JP2003284554A - New recombinant feline herpes virus type 1 and polyvalent vaccine using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vector virus having at least two kinds of foreign nucleic acid sequences inserted thereinto by identifying the gene regions in a genome at which inserted foreign genes can be expressed without affecting the replication of a feline herpes virus type 1 (FHV-1), and to provide an attenuated recombinant FHV-1 utilizable as a vaccine. <P>SOLUTION: The attenuated recombinant FHV-1 is obtained by inserting at least two kinds of foreign genes into two different gene regions not fatally affecting the proliferation of the virus in the genome of the feline herpes virus type 1. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to use of feline herpesvirus type 1 (feline herpesvirus type 1, hereinafter abbreviated as FHV-1) as a vector virus.

[0002]

2. Description of the Related Art Regarding the gene of a virus whose cat is a host, for example, herpes viridae (Herpesvidae) has been known.
ridae's Alphaherpes vilinae (Alphaherpesvi)
A part of the genomic DNA of FHV-1, a herpesvirus that induces viral rhinotracheitis in cats, belongs to the subfamily, and is artificially deleted using genetic engineering techniques. A recombinant viral vector that does not exist in nature has been created by introducing an exogenous gene so as to be expressed in cells or in the animal body. Such a recombinant viral vector produces a product derived from an exogenous gene in addition to a viral antigen derived from FHV-1 by infecting cells or animals, and when inoculated into an animal,
It is known to confer immunity to foreign gene products in addition to FHV-1 (N. Yokoyama et al., 1996, Arc
h. Virol. 141: 481-494; N. Yokoyama et al., 1996,
Arch. Virol. 141: 2339-2351).

As a specific example of such an FHV-1 vector, a recombinant viral vector in which a thymidine kinase (hereinafter abbreviated as TK) gene region of the viral genome is deleted and an exogenous gene is inserted into the defective site is known. Has been
(JH Nunberg et al., 1989, J. Virol. 63: 3240-324
9; N. Yokoyama et al., 1995, J. Vet. Med. Sci. 5
7: 709-714). Further, it has been confirmed that such recombinant FHV-1 expresses a protein derived from an exogenous gene introduced into the TK gene region in addition to the FHV-1 protein without impairing the replication ability of the virus. (GH Cole e
t al., J. Virol. 1990, 64: 4930-4938; RC Wardle
y et al., 1992, JG Virol. 73: 1811-1818). Furthermore, Yokoyama et al. (N. Yokoyama et al., 1996, Arch. Vir
ol. 141: 2339-2351), recombinant FHV-1 in which a foreign gene was inserted in the TK gene region did not show pathogenicity to cats, and cats inoculated with the recombinant FHV-1 were foreign. It has been reported to produce antibodies to the gene product.

On the other hand, an open reading frame 2 positioned downstream of the U _{L (Unique} long) gC gene regions within the region as a foreign gene insertion site other than the TK gene region in FHV-1 genome (open reading frame 2, ORF2 )It has been known. Willemse et al. (MJ Willemse et al., 199
4, J. Gen. Virol. 75: 3107-3116) is a recombinant FHV-1 in which a gene encoding a foreign gene, β-galactosidase (hereinafter abbreviated as LacZ), is inserted into ORF2.
It has been reported that it possessed a virus replication capacity similar to that of FHV-1. Further, FHV-1 genome _{U S} (unique short)
U _S 8.5 in the region, gene region, such as gI and gE It is also known that not necessarily required for replication of FHV-1.

[0005] With respect to the recombinant FHV-1 having inserted a foreign gene into these gene regions, LacZ in U _S 8.5 gene region
It was reported that recombinant FHV-1 with the inserted gene retains the same replication ability as attenuated FHV-1 (MJ Willemse et a
l., 1995, Virology 208: 704-711) while gI
It has been reported that recombinant FHV-1 in which LacZ is inserted into the gE gene region and gF gene region have markedly reduced replication ability as compared with attenuated FHV-1 (MJ Willemse et al., 1996, Vaccine 14:
1-5; MD Sussman et al., 1995, Virology 214: 1
2-20). These U _S genes in addition to the recombinant FHV-1 having a foreign gene insertion site, the FHV-1 genome ORF5 (nucleotide numbers
5869-7113), a recombinant FHV-1 in which an exogenous gene is inserted has been proposed (Japanese Patent Publication No. 2000-501927). However, the safety of these recombinant viral vectors to animals has not been investigated, and it is unknown whether they are nonpathogenic to the host cat. For reference, these exogenous gene insertion sites are shown in a part of FIG. 1 together with the SalI map.

Regarding recombinant FHV-1, several sites in the FHV-1 genome have been identified as foreign gene insertion sites as described above, but multiple foreign genes are simultaneously inserted into multiple gene insertion sites. No recombinant FHV-1 vector was prepared.

On the other hand, in consideration of its use as a vaccine, if it is assumed that essentially one kind of foreign gene is inserted into one gene insertion site, the recombinant FHV produced by the conventional techniques will be produced. Although the -1 vector can be used as a divalent vaccine including FHV-1 itself, it does not always sufficiently respond to the current situation of various vaccines for cats, which are in high demand for multivalentization.

By the way, in order to maintain the life cycle of a virus, phosphorylation of viral proteins is required, and phosphorylation is a cellular or viral protein kinase (protein phosphorylating enzyme: serine / threonine kinase and tyrosine kinase). Is believed to be in charge. Similar to FHV-1, herpes simplex virus type 1 (HSV-1), herpes zoster virus (VZV), or Epstein-Barr virus (EBV) classified in the gammaherpesvirus subfamily that belongs to the alphaherpesvirus subfamily.
For, the presence of a gene sequence encoding a protein kinase in the viral genome is known, and the amino acid sequence encoded by the gene is highly conserved among herpesviruses.

In particular, catalytic domains I to VI of protein kinase have been reported to be highly conserved in various herpesviruses (RF Smith, and TF Smith. 198).
9. J. Virol. 63: 450-455). In addition, the typical amino acid sequence of domain I in the catalytic domain of herpesvirus protein kinase is GXGXXGXV (X is an amino acid with low conservation), and such amino acid sequences are HSV-1, VZV, EB.
V, human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) have been found to be highly conserved (MS Chee, GJ Lawrence, and B.
G. Barrel. 1989.J. Gen. Virol. 70: 1151-1160).

[0010]

However, in the prior art, except for the TK gene-deficient recombinant FHV-1 vector,
It is sufficiently attenuated and safe for cats, retains sufficient replication capacity for production as a vaccine virus, and can simultaneously confer immunity to three or more pathogenic gene products including FHV-1 itself. There has been no case of producing a recombinant FHV-1 vector. That is, in the known recombinant FHV-1 vector, the exogenous gene insertion site is essentially
Since it is limited to one region of the V-1 genome, it can be expected that the cat inoculated with the recombinant FHV-1 is only immunized against one foreign antigen other than FHV-1.

Therefore, for example, in order to confer protective immunity against infection with three pathogenic microorganisms including FHV-1 in cats, recombinant FHV-1 in which one kind of foreign gene is inserted is inoculated, and further, A vaccine consisting of one target pathogenic microorganism or an antigen derived from it must be inoculated. This means that not only diversification of products and manufacturing processes and rising manufacturing costs are unavoidable in the field of vaccine production, but also side effects due to inoculation of multiple vaccines consisting of pathogenic microorganisms are easily expected. .

[0012] On the other hand, a problem when the vector virus is inoculated and introduced into a target animal is homologous gene recombination between the vector virus and the naturally infected virus in the animal individual, and the gene recombination attenuates the vector virus. May become pathogenic.
In order to reduce the acquisition of pathogenicity of vector viruses by such gene recombination, gene mutations or foreign gene fragments are introduced at multiple sites of the vector virus genome, and homology between the vector virus genome and the pathogenic virus genome is It is necessary to prevent the vector virus from converting into a pathogenic virus by means of sex gene recombination unless the gene sequences at multiple sites in the vector virus genome are simultaneously converted into the pathogenic virus type. However, in the present situation, the attenuated recombinant FHV-1 vector in which such multiple gene regions are converted into a gene sequence different from the pathogenic virus or an exogenous gene is simultaneously introduced into the multiple gene regions is mainly used. The vector virus has not been created because it impairs replication.

Vaccines currently used to prevent viral infections and bacterial infections of cats, specifically feline calicivirus, feline panleukopenia virus, feline leukemia virus, rabies virus, FHV-1 and As a vaccine for preventing infections caused by chlamydia, both live attenuated vaccines and inactivated vaccines are administered by injection. However, inoculation by injection is not always the best inoculation method, because the inoculation is complicated and the animal suffers pain. On the contrary, the transmucosal inoculation method such as eye drops, nasal and oral inoculation is easy to inoculate, and does not cause pain to the inoculated animal and further induces mucosal immunity. Compared with many points.

[0014]

[Means for Solving the Problems] The inventors of the present invention have studied to solve the above problems, and have known TK-deficient attenuated recombinant FHV-
1 vector (JP-A-9-267) was further improved. That is, as a new foreign gene insertion site that does not have a lethal effect on FHV-1 replication / proliferation, Sal of the FHV-1 genome is used.
BamH in I fragment located U _L region in the I cleaved DNA fragments
By identifying the I cleavage site (hereinafter abbreviated as I fragment), it was found that recombinant FHV-1 retains virus replication ability and the inserted foreign gene product is expressed. The present invention has been completed by clarifying that the vicinity of the BamHI site in this I fragment is a gene region encoding protein kinase.

Further, a recombinant FHV-1 which can be used as a vector was prepared by inserting different foreign genes into both gene regions of the I fragment and the known TK gene. As a result, the virus (virus vector) is capable of replicating in cells infected with it or in cats inoculated transmucosally, and recombinant FHV-1 in infected cells or cats.
The present invention has been completed by finding that at least two types of foreign gene products inserted into the genome are produced.

That is, the attenuated recombinant feline herpes of the present invention
Pesvirus type 1 is a genotype of feline herpesvirus type 1.
At least two types of foreign matter in two different areas
The gene is inserted so that it can be expressed.
Cats in which two foreign genes are inserted so that they can be expressed
Two different regions within the herpesvirus 1 genome
Has a lethal effect on the growth of feline herpesvirus 1
It is characterized by two areas that are not given. Also before
To give a specific example of this area, this area
Unique among SalI-cleaved DNA fragments of Svirus type 1 genome
Cron (U_L) Region I fragment and thymidine
Two gene regions, a gene region encoding a nase,
Alternatively, the uniqueness of the feline herpesvirus type 1 genome
Long (U _L) Area of protein kinase
Coding gene region and thymidine kinase
There are two gene regions including a gene region.

The protein kinase present in the unique long ( _UL ) region of the feline herpesvirus type 1 genome contains the amino acid sequence shown by SEQ ID NO: 2 in a part thereof, and therefore, the The insertion site is not limited to the gene region encoded by the I fragment, but the entire gene region encoding this protein kinase is targeted.

In the present invention, "expressively" means that an exogenous gene is linked to a gene expression regulatory sequence such as a promoter or enhancer so that the inserted exogenous gene expresses its gene product in cells. It means that a gene fragment consisting of a gene or a part of its sequence is inserted. A sequence such as a promoter, which is a gene expression regulatory sequence for enabling expression of this foreign gene, may be inserted together with the foreign gene as a part of the foreign gene to be inserted, or feline herpesvirus type 1 An exogenous gene, which is a structural gene, may be inserted and arranged so that (FHV-1) itself utilizes a gene expression regulatory sequence such as a promoter possessed in the genome. That is, the foreign gene to be inserted in the present invention may be a structural gene alone or a gene cassette in which the structural gene and a gene expression regulatory sequence such as a promoter are combined.
Preferably, a gene cassette in which a structural gene and a gene expression regulatory sequence are preliminarily linked is used because the expression of the gene product of the inserted foreign gene can be ensured and the flexibility of the insertion site can be secured.

When an exogenous gene is inserted into the I fragment,
The existence of a gene expression regulatory sequence such as a promoter required for the expression of the foreign gene in the fragment, particularly at the BamHI recognition site, which is the foreign gene insertion site, is unknown. Also, if Ba
Even if a gene expression regulatory sequence is present near the mHI recognition site, the mechanism for inserting an exogenous gene is homologous gene recombination that occurs between the transfer vector and the FHV-1 genome. For these reasons, the exogenous gene is not always inserted into the site where the expression of the exogenous gene is efficiently regulated by the gene expression regulatory sequence. Therefore, it is preferable to insert an exogenous gene as a gene cassette containing a gene expression regulatory sequence such as a promoter. Also,
Also in the insertion of an exogenous gene into a gene region encoding a protein kinase, it is preferable to insert the exogenous gene as a gene cassette containing a gene expression regulatory sequence such as a promoter for the same reason.

The gene expression regulatory sequences such as promoters include FHV-1-derived gC promoter and gB promoter, the immediate early (IMV) of human cytomegalovirus (HCMV).
E) Promoter or Marek's disease virus (MDV)
Derived RNA 1.8 promoter (G. Bradley et al., 198
9, J. Virol. 63: 2534-2542). Of these, the FHV-1-derived gC promoter produces an exogenous gene product at the same level as when FHV-1 was naturally infected,
In addition, the immediate early promoter of human cytomegalovirus is particularly preferable in that it actively produces an exogenous gene product due to its strong promoter activity, and that the expressed exogenous gene product provides high immunogenicity. Is.

On the other hand, in the insertion of an exogenous gene into the gene region encoding thymidine kinase, gene expression regulatory sequences such as promoters and enhancers for the expression of thymidine kinase are present near the insertion site of the exogenous gene. Similarly, insertion as a gene cassette containing a gene expression regulatory sequence is preferable in that the expression of the foreign gene can be regulated more appropriately and efficiently. In this case, examples of gene expression regulatory sequences such as promoters include FHV-1-derived gC promoter and gB promoter, human cytomegalovirus immediate-early promoter, MDV-derived RNA 1.8 promoter, and the like. Of these, the gC promoter and the human cytomegalovirus immediate-early promoter are particularly preferable for the reasons described above.

On the other hand, the exogenous gene insertably expressible in two different regions in the feline herpesvirus type 1 genome is derived from a pathogenic microorganism and is a polypeptide that induces an infection protective ability in animals. A gene fragment comprising a coding gene or a partial sequence of the gene, or, for example, interferon γ, interferon α, interferon β, interferon ω, interleukin 12, interleukin 18, interleukin 10, TNFα It is characterized in that it is a gene fragment which is derived from various cytokines and codes for a product showing a therapeutic effect on animals or a partial sequence of the gene.

The present invention also includes a vaccine or therapeutic composition containing the attenuated recombinant feline herpesvirus type 1, which vaccine or therapeutic composition is used by transmucosal inoculation. It has a feature.

Further, the present invention provides a method for preparing an attenuated recombinant feline herpesvirus type 1 in which at least two kinds of foreign genes are expressibly inserted into two different regions in the feline herpesvirus type 1 genome. including. This preparation method comprises the steps of (1) preparing at least two types of transfer vectors in which different expressible foreign genes are inserted into gene fragments consisting of two different gene regions of the feline herpesvirus type 1 genome. ,
(2) Feline herpesvirus 1 in which an exogenous gene has been inserted into a feline cell, preferably a feline cell line, using at least two types of transfer vectors obtained
Of a gene fragment consisting of different gene regions of the type genome and infecting feline herpesvirus type 1 to cause homologous gene recombination between the introduced gene fragment and the feline herpesvirus type 1 genome And (3) selecting a feline herpesvirus type 1 in which homologous gene recombination has occurred.
In this method, homologous gene recombination between a plurality of introduced gene fragments and the feline herpesvirus type 1 genome is performed by a single operation.

Further, the attenuated recombinant feline herpesvirus type 1 of the present invention superinfects feline cells, preferably feline cell lines, with recombinant viruses having foreign genes inserted in different regions of the genome. Can also be obtained. This preparation method comprises the steps of (1) preparing at least two types of transfer vectors in which different expressible foreign genes are inserted into gene fragments consisting of two different gene regions of the feline herpesvirus type 1 genome. (2) A feline herpesvirus type 1 genome in which an exogenous gene is inserted into a feline cell, preferably a feline cell line, using each of the obtained transfer vectors of at least two types. Introducing a gene fragment consisting of different gene regions and infecting feline herpesvirus 1 with the introduced gene fragment and feline herpesvirus 1
(3) selecting a feline herpesvirus type 1 in which homologous gene recombination has occurred, and selecting one gene in the feline herpesvirus type 1 genome Obtaining at least two types of recombinant feline herpesviruses 1 in which the regions are recombined, and (4) obtaining the respective recombinant feline herpesviruses 1 in feline cells, preferably feline strains (5) Feline herpesvirus type 1 in which homologous gene recombination has occurred, wherein the homologous gene recombination occurs between at least two types of recombinant feline herpesvirus type 1 genomes And a step of selecting. This method includes a step of preparing and isolating at least two types of recombinant feline herpesvirus type 1 in which an exogenous gene is inserted into a single gene region in the feline herpesvirus type 1 genome.

The above-mentioned cells to be infected with feline herpesvirus type 1 mean cells derived from cats. The feline cell line is a cell consisting of a single cell type and characterized by being continuously culturable by a tissue culture technique. As such a feline cell line,
For example, CrFK cells (RACra which is a cell line derived from cat kidney)
ndell et al., 1973, In Vitro 9: 176-185), MYA-1 cells, which are interleukin 2-dependent cell line lymphocytes (T.
Miyazawa et al., 1989, Arch. Virol. 108: 131-13
5), FeT-J cells, which are interleukin-independent CD4-positive cell line lymphocytes (T. Hohdatsu et al., 1996, J. Gen. Vi.
rol. 77: 93-100). Of these, CrFK cells are preferable because they are highly sensitive to FHV-1 and have good FHV-1 proliferation.

The above-mentioned feline herpesvirus type 1
In the preparation method, a cat herpe into which an exogenous gene is inserted
Two distinct genetic regions within the Svirus type 1 genome
Is the SalI-cut DNA fragment of the feline herpesvirus type 1 genome.
Unique long (U_L) I fragment existing in the region
And the gene region encoding thymidine kinase.
Gene region, or feline herpesvirus type 1 geno
Unique long (U _L) Protein present in the region
A gene region encoding a kinase and thymidine kinase
Two gene regions, one that encodes
It is a good thing.

The present invention also provides a method for expressing or producing an exogenous gene in a cell by infecting a cell using the thus obtained attenuated recombinant feline herpesvirus type 1 as a vector. Is what
Also used as a multivalent vaccine, transmucosally inoculated into animals,
An immunization method of immunizing an inoculated animal is also provided.

The cells to be infected here are preferably those having a receptor for feline herpesvirus type 1. Such cells include cat-derived cells, preferably feline cell lines. Further, as a vaccinated animal, an animal of the feline family, preferably an animal in which infection is established against feline herpesvirus type 1, such as a cat, is a preferable target. The immunity to be given includes systemic humoral immunity and cellular immunity as well as local immunity based on mucosal inoculation. On the other hand, even in animals other than felines, the recombinant feline herpesvirus 1 of the present invention
The mold can also be vaccinated to immunize. That is, even in animals that are insensitive to feline herpesvirus 1 such as animals other than cats, at least by inoculating this recombinant feline herpesvirus 1
The foreign gene inserted into the FHV-1 genome can be expressed and produced in the inoculated animal. As a result, when the foreign gene encodes an antigenic protein of a pathogenic microorganism, the inoculated animal is endowed with immunity to the pathogenic microorganism.

As described above, the present invention has found a new foreign gene insertion site, and thus, there is no example so far.
This is a new FHV-1 vector in which an exogenous gene can be inserted into two gene regions in the FHV-1 genome.

Further, of foreign genes inserted into two different regions in the feline herpesvirus type 1 genome, a gene encoding a polypeptide which induces the defense against infection in animals or the gene thereof. The gene fragment consisting of a part of the sequence of the gene includes a gene encoding a protein having an antigenicity produced by an animal belonging to the feline family, mainly various cats that infect cats, or a gene fragment consisting of a part of the sequence of the gene. And antigenic proteins produced by various pathogenic microorganisms that infect animals other than cats. Examples of these include feline leukemia virus coat and core proteins, feline panleukopenia virus VP2 protein, feline immunodeficiency virus coat and core proteins, feline calicivirus capsid protein, and chlamydia major Outer membrane protein MOMP
-1, a gene encoding a coat protein and nucleocapsid protein of rabies virus, a structural protein of canine distemper virus or canine parvovirus, or a gene fragment consisting of a part of the sequence thereof.

