JPH1066578A - Recombinant canine herpes virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new recombinant virus useful for mass production of valuable proteins, vaccines for animals, as e.g. a vector virus, by inserting a nucleic acid sequence nonexistent on a canine herpes virus genome onto the genome. SOLUTION: This new recombinant canine herpes virus is obtained by inserting a nucleic acid sequence nonexistent on a canine herpes virus genome onto a domain having no lethal effect on the viral proliferation on the genome. This virus is useful for the mass production system for valuable proteins and producing vaccines or medicines for animals through its expression, as e.g. a vector virus, in infected cells following linking to an extraneous nucleic acid sequence. Specifically, this recombinant virus is obtained by transferring a recombinant DNA into a canine herpes virus. In this case, the recombinant DNA is prepared by the following process: a genome DNA extracted from a canine herpes virus, the thymidine kinase gene in the genome is cloned, an extraneous nucleic acid sequence is linked to the domain and further bound to e.g. a promoter acting in a eukaryocyte.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the use of canine herpes virus (CHV) as a vector virus. More specifically, the present invention relates to the downstream of various promoters (including the promoter of CHV itself) operating in eukaryotic cells. By inserting one or more expression units linked to a foreign nucleic acid sequence or a part thereof into a CHV genome, a recombinant CHV expressing a product derived from the inserted foreign nucleic acid sequence in infected cells can be obtained. The present invention relates to a method of producing the recombinant CHV, a recombinant CHV produced by the present method, and a method of inoculating an animal with the recombinant CHV to obtain the effect of the inserted foreign nucleic acid sequence.

[0002]

BACKGROUND OF THE INVENTION Virology has been the subject of current virology with much research on its pathogenicity in the early years of its research. Virology, coupled with research into pathogenicity elucidation, aims to make viruses into vaccines,
It has been attenuated or detoxified by a method that requires a long time, that is, long-term passage using animals or tissue culture cells, and is not so reliable. Subsequently, when genetic engineering technology was introduced into virology, it became possible to elucidate the pathogenicity or vaccination of viruses at the molecular level, and vaccines using this technology have been actively developed. On the other hand, using this technique, the production of a recombinant virus in which a nucleic acid sequence (foreign nucleic acid sequence) not present on the genome of a naturally occurring virus (wild-type virus) is inserted, that is, the vectorization of a virus is actively performed. It has become. Recombinant viruses may be used, for example, by constructing a foreign nucleic acid sequence to express in a cell and incorporating it into a vector virus and infecting the recombinant virus in vitro or in a cell of an animal body. It is used to elucidate the function of the product, to produce the gene product, or to inoculate the animal body and to obtain the effect of the foreign nucleic acid sequence introduced into the vector virus. Specifically, the effect when inoculated into an animal body is, for example, a vaccine effect when a gene encoding a protective antigen of various pathogenic microorganisms is introduced as a foreign nucleic acid sequence, a gene encoding a bioactive substance is a foreign nucleic acid sequence. The therapeutic effect when introduced as such is considered. Once a method for producing a virus vector, in other words, a method for producing a recombinant virus, is established, it is possible to produce various types of recombinant viruses according to the purpose using the same method.
In addition, despite the wide range of effects that can be achieved, the methods for producing recombinant viruses that are responsible for them are often the same as the methods for producing wild-type viruses, and many objectives can be achieved with a single production method. Recovery and maintenance of the target animal's health,
It is expected to contribute to promotion.

[0003]

Although some viruses have been vectorized, only adenoviruses are known as vector viruses for canines.

[0004] Viruses belonging to the family Herpesviridae, such as herpes simplex virus type 1 (Malik et al., 1992,
Virology, 190: 702-715), Aujeszky's disease virus (Thomsen et al., 1987, Gene, 57: 261-265), bovine herpesvirus type 1 (Kit et al., 1992, Arch. Viro)
l., 124: 1-20), feline herpesvirus 1 (Coleet)
al., 1990, J. Virol., 64: 4930-4938) have already been developed as vector viruses for expressing foreign nucleic acid sequences in animals. When a herpesviridae virus is developed as a vector virus, the foreign nucleic acid sequence of interest is often inserted into a nonessential gene region identified on the herpesvirus genome, that is, a gene region not essential for the growth of the virus. Gen, one of the inventors of the present invention, conducted intensive studies and revealed the nucleotide sequence of the thymidine kinase gene, which is considered to be one of the candidates for a foreign nucleic acid sequence insertion site in CHV. or,
This region is described in Remond et al. (1995, Virus Res., 39: 341-354).
) Is also analyzing its structure. Furthermore, L
(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. (1996, J. Gen. Viro
l., 77: 37-48) are vigorously determining the nucleotide sequence of a region called UL of the CHV genome. But CH
Although the analysis of the gene structure of V has been energetically advanced in recent years as described above, there is currently no knowledge as to whether or not this CHV can be used as a vector virus.

[0005] In view of such circumstances, the inventors of the present invention aimed at developing a virus vector versatile for canines, aiming at vectorization of canine herpes virus (CHV), and completing the present invention. Reached.

That is, a first object of the present invention is to provide a CHV
To show that there are a plurality of sites for inserting a foreign nucleic acid sequence on a genomic gene, that is, to show that CHV is sufficiently usable as a vector virus, and to provide some methods for determining this position. It is in.

A second object is to establish several 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 for 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 a living body with recombinant CHV. is there.

A fourth object of the present invention 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 use of the present invention. .

Another object of the present invention is to provide a recombinant CHV produced as described above.

[0011]

In order to achieve the above object, the present invention provides a recombinant canine herpes in which a nucleic acid sequence not present on the canine herpesvirus genome (hereinafter referred to as "foreign nucleic acid sequence") is inserted into the genome. Provide a virus. 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 thereto.

The foreign nucleic acid sequence can be introduced into the CHV genome using, for example, the technique of homologous recombination. Therefore, in the present invention, as a first step, any DNA fragment of the CHV genome is cloned on a plasmid DNA,
It was used as a transfer vector described later. Genetic engineering techniques used in cloning and subsequent steps are described in Sambrook et al. (1989, Molecular Cloning: Alabo
ratory manual, 2ndedition, Cold Spring Harbor La
boratory Press, New York).

In the next step, a mutation is introduced into an arbitrary position of the cloned DNA fragment. In this case, if a mutation is introduced in a manner that impairs the function of the gene at that position, it can be determined whether or not the gene is a non-essential gene.

Next, the plasmid DN into which this mutation has been introduced
A is transfected into CHV host cells and cultured for a certain period of time, and then infected with a wild-type virus. Thus D
Culture of cells infected with NA and infected with a wild-type virus is expected to produce CHV when cultured for a certain period of time. In this process, CHV genomic DNA undergoes homologous recombination with a mutated plasmid DNA during its growth. And a virus particle having this recombinant virus genome can be expected. Plasmid D contains some of the viruses produced under these circumstances.
By analyzing whether there is a virus incorporating the mutation introduced on the NA or not, it is possible to determine whether the mutation introduction site on the CHV genome is a site into which a foreign nucleic acid sequence can be inserted. By this method, a plurality of exogenous nucleic acid sequence insertable sites which may be present can be sequentially found.

Once the foreign nucleic acid sequence insertion site on the CHV genome is known, the same technique is applied or improved to obtain the desired foreign nucleic acid sequence in a nucleic acid structure for achieving the purpose, for example, a polynucleic acid in a eukaryotic cell. It is possible to construct a recombinant CHV in which the peptide is constructed with a structure capable of expressing the peptide, and into which the peptide is inserted. The number of foreign nucleic acid sequences that can be inserted into the CHV genome need not be one, and a recombinant CHV into which a plurality of foreign nucleic acid sequences have been inserted can be produced depending on the purpose.

