WO1998008936A1

WO1998008936A1 - Recombinant canine herpesvirus

Info

Publication number: WO1998008936A1
Application number: PCT/JP1997/000236
Authority: WO
Inventors: Xuenan Xuan; Kotaro Tuchiya; Susumu Ueda; Takeshi Mikami; Haruki Otsuka
Original assignee: Nippon Institute For Biological Science
Priority date: 1996-08-28
Filing date: 1997-01-31
Publication date: 1998-03-05
Also published as: JPH1066578A; AU1557297A

Abstract

A recombinant canine herpesvirus usable as a vector virus carrying a nucleic acid sequence introduced thereinto, which sequence does not occur in a canine herpesvirus vector. A recombinant canine herpesvirus constructed by inserting a nucleic acid sequence which does not occur in canine herpesvirus genome into a region on the genome exerting no lethal effect on the growth of the virus.

Description

Specification

Recombinant canine virus

Technical field

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.

Background art

In the early days of virology, much research was done on its pathogenicity and the foundation of the current virology has been laid. Virology, coupled with research into pathogenicity elucidation, aims at pectinization of the virus, requires a long time of long-term passage using animals or tissue culture cells, is not so reliable, and is attenuated by methods. Have been detoxified. Later, when genetic engineering technology was introduced into virology, it became possible to elucidate the pathogenesis or vaccination of viruses at the molecular level, and vaccines using this technology are being actively developed. On the other hand, the use of this technology to create a recombinant virus in which a nucleic acid sequence (foreign nucleic acid sequence) not present in the genome of a naturally-occurring virus (wild-type virus) has been introduced, that is, a viral vector has been actively used. It has come to be done. Recombinant viruses can be used, for example, by constructing a foreign nucleic acid sequence so as to express it in a cell, incorporating it into a vector virus, and infecting the recombinant virus in a test tube or a cell of an animal body. In order to elucidate the function of the product and to produce a gene product, or to inoculate the animal body and inoculate the animal into a foreign nucleic acid sequence introduced into a vector virus Used to get a column effect. Specifically, the effects of inoculation into an animal body include, for example, a vaccine effect when a gene encoding a protective antigen of various pathogenic microorganisms is introduced as a foreign nucleic acid sequence, and a gene coding for a bioactive substance. The therapeutic effect when introduced as a nucleic acid sequence is considered. Once the method of virus vectorization, in other words, the method of producing a recombinant virus, is established, it is possible to produce various types of recombinant viruses according to the purpose using the same method. Moreover, despite the wide range of effects that can be achieved, the method of producing recombinant virus that plays a role in many cases is similar to the method of producing wild-type virus, and many objectives can be achieved with a single production method. It is expected to contribute to the recovery, maintenance and promotion of the health of the target animals.

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. When a virus of the family Herpesvirus is developed as a vector virus, the foreign nucleic acid sequence of interest is located in a non-essential gene region identified on the genome of the herpes virus, ie, a gene region that is not essential for the propagation of the virus. Gen, one of the inventors of the present invention, conducted intensive studies and found that the nucleotide sequence of the thymidine kinase gene, which is considered to be one of the candidates for the foreign nucleic acid sequence insertion site in C CV Revealed. The structure of this region is being analyzed by Remond et al. (1995, Virus Res., 39: 341-354). Furthermore, Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039) have determined the nucleotide sequences of the gB, gC, and gD genes, which are CHV structural proteins, and have described Remond et al. Gen. Virol., 77: 37-48) has been energetically determining the nucleotide sequence of a region called UL in the CΗV genome. However, although the analysis of the gene structure of CHV has been energetically advanced in recent years as described above, this CHV is used as a vector virus. There is currently no knowledge of whether it can be used.

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

That is, a first object of the present invention is to show that there are a plurality of sites for inserting a foreign nucleic acid sequence on a CHV genomic gene, that is, that CHV can be sufficiently used as a vector virus, It also aims to provide some ways to determine this position.

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.

Further, 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).

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

Next, the plasmid DNA into which this mutation has been introduced is transfected into CHV host cells, cultured for a certain period of time, and then infected with a wild-type virus. When cells transfected with the DNA and infected with the wild-type virus are cultured for a certain period of time, the cells are expected to produce CHV. In this process, the CHV genome DNA is transformed into a plasmid that has been mutated during its propagation. New generation of a recombinant virus genome by homologous recombination with DNA and DNA particles having the recombinant virus genome can be expected. By analyzing the presence or absence of a virus that incorporates the mutation introduced on plasmid DNA in the virus produced under such circumstances, the mutation-introduced site on the CHV genome can be used to identify the foreign nucleic acid sequence. It can be determined whether the site is an insertable site. By this method, there may be a plurality of sites where foreign nucleic acid sequence can be inserted.

Once the foreign nucleic acid sequence on the CHV genome has been identified, a similar technique can be applied or improved to achieve the desired foreign nucleic acid sequence. It is possible to construct a recombinant CHV that is constructed with a structure that expresses and that inserts it. The number of foreign nucleic acid sequences that can be inserted into the CHV genome does not need to be one, and a recombinant CHV in which a plurality of foreign nucleic acids are inserted can be produced depending on the purpose.

CHV is pathogenic in puppies up to 2 weeks of age, but rarely in later dogs. Use of CHV with vector as a vector virus will effectively contribute to the recovery, maintenance and promotion of the health of dogs and other animals susceptible to recombinant CHV Can be given. BRIEF DESCRIPTION OF THE FIGURES

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. BEST MODE FOR CARRYING OUT THE INVENTION

To complete the present invention, the basic techniques used are genetic engineering techniques and virology techniques. The former technology was described by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York), and the latter technology was developed by the National Institute of Health, National Institute of Health (1973). , Revised second edition, Maruzen Co., Ltd.).

The first step in carrying out the present invention involves cloning the DNA fragment of the CHV genome. Any CHV strain can be used as the CHV used in the present invention, and examples thereof include strain D004 (ATCC VR-552) and strain F205 (ATCC VR-647). This process has already been described in Remond et al. C1995, Virus Res., 39: 341-354), Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039) or Reraond et al. (1996, J. Gen. Virol. , 77: 37-48) have been established and can be carried out according to their reported method. Alternatively, a synthetic DNA primer is arbitrarily prepared with reference to the nucleotide sequence known to date, and the desired fragment is amplified by polymerase-chain reaction (PCR) to obtain an appropriate plasmid DN. It can also be cloned on A.

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.

