virus G protein) Protein, canine distemper virus F protein, canine distemper virus H protein, or Aujeszky's disease virus thymidine kinase expression unit) It is expected that homologous recombination will occur and that a recombinant virus incorporating two expression units will appear. The recombinant virus emerging in this manner forms a blue black on the black-assay using X-gal due to the beta-gal gene product. In fact, virus plasmids that form blue black by introducing their respective plasmids were obtained. These virus clones, which were simultaneously introduced with their respective viral protein expression units, were found to be resistant to recombinant virus-infected cells. Fluorescent antibody method using rabies virus ゥ heron serum or anti-innus temper virus ゥ sera serum, or added to recombinant virus that incorporates a unit of expression of thymidine kinase of oeskinosis virus during virus propagation in tissue culture Confirmed by sensitivity to 5-1000-2'-DEOXYURI INE (IDU) at a concentration of 50 £ / 1111. In addition, the anti-indistemper virus peregret serum was prepared by inoculating subcutaneous persimmons of purified canine distemper virus with Freund's adjuvant. This example shows that two or more independent expression units can be inserted into a single site of a foreign nucleic acid sequence insertion site, and provides, for example, a method for producing a multivalent vaccine. The recombinant viruses obtained in this example were the plasmids pCTKdlEH / Lac Z / RV-G, pCTKdlEH / Lac Z / CDV-F, pCTKdlEH / Lac Z / CDV-H, pCTKdlEH / Lac Z used for homologous recombination. / PTK corresponding to CHV (D) dlTK / Lac Z / RV-G, CHV (D) dlTK / Lac Z / CDV -F, CHV (D) dlT / Lac Z / CDV -H, or CHV ( D) It was named dlTK / Lac Z / PTK.
( 1 8 ) 狂犬病ウィルス G夕ンパク質発現単位、 ィヌジステンパーウィルス Fタ ンパク質発現単位あるいはィヌジステンパーウィルス F夕ンパク質発現単位を C H Vゲノムのチミジンキナーゼ遣伝子領域に組み込んだ組換え C H Vの作出 本実施例においても基本的な操作は実施例 ( 1 5 ) と同様に行ったが、 プラス ミ ド D N Aを導入した後に感染させるウィルスとして実施例 (1 7 ) で作出した 組換えウィルス CHV(D)dlTK/Lac Z/PTKを使用した。 このウィルスはォ一エスキ一病 ウィルスのチミ ジンキナーゼ遺伝子産物を発現し実施例 (1 7 ) で述べたように IDU に感受性を示す。 逆にこの遺伝子を欠損した場合は、 この薬剤に耐性となる。 この性質は C H Vのチミ ジンキナーゼ遺伝子產物では弱い。 そこで実施例 (6 ) で作出したプラスミ ド pCTKdlEH/RV- G 、 実施例 ( 1 3 ) で作出したプラス ミ ド
pCT dlEH/CDV-F, あるいは同じく実施例 (1 3 ) で作出したプラスミ ド pCTKdlEH 'CDV - !fをエレク トロポレーシヨン法により導入した MDCK細胞に、 組換えウィルス CHV (D)dlTK/Lac Z/PTKを感染させ、 出現してくるウィルスをあらかじめ IDU存在下で MDCK細胞に感染させ IDU に耐性を示す新たな組換えウィルスを選択的に増殖させ る。 すなわち、 得られたウィルス液をプラスチック製 12穴プレート上の MDCK細胞 シー トに接種し、 1時間吸着した後、 50 ju g/mlの IDU と 5¾FBS を含むイーグルの最 小必須培地をゥェル当たり lral添加し 37°Cの炭酸ガス培養器内で 24〜48時間培養 し、 凍結融解を 3回行 tゝさらに超音波破砕機で感染細胞を破砕して 3000回転で 5分 間遠心した上清を回収した。 このウィルス液をさらにもう一度、 IDU存在下での選 択培養を行い、 得られたウィルス液について X- gal 存在下でのプラックアツセィを 行った。 本実施例で出現が期待される組換えウィルスはォーエスキー病ゥィルス チミジンキナーゼ発現単位に加えて beta- gal発現単位も欠失するように構築されて いるため、 ブラックアツセィでは白色のブラックとして出現する。 実際、 本実施 例での選択培養を行った後のウィルス液では白色のブラックを形成する組換えゥ ィルスの割合が全ウィルスの 3分の 1と非常に高かった。 本実施例は組換えウイ ルス CHV(D)dlTK/Lac Z/PTKがプラックの色による選択に加えて IDU による選択培養 を可能にし、 容易に新たな組換えウィルスをクローニングする方法を提示してい る。 得られた組換えウィルスは相同組換えに使用したプラスミ ド pCTKdlEH/RV- G、 pCTKdlEH/CDV - F、 あるいは pCTKdlEH/CDV- Hに対応して CHV(D)dlTK/RV - G 、 CHV(D) dlTK/CDV- F、 あるいは CHV(D)dlTK/CDV- Hと命名した。 (18) Recombinant CHV in which rabies virus G protein expression unit, inudistemper virus F protein expression unit, or inudistemper virus F protein expression unit are incorporated into the thymidine kinase gene transfer region of the CHV genome. In this example, the basic operation was performed in the same manner as in Example (15), except that the recombinant virus CHV produced in Example (17) was used as a virus to be infected after introduction of plasmid DNA. (D) dlTK / Lac Z / PTK was used. This virus expresses the thymidine kinase gene product of the oeschis disease virus and is sensitive to IDU as described in Example (17). Conversely, when this gene is deleted, it becomes resistant to this drug. This property is weak in the thymidine kinase gene of CHV. Therefore, the plasmid pCTKdlEH / RV-G created in Example (6), the plasmid created in Example (13), Recombinant virus CHV (D) dlTK / Lac Z / PTK was introduced into MDCK cells into which pCT dlEH / CDV-F or the plasmid pCTKdlEH 'CDV-! f produced in Example (13) was introduced by electroporation. And infect the MDCK cells with the emerging virus in the presence of IDU in advance to selectively grow a new recombinant virus that is resistant to IDU. That is, the obtained virus solution was inoculated on an MDCK cell sheet on a plastic 12-well plate, adsorbed for 1 hour, and then the minimum essential medium of Eagle containing 50 jug / ml of IDU and 5¾FBS was applied to each well. Incubate in a carbon dioxide incubator at 37 ° C for 24 to 48 hours, freeze-thaw three times, crush the infected cells with an ultrasonic homogenizer, and centrifuge at 3,000 rpm for 5 minutes. Collected. This virus solution was again subjected to selective culture in the presence of IDU, and the resulting virus solution was subjected to plaque assay in the presence of X-gal. Since the recombinant virus expected to appear in this example is constructed so as to delete the beta-gal expression unit in addition to the Aujeszky's disease virus thyridine kinase expression unit, it appears as white black in Black Athesi. . In fact, in the virus solution after the selective culture in this example, the ratio of the recombinant virus forming white black was as high as one third of the total virus. In this example, the recombinant virus CHV (D) dlTK / Lac Z / PTK enables selection culture by IDU in addition to selection by plaque color, and provides a method for easily cloning a new recombinant virus. You. The resulting recombinant virus was used in response to the plasmids pCTKdlEH / RV-G, pCTKdlEH / CDV-F, or pCTKdlEH / CDV-H used for homologous recombination, CHV (D) dlTK / RV-G, CHV (D ) Named dlTK / CDV-F or CHV (D) dlTK / CDV-H.
( 1 9 ) beta- gal発現単位を C H Vゲノムの 0RF2領域に組み込んだ組換え C H V の作出 (19) Creation of recombinant CHV with beta-gal expression unit integrated into 0RF2 region of CHV genome
本実施例においても基本的な操作は実施例 (1 5 ) と同様に行った。 本実施例 ではエレク トロポレーシヨン法によって MDCK細胞内に導入するプラスミ ドには実施 例 (1 4 ) で作出したプラスミ ド pORF2/Lac Z を使用し、 このプラスミ ドを導入さ れた細胞に感染させるウィルスとして C H V . D F D— 6株を使用した。 本実施 例では C H Vゲノム上の 0RF2遺伝子を分断し、 その部位に beta- gal発現単位が相同 組換えにより挿入されることが期待される。 X-gal 存在下でのブラックアツセィに
より青色のブラックが出現しこれをクローニングし、 CH(D)dlORF2/Lac Z と命名し た。 本実施例は C H Vゲノム上の 0RF2領域もまた外来核酸配列挿入可能部位である ことを示している。 In this example, the basic operation was performed in the same manner as in Example (15). In this example, the plasmid pORF2 / LacZ created in Example (14) was used as the plasmid to be introduced into MDCK cells by the electroporation method, and the virus that infects cells into which this plasmid was introduced was used. CHV. DFD-6 strains were used. In this example, it is expected that the 0RF2 gene on the CHV genome is disrupted, and the beta-gal expression unit is inserted into the site by homologous recombination. In the presence of X-gal A bluer black appeared and was cloned and named CH (D) dlORF2 / Lac Z. This example shows that the 0RF2 region on the CHV genome is also a site into which a foreign nucleic acid sequence can be inserted.