Although CHV is pathogenic in puppies up to 2 weeks of age, it is rarely pathogenic in later dogs, and if such, it has little pathogenicity. Use of a CHV having the above characteristics as a vector virus can effectively contribute to the recovery, maintenance, and promotion of the health of dogs and other animals susceptible to recombinant CHV.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In completing the present invention,
The basic techniques used are genetic engineering techniques and virology techniques. The former technique is described in Sambrook et al. (1989, Mo.
lecular Cloning: A laboratory manual, 2nd editio
n, Cold Spring Harbor Laboratory Press, New Yor
k), and the latter technique has been compiled by the National Institute of Preventive Health Alumni Association (1973, General Review of Experimental Viruses, Revised 2nd Edition, Maruzen Co., Ltd.).

In the first step of carrying out the present invention, a DNA fragment of the CHV genome is cloned. Any CHV strain can be used as the CHV used in the present invention. For example, strain D004 (ATCC VR-552) or strain F205 (ATCC VR-552) can be used.
VR-647). This process is already R
emond et al. (1995, Virus Res., 39: 341-354), Limbach
(1994, J. Gen. Virol., 75: 2029-2039) or R
emond et al. (1996, J. Gen. Virol., 77: 37-48) can be used according to the method reported by them. Alternatively, a synthetic DNA primer can be arbitrarily prepared with reference to a base sequence known to date, and a desired fragment can be amplified by a polymerase chain reaction (PCR) and cloned into an appropriate plasmid DNA. .

Plasmid DNA carrying a 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 can be used as a source of a promoter that operates in eukaryotic cells. Also. As described above, a transfer vector is used when a foreign nucleic acid sequence is inserted into the CHV genome by homologous recombination. The nucleic acid sequence before and after the insertion site on the CHV genome is located on both sides of the foreign nucleic acid sequence, and the genomic sequence is determined by these sequences. Homologous recombination with DNA can be caused. These sequences must be of sufficient length to allow homologous recombination with the CHV genome.

In the next step, a mutation is introduced at any position of the CHV genomic DNA fragment on the transfer vector. Mutation can be introduced by methods such as deletion, insertion, or base substitution. Malik et al. (1992,
Virology, 190: 702-715), Thomsen et al. (1987, Gen.
e, 57: 261-265), Kit et al. (1992, Arch. Virol., 124).
: 1-20), or Cole et al. (1990, J. Virol., 64: 493).
0-4938). Specifically, for example, a β-galactosidase (beta-gal) expression unit having a promoter and a polyadenylation signal is prepared from the plasmid pCH110 (Pharmacia) and inserted into an arbitrary position of the CHV genome fragment on the transfer vector. Thus, a mutation can be introduced. The term expression unit refers to a unit of nucleic acid structure for autonomously expressing a foreign nucleic acid sequence in a eukaryotic cell, and specifically has the same direction as the direction of the foreign nucleic acid sequence, for example, the direction encoding a polypeptide. A structural unit of a nucleic acid sequence in which a promoter operating in a eukaryotic cell is arranged upstream of a foreign nucleic acid sequence in a directional manner and a polyadenylation signal is arranged downstream thereof. The plasmid DNA into which the mutation was introduced was CHV
Along with the genomic DNA of Malik et al. (1992, Virol
ogy, 190: 702-715), Thomsen et al. (1987, Gene, 5).
7: 261-265), Kit et al. (1992, Arch. Virol., 124: 1).
-20), or Cole et al. (1990, J. Virol., 64: 4930-4)
938), and co-transfected into CHV host cells such as MDCK cells. 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 the cells are cultured for several days, in addition to the parental virus in the culture supernatant and the cells, the production of a recombinant virus which is thought to be regenerated by homologous recombination between the CHV genomic DNA and the transfer vector DNA into which the mutation has been introduced is produced. Can be expected. From this, the virus is recovered and the virus clone is separated by a plaque assay. Methods appropriate for the mutations introduced into these viral clones on plasmid DNA, such as those described by Malik et al. (1992, Virology, 190: 702-715), Thom
Sen 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) to analyze whether or not a mutation-introduced virus clone, that is, a recombinant virus, is present. Specifically, for example, the above beta
If a mutation was introduced using the -gal expression unit, 5-bromo-4-chloro-3-indoly
By mixing or layering l-β-D-galactoside (X-gal) in agar or methylcellulose medium, the appearance of the recombinant virus can be easily confirmed as a blue plaque. The probability of introduction of the mutation is sometimes 1 in 1,000 virus clones produced,
The appearance of recombinant CHV can be easily confirmed by using -gal expression unit and X-gal. 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 a plurality of foreign nucleic acid sequence insertion sites present on the CHV genome in the same manner. I can find it. 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 desired nucleic acid sequence into that site. The nucleic acid sequence of interest is constructed, for example, in the structure of the expression unit described above. For example, a promoter for the immediate early gene of human cytomegalovirus, a promoter for the early gene of Simian Virus 40, or a promoter naturally present on the CHV genome are used as the promoter that operates in eukaryotic cells used at this time. 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. The autonomous expression unit thus constructed is inserted into a foreign nucleic acid sequence insertion site on the transfer vector. In order to facilitate selection of the thus constructed transfer vector and CHV or, for example, a recombinant virus, a recombinant CHV in which a beta-gal expression unit has been previously inserted into the same foreign nucleic acid sequence insertion site.
To induce homologous recombination in the cells as described above,
Thereafter, a recombinant virus clone can be selected by applying the method of searching for a foreign nucleic acid sequence insertion site.

The effect of the recombinant CHV produced as described above can be exerted by infecting its host cells, for example, MDCK cells (ATCC CCL34). For example, if the inserted foreign nucleic acid sequence is a rabies virus G protein expression unit, it can be used to produce that protein using tissue culture. Furthermore, the effect of recombinant CHV can be directly obtained in an animal by inoculating the recombinant virus directly into an animal, for example, inoculating intranasally, subcutaneously, intradermally, intravenously, intramuscularly or orally. . For example, when an animal is inoculated with a recombinant CHV into which a rabies virus G protein expression unit has been inserted, an effect of obtaining the ability of the inoculated animal to protect against rabies virus infection can be obtained. This paves the way for vaccine production using recombinant CHV.

As described above, the first use of CHV as a vector virus is considered to be the vector vaccine described above. The foreign nucleic acid sequence that can be inserted for the purpose of this use includes, in addition to the above-mentioned rabies virus G protein gene, for example, the H protein gene and F protein gene of canine distemper virus, and the hexon of canine adenovirus type 1 or type 2. Protein gene or fiber protein gene, parainfluenza virus F protein gene or HN
Examples of the gene include a protein gene and a gene encoding a protective antigen for infection of a pathogen to a host animal to which CHV infection is allowed, such as a dog, such as a VP1 or VP2 protein gene of canine parvovirus.

Further, when CHV is used as a vector virus, it can be used for therapy. In this case, a foreign nucleic acid sequence such as lymphokine,
A case where a gene encoding a bioactive substance such as interferon or cytokine is inserted can be exemplified.

[0024]

The present invention will be described more specifically with reference to the following examples.