In the next step, a mutation is introduced at any position of the CHV genomic DNA fragment on this transfer vector. Mutations can be introduced by methods such as deletion, insertion, or base substitution. Malik et al. (1992, Virology, 190: 702-715), Thorasen et al. (1987, Gene, 57: 261-265). ), Kit et al. (1992, Arch. Virol., 124: 1-20) or Cole et al. (1990, J. Virol., 64: 4930-4938). Specifically, for example, a / 3-galactosidase (beta-gal) expression unit having a promoter and a polyadenylation signal is prepared from plasmid pCHl10 (Pharmacia), and this is used to prepare the CHV genome on the transfer vector. Mutations can be introduced by inserting the fragment at any position. The term expression unit refers to a unit of a nucleic acid structure for autonomously expressing a foreign nucleic acid sequence in a eukaryotic cell, and specifically encodes the direction of the foreign nucleic acid sequence, for example, a polypeptide. A structural unit of a nucleic acid sequence having the same direction as that of a foreign nucleic acid sequence and operating in a eukaryotic cell upstream of a foreign nucleic acid sequence, and a polyadenylation signal arranged downstream thereof. The plasmid DNA into which the mutation has been introduced can be used together with the genomic DNA of CHV, for example, Malik et al. (1992, Virology, 190: 702-715), Thomsen et al. (1987, Gene, 57: 261-265), Kit et al. Virol., 124: 1-20), or according to the method of Cole et al. (1990, J. Virol., 64: 4930-4938), cotransfect into a host cell of CHV such as MDCK cell. Alternatively, cells may be transfected with the above-described plasmid into which a mutation has been introduced in advance, and then infected with CHV. When these cells are cultured for several days, in addition to the parental virus in the culture supernatant and cells, a group that is likely to be regenerated by homologous recombination between the CHV genomic DNA and the mutated transfer vector DNA Production of a recombinant virus can be expected. The virus is recovered from this and virus clones are cloned by the Blackassay method. Methods appropriate for the mutations introduced into these viral clones on plasmid DNA, such as For example, Malik et al. (1992, Virology, 190: 702-715), Thomsen et al. (1987, Gene, 57: 261-265), Kit et al. (1992, Arch. Virol., 124: 1-20), or Cole et al. 1990, J. Virol., 64: 4930-4938), and analyze whether or not a mutation-introduced virus clone, that is, a recombinant virus, is present. Specifically, for example, when a mutation is introduced using the above-mentioned beta-gal expression unit, 5-bromo-4--4-chloro-3-indolyl-yS-D-galactoside ( By mixing or overlaying X-gal) in agar or methylcellulose medium, the appearance of recombinant virus can be easily confirmed as a blue black. When the mutation is introduced, it can be as high as 1 in 1,000 virus clones produced, and the use of beta-gal expression units and X-gal facilitates the appearance of recombinant CHV. You can check. Once it is confirmed that a mutation can be introduced even with such a low probability, it means that the site has been identified as a foreign nucleic acid sequence insertion site, and the foreign nucleic acid sequence insertion site present on the CHV genome is identified in the same manner. You can find more than one.

Once a foreign nucleic acid sequence insertion site on the CHV genome is found, the next step in the present invention is to insert the target nucleic acid sequence into that site. The target nucleic acid sequence is constructed, for example, with the structure of the expression unit described above. Promoters that operate in eukaryotic cells used at this time include, for example, the promoter of the immediate early gene of human cytomegalovirus, the promoter of the early gene of Simian Virus 40, or the promoter that naturally exists on the CHV genome. A nucleic acid sequence of interest, for example, a sequence of an open reading frame (hereinafter abbreviated as “ORF”) encoding a rabies virus G protein is ligated downstream of this. The autonomous expression unit thus constructed is inserted into the foreign nucleic acid sequence insertion site on the transfer vector. In order to select the thus constructed transfer vector and CHV or, for example, a recombinant virus, use a recombinant CHV in which a beta-gal expression unit has been inserted into the same foreign nucleic acid sequence insertion site in advance. In the same manner as described above, homologous recombination is induced in cells, and then a clone of a recombinant virus can be selected by applying a method in which a foreign nucleic acid sequence insertion site has been searched.

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

As described above, the use of CHV as a vector virus is primarily considered to be the vector vaccine described above. In addition, foreign nucleic acid sequences that may be introduced for the purpose of this use include, in addition to the above-mentioned rabies virus G protein gene, for example, the H- and F-protein genes of canine distemper virus, Inducible adenovirus type 1 or 2 hexon protein gene or fiber protein gene, parainfluenza virus F protein protein or HN protein gene, canine parvovirus VP 1 or Examples of the gene include a gene encoding a protective antigen of a pathogen to a host animal that is capable of infecting CHV, such as a dog, such as a VP2 protein gene.

In addition, when CHV is used as a vector virus, it can be used for therapeutic applications. In this case, a foreign nucleic acid sequence encoding a physiologically active substance such as lymphokine, interferon, or cytokine is used. A case where a child is introduced can be exemplified. Example

Hereinafter, the present invention will be described more specifically with reference to examples.

(1) Extraction of C H V genome DNA

MDCK cells suspended in Eagle's minimum essential medium supplemented with 5% fetal bovine serum (FBS) are seeded on a 6 cm diameter tissue culture plastic dish to form a monolayer cell sheet. Incubate in a carbon dioxide incubator II (5 ° C, 37 ° C, in the presence of carbon dioxide) until complete At the time when the cell sheet is formed, the CHV and YP11 strains (with the University of Tokyo, Veterinary Microbiology Division II) or DFD-6 strains (Nissei Laboratories) that have an infectivity higher than the number of cells forming the sheet, excluding the culture solution The virus was adsorbed to the cells. After adsorption, Eagle's minimum essential medium supplemented with 2 ml of FBS was added, and the cells were cultured again in a carbon dioxide incubator for 22 to 23 hours. After the culture, remove the culture medium, wash the MDCK cells once with phosphate buffered saline, add 1 ml of 0.1 M Tris-monohydrochloride buffer (pH 9) containing 1¾SDS, 0.1 M NaCl and ImM EDTA, and add 10 ml at room temperature. Let stand for minutes. Thereafter, 50 ^ 1 of a 20 mg / ml protease solution was added, and the mixture was allowed to stand at 37 ° C. This solution was recovered and extracted twice with an equal volume of a phenol-chloroform solution (1: 1) to recover an aqueous layer. When 0.1 ml of a 3M NaCl solution and 2 ml of ethanol are added to the collected aqueous solution, a filamentous DNA precipitates.Then, this is wound around a glass rod, collected, washed with a 70% ethanol solution, and dried. Dissolve in 0.1 ml of distilled water and store at 4 ° C until use. With this operation, 30 to 50 jug of DNA was recovered.