(20) 狂犬病ウィルス Gタンパク質発現単位を組み込んだ組換え C H Vによる狂 犬病ウィルスに対する中和抗体価のィヌにおける誘導 (20) Induction of neutralizing antibody titer against rabies virus in dogs by recombinant CHV incorporating rabies virus G protein expression unit
実施例 (1 6 ) で作出した組換えウィルス CHV(Y)dlTK/RV- G、 C H V , Y P 11 株あるいは市販の不活化狂犬病ワクチン (日生研株式会社) を 1群 2ないし 3匹 のィヌに接種し、 狂犬病ウィルスに対する中和抗体の出現とその抗体価を観察し た。 組換え C H Vは 2 X 103· 75TCID5()を両側の鼻腔内へ、 非組換え C H Vは 2 X 104· 5 TCID ()を両側の鼻腔内へ、 不活化狂犬病ワクチンはその処方箋に従い皮下へ 接種した。 経時的に採取した接種前および接種後の血清を 2倍階段希釈しこれに 等量の 100 TCID5()の狂犬病ウィルスを含む液を混合し 37°Cで 1時間反応させた後、 9 6穴プレー トに 100 ずつ分注し、 これにさらに HmLu- C3 細胞浮遊液 50 1 を添加して炭酸ガス培養器で培養した。 細胞変性効果の出現の有無により血清中 の抗体価を測定した。 接種後の中和抗体価の動きを表 1 に示した。 The recombinant virus CHV (Y) dlTK / RV-G, CHV, YP11 strain produced in Example (16) or a commercially available inactivated rabies vaccine (Nisseiken Co., Ltd.) was used in two or three dogs per group. Then, the appearance of neutralizing antibodies against rabies virus and the antibody titers were observed. Recombinant CHV can to 2 X 10 3 · 75 TCID 5 () on both sides of the nasal cavity, non-recombinant CHV is to 2 X 10 4 · 5 TCID ( ) on both sides of the nasal cavity, inactivated rabies vaccine according to its prescription It was inoculated subcutaneously. Two-fold serial dilutions of the pre-inoculation and post-inoculation sera collected over time, mixed with an equal volume of a solution containing 100 TCID 5 () rabies virus, and allowed to react at 37 ° C for 1 hour. Aliquots of 100 were added to the wells, and HmLu-C3 cell suspension 501 was added to the wells and cultured in a carbon dioxide incubator. The antibody titer in the serum was determined based on the appearance of the cytopathic effect. Table 1 shows the movement of the neutralizing antibody titer after inoculation.
表 1 狂犬病ゥィルスに対する中和抗体価の推移 Table 1 Changes in neutralizing antibody titers against rabies virus
表 1に示すように組み換えゥィルス接種群では接種後 1週目より狂犬病ウイル スに対する中和抗体価が認められ、 接種 4週目における中和抗体価は市販の狂犬 病ワクチンより高かった。 この結果は、 本発明で作出された組換えウィルスが組
み換えベクターワクチンとして有用であることを示している。 産業上の利用可能性 As shown in Table 1, neutralizing antibody titers against rabies virus were observed in the recombinant virus-inoculated group 1 week after inoculation, and the neutralizing antibody titer at 4 weeks after inoculation was higher than that of the commercially available rabies vaccine. This result indicates that the recombinant virus produced in the present invention This shows that it is useful as a recombinant vector vaccine. Industrial applicability
本発明によれば、 C H Vに対する遺伝子組換え技術を用いることにより、 有用 タンパク質の大量生産システム、 動物用ワクチンおよび治療薬の製造のための合 理的な方法を提供し、 動物の健康の回復、 維持、 増進に貢献するという効果が得 られる。
According to the present invention, the use of genetic recombination technology for CHV provides a mass production system of useful proteins, a rational method for the production of veterinary vaccines and therapeutic agents, restoration of animal health, This has the effect of contributing to maintenance and promotion.