Further, a gene fragment consisting of a gene encoding a gene product showing a therapeutic effect on animals or a partial sequence of the gene includes a viral infection, a bacterial infection or a viral infection in a cat or another animal. There are genes that encode TH1-type and TH2-type cytokines that have a protective effect against tumors and a therapeutic effect. Such examples include interferon γ, interferon α, interferon β, interferon ω, interleukin 12, interleukin 18, interleukin 10,
Examples of the gene fragment include a gene encoding TNFα and the like or a partial sequence thereof.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION The attenuated recombinant feline herpesvirus type 1 of the present invention is a feline herpesvirus type 1 genome in which two different regions are expressibly inserted into two different regions. And can be used as a vector or vaccine.

Such a recombinant virus can be obtained by preparing a different transfer vector and homologous gene recombination between the gene sequence of this transfer vector and the feline herpesvirus type 1 genome. That is, in order to obtain feline herpesvirus type 1 in which an exogenous gene is inserted into two different regions in the genome as in the present invention, two types of transfer vectors are used to homologous genes in each region in the genome. By homologous recombination either by causing recombination or by once preparing feline herpesvirus type 1 in which an exogenous gene has been inserted into different regions of the genome, and then superinfecting these viruses. Can also be obtained. The latter is a preferable method because each recombinant virus grows in infected cells, and thus the probability of homologous gene recombination increases.

The region into which the foreign gene is inserted is
It must not be a region that encodes a gene essential for virus growth and must be a region that allows expression of the inserted foreign gene. As an insertion region of an exogenous gene satisfying such a condition, an I fragment portion present in the unique long ( _UL ) region of the SalI-cut DNA fragment of the feline herpesvirus type 1 genome and a gene region encoding thymidine kinase are included. Two gene regions or a unique long feline herpesvirus type 1 genome
Two gene regions, that is, a gene region encoding a protein kinase existing in the ( _UL ) region and a gene region encoding a thymidine kinase are exemplified.

When an exogenous gene is inserted into the I fragment,
The insertion site of the exogenous gene may be any part of the I fragment. In particular, SEQ ID NO: 1 of which the sequence is fixed among I fragments
The portion (PstI-HindHI in the I fragment, sequence region, FIG. 1) indicated by is a preferable region because insertion of an exogenous gene is facilitated.

In the case where an exogenous gene is inserted in the gene region part encoding the protein kinase, the protein kinase is encoded by the gene sequence of the I fragment part, and the amino acid sequence shown in SEQ ID NO: 2 is used as a part thereof. It includes. Therefore, the insertion site of the exogenous gene may be not only the structural gene encoding this protein kinase, but also the regulatory gene part of the protein kinase, and is not limited to the I fragment part, but encodes a protein kinase. The range of all genes is of interest.

Feline herpesvirus type 1 genome and SalI
A map of the case of digestion with the above is shown in FIG. 1 together with the conventionally known insertion site of a foreign gene. In the figure, the _{U L} is unique long region, the _{U S} is unique short region,
IR _S indicates the internal repeat short area, TR _S
FIG. 6 is a diagram showing a terminal repeat short region.
On the other hand, each of the letters A to N represents the name of each fragment when cleaved with SalI (PA Rota et al., 1986, Vir.
ology 154: 168-179; A. Grail et al., 1991, Arch.
Virol. 116: 209-220). Particularly preferred foreign gene insertion regions in the present invention are the sequence region in this I fragment, particularly the BamHI site, and the other region.
It is the thymidine kinase region indicated by TK in the fragment.

Then, when the gene sequence in the vicinity of the BamHI site in the I fragment which is the insertion region of this unique foreign gene was examined, it was found that
It constituted a part of the gene sequence encoding the kinase. Therefore, a gene region encoding protein kinase is preferable as an insertion site for an exogenous gene.

It has been confirmed that the foreign gene inserted into these regions expresses a gene product protein in infected cells. In addition, the recombinant FHV-1 constructed in the present invention is a virus that is more attenuated (non-pathogenic) by deleting the thymidine kinase of the attenuated FHV-1 used in the production of the recombinant virus. Has become.

Therefore, the attenuated recombinant feline herpesvirus type 1 of the present invention can be used as a vector or as a vaccine. It has been confirmed that when used as a vaccine, it induces production of an antibody against a protein produced by the expression of at least two kinds of inserted foreign genes and an antibody against feline herpesvirus type 1. Since this vaccine is a herpes virus, transmucosal administration, that is, nasal, instillation and oral administration is a particularly preferable administration method, but administration by injection which has been conventionally used is also effective.

The vaccine can be prepared by suspending the recombinant FHV-1 of the present invention in an isotonic phosphate buffer. The amount of virus to be inoculated to an animal varies depending on the titer and immunogenicity of the virus. For example, when a cat is transmucosally inoculated, the amount of vaccine solution to be administered is 1-2.
ml is common. It is preferable to adjust the amount of the administered solution to a virus amount that can induce immunity.

As an example of the present invention, the case where a gene fragment encoding feline calicivirus capsid protein and a gene encoding β-galactosidase are used as an exogenous gene will be described below.

The recombinant FHV-1 of the present invention, as schematically shown in FIG. 2, is a Glycoprotein C promoter derived from FHV-1 (hereinafter referred to as g
Recombination in which a gene fragment (hereinafter abbreviated as gC-Cap) encoding a feline calicivirus (hereinafter abbreviated as FCV) capsid protein accompanied by a C promoter is incorporated
FHV-1 (dTK-gC / Cap-FHV, FIG. 2A, JP-A-9-267,
) And FHV-1 I fragment to the cytomegalovirus (CM
V) A feline kidney-derived cell line was superinfected with recombinant FHV-1 (Lac FHV, see FIG. 2B) incorporating a β-galactosidase gene (hereinafter, abbreviated as Pro-LacZ) fragment with an immediate early promoter derived from V), GC-Cap fragment and Pro-in the same viral genome emerged by homologous recombination between both viruses
Recombinant FHV-1 with integrated LacZ fragment (FHV-Cap / Lac,
2C).

The method for producing recombinant FHV-1 will be described below.

First, a transfer vector containing a Pro-LacZ fragment was prepared as follows. That is, FH
V-1 genomic DNA, which is one of the SalI-cleaved DNA fragments, was incorporated into the plasmid vector pSal-I (Faculty of Agriculture, University of Tokyo, distributed by Dr. Takayuki Miyazawa). A plasmid containing the newly constructed I fragment recloned in a plasmid vector
A LacZ gene fragment (Pro-Lac) in which a commercially available eukaryotic expression vector pCMVβ (CLONTECH) -derived immediate early promoter was attached to a unique BamHI cleavage site within the I fragment of pdBSI
Z) was incorporated to prepare a transfer vector pdBSI-LacZ.

Escherichia coli, which is a transformant containing this pdBSI-LacZ, was deposited as E-dBSI-LacZ under the deposit number FERM P-17935 at the Patent Microorganism Depositary Center, Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology, June 2000. Deposit on 30th (deposit number FERM
P-17935) and changed to international deposit as of October 3, 2001 (accession number FERM BP-7761). At present, the name of the depositary institution has been changed to “Independent Administrative Agency National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center”.

Next, the transformant constructed as described above
PdBSI-LacZ DNA was extracted from E-dBSI-LacZ and CrFK cells (Cran
dell feline kidney cells, feline kidney-derived cell lines, and a gift from Dr. Takayuki Miyazawa, Faculty of Agriculture, The University of Tokyo) were transfected by the transfection method, and then the CrFK cells were infected with attenuated FHV-1. After that, homologous gene recombination was caused between the FHV-1 genomic DNA-derived I fragment in pdBSI-LacZ and FHV-1 genomic DNA, and recombinant F in which Pro-LacZ was inserted into the I fragment region of FHV-1.
HV-1 was produced. Recombinant FHV-1 produced in this way
Was designated as LacZ-FHV (Fig. 2B).

Next, the EcoRV-SmaI gene fragment in the TK gene region was deleted from the FHV-1 genome by using a genetic engineering technique, and the FHV-1-derived gC promoter and F
Linked to the gene encoding CV capsid protein
Recombinant FHV-1 with inserted DNA fragment (dTK-gC / Cap-FHV, Fig. 2
A, Distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, The University of Tokyo; References, N. Yokoyama et al., 1998, J. Vet. Med. Sci. 6
0: 717-723) and LacZ-FHV described above were co-infected into CrFK cells to induce homologous gene recombination between these two types of recombinant FHV-1 and BamHI in the I fragment of the FHV-1 genome. La in the part
Recombinant FHV-1 in which the FCV capsid protein gene was incorporated into the cZ and TK gene regions, respectively, was produced.

The recombinant FHV-1 thus obtained, that is, the recombinant FHV-1 capable of producing the FCV capsid protein and β-galactosidase, was obtained as FHV-Cap / LacZ.
(Fig. 2C).

The above operation is performed by Sam Block et al. (J. Sambro
ok et al., 1989, Molecular cloning: a laboratory m
anual. 2nd edition, Cold Spring Harbor, New York:
It can be performed by known gene manipulation techniques and cell engineering techniques described by Cold Spring Harbor Press).

The FHV-Cap / LacZ prepared as described above has the ability to replicate in a feline-derived cell line and in a feline individual, and when inoculated into the nasal cavity, eyes or oral cavity of a feline, the feline has no clinical signs. It was confirmed to be safe for cats because it did not cause side effects.

As described above, the SalI-cut DNA of the FHV-1 genome
Found that BamHI recognition site in the I fragment located U _L region of the fragment is not essential for replication of FHV-1, integrate the foreign gene into the I fragment, a gene further from the foreign gene incorporated The present invention is the first to discover that a product is produced in cells of animal origin and in the body of an animal.

In addition, a recombinant FHV-1 was constructed by incorporating an exogenous gene into the two gene regions of the I fragment and the TK gene existing in the _UL region of the SalI digested DNA fragment of the FHV-1 genome, Further, the present invention is the first to find that the recombinant FHV-1 replicates in the feline cell line and the feline body.

Furthermore, the nucleotide sequence near the BamHI digestion site in the SalI I fragment newly found as the insertion site of the foreign gene was determined, and the amino acid sequence predicted from the nucleotide sequence was analyzed. As a result, the vicinity of the BamHI digestion site was determined. Have a similar amino acid sequence to that of catalytic domains I to VI of protein kinases of other herpesviruses. The nucleotide sequence of the SalI I fragment in the FHV-1 genome has not been determined so far, and the present invention is the first time that it was found that the nucleotide sequence encoding a protein kinase is present in the I fragment. .

[0056] Further, also beginning the present invention was discovered that protein kinase region of the U _L region of the FHV-1 genome is effective as the foreign gene insertion site.

[0057]

The present invention will be described in more detail with reference to the following examples. 3 and 4 show the outline of the experimental operation carried out in the present invention. In addition, FIG. 3 shows the LacZ insertion transfer vector pd.
An outline up to the production of BSI-LacZ is shown in FIG. 4, in which DNA from this transfer vector pdBSI-LacZ is used to insert recombinant FHV-1 FHV-1 into which LacZ and FCV capsid genes have been inserted.
It shows an outline of the method for producing V-Cap / Lac.

1. Construction of transfer vector for producing recombinant FHV-1 vector (1) Construction of plasmid incorporating FHV-1 genomic I fragment pSal which is a plasmid vector containing I fragment of SalI library of FHV-1 genomic DNA -I (distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, University of Tokyo) was digested with SalI, and then commercially available QIAquick
Using a Gel Extraction Kit (QIAGEN), the I fragment of the FHV-1 genome was separated and prepared according to the attached manual.

On the other hand, the plasmid vector for incorporating this I fragment was a commercially available plasmid vector pUC18 (Amersham Pharmacia Biotech) opened by digestion with BamHI.
In the presence of T4 DNA polymerase (Takara Shuzo) at 37 ° C for 5
Blunt ends were obtained by incubation for a minute. Then, in the presence of T4 DNA ligase (GIBCO BRL), 16 ° C, 1
It was prepared by ligating BamHI cleavage sites blunted by incubation for 7 hours. The obtained plasmid vector lacking the BamHI recognition site was named pUC-dB. In the figure, the BamHI surrounded by a square represents the BamHI site that disappeared by such an operation.

Next, pUC-dB was digested with SalI to open the ring, and at the SalI digestion site, I of the FHV-1 genome separated and prepared above was prepared.
The fragments were ligated using T4 DNA ligase. The plasmid obtained by incorporating the I fragment of the FHV-1 genome into the SalI digestion site of pUC-dB thus obtained was designated as pdBSI.

(2) Preparation of Transformant Containing pdBSI E. coli XL-1 B Competent with pdBSI DNA Solution
E. coli was transformed by mixing it with lue (manufactured by Stratagene) and introducing the plasmid DNA into E. coli according to the attached manual. LB agar medium containing 50 μg / ml of ampicillin (Agar medium containing 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 g of sodium chloride, and 15 g of Bacto agar in 1 L). C. for 17 hours at 37.degree. C. to obtain ampicillin-resistant transformant clones grown on an agar medium. The plasmid DNA extracted from each transformant clone was then digested with SalI and analyzed by electrophoresis on a 0.8% agarose gel (Fig. 6, lane 1) to give about 6,800.
Selecting transformant clones having the base pair I fragment,
The clone was designated as E-dBSI.

(3) Construction of Transfer Vector pCMVβ (CLONETECH), which expresses β-galactosidase in a commercially available eukaryotic expression plasmid vector, was digested with PstI and CM was prepared using the above QIAquick Gel Extraction Kit.
A gene fragment (Pro-LacZ) containing the LacZ gene with an immediate early promoter derived from V was isolated and purified. Then, the PstI digestion site was blunted by incubating the Pro-LacZ in the presence of T4 DNA polymerase at 37 ° C for 5 minutes.

On the other hand, the above-mentioned pdBSI, which is a plasmid for incorporating Pro-LacZ, was opened by digestion with BamHI having a recognition site at one site in the I fragment, and the BamHI site was transformed with T4 as described above. It was smoothed by DNA polymerase treatment.

Next, blunt-ended Pro-LacZ and ring-opened blunt-ended pdBSI were treated with T4 DNA ligase in the presence of 16
Ligation was carried out by incubation at 0 ° C for 17 hours. The transfer vector in which Pro-LacZ was incorporated into the blunted BamHI site in the I fragment of the FHV-1 genome-derived SalI library thus obtained was named pdBSI-LacZ (FIG. 3).

The constructed transfer vector pdBSI-La
A schematic diagram of cZ is shown in FIG. In the figure, "LacZ" represents a LacZ gene fragment, PstI and BamHI surrounded by a square represent blunt-ended PstI recognition site and BamHI recognition site, respectively, and "Pro" represents human cytomegalovirus (HCM
V) indicates the immediate early promoter, and the arrow indicates the orientation of the LacZ gene fragment and the promoter.

2. Preparation of transformants containing pdBSI-LacZ pdBSI-LacZ DNA solution commercially available E. coli X
E. coli was transformed by mixing with L-2 Blue MRF 'and introducing the plasmid DNA into E. coli according to the attached manual, and the transformant was further incubated at 37 ° C using LB agar medium containing 50 μg / ml ampicillin. By culturing for 17 hours, an ampicillin-resistant transformant clone grown on an agar medium was obtained.

Next, the plasmid DNA extracted from each transformant clone was digested with SalI, and analyzed by electrophoresis on 0.8% agarose gel to give about 6,600 base pairs and about 4,700 base pairs.
A clone with each of the I fragment divided into two base pairs (based on the SalI site in the Pro-LacZ cassette inserted in the I fragment, see FIG. 5) and the DNA fragment of the vector plasmid pUC18 of about 2,600 base pairs. Were selected (FIG. 6, lane 2, digestion of pdBSI-LacZ with SalI). In addition, NotI digestion of selected clones revealed that the clones had an L of 3,474 base pairs.
It was confirmed to have an acZ DNA fragment (FIG. 6, lane 3, digestion of pdBSI-LacZ with NotI). In the electrophoresis results shown in FIG. 6, lane 1 is pdBSI in which the I fragment without Pro-LacZ was inserted was digested with SalI, and the I fragment of about 7,000 base pairs and about 2,600 base pairs were obtained. 2 and the DNA fragment of the vector plasmid pUC18 of the above (FIG. 5). Further, in FIG. 6, lane M is a molecular weight marker and shows the approximate number of base pairs.

Transformed E. coli containing the transfer vector pdBSI-LacZ selected in this manner was transformed into E-dB.
Named SI-LacZ, deposited at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry (accession number FERM P-1
7935), and changed to international deposit on October 3, 2001 (accession number FERM BP-7716).

3. Preparation of LacZ-inserted recombinant FHV-1 The above transfer vector pdBSI-LacZ was added to CrFK cells.
By introducing DNA by the transfection method and further infecting CrFK cells into which the gene has been introduced with attenuated FHV-1, by homologous gene recombination between pdBSI-LacZ DNA and the attenuated FHV-1 genome in the cell. , Recombinant FHV-1 having an I fragment in which the LacZ gene was integrated at the BamHI cleavage site within the I fragment which is one of the SalI digested DNA fragments of the FHV-1 genome was obtained. The details will be described below.

3 μg of pdBSI-LacZ DNA was mixed with 10 μl of a commercially available transfection reagent, LipofectAMINE (manufactured by GIBCO BRL), and reacted at room temperature for 45 minutes. Then, using a 6-well plate for tissue culture, the minimum essential medium of Dulbecco containing 10% fetal bovine serum (hereinafter, abbreviated as D-MEM, manufactured by Nissui), at 37 ° C, after culturing in the presence of 5% carbon dioxide, D-MEM 3
To 1 × 10 ⁶ CrFK cells washed twice with ml, pdBSI-L
A reaction solution of acZ DNA and a transfection reagent was added, and pdBSI-LacZ DNA was introduced into CrFK cells.

Next, the gene-introduced CrFK cells were transferred to 37
After culturing for 24 hours in the presence of 5% carbon dioxide at ℃, CrFK cells were infected with attenuated FHV-1 at a MOI (multiplicity of infection, virus infectivity) of 0.01. Like this pdBS
After gene transfer of I-LacZ DNA, CrFK cells infected with attenuated FHV-1 were cultured for about 3 days, and when cytopathic effect (cytopathic effect, hereinafter abbreviated as CPE) was observed in most cells The cells were disrupted by freeze-thawing the cell suspension three times, and finally the virus supernatant containing both attenuated FHV-1 and recombinant FHV-1 was added to the centrifugal supernatant of the disrupted cell suspension. Recovered. Screening of recombinant FHV-1 from this virus solution was performed by the plaque selection method shown below.

That is, the collected virus solution was added to D-MEM for 1
Serial dilution from 0 ^-1 to 10 ^-5, 300 μl of each diluted virus solution
1 × 10 ⁶ CrF grown in 6-well tissue culture plate
After adding to K cells and adsorbing the virus at 37 ° C for 1 hour, 2%
D-MEM soft agar medium containing fetal bovine serum and 1% agarose 1
ml was layered and cultured for 2 days. Then 0.01% Neutral Red and 1 mg / ml X-gal (5-bromo-4-chloro-3-in
dolyl β-D-galactopyranoside, manufactured by SIGMA), D-MEM soft agar medium 1 containing 1% fetal bovine serum and 1% agarose 1
ml was overlaid and the cells were cultured for a further 24 hours and blue plaques formed by the propagated recombinant virus were isolated. After that, the same plaque selection method was repeated twice and LacZ
Recombinant FHV-1 in which I was integrated into the I fragment in the genome was selected. The thus obtained LacZ-inserted recombinant FHV-1 was ligated to Lac
-FHV (see Figure 2B).

4. Preparation of recombinant FHV-1 with LacZ and FCV capsid protein gene insertion (1) Preparation of TK-deficient / FCV capsid protein gene insertion FHV-1 FCV with FHV-1-derived gC promoter attached to the TK gene region in a tissue culture flask Recombinant FHV-1 incorporating the capsid protein gene (hereinafter abbreviated as dTK-gC / Cap-FHV, distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, University of Tokyo) 1 × 10
MOI: 0.01 was added to ⁷ CrFK cells, and the cells were adsorbed and infected by incubation at 37 ° C for 1 hour. Then
The cells were cultured in D-MEM medium containing 2% fetal bovine serum at 37 ° C in the presence of 5% carbon dioxide for about 3 days until CPE was observed in most cells. Subsequently, the cells were disrupted by freeze-thawing the cell suspension three times, and the recombinant FHV in which the capsid protein gene of FCV was inserted into the TK gene region that was deleted in the centrifugal supernatant of the disrupted cell suspension. Got -1. The recombinant FHV-1 thus obtained was dTK-gC / Cap-FHV (Fig. 2A).
(See).