(2) Cloning of fragment containing thymidine kinase gene (TK) from CHV genome DNA

Figure 1 shows the procedure for cloning the gene fragment. That is, the CHV genomic DNA 201 recovered in Example 1 above was treated with 50 units of the restriction enzyme Xbal overnight at 37, followed by agarose gel electrophoresis, and the agarose portion containing the 6.5 kilobase (kb) fragment was removed. The DNA was collected and collected using a Gene Clean Kit (BI0101). This was ligated to a plasmid pBluescript I1SKC +) that had been digested with Xbal and dephosphorylated with calf intestinal alkaline phosphatase (C1AP) using ligation kit (Takara Shuzo Co., Ltd.), and transformed into Escherichia coli DH5a. It was introduced by a transformation reaction. This was inoculated on an LB agar medium containing 50 g / ml of ampicillin to isolate transformed E. coli, and the plasmid DNA of each clone was prepared by the Algari method. Plasmid DNA into which the above 6.50 kb fragment was incorporated was selected from these by cleavage with the restriction enzyme Xbal and analysis by agarose electrophoresis. The direction of insertion of gene fragments into plasmid DNA is as shown in the figure. Although clones in the direction opposite to the ^ ^ direction were obtained, in this example, clones inserted in the directions shown in the figure were cloned and used in the following examples. Whether the 6.50 kbp fragment cloned on this recombinant plasmid was the target gene was determined by a HincII-cleaved 1.5 kb fragment containing the thymidine kinase gene derived from feline herpesvirus C7301 (Yokoyama et al. al., 1995, J. Vet. Med. Sci., 57: 709-714), and confirmed by the hybridization method according to the method of Maeda et al. (1992, Arch. Virol., 127: 387-397). The obtained 9.46 kb recombinant plasmid was designated as pBS / CTKXbaI6.5.

(3) Cloning of gC and ORF2 genes from CHV genomic DNA FIG. 2 shows the procedure for cloning gene fragments. Specifically, referring to the report of Limbach et al. (1994, J. Gen. Virol., 75: 2029-2039), the following synthetic DN DN primer consisting of 20 residues:

5 '-CGAGCCCTAATTATTGGTTT- 3' and

5 '-TACAACTGTTTAATAAAGAC-3'

Was prepared, and the DNA extracted in Example (1) was used as a 铸 type to perform a polymerase zethiane reaction (PCR). The reaction mixture contains 1 Ug of genomic DNA of the CHV YP11 strain in 50 β \, 2 M each of the above-mentioned synthetic DNA primer, 800 deoxynucleotides each, and lOXTaq polymerase attached to 51 enzymes. After mixing buffer and 1.25 units of Taq polymerase (Perkin 'Elma Japan Co., Ltd.) and overlaying liquid paraffin, 25 cycles of PCR were performed at 94 ° C for 1 minute, 50 ° C for 1 minute, and 722 minutes for 1 cycle. I went. A 2.28 kb DNA fragment predicted from the nucleotide sequence was separated by agarose gel electrophoresis and recovered by Gene Clean Kit. This DNA fragment contains 0RF2, which encodes gC protein, in addition to 0RF2. Dissolve this DNA fragment in 70 mM Tris-HCl buffer, pH7.6 containing 50 // 1 of 10 mM magnesium chloride, 5πι スレ dithiothreitol, and lmM ATP, add 20 units of T4 polynucleotide kinase, and heat at 37 ° C. The reaction was performed for 1 hour to phosphorylate the 5 'end of the DNA fragment. The 2.36 kb fragment of plasmid plIC19, digested with the restriction enzyme PvuII and treated with CIAP, was ligated with the above phosphorylated DNA fragment using a ligation kit, and used to transform E. coli DH5a, which had been transformed into a combination. The transformed E. coli was isolated by inoculating the LB agar medium containing 50 / g / ml of ampicillin from the transformed E. coli. A was recovered. Although the gene fragment was inserted into plasmid DNA in the direction opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 4.65 kbp plasmid was named p0RF2.

(4) Construction of transfer vector from plasmid pBSZCTKXbaI6.5

Fig. 3 shows the procedure for constructing the transfer vector. That is, the pBS / CT baI6.5 obtained in Example (2) was digested with the restriction enzyme Xbal, the DNA ends were smoothed with the Klenow enzyme, and then the 6.50 kb fragment was purified by agarose gel electrophoresis and gene clean kit. Recovered. This was ligated with a 2.36 kb fragment of plasmid pUC19 digested with the restriction enzyme PvuII and treated with CIAP using a ligation kit, and introduced into a combined E. coli DH5α, and 50 μg / ml of ampicillin was added. The transformed Escherichia coli was inoculated by inoculating the LB agar medium containing the plasmid, and recovered from Escherichia coli holding the plasmid DNA having the 6.50 kb fragment incorporated therein. Although the insertion direction of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was used in the following examples. The obtained 8.87 kb plasmid was designated as pUC (dl-P) CTKXbal6.5.

This plasmid pUC (dl-P) CTKXbaI6.5 was digested with the restriction enzymes EcoRl and Hindlll, split into two fragments of 8.36 kb and 0.50 kb, separated by agarose gel electrophoresis, and 8.36 kbp by Gene Clean Kit. A fragment was obtained. Separately, pUC19 was digested with restriction enzymes EcoRI and Hindi II to prepare a 57 base pair (bp) fragment containing a multi-cloning site, and ligated to the above 8.36 kbp fragment using a ligation kit. This was introduced into the transformed E. coli 5α by a transformation reaction, and the transformed E. coli was isolated by inoculation on an LB agar medium containing 50 g / nil of ampicillin. Retains integrated plasmid DNA This was recovered from Escherichia coli. The 8.41 kb plasmid obtained was named pCTKdlEH / MCS.

(5) Insertion of a galactosidase (beta-gal) expression unit into the transfer vector 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). Furthermore, after cutting the transfer vector (11 (11 £) 1 / »^ 3 with the restriction enzyme ^ 11 £ 111 1) created in Example (4), the ends are blunted with Klenow enzyme, and then subjected to CIAP treatment. The transfer vector and the 4.24 kbp beta-gal expression unit recovered above were ligated using a ligation kit, and introduced into a competent Escherichia coli DH5 cell by a transformation reaction. The transformed E. coli was isolated by inoculating the LB agar medium containing the E. coli, and recovered from the E. coli harboring the transfer vector DNA containing the beta-gal expression unit. As a result, clones in the direction opposite to the direction shown in the figure were also obtained due to ligation between smooth ends, but in this example, clones in the direction shown in the figure were cloned, and Was used in the real 施例. Plasmid obtained 12. 66Kbp was named pCTKdlEH / Lac Z.

