JPH09267A - Recombined feline herpesvirus 1 type capable of mutating thymidine kinase gene and vaccine containing the same - Google Patents

Abstract

PURPOSE: To obtain a recombination type virus in which the EcoRV-SmaI site of thymidine kinase gene in feline Herpesvirus 1 type genom is defected, which is stable and sufficient in attenuation, and which is useful as a vaccine for feline virus infections diseases, etc. CONSTITUTION: This virus is a new recombined feline Herpesvirus 1 type (FHV-1) in which a gene coding thymidine kinase in the genom of feline Herpesvirus 1 type belonging to the Alphaherpesvirinae subfamily of Herpesviridae has a DNA sequence at least capable of being hybridized with a DNA sequence of the formula, which has a EcoRV-cleaved site and a SmaI- cleaved site at the No.618 position and the No.1074 position, respectively, which lacks at least the EcoRV-SmaI site, and which is stable and sufficient in attenuation and is useful as a vaccine for feline infectious diseases, etc. The virus is obtained by removing the EcoRV-SmaI of the thymidine kinase gene of the genom and, if necessary, subsequently inserting an exogenote.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant feline herpesvirus type 1 (hereinafter also referred to as FHV-1) which is safe and useful as a vaccine against feline viral diseases and a group using FHV-1 as a vector. The present invention relates to the development of a modified multivalent vaccine, a cultured cell and a cell culture containing the virus, a vaccine containing the virus, and an immunization method using the vaccine. More specifically, it relates to attenuated feline herpesvirus type 1. Recombinant feline herpesvirus type 1, which is sufficient, retains the ability to proliferate the virus, has excellent immunogenicity, and has clear biological and genetic markers, cultured cells containing the virus, and cell culture The present invention relates to a product, a vaccine containing the virus, and an immunization method using the vaccine.

[0002]

BACKGROUND ART There are various viruses as important infectious pathogens of cats. One of them is Herpesvir
There is a feline herpesvirus type 1 belonging to the subfamily Alphaherpesvirinae of idae. Feline herpesvirus type 1 is viral feline rhinotracheitis (FV
R), which is one of the most important pathogens for cats, which mainly causes upper respiratory diseases. In particular, newborn or debilitated cat infection with FHV-1 results in severe systemic disease and high mortality [Povey, RC1979. A review of felin.
e rhinotracheitis (feline herpesvirus I infection) C
omp. Immunol. Microbiol. Infect. Dis. 2: 373-38
7.].

Many FHV-1 strains have been isolated in various regions of the world and have been comparatively examined for antigenicity by several serological means. As a result, the various FH
V-1 isolates were indistinguishable in their antigenicity [Bittle, JL, York, CJ, Newberne, JW and M
artin, M. 1960. Serologic relationship of new feli
ne cytopathogenic viruses. Am. J. Vet. Res. 21: 54
7-550, Crandel, RA, Genaway, JR, Niemann, W.
H. and Maurer, FD 1960. Comparative studyof thre
e isolates with the original feline viral rhinotra
cheitis virus.Am. J. Vet. Res. 21: 504-506., Johns
on, RH and Thomas, RG 1966. Feline Viral rhino
tracheitis in Britain. Vet. Rec. 79: 188-190., Moc
hizuki, M., Konishi, S. and Ogata, M. 1977. Sero-di
agnostic aspects of feline herpesvirus infection.
Jpn. J. Vet. Sci. 39: 191-194., Studdert, MJ and
Martin, MC 1970. Virus diseases of the respirato
ry tract of cats: 1.Isolation of feline rhinotrach
eitis virus. Aust. Vet. J. 46: 99-104., Tan, RJ
S. 1970. Serological comparisons of feline respira
tory viruses. Jpn. J. Med. Sci. Biol. 23: 419-42
Four.〕.

However, regarding the cleavage patterns of the various FHV-1 DNA restriction endonucleases (restriction enzymes), there was a slight change in the electrophoretic mobility of some fragments, but no significant change was observed. It was not issued (Grail, A. and Harbour, DA 1990. Restrictio
n endonuclease analysis of DNA from isolates felin
e herpesvirus type 1. Jpn. J. Vet. Sci. 52: 1007-1
013., Herrmann, SC, Gaskell, RM, Ehlers, B. an
d Ludwig, H. 1984. Characterization of thefeline h
erpesvirus genome and molecular epidemiology of is
olates from natural outbreaks and latent infection
s. pp 321-326. In: Latent herpesvirus infection in
veterinary medicine (Wittmann, A., Gaskell, RM,
Ehlers, B. And Rziha, HJ eds.), Martinus Nijihof
f, Boston.]. From these data, FHV-1
It is found to be a homologous population compared to other α-herpesviruses.