Claims
1 . ィヌヘルぺスウィルスゲノム上に存在しない核酸配列をそのゲノム上に挿入 したことを特徴とする組換えィヌヘルぺスウィルス。1. A recombinant canine virus, wherein a nucleic acid sequence that does not exist on the canine virus genome is inserted into the genome.
. 請求項 1 において、 核酸配列の揷入を相同組換え法によって行うことを特徴 とする組換えィヌヘルぺスウィルス。 The recombinant canine virus according to claim 1, wherein the nucleic acid sequence is introduced by a homologous recombination method.
. 請求項 1のィヌヘルぺスウィルスゲノム上に存在しない核酸配列を揷入する 部位がィヌヘルぺスウィルスゲノム上のウィルス増殖に致死的な影響を与えな い領域であることを特徴とする組換えィヌヘルぺスウィルス。 The recombination characterized in that the site for introducing a nucleic acid sequence not present on the canine virus genome according to claim 1 is a region on the canine virus genome that does not have a lethal effect on virus growth. Influenza virus.
. 請求項 3のウィルス増殖に致死的な影響を与えない領域がチミ ジンキナーゼ 遺伝子領域であることを特徴とする組換えィヌヘルぺスウィルス。 4. The recombinant canine herpes virus according to claim 3, wherein the region having no lethal effect on virus growth is the thymidine kinase gene region.
5 . 請求項 3のウィルス増殖に致死的な影響を与えない領域が g C遺伝子の下流 に存在する O R F 2領域であることを特徴とする組換えィヌヘルぺスウィルス。 . 請求項 1のィヌヘルぺスウィルスゲノム上に存在しない核酸配列が、 真核紬 胞内で作動するプロモーターとポリアデ二レ一ションシグナルの間に同一方向 で連結された自律的な発現単位として構築され、 揷入されたことを特徴とする 組換えィヌヘルぺスウイルス。 5. The recombinant canine virus according to claim 3, wherein the region that does not have a lethal effect on virus growth is the ORF2 region located downstream of the gC gene. The nucleic acid sequence that is not present on the canine virus genome of claim 1 is constructed as an autonomous expression unit connected in the same direction between a promoter operating in a eukaryotic cell and a polyadenylation signal. And a recombinant canine herpes virus.
7 . 請求項 6の自律的な発現単位が複数個挿入されたことを特徴とする組換えィ ヌヘルぺスゥィルス。 7. A recombinant Indobacterium virus, wherein a plurality of the autonomous expression units of claim 6 are inserted.
8 . 請求項 6の真核細胞内で作動するプロモーターとしてヒ トサイ トメガロウイ ルスの前初期遺伝子のプロモーターを用いたことを特徵とする組換えィヌヘル ぺスウイルス。 8. A recombinant canine virus, characterized in that the promoter of the immediate early gene of human megalovirus is used as the promoter operable in eukaryotic cells according to claim 6.
9. 請求項 6の真核細胞内で作動するプロモータ一として Simian Virus 40 の初期 遺伝子のプロモーターを用いたことを特徴とする組換えィヌヘルぺスウィルス。 9. A recombinant canine virus, wherein the promoter of the early gene of Simian Virus 40 is used as one of the promoters operating in eukaryotic cells according to claim 6.
1 0 . 請求項 1、 6ないし 9のいずれか一項に記載したィヌヘルぺスウィルスゲ ノム上に存在しない核酸配列が、 病原微生物に由来し動物に対して感染防御能 を誘導する抗原をコードしている核酸配列であることを特徴とする組換えィヌ ヘルぺスゥィルス。
10. The nucleic acid sequence which is not present on the canine virus virus genome according to any one of claims 1, 6 to 9, encodes an antigen which is derived from a pathogenic microorganism and induces an ability to protect an animal against infection. A recombinant nucleic acid sequence.
1 . 請求項 1 0の感染防御能を誘導する抗原をコードしている核酸配列が狂犬 病ウィルス Gタンパク質をコードしている核酸配列であることを特徴とする組 換えィヌヘルぺスゥィルス。 1. The recombinant dog strain of claim 10, wherein the nucleic acid sequence encoding an antigen which induces the ability to protect against infection according to claim 10 is a nucleic acid sequence encoding a rabies virus G protein.