(1) Extraction of CHV Genomic DNA MDCK cells suspended in Eagle's minimum essential medium supplemented with 5% fetal bovine serum (FBS) were inoculated into a 6 cm-diameter plastic dish for tissue culture. Culture in a carbon dioxide incubator (37 ° C in the presence of 5% carbon dioxide) until a cell sheet is formed. At the time when the cell sheet is formed, CH having an infectivity greater than or equal to the number of cells forming the sheet, excluding the culture solution
V.YP11 strain (University of Tokyo, possessed by the Department of Veterinary Microbiology) or DFD-6 strain (Nisseiken Co., Ltd.) was inoculated and allowed to stand for 1 hour to adsorb the virus to the cells. 2% of 1% after adsorption
Of Eagle's minimum essential medium to which FBS was added, and cultured again for 22 to 23 hours in a carbon dioxide incubator. After culture
The culture medium was removed, and the MDCK cells were washed once with a phosphate buffered saline, and 1 ml of 0.1 M Tris-HCl buffer (pH 9) containing 1% SDS, 0.1 M NaCl, and 1 mM EDTA was added, and the mixture was allowed to stand at room temperature for 10 minutes. Was placed. Thereafter, 50 μl of a 20 mg / ml pronase solution was added, and 3
It was left at 7 ° C. overnight. This solution was recovered and extracted twice with an equal volume of a phenol / chloroform solution (1: 1) to recover an aqueous layer. 0.1 ml of 3M NaC is added to the collected aqueous solution.
l When the solution and 2 ml of ethanol are added, a filamentous DNA precipitates out.
Washed with 70% ethanol solution, dried, dissolved in 0.1 ml of distilled water and stored at 4 ° C. until use. 30 with this operation
5050 μg of DNA could be recovered.

(2) Thymidine from CHV genomic DNA
Cloning of Fragment Containing Kinase Gene (TK) FIG. 1 shows a procedure for cloning a gene fragment. That is, the CHV genome D recovered in Example (1) above
After treating 20 μl of NA at 37 ° C. overnight with 50 units of restriction enzyme XbaI, agarose gel electrophoresis was performed, and 6.5 kilobases (k
b) The agarose portion containing the fragment was recovered, and the DNA contained therein was recovered using a GeneClean kit (BIO 101). This was ligated with a plasmid pBluescript IISK (+) digested with XbaI and dephosphorylated with calf intestinal alkaline phosphatase (CIAP) using a ligation kit (Takara Shuzo Co., Ltd.) to form competent E. coli D
H5α was introduced by a transformation reaction. This was inoculated on an LB agar medium containing 50 μg / ml of ampicillin to isolate transformed E. coli, and the plasmid DNA of each clone was prepared by an alkaline method. A plasmid DNA into which the above-mentioned 6.50 kb fragment was incorporated was selected from these by cleavage with the restriction enzyme XbaI and analysis by agarose electrophoresis. Although clones in which the gene fragment was inserted into the plasmid DNA in the direction opposite to the direction shown in the figure were obtained, in this example, the clone inserted in the direction shown in the figure was cloned, and the following examples were used. Used in Whether the 6.50 kbp fragment cloned on this recombinant plasmid is the gene of interest includes the thymidine kinase gene derived from feline herpesvirus C7301 strain
HincII digested 1.5 kb fragment (Yokoyama et al., 1995, J. Vet.
Med. Sci., 57: 709-714) using Maeda et al. (1992, Arc
h. Virol., 127: 387-397). The obtained 9.46 kb recombinant plasmid was designated as pBS / CTKXbaI6.5.

(3) gC from CHV genomic DNA
It showed cloning procedure of the gene fragment for cloning Figure 2 beauty ORF2 gene. That is, Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039)
), The following synthetic DN consisting of 20 residues
A primers 5′-CGAGCCCTAATTATTGGTTT-3 ′ and 5′-TACAACTGTTTAATAAAGAC-3 ′ were prepared, and a polymerase chain reaction (PCR) was performed using the DNA extracted in the above Example (1) as a template. The reaction solution contained 1 μg of genomic DNA of CHV YP11 strain in 50 μl.
g, 2 μM each of the above synthetic DNA primers, 800 μ each
M deoxynucleotide, attached to 5 μl enzyme
10X Taq polymerase buffer and 1.25 units of Taq
Polymerase (Perkin-Elmer Japan) was mixed, and liquid paraffin was overlaid.
PCR was carried out for 25 cycles at a temperature of 72 ° C. for 2 minutes. A 2.28 kb DNA fragment predicted from the nucleotide sequence was separated by agarose electrophoresis and recovered by Gene Clean Kit. This DNA fragment contains ORF2 as well as ORF encoding gC protein. This DNA fragment
50 μl of 70 mM Tris-HCl buffer containing 10 mM magnesium chloride, 5 mM dithiothreitol, 1 mM ATP, pH 7.6
And 20 units of T4 polynucleotide kinase was added thereto, followed by reaction at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment. The 2.36 kb fragment of plasmid pUC19 digested with the restriction enzyme PvuII and treated with CIAP was ligated with the above phosphorylated DNA fragment using a ligation kit, and introduced into competent Escherichia coli DH5α by a transformation reaction.
Transformed E. coli was isolated by inoculating on LB agar medium containing μg / ml ampicillin.
Plasmid DNA was recovered from Escherichia coli holding the plasmid DNA into which the 28 kbp fragment had been incorporated. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. Got
The 4.65 kbp plasmid was named pORF2.

(4) The plasmid pBS / CTKXbaI6.5
Construction of Transfer Vector FIG. 3 shows the procedure for constructing the transfer vector. That is, pBS / CTKXbaI6.5 obtained in Example (2) was digested with the restriction enzyme XbaI and the DNA ends were blunt-ended with the Klenow enzyme, and then a 6.50 kb fragment was recovered by agarose electrophoresis and a gene clean kit. 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, introduced into competent Escherichia coli DH5α, and inoculated on LB agar medium containing 50 μg / ml ampicillin. The transformed Escherichia coli was separated and recovered from the Escherichia coli holding the plasmid DNA in which the 6.50 kb fragment was incorporated. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. The resulting 8.87kb plasmid was pUC (dl-P) CTKXb
Named aI6.5.

The plasmid pUC (dl-P) CTKXbaI6.5 was digested with restriction enzymes EcoRI and HindIII to obtain 8.36 kb and 0.50 kb of the plasmid pUC (dl-P) CTKXbaI6.5.
The fragment was divided into two fragments, separated by agarose electrophoresis, and an 8.36 kbp fragment was obtained using a GeneClean kit. Separately, pUC19 was digested with restriction enzymes EcoRI and HindIII to prepare a 57 base pair (bp) fragment containing a multiple cloning site, and ligated to the above 8.36 kbp fragment using a ligation kit. Escherichia coli DH5α which made this competent
Into a LB agar medium containing 50 μg / ml ampicillin to isolate transformed Escherichia coli, which was isolated from an Escherichia coli having a plasmid DNA having a multicloning site integrated with an 8.36 kb fragment. Was recovered. The obtained 8.41 kb plasmid was designated as pCTKdlEH / MCS.

(5) Transfer vector pCTKdlEH / M
Insertion of β-galactosidase (beta-gal) expression unit into CS
The procedure for constructing the input plasmid is shown in FIG. That is, plasmid pCH110 (Pharmacia) was replaced with restriction enzyme Tth111I.
Digestion with BamHI, treatment with Klenow enzyme to blunt the ends of the DNA fragment, and DNA containing the 4.24 kbp beta-gal gene
Fragments were recovered by agarose electrophoresis and GeneClean kit. This fragment contains the SV40 early promoter (prom) upstream of the ORF (beta-gal-ORF) encoding the beta-gal protein, and also contains the SV40 polyadenylation signal (poly (A)) downstream. It is collected as a beta-gal expression unit (Lac Z cassette). Furthermore, the transfer vector pCTKdlEH created in Example (4)
/ MCS is cut with restriction enzyme HindIII, blunt-ended with Klenow enzyme, and subjected to CIAP treatment. This transfer vector and the 4.24 kbp beta-gal expression unit recovered above were ligated using a ligation kit, introduced into competent Escherichia coli DH5α by a transformation reaction, and placed on an LB agar medium containing 50 μg / ml ampicillin. The transformed Escherichia coli was isolated by inoculation, and recovered from the Escherichia coli holding the transfer vector DNA into which the beta-gal expression unit was incorporated. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. The obtained 12.66 kbp plasmid was designated as pCTKdlEH / LacZ.