(6) Insertion of mad virus G protein expression unit into transfer vector pCTKdlEH / MCS

The procedure for constructing the plasmid is shown in Fig. 5 and Fig. 6. That is, the plasmid pUC-ARAG disclosed in JP-A-3-139286 was renamed pUCRaGl.25 and used in this example. As shown in Fig. 5, this was cut with the restriction enzymes EcoRI and HindII and treated with Klenow enzyme to make the DNA ends blunt. After that, the 2.0 kb rabies virus G protein gene was subjected to agarose gel electrophoresis and gene Purified by clean kit did. This was digested with the restriction enzyme Xbal, treated with Klenow enzyme to make the DNA end blunted, and ligated with a 3,90 kb fragment of plasmid (pCDM8Gnvitorogen), which had been treated with CIAP, using a ligation kit to form a combination. Escherichia coli MC1061 / P3 was introduced by a transformation reaction, and the transformed Escherichia coli was isolated by inoculation on LB agar medium containing 7.5 g / ml tetracycline, and the rabies virus G protein gene was integrated. This was recovered from Escherichia coli retaining plasmid DNA. As shown in the figure, clones in which the G protein 遗 gene is integrated in the same direction with respect to the human cytomegalovirus immediate-early promoter (CMV-prom) on PCM8 are plasmid DNA with the restriction enzymes Hindlll and EcoRl. Was selected as a clone in which a 5.95 kb fragment appeared when cleaved. As shown in Fig. 6, the plasmid pCDM8-G prepared in this manner was digested with restriction enzymes Mlul and BamHI, treated with Klenow enzyme, and the DNA ends were blunt-ended. The G protein expression unit (G cassette) was purified using agarose electrophoresis and a gene clean kit. Separately, the transfer vector-pCTKdlEH / MCS prepared in Example (4) was digested with the restriction enzyme Xbal, followed by blunt-end blunting with Klenow enzyme and CIAP treatment, and the 3.42 kb rabies virus obtained above. The G protein expression unit was ligated using a ligation kit, introduced into a transformed E. coli DH5a by a transformation reaction, and inoculated on an LB agar medium containing 50 g, ml of ampicillin, and transformed. And the plasmid DNA was recovered from Escherichia coli holding the plasmid DNA into which the above 3.42 kb fragment had been incorporated. Although the direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The resulting 11.84 kb plasmid was designated pCTKdlEH / RV-G.

(7) Insertion of beta-gal expression unit and rabies virus G protein II expression unit into transfer vector pCTKdlEH / MCS

Figure 7 shows the procedure for constructing the plasmid. That is, pCTKdlEH / Lac Z prepared in Example (5) was digested with restriction enzyme Xbal, followed by blunt-ending with Klenow enzyme and CIAP treatment. The rabies virus G protein expression unit of 3.42 kb with blunt-ended DNA ends prepared in Example (6) was ligated using a ligation kit, and transformed into a competent Escherichia coli DH5a. The transformed E. coli was isolated by inoculating the LB agar medium containing 50 ^ g / ml of ampicillin, and the plasmid DNA was isolated from the E. coli harboring the 3.42 kbp fragment-incorporated plasmid DNA. Was recovered. The direction of insertion of the gene fragment into Plasmid DNA was the same as that shown in the figure due to ligation of blunt ends. Was cloned and used in the following Examples. The obtained 16.08 kb plasmid was designated as pCTKdlEH / Lac Z / RV-G.

(8) Cloning of canine distemper virus F protein protein gene

Vero cells are cultured in three roller bottles to form a monolayer cell sheet, which is then infected with Inu Distemper Virus (CDV) strain HK (Nissei Laboratories) and stored at 37 ° C. Cultured for days. After the culture, the culture supernatant was collected and centrifuged at 4000 rpm for 10 minutes to remove cell debris in the culture supernatant. This solution is centrifuged at 19,000 rpm for 2 hours using a Type 19 rotor (Beckman), and the precipitate is collected and suspended in 10 mM Tris-chloride buffer (TE buffer, pH 7.4) containing 5 ml of IIDM EDTA. I let it. This was further centrifuged at 3000 rpm for 10 minutes, and the supernatant was overlaid on a 60% («'/ W) sucrose solution and a 24% (W / W) sucrose solution, and the SW28 rotor (Beckman) was used. The mixture was centrifuged at 25,000 rpm for 90 minutes, and the component precipitated at the boundary between the 60¾ (I / W) sucrose solution and the 24¾ (W / W) sucrose solution was recovered as a virus fraction. The recovered components were further diluted 3 times or more with TE buffer, and centrifuged again at 25,000 rpm for 90 minutes using a SW28 rotor to collect the precipitate as a purified virus. The precipitate was suspended in 2 ml of TE buffer, and the protein concentration was measured by the oral method using a part of the precipitate, and the remaining part was stored at -80 ° C until use.

To recover the CDV genomic RNA, 250 mg (1.5 μl of 1 M Tris-HCl buffer (pH 8), 6 μl of 0.5 M EDTA, 3 μl of lOmg / ml Proteinase K solution and 37.5 «1 in 10% SDS solution were added and reacted for 30 minutes at 37. This was extracted twice with a phenol: chloroform solution (1: 1), and the aqueous layer was recovered. 30 1 3M sodium acetate Solution (pH5.2) and lml of ethanol were added. The mixture was allowed to stand at -80 ° C for 20 minutes, centrifuged at 15,000 rpm for 10 minutes to collect the precipitate, and dissolved in 100 ^ 1 TE buffer (pH8). Was.

The RNA concentration was determined by measuring the absorbance at 280 nm.

The cloning procedure of the CDV F protein gene is shown in FIGS. 8 and 9. That is, cDNA was synthesized from CDV genomic RNA as follows. That is, for 8 g of RNA, 51 of 10X reverse transcription reaction buffer (0.5 M Tris-HCl buffer, pH 8.3, 0.5 M KC 1.80 mM magnesium chloride, 0.1 M dithioleitol) ΙΟμΜ Random primer 1, 11 ribonuclease inhibitor (38 units / 1, Wako Pure Chemical Industries, Ltd.), to make a total amount of 431, leave at 90 ° C for 5 minutes, quench in ice water, In addition, 1 ^ 1 ribonuclease inhibitor, 51 deoxynucleotide mixture (each lOmM) and \ μ! Reverse transcriptase (29 units, 1 by Seikagaku Corporation) The mixture was reacted at C for 90 minutes to synthesize cDNA. After the reaction, the random primer was removed using Ultrafree C3TK (Millipore) to obtain the final solution volume.