The recombinant FHV-1 dTK-gC / Cap-
The transfer vector (named pfTK (gCp) -Cap) incorporating the feline calicivirus capsid protein gene and the FHV-1 derived gC promoter used to generate FHV had the structure shown in FIG. In the figure, the shaded area shows a part of the multiple cloning site derived from the plasmid vector (pBluescript KS-) that was incorporated during the construction of pfTK (gCp) -Cap, and "Pro" was derived from FHV-1. gC promoter, "FCV Capsid" for feline calicivirus capsid protein gene, "SalIA" for FHV-
In the SalIA fragment derived from one genome, "TK" represents the thymidine kinase gene, and the dotted line in the thymidine kinase gene ("TK") represents the deleted gene region. The arrows in the figure indicate the orientation of the feline calicivirus capsid gene and promoter.

(2) Preparation of FHV-Cap / Lac Laxed 1 × 10 ⁶ CrFK cells in a ^6-well tissue culture plate.
c-FHV was added at MOI: 0.01, and the recombinant virus was adsorbed and infected with CrFK cells by incubation at 37 ° C for 1 hour, and then in D-MEM medium containing 2% fetal bovine serum for 24 hours. By culturing, CrFK cells infected with Lac-FHV were grown. Next, dTK-gC / Cap-FHV was added to the Lac-FHV-infected CrFK cells.
Virus solution was added to MOI: 0.01, and dTK-gC / Cap FHV was adsorbed to Lac-FHV-infected CrFK cells by the same procedure as above.
Super-infected. In this way Lac-FHV and dTK-gC / Cap-FH
About 3 times until CrFK cells superinfected with V until CPE is detected
After culturing for a day, the cells were disrupted by a freeze-thaw operation similar to the above, and the recombinant virus, that is, Lac-FHV and dTK-gC / Cap-FHV was partially gene-generated in the centrifugal supernatant of the cell disruption solution. By recombination, a virus solution containing recombinant FHV-1 in which the LacZ gene and the FCV capsid protein gene associated with the gC promoter were integrated in one viral genome was obtained. The above recombinant FHV-1 was selected from this virus solution by the plaque selection method described below.

That is, after serially diluting the virus solution from 10 ⁻¹ to 10 ⁻⁵ , 300 μl of each diluted virus solution was added to 1 × 10 ⁶ CrFK cells in a ^6-well tissue culture plate, and the dilution was performed at 37 ° C. After adsorbing and infecting the cells with the virus for a period of time, 100 mg of arabinofuranosyluracil (arabinofuranocyluracil), 2
1 ml of D-MEM soft agar medium containing 1% of fetal bovine serum and 1% of agarose was overlaid and cultured at 37 ° C. in the presence of 5% carbon dioxide. Two days later, D-MEM containing 0.01% neutral red, 1 mg / ml X-gal, 100 mg arabinofuranosyluracil, 1% fetal bovine serum and 1% agarose on D-MEM soft agar medium. Overlay 1 ml of MEM soft agar medium and continue culturing,
After 24 hours, blue plaques formed by the propagated recombinant virus were isolated. After that, the same plaque selection method was repeated twice, and the LacZ gene was inserted into the I fragment of the viral genome, and the FCV capsid protein gene with the FHV-1-derived gC promoter was inserted into the deleted TK gene region. The replacement FHV-1 was selected. The recombinant FHV-1 thus obtained was named FHV-Cap / Lac (see FIG. 2C).

Regarding the FHV-Cap / Lac obtained here, the certificate of refusal of acceptance by the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, dated December 2, 1999 (1999 (November 29th application) was issued,
Although not deposited at the center, this recombinant virus FHV-Cap / Lac is stored at Tsukuba Central Research Institute, Kyoritsu Pharmaceutical Co., Ltd., and can be distributed if necessary. Also, regarding this FHV-Cap / Lac, currently ATCC (Amer
The ican Type Culture Collection) is being prepared for deposit.

5. Expression of foreign protein from FHV-Cap / Lac in cell lines Expression of FCV capsid protein and β-galactosidase derived from FHV-Cap / Lac in CrFK cells infected with recombinant FHV-1, FHV-Cap / Lac Was analyzed by the fluorescent antibody method shown below.

That is, FHV-Cap was added to 1 × 10 ⁶ CrFK cells.
/ Lac was added at MOI: 0.01 to adsorb and infect the virus in the same manner as above, and then in D-MEM medium containing 2% fetal bovine serum in the presence of 5% carbon dioxide at 37 ° C for 24 hours. Cultured. The cultured cells were then treated with phosphate buffer (137 mM sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium hydrogen phosphate, and 1.47 mM potassium dihydrogen phosphate, pH.
7.4) and fixed with cold acetone.
Expression of the FCV capsid protein and β-galactosidase in the FHV-Cap / Lac-infected CrFK cells fixed in this manner was analyzed by the fluorescent antibody method described below.

Expression analysis of the FCV capsid protein was carried out by the following procedure. First, fixed CrFK cells were allowed to react with mouse anti-FCV capsid protein monoclonal antibody diluted 1: 100 with phosphate buffer solution (distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, University of Tokyo) for 1 hour at 37 ° C, and then phosphate buffer solution. With 3 cells
After washing twice, dilute 1: 1,000 tetramethylrhodamine-5 (-6) -isothiocyanate (Tetramethylrhodamin
The mixture was allowed to react with e-5- (and-6) -isothiocyanate, TRITC) -labeled goat anti-mouse IgG antibody (manufactured by ICN Pharmaceuticals) at 37 ° C for 1 hour. Then, the cells were washed three times with a phosphate buffer and observed under a fluorescence microscope to observe the expression of the capsid protein of FCV. In addition, CrFK cells not infected with FHV-Cap / Lac were treated in the same manner as above to serve as a negative control.

As a result of microscopic examination, as shown in FIG. 8B, red specific fluorescence which was not detected in the negative control FHV-Cap / Lac-uninfected CrFK cells was detected in FHV-Cap / Lac-infected CrFK cells, and CrFK was detected. It was revealed that the FCV capsid protein was expressed and produced from FHV-Cap / Lac in cells.

On the other hand, the expression of β-galactosidase from FHV-Cap / Lac was confirmed by FHV-Cap / Lac-infected C prepared as described above.
Rabbit anti-β-galactosidase serum (Chemicon International) diluted 1: 100 as primary antibody in rFK cells.
And fluorescein isothiocyanat diluted 1: 2,000 as a secondary antibody.
e, FITC) labeled goat anti-rabbit IgG serum (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted in the same manner as above, and fluorescence-labeled β-galactosidase was detected under a microscope using a fluorescence microscope. As in the above, CrFK cells not infected with FHV-Cap / Lac were used as a negative control.

As a result, as shown in FIG. 8A, FHV-Cap was not detected in the negative control FHV-Cap / Lac non-infected CrFK cells, and
The β-galactosidase labeled with green fluorescence was detected only in CrFK cells infected with / Lac.
It was revealed that β-galactosidase was expressed and produced from V-Cap / Lac.

In the double staining method in which the fluorescent antibody method for detecting the FCV capsid protein and the fluorescent antibody method for detecting β-galactosidase were simultaneously performed, the FCV capsid protein labeled with red fluorescence and the green fluorescence were detected. Since the labeled β-galactosidase was detected on the same cells, it was revealed that both FCV capsid protein and β-galactosidase were simultaneously expressed and produced in FHV-Cap / Lac-infected CrFK cells.

From the above results, FHV-C which is recombinant FHV-1
It was demonstrated that ap / Lac replicates in the feline cell line and simultaneously expresses the capsid protein gene of FCV inserted as an exogenous gene and the capsid protein from the LacZ gene and β-galactosidase, respectively.

6. Production of antibodies in cats (1) Preparation of FHV-Cap / Lac As described above, MOI of FHV-Cap / Lac was applied to 1 × 10 ⁷ CrFK cells.
Adsorbed and infected with 0.01. Then, the infected CrFK cells were cultured in D-MEM medium containing 2% fetal bovine serum at 37 ° C. in the presence of 5% carbon dioxide gas for about 4 days until CPE was observed in most cells. Subsequently, the cultured cell suspension was freeze-thawed three times to disrupt the cells, and FHV-Cap / Lac was obtained in the centrifugal supernatant of the disrupted cell suspension.

(2) Detection of anti-β-galactosidase antibody, anti-FHV-1 antibody, and anti-capsid antibody in FHV-Cap / Lac inoculated cats 2 × 10 ⁶ TCID ₅₀ / ml FHV-Cap / Lac 2 ml for 1 year old Three male cats were inoculated nasally, orally, and by instillation, respectively, and after 3 weeks, each cat was additionally inoculated under the same conditions. Five days after this additional inoculation, blood was collected from the jugular vein of each cat, and anti-β-galactosidase antibody in the serum prepared from the blood was detected by the indirect fluorescent antibody method, and anti-FHV-1 antibody against FHV-1 was detected. The sum test and the anti-capsid antibody were detected by the indirect fluorescent antibody method. The indirect fluorescent antibody method, the neutralization test method and their results will be described below.

(2-1) Detection of anti-β-galactosidase antibody β-galactosidase expression plasmid vector pCMVβ 0.3
μg was reacted with 2 μl of commercially available transfection reagent, LipofectAMINE (manufactured by GIBCO BRL) for 45 minutes at room temperature, and the reaction product was placed on an 8-well culture slide glass (manufactured by BECTON DIKINSON). The pCMVβ DNA was introduced into the cells in addition to the 8 × 10 ⁴ CrFK cells cultured in ( ⁴ ). Then, the gene-introduced CrFK cells were incubated at 37 ° C.,
After culturing in the presence of 5% carbon dioxide for 24 hours, washing with phosphate buffer 3 times, the cells were fixed in chilled acetone.

On the other hand, prior to the indirect fluorescent antibody method, FHV-Ca was used.
The serum of the cat inoculated with p / Lac was mixed with an equal volume of the supernatant of the CrFK cell lysate and centrifuged, and the nonspecific antigen-antibody reaction was reduced by the absorption operation at 37 ° C for 30 minutes. This cat serum was diluted 1:20 with phosphate buffer and used as the primary antibody.

After reacting the acetone-fixed CrFK cells prepared as described above with 100 μl of 1:20 diluted feline serum at 37 ° C. for 1 hour,
The cells were washed three times with phosphate buffer and further diluted 1: 1,000 with phosphate buffer to give FITC-labeled goat anti-cat IgG antibody (ICN / CA
It was reacted with PPEL) at 37 ° C for 1 hour. Then, the cells were washed three times with phosphate buffer, and the fluorescence-labeled β-galactosidase in CrFK cells was detected by examining under a fluorescence microscope. Not transfected with pCMVβ
CrFK cells and feline serum before FHV-Cap / Lac inoculation were each subjected to the same indirect fluorescent antibody method as described above to serve as a negative control.

As shown in FIG. 9, pCMV used as a negative control
CrFK cells without β DNA introduced and FHV-Cap / Lac
It was not detected when the pre-inoculation cat serum was used (Fig. 9).
A), pCMVβ DN reacted with FHV-Cap / Lac inoculated cat serum
Fluorescently labeled β-galactosidase was detected only in A-introduced CrFK cells (Fig. 9B). As a result, the inoculated FHV-Cap / Lac replicated in the cat body, and the FHV-Cap / Lac
It was revealed that β-galactosidase was produced from ac.

(2-2) Measurement of anti-FHV-1 antibody FHV-Cap / Lac inoculated cat serum diluted 2-fold serially with D-MEM medium
Tissue culture plates in 100 [mu] l and ^{_{6 × 10 2 TCID 5 0 /}} ml 96 -well and FHV-1 solution 100 [mu] l, prepared, 37 ° C., after 1 hour incubation in the presence of 5% carbon dioxide gas, the 100 [mu] l 2 × 1
0 In addition to the ^five 24 CrFK cells fiber culture in the sheet of holes, further 37 ° C., then 1 hour incubation in the presence of 5% carbon dioxide were absorbed and infect FHV-1 into CrFK cells.

Then, the cell culture medium was removed, and 300 μl of D-MEM soft agar medium containing 2% fetal bovine serum and 1% agarose was layered on the CrFK cells and cultured for 2 days.
The plaques caused by the proliferating virus that appeared when the cells were overlaid with 300 μl of D-MEM soft agar medium containing Neutral Red, 1% fetal bovine serum and 1% agarose and cultured for 24 hours were counted. The number of plaques that appeared at this time was FHV-Ca.
CrFK infected with FHV-1 treated with p / Lac uninoculated cat serum
The dilution ratio of feline serum that was less than half the number of plaques that appeared in the cells was determined, and the reciprocal of the highest dilution ratio of the feline serum was used as the neutralizing antibody titer. In the above measurement of neutralizing antibody titer, FHV-Cap / Lac was inoculated 3
Head cat sera showed 8-fold, 16-fold, and 16-fold neutralizing antibody titers, respectively. As a result, FH inoculated in the cat body
It was confirmed that V-Cap / Lac was duplicated.

(2-3) Measurement of anti-calicivirus capsid protein antibody 100 μl of a virus solution of feline calicivirus F4 strain of 8 × 10 ³ TCID ₅₀ / ml was added to an 8-well culture slide glass (BECTON D).
8 × 10 ⁴ CrFK cells cultivated on IKINSON) were added and incubated for 1 hour at 37 ° C. in the presence of 5% carbon dioxide to adsorb and infect the feline calicivirus F4 strain. Then, after washing the cells with D-MEM medium, the cells were washed with D-MEM medium containing 2% fetal bovine serum at 37 ° C. in the presence of 5% carbon dioxide gas.
Cells were cultured for days. The cultured feline calicivirus F4 strain infected cells were washed three times with a phosphate buffer and then fixed in chilled acetone.

On the other hand, prior to the indirect antibody method, FHV-Cap / La
c The serum of the inoculated cat was mixed with an equal volume of the supernatant of the CrFK cell lysate, and the nonspecific antigen-antibody reaction was reduced by the absorption operation at 37 ° C for 30 minutes. This cat serum was then treated with phosphate buffer
It was diluted 1: 8 and used as the primary antibody.

Acetone-fixed feline calicivirus-infected CrFK cells prepared as described above and 100 μl of 1: 8 diluted FHV-Cap / Lac inoculated feline serum were reacted at 37 ° C. for 1 hour, and then the cells were washed 3 times with phosphate buffer. In addition, FITC-labeled goat anti-cat IgG antibody (manufactured by ICN / CAPPEL) diluted 1: 1,000 with phosphate buffer and 37
The reaction was carried out at 0 ° C for 1 hour. The cells were then washed three times with phosphate buffer and examined under a fluorescence microscope to detect fluorescently labeled feline calicivirus capsid protein in CrFK cells. In addition, the indirect fluorescent antibody method similar to the above was performed using each of the CrFK cells not infected with feline calicivirus and the cat serum before inoculation of FHV-Cap / Lac, and used as a negative control. The results are shown in Fig. 10.

As shown in FIG. 10A, as a negative control, fluorescence-labeled feline calicivirus capsid protein was detected when feline serum before inoculation with FHV-Cap / Lac was reacted with feline calicivirus-infected CrFK cells. Was not done. Also, when the cat serum inoculated with FHV-Cap / Lac was reacted with feline calicivirus-uninfected CrFK cells, the fluorescently labeled feline calicivirus capsid protein as described above was not detected (not shown). That is, the fluorescently labeled feline calicivirus capsid protein was detected only when FHV-Cap / Lac inoculated cat serum was reacted with feline calicivirus-infected CrFK cells, as shown in FIG. 10B. It was From this, it was revealed that the inoculated FHV-Cap / Lac replicated in the cat body, and the feline calicivirus F4 strain capsid protein was produced from the FHV-Cap / Lac.

From the above results, when FHV-Cap / Lac was transmucosally inoculated into cats, the inoculated cats were inserted into the thymidine kinase region as well as producing antibodies against FHV-1 while maintaining a healthy condition. And antibodies against feline calicivirus capsid protein, a product of a foreign gene, and antibodies against β-galactosidase, a product of a foreign gene inserted in the BamHI region of the I fragment. Therefore, the recombination in the present invention
It can be seen that the insertion sites of foreign genes in FHV-1 do not affect the replication of the virus itself, and the foreign genes integrated into these insertion sites produce proteins separately.

That is, it is possible to immunize cats with at least two foreign gene products in addition to FHV-1 by nasally, orally and instilling transmucosally with this recombinant virus. Yes, in fact, as a result of intranasal, oral and instillation of this recombinant viral vector FHV-Cap / Lac in cats, the inoculated cats are calicivirus capsid proteins, β-galactosidase and viral vectors while maintaining their health. Antibodies to FHV-1 were produced.

7. Feline herpesvirus type 1 genome Sa
Determination of partial base sequence of lI I fragment (1) Determination of sequence The partial base sequence of SalI I fragment derived from FHV-1 genome incorporated in plasmid pdBSI (see Fig. 1) was used as a DNA sequencer.
(Manufactured by Pharmacia). The details will be described below.

1 from SalI I fragment of FHV-1 integrated in pdBSI
A 969 base pair partial DNA fragment (PstI-HindIII DNA fragment) was cut out, and a commercially available cloning vector pB1uescrip
tIISK (+) (manufactured by Stratagene) was incorporated into the PstI-HindIII digestion site. The plasmid containing the partial base sequence of the SalI I fragment of FHV-1 obtained was named pSal-Hind.
Then, using the pSal-Hind plasmid DNA as a template, PstI-Hin
A forward primer (M13 containing a sequence derived from a plasmid vector existing upstream or downstream of the dIII DNA fragment)
Universal primer, 5'-CGACGTTGTAAAACGACGGCCAGT,
SEQ ID NO: 3) and reverse primer (M13 Reverse pr
imer, 5'-CAGGAAACAGCTATGAC, SEQ ID NO: 4) was used to analyze the PstI-HindIII DNA fragment by the cycle sequencing method.
A base sequence of about 600 bases was determined from each of the 5'end and the 3'end. In addition, of the determined Pstl-HindIII DNA fragment
4 new primers (Primer X1: SEQ ID NO: 5, Primer X2: SEQ ID NO: 6, Primer X3: SEQ ID NO: 7 and Primer X4: SEQ ID NO: 8) based on the base sequences of the 5 ′ end side and the 3 ′ end side Synthesized and PstI-Hind by primer walking method
The total base sequence of the III DNA fragment was determined to be 1969 bases. The determined nucleotide sequence of the PstI-HindIII DNA fragment within the SalI I fragment derived from the FHV-1 genome is shown in SEQ ID NO: 1. Also, SEQ ID NO: 1
The amino acid sequence predicted from the nucleotide sequence shown in is shown in SEQ ID NO: 2.

(2) Sa of the feline herpesvirus type 1 genome
S the nucleotide sequence of the li I fragment present in the U _L region of the FHV-1 genome shown in comparative analysis SEQ ID NO: 2 amino acid sequences encoding
The amino acid sequence predicted from the nucleotide sequence near the BamHI digestion site in the alI I fragment (PstI-HindIII DNA fragment) was determined using herpes simplex virus type 1 (HSV-1), equine herpesvirus type 1 (EHV-1) and equine herpes. the amino acid sequence of protein kinase present in the U _L region of the viral type 4 (EHV-4) (RF Smith , and TF Smith. 1989. J. Virol. 6
3: 450-455, GenBank accession number: EHV-1, NC001491
EHV-4, NC001844) and shown in FIGS. 11 and 12. As is clear from the comparison of the amino acid sequences shown in FIGS. 11 and 12, the amino acid sequences of the catalytic domains I to VI of the protein kinases surrounded by squares are FHV-1, H
It was highly conserved among herpesviruses such as SV-1, EHV-1 and EHV-4. In particular, PstI in the SalI I fragment of FHV-1
-In the amino acid sequence encoded by the HindIII DNA fragment, the amino acids corresponding to the constituent amino acids of each catalytic domain of protein kinase found in the sequence are HSV-
Even when the amino acid is different from the constituent amino acids of each catalytic domain of 1 protein kinase, there were many cases in which it was the same as the constituent amino acids of each catalytic domain of protein kinase of EHV-1 and EHV-4.

[0103] From the above analysis, the base sequence of BamHI digestion site near the U _L region SalII fragment which is foreign gene insertion site found in this invention was revealed to encode protein kinase.

[0104] On the other hand, protein kinase of the U _L region of the pseudorabies virus belonging to the same alphaherpesvirinae and FHV-1 is, is finding that it is not essential to the growth or replication of the virus has been reported (N .de Win
d, J. Domen, and A. Berns. 1992. J. Virol. 66: 5200-
5209).