(6) Transfer vector pCTKdlEH / M
The procedure for constructing the plasmid for inserting the rabies virus G protein expression unit into CS is shown in FIGS. That is, the plasmid pUC-ΔRaG disclosed in JP-A-3-139286 was renamed pUCRaG1.25 and used in this example. As shown in FIG. 5, this was digested with restriction enzymes EcoRI and HindIII.
Then, the DNA terminal was blunt-ended by treatment with Klenow enzyme, and the 2.04 kb rabies virus G protein gene was purified by agarose electrophoresis and a gene clean kit. This was digested with the restriction enzyme XbaI, treated with Klenow enzyme to blunt the DNA ends and ligated with a 3.90 kb fragment of plasmid pCDM8 (Invitorogen), which had been treated with CIAP, using a ligation kit, and made competent Escherichia coli MC1061 / Introduced into P3 by a transformation reaction,
The transformed Escherichia coli was isolated by inoculation on an LB agar medium containing 7.5 μg / ml tetracycline, and recovered from the Escherichia coli carrying the plasmid DNA incorporating the rabies virus G protein gene. As shown in the figure, a clone in which the G protein gene was integrated in the same direction as the human cytomegalovirus immediate-early promoter (CMV-prom) on pCDM8 was obtained by cutting the plasmid DNA with the restriction enzymes HindIII and EcoRI. It was selected as a clone in which a 5.95 kb fragment appeared.
Further, as shown in FIG. 6, the plasmid pCDM8-G thus prepared was digested with restriction enzymes MluI and BamHI, and treated with Klenow enzyme to make the DNA ends blunt.
kb rabies virus G protein expression unit (G cassett
e) was purified using agarose electrophoresis and GeneClean kit. Separately, the transfer vector pCTKdlEH / MCS prepared in Example (4) was digested with the restriction enzyme XbaI, followed by blunt-ending with Klenow enzyme and CIAP treatment, and expression of the 3.42 kb rabies virus G protein obtained above. The units were ligated using a ligation kit, introduced into a competent Escherichia coli DH5α by a transformation reaction, and transformed into Escherichia coli by inoculation on an LB agar medium containing 50 μg / ml ampicillin, and the above 3.42 kb was separated. Plasmid DN incorporating the fragment
Plasmid DNA was recovered from E. coli holding A.
In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. The obtained 11.84 kb plasmid was designated as pCTKdlEH / RV-G.

(7) Transfer vector pCTKdlEH / M
Beta-gal expression unit and rabies virus G tamper to CS
FIG. 7 shows the procedure for constructing the plasmid for inserting the expression unit . That is, pCTKdlEH / Lac Z prepared in Example (5) was cleaved with a restriction enzyme XbaI, followed by blunting of the ends with Klenow enzyme and CIAP treatment,
The rabies virus G protein expression unit prepared by blunting the 3.42 kb DNA end prepared in Example (6) was ligated thereto using a ligation kit, and introduced into competent Escherichia coli DH5α by a transformation reaction. The transformed Escherichia coli was isolated by inoculating the LB agar medium containing ml of ampicillin, and the plasmid DNA was recovered from the Escherichia coli holding the plasmid DNA incorporating the 3.42 kbp fragment. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. 16.08 kb obtained
The plasmid p was named pCTKdlEH / Lac Z / RV-G.

(8) Canine distemper virus F tamper
Cloning of protein genes Vero cells are cultured in three roller bottles to form a monolayer cell sheet, which is then subjected to canine distemper virus (CD
V) Infect HK strain (Nisseiken Co., Ltd.) and
Cultured for days. After the culture, collect the culture supernatant and
Centrifugation was performed for 10 minutes to remove cell debris in the culture supernatant.
This solution was washed with a Type 19 rotor (Beckman).
Centrifuge at 000 rpm for 2 hours, collect the precipitate, and add 5 ml of 1 mM ED
10 mM Tris-HCl buffer containing TA (TE buffer, pH 7.4)
Floated. This is further centrifuged at 3000 rpm for 10 minutes,
The supernatant was layered on a 60% (W / W) sucrose solution and a 24% (W / W) sucrose solution, and centrifuged at 25,000 rpm for 90 minutes using a SW28 rotor (Beckman), and 60% The component precipitated at the boundary between the (W / W) sucrose solution and the 24% (W / W) sucrose solution was collected as a virus fraction. The recovered components are further diluted 3 times or more with TE buffer, and
The mixture was centrifuged at 25,000 rpm for 90 minutes using a SW28 rotor, and the precipitate was collected as a purified virus. The precipitate was suspended in 2 ml of TE buffer, and the protein concentration was measured by the Lowry method using a part of the precipitate, and the remaining part was stored at -80 ° C until use.

To recover CDV genomic RNA, 1.5 μl of 1 M Tris-HCl buffer (pH 8) was added to 250 μl of the purified virus solution containing 1.5 mg as a protein amount.
A 0.5MEDTA solution, 3 µl of a 10 mg / ml proteinase K solution and 37.5 µl of a 10% SDS solution were added, and reacted at 37 ° C for 30 minutes. This is phenol: chloroform solution
The extraction operation was performed twice with (1: 1), the aqueous layer was recovered, 30 μl of a 3M sodium acetate solution (pH 5.2) and 1 ml of ethanol were added thereto, and the mixture was allowed to stand at −80 ° C. for 20 minutes. After 15000
The precipitate was collected by centrifugation for 10 minutes by rotation, and dissolved in 100 μl of TE buffer (pH 8). RNA concentration was determined by measuring absorbance at 280 nm.

The procedure for cloning the F protein gene of CDV is shown in FIG. 8 and FIG. That is, cDNA was synthesized from CDV genomic RNA as follows.
That is, for 8 μg of RNA, 5 μl of 10 × reverse transcription reaction buffer (0.5 M Tris-HCl buffer, pH 8.3, 0.5 M
KCl, 80 mM magnesium chloride, 0.1 M dithiothreitol), 2 μl of 10 μM random primer, 1 μl of ribonuclease inhibitor (38 unit / μl, Wako Pure Chemical Industries, Ltd.) to make a total volume of 43 μl, and at 90 ° C. for 5 minutes After standing, the mixture was rapidly cooled in ice water, and 1 μl of a ribonuclease inhibitor, 5 μl of a mixed solution of deoxynucleotides (10 mM each), and 1 μl of a reverse transcriptase (29 units / μl, manufactured by Seikagaku Corporation) were added. Reaction at 42 ° C for 90 minutes cDN
A was synthesized. After the completion of the reaction, the random primer was removed using Ultrafree C3TK (Millipore) to obtain a final solution volume of 20 μl.