On the other hand, F which is less toxic in the field
HV-1 strains exist and these strains have been selected and developed for use as live attenuated vaccines. These are Bittle, JL, and Rubic, WJ 1974. Stud
ies of feline viral rhinotracheitis vaccine.Vet.
Med. Small. Anim. Clin. 69: 1503-1505., Bittle, J.
L., and Rubic, WJ 1975. Immunogenic and protecti
ve effects of the F-2 strain of feline viral rhino
tracheitis virus. Am. J. Vet. Res. 36: 89-91., Bi
ttle, JL and Rubic, WJ 1975. A feline caliciv
irus vaccine combined with feline viral rhinotrach
eitis and feline panleukopenia vaccine. Feline. Pr
ac. 5: 13-15., Davis, EV and Beckenbauer, WH
1976. Studies on the safety and efficacy of an int
ranasal feline rhinotracheitis-calici virus vaccin
e. Vet. Med. Small Anim. Clin. 71: 1405-1410., Edwa
rds, BG, Buell, DJ and Acree, WM 1977. Evalu
ation of a new feline rhinotracheitis virus vaccin
e. Vet. Med Small Anim. Clin. 72: 205-209.,

Edwards, BG, Buell, DJ, Acree, W.
M. and Payne, JB 1977. Evaluation of feline rhino
tracheitis / feline panleukopenia combination vacc
ines by consecutive virulent challenge. Feline Pla
c. 7: 45-50., Orr, CM, Gaskell, CJ and Gaskel
l, RM 1980. Interaction of an intranasal combine
dfeline viral thinotracheitis, feline calicivirus
vaccine and the FVR carrier state.Vet. Rec. 23: 1
64-166., Povey, RC and Wilson, MR 1978.A comp
arison of inactivated feline viral rhinotracheitis
and feline caliciviral disease vaccines with live
-modified viral vaccines. Feline. Prac. 8: 35-4
2., 37. Scott, FW 1975. Evaluation of a feline v
iral rhinotracheitis vaccine. Feline Prac. 5: 17-2
2., Scott, FW 1977. Evaluation of a feline viral
Rhinotracheitis-feline calicivirus disease vaccin
e. Am.J.Vet.Res. 38: 229-234., Slater, E. and Yo
rk, C. 1976. Comparative studies on parental and i
ntranasal inoculation of an attenuated feline herp
esvirus. Dev. Biol. Standard. 33: 410-416., Wilso
n, JHG 1978. Intranasal vaccination against upp
er respiratory tract disease (URD) in the cat. II.
Results of field studies under enzootic conditions
in the Netherlands with a combined vaccine contai
ning live attenuated calici- and herpesvirus.Com
p. Immunol. Microbiol. Infect. Dis. 1: 43-48. However, there was no biological marker in the vaccine.

[0007] As a method for preventing infection of a cat by a pathogenic microorganism, there is a method of administering the above-mentioned attenuated live vaccine or inactivated vaccine. But,
These vaccines have various drawbacks. Attenuated live vaccines may be poorly attenuated, which causes the virus to become virulent after vaccination, causing vaccinated animals to become sick or even transmitted to other individuals, resulting in extremely serious problems. Problems occur. The F2 strain is one of the current live vaccines. This F2 strain was attenuated by low temperature passage. Maeda et al. Have reported the possibility that immunoblot analysis of F2 strain, cleavage pattern analysis with MluI, etc. can serve as markers. However, the current vaccine strains have no biological or genetic basis to demonstrate attenuation and no established markers.

The inactivated vaccine has a low level of immunogenicity because the antigen determinant for inducing neutralizing antibody is denatured by the inactivation treatment. Therefore, it has been necessary to immunize twice or more without functioning as a vaccine. Furthermore, conventional live vaccines or inactivated vaccines have no biological marker, and it is unclear whether the animal that is an infection or virus carrier is due to a field virus strain or vaccinated. This causes a problem that it becomes impossible to grasp the spread state of the virulent virus which is an outdoor strain, and it becomes impossible to take measures to prevent the spread of the virulent virus.

On the other hand, thymidine kinase (TK) encoded in the genome of many α-herpesviruses.
The gene, in actively dividing tissue culture cells,
It is known that it is not essential for virus growth. However, TK synthesized by α-herpesvirus seems to promote the growth of the virus in non-dividing cells [Field, HJ and Wildy,
P. 1978. The pathogenicity of thymidine kinase-def
icient mutants of herpes simplex virus. J. Hyg. 81:
267-277.].