[0105] and the analysis result of the above amino acid sequence, the protein kinase of U _L region of the herpesvirus Together, finding that it is not essential for replication and proliferation of the virus, genes of BamHI near the I fragment portion, a portion of the gene encoding the protein kinase of U _L region, moreover, found to be protein kinase is the gene product are those that are not essential for replication and proliferation of the virus, heading in the present invention exogenous gene insertion site is not limited to BamHI digestion site within the SalI I fragment of FHV-1 genome, apparently may be as long as the protein kinase gene region of the FHV-1 genome U _L region Became.

[0106]

INDUSTRIAL APPLICABILITY According to the present invention, recombination capable of expressing and producing at least two kinds of foreign gene products
This recombinant FHV-1 vector can be used as a safe and effective multivalent vaccine virus that can produce an FHV-1 vector.

[Brief description of drawings]

FIG. 1 is a diagram showing a map of the feline herpesvirus type 1 genome and digestion with SalI. Further, in the figure, together with the foreign gene insertion region and the sequence region of the present invention, a conventionally known foreign gene insertion region is also shown.

FIG. 2 is a diagram schematically showing the insertion site of the recombinant FHV-1 of the present invention in the genome.

[Fig. 3] LacZ insertion transfer vector (pdBSI-LacZ)
It is a figure showing the outline of the manufacturing method of.

FIG. 4 is a diagram showing an outline of a method for producing LacZ / FCV capsid gene-inserted recombinant FHV-1.

FIG. 5 is a diagram showing a schematic diagram of the constructed transfer vector pdBSI-LacZ.

FIG. 6 is a diagram showing analysis results of various plasmid vectors by agarose gel electrophoresis.

FIG. 7: TK-deficient feline calicivirus capsid protein gene inserted into the feline calicivirus capsid protein gene and FHV-1 derived gC promoter used to generate recombinant FHV-1 dTK-gC / Cap-FHV FIG. 3 is a schematic diagram showing the structure of a transfer vector pfTK (gCp) -Cap.

FIG. 8 shows the detection results of β-galactosidase and FCV capsid protein in FHV-Cap / Lac-infected CrFK cells by the indirect fluorescent antibody method. In the figure, A shows the result of β-galactosidase labeled with FITC, and B shows TRITC.
The results of labeled FCV capsid protein are shown.

FIG. 9: FHV-Cap / Lac inoculated cat serum anti-serum by indirect fluorescent antibody method using FHV-Cap / Lac cat serum 5 days after inoculation as the primary antibody and FITC-labeled goat anti-cat IgG antibody as the secondary antibody It is the figure which showed the detection result of (beta) galactosidase antibody. In the figure, A shows the result of CrFK cells into which pCMVβ DNA was not introduced (negative control), and B shows Cr into which pCMVβ DNA was introduced.
The results with FK cells are shown.

FIG. 10 is a diagram showing the results of detecting an anti-calcicivirus capsid protein antibody by an indirect fluorescent antibody method. In the figure, A is a negative control obtained by reacting feline serum before inoculation with FHV-Cap / Lac with feline calicivirus-infected CrFK cells, and B is a control.
FHV-Cap / Lac inoculated cat serum and feline calicivirus-infected CrF
The result when reacting with K cells is shown.

[11] S present in the U _L region of the FHV-1 genome of the invention
The amino acid sequence predicted from the nucleotide sequence near the BamHI digestion site in the alI I fragment (PstI-HindIII DNA fragment) was determined using herpes simplex virus type 1 (HSV-1), equine herpesvirus type 1 (EHV-1) and equine herpes. shows a comparison of the amino acid sequence of protein kinase present in the U _L region of the viral type 4 (EHV-4). In the figure, the boxed area indicates the catalytic domains I to VI of protein kinase, and “*” indicates that the amino acid of FHV-1 is HSV-1, EHV-1 or E.
It is shown to be the same as any of the amino acids of HV-4.

FIG. 12 is a continuation of FIG.

[Sequence list]                                SEQUENCE LISTING         <110> Kyoritsu Seiyaku, Corporation <120> Recombinant feline herpesvirus type 1 and multivalent vaccine usin g thereof <130> KRS-12 <140> --- <141> 2002-03-29 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 1969 <212> DNA <213> Feline herpesvirus type 1 <220> <221> CDS <222> (361) .. (1968) <400> 1 agcttaccaa cgtgaatcaa tatttaaggc acgtacgcta gacatgatcc gtggagggat 60 agataggcgt gatcctattt ttgttcacac atttacgtca gcaaaacggg ccgggcaggc 120 cttgacagac aactacacac atactctagg gtggaggcga tagaacaaaa gacacaatca 180 ctgcgactgc gcattgaggc acagacgccg tcgaagaagt actgcatagc catcgacggt 240 atctccagtc agacttcact gataaaattg atgctgtgga tgataagata tttgaaaagg 300 aaaatcaatt aaccgaaact attctagaag tagatccaga cccgaaactc tggaacgtgg 360 atg gct aga cga ggc gga cga agc gct act gac gag atg gat gtt ggc 408 Met Ala Arg Arg Gly Gly Arg Ser Ala Thr Asp Glu Met Asp Val Gly   1 5 10 15 gga agc tcc caa ggc gac cca ctg tca cat gga cca ata ctc tca cct 456 Gly Ser Ser Gln Gly Asp Pro Leu Ser His Gly Pro Ile Leu Ser Pro              20 25 30 atc aca aga cca tca tcg ggg gtc cgg gaa ggg ggg gga cat tgc aac 504 Ile Thr Arg Pro Ser Ser Gly Val Arg Glu Gly Gly Gly His Cys Asn          35 40 45 acg gcc gat ccc cac tct caa gga aac cat atc aaa cgg ggt ata tgt 552 Thr Ala Asp Pro His Ser Gln Gly Asn His Ile Lys Arg Gly Ile Cys      50 55 60 aaa cca gga gtc tct gga tcc ggt aac aca gcc gat agc gct cac aaa 600 Lys Pro Gly Val Ser Gly Ser Gly Asn Thr Ala Asp Ser Ala His Lys  65 70 75 80 cac ctc acc atg tcc cca agg agg ctg cgc cca tta cca cac aga gaa 648 His Leu Thr Met Ser Pro Arg Arg Leu Arg Pro Leu Pro His Arg Glu                  85 90 95 ggg atc tta cgt cac cgg att aaa gaa gaa tgt caa gac ttt cag gct 696 Gly Ile Leu Arg His Arg Ile Lys Glu Glu Cys Gln Asp Phe Gln Ala             100 105 110 ggt aac gga gaa ggt aaa atc agg gca aac acc gcc atc gac aga tat 744 Gly Asn Gly Glu Gly Lys Ile Arg Ala Asn Thr Ala Ile Asp Arg Tyr         115 120 125 ttt aca cga gcg aga cgt atc ttc aaa tat aca ccc aga cgc atg tct 792 Phe Thr Arg Ala Arg Arg Ile Phe Lys Tyr Thr Pro Arg Arg Met Ser     130 135 140 agt aga cga ggc ggg aga acc act ccc cca tgt atg gct ggg tgg gct 840 Ser Arg Arg Gly Gly Arg Thr Thr Pro Pro Cys Met Ala Gly Trp Ala 145 150 155 160 tcc ccc tct ggt gga cga tat gac ggg ctc att cga ggg gac tcc aac 888 Ser Pro Ser Gly Gly Arg Tyr Asp Gly Leu Ile Arg Gly Asp Ser Asn                 165 170 175 aat gga cgg acc gat ata cca aat acc ctg act cga att cct ata cat 936 Asn Gly Arg Thr Asp Ile Pro Asn Thr Leu Thr Arg Ile Pro Ile His             180 185 190 gag gta tgt acc cca tta aca aca aat ccc ggc aac agg tca tct att 984 Glu Val Cys Thr Pro Leu Thr Thr Asn Pro Gly Asn Arg Ser Ser Ile         195 200 205 ttg aaa att agg aaa att aag cgt gtt acg atc cct gtg ttc tca gtg 1032 Leu Lys Ile Arg Lys Ile Lys Arg Val Thr Ile Pro Val Phe Ser Val     210 215 220 tca gca gaa atg cat tac tct aag gtg gca cta gga gaa cca ccg aag 1080 Ser Ala Glu Met His Tyr Ser Lys Val Ala Leu Gly Glu Pro Pro Lys 225 230 235 240 ttc ggg ggg gct ggt ggg tat gga gaa gta cag att tat cga caa aca 1128 Phe Gly Gly Ala Gly Gly Tyr Gly Glu Val Gln Ile Tyr Arg Gln Thr                 245 250 255 ggt ctg gcc atc aaa aca tca tca agt cca tcg tgt ttt gaa cat gaa 1176 Gly Leu Ala Ile Lys Thr Ser Ser Ser Pro Ser Cys Phe Glu His Glu             260 265 270 tta tta gtc act tta tta gcc ggg gag agc tct cta cgc gct aga tca 1224 Leu Leu Val Thr Leu Leu Ala Gly Glu Ser Ser Leu Arg Ala Arg Ser         275 280 285 tcc ata ggc ata act ggg ata att tac ccc gtt gca ttt tca tta acc 1272 Ser Ile Gly Ile Thr Gly Ile Ile Tyr Pro Val Ala Phe Ser Leu Thr     290 295 300 gaa cac caa atg gta ttc aaa gcg tat gat atg gat ctg aat gta tat 1320 Glu His Gln Met Val Phe Lys Ala Tyr Asp Met Asp Leu Asn Val Tyr 305 310 315 320 tgt aat aaa cta tca tcc gct gga ccc cca aca tca aat ata ctt aat 1368 Cys Asn Lys Leu Ser Ser Ala Gly Pro Pro Thr Ser Asn Ile Leu Asn                 325 330 335 gcg atg gaa cat gcg ttc atc ggg ttg ggt aag gct gtg gca tac ctg 1416 Ala Met Glu His Ala Phe Ile Gly Leu Gly Lys Ala Val Ala Tyr Leu             340 345 350 aac acc aaa tgc ggc tta acg cat ttg gat atc aaa tgt gga aat ata 1464 Asn Thr Lys Cys Gly Leu Thr His Leu Asp Ile Lys Cys Gly Asn Ile         355 360 365 ttc gtc aac aca aaa aat tgt gtt ata aaa gat tat gtc ata gcc gat 1512 Phe Val Asn Thr Lys Asn Cys Val Ile Lys Asp Tyr Val Ile Ala Asp     370 375 380 ttt agt ctg atg act cta aac aca aat tct acc gta atg cgg gcg gag 1560 Phe Ser Leu Met Thr Leu Asn Thr Asn Ser Thr Val Met Arg Ala Glu 385 390 395 400 ttt gaa att ccc act ggg gat gcg tca aat aag gtc cta cgc ctt tca 1608 Phe Glu Ile Pro Thr Gly Asp Ala Ser Asn Lys Val Leu Arg Leu Ser                 405 410 415 cga ggg gcg gcg aca act ata ttt agt ctg gta ttg ggt cat gga cat 1656 Arg Gly Ala Ala Thr Thr Ile Phe Ser Leu Val Leu Gly His Gly His             420 425 430 aac caa ccc acg gag ata ctg gtt gac ttt att aat aac agt gga ctg 1704 Asn Gln Pro Thr Glu Ile Leu Val Asp Phe Ile Asn Asn Ser Gly Leu         435 440 445 gct cga cac cgc ggc cca tta gac agt gac gtt ggt gta gct gtt gac 1752 Ala Arg His Arg Gly Pro Leu Asp Ser Asp Val Gly Val Ala Val Asp     450 455 460 ttg tat gct ctt gga cag gtg cta ttg gaa ctg ctt ttg act gga tgc 1800 Leu Tyr Ala Leu Gly Gln Val Leu Leu Glu Leu Leu Leu Thr Gly Cys 465 470 475 480 ctt tcc cct cgg tta ccg gtc ccc att ctt aga aat acg aca tat tac 1848 Leu Ser Pro Arg Leu Pro Val Pro Ile Leu Arg Asn Thr Thr Tyr Tyr                 485 490 495 tac tac cta cac cag gtg acc gtg gaa tat gcc ttg gat ctc tta gca 1896 Tyr Tyr Leu His Gln Val Thr Val Glu Tyr Ala Leu Asp Leu Leu Ala             500 505 510 tat ctg cgc act ata ccc cca tat att tcc ttc ttc acc tat tac aac 1944 Tyr Leu Arg Thr Ile Pro Pro Tyr Ile Ser Phe Phe Thr Tyr Tyr Asn         515 520 525 aat tca tgg tgt tcc ata ccc tgc a 1969 Asn Ser Trp Cys Ser Ile Pro Cys     530 535 <210> 2 <211> 536 <212> PRT <213> Feline herpesvirus type 1 <400> 2 Met Ala Arg Arg Gly Gly Arg Ser Ala Thr Asp Glu Met Asp Val Gly   1 5 10 15 Gly Ser Ser Gln Gly Asp Pro Leu Ser His Gly Pro Ile Leu Ser Pro              20 25 30 Ile Thr Arg Pro Ser Ser Gly Val Arg Glu Gly Gly Gly His Cys Asn          35 40 45 Thr Ala Asp Pro His Ser Gln Gly Asn His Ile Lys Arg Gly Ile Cys      50 55 60 Lys Pro Gly Val Ser Gly Ser Gly Asn Thr Ala Asp Ser Ala His Lys  65 70 75 80 His Leu Thr Met Ser Pro Arg Arg Leu Arg Pro Leu Pro His Arg Glu                  85 90 95 Gly Ile Leu Arg His Arg Ile Lys Glu Glu Cys Gln Asp Phe Gln Ala             100 105 110 Gly Asn Gly Glu Gly Lys Ile Arg Ala Asn Thr Ala Ile Asp Arg Tyr         115 120 125 Phe Thr Arg Ala Arg Arg Ile Phe Lys Tyr Thr Pro Arg Arg Met Ser     130 135 140 Ser Arg Arg Gly Gly Arg Thr Thr Pro Pro Cys Met Ala Gly Trp Ala 145 150 155 160 Ser Pro Ser Gly Gly Arg Tyr Asp Gly Leu Ile Arg Gly Asp Ser Asn                 165 170 175 Asn Gly Arg Thr Asp Ile Pro Asn Thr Leu Thr Arg Ile Pro Ile His             180 185 190 Glu Val Cys Thr Pro Leu Thr Thr Asn Pro Gly Asn Arg Ser Ser Ile         195 200 205 Leu Lys Ile Arg Lys Ile Lys Arg Val Thr Ile Pro Val Phe Ser Val     210 215 220 Ser Ala Glu Met His Tyr Ser Lys Val Ala Leu Gly Glu Pro Pro Lys 225 230 235 240 Phe Gly Gly Ala Gly Gly Tyr Gly Glu Val Gln Ile Tyr Arg Gln Thr                 245 250 255 Gly Leu Ala Ile Lys Thr Ser Ser Ser Pro Ser Cys Phe Glu His Glu             260 265 270 Leu Leu Val Thr Leu Leu Ala Gly Glu Ser Ser Leu Arg Ala Arg Ser         275 280 285 Ser Ile Gly Ile Thr Gly Ile Ile Tyr Pro Val Ala Phe Ser Leu Thr     290 295 300 Glu His Gln Met Val Phe Lys Ala Tyr Asp Met Asp Leu Asn Val Tyr 305 310 315 320 Cys Asn Lys Leu Ser Ser Ala Gly Pro Pro Thr Ser Asn Ile Leu Asn                 325 330 335 Ala Met Glu His Ala Phe Ile Gly Leu Gly Lys Ala Val Ala Tyr Leu             340 345 350 Asn Thr Lys Cys Gly Leu Thr His Leu Asp Ile Lys Cys Gly Asn Ile         355 360 365 Phe Val Asn Thr Lys Asn Cys Val Ile Lys Asp Tyr Val Ile Ala Asp     370 375 380 Phe Ser Leu Met Thr Leu Asn Thr Asn Ser Thr Val Met Arg Ala Glu 385 390 395 400 Phe Glu Ile Pro Thr Gly Asp Ala Ser Asn Lys Val Leu Arg Leu Ser                 405 410 415 Arg Gly Ala Ala Thr Thr Ile Phe Ser Leu Val Leu Gly His Gly His             420 425 430 Asn Gln Pro Thr Glu Ile Leu Val Asp Phe Ile Asn Asn Ser Gly Leu         435 440 445 Ala Arg His Arg Gly Pro Leu Asp Ser Asp Val Gly Val Ala Val Asp     450 455 460 Leu Tyr Ala Leu Gly Gln Val Leu Leu Glu Leu Leu Leu Thr Gly Cys 465 470 475 480 Leu Ser Pro Arg Leu Pro Val Pro Ile Leu Arg Asn Thr Thr Tyr Tyr                 485 490 495 Tyr Tyr Leu His Gln Val Thr Val Glu Tyr Ala Leu Asp Leu Leu Ala             500 505 510 Tyr Leu Arg Thr Ile Pro Pro Tyr Ile Ser Phe Phe Thr Tyr Tyr Asn         515 520 525 Asn Ser Trp Cys Ser Ile Pro Cys     530 535 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: M13 Universal       primer <400> 3 cgacgttgta aaacgacggc cagt 24 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: M13 Reverse       primer <400> 4 caggaaacag ctatgac 17 <210> 5 <211> 22 <212> DNA <213> Primer X1 <400> 5 tagtagacga ggcgggagaa cc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer X2 <400> 6 aatagatgac ctgttgccgg ga 22 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer X3 <400> 7 tgcgttcatc gggttgggta agg 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer X4 <400> 8 accttatttg acgcatcccc agtg 24

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[Procedure amendment]

[Submission date] May 13, 2002 (2002.5.1)
3)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Novel recombinant feline herpesvirus type 1 and multivalent vaccine using the same

[Claims]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to use of feline herpesvirus type 1 (feline herpesvirus type 1, hereinafter abbreviated as FHV-1) as a vector virus.

[0002]

2. Description of the Related Art Regarding the gene of a virus whose cat is a host, for example, herpes viridae (Herpesvidae) has been known.
ridae's Alphaherpes vilinae (Alphaherpesvi)
A part of the genomic DNA of FHV-1, a herpesvirus that induces viral rhinotracheitis in cats, belongs to the subfamily, and is artificially deleted using genetic engineering techniques. A recombinant viral vector that does not exist in nature has been created by introducing an exogenous gene so as to be expressed in cells or in the animal body. Such a recombinant viral vector produces a product derived from an exogenous gene in addition to a viral antigen derived from FHV-1 by infecting cells or animals, and when inoculated into an animal,
It is known to confer immunity to foreign gene products in addition to FHV-1 (N. Yokoyama et al., 1996, Arc
h. Virol. 141: 481-494; N. Yokoyama et al., 1996,
Arch. Virol. 141: 2339-2351).

As a specific example of such an FHV-1 vector, a recombinant viral vector in which a thymidine kinase (hereinafter abbreviated as TK) gene region of the viral genome is deleted and an exogenous gene is inserted into the defective site is known. Has been
(JH Nunberg et al., 1989, J. Virol. 63: 3240-324
9; N. Yokoyama et al., 1995, J. Vet. Med. Sci. 5
7: 709-714). Further, it has been confirmed that such recombinant FHV-1 expresses a protein derived from an exogenous gene introduced into the TK gene region in addition to the FHV-1 protein without impairing the replication ability of the virus. (GH Cole e
t al., J. Virol. 1990, 64: 4930-4938; RC Wardle
y et al., 1992, JG Virol. 73: 1811-1818). Furthermore, Yokoyama et al. (N. Yokoyama et al., 1996, Arch. Vir
ol. 141: 2339-2351), recombinant FHV-1 in which a foreign gene was inserted in the TK gene region did not show pathogenicity to cats, and cats inoculated with the recombinant FHV-1 were foreign. It has been reported to produce antibodies to the gene product.

On the other hand, an open reading frame 2 positioned downstream of the U _{L (Unique} long) gC gene regions within the region as a foreign gene insertion site other than the TK gene region in FHV-1 genome (open reading frame 2, ORF2 )It has been known. Willemse et al. (MJ Willemse et al., 199
4, J. Gen. Virol. 75: 3107-3116) is a recombinant FHV-1 in which a gene encoding a foreign gene, β-galactosidase (hereinafter abbreviated as LacZ), is inserted into ORF2.
It has been reported that it possessed a virus replication capacity similar to that of FHV-1. Further, FHV-1 genome _{U S} (unique short)
U _S 8.5 in the region, gene region, such as gI and gE It is also known that not necessarily required for replication of FHV-1.