In order to synthesize the CDV F protein gene by the PCR method, the Ac sequence in which the entire nucleotide sequence of CDV is registered from the nucleotide sequence database of the EMBL data library.
Obtain the data of cession Number L13194 and refer to this data for two synthetic primers, MP2: 5'-CAA (or G) CCATCAGTCCCACAG (or
A) GA-3 'and FRP2: 5'-GAGATATATGACCAGAATACT-3' were synthesized. For 2 μl of cDNA synthesized above
2 μl of 10XPfu polymerase buffer (supplied with polymerase), 0.5 μl of each 10 μM synthetic primer,
5 μl deoxynucleotide mixture (2 mM each), 14 μl
l of distilled water, 0.5 μl of Pfu polymerase (2.5 units /
μl) and overlay with liquid paraffin.
30 cycles of PCR were carried out for 30 seconds at 56 ° C. for 30 seconds and 5 minutes at 72 ° C., to obtain an expected 2.59 kb DNA fragment as a PCR product. Phenol: chloroform
Perform extraction operation with (1: 1), remove primer and deoxynucleotide with Ultrafree C3TK, and remove
Dissolve in 30 μl of 70 mM Tris-HCl buffer containing 10 mM magnesium chloride, 5 mM dithiothreitol, and 6 mM ATP, pH 7.6, add 10 units of T4 polynucleotide kinase, react at 37 ° C. for 1 hour, and react for 1 hour at 37 ° C. 'The end was phosphorylated. The phosphorylated DNA fragment was ligated using a ligation kit to plasmid pUC19, which had been cleaved with restriction enzyme SmaI and dephosphorylated with bacterial alkaline phosphatase (BAP), and transformed into competent E. coli XL1-Blue by a ligation kit. The transformed Escherichia coli was isolated by inoculating the LB agar medium containing 50 μg / ml of ampicillin, and the plasmid DN was isolated from Escherichia coli having the plasmid DNA incorporating the 2.59 kb fragment.
A was recovered. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. The resulting 5.27 kb plasmid was pUC1
It was named 9-MP2-FRP2.

Further, as shown in FIG. 9, a synthetic primer FPfnl-2: 5′-CAACACAGCCAAGCCCCATG-3 ′ was synthesized to reclone only the ORF region (F-ORF) encoding the F protein, and 5 μl of the plasmid was synthesized. pUC19-MP2-FRP2 (1 ng / μ
1) 2 μl of 10XPfu polymerase buffer (supplied with the polymerase), 0.5 μl of each of 10 μM synthetic primers FPfnl-2 and FRP2, 1 μl of deoxynucleotide mixture (10 mM each), 10.5 μl of distilled water,
After adding μl of Pfu polymerase and overlaying liquid paraffin, 20 cycles of PCR were performed at 94 ° C for 60 seconds, 56 ° C for 30 seconds and 72 ° C for 3 minutes, and the expected 2.03 kb DNA was obtained.
The fragment was obtained as a PCR product. The reaction solution is phenol:
After performing extraction with chloroform (1: 1) and removing primers and deoxynucleotides with ultra-free C3TK, this was added to 30 μl of a 70 mM Tris-HCl buffer (pH 7.0) containing 10 mM magnesium chloride, 5 mM dithiothreitol, and 6 mM ATP. 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 is
Plasmid pUC19, cut with restriction enzyme SmaI and dephosphorylated with bacterial alkaline phosphatase (BAP), was ligated using a ligation kit, introduced into competent Escherichia coli MC1061 by a transformation reaction, and 50 μg / ml of ampicillin was introduced. The transformed Escherichia coli was isolated by inoculating the LB agar medium containing the plasmid, and the plasmid DNA was recovered from the Escherichia coli holding the plasmid DNA into which the 2.03 kb fragment was incorporated. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. The obtained 4.71 kbp plasmid was named pUC19-FPfnl-2-FRP2. The nucleotide sequence of the cloned DNA fragment was determined using a commercially available cycle sequencing kit (Perkin Elmer Japan) and its sequencer, and the previously reported CDV and F protein gene data (EMBL data library Accession Number L13194
As a result, it was determined that the target gene could be cloned since the homology was 90% or more.

Further, the plasmid pUC19-FPfnl-2-FRP2 was digested with restriction enzymes BamHI and SacI, and DNase was digested with Klenow enzyme.
After blunting the A-terminus, a 2.04 kb DNA fragment was separated by agarose electrophoresis and recovered by Gene Clean Kit. This was cleaved with the restriction enzyme SmaI and dephosphorylated by BAP treatment, pAcYMl (Matsuura et al.
l. 1989, Virology, 173: 674-682), using a ligation kit to make competent E. coli.
Transformed Escherichia coli was introduced into MC1061 by a transformation reaction, and inoculated on an LB agar medium containing 50 μg / ml of ampicillin, the transformed Escherichia coli was separated, and the plasmid DNA was isolated from Escherichia coli holding the plasmid DNA incorporating the 2.04 kb fragment. Collected. Plasmid DNA of gene fragment
Although the clone was inserted in the opposite direction to that shown in the figure due to ligation between blunt ends,
In this example, clones in the directions shown in the figures were cloned and used in the following examples. The obtained 11.24 kb plasmid was designated as pAcHK-F.

(9) Canine distemper virus H tamper
Figure Cloning procedure for H protein gene cloning CDV clauses protein gene 8
It was shown to. That is, the H protein gene of CDV is
Two synthetic primers, HPfnl: 5'-CTCAGGTAGTCCAGCAATGCTC-3, referencing the data of Accession Number L13194 in which the entire nucleotide sequence of CDV is registered from the nucleotide sequence database of the EMBL data library in order to synthesize by the CR method. 'And HRP5: 5'-ATGCTGGAGATGGTTTAATTCAATC-3' was synthesized, 2 µl of cDNA synthesized in Example (8) was used for 2 µl of 10XPfu polymerase buffer (attached to the polymerase), and 0.5 µl of each 10 µM synthetic primer. , 5μl of deoxynucleotide mixture (2m each)
M), 14 μl of distilled water and 0.5 μl of Pfu polymerase were added, and liquid paraffin was overlaid.
30 cycles of PCR were performed at 72 ° C. for 5 minutes per second, and an expected 1.93 kb DNA fragment was obtained as a PCR product. The reaction mixture was extracted with phenol: chloroform (1: 1), and the primers and deoxynucleotides were removed with Ultrafree C3TK.
10 mM magnesium chloride, 5 mM dithiothreitol, 6 mM
Dissolve in 70 mM Tris-HCl buffer containing ATP, pH 7.6,
Add 10 units of T4 polynucleotide kinase, 37 ° C
For 1 hour to phosphorylate the 5 'end of the DNA fragment.
The phosphorylated DNA fragment is digested with restriction enzyme SmaI and BAP
The plasmid pUC19 dephosphorylated in step 1 was ligated with a ligation kit to obtain competent E. coli XLl-
Transformed Escherichia coli was introduced into the LB agar medium containing 50 μg / ml of ampicillin, and the transformed E. coli was isolated by inoculation into Blue.
) Was recovered from E. coli harboring the plasmid DNA into which the plasmid DNA was incorporated. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. 4.62kb obtained
Was named pUC19-HPfnl-HRP5.

The nucleotide sequence of the cloned DNA fragment was determined using a commercially available cycle sequencing kit (Perkin Elmer Japan) and its sequencer, and the previously reported CDV and H protein gene data (EMBL data) Library Acce
ssion Number L13194)
Based on the above homology, it was determined that the target gene could be cloned.

(10) Transfer vector pCTKdlEH
-Gal expression unit and canine distemper ui in M / MCS
FIG. 10 shows the procedure for constructing the insertion plasmid for the Russ F protein expression unit . That is, the plasmid pAcHK-F created in Example (8) was replaced with the restriction enzyme Ba.
After digestion with mHI and blunting of the DNA end using T4 DNA polymerase, a 2.06 kb F protein gene fragment was recovered using agarose electrophoresis and Gene Clean Kit. Separately, in order to remove the rabies virus G protein gene from the plasmid pCTKdlEH / LacZ / RV-G created in Example (7), this was cut with the restriction enzyme XbaI, followed by blunting with Klenow enzyme and CIAP treatment. And the 2.06 kb F protein gene fragment recovered above was ligated using a ligation kit, introduced into competent E. coli DH5α by a transformation reaction, and placed on an LB agar medium containing 50 μg / ml ampicillin. Transformed Escherichia coli was isolated by inoculation, and plasmid DNA was recovered from Escherichia coli holding the plasmid DNA into which the F protein gene fragment was incorporated. As for the direction of insertion of the gene fragment into the plasmid DNA, a clone was obtained in the opposite direction to the direction shown in the figure due to ligation of blunt ends, but as shown in the figure, 3.92 kb on the plasmid
A clone having an EcoRI site present in the F protein ORF at the position of was selected by analyzing the cleavage image with the restriction enzyme EcoRI. Due to this construction, the F protein ORF is converted to the human cytomegalovirus immediate early promoter (CMV-pro
m) is incorporated in the same direction as that of m) and functions as an expression unit (F cassette) as in the case of the G protein expression unit. The resulting 16.09 kb plasmid was pCTKdlEH / Lac Z
/ CDV-F.