[0010] Recently, many studies have been conducted to attenuate the mutations in the TK gene. That is,
Human herpes simplex virus (H)
SV), Aujeszky's disease virus (pseudorabies viru)
s, PRV) [Kit, S., Kit, M. and Pirtle, EC 198
5. Attenuated properties of thymidine kinase-negat
ive deletion mutant of pseudorabies virus. Am. J.
Vet. Res. 46: 1359-1367., Tenser, RB, Ressel,
S., J., Fralish, FA and Jones, JC 1983.The rol
e of pseudorabies virus thymidine kinase expressio
n in tregeminalgenglion infection. J. Gen. Virol.
64: 1396-1373.], Bovine herpesvirus type 1 (BHV
-1) [Kit, S., Qavi, H., Gaines, JD, Billingsl
ey, P. and McConnell, S. 1985. Thymidine kinase-ne
gative bovine herpesvirus type1 mutant is stable a
nd highly attenuated in calves. Arch. Virol. 86: 5
3-83.], And equine herpesvirus type 1 (EHV-
1) [Slater, JD, Gibson, JS and Field, HJ 1
993. Pathogenicity of a thymidine kinase-deficient
mutant of equine herpesvirus 1 in mice and specif
ic pathogen-free foals. J. Gen. Virol 74: 819-82
3.] TK gene-deficient mutants are significantly attenuated in their natural host or mouse, and reactivation after latently appearing in nerve cells is unlikely to occur [Efstathiou, S., Kemp, S., Darby, G.and Minson,
AC 1989. The role of herpes simplex virus type 1
thymidinekinase in pathogenesis. J. Gen. Virol. 70:
869-879.]. Such TK gene mutants have been proposed as the basis for vaccine production.

In Japanese Patent Publication No. 6-511392, there is FH
Disclosed are FHV-1 mutants having mutations in the sections of open reading frames 1-6 located to the right of the Us region of the V genome. However,
This FHV-1 mutant may still be insufficiently attenuated by the virus, and may become ill when inoculated with the virus. Furthermore, the strain described in the above-mentioned publication needs to be prepared by intentionally inserting a clear biological marker into the attenuated strain, and the operation is complicated. Further, in the technique described in the above publication, when a heterologous gene is inserted into the above section and expressed, a foreign promoter has to be inserted upstream of the foreign gene, and the operation is more complicated. When an exogenous promoter is used, the expression of the inserted gene may be abnormal, and there is a problem in safety as a recombinant multivalent vaccine.

[0012] Although the above-mentioned publication discloses recombinant FHV-1 in which a heterologous DNA encoding an antigen of a feline pathogen is inserted, the antigen is in vitro and in vivo.
It is not clear whether or not it is expressed in E. coli, and its actual usefulness as a recombinant multivalent vaccine when a recombinant FHV-1 is inoculated into a cat is unknown.

Nunberg, J. et al. H. (Nunberg,
JK, Wright, DK, Cole GE, Petrovskis, EA, P
ost, LE, Compton, T. and Gilbert, JH 1989. Ide
ntification of the thymidine kinase gene of feline
herpesvirus: Use of degenerate oligonucleotides
in the polymerase chain reaction to isolate herpes
virus gene homologus. J. Virol. 63: 3240-324
9.), EcoRV-Hind of TK gene of FHV-1
We have reported recombinant feline herpesvirus type 1 in which the III or EcoRI-HindIII site has been deleted. Nunberg et al., In the aforementioned reference, first used FHV-1 strain UC-D isolated in the United States for FHV-1.
The TK gene of V-1 was identified. The TK gene has 3
It was 1029 bp (base pairs) in length encoding a putative TK protein composed of 43 amino acids. They found that EcoRV-HindI in the TK gene
The II fragments deficient, TK ^- recombinant FH showing the phenotype of
V-1 was created.

On the other hand, Cole et al. (Cole, GE, Stac
y-Phipps, S. and Nunberg, JH 1990. Recombinant f
eline herpesvirus expressing feline leukemia virus
envelope and gag proteins. J. Virol. 64: 4930-493
7.), EcoRV- of TK gene of FHV-1
HindIII or EcoRI-HindIII
It has been reported that the expression cassettes of the genes encoding the feline leukemia virus envelope and gag protein are inserted into the region where the site was deleted, and those proteins are expressed in cultured cells. Here, an expression cassette using the immediate early promoter of human cytomegalovirus is inserted into the TK gene of FHV-1.