To synthesize the CDV F protein gene by PCR, we obtained the Accession Number L13194 data, which contains the entire CDV nucleotide sequence, from the nucleotide sequence database of the EMBL Data Library. Synthetic primers,

MP2: 5'-CAA (or G) CCATCAGTCCCACAG (or Λ) GA-3 '

FRP2: 5'-GAGATATATGACCAGAATACT-3 '

Was synthesized. LOXPfu polymerase buffer (attached to polymerase) for 2 ^ 1 cDNA synthesized above, 0.5 01 of 10 / M synthetic primer and 51 deoxynucleotides mixed Solution (2 mM each), 14 // 1 distilled water, 0.5 μΐ Pfu polymerase (2.5 unit /// l), and overlay with liquid paraffin. 30 cycles of PCR were performed with 5 minutes as one cycle, and the expected 2.59 kb DNA fragment was obtained as a PCR product. The reaction mixture was extracted with phenol: chloroform (1: 1) to remove the primers and deoxynucleotides with Ultrafree C3TK. Then, the reaction mixture was added to 30/1 lOmM magnesium chloride, 5 mM dithiothreitol, 6 mM Dissolve in 70 mM Tris-HCl buffer containing ATP, pH 7.6, Ten units of T4 polynucleotide kinase were added and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment. The phosphorylated DNA fragment was digested with the restriction enzyme Sraa and ligated using a ligation kit to plasmid pUC19, which was dephosphorylated with alkaline phosphatase (BAP) derived from Pseudomonas aeruginosa. Transformed E. coli was introduced into XU-Blue by a transformation reaction and inoculated on LB agar medium containing 50 ^ g / ml of ampicillin to isolate transformed Escherichia coli. Plasmid DNA incorporating the above 2.59 kb fragment was isolated. Plasmid DNA was recovered from the retained E. coli. Although the gene fragment was inserted into the plasmid DNA in the direction opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 5.27 kb plasmid was designated as pUCl9-MP2-FRP2.

Furthermore, as shown in Fig. 9, synthetic primers were used to reclone only the 0RF region encoding the F protein (F-0RF).

FPfnl-2: 5 '-CAACACAGCCAAGCCCCATG-3'

Of 5 // 1 plasmid pUC19-MP2-FRP2 (\ ng / μΐ) to 21 lOXPfu polymerase buffer (attached to the polymerase), 0.51 each of 10 M synthetic primer-FPfn Mixture of DNA 2 and FRP2, ΙβΙ

(Each lOmM), 10.51 distilled water and 0.51 Pfu polymerase were added, and the flow buffer was overlaid.Then, 94 ° C for 60 seconds, 56 ° C for 30 seconds, and 72 ° C for 3 minutes were defined as one cycle. The PCR was performed for 20 cycles, and the expected 2.03 kb DNA fragment was obtained as a PCR product. The reaction mixture was extracted with phenol: chloroform (1: 1) to remove the primers and deoxynucleotides with Ultrafree C3TK. 70 mM Tris-HCl buffer containing ATP

(pH 7.6), 10 units of T4 polynucleotide kinase were added, and the mixture was reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of the DNA fragment. The phosphorylated DNA fragment was ligated using a ligation kit to plasmid PUC19, which had been cleaved with the restriction enzyme Smal and dephosphorylated with bacterial alkaline phosphatase (BAP), and ligated using a ligation kit to form E. coli MC1061 Introduced by transformation and containing 50 g'ml of ampicillin Transformed Escherichia coli was inoculated by inoculating a natural medium, and plasmid DNA was recovered from Escherichia coli holding the plasmid DNA in which the 2.03 kb fragment was incorporated. Clones were inserted in the opposite direction to the direction shown in the figure due to the ligation of blunt ends between gene fragments into plasmid DNA.In this example, clones in the direction shown in the figure were cloned. However, it was used in the following examples. The obtained 4.71 kbp plasmid was named pUC19-FPfn2-FRP2.

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.

Furthermore, the plasmid pUC19-FPfn2-FRP2 was digested with the restriction enzymes BamHI and Sacl, the DNA ends were blunt-ended using Klenow enzyme, and the 2.04 kb DNA fragment was separated by agarose electrophoresis to generate a gene clean kit. Collected by the This was ligated to the plasmid pAcYMl (Matsuura et al., 1989, Virology, 173: 674-682), which had been cleaved with the restriction enzyme Smal and dephosphorylated by BAP treatment, using a ligature kit and made competent. Transformed E. coli was introduced into E. coli MC1061 by a transformation reaction and inoculated on LB agar medium containing 50 g / ml ampicillin.The transformed E. coli was separated, and the plasmid DNA containing the 2,04 kb fragment was retained. Plasmid DNA was recovered from the resulting E. coli. Although the direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained. Clones were cloned and used in the examples below. The obtained 11.24 kb plasmid was designated as pAcHK-F.

(9) Cloning of canine distemper virus H protein protein gene

The procedure for cloning the H protein gene of CDV is shown in FIG. In other words, in order to synthesize the CDV H protein gene by PCR, the entire nucleotide sequence of CDV is registered from the nucleotide sequence database of the EMBL data library. Two synthetic primers, referring to the data of Number L13194,

HPfnl: 5 '-(^: 80-chome 461 (ji-hachi 4-8-chome 6 (: 1 ^> (;-3'

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. coli harboring the plasmid DNA incorporating the 1.93 kb fragment (H-ORF) was introduced. More plasmid DNA was recovered. Although the direction of insertion of the gene fragment into plasmid DNA was opposite to that shown in the figure due to ligation of blunt ends, clones in the direction shown in the figure were obtained in this example. Was cloned and used in the following Examples. The obtained 4.62 kb plasmid was designated as pUC19-HPfn HRP5.

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. C1AP treatment was performed, and the 2.06 kb F protein gene fragment recovered above was ligated using a ligation kit, and introduced into a competent Escherichia coli DH5 cell by a transformation reaction to give 50 g / ml. The transformed Escherichia coli was isolated by inoculating the LB agar medium containing ampicillin, and the plasmid DNA was recovered from Escherichia coli carrying the plasmid DNA in which the F protein gene fragment was incorporated. The direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but as shown in the figure, 3. A clone having an EcoRI site present in the ORF of the F protein at a position of 92 kb was selected by analysis of a cleavage image with the restriction enzyme EcoRI. By this construction, the F protein ORF is integrated in the same direction with respect to the human cytomegalovirus immediate-early promoter (CMV-prom), and the expression unit (F cassette as in the case of the G protein expression unit). ). The obtained 16.09 kb plasmid was designated as pCTKdlEH / Lac Z / CDV-F.