(11) Transfer vector pCTKdlEH
-Gal expression unit and canine distemper ui in M / MCS
FIG. 11 shows the procedure for constructing the insertion plasmid for the Rus H protein expression unit . That is, the plasmid pUC19-HPfnl-HRP5 produced in Example (9) was digested with restriction enzymes BamHI and PvuII, and the plasmid was digested with Klenow enzyme.
After blunting the 5 'end of NA, a 2.05 kb H protein gene fragment was recovered using agarose electrophoresis and a GeneClean kit. Separately from this, the plasmid pCTKdlEH / LacZ / RV-G prepared in Example (7) was used to prepare Example (1).
In the same manner as in 0), this was digested with the restriction enzyme XbaI, subjected to blunt-end blunting with Klenow enzyme and CIAP treatment, and the 2.05 kb H protein gene fragment recovered above was ligated using a ligation kit. Escherichia coli DH5α
Into the LB agar medium containing 50 μg / ml ampicillin to isolate transformed Escherichia coli, and recover plasmid DNA from Escherichia coli carrying the plasmid DNA incorporating the H protein gene fragment did. Plasmid DN of gene fragment
A clone was obtained in the direction opposite to the direction shown in the figure due to ligation of blunt ends in the direction of insertion into A. However, as shown in the figure, the H protein was located at 3.72 kb and 4.16 kb on the plasmid. A clone having an EcoRI site present in the ORF was selected by analyzing the cleavage image with the restriction enzyme EcoRI, and the H protein expression unit (Hcass) was placed on the plasmid.
ette). The resulting 16.08 kb plasmid was pC
It was named TKdlEH / Lac Z / CDV-H.

(12) Transfer vector pCTKdlEH
-Gal expression unit and Aujeszky's disease virus in MDS / MCS
FIG. 12 shows the procedure for constructing the insertion plasmid for the stymidine kinase expression unit . That is,
PstI-K of the Aujeszky's disease virus genome containing the 2.8kb Aujeszky's disease virus thymidine kinase gene (PTK)
Plasmid pPTK, into which the pnI fragment was cloned, was purchased from S.K.
Dr. t (Divisionof Biochemical Virology, Baylor Co.
llege of Medicine, Houston, Texas, USA). This was digested with EcoRI and HindIII and blunt-ended with Klenow enzyme, followed by agarose electrophoresis and Gene Clean Kit for 2.79.
A kb of Aujeszky's disease virus thymidine kinase gene fragment was recovered. This fragment has a native promoter of the thymidine kinase gene and a polyadenylation signal, and operates as an expression unit. Separately, pCTKdlEH / Lac Z prepared in Example (5) was cleaved with restriction enzyme BamHI, followed by blunt-end by Klenow enzyme and dephosphorylation by CIAP treatment, and the 2.79 kb recovered above. The thymidine kinase gene fragment was ligated using a ligation kit, introduced into competent Escherichia coli DH5α by a transformation reaction, and LB containing 50 μg / ml ampicillin was added.
The transformed Escherichia coli was isolated by inoculation on an agar medium, and the plasmid DN was isolated from Escherichia coli having the plasmid DNA incorporating the F protein gene fragment.
A was recovered. In the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure were obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned. It was used in the following examples. The resulting 15.42 kb plasmid was pC
It was named TKdlEH / Lac Z / PTK.

(13) Transfer vector pCTKdlEH
Expression of canine distemper virus F protein to MCS / MCS
Position or canine distemper virus H protein expression
FIG. 13 shows the procedure for constructing the plasmid into which the F protein expression unit was inserted, and FIG. 14 shows the procedure for constructing the plasmid into which the H protein expression unit was inserted. That is, rabies virus G was obtained from pCTKdlEH / RV-G produced in Example (6).
This is used to remove the protein gene.
A 9.79 kb fragment prepared by digestion with Klenow enzyme and blunt-ended with CIAP and CIAP treatment was prepared, and this was used to prepare the 2.06 kb F protein gene fragment prepared in Example (10), or the 2.05 kb fragment prepared in Example (11). Each of the kb H protein gene fragments was ligated using a ligation kit, introduced into a competent Escherichia coli DH5α by a transformation reaction, and inoculated into an LB agar medium containing 50 μg / ml ampicillin to transform Escherichia coli. Separate and
Plasmid DN incorporating F protein gene fragment
Plasmid DNA was recovered from E. coli holding A.
In each case, clones were inserted in the opposite direction to the direction shown in the figure due to the ligation of blunt ends in the direction of insertion of the gene fragment into the plasmid DNA, but the EcoRI site was observed at the position shown in the figure. The clones to be obtained were selected by analyzing the cleavage images with the restriction enzyme EcoRI. F
The 11.85kbp plasmid with the protein gene incorporated therein has the pCTKdlEH / CDV-F, H protein gene incorporated therein.
The 1.84 kb plasmid was designated as pCTKdlEH / CDV-H.

(14) To transfer vector pORF2
FIG. 15 shows the procedure for constructing the plasmid for inserting the beta-gal expression unit . That is, the plasmid pORF2 created in Example (3) was replaced with the restriction enzyme PvuI.
The fragment was digested with I, treated with CIAP, and a 4.24 kb beta-gal expression unit prepared in Example (5) and having a blunted DNA end was ligated using a ligation kit to obtain a competent Escherichia coli DH5α. Into the transformant
The transformed E. coli was isolated by inoculating on LB agar medium containing μg / ml ampicillin.
Plasmid DNA was recovered from Escherichia coli holding the plasmid DNA into which the 24 kb fragment had been incorporated. As for the direction of insertion of the gene fragment into the plasmid DNA, clones in the direction opposite to the direction shown in the figure can be obtained due to ligation of blunt ends, but in this example, clones in the direction shown in the figure were cloned, and Was used in Examples. 8.
The 89 kb plasmid was designated as pORF2 / Lac Z.