[0005] With respect to the recombinant FHV-1 having inserted a foreign gene into these gene regions, LacZ in U _S 8.5 gene region
It was reported that recombinant FHV-1 with the inserted gene retains the same replication ability as attenuated FHV-1 (MJ Willemse et a
l., 1995, Virology 208: 704-711) while gI
It has been reported that recombinant FHV-1 in which LacZ is inserted into the gE gene region and gF gene region have markedly reduced replication ability as compared with attenuated FHV-1 (MJ Willemse et al., 1996, Vaccine 14:
1-5; MD Sussman et al., 1995, Virology 214: 1
2-20). These U _S genes in addition to the recombinant FHV-1 having a foreign gene insertion site, the FHV-1 genome ORF5 (nucleotide numbers
5869-7113), a recombinant FHV-1 in which an exogenous gene is inserted has been proposed (Japanese Patent Publication No. 2000-501927). However, the safety of these recombinant viral vectors to animals has not been investigated, and it is unknown whether they are nonpathogenic to the host cat. For reference, these exogenous gene insertion sites are shown in a part of FIG. 1 together with the SalI map.

Regarding recombinant FHV-1, several sites in the FHV-1 genome have been identified as foreign gene insertion sites as described above, but multiple foreign genes are simultaneously inserted into multiple gene insertion sites. No recombinant FHV-1 vector was prepared.

On the other hand, in consideration of its use as a vaccine, if it is assumed that essentially one kind of foreign gene is inserted into one gene insertion site, the recombinant FHV produced by the conventional techniques will be produced. Although the -1 vector can be used as a divalent vaccine including FHV-1 itself, it does not always sufficiently respond to the current situation of various vaccines for cats, which are in high demand for multivalentization.

By the way, in order to maintain the life cycle of a virus, phosphorylation of viral proteins is required, and phosphorylation is a cellular or viral protein kinase (protein phosphorylating enzyme: serine / threonine kinase and tyrosine kinase). Is believed to be in charge. Similar to FHV-1, herpes simplex virus type 1 (HSV-1), herpes zoster virus (VZV), or Epstein-Barr virus (EBV) classified in the gammaherpesvirus subfamily that belongs to the alphaherpesvirus subfamily.
For, the presence of a gene sequence encoding a protein kinase in the viral genome is known, and the amino acid sequence encoded by the gene is highly conserved among herpesviruses.

In particular, catalytic domains I to VI of protein kinase have been reported to be highly conserved in various herpesviruses (RF Smith, and TF Smith. 198).
9. J. Virol. 63: 450-455). In addition, the typical amino acid sequence of domain I in the catalytic domain of herpesvirus protein kinase is GXGXXGXV (X is an amino acid with low conservation), and such amino acid sequences are HSV-1, VZV, EB.
V, human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) have been found to be highly conserved (MS Chee, GJ Lawrence, and B.
G. Barrel. 1989.J. Gen. Virol. 70: 1151-1160).

[0010]

However, in the prior art, except for the TK gene-deficient recombinant FHV-1 vector,
It is sufficiently attenuated and safe for cats, retains sufficient replication capacity for production as a vaccine virus, and can simultaneously confer immunity to three or more pathogenic gene products including FHV-1 itself. There has been no case of producing a recombinant FHV-1 vector. That is, in the known recombinant FHV-1 vector, the exogenous gene insertion site is essentially
Since it is limited to one region of the V-1 genome, it can be expected that the cat inoculated with the recombinant FHV-1 is only immunized against one foreign antigen other than FHV-1.

Therefore, for example, in order to confer protective immunity against infection with three pathogenic microorganisms including FHV-1 in cats, recombinant FHV-1 in which one kind of foreign gene is inserted is inoculated, and further, A vaccine consisting of one target pathogenic microorganism or an antigen derived from it must be inoculated. This means that not only diversification of products and manufacturing processes and rising manufacturing costs are unavoidable in the field of vaccine production, but also side effects due to inoculation of multiple vaccines consisting of pathogenic microorganisms are easily expected. .

[0012] On the other hand, a problem when the vector virus is inoculated and introduced into a target animal is homologous gene recombination between the vector virus and the naturally infected virus in the animal individual, and the gene recombination attenuates the vector virus. May become pathogenic.
In order to reduce the acquisition of pathogenicity of vector viruses by such gene recombination, gene mutations or foreign gene fragments are introduced at multiple sites of the vector virus genome, and homology between the vector virus genome and the pathogenic virus genome is It is necessary to prevent the vector virus from converting into a pathogenic virus by means of sex gene recombination unless the gene sequences at multiple sites in the vector virus genome are simultaneously converted into the pathogenic virus type. However, in the present situation, the attenuated recombinant FHV-1 vector in which such multiple gene regions are converted into a gene sequence different from the pathogenic virus or an exogenous gene is simultaneously introduced into the multiple gene regions is mainly used. The vector virus has not been created because it impairs replication.

Vaccines currently used to prevent viral infections and bacterial infections of cats, specifically feline calicivirus, feline panleukopenia virus, feline leukemia virus, rabies virus, FHV-1 and As a vaccine for preventing infections caused by chlamydia, both live attenuated vaccines and inactivated vaccines are administered by injection. However, inoculation by injection is not always the best inoculation method, because the inoculation is complicated and the animal suffers pain. On the contrary, the transmucosal inoculation method such as eye drops, nasal and oral inoculation is easy to inoculate, and does not cause pain to the inoculated animal and further induces mucosal immunity. Compared with many points.

[0014]

[Means for Solving the Problems] The inventors of the present invention have studied to solve the above problems, and have known TK-deficient attenuated recombinant FHV-
1 vector (JP-A-9-267) was further improved. That is, as a new foreign gene insertion site that does not have a lethal effect on FHV-1 replication / proliferation, Sal of the FHV-1 genome is used.
BamH in I fragment located U _L region in the I cleaved DNA fragments
By identifying the I cleavage site (hereinafter abbreviated as I fragment), it was found that recombinant FHV-1 retains virus replication ability and the inserted foreign gene product is expressed. The present invention has been completed by clarifying that the vicinity of the BamHI site in this I fragment is a gene region encoding protein kinase.

Further, a recombinant FHV-1 which can be used as a vector was prepared by inserting different foreign genes into both gene regions of the I fragment and the known TK gene. As a result, the virus (virus vector) is capable of replicating in cells infected with it or in cats inoculated transmucosally, and recombinant FHV-1 in infected cells or cats.
The present invention has been completed by finding that at least two types of foreign gene products inserted into the genome are produced.

That is, the attenuated recombinant feline herpes of the present invention
Pesvirus type 1 is a genotype of feline herpesvirus type 1.
At least two types of foreign matter in two different areas
The gene is inserted so that it can be expressed.
Cats in which two foreign genes are inserted so that they can be expressed
Two different regions within the herpesvirus 1 genome
Has a lethal effect on the growth of feline herpesvirus 1
It is characterized by two areas that are not given. Also before
To give a specific example of this area, this area
Unique among SalI-cleaved DNA fragments of Svirus type 1 genome
Cron (U_L) Region I fragment and thymidine
Two gene regions, a gene region encoding a nase,
Alternatively, the uniqueness of the feline herpesvirus type 1 genome
Long (U _L) Area of protein kinase
Coding gene region and thymidine kinase
There are two gene regions including a gene region.

The protein kinase present in the unique long ( _UL ) region of the feline herpesvirus type 1 genome contains the amino acid sequence shown by SEQ ID NO: 2 in a part thereof, and therefore, the The insertion site is not limited to the gene region encoded by the I fragment, but the entire gene region encoding this protein kinase is targeted.

In the present invention, "expressively" means that an exogenous gene is linked to a gene expression regulatory sequence such as a promoter or enhancer so that the inserted exogenous gene expresses its gene product in cells. It means that a gene fragment consisting of a gene or a part of its sequence is inserted. A sequence such as a promoter, which is a gene expression regulatory sequence for enabling expression of this foreign gene, may be inserted together with the foreign gene as a part of the foreign gene to be inserted, or feline herpesvirus type 1 An exogenous gene, which is a structural gene, may be inserted and arranged so that (FHV-1) itself utilizes a gene expression regulatory sequence such as a promoter possessed in the genome. That is, the foreign gene to be inserted in the present invention may be a structural gene alone or a gene cassette in which the structural gene and a gene expression regulatory sequence such as a promoter are combined.
Preferably, a gene cassette in which a structural gene and a gene expression regulatory sequence are preliminarily linked is used because the expression of the gene product of the inserted foreign gene can be ensured and the flexibility of the insertion site can be secured.

When an exogenous gene is inserted into the I fragment,
The existence of a gene expression regulatory sequence such as a promoter required for the expression of the foreign gene in the fragment, particularly at the BamHI recognition site, which is the foreign gene insertion site, is unknown. Also, if Ba
Even if a gene expression regulatory sequence is present near the mHI recognition site, the mechanism for inserting an exogenous gene is homologous gene recombination that occurs between the transfer vector and the FHV-1 genome. For these reasons, the exogenous gene is not always inserted into the site where the expression of the exogenous gene is efficiently regulated by the gene expression regulatory sequence. Therefore, it is preferable to insert an exogenous gene as a gene cassette containing a gene expression regulatory sequence such as a promoter. Also,
Also in the insertion of an exogenous gene into a gene region encoding a protein kinase, it is preferable to insert the exogenous gene as a gene cassette containing a gene expression regulatory sequence such as a promoter for the same reason.

The gene expression regulatory sequences such as promoters include FHV-1-derived gC promoter and gB promoter, the immediate early (IMV) of human cytomegalovirus (HCMV).
E) Promoter or Marek's disease virus (MDV)
Derived RNA 1.8 promoter (G. Bradley et al., 198
9, J. Virol. 63: 2534-2542). Of these, the FHV-1-derived gC promoter produces an exogenous gene product at the same level as when FHV-1 was naturally infected,
In addition, the immediate early promoter of human cytomegalovirus is particularly preferable in that it actively produces an exogenous gene product due to its strong promoter activity, and that the expressed exogenous gene product provides high immunogenicity. Is.

On the other hand, in the insertion of an exogenous gene into the gene region encoding thymidine kinase, gene expression regulatory sequences such as promoters and enhancers for the expression of thymidine kinase are present near the insertion site of the exogenous gene. Similarly, insertion as a gene cassette containing a gene expression regulatory sequence is preferable in that the expression of the foreign gene can be regulated more appropriately and efficiently. In this case, examples of gene expression regulatory sequences such as promoters include FHV-1-derived gC promoter and gB promoter, human cytomegalovirus immediate-early promoter, MDV-derived RNA 1.8 promoter, and the like. Of these, the gC promoter and the human cytomegalovirus immediate-early promoter are particularly preferable for the reasons described above.

On the other hand, the exogenous gene insertably expressible in two different regions in the feline herpesvirus type 1 genome is derived from a pathogenic microorganism and is a polypeptide that induces an infection protective ability in animals. A gene fragment comprising a coding gene or a partial sequence of the gene, or, for example, interferon γ, interferon α, interferon β, interferon ω, interleukin 12, interleukin 18, interleukin 10, TNFα It is characterized in that it is a gene fragment which is derived from various cytokines and codes for a product showing a therapeutic effect on animals or a partial sequence of the gene.

The present invention also includes a vaccine or therapeutic composition containing the attenuated recombinant feline herpesvirus type 1, which vaccine or therapeutic composition is used by transmucosal inoculation. It has a feature.

Further, the present invention provides a method for preparing an attenuated recombinant feline herpesvirus type 1 in which at least two kinds of foreign genes are expressibly inserted into two different regions in the feline herpesvirus type 1 genome. including. This preparation method comprises the steps of (1) preparing at least two types of transfer vectors in which different expressible foreign genes are inserted into gene fragments consisting of two different gene regions of the feline herpesvirus type 1 genome. ,
(2) Feline herpesvirus 1 in which an exogenous gene has been inserted into a feline cell, preferably a feline cell line, using at least two types of transfer vectors obtained
Of a gene fragment consisting of different gene regions of the type genome and infecting feline herpesvirus type 1 to cause homologous gene recombination between the introduced gene fragment and the feline herpesvirus type 1 genome And (3) selecting a feline herpesvirus type 1 in which homologous gene recombination has occurred.
In this method, homologous gene recombination between a plurality of introduced gene fragments and the feline herpesvirus type 1 genome is performed by a single operation.

Further, the attenuated recombinant feline herpesvirus type 1 of the present invention superinfects feline cells, preferably feline cell lines, with recombinant viruses having foreign genes inserted in different regions of the genome. Can also be obtained. This preparation method comprises the steps of (1) preparing at least two types of transfer vectors in which different expressible foreign genes are inserted into gene fragments consisting of two different gene regions of the feline herpesvirus type 1 genome. (2) A feline herpesvirus type 1 genome in which an exogenous gene is inserted into a feline cell, preferably a feline cell line, using each of the obtained transfer vectors of at least two types. Introducing a gene fragment consisting of different gene regions and infecting feline herpesvirus 1 with the introduced gene fragment and feline herpesvirus 1
(3) selecting a feline herpesvirus type 1 in which homologous gene recombination has occurred, and selecting one gene in the feline herpesvirus type 1 genome Obtaining at least two types of recombinant feline herpesviruses 1 in which the regions are recombined, and (4) obtaining the respective recombinant feline herpesviruses 1 in feline cells, preferably feline strains (5) Feline herpesvirus type 1 in which homologous gene recombination has occurred, wherein the homologous gene recombination occurs between at least two types of recombinant feline herpesvirus type 1 genomes And a step of selecting. This method includes a step of preparing and isolating at least two types of recombinant feline herpesvirus type 1 in which an exogenous gene is inserted into a single gene region in the feline herpesvirus type 1 genome.

The above-mentioned cells to be infected with feline herpesvirus type 1 mean cells derived from cats. The feline cell line is a cell consisting of a single cell type and characterized by being continuously culturable by a tissue culture technique. As such a feline cell line,
For example, CrFK cells (RACra which is a cell line derived from cat kidney)
ndell et al., 1973, In Vitro 9: 176-185), MYA-1 cells, which are interleukin 2-dependent cell line lymphocytes (T.
Miyazawa et al., 1989, Arch. Virol. 108: 131-13
5), FeT-J cells, which are interleukin-independent CD4-positive cell line lymphocytes (T. Hohdatsu et al., 1996, J. Gen. Vi.
rol. 77: 93-100). Of these, CrFK cells are preferable because they are highly sensitive to FHV-1 and have good FHV-1 proliferation.

The above-mentioned feline herpesvirus type 1
In the preparation method, a cat herpe into which an exogenous gene is inserted
Two distinct genetic regions within the Svirus type 1 genome
Is the SalI-cut DNA fragment of the feline herpesvirus type 1 genome.
Unique long (U_L) I fragment existing in the region
And the gene region encoding thymidine kinase.
Gene region, or feline herpesvirus type 1 geno
Unique long (U _L) Protein present in the region
A gene region encoding a kinase and thymidine kinase
Two gene regions, one that encodes
It is a good thing.

The present invention also provides a method for expressing or producing an exogenous gene in a cell by infecting a cell using the thus obtained attenuated recombinant feline herpesvirus type 1 as a vector. Is what
Also used as a multivalent vaccine, transmucosally inoculated into animals,
An immunization method of immunizing an inoculated animal is also provided.

The cells to be infected here are preferably those having a receptor for feline herpesvirus type 1. Such cells include cat-derived cells, preferably feline cell lines. Further, as a vaccinated animal, an animal of the feline family, preferably an animal in which infection is established against feline herpesvirus type 1, such as a cat, is a preferable target. The immunity to be given includes systemic humoral immunity and cellular immunity as well as local immunity based on mucosal inoculation. On the other hand, even in animals other than felines, the recombinant feline herpesvirus 1 of the present invention
The mold can also be vaccinated to immunize. That is, even in animals that are insensitive to feline herpesvirus 1 such as animals other than cats, at least by inoculating this recombinant feline herpesvirus 1
The foreign gene inserted into the FHV-1 genome can be expressed and produced in the inoculated animal. As a result, when the foreign gene encodes an antigenic protein of a pathogenic microorganism, the inoculated animal is endowed with immunity to the pathogenic microorganism.

As described above, the present invention has found a new foreign gene insertion site, and thus, there is no example so far.
This is a new FHV-1 vector in which an exogenous gene can be inserted into two gene regions in the FHV-1 genome.

Further, of foreign genes inserted into two different regions in the feline herpesvirus type 1 genome, a gene encoding a polypeptide which induces the defense against infection in animals or the gene thereof. The gene fragment consisting of a part of the sequence of the gene includes a gene encoding a protein having an antigenicity produced by an animal belonging to the feline family, mainly various cats that infect cats, or a gene fragment consisting of a part of the sequence of the gene. And antigenic proteins produced by various pathogenic microorganisms that infect animals other than cats. Examples of these include feline leukemia virus coat and core proteins, feline panleukopenia virus VP2 protein, feline immunodeficiency virus coat and core proteins, feline calicivirus capsid protein, and chlamydia major Outer membrane protein MOMP
-1, a gene encoding a coat protein and nucleocapsid protein of rabies virus, a structural protein of canine distemper virus or canine parvovirus, or a gene fragment consisting of a part of the sequence thereof.

Further, a gene fragment consisting of a gene encoding a gene product showing a therapeutic effect on animals or a partial sequence of the gene includes a viral infection, a bacterial infection or a viral infection in a cat or another animal. There are genes that encode TH1-type and TH2-type cytokines that have a protective effect against tumors and a therapeutic effect. Such examples include interferon γ, interferon α, interferon β, interferon ω, interleukin 12, interleukin 18, interleukin 10,
Examples of the gene fragment include a gene encoding TNFα and the like or a partial sequence thereof.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION The attenuated recombinant feline herpesvirus type 1 of the present invention is a feline herpesvirus type 1 genome in which two different regions are expressibly inserted into two different regions. And can be used as a vector or vaccine.

Such a recombinant virus can be obtained by preparing a different transfer vector and homologous gene recombination between the gene sequence of this transfer vector and the feline herpesvirus type 1 genome. That is, in order to obtain feline herpesvirus type 1 in which an exogenous gene is inserted into two different regions in the genome as in the present invention, two types of transfer vectors are used to homologous genes in each region in the genome. By homologous recombination either by causing recombination or by once preparing feline herpesvirus type 1 in which an exogenous gene has been inserted into different regions of the genome, and then superinfecting these viruses. Can also be obtained. The latter is a preferable method because each recombinant virus grows in infected cells, and thus the probability of homologous gene recombination increases.

The region into which the foreign gene is inserted is
It must not be a region that encodes a gene essential for virus growth and must be a region that allows expression of the inserted foreign gene. As an insertion region of an exogenous gene satisfying such a condition, an I fragment portion present in the unique long ( _UL ) region of the SalI-cut DNA fragment of the feline herpesvirus type 1 genome and a gene region encoding thymidine kinase are included. Two gene regions or a unique long feline herpesvirus type 1 genome
Two gene regions, that is, a gene region encoding a protein kinase existing in the ( _UL ) region and a gene region encoding a thymidine kinase are exemplified.

When an exogenous gene is inserted into the I fragment,
The insertion site of the exogenous gene may be any part of the I fragment. In particular, SEQ ID NO: 1 of which the sequence is fixed among I fragments
The portion (PstI-HindHI in the I fragment, sequence region, FIG. 1) indicated by is a preferable region because insertion of an exogenous gene is facilitated.

In the case where an exogenous gene is inserted in the gene region part encoding the protein kinase, the protein kinase is encoded by the gene sequence of the I fragment part, and the amino acid sequence shown in SEQ ID NO: 2 is used as a part thereof. It includes. Therefore, the insertion site of the exogenous gene may be not only the structural gene encoding this protein kinase, but also the regulatory gene part of the protein kinase, and is not limited to the I fragment part, but encodes a protein kinase. The range of all genes is of interest.

Feline herpesvirus type 1 genome and SalI
A map of the case of digestion with the above is shown in FIG. 1 together with the conventionally known insertion site of a foreign gene. In the figure, the _{U L} is unique long region, the _{U S} is unique short region,
IR _S indicates the internal repeat short area, TR _S
FIG. 6 is a diagram showing a terminal repeat short region.
On the other hand, each of the letters A to N represents the name of each fragment when cleaved with SalI (PA Rota et al., 1986, Vir.
ology 154: 168-179; A. Grail et al., 1991, Arch.
Virol. 116: 209-220). Particularly preferred foreign gene insertion regions in the present invention are the sequence region in this I fragment, particularly the BamHI site, and the other region.
It is the thymidine kinase region indicated by TK in the fragment.