(11) Insertion of beta-gal expression unit and canine distemper virus H protein expression unit into transfer vector-pCTKdlEH / MCS

Figure 11 shows the procedure for constructing the plasmid. In other words, the plasmid created in the embodiment (9)

After digestion with DNA and PvuII and blunting the 5 'end of the DNA using Klenow enzyme, a 2.5 kb H protein gene fragment was recovered using agarose electrophoresis and a gene clean kit. Separately from this, the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7) was used in the same manner as in Example (10). After digestion with the restriction enzyme Xbal, the ends were blunt-ended with Klenow enzyme and treated with CIAP, and the 2.5 kb H protein gene fragment recovered above was ligated using a ligation kit to obtain a recombinant E. coli DH5. a) Transformed Escherichia coli was isolated by inoculating a LB agar medium containing ampicillin into the LB agar medium containing the H protein gene fragment. Was recovered. The direction of insertion of the gene fragment into plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but a clone of 3.72 kb on the plasmid was obtained as shown in the figure. 4. A clone having an EcoRI site present in the H protein ORF at the position of 16 kb was selected by analyzing the cleavage image with the restriction enzyme EcoRI, and an H protein expression unit (11 cassette) was constructed on the plasmid. The obtained 16.08 kb plasmid was designated as pCTKdlEH / LacZ / CDVH.

(12) Insertion of beta-gal expression unit and oeskey disease virus thymidine kinase expression unit into transfer vector pCTKdlEH / MCS

Figure 12 shows the procedure for constructing the plasmid. That is, a plasmid pPTK obtained by cloning a Pst1-Kpnl fragment of the oeschis disease virus genome containing the 2.8 kb oeschis disease virus thymidine kinase gene (PTK) was obtained from Dr. S. Kit of the United States. Biochemical Virology, Baylor College of Medicine, Houston, Texas, USA). This is digested with EcoRI and Hindlll and the DNA ends are blunted using Klenow enzyme.Then, using agarose gel electrophoresis and Gene Clean Kit, the 2.79 kb Aujeszky's disease virus thymidine kinase gene is digested. The fragments were recovered. This fragment has the original promoter and polyadenylation signal of the thymidine kinase gene and operates as an expression unit. Separately, pCTKdlEH / Lac Z prepared in Example (5) was digested with the restriction enzyme BamHI, followed by blunt-end blunting with Kleno monoenzyme and dephosphorylation by CIAP treatment. A 79 kb thymidine kinase gene fragment was ligated using a ligation kit, introduced into a transformed Escherichia coli により 5α by a transformation reaction, and inoculated on an LB agar medium containing 50 g / ral ampicillin. Isolate the formed E. coli and Blasmid DNA was recovered from E. coli harboring plasmid DNA in which the protein fragment was integrated. The direction of insertion of the gene fragment into the plasmid DNA was opposite to the direction shown in the figure due to ligation of blunt ends, but in this example, the clone in the direction shown in the figure was obtained. Was cloned and used in the following Examples. The obtained 15.42 kb plasmid was designated as pCTKdlEH / Lac Z / PTK.

(13) Transfer vector expression unit of canine distemper virus F protein or expression unit of canine distemper virus H protein to pCTKdlEH / MCS

Fig. 13 shows the procedure for constructing a plasmid into which the F protein expression unit was inserted, and Fig. 14 shows the procedure for constructing a plasmid into which the H protein expression unit was inserted. In other words, in order to remove the rabies virus G protein gene from pCTKdlEH / RV-G created in Example (6), it was cut with the restriction enzyme Xba1, followed by blunt-end with Cleno enzyme and CIAP. A treated 9.79 kb fragment was prepared, and this was combined with the 2.06 kb F protein gene fragment prepared in Example (10) or the 2.05 kb H protein gene fragment prepared in Example (11). Were ligated using a ligation kit, introduced into the transformed E. coli DH5a by a transformation reaction, and inoculated on an LB agar medium containing 50 g / nil of ampicillin to separate transformed E. coli. The plasmid DNA was recovered from Escherichia coli harboring the plasmid DNA into which the protein gene fragment was integrated. In each case, clones were inserted in the opposite direction to the direction shown in the figure due to ligation of blunt ends with the insertion direction of the gene fragment into the plasmid DNA. Clones with sites were selected by analysis of cleavage images with the restriction enzyme EcoRI. The 11.85kbp plasmid incorporating the F protein gene was designated pCTKdlEH / CDV-F, and the 11.84kb plasmid incorporating the H protein gene was designated pCTKdlEH / CDV-H.

(14) Insert beta-gal expression unit into transfer vector p0RF2

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.

(15) Creation of recombinant CHV incorporating beta-gal expression unit into thymidine kinase gene region of CHV genome

A sheet of MDCK cells formed in a 75 cm ² plastic culture flask is digested with a physiological buffered saline solution containing 0.25% tribsine and lmM EDTA, the cells are detached from the surface of the incubator, and a lidar containing 20 ml of 5% FBS And centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded to collect the cell precipitate. The cells were suspended in 20 ml of Eagle's minimum essential medium without FBS, centrifuged again at 1500 rpm for 5 minutes, and the centrifuged supernatant was discarded to collect the cell precipitate. The cell pellet was suspended in 5001 ET buffer (RPM1640 medium containing 10 mM D-glucose and 5 mM dithiothreitol) and transferred to a cuvette (BioRad) for electoral poration. To this was added 30 zl of the plasmid pCTKdlEH / Lac Z prepared in Example (5) and adjusted to lmg / ml, allowed to stand in ice water for 5 minutes, and then treated with Gene Pulser (Bio-Rad) at 960 バイオ FD ( An electric pulse was applied under the conditions of microfarads) and 0.3 kV (kilovolt) / cm, and the DNA was again introduced into the cells by leaving still in ice water for 10 minutes. The cells were transferred to a plastic culture dish of 6 cm in diameter, to which 5 ml of the minimum essential culture medium of Eagle containing 10% FBS was added, and cultured in 37 carbon dioxide incubators for 6 to 12 hours. After the culture, the culture solution was removed, CHV and YP11 strains having an infectious titer equivalent to the number of cells were inoculated, and allowed to stand for 1 hour to adsorb the virus to the cells. After the adsorption, 2 ml of Eagle's minimum essential medium to which FBS was added was added dropwise, and the cells were cultured again in a carbon dioxide incubator at 37 ° C for 48 hours. During the culture of the CHV parent virus genome during this culture, the thymidine kinase gene In the offspring region, it is expected that homologous recombination with the plasmid DNA already introduced into the cells by the electroporation method will occur. The appearance of the virus is expected. After this culture, the cells were freeze-thawed three times, and the infected cells were disrupted with an ultrasonic disrupter, centrifuged at 3,000 rpm for 5 minutes, and the supernatant was recovered as a virus solution and stored at -80 ° C until use. .