(15) The expression unit of beta-gal is CHV genome
CH Incorporated into Thymidine Kinase Gene Region
Production of V MD formed in 75 cm ² plastic culture flask
The CK cell sheet is digested with physiological buffered saline solution containing 0.25% trypsin and 1 mM EDTA, the cells are detached from the surface of the incubator, dispersed in 20 ml of Eagle's minimum essential medium containing 5% FBS, and centrifuged at 1500 rpm for 5 minutes Then, the centrifugal supernatant was discarded and the cell precipitate was collected. 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 cells were precipitated with 500 μl of ET buffer (10 mM D-glucose,
The suspension was suspended in an RPMI1640 medium containing 5 mM dithiothreitol) and transferred to a cuvette (Bio-Rad) for electroporation. 30 μl of the plasmid pCTKdlEH / Lac Z prepared in Example (5) and adjusted to 1 mg / ml was added thereto, and allowed to stand in ice water for 5 minutes. Then, 960 μFD (microfarad), 0.3 kV with Gene Pulser (Bio-Rad) was added. An electric pulse was applied under the conditions of (kilovolt) / cm, and the DNA was introduced into the cells by resting again in ice water for 10 minutes. The cells were transferred to a 6 cm diameter plastic culture dish, to which 5 ml of Eagle's minimum essential medium containing 10% FBS was added, and cultured in a carbon dioxide incubator at 37 ° C for 6 to 12 hours. After the culture, the culture solution was removed, the CHV, YP11 strain having an infectious titer equivalent to the cell number was 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 supplemented with 1% FBS was added dropwise, and the cells were cultured again in a carbon dioxide incubator at 37 ° C for 48 hours. C during this culture
During the replication of the genome of the parent HV virus, the plasmid D which has already been introduced into the cells by electroporation in the region of the thymidine kinase gene on this genome.
It can be expected that homologous recombination with NA will occur, which is expected to lead to the emergence of a recombinant virus having a nascent recombinant CHV genome. After this culture, freeze-thaw for 3 hours.
The infected cells were disrupted with an ultrasonic disrupter and 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 plaque 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 after removing the inoculated virus solution, 0.6% agarose was added. And 2% FBS
Overlay 2 ml of Eagle's minimum essential medium per well containing 1 ml per well when agarose has solidified.
Eagle's minimum essential medium containing 1% FBS was added, and the cells were cultured in a carbon dioxide incubator at 37 ° C for 48 hours. After culturing, the liquid layer was removed, and 1 ml of Eagle's minimum essential medium containing 600 μg / ml of X-gal and 2% FBS 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 removed again, and a blue plaque was collected using a Pasteur pipette by enzymatic reaction using the beta-gal gene product X-gal as a substrate, and 1 ml of 1% FBS was added. The cells were suspended in the minimum essential medium of the Eagle containing them, subjected to freeze-thaw and sonication three times, and the same plaque assay was performed again to purify the virus clones. In this embodiment, C
This indicates that the thymidine kinase gene region on the HV 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.

(16) Rabies virus G protein expression
Units are assembled into the thymidine kinase gene region of the CHV genome.
Production of the incorporated recombinant CHV The basic operation was performed in the same manner as in Example (15). In this example, the plasmid pCTKdlEH / RV-G created in Example (6) was used as a plasmid to be introduced into MDCK cells by the electroporation method, and this plasmid was used as a virus to infect cells into which the plasmid was introduced. The recombinant virus CHV (Y) dlTK / LacZ produced in (15) was used.
In this case, CHV (Y) dlTK / Lac used as parent virus
It can be expected that the Z beta-gal expression unit and the rabies virus G protein expression unit on the plasmid DNA will be replaced by homologous recombination. For this reason, the recombinant CHV into which the rabies virus G protein expression unit has been incorporated has a phenotype of forming a blue plaque of the parent virus when the plaque assay using X-gal shown in Example (15) is performed. It mutates to the phenotype of forming white plaques. In fact, in this example, the recombinant virus forming a white plaque was cloned by a plaque assay, and the expression of the rabies virus G protein was confirmed by a fluorescent antibody method using antirabies virus rabbit serum against the recombinant virus-infected cells. Was. The anti-rabies virus rabbit serum was prepared by inoculating the purified rabies virus subcutaneously with rabbits together with a french adjuvant. In this example, the recombinant virus CHV (Y) dlTK / Lac
Z enables a method of selecting by color of virus plaque, indicating that it is effective in producing a new recombinant CHV of interest. The recombinant virus obtained in this example was named CHV (Y) dlTK / RV-G.

(17) beta-gal expression unit and rabies virus
G protein expression unit, beta-gal expression unit and canine
Temper virus F protein expression unit, beta-gal expression
Unit and canine distemper virus H protein expression unit
Position or beta-gal expression unit and Aujeszky's disease virus
Thymidine kinase expression unit is converted to thymidine of CHV genome
Production of Recombinant CHV Incorporated in Kinase Gene Region In this example, basic operations were performed in the same manner as in Example (15). In this example, the plasmid introduced into MDCK cells by the electroporation method includes the plasmid pCTKdlEH / LacZ / RV-G created in Example (7) and the plasmid pCTKdlEH / LacZ / created in Example (10). CDV-
F, plasmid pCTKdlEH / Lac Z / produced in Example (11)
CDV-H or plasmid pCT created in Example (12)
Using KdlEH / Lac Z / PTK, CHV, DFD-6 were used as viruses to infect cells transfected with this plasmid.
The strain was used. In this embodiment, the parent virus CH
V, the thymidine kinase gene of the DFD-6 strain and the bivalent expression units of each plasmid, ie, beta-gal expression unit and each virus protein expression unit (rabies virus G protein, canine distemper virus F protein, canine distemper virus H protein) Or Aujeszky's disease virus thymidine kinase expression unit)
It is expected that a homologous recombination occurs between the two, and that a recombinant virus incorporating two expression units will appear. The recombinant virus thus emerging forms a blue plaque in the plaque assay using X-gal due to the beta-gal gene product. In fact, virus clones that form blue plaques by introducing the respective plasmids were obtained,
These cells were simultaneously transfected with their respective viral protein expression units by the fluorescent antibody method using anti-rabies virus rabbit serum or anti-canine distemper virus rabbit serum against recombinant virus-infected cells, or Aujeszky's disease virus thymidine kinase. In the case of a recombinant virus incorporating an expression unit, 5-IODO-2'-DE at a concentration of 50 μg / ml added during virus growth in tissue culture
Confirmed by sensitivity to OXYURIDINE (IDU). The anti-canine distemper virus rabbit serum was prepared by inoculating a purified canine distemper virus subcutaneously into rabbits together with a french adjuvant. This example shows that two or more independent expression units can be inserted into one foreign nucleic acid sequence insertion site,
For example, it provides a method for producing a multivalent vaccine. The recombinant virus obtained in this example is the plasmid pCTKdlEH / Lac Z / RV-G used for homologous recombination, pCTKdlEH / Lac Z / C
CHV (D) dlTK / Lac Z / RV-G, CHV (D) dlTK / Lac Z / CDV corresponding to DV-F, pCTKdlEH / Lac Z / CDV-H, pCTKdlEH / Lac Z / PTK
-F, CHV (D) dlTK / Lac Z / CDV-H, or CHV (D) dlTK / Lac
Named Z / PTK.

(18) Rabies virus G protein expression
Unit, canine distemper virus F protein expression unit
Alternatively, canine distemper virus F protein expression
Position in the thymidine kinase gene 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 produced in Example (17) was used as a virus to be infected after introduction of plasmid DNA. CHV (D) dlTK / Lac Z / PTK was used. This virus expresses the thymidine kinase gene product of Aujeszky's 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 product of CHV. Therefore, the plasmid pCTKdlEH / RV-G created in Example (6), the plasmid pCTKdlEH / CDV-F created in Example (13), or the plasmid pCTKdlEH / CDV-H similarly created in Example (13) was electroporated. Recombinant virus CH was added to MDCK cells introduced by the
Infect V (D) dlTK / Lac Z / PTK, infect the emerging virus into MDCK cells in the presence of IDU in advance, and selectively grow a new recombinant virus 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 1 ml of Eagle's minimum essential medium containing 50 μg / ml IDU and 5% FBS was added per well. The cells were cultured in a carbon dioxide incubator at 37 ° C. for 24 to 48 hours, freeze-thawed three times, and the infected cells were crushed with an ultrasonic crusher and centrifuged at 3,000 rpm for 5 minutes to recover the supernatant. This virus solution is re-subjected to ID
The selective culture was performed in the presence of U, and the resulting virus solution was subjected to a plaque assay in the presence of X-gal. The recombinant virus expected to appear in this example is beta-gal in addition to the Aujeszky's disease virus thymidine kinase expression unit.
Since the expression unit is also constructed so as to be deleted, it appears as a white plaque in the plaque assay. In fact,
In the virus solution after the selective culture in this example, the ratio of the recombinant virus forming a white plaque was as high as one third of the whole virus. In this example, a recombinant virus CHV (D) dlTK / Lac Z / PTK enables selective culture by IDU in addition to selection by plaque color, and provides a method for easily cloning a new recombinant virus. .
The resulting recombinant virus was the plasmid pCTKdlEH / RV-G, pCTKdlEH / CDV-F, or pCTKdl used for homologous recombination.
CHV (D) dlTK / RV-G, CHV (D) dlTK / CDV corresponding to EH / CDV-H
-F or CHV (D) dlTK / CDV-H.