Then, when the gene sequence in the vicinity of the BamHI site in the I fragment which is the insertion region of this unique foreign gene was examined, it was found that
It constituted a part of the gene sequence encoding the kinase. Therefore, a gene region encoding protein kinase is preferable as an insertion site for an exogenous gene.

It has been confirmed that the foreign gene inserted into these regions expresses a gene product protein in infected cells. In addition, the recombinant FHV-1 constructed in the present invention is a virus that is more attenuated (non-pathogenic) by deleting the thymidine kinase of the attenuated FHV-1 used in the production of the recombinant virus. Has become.

Therefore, the attenuated recombinant feline herpesvirus type 1 of the present invention can be used as a vector or as a vaccine. It has been confirmed that when used as a vaccine, it induces production of an antibody against a protein produced by the expression of at least two kinds of inserted foreign genes and an antibody against feline herpesvirus type 1. Since this vaccine is a herpes virus, transmucosal administration, that is, nasal, instillation and oral administration is a particularly preferable administration method, but administration by injection which has been conventionally used is also effective.

The vaccine can be prepared by suspending the recombinant FHV-1 of the present invention in an isotonic phosphate buffer. The amount of virus to be inoculated to an animal varies depending on the titer and immunogenicity of the virus. For example, when a cat is transmucosally inoculated, the amount of vaccine solution to be administered is 1-2.
ml is common. It is preferable to adjust the amount of the administered solution to a virus amount that can induce immunity.

As an example of the present invention, the case where a gene fragment encoding feline calicivirus capsid protein and a gene encoding β-galactosidase are used as an exogenous gene will be described below.

The recombinant FHV-1 of the present invention, as schematically shown in FIG. 2, is a Glycoprotein C promoter derived from FHV-1 (hereinafter referred to as g
Recombination in which a gene fragment (hereinafter abbreviated as gC-Cap) encoding a feline calicivirus (hereinafter abbreviated as FCV) capsid protein accompanied by a C promoter is incorporated
FHV-1 (dTK-gC / Cap-FHV, FIG. 2A, JP-A-9-267,
) And FHV-1 I fragment to the cytomegalovirus (CM
V) A feline kidney-derived cell line was superinfected with recombinant FHV-1 (Lac FHV, see FIG. 2B) incorporating a β-galactosidase gene (hereinafter, abbreviated as Pro-LacZ) fragment with an immediate early promoter derived from V), GC-Cap fragment and Pro-in the same viral genome emerged by homologous recombination between both viruses
Recombinant FHV-1 with integrated LacZ fragment (FHV-Cap / Lac,
2C).

The method for producing recombinant FHV-1 will be described below.

First, a transfer vector containing a Pro-LacZ fragment was prepared as follows. That is, FH
V-1 genomic DNA, which is one of the SalI-cleaved DNA fragments, was incorporated into the plasmid vector pSal-I (Faculty of Agriculture, University of Tokyo, distributed by Dr. Takayuki Miyazawa). A plasmid containing the newly constructed I fragment recloned in a plasmid vector
A LacZ gene fragment (Pro-Lac) in which a commercially available eukaryotic expression vector pCMVβ (CLONTECH) -derived immediate early promoter was attached to a unique BamHI cleavage site within the I fragment of pdBSI
Z) was incorporated to prepare a transfer vector pdBSI-LacZ.

Escherichia coli, which is a transformant containing this pdBSI-LacZ, was deposited as E-dBSI-LacZ under the deposit number FERM P-17935 at the Patent Microorganism Depositary Center, Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology, June 2000. Deposit on 30th (deposit number FERM
P-17935) and changed to international deposit as of October 3, 2001 (accession number FERM BP-7761). At present, the name of the depositary institution has been changed to “Independent Administrative Agency National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center”.

Next, the transformant constructed as described above
PdBSI-LacZ DNA was extracted from E-dBSI-LacZ and CrFK cells (Cran
dell feline kidney cells, feline kidney-derived cell lines, and a gift from Dr. Takayuki Miyazawa, Faculty of Agriculture, The University of Tokyo) were transfected by the transfection method, and then the CrFK cells were infected with attenuated FHV-1. After that, homologous gene recombination was caused between the FHV-1 genomic DNA-derived I fragment in pdBSI-LacZ and FHV-1 genomic DNA, and recombinant F in which Pro-LacZ was inserted into the I fragment region of FHV-1.
HV-1 was produced. Recombinant FHV-1 produced in this way
Was designated as LacZ-FHV (Fig. 2B).

Next, the EcoRV-SmaI gene fragment in the TK gene region was deleted from the FHV-1 genome by using a genetic engineering technique, and the FHV-1-derived gC promoter and F
Linked to the gene encoding CV capsid protein
Recombinant FHV-1 with inserted DNA fragment (dTK-gC / Cap-FHV, Fig. 2
A, Distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, The University of Tokyo; References, N. Yokoyama et al., 1998, J. Vet. Med. Sci. 6
0: 717-723) and LacZ-FHV described above were co-infected into CrFK cells to induce homologous gene recombination between these two types of recombinant FHV-1 and BamHI in the I fragment of the FHV-1 genome. La in the part
Recombinant FHV-1 in which the FCV capsid protein gene was incorporated into the cZ and TK gene regions, respectively, was produced.

The recombinant FHV-1 thus obtained, that is, the recombinant FHV-1 capable of producing the FCV capsid protein and β-galactosidase, was obtained as FHV-Cap / LacZ.
(Fig. 2C).

The above operation is performed by Sam Block et al. (J. Sambro
ok et al., 1989, Molecular cloning: a laboratory m
anual. 2nd edition, Cold Spring Harbor, New York:
It can be performed by known gene manipulation techniques and cell engineering techniques described by Cold Spring Harbor Press).

The FHV-Cap / LacZ prepared as described above has the ability to replicate in a feline-derived cell line and in a feline individual, and when inoculated into the nasal cavity, eyes or oral cavity of a feline, the feline has no clinical signs. It was confirmed to be safe for cats because it did not cause side effects.

As described above, the SalI-cut DNA of the FHV-1 genome
Found that BamHI recognition site in the I fragment located U _L region of the fragment is not essential for replication of FHV-1, integrate the foreign gene into the I fragment, a gene further from the foreign gene incorporated The present invention is the first to discover that a product is produced in cells of animal origin and in the body of an animal.

In addition, a recombinant FHV-1 was constructed by incorporating an exogenous gene into the two gene regions of the I fragment and the TK gene existing in the _UL region of the SalI digested DNA fragment of the FHV-1 genome, Further, the present invention is the first to find that the recombinant FHV-1 replicates in the feline cell line and the feline body.

Furthermore, the nucleotide sequence near the BamHI digestion site in the SalI I fragment newly found as the insertion site of the foreign gene was determined, and the amino acid sequence predicted from the nucleotide sequence was analyzed. As a result, the vicinity of the BamHI digestion site was determined. Have a similar amino acid sequence to that of catalytic domains I to VI of protein kinases of other herpesviruses. The nucleotide sequence of the SalI I fragment in the FHV-1 genome has not been determined so far, and the present invention is the first time that it was found that the nucleotide sequence encoding a protein kinase is present in the I fragment. .

[0056] Further, also beginning the present invention was discovered that protein kinase region of the U _L region of the FHV-1 genome is effective as the foreign gene insertion site.

[0057]

The present invention will be described in more detail with reference to the following examples. 3 and 4 show the outline of the experimental operation carried out in the present invention. In addition, FIG. 3 shows the LacZ insertion transfer vector pd.
An outline up to the production of BSI-LacZ is shown in FIG. 4, in which DNA from this transfer vector pdBSI-LacZ is used to insert recombinant FHV-1 FHV-1 into which LacZ and FCV capsid genes have been inserted.
It shows an outline of the method for producing V-Cap / Lac.

1. Construction of transfer vector for producing recombinant FHV-1 vector (1) Construction of plasmid incorporating FHV-1 genomic I fragment pSal which is a plasmid vector containing I fragment of SalI library of FHV-1 genomic DNA -I (distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, University of Tokyo) was digested with SalI, and then commercially available QIAquick
Using a Gel Extraction Kit (QIAGEN), the I fragment of the FHV-1 genome was separated and prepared according to the attached manual.

On the other hand, the plasmid vector for incorporating this I fragment was a commercially available plasmid vector pUC18 (Amersham Pharmacia Biotech) opened by digestion with BamHI.
In the presence of T4 DNA polymerase (Takara Shuzo) at 37 ° C for 5
Blunt ends were obtained by incubation for a minute. Then, in the presence of T4 DNA ligase (GIBCO BRL), 16 ° C, 1
It was prepared by ligating BamHI cleavage sites blunted by incubation for 7 hours. The obtained plasmid vector lacking the BamHI recognition site was named pUC-dB. In the figure, the BamHI surrounded by a square represents the BamHI site that disappeared by such an operation.

Next, pUC-dB was digested with SalI to open the ring, and at the SalI digestion site, I of the FHV-1 genome separated and prepared above was prepared.
The fragments were ligated using T4 DNA ligase. The plasmid obtained by incorporating the I fragment of the FHV-1 genome into the SalI digestion site of pUC-dB thus obtained was designated as pdBSI.

(2) Preparation of Transformant Containing pdBSI E. coli XL-1 B Competent with pdBSI DNA Solution
E. coli was transformed by mixing it with lue (manufactured by Stratagene) and introducing the plasmid DNA into E. coli according to the attached manual. LB agar medium containing 50 μg / ml of ampicillin (Agar medium containing 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 g of sodium chloride, and 15 g of Bacto agar in 1 L). C. for 17 hours at 37.degree. C. to obtain ampicillin-resistant transformant clones grown on an agar medium. The plasmid DNA extracted from each transformant clone was then digested with SalI and analyzed by electrophoresis on a 0.8% agarose gel (Fig. 6, lane 1) to give about 6,800.
Selecting transformant clones having the base pair I fragment,
The clone was designated as E-dBSI.

(3) Construction of Transfer Vector pCMVβ (CLONETECH), which expresses β-galactosidase in a commercially available eukaryotic expression plasmid vector, was digested with PstI and CM was prepared using the above QIAquick Gel Extraction Kit.
A gene fragment (Pro-LacZ) containing the LacZ gene with an immediate early promoter derived from V was isolated and purified. Then, the PstI digestion site was blunted by incubating the Pro-LacZ in the presence of T4 DNA polymerase at 37 ° C for 5 minutes.

On the other hand, the above-mentioned pdBSI, which is a plasmid for incorporating Pro-LacZ, was opened by digestion with BamHI having a recognition site at one site in the I fragment, and the BamHI site was transformed with T4 as described above. It was smoothed by DNA polymerase treatment.

Next, blunt-ended Pro-LacZ and ring-opened blunt-ended pdBSI were treated with T4 DNA ligase in the presence of 16
Ligation was carried out by incubation at 0 ° C for 17 hours. The transfer vector in which Pro-LacZ was incorporated into the blunted BamHI site in the I fragment of the FHV-1 genome-derived SalI library thus obtained was named pdBSI-LacZ (FIG. 3).

The constructed transfer vector pdBSI-La
A schematic diagram of cZ is shown in FIG. In the figure, "LacZ" represents a LacZ gene fragment, PstI and BamHI surrounded by a square represent blunt-ended PstI recognition site and BamHI recognition site, respectively, and "Pro" represents human cytomegalovirus (HCM
V) indicates the immediate early promoter, and the arrow indicates the orientation of the LacZ gene fragment and the promoter.

2. Preparation of transformants containing pdBSI-LacZ pdBSI-LacZ DNA solution commercially available E. coli X
E. coli was transformed by mixing with L-2 Blue MRF 'and introducing the plasmid DNA into E. coli according to the attached manual, and the transformant was further incubated at 37 ° C using LB agar medium containing 50 μg / ml ampicillin. By culturing for 17 hours, an ampicillin-resistant transformant clone grown on an agar medium was obtained.

Next, the plasmid DNA extracted from each transformant clone was digested with SalI, and analyzed by electrophoresis on 0.8% agarose gel to give about 6,600 base pairs and about 4,700 base pairs.
A clone with each of the I fragment divided into two base pairs (based on the SalI site in the Pro-LacZ cassette inserted in the I fragment, see FIG. 5) and the DNA fragment of the vector plasmid pUC18 of about 2,600 base pairs. Were selected (FIG. 6, lane 2, digestion of pdBSI-LacZ with SalI). In addition, NotI digestion of selected clones revealed that the clones had an L of 3,474 base pairs.
It was confirmed to have an acZ DNA fragment (FIG. 6, lane 3, digestion of pdBSI-LacZ with NotI). In the electrophoresis results shown in FIG. 6, lane 1 is pdBSI in which the I fragment without Pro-LacZ was inserted was digested with SalI, and the I fragment of about 7,000 base pairs and about 2,600 base pairs were obtained. 2 and the DNA fragment of the vector plasmid pUC18 of the above (FIG. 5). Further, in FIG. 6, lane M is a molecular weight marker and shows the approximate number of base pairs.

Transformed E. coli containing the transfer vector pdBSI-LacZ selected in this manner was transformed into E-dB.
Named SI-LacZ, deposited at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry (accession number FERM P-1
7935), and changed to international deposit on October 3, 2001 (accession number FERM BP-7716).

3. Preparation of LacZ-inserted recombinant FHV-1 The above transfer vector pdBSI-LacZ was added to CrFK cells.
By introducing DNA by the transfection method and further infecting CrFK cells into which the gene has been introduced with attenuated FHV-1, by homologous gene recombination between pdBSI-LacZ DNA and the attenuated FHV-1 genome in the cell. , Recombinant FHV-1 having an I fragment in which the LacZ gene was integrated at the BamHI cleavage site within the I fragment which is one of the SalI digested DNA fragments of the FHV-1 genome was obtained. The details will be described below.

3 μg of pdBSI-LacZ DNA was mixed with 10 μl of a commercially available transfection reagent, LipofectAMINE (manufactured by GIBCO BRL), and reacted at room temperature for 45 minutes. Then, using a 6-well plate for tissue culture, the minimum essential medium of Dulbecco containing 10% fetal bovine serum (hereinafter, abbreviated as D-MEM, manufactured by Nissui), at 37 ° C, after culturing in the presence of 5% carbon dioxide, D-MEM 3
To 1 × 10 ⁶ CrFK cells washed twice with ml, pdBSI-L
A reaction solution of acZ DNA and a transfection reagent was added, and pdBSI-LacZ DNA was introduced into CrFK cells.

Next, the gene-introduced CrFK cells were transferred to 37
After culturing for 24 hours in the presence of 5% carbon dioxide at ℃, CrFK cells were infected with attenuated FHV-1 at a MOI (multiplicity of infection, virus infectivity) of 0.01. Like this pdBS
After gene transfer of I-LacZ DNA, CrFK cells infected with attenuated FHV-1 were cultured for about 3 days, and when cytopathic effect (cytopathic effect, hereinafter abbreviated as CPE) was observed in most cells The cells were disrupted by freeze-thawing the cell suspension three times, and finally the virus supernatant containing both attenuated FHV-1 and recombinant FHV-1 was added to the centrifugal supernatant of the disrupted cell suspension. Recovered. Screening of recombinant FHV-1 from this virus solution was performed by the plaque selection method shown below.

That is, the collected virus solution was added to D-MEM for 1
Serial dilution from 0 ^-1 to 10 ^-5, 300 μl of each diluted virus solution
1 × 10 ⁶ CrF grown in 6-well tissue culture plate
After adding to K cells and adsorbing the virus at 37 ° C for 1 hour, 2%
D-MEM soft agar medium containing fetal bovine serum and 1% agarose 1
ml was layered and cultured for 2 days. Then 0.01% Neutral Red and 1 mg / ml X-gal (5-bromo-4-chloro-3-in
dolyl β-D-galactopyranoside, manufactured by SIGMA), D-MEM soft agar medium 1 containing 1% fetal bovine serum and 1% agarose 1
ml was overlaid and the cells were cultured for a further 24 hours and blue plaques formed by the propagated recombinant virus were isolated. After that, the same plaque selection method was repeated twice and LacZ
Recombinant FHV-1 in which I was integrated into the I fragment in the genome was selected. The thus obtained LacZ-inserted recombinant FHV-1 was ligated to Lac
-FHV (see Figure 2B).

4. Preparation of recombinant FHV-1 with LacZ and FCV capsid protein gene insertion (1) Preparation of TK-deficient / FCV capsid protein gene insertion FHV-1 FCV with FHV-1-derived gC promoter attached to the TK gene region in a tissue culture flask Recombinant FHV-1 incorporating the capsid protein gene (hereinafter abbreviated as dTK-gC / Cap-FHV, distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, University of Tokyo) 1 × 10
MOI: 0.01 was added to ⁷ CrFK cells, and the cells were adsorbed and infected by incubation at 37 ° C for 1 hour. Then
The cells were cultured in D-MEM medium containing 2% fetal bovine serum at 37 ° C in the presence of 5% carbon dioxide for about 3 days until CPE was observed in most cells. Subsequently, the cells were disrupted by freeze-thawing the cell suspension three times, and the recombinant FHV in which the capsid protein gene of FCV was inserted into the TK gene region that was deleted in the centrifugal supernatant of the disrupted cell suspension. Got -1. The recombinant FHV-1 thus obtained was dTK-gC / Cap-FHV (Fig. 2A).
(See).

The recombinant FHV-1 dTK-gC / Cap-
The transfer vector (named pfTK (gCp) -Cap) incorporating the feline calicivirus capsid protein gene and the FHV-1 derived gC promoter used to generate FHV had the structure shown in FIG. In the figure, the shaded area shows a part of the multiple cloning site derived from the plasmid vector (pBluescript KS-) that was incorporated during the construction of pfTK (gCp) -Cap, and "Pro" was derived from FHV-1. gC promoter, "FCV Capsid" for feline calicivirus capsid protein gene, "SalIA" for FHV-
In the SalIA fragment derived from one genome, "TK" represents the thymidine kinase gene, and the dotted line in the thymidine kinase gene ("TK") represents the deleted gene region. The arrows in the figure indicate the orientation of the feline calicivirus capsid gene and promoter.

(2) Preparation of FHV-Cap / Lac Laxed 1 × 10 ⁶ CrFK cells in a ^6-well tissue culture plate.
c-FHV was added at MOI: 0.01, and the recombinant virus was adsorbed and infected with CrFK cells by incubation at 37 ° C for 1 hour, and then in D-MEM medium containing 2% fetal bovine serum for 24 hours. By culturing, CrFK cells infected with Lac-FHV were grown. Next, dTK-gC / Cap-FHV was added to the Lac-FHV-infected CrFK cells.
Virus solution was added to MOI: 0.01, and dTK-gC / Cap FHV was adsorbed to Lac-FHV-infected CrFK cells by the same procedure as above.
Super-infected. In this way Lac-FHV and dTK-gC / Cap-FH
About 3 times until CrFK cells superinfected with V until CPE is detected
After culturing for a day, the cells were disrupted by a freeze-thaw operation similar to the above, and the recombinant virus, that is, Lac-FHV and dTK-gC / Cap-FHV was partially gene-generated in the centrifugal supernatant of the cell disruption solution. By recombination, a virus solution containing recombinant FHV-1 in which the LacZ gene and the FCV capsid protein gene associated with the gC promoter were integrated in one viral genome was obtained. The above recombinant FHV-1 was selected from this virus solution by the plaque selection method described below.

That is, after serially diluting the virus solution from 10 ⁻¹ to 10 ⁻⁵ , 300 μl of each diluted virus solution was added to 1 × 10 ⁶ CrFK cells in a ^6-well tissue culture plate, and the dilution was performed at 37 ° C. After adsorbing and infecting the cells with the virus for a period of time, 100 mg of arabinofuranosyluracil (arabinofuranocyluracil), 2
1 ml of D-MEM soft agar medium containing 1% of fetal bovine serum and 1% of agarose was overlaid and cultured at 37 ° C. in the presence of 5% carbon dioxide. Two days later, D-MEM containing 0.01% neutral red, 1 mg / ml X-gal, 100 mg arabinofuranosyluracil, 1% fetal bovine serum and 1% agarose on D-MEM soft agar medium. Overlay 1 ml of MEM soft agar medium and continue culturing,
After 24 hours, blue plaques formed by the propagated recombinant virus were isolated. After that, the same plaque selection method was repeated twice, and the LacZ gene was inserted into the I fragment of the viral genome, and the FCV capsid protein gene with the FHV-1-derived gC promoter was inserted into the deleted TK gene region. The replacement FHV-1 was selected. The recombinant FHV-1 thus obtained was named FHV-Cap / Lac (see FIG. 2C).