The recombinant virus present in the virus solution recovered above was cloned by the following black assay. That is, the MDCK cell sheet formed on a plastic 6-well plate was inoculated with a 10-fold serially diluted virus solution, allowed to stand for 1 hour to adsorb the virus to the cells, and the inoculated virus solution was removed. Overlay 2 ml of Eagle's minimum essential medium containing 6 agarose and 2 FBS per well, and add 1 ml of Eagle's minimum essential medium containing 1 ml of FBS per well when the agarose solidifies, and add 37 ° C CO2 incubator. For 48 hours. After the culture, the liquid layer was removed, and 1 ml of Eagle's minimum essential medium containing 600 g / inl of X-gal and 2¾FBS was added per well, followed by further culturing in a carbon dioxide incubator at 37 ° C for 12 hours. After this culture, the liquid layer was again removed, and a blue-colored plaque was recovered using a Pasteur pipet by enzymatic reaction using X-gal, a beta-gal gene product, as a substrate. The cells were suspended in Eagle's minimum essential medium containing 1% FBS, subjected to three freeze-thaw cycles and sonication, and again subjected to the same black assay to purify the virus clones. This example shows that the thymidine kinase gene region on the CHV genome is a site into which a foreign nucleic acid sequence can be inserted. The recombinant virus obtained in this example was named CHV (Y) dlTK / Lac Z.

(16) Production of recombinant CHV incorporating rabies virus G protein expression unit into thymidine kinase gene region of CHV genome

The basic operation was performed in the same manner as in Example (15). In this example, the plasmid pCTKdlEH / RV-G produced in Example (6) was used as the plasmid to be introduced into MDCK cells by the electroporation method, and the plasmid-infected cells were infected. The recombinant virus CHV (Y) dlTK / LacZ produced in Example (15) was used as the virus. In this case, the homologous recombination between the beta-gal expression unit of CHV (Y) dlTK / Lac Z used as the parent virus and the rabies virus G protein expression unit on plasmid DNA was performed. We can expect to be replaced more. Therefore, the recombinant CHV into which the rabies virus G protein expression unit is integrated has the phenotype of forming the parent virus blue black when black-assay using X-gal shown in Example (15) is performed. The phenotype changes from white to black. In fact, in this example, the recombinant virus forming a white plaque was cloned by BlackAssy, and the expression of the rabies virus G protein was confirmed by the fluorescent antibody method using the antirabies virus ゥ sera serum against the recombinant virus-infected cells. Was. In addition, anti-rabies virus egret serum was prepared by inoculating subcutaneous rabbits with purified rabies virus together with Freund's adjuvant. This example shows that the recombinant virus CHV (Y) dlTK / Lac Z enables a screening method based on the color of virus black and is effective in producing a new recombinant CHV of interest. The recombinant virus obtained in this example was named CHV (Y) dlTK / RV-G.

(17) Beta-gal expression unit and the canine disease virus G protein expression unit, beta-gal expression unit and the Inu distemper virus F protein expression unit, beta-gal expression unit and the Inu distemper virus H protein expression unit Or Recombinant CHV in which the beta-gal expression unit and the Aujeszky's disease virus thymidine kinase expression unit are integrated into the thymidine kinase gene region of the CHV genome

In this example, the basic operation was performed in the same manner as in Example (15). In this example, the plasmid pCTKdlEH / Lac Z / RV-G created in Example (7) and the plasmid _P CTKdlEH created in Example (10) were introduced into MDCK cells by the electroporation method. / Lac Z / CDV-F, the plasmid pCTKdlEH / Lac Z 'CDV-H created in Example (11) or the plasmid pCTKdlEH / Lac Ζ,' ΡΤΚ created in Example (12). CHV, DFD-6 strain was used as a virus to infect cells into which this plasmid had been introduced. In this example, the parent virus CHV, the thymidine kinase gene of the DFD-6 strain and the bivalent expression unit of each plasmid, ie, beta-gal expression unit and each virus protein expression unit (rabies virus G protein) Protein, canine distemper virus F protein, canine distemper virus H protein, or Aujeszky's disease virus thymidine kinase expression unit) It is expected that homologous recombination will occur and that a recombinant virus incorporating two expression units will appear. The recombinant virus emerging in this manner forms a blue black on the black-assay using X-gal due to the beta-gal gene product. In fact, virus plasmids that form blue black by introducing their respective plasmids were obtained. These virus clones, which were simultaneously introduced with their respective viral protein expression units, were found to be resistant to recombinant virus-infected cells. Fluorescent antibody method using rabies virus ゥ heron serum or anti-innus temper virus ゥ sera serum, or added to recombinant virus that incorporates a unit of expression of thymidine kinase of oeskinosis virus during virus propagation in tissue culture Confirmed by sensitivity to 5-1000-2'-DEOXYURI INE (IDU) at a concentration of 50 £ / 1111. In addition, the anti-indistemper virus peregret serum was prepared by inoculating subcutaneous persimmons of purified canine distemper virus with Freund's adjuvant. This example shows that two or more independent expression units can be inserted into a single site of a foreign nucleic acid sequence insertion site, and provides, for example, a method for producing a multivalent vaccine. The recombinant viruses obtained in this example were the plasmids pCTKdlEH / Lac Z / RV-G, pCTKdlEH / Lac Z / CDV-F, pCTKdlEH / Lac Z / CDV-H, pCTKdlEH / Lac Z used for homologous recombination. / PTK corresponding to CHV (D) dlTK / Lac Z / RV-G, CHV (D) dlTK / Lac Z / CDV -F, CHV (D) dlT / Lac Z / CDV -H, or CHV ( D) It was named dlTK / Lac Z / PTK.