(19) Using the beta-gal expression unit in the CHV genome
In this example, the basic operation was performed in the same manner as in Example (15). In this example, the plasmid introduced into MDCK cells by the electroporation method is the same as that in Example (1).
The plasmid pORF2 / LacZ created in 4) was used, and the CHV, DFD-6 strain was used as a virus to infect cells into which this plasmid had been introduced. In this embodiment, CH
The ORF2 gene on the V genome is disrupted and beta-gal
It is expected that the expression unit will be inserted by homologous recombination. A plaque assay in the presence of X-gal revealed a blue plaque which was cloned and cloned into CH (D) dlORF2 / La
Named cZ. This example shows that the ORF2 region on the CHV genome is also a site into which a foreign nucleic acid sequence can be inserted.

(20) Rabies virus G protein expression unit
Against rabies virus caused by recombinant CHV
Induction of a neutralizing antibody titer in dogs Recombinant virus CHV (Y) dlTK / R produced in Example (16)
VG, CHV, YP11 strain or a commercially available inactivated rabies vaccine (Nisseiken Co., Ltd.) was inoculated into 2 to 3 dogs per group, and the appearance of neutralizing antibodies against rabies virus and their antibody titers were observed. Recombinant CHV was inoculated with 2 × 10 ^3.75 TCID ₅₀ into both nasal passages, non-recombinant CHV was with 2 × 10 ^4.5 TCID ₅₀ into both nasal passages, and inactivated rabies vaccine was injected subcutaneously according to its prescription. The sera collected before and after the inoculation, which were collected with time, were serially diluted two-fold, mixed with an equal volume of a solution containing 100 TCID ₅₀ of rabies virus, and allowed to react at 37 ° C. for 1 hour. The suspension was further added with 50 μl of the HmLu-C3 cell suspension, and cultured in a carbon dioxide incubator. The antibody titer in the serum was determined by the presence or absence of the appearance of the cytopathic effect. Table 1 shows the movement of neutralizing antibody titer after inoculation.
It was shown to.

[0053]

[Table 1]

As shown in Table 1, in the group inoculated with the recombinant virus, a neutralizing antibody titer against the rabies virus was observed from 1 week after the inoculation, and the neutralizing antibody titer at 4 weeks after the inoculation was higher than that of the commercially available rabies vaccine. This result indicates that the recombinant virus produced in the present invention is useful as a recombinant vector vaccine.

[0055]

Industrial Applicability 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 production of animal vaccines and therapeutic agents, and Recovery and maintenance of health,
The effect of contributing to promotion is obtained.

[Brief description of the drawings]

FIG. 1. DNA containing CHV and thymidine kinase genes
Cloning of fragments. 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: Cloning of DNA fragments containing CHV, gC and ORF2 genes.

FIG. 3. Construction of a transfer vector derived from the thymidine kinase gene region.

FIG. 4. Construction of a transfer vector incorporating a beta-gal expression unit in the thymidine kinase gene region.

FIG. 5. Construction of a 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 shows construction of a transfer vector in which a beta-gal expression unit and a rabies virus G protein expression unit have been incorporated into the thymidine kinase gene region.

FIG. 8: Cloning of CDV, F protein gene and H protein gene.

FIG. 9 is a continuation of FIG. 8;

FIG. 10 shows construction of a transfer vector in which a beta-gal expression unit and CDV and F protein expression units have been incorporated into the thymidine kinase gene region.

FIG. 11 shows construction of a transfer vector in which a beta-gal expression unit and CDV and H protein expression units have been incorporated into the thymidine kinase gene region.

FIG. 12 shows construction of a transfer vector in which a beta-gal expression unit and an Aujeszky's disease virus thymidine kinase expression unit have been incorporated into the thymidine kinase gene region.

FIG. 13 shows construction of a transfer vector in which CDV and F protein expression units have been incorporated into the thymidine kinase gene region.

FIG. 14 shows construction of a transfer vector in which CDV and H protein expression units have been incorporated into the thymidine kinase gene region.

FIG. 15 shows construction of a transfer vector in which a beta-gal expression unit has been incorporated into the ORF2 region.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12N 7/00 C12P 21/02 C C12P 21/02 C12N 5/00 B // (C12N 5/10 (C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Osamu Otsuka 2-10-38 Ofuna, Kamakura City, Kanagawa Prefecture

Claims (19)

[Claims] 1. A recombinant canine herpesvirus, wherein a nucleic acid sequence not present on the canine herpesvirus genome has been inserted into the genome. 2. The recombinant canine herpesvirus according to claim 1, wherein the nucleic acid sequence is inserted by a homologous recombination method. 3. The recombination characterized in that the site for inserting a nucleic acid sequence not present on the canine herpesvirus genome according to claim 1 is a region on the canine herpesvirus genome that does not have a lethal effect on virus growth. Canine herpes virus. 4. The recombinant canine herpesvirus according to claim 3, wherein the region having no lethal effect on the virus growth is the thymidine kinase gene region. 5. The ORF2 according to claim 3, wherein the region having no lethal effect on the virus growth is located downstream of the gC gene.
A recombinant canine herpes virus, which is a region. 6. The nucleic acid sequence of claim 1, which is not present on the canine herpes virus genome, is constructed as an autonomous expression unit connected in the same direction between a promoter and a polyadenylation signal operable in eukaryotic cells. And a recombinant canine herpes virus. 7. A recombinant canine herpes virus, wherein a plurality of the autonomous expression units according to claim 6 are inserted. 8. A recombinant canine herpes virus, wherein the promoter of the immediate early gene of human cytomegalovirus is used as the promoter operable in eukaryotic cells according to claim 6. 9. A recombinant canine herpes virus, wherein the promoter of the early gene of SimianVirus 40 is used as the promoter operable in eukaryotic cells according to claim 6. 10. The nucleic acid sequence which is not present on the canine herpesvirus 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 canine herpesvirus. 11. The recombinant canine herpesvirus 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 rabies virus G protein. 12. The recombinant canine herpesvirus according to claim 10, wherein the nucleic acid sequence encoding an antigen for inducing protective ability against infection is a nucleic acid sequence encoding a canine distemper virus H protein. 13. The recombinant canine herpesvirus 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. 14. The recombinant canine herpesvirus according to any one of claims 1 to 13, wherein the recombinant canine herpesvirus has a β-galactosidase gene or a thymidine kinase gene sensitive to 5-IODO-2′-DEOXYURIDINE. A method of selecting using a method. 15. A method comprising inoculating an animal with the recombinant canine herpesvirus according to claim 10 to confer protection against infectious diseases. A method for preparing a vaccine, comprising the recombinant canine herpesvirus according to any one of claims 10 to 13. A vaccine comprising the recombinant canine herpesvirus according to any one of claims 10 to 13. 18. The recombinant according to claim 1, wherein the nucleic acid sequence not present on the canine herpesvirus genome is a nucleic acid sequence encoding a product having a therapeutic effect on animals. Canine herpes virus. 19. A method for treating a disease by inoculating an animal with the recombinant canine herpesvirus of claim 18.