Regarding the FHV-Cap / Lac obtained here, the certificate of refusal of acceptance by the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, dated December 2, 1999 (1999 (November 29th application) was issued,
Although not deposited at the center, this recombinant virus FHV-Cap / Lac is stored at Tsukuba Central Research Institute, Kyoritsu Pharmaceutical Co., Ltd., and can be distributed if necessary. Also, regarding this FHV-Cap / Lac, currently ATCC (Amer
The ican Type Culture Collection) is being prepared for deposit.

5. Expression of foreign protein from FHV-Cap / Lac in cell lines Expression of FCV capsid protein and β-galactosidase derived from FHV-Cap / Lac in CrFK cells infected with recombinant FHV-1, FHV-Cap / Lac Was analyzed by the fluorescent antibody method shown below.

That is, FHV-Cap was added to 1 × 10 ⁶ CrFK cells.
/ Lac was added at MOI: 0.01 to adsorb and infect the virus in the same manner as above, and then in D-MEM medium containing 2% fetal bovine serum in the presence of 5% carbon dioxide at 37 ° C for 24 hours. Cultured. The cultured cells were then treated with phosphate buffer (137 mM sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium hydrogen phosphate, and 1.47 mM potassium dihydrogen phosphate, pH.
7.4) and fixed with cold acetone.
Expression of the FCV capsid protein and β-galactosidase in the FHV-Cap / Lac-infected CrFK cells fixed in this manner was analyzed by the fluorescent antibody method described below.

Expression analysis of the FCV capsid protein was carried out by the following procedure. First, fixed CrFK cells were allowed to react with mouse anti-FCV capsid protein monoclonal antibody diluted 1: 100 with phosphate buffer solution (distributed by Dr. Takayuki Miyazawa, Faculty of Agriculture, University of Tokyo) for 1 hour at 37 ° C, and then phosphate buffer solution. With 3 cells
After washing twice, dilute 1: 1,000 tetramethylrhodamine-5 (-6) -isothiocyanate (Tetramethylrhodamin
The mixture was allowed to react with e-5- (and-6) -isothiocyanate, TRITC) -labeled goat anti-mouse IgG antibody (manufactured by ICN Pharmaceuticals) at 37 ° C for 1 hour. Then, the cells were washed three times with a phosphate buffer and observed under a fluorescence microscope to observe the expression of the capsid protein of FCV. In addition, CrFK cells not infected with FHV-Cap / Lac were treated in the same manner as above to serve as a negative control.

As a result of microscopic examination, as shown in FIG. 8B, red specific fluorescence which was not detected in the negative control FHV-Cap / Lac-uninfected CrFK cells was detected in FHV-Cap / Lac-infected CrFK cells, and CrFK was detected. It was revealed that the FCV capsid protein was expressed and produced from FHV-Cap / Lac in cells.

On the other hand, the expression of β-galactosidase from FHV-Cap / Lac was confirmed by FHV-Cap / Lac-infected C prepared as described above.
Rabbit anti-β-galactosidase serum (Chemicon International) diluted 1: 100 as primary antibody in rFK cells.
And fluorescein isothiocyanat diluted 1: 2,000 as a secondary antibody.
e, FITC) labeled goat anti-rabbit IgG serum (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted in the same manner as above, and fluorescence-labeled β-galactosidase was detected under a microscope using a fluorescence microscope. As in the above, CrFK cells not infected with FHV-Cap / Lac were used as a negative control.

As a result, as shown in FIG. 8A, FHV-Cap was not detected in the negative control FHV-Cap / Lac non-infected CrFK cells, and
The β-galactosidase labeled with green fluorescence was detected only in CrFK cells infected with / Lac.
It was revealed that β-galactosidase was expressed and produced from V-Cap / Lac.

In the double staining method in which the fluorescent antibody method for detecting the FCV capsid protein and the fluorescent antibody method for detecting β-galactosidase were simultaneously performed, the FCV capsid protein labeled with red fluorescence and the green fluorescence were detected. Since the labeled β-galactosidase was detected on the same cells, it was revealed that both FCV capsid protein and β-galactosidase were simultaneously expressed and produced in FHV-Cap / Lac-infected CrFK cells.

From the above results, FHV-C which is recombinant FHV-1
It was demonstrated that ap / Lac replicates in the feline cell line and simultaneously expresses the capsid protein gene of FCV inserted as an exogenous gene and the capsid protein from the LacZ gene and β-galactosidase, respectively.

6. Production of antibodies in cats (1) Preparation of FHV-Cap / Lac As described above, MOI of FHV-Cap / Lac was applied to 1 × 10 ⁷ CrFK cells.
Adsorbed and infected with 0.01. Then, the infected CrFK cells were cultured in D-MEM medium containing 2% fetal bovine serum at 37 ° C. in the presence of 5% carbon dioxide gas for about 4 days until CPE was observed in most cells. Subsequently, the cultured cell suspension was freeze-thawed three times to disrupt the cells, and FHV-Cap / Lac was obtained in the centrifugal supernatant of the disrupted cell suspension.

(2) Detection of anti-β-galactosidase antibody, anti-FHV-1 antibody, and anti-capsid antibody in FHV-Cap / Lac inoculated cats 2 × 10 ⁶ TCID ₅₀ / ml FHV-Cap / Lac 2 ml for 1 year old Three male cats were inoculated nasally, orally, and by instillation, respectively, and after 3 weeks, each cat was additionally inoculated under the same conditions. Five days after this additional inoculation, blood was collected from the jugular vein of each cat, and anti-β-galactosidase antibody in the serum prepared from the blood was detected by the indirect fluorescent antibody method, and anti-FHV-1 antibody against FHV-1 was detected. The sum test and the anti-capsid antibody were detected by the indirect fluorescent antibody method. The indirect fluorescent antibody method, the neutralization test method and their results will be described below.

(2-1) Detection of anti-β-galactosidase antibody β-galactosidase expression plasmid vector pCMVβ 0.3
μg was reacted with 2 μl of commercially available transfection reagent, LipofectAMINE (manufactured by GIBCO BRL) for 45 minutes at room temperature, and the reaction product was placed on an 8-well culture slide glass (manufactured by BECTON DIKINSON). The pCMVβ DNA was introduced into the cells in addition to the 8 × 10 ⁴ CrFK cells cultured in ( ⁴ ). Then, the gene-introduced CrFK cells were incubated at 37 ° C.,
After culturing in the presence of 5% carbon dioxide for 24 hours, washing with phosphate buffer 3 times, the cells were fixed in chilled acetone.

On the other hand, prior to the indirect fluorescent antibody method, FHV-Ca was used.
The serum of the cat inoculated with p / Lac was mixed with an equal volume of the supernatant of the CrFK cell lysate and centrifuged, and the nonspecific antigen-antibody reaction was reduced by the absorption operation at 37 ° C for 30 minutes. This cat serum was diluted 1:20 with phosphate buffer and used as the primary antibody.

After reacting the acetone-fixed CrFK cells prepared as described above with 100 μl of 1:20 diluted feline serum at 37 ° C. for 1 hour,
The cells were washed three times with phosphate buffer and further diluted 1: 1,000 with phosphate buffer to give FITC-labeled goat anti-cat IgG antibody (ICN / CA
It was reacted with PPEL) at 37 ° C for 1 hour. Then, the cells were washed three times with phosphate buffer, and the fluorescence-labeled β-galactosidase in CrFK cells was detected by examining under a fluorescence microscope. Not transfected with pCMVβ
CrFK cells and feline serum before FHV-Cap / Lac inoculation were each subjected to the same indirect fluorescent antibody method as described above to serve as a negative control.

As shown in FIG. 9, pCMV used as a negative control
CrFK cells without β DNA introduced and FHV-Cap / Lac
It was not detected when the pre-inoculation cat serum was used (Fig. 9).
A), pCMVβ DN reacted with FHV-Cap / Lac inoculated cat serum
Fluorescently labeled β-galactosidase was detected only in A-introduced CrFK cells (Fig. 9B). As a result, the inoculated FHV-Cap / Lac replicated in the cat body, and the FHV-Cap / Lac
It was revealed that β-galactosidase was produced from ac.

(2-2) Measurement of anti-FHV-1 antibody FHV-Cap / Lac inoculated cat serum diluted 2-fold serially with D-MEM medium
Tissue culture plates in 100 [mu] l and ^{_{6 × 10 2 TCID 5 0 /}} ml 96 -well and FHV-1 solution 100 [mu] l, prepared, 37 ° C., after 1 hour incubation in the presence of 5% carbon dioxide gas, the 100 [mu] l 2 × 1
0 In addition to the ^five 24 CrFK cells fiber culture in the sheet of holes, further 37 ° C., then 1 hour incubation in the presence of 5% carbon dioxide were absorbed and infect FHV-1 into CrFK cells.

Then, the cell culture medium was removed, and 300 μl of D-MEM soft agar medium containing 2% fetal bovine serum and 1% agarose was layered on the CrFK cells and cultured for 2 days.
The plaques caused by the proliferating virus that appeared when the cells were overlaid with 300 μl of D-MEM soft agar medium containing Neutral Red, 1% fetal bovine serum and 1% agarose and cultured for 24 hours were counted. The number of plaques that appeared at this time was FHV-Ca.
CrFK infected with FHV-1 treated with p / Lac uninoculated cat serum
The dilution ratio of feline serum that was less than half the number of plaques that appeared in the cells was determined, and the reciprocal of the highest dilution ratio of the feline serum was used as the neutralizing antibody titer. In the above measurement of neutralizing antibody titer, FHV-Cap / Lac was inoculated 3
Head cat sera showed 8-fold, 16-fold, and 16-fold neutralizing antibody titers, respectively. As a result, FH inoculated in the cat body
It was confirmed that V-Cap / Lac was duplicated.

(2-3) Measurement of anti-calicivirus capsid protein antibody 100 μl of a virus solution of feline calicivirus F4 strain of 8 × 10 ³ TCID ₅₀ / ml was added to an 8-well culture slide glass (BECTON D).
8 × 10 ⁴ CrFK cells cultivated on IKINSON) were added and incubated for 1 hour at 37 ° C. in the presence of 5% carbon dioxide to adsorb and infect the feline calicivirus F4 strain. Then, after washing the cells with D-MEM medium, the cells were washed with D-MEM medium containing 2% fetal bovine serum at 37 ° C. in the presence of 5% carbon dioxide gas.
Cells were cultured for days. The cultured feline calicivirus F4 strain infected cells were washed three times with a phosphate buffer and then fixed in chilled acetone.

On the other hand, prior to the indirect antibody method, FHV-Cap / La
c The serum of the inoculated cat was mixed with an equal volume of the supernatant of the CrFK cell lysate, and the nonspecific antigen-antibody reaction was reduced by the absorption operation at 37 ° C for 30 minutes. This cat serum was then treated with phosphate buffer
It was diluted 1: 8 and used as the primary antibody.

Acetone-fixed feline calicivirus-infected CrFK cells prepared as described above and 100 μl of 1: 8 diluted FHV-Cap / Lac inoculated feline serum were reacted at 37 ° C. for 1 hour, and then the cells were washed 3 times with phosphate buffer. In addition, FITC-labeled goat anti-cat IgG antibody (manufactured by ICN / CAPPEL) diluted 1: 1,000 with phosphate buffer and 37
The reaction was carried out at 0 ° C for 1 hour. The cells were then washed three times with phosphate buffer and examined under a fluorescence microscope to detect fluorescently labeled feline calicivirus capsid protein in CrFK cells. In addition, the indirect fluorescent antibody method similar to the above was performed using each of the CrFK cells not infected with feline calicivirus and the cat serum before inoculation of FHV-Cap / Lac, and used as a negative control. The results are shown in Fig. 10.

As shown in FIG. 10A, as a negative control, fluorescence-labeled feline calicivirus capsid protein was detected when feline serum before inoculation with FHV-Cap / Lac was reacted with feline calicivirus-infected CrFK cells. Was not done. Also, when the cat serum inoculated with FHV-Cap / Lac was reacted with feline calicivirus-uninfected CrFK cells, the fluorescently labeled feline calicivirus capsid protein as described above was not detected (not shown). That is, the fluorescently labeled feline calicivirus capsid protein was detected only when FHV-Cap / Lac inoculated cat serum was reacted with feline calicivirus-infected CrFK cells, as shown in FIG. 10B. It was From this, it was revealed that the inoculated FHV-Cap / Lac replicated in the cat body, and the feline calicivirus F4 strain capsid protein was produced from the FHV-Cap / Lac.

From the above results, when FHV-Cap / Lac was transmucosally inoculated into cats, the inoculated cats were inserted into the thymidine kinase region as well as producing antibodies against FHV-1 while maintaining a healthy condition. And antibodies against feline calicivirus capsid protein, a product of a foreign gene, and antibodies against β-galactosidase, a product of a foreign gene inserted in the BamHI region of the I fragment. Therefore, the recombination in the present invention
It can be seen that the insertion sites of foreign genes in FHV-1 do not affect the replication of the virus itself, and the foreign genes integrated into these insertion sites produce proteins separately.

That is, it is possible to immunize cats with at least two foreign gene products in addition to FHV-1 by nasally, orally and instilling transmucosally with this recombinant virus. Yes, in fact, as a result of intranasal, oral and instillation of this recombinant viral vector FHV-Cap / Lac in cats, the inoculated cats are calicivirus capsid proteins, β-galactosidase and viral vectors while maintaining their health. Antibodies to FHV-1 were produced.

7. Feline herpesvirus type 1 genome Sa
Determination of partial base sequence of lI I fragment (1) Determination of sequence The partial base sequence of SalI I fragment derived from FHV-1 genome incorporated in plasmid pdBSI (see Fig. 1) was used as a DNA sequencer.
(Manufactured by Pharmacia). The details will be described below.

1 from SalI I fragment of FHV-1 integrated in pdBSI
A 969 base pair partial DNA fragment (PstI-HindIII DNA fragment) was cut out, and a commercially available cloning vector pB1uescrip
tIISK (+) (manufactured by Stratagene) was incorporated into the PstI-HindIII digestion site. The plasmid containing the partial base sequence of the SalI I fragment of FHV-1 obtained was named pSal-Hind.
Then, using the pSal-Hind plasmid DNA as a template, PstI-Hin
A forward primer (M13 containing a sequence derived from a plasmid vector existing upstream or downstream of the dIII DNA fragment)
Universal primer, 5'-CGACGTTGTAAAACGACGGCCAGT,
SEQ ID NO: 3) and reverse primer (M13 Reverse pr
imer, 5'-CAGGAAACAGCTATGAC, SEQ ID NO: 4) was used to analyze the PstI-HindIII DNA fragment by the cycle sequencing method.
A base sequence of about 600 bases was determined from each of the 5'end and the 3'end. In addition, of the determined Pstl-HindIII DNA fragment
4 new primers (Primer X1: SEQ ID NO: 5, Primer X2: SEQ ID NO: 6, Primer X3: SEQ ID NO: 7 and Primer X4: SEQ ID NO: 8) based on the base sequences of the 5 ′ end side and the 3 ′ end side Synthesized and PstI-Hind by primer walking method
The total base sequence of the III DNA fragment was determined to be 1969 bases. The determined nucleotide sequence of the PstI-HindIII DNA fragment within the SalI I fragment derived from the FHV-1 genome is shown in SEQ ID NO: 1. Also, SEQ ID NO: 1
The amino acid sequence predicted from the nucleotide sequence shown in is shown in SEQ ID NO: 2.

(2) Sa of the feline herpesvirus type 1 genome
S the nucleotide sequence of the li I fragment present in the U _L region of the FHV-1 genome shown in comparative analysis SEQ ID NO: 2 amino acid sequences encoding
The amino acid sequence predicted from the nucleotide sequence near the BamHI digestion site in the alI I fragment (PstI-HindIII DNA fragment) was determined using herpes simplex virus type 1 (HSV-1), equine herpesvirus type 1 (EHV-1) and equine herpes. the amino acid sequence of protein kinase present in the U _L region of the viral type 4 (EHV-4) (RF Smith , and TF Smith. 1989. J. Virol. 6
3: 450-455, GenBank accession number: EHV-1, NC001491
EHV-4, NC001844) and shown in FIGS. 11 and 12. As is clear from the comparison of the amino acid sequences shown in FIGS. 11 and 12, the amino acid sequences of the catalytic domains I to VI of the protein kinases surrounded by squares are FHV-1, H
It was highly conserved among herpesviruses such as SV-1, EHV-1 and EHV-4. In particular, PstI in the SalI I fragment of FHV-1
-In the amino acid sequence encoded by the HindIII DNA fragment, the amino acids corresponding to the constituent amino acids of each catalytic domain of protein kinase found in the sequence are HSV-
Even when the amino acid is different from the constituent amino acids of each catalytic domain of 1 protein kinase, there were many cases in which it was the same as the constituent amino acids of each catalytic domain of protein kinase of EHV-1 and EHV-4.

[0103] From the above analysis, the base sequence of BamHI digestion site near the U _L region SalII fragment which is foreign gene insertion site found in this invention was revealed to encode protein kinase.

[0104] On the other hand, protein kinase of the U _L region of the pseudorabies virus belonging to the same alphaherpesvirinae and FHV-1 is, is finding that it is not essential to the growth or replication of the virus has been reported (N .de Win
d, J. Domen, and A. Berns. 1992. J. Virol. 66: 5200-
5209).

[0105] and the analysis result of the above amino acid sequence, the protein kinase of U _L region of the herpesvirus Together, finding that it is not essential for replication and proliferation of the virus, genes of BamHI near the I fragment portion, a portion of the gene encoding the protein kinase of U _L region, moreover, found to be protein kinase is the gene product are those that are not essential for replication and proliferation of the virus, heading in the present invention exogenous gene insertion site is not limited to BamHI digestion site within the SalI I fragment of FHV-1 genome, apparently may be as long as the protein kinase gene region of the FHV-1 genome U _L region Became.

[0106]

INDUSTRIAL APPLICABILITY According to the present invention, recombination capable of expressing and producing at least two kinds of foreign gene products
This recombinant FHV-1 vector can be used as a safe and effective multivalent vaccine virus that can produce an FHV-1 vector.