(18) Recombinant CHV in which rabies virus G protein expression unit, inudistemper virus F protein expression unit, or inudistemper virus F protein expression unit are incorporated into the thymidine kinase gene transfer region of the CHV genome. In this example, the basic operation was performed in the same manner as in Example (15), except that the recombinant virus CHV produced in Example (17) was used as a virus to be infected after introduction of plasmid DNA. (D) dlTK / Lac Z / PTK was used. This virus expresses the thymidine kinase gene product of the oeschis disease virus and is sensitive to IDU as described in Example (17). Conversely, when this gene is deleted, it becomes resistant to this drug. This property is weak in the thymidine kinase gene of CHV. Therefore, the plasmid pCTKdlEH / RV-G created in Example (6), the plasmid created in Example (13), Recombinant virus CHV (D) dlTK / Lac Z / PTK was introduced into MDCK cells into which pCT dlEH / CDV-F or the plasmid pCTKdlEH 'CDV-! f produced in Example (13) was introduced by electroporation. And infect the MDCK cells with the emerging virus in the presence of IDU in advance to selectively grow a new recombinant virus that is resistant to IDU. That is, the obtained virus solution was inoculated on an MDCK cell sheet on a plastic 12-well plate, adsorbed for 1 hour, and then the minimum essential medium of Eagle containing 50 jug / ml of IDU and 5¾FBS was applied to each well. Incubate in a carbon dioxide incubator at 37 ° C for 24 to 48 hours, freeze-thaw three times, crush the infected cells with an ultrasonic homogenizer, and centrifuge at 3,000 rpm for 5 minutes. Collected. This virus solution was again subjected to selective culture in the presence of IDU, and the resulting virus solution was subjected to plaque assay in the presence of X-gal. Since the recombinant virus expected to appear in this example is constructed so as to delete the beta-gal expression unit in addition to the Aujeszky's disease virus thyridine kinase expression unit, it appears as white black in Black Athesi. . In fact, in the virus solution after the selective culture in this example, the ratio of the recombinant virus forming white black was as high as one third of the total virus. In this example, the recombinant virus CHV (D) dlTK / Lac Z / PTK enables selection culture by IDU in addition to selection by plaque color, and provides a method for easily cloning a new recombinant virus. You. The resulting recombinant virus was used in response to the plasmids pCTKdlEH / RV-G, pCTKdlEH / CDV-F, or pCTKdlEH / CDV-H used for homologous recombination, CHV (D) dlTK / RV-G, CHV (D ) Named dlTK / CDV-F or CHV (D) dlTK / CDV-H.

(19) Creation of recombinant CHV with beta-gal expression unit integrated into 0RF2 region of CHV genome

In this example, the basic operation was performed in the same manner as in Example (15). In this example, the plasmid pORF2 / LacZ created in Example (14) was used as the plasmid to be introduced into MDCK cells by the electroporation method, and the virus that infects cells into which this plasmid was introduced was used. CHV. DFD-6 strains were used. In this example, it is expected that the 0RF2 gene on the CHV genome is disrupted, and the beta-gal expression unit is inserted into the site by homologous recombination. In the presence of X-gal A bluer black appeared and was cloned and named CH (D) dlORF2 / Lac Z. This example shows that the 0RF2 region on the CHV genome is also a site into which a foreign nucleic acid sequence can be inserted.

(20) Induction of neutralizing antibody titer against rabies virus in dogs by recombinant CHV incorporating rabies virus G protein expression unit

The recombinant virus CHV (Y) dlTK / RV-G, CHV, YP11 strain produced in Example (16) or a commercially available inactivated rabies vaccine (Nisseiken Co., Ltd.) was used in two or three dogs per group. Then, the appearance of neutralizing antibodies against rabies virus and the antibody titers were observed. Recombinant CHV can to ^{^{_{2 X 10 3 · 75 TCID 5}}} () on both sides of the nasal cavity, non-recombinant CHV is to ^{^{_{2 X 10 4 · 5 TCID (}}} ) on both sides of the nasal cavity, inactivated rabies vaccine according to its prescription It was inoculated subcutaneously. Two-fold serial dilutions of the pre-inoculation and post-inoculation sera collected over time, mixed with an equal volume of a solution containing 100 TCID _{5 ()} rabies virus, and allowed to react at 37 ° C for 1 hour. Aliquots of 100 were added to the wells, and HmLu-C3 cell suspension 501 was added to the wells and cultured in a carbon dioxide incubator. The antibody titer in the serum was determined based on the appearance of the cytopathic effect. Table 1 shows the movement of the neutralizing antibody titer after inoculation.

Table 1 Changes in neutralizing antibody titers against rabies virus

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

According to the present invention, the use of genetic recombination technology for CHV provides a mass production system of useful proteins, a rational method for the production of veterinary vaccines and therapeutic agents, restoration of animal health, This has the effect of contributing to maintenance and promotion.

Claims

The scope of the claims

1. A recombinant canine virus, wherein a nucleic acid sequence that does not exist on the canine virus genome is inserted into the genome.

The recombinant canine virus according to claim 1, wherein the nucleic acid sequence is introduced by a homologous recombination method.

The recombination characterized in that the site for introducing a nucleic acid sequence not present on the canine virus genome according to claim 1 is a region on the canine virus genome that does not have a lethal effect on virus growth. Influenza virus.

4. The recombinant canine herpes virus according to claim 3, wherein the region having no lethal effect on virus growth is the thymidine kinase gene region.

5. The recombinant canine virus according to claim 3, wherein the region that does not have a lethal effect on virus growth is the ORF2 region located downstream of the gC gene. The nucleic acid sequence that is not present on the canine virus genome of claim 1 is constructed as an autonomous expression unit connected in the same direction between a promoter operating in a eukaryotic cell and a polyadenylation signal. And a recombinant canine herpes virus.

7. A recombinant Indobacterium virus, wherein a plurality of the autonomous expression units of claim 6 are inserted.

8. A recombinant canine virus, characterized in that the promoter of the immediate early gene of human megalovirus is used as the promoter operable in eukaryotic cells according to claim 6.

9. A recombinant canine virus, wherein the promoter of the early gene of Simian Virus 40 is used as one of the promoters operating in eukaryotic cells according to claim 6.

10. The nucleic acid sequence which is not present on the canine virus virus genome according to any one of claims 1, 6 to 9, encodes an antigen which is derived from a pathogenic microorganism and induces an ability to protect an animal against infection. A recombinant nucleic acid sequence.

1. The recombinant dog strain of claim 10, wherein the nucleic acid sequence encoding an antigen which induces the ability to protect against infection according to claim 10 is a nucleic acid sequence encoding a rabies virus G protein.

2. The recombinant canine virus according to claim 10, wherein the nucleic acid sequence encoding an antigen that induces the protective ability against infection is a nucleic acid sequence encoding a canine distemper virus H protein.

3. The recombinant dog according to claim 10, wherein the nucleic acid sequence encoding an antigen that induces the protective ability against infection is a nucleic acid sequence encoding a canine distemper virus F protein.ぺ virus.

4. The recombinant canine virus according to any one of claims 1 to 13, wherein a thymidine kinase gene which is sensitive to a / 3-galactosidase gene or 5-I0D0-2'-DEOXYURIDINE is inserted. A method characterized in that the selection is carried out using a canine virus.

5. A method comprising inoculating an animal with the recombinant canine herpesvirus according to any one of claims 10 to 13 to confer protection against infectious diseases.

6. A method for preparing a vaccine, comprising the recombinant canine virus according to any one of claims 10 to 13.

7. A vaccine comprising the recombinant canine virus according to any one of claims 10 to 13.

8. The recombination characterized in that the nucleic acid sequence that is not present on the canine virus genome of claim 1 and claim 6 is a nucleic acid sequence encoding a product having a therapeutic effect on animals. Influenza virus.

9. A method comprising treating an animal by inoculating an animal with the recombinant canine virus of claim 18.