WO1998008936A1 - Herpes-virus canin recombine - Google Patents

Herpes-virus canin recombine Download PDF

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Publication number
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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virus
nucleic acid
recombinant
acid sequence
canine
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PCT/JP1997/000236
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English (en)
Japanese (ja)
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Xuenan Xuan
Kotaro Tuchiya
Susumu Ueda
Takeshi Mikami
Haruki Otsuka
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Nippon Institute For Biological Science
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Priority to AU15572/97A priority Critical patent/AU1557297A/en
Publication of WO1998008936A1 publication Critical patent/WO1998008936A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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

L'invention concerne un herpes-virus canin recombiné pouvant être utilisé comme virus vecteur dans lequel a été introduite une séquence d'acide nucléique qui, normalement, n'apparaît pas dans un vecteur d'herpes-virus canin. L'invention concerne également un herpes-virus canin recombiné, formé par insertion d'une séquence d'acide nucléique, qui, normalement, n'apparaît pas dans le génome de l'herpes-virus canin, dans une région du génome n'ayant pas d'effet létal sur la croissance du virus.
PCT/JP1997/000236 1996-08-28 1997-01-31 Herpes-virus canin recombine WO1998008936A1 (fr)

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AU15572/97A AU1557297A (en) 1996-08-28 1997-01-31 Recombinant canine herpesvirus

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03139286A (ja) * 1989-10-25 1991-06-13 Nippon Seibutsu Kagaku Kenkyusho 狂犬病ウイルスｇ蛋白質を産生する組み換えバキュロウイルス及びｇ蛋白質の製造方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03139286A (ja) * 1989-10-25 1991-06-13 Nippon Seibutsu Kagaku Kenkyusho 狂犬病ウイルスｇ蛋白質を産生する組み換えバキュロウイルス及びｇ蛋白質の製造方法

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Title
ARCH. VIROL., (1992), Vol. 124, KIT S. et al., "Expression of Porcine Pseudorabies Virus Gene by a Bovine Herpesvirus-1 (Infectious Bovine Rhinotracheitis Virus)", p. 1-20. *
GENE, (1987), Vol. 57, DARRELL R. THOMSEN et al., "Pseudorabies Virus as a Live Virus Vector for Expression of Foreign Genes", p. 261-265. *
J. GEN. VIROL., (1994), Vol. 75, No. 8, LIMBACH KEITH J. et al., "Nucleotide Sequence of the Genes Encoding the Canine Herpesvirus gB, gC and gD Homologues", p. 2029-2039. *
J. GEN. VIROL., (January 1996), Vol. 77, No. 1, REMOND MICHELLE et al., "Gene Organization in the UL Region and Inverted Repeats of the Canine Herpesvirus Genome", p. 37-48. *
J. GEN. VIROL., (March 1996), Vol. 77, No. 3, HARDER T.C. et al., "Canine Distemper Virus from Diseased Large Felids: Biological Properties and Phylogenetic Relationships", p. 397-405. *
J. VIROL., (1990), Vol. 64, No. 10, GEORGETTE E. COLE et al., "Recombinant Feline Herpesviruses Expressing Feline Leukemia Virus Envelope and Gag Proteins", p. 4930-4938. *
VIROLOGY, (1992), Vol. 190, No. 2, AJAY K. MALIK et al., "Genetic Analysis of the Herpes Simplex Virus Type 1 UL9 Gene: Isolation of a LacZ Insertion Mutant and Expression in Eukaryotic Cells", p. 702-715. *
VIRUS RES., (1987), Vol. 8, No. 4, BARRETT T. et al., "The Nucleotide Sequence of the Gene Encoding the F Protein of Canine Distemper Virus: A Comparison of the Deduced Amino Acid Sequence with Other Paramyxoviruses", p. 373-386. *
VIRUS RES., (1995), Vol. 39, REMOND MICHELE et al., "Sequence of the Canine Herpesvirus Thymidine Kinase Gene: Taxon-Preferred Amino Acid Residues in the alphaHerpesviral Thymidine Kinases", p. 341-354. *

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