2 . 請求項 1 0の感染防御能を誘導する抗原をコードしている核酸配列がィヌ ジステンパーウィルス Hタンパク質をコードしている核酸配列であることを特 徴とする組換えィヌヘルぺスウィルス。 2. The recombinant canine virus according to claim 10, wherein the nucleic acid sequence encoding an antigen that induces the protective ability against infection is a nucleic acid sequence encoding a canine distemper virus H protein.
3 . 請求項 1 0の感染防御能を誘導する抗原をコードしている核酸配列がィヌ ジステンパーウィルス F夕ンパク質をコ一ドしている核酸配列であることを特 徴とする組換えィヌヘルぺスウィルス。 3. The recombinant dog according to claim 10, wherein the nucleic acid sequence encoding an antigen that induces the protective ability against infection is a nucleic acid sequence encoding a canine distemper virus F protein.ぺ virus.
4 . 請求項 1ないし 1 3のいずれかの組換えィヌヘルぺスウィルスを、 /3—ガ ラク トシダーゼ遺伝子あるいは 5-I0D0- 2 ' - DEOXYURIDINEに感受性を示すチミジン キナーゼ遺伝子を揷入された組換えィヌヘルぺスウィルスを用いて選択するこ とを特徴とする方法。 4. The recombinant canine virus according to any one of claims 1 to 13, wherein a thymidine kinase gene which is sensitive to a / 3-galactosidase gene or 5-I0D0-2'-DEOXYURIDINE is inserted. A method characterized in that the selection is carried out using a canine virus.
5 . 請求項 1 0〜 1 3の組換えィヌヘルぺスウィルスを動物に接種して感染症 に対する防御能を賦与することを特徴とする方法。 5. A method comprising inoculating an animal with the recombinant canine herpesvirus according to any one of claims 10 to 13 to confer protection against infectious diseases.
6 . 請求項 1 0〜 1 3のいずれか一項記載の組換えィヌヘルぺスウィルスを含 有することを特徴とするワクチンの調製方法。 6. A method for preparing a vaccine, comprising the recombinant canine virus according to any one of claims 10 to 13.
7 . 請求項 1 0〜 1 3のいずれか一項記載の組換えィヌヘルぺスウィルスを含 有することを特徴とするワクチン。 7. A vaccine comprising the recombinant canine virus according to any one of claims 10 to 13.
8 . 請求項 1および請求項 6のィヌヘルぺスウィルスゲノム上に存在しない核 酸配列が、 動物に对して治療効果を持つ産物をコードしている核酸配列である ことを特徵とする組換えィヌヘルぺスウィルス。 8. The recombination characterized in that the nucleic acid sequence that is not present on the canine virus genome of claim 1 and claim 6 is a nucleic acid sequence encoding a product having a therapeutic effect on animals. Influenza virus.
9 . 請求項 1 8の組換えィヌヘルぺスウィルスを動物に接種して疾病の治療を することを特徴とする方法。
9. A method comprising treating an animal by inoculating an animal with the recombinant canine virus of claim 18.
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AU15572/97A AU1557297A (en) | 1996-08-28 | 1997-01-31 | Recombinant canine herpesvirus |
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JP8226832A JPH1066578A (en) | 1996-08-28 | 1996-08-28 | Recombinant canine herpes virus |
JP8/226832 | 1996-08-28 |
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WO1998008936A1 true WO1998008936A1 (en) | 1998-03-05 |
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AU (1) | AU1557297A (en) |
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Publication number | Priority date | Publication date | Assignee | Title |
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JPH03139286A (en) * | 1989-10-25 | 1991-06-13 | Nippon Seibutsu Kagaku Kenkyusho | Recombinant baculovirus to produce rabies virus g protein and production of g protein |
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1996
- 1996-08-28 JP JP8226832A patent/JPH1066578A/en active Pending
-
1997
- 1997-01-31 WO PCT/JP1997/000236 patent/WO1998008936A1/en active Application Filing
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JPH03139286A (en) * | 1989-10-25 | 1991-06-13 | Nippon Seibutsu Kagaku Kenkyusho | Recombinant baculovirus to produce rabies virus g protein and production of g protein |
Non-Patent Citations (9)
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AU1557297A (en) | 1998-03-19 |
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