[0107]

[Sequence list]                                SEQUENCE LISTING         <110> Kyoritsu Seiyaku, Corporation <120> Recombinant feline herpesvirus type 1 and multivalent vaccine usin g thereof <130> KRS-12 <140> --- <141> 2002-03-29 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 1969 <212> DNA <213> Feline herpesvirus type 1 <220> <221> CDS <222> (361) .. (1968) <400> 1 agcttaccaa cgtgaatcaa tatttaaggc acgtacgcta gacatgatcc gtggagggat 60 agataggcgt gatcctattt ttgttcacac atttacgtca gcaaaacggg ccgggcaggc 120 cttgacagac aactacacac atactctagg gtggaggcga tagaacaaaa gacacaatca 180 ctgcgactgc gcattgaggc acagacgccg tcgaagaagt actgcatagc catcgacggt 240 atctccagtc agacttcact gataaaattg atgctgtgga tgataagata tttgaaaagg 300 aaaatcaatt aaccgaaact attctagaag tagatccaga cccgaaactc tggaacgtgg 360 atg gct aga cga ggc gga cga agc gct act gac gag atg gat gtt ggc 408 Met Ala Arg Arg Gly Gly Arg Ser Ala Thr Asp Glu Met Asp Val Gly   1 5 10 15 gga agc tcc caa ggc gac cca ctg tca cat gga cca ata ctc tca cct 456 Gly Ser Ser Gln Gly Asp Pro Leu Ser His Gly Pro Ile Leu Ser Pro              20 25 30 atc aca aga cca tca tcg ggg gtc cgg gaa ggg ggg gga cat tgc aac 504 Ile Thr Arg Pro Ser Ser Gly Val Arg Glu Gly Gly Gly His Cys Asn          35 40 45 acg gcc gat ccc cac tct caa gga aac cat atc aaa cgg ggt ata tgt 552 Thr Ala Asp Pro His Ser Gln Gly Asn His Ile Lys Arg Gly Ile Cys      50 55 60 aaa cca gga gtc tct gga tcc ggt aac aca gcc gat agc gct cac aaa 600 Lys Pro Gly Val Ser Gly Ser Gly Asn Thr Ala Asp Ser Ala His Lys  65 70 75 80 cac ctc acc atg tcc cca agg agg ctg cgc cca tta cca cac aga gaa 648 His Leu Thr Met Ser Pro Arg Arg Leu Arg Pro Leu Pro His Arg Glu                  85 90 95 ggg atc tta cgt cac cgg att aaa gaa gaa tgt caa gac ttt cag gct 696 Gly Ile Leu Arg His Arg Ile Lys Glu Glu Cys Gln Asp Phe Gln Ala             100 105 110 ggt aac gga gaa ggt aaa atc agg gca aac acc gcc atc gac aga tat 744 Gly Asn Gly Glu Gly Lys Ile Arg Ala Asn Thr Ala Ile Asp Arg Tyr         115 120 125 ttt aca cga gcg aga cgt atc ttc aaa tat aca ccc aga cgc atg tct 792 Phe Thr Arg Ala Arg Arg Ile Phe Lys Tyr Thr Pro Arg Arg Met Ser     130 135 140 agt aga cga ggc ggg aga acc act ccc cca tgt atg gct ggg tgg gct 840 Ser Arg Arg Gly Gly Arg Thr Thr Pro Pro Cys Met Ala Gly Trp Ala 145 150 155 160 tcc ccc tct ggt gga cga tat gac ggg ctc att cga ggg gac tcc aac 888 Ser Pro Ser Gly Gly Arg Tyr Asp Gly Leu Ile Arg Gly Asp Ser Asn                 165 170 175 aat gga cgg acc gat ata cca aat acc ctg act cga att cct ata cat 936 Asn Gly Arg Thr Asp Ile Pro Asn Thr Leu Thr Arg Ile Pro Ile His             180 185 190 gag gta tgt acc cca tta aca aca aat ccc ggc aac agg tca tct att 984 Glu Val Cys Thr Pro Leu Thr Thr Asn Pro Gly Asn Arg Ser Ser Ile         195 200 205 ttg aaa att agg aaa att aag cgt gtt acg atc cct gtg ttc tca gtg 1032 Leu Lys Ile Arg Lys Ile Lys Arg Val Thr Ile Pro Val Phe Ser Val     210 215 220 tca gca gaa atg cat tac tct aag gtg gca cta gga gaa cca ccg aag 1080 Ser Ala Glu Met His Tyr Ser Lys Val Ala Leu Gly Glu Pro Pro Lys 225 230 235 240 ttc ggg ggg gct ggt ggg tat gga gaa gta cag att tat cga caa aca 1128 Phe Gly Gly Ala Gly Gly Tyr Gly Glu Val Gln Ile Tyr Arg Gln Thr                 245 250 255 ggt ctg gcc atc aaa aca tca tca agt cca tcg tgt ttt gaa cat gaa 1176 Gly Leu Ala Ile Lys Thr Ser Ser Ser Pro Ser Cys Phe Glu His Glu             260 265 270 tta tta gtc act tta tta gcc ggg gag agc tct cta cgc gct aga tca 1224 Leu Leu Val Thr Leu Leu Ala Gly Glu Ser Ser Leu Arg Ala Arg Ser         275 280 285 tcc ata ggc ata act ggg ata att tac ccc gtt gca ttt tca tta acc 1272 Ser Ile Gly Ile Thr Gly Ile Ile Tyr Pro Val Ala Phe Ser Leu Thr     290 295 300 gaa cac caa atg gta ttc aaa gcg tat gat atg gat ctg aat gta tat 1320 Glu His Gln Met Val Phe Lys Ala Tyr Asp Met Asp Leu Asn Val Tyr 305 310 315 320 tgt aat aaa cta tca tcc gct gga ccc cca aca tca aat ata ctt aat 1368 Cys Asn Lys Leu Ser Ser Ala Gly Pro Pro Thr Ser Asn Ile Leu Asn                 325 330 335 gcg atg gaa cat gcg ttc atc ggg ttg ggt aag gct gtg gca tac ctg 1416 Ala Met Glu His Ala Phe Ile Gly Leu Gly Lys Ala Val Ala Tyr Leu             340 345 350 aac acc aaa tgc ggc tta acg cat ttg gat atc aaa tgt gga aat ata 1464 Asn Thr Lys Cys Gly Leu Thr His Leu Asp Ile Lys Cys Gly Asn Ile         355 360 365 ttc gtc aac aca aaa aat tgt gtt ata aaa gat tat gtc ata gcc gat 1512 Phe Val Asn Thr Lys Asn Cys Val Ile Lys Asp Tyr Val Ile Ala Asp     370 375 380 ttt agt ctg atg act cta aac aca aat tct acc gta atg cgg gcg gag 1560 Phe Ser Leu Met Thr Leu Asn Thr Asn Ser Thr Val Met Arg Ala Glu 385 390 395 400 ttt gaa att ccc act ggg gat gcg tca aat aag gtc cta cgc ctt tca 1608 Phe Glu Ile Pro Thr Gly Asp Ala Ser Asn Lys Val Leu Arg Leu Ser                 405 410 415 cga ggg gcg gcg aca act ata ttt agt ctg gta ttg ggt cat gga cat 1656 Arg Gly Ala Ala Thr Thr Ile Phe Ser Leu Val Leu Gly His Gly His             420 425 430 aac caa ccc acg gag ata ctg gtt gac ttt att aat aac agt gga ctg 1704 Asn Gln Pro Thr Glu Ile Leu Val Asp Phe Ile Asn Asn Ser Gly Leu         435 440 445 gct cga cac cgc ggc cca tta gac agt gac gtt ggt gta gct gtt gac 1752 Ala Arg His Arg Gly Pro Leu Asp Ser Asp Val Gly Val Ala Val Asp     450 455 460 ttg tat gct ctt gga cag gtg cta ttg gaa ctg ctt ttg act gga tgc 1800 Leu Tyr Ala Leu Gly Gln Val Leu Leu Glu Leu Leu Leu Thr Gly Cys 465 470 475 480 ctt tcc cct cgg tta ccg gtc ccc att ctt aga aat acg aca tat tac 1848 Leu Ser Pro Arg Leu Pro Val Pro Ile Leu Arg Asn Thr Thr Tyr Tyr                 485 490 495 tac tac cta cac cag gtg acc gtg gaa tat gcc ttg gat ctc tta gca 1896 Tyr Tyr Leu His Gln Val Thr Val Glu Tyr Ala Leu Asp Leu Leu Ala             500 505 510 tat ctg cgc act ata ccc cca tat att tcc ttc ttc acc tat tac aac 1944 Tyr Leu Arg Thr Ile Pro Pro Tyr Ile Ser Phe Phe Thr Tyr Tyr Asn         515 520 525 aat tca tgg tgt tcc ata ccc tgc a 1969 Asn Ser Trp Cys Ser Ile Pro Cys     530 535 <210> 2 <211> 536 <212> PRT <213> Feline herpesvirus type 1 <400> 2 Met Ala Arg Arg Gly Gly Arg Ser Ala Thr Asp Glu Met Asp Val Gly   1 5 10 15 Gly Ser Ser Gln Gly Asp Pro Leu Ser His Gly Pro Ile Leu Ser Pro              20 25 30 Ile Thr Arg Pro Ser Ser Gly Val Arg Glu Gly Gly Gly His Cys Asn          35 40 45 Thr Ala Asp Pro His Ser Gln Gly Asn His Ile Lys Arg Gly Ile Cys      50 55 60 Lys Pro Gly Val Ser Gly Ser Gly Asn Thr Ala Asp Ser Ala His Lys  65 70 75 80 His Leu Thr Met Ser Pro Arg Arg Leu Arg Pro Leu Pro His Arg Glu                  85 90 95 Gly Ile Leu Arg His Arg Ile Lys Glu Glu Cys Gln Asp Phe Gln Ala             100 105 110 Gly Asn Gly Glu Gly Lys Ile Arg Ala Asn Thr Ala Ile Asp Arg Tyr         115 120 125 Phe Thr Arg Ala Arg Arg Ile Phe Lys Tyr Thr Pro Arg Arg Met Ser     130 135 140 Ser Arg Arg Gly Gly Arg Thr Thr Pro Pro Cys Met Ala Gly Trp Ala 145 150 155 160 Ser Pro Ser Gly Gly Arg Tyr Asp Gly Leu Ile Arg Gly Asp Ser Asn                 165 170 175 Asn Gly Arg Thr Asp Ile Pro Asn Thr Leu Thr Arg Ile Pro Ile His             180 185 190 Glu Val Cys Thr Pro Leu Thr Thr Asn Pro Gly Asn Arg Ser Ser Ile         195 200 205 Leu Lys Ile Arg Lys Ile Lys Arg Val Thr Ile Pro Val Phe Ser Val     210 215 220 Ser Ala Glu Met His Tyr Ser Lys Val Ala Leu Gly Glu Pro Pro Lys 225 230 235 240 Phe Gly Gly Ala Gly Gly Tyr Gly Glu Val Gln Ile Tyr Arg Gln Thr                 245 250 255 Gly Leu Ala Ile Lys Thr Ser Ser Ser Pro Ser Cys Phe Glu His Glu             260 265 270 Leu Leu Val Thr Leu Leu Ala Gly Glu Ser Ser Leu Arg Ala Arg Ser         275 280 285 Ser Ile Gly Ile Thr Gly Ile Ile Tyr Pro Val Ala Phe Ser Leu Thr     290 295 300 Glu His Gln Met Val Phe Lys Ala Tyr Asp Met Asp Leu Asn Val Tyr 305 310 315 320 Cys Asn Lys Leu Ser Ser Ala Gly Pro Pro Thr Ser Asn Ile Leu Asn                 325 330 335 Ala Met Glu His Ala Phe Ile Gly Leu Gly Lys Ala Val Ala Tyr Leu             340 345 350 Asn Thr Lys Cys Gly Leu Thr His Leu Asp Ile Lys Cys Gly Asn Ile         355 360 365 Phe Val Asn Thr Lys Asn Cys Val Ile Lys Asp Tyr Val Ile Ala Asp     370 375 380 Phe Ser Leu Met Thr Leu Asn Thr Asn Ser Thr Val Met Arg Ala Glu 385 390 395 400 Phe Glu Ile Pro Thr Gly Asp Ala Ser Asn Lys Val Leu Arg Leu Ser                 405 410 415 Arg Gly Ala Ala Thr Thr Ile Phe Ser Leu Val Leu Gly His Gly His             420 425 430 Asn Gln Pro Thr Glu Ile Leu Val Asp Phe Ile Asn Asn Ser Gly Leu         435 440 445 Ala Arg His Arg Gly Pro Leu Asp Ser Asp Val Gly Val Ala Val Asp     450 455 460 Leu Tyr Ala Leu Gly Gln Val Leu Leu Glu Leu Leu Leu Thr Gly Cys 465 470 475 480 Leu Ser Pro Arg Leu Pro Val Pro Ile Leu Arg Asn Thr Thr Tyr Tyr                 485 490 495 Tyr Tyr Leu His Gln Val Thr Val Glu Tyr Ala Leu Asp Leu Leu Ala             500 505 510 Tyr Leu Arg Thr Ile Pro Pro Tyr Ile Ser Phe Phe Thr Tyr Tyr Asn         515 520 525 Asn Ser Trp Cys Ser Ile Pro Cys     530 535 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: M13 Universal       primer <400> 3 cgacgttgta aaacgacggc cagt 24 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: M13 Reverse       primer <400> 4 caggaaacag ctatgac 17 <210> 5 <211> 22 <212> DNA <213> Primer X1 <400> 5 tagtagacga ggcgggagaa cc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer X2 <400> 6 aatagatgac ctgttgccgg ga 22 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer X3 <400> 7 tgcgttcatc gggttgggta agg 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer X4 <400> 8 accttatttg acgcatcccc agtg 24

[Brief description of drawings]

FIG. 1 is a diagram showing a map of the feline herpesvirus type 1 genome and digestion with SalI. Further, in the figure, together with the foreign gene insertion region and the sequence region of the present invention, a conventionally known foreign gene insertion region is also shown.

FIG. 2 is a diagram schematically showing the insertion site of the recombinant FHV-1 of the present invention in the genome.

[Fig. 3] LacZ insertion transfer vector (pdBSI-LacZ)
It is a figure showing the outline of the manufacturing method of.

FIG. 4 is a diagram showing an outline of a method for producing LacZ / FCV capsid gene-inserted recombinant FHV-1.

FIG. 5: Constructed transfer vector pdBSI-LacZ
It is the figure which showed the schematic diagram of.

FIG. 6 is a diagram showing analysis results of various plasmid vectors by agarose gel electrophoresis.

FIG. 7: TK-deficient feline calicivirus capsid protein gene inserted into the feline calicivirus capsid protein gene and FHV-1 derived gC promoter used to generate recombinant FHV-1 dTK-gC / Cap-FHV FIG. 3 is a schematic diagram showing the structure of a transfer vector pfTK (gCp) -Cap.

FIG. 8 shows the detection results of β-galactosidase and FCV capsid protein in FHV-Cap / Lac-infected CrFK cells by the indirect fluorescent antibody method. In the figure, A shows the result of β-galactosidase labeled with FITC, and B shows TRITC.
The results of labeled FCV capsid protein are shown.

FIG. 9: FHV-Cap / Lac inoculated cat serum anti-serum by indirect fluorescent antibody method using FHV-Cap / Lac cat serum 5 days after inoculation as the primary antibody and FITC-labeled goat anti-cat IgG antibody as the secondary antibody It is the figure which showed the detection result of (beta) galactosidase antibody. In the figure, A shows the result of CrFK cells into which pCMVβ DNA was not introduced (negative control), and B shows Cr into which pCMVβ DNA was introduced.
The results with FK cells are shown.

FIG. 10 is a diagram showing the results of detecting an anti-calcicivirus capsid protein antibody by an indirect fluorescent antibody method. In the figure, A is a negative control obtained by reacting feline serum before inoculation with FHV-Cap / Lac with feline calicivirus-infected CrFK cells, and B is a control.
FHV-Cap / Lac inoculated cat serum and feline calicivirus-infected CrF
The result when reacting with K cells is shown.

[11] S present in the U _L region of the FHV-1 genome of the invention
The amino acid sequence predicted from the nucleotide sequence near the BamHI digestion site in the alI I fragment (PstI-HindIII DNA fragment) was determined using herpes simplex virus type 1 (HSV-1), equine herpesvirus type 1 (EHV-1) and equine herpes. shows a comparison of the amino acid sequence of protein kinase present in the U _L region of the viral type 4 (EHV-4). In the figure, the boxed area indicates the catalytic domains I to VI of protein kinase, and “*” indicates that the amino acid of FHV-1 is HSV-1, EHV-1 or E.
It is shown to be the same as any of the amino acids of HV-4.

FIG. 12 is a continuation of FIG.

Continued front page    F-term (reference) 4B024 AA01 BA32 CA04 DA02 DA06                       EA04 GA11 GA18 GA19 HA08                 4B065 AA26X AA90X AA95X AA95Y                       AB01 BA02 BA14 CA45                 4C084 AA02 BA44 DA01 MA01 NA14                       ZB322 ZC612                 4C087 AA01 BB65 BC83 CA12 MA01                       MA05 NA13 ZB32 ZC61

Claims (19)

[Claims] 1. An attenuated recombinant feline herpesvirus type 1 in which at least two types of foreign genes are expressibly inserted into two different regions in the feline herpesvirus type 1 genome. 2. The attenuated recombination of claim 1, wherein the two different regions in the feline herpesvirus type 1 genome are two regions that do not have a lethal effect on feline herpesvirus type 1 growth. Feline herpesvirus type 1. 3. Within the genome of feline herpesvirus type 1
Two different regions represent the feline herpesvirus type 1 genome
Unique long (U _L) In the area
Existing I fragment and thymidine kinase encoding fragment
The two gene regions of the gene region and the gene region according to claim 1.
Attenuated recombinant feline herpesvirus type 1. 4. Two regions that do not have a lethal effect on the growth of feline herpesvirus type 1, two different regions in the genome of feline herpesvirus type 1, are SalI-cut DNA of the feline herpesvirus type 1 genome. The attenuated recombinant feline herpesvirus type 1 according to claim 2, which comprises two gene regions, an I fragment portion existing in a unique long ( _UL ) region and a gene region encoding thymidine kinase. 5. A gene region encoding a protein kinase present in a unique long ( _UL ) region of the feline herpesvirus type 1 genome, and two different regions in the feline herpesvirus type 1 genome, and a thymidine kinase. The attenuated recombinant feline herpesvirus type 1 according to claim 1, which is two gene regions including a coding gene region. 6. A gene region encoding a protein kinase existing in a unique long ( _UL ) region of the feline herpesvirus type 1 genome, wherein two regions that do not have a lethal effect on the proliferation of feline herpesvirus type 1 are present. And the gene region encoding thymidine kinase, the attenuated recombinant feline herpesvirus type 1 according to claim 2. 7. The foreign gene is derived from a pathogenic microorganism,
7. Attenuation according to any one of claims 1 to 6, which is a gene fragment comprising a gene encoding a polypeptide that induces an infection protective ability in animals or a partial sequence of the gene. Recombinant feline herpesvirus 1
Type. 8. The method according to claim 1, wherein the exogenous gene is a gene derived from various cytokines and encoding a product showing a therapeutic effect on animals, or a gene fragment consisting of a partial sequence of the gene. Item 7. The attenuated recombinant feline herpesvirus type 1 according to any one of Items 6. 9. A multivalent vaccine comprising the attenuated recombinant feline herpesvirus type 1 of claim 7. 10. The multivalent vaccine according to claim 9, wherein the vaccination method is transmucosal vaccination or vaccination. 11. A therapeutic composition comprising the attenuated recombinant feline herpesvirus type 1 of claim 8. 12. The therapeutic composition according to claim 11, wherein the method of administration of the therapeutic composition is transmucosal administration or administration by injection. 13. A step of preparing at least two types of transfer vectors in which different expressible foreign genes are respectively inserted into gene fragments consisting of two different gene regions of the feline herpesvirus type 1 genome, Using at least two types of transfer vectors, a gene fragment consisting of different gene regions of the feline herpesvirus type 1 genome into which an exogenous gene has been inserted is introduced into feline cells, and feline herpesvirus type 1 is infected. The step of causing homologous gene recombination between the introduced gene fragment and the feline herpesvirus type 1 genome, and selecting the feline herpesvirus type 1 in which homologous gene recombination has occurred. At least two types of feline herpesvirus 1 are found in two different regions in the genome. A method for preparing an attenuated recombinant feline herpesvirus type 1 in which an exogenous gene is inserted so that it can be expressed. 14. A step of preparing at least two types of transfer vectors in which different expressible foreign genes are respectively inserted into gene fragments consisting of two different gene regions of the feline herpesvirus type 1 genome, Feline herpesvirus 1 into which an exogenous gene has been individually inserted using each of the transfer vectors of at least two types
A gene fragment consisting of different gene regions of the
The step of introducing feline herpesvirus type 1 into a feline cell and infecting the feline herpesvirus type 1 to cause homologous gene recombination between the introduced gene fragment and the feline herpesvirus type 1 genome; Selecting the resulting feline herpesvirus type 1 to obtain at least two types of recombinant feline herpesvirus type 1 in which one gene region in the genome of feline herpesvirus type 1 is recombined, respectively; A step of superinfecting a feline cell with each recombinant feline herpesvirus type 1 and causing homologous gene recombination between at least two types of recombinant feline herpesvirus type 1 genomes, and homology Selecting the feline herpesvirus type 1 in which the gene recombination has occurred. A method for preparing an attenuated recombinant feline herpesvirus type 1 in which at least two types of foreign genes are expressibly inserted into a region. 15. Two different gene regions within the genome of feline herpesvirus 1 are feline herpesvirus 1
Unique long of SalI-cleaved DNA fragments of type genome
15. The preparation method according to claim 13 or claim 14, which comprises two gene regions, an I fragment portion existing in the ( _UL ) region and a gene region encoding thymidine kinase. 16. The feline herpesvirus-1 comprises two different gene regions within the feline herpesvirus-1 genome.
15. The preparation method according to claim 13 or 14, which comprises two gene regions, a gene region encoding a protein kinase existing in a unique long ( _UL ) region of the type genome and a gene region encoding thymidine kinase. . 17. A method for introducing an exogenous gene, which comprises the step of infecting a cell with the attenuated recombinant feline herpesvirus type 1 according to any one of claims 1 to 8. 18. An immunization method for immunizing an inoculated animal by transmucosally inoculating or injecting an animal with the attenuated recombinant feline herpesvirus type 1 according to claim 7. 19. The transfer vector deposited under accession number FERM BP-7761.