In addition to the above, [Kit, S., Kit, M. and P
irtle, EC 1985. Attenuated properties of thymidi
ne kinase-negative deletion mutant of pseudorabies
virus. Am. J. Vet. Res. 46: 1359-1367., Kit, S.,
Kit, M. and McConnell, S. 1986. Intramuscular and
intravaginal vaccination of pregnant cows withthym
idine kinase-negative, temperature-resistant infec
tious bovine rhinotracheitis virus (bovine herpesvi
rus 1). Vaccine. 4: 55-61.], the inactivation (TK-) of the Aujeszky's disease virus of the α-herpesvirus subfamily and bovine herpesvirus type 1 (TK ⁻ ) is associated with the virus pathogenicity and reactivation. It was shown to reduce activation. TK
Mutants or recombinant deletions of genes have been proposed as effective agents for immune defense or as viral vectors for recombinant vaccines expressing heterologous immune antigens.

However, according to the technique of Cole et al., The portions of the feline leukemia virus envelope and the portion into which the expression cassette of the gene encoding the gag protein is inserted are restricted to the restriction enzymes EcoRV, HindIII, and EcoR.
Since it has a cleavage end due to I or the like, the foreign gene to be inserted must be matched with the cleavage end or reconstructed into a sequence at the insertable end, which is extremely complicated operation. In addition, since a human cytomegalovirus-derived promoter is used for the expression of the inserted gene, appropriate expression of the inserted gene may cause abnormalities, which poses a problem in safety as a recombinant multivalent vaccine. is there. Furthermore, it is not demonstrated in the above-mentioned literature that the recombinant virus has properties as a practical vaccine, that is, the ability to grow in vivo in cats, immunogenicity and weak toxicity (safety). However, it was doubtful whether it would be useful as a vaccine.

Also, Wardley, RC et al.
Infection experiments on cats have been reported using the recombinant virus (in which the expression cassette of the gene encoding the feline leukemia virus envelope and gag protein is inserted into the TK gene of FHV-1) by W. et al.
y, RC et al, 1992, J. Gen. Virol. 73: 1811-181
8). However, with this technique, there is no detailed report on the pathogenicity and growth of TK-deficient FHV-1 strains in cats. Furthermore, since the baculovirus-expressed protein was administered together with the bivalent vaccine to achieve 100% protection, the recombinant FHV-1 strain was not administered alone. Therefore, the recombinant FHV-1 strain had insufficient immunogenicity.

[0018]

Therefore, in the above-mentioned various conventional techniques, the recombinant virus has insufficient characteristics as a live vaccine, or it involves complicated operations for production, and further, The fact is that practical use as a live vaccine has not been disclosed, and there has been no recombinant virus that satisfies all these characteristics.

Therefore, the object of the present invention is to achieve stable and sufficient attenuation by gene manipulation, and to sufficiently maintain the ability to grow in cultured cells in vitro and the ability to grow cats in vivo. It is an object of the present invention to provide a recombinant feline herpesvirus type 1 which has excellent immunogenicity and clearly has biological and genetic markers that can be distinguished from field strains, and a vaccine containing the same. Another object of the present invention is to achieve stable and sufficient attenuation by gene manipulation, to sufficiently maintain the ability to grow in cultured cells in vitro and the ability to grow in vivo in cats, and FHV-1. Has excellent immunogenicity, clearly has biological and genetic markers that can be distinguished from field strains, and the protein that is a product of a foreign gene such as a heterologous immunogen is stably and appropriately expressed, and It is an object of the present invention to provide a recombinant feline herpesvirus type 1 which does not need to intentionally insert a foreign heterologous promoter for its expression, and a vaccine containing the same. Other objects of the present invention will be apparent from the following description.

[0020]

The present inventors repeated various studies in order to solve the problems of the above-mentioned conventional techniques and achieve the object of the present invention. As a result, it has been found that the above-mentioned object can be achieved by the following constitution. (1) Alphabes of Herpesviridae
Recombinant feline herpesvirus type 1 characterized in that at least the EcoRV-SmaI site of the gene encoding thymidine kinase in the genome of feline herpesvirus type 1 belonging to the subfamily rpesvirinae is deleted. (2) Alphabes of Herpesviridae
A recombinant feline herpesvirus type 1 characterized in that an exogenous gene is inserted into the SmaI site in the gene encoding thymidine kinase in the genome of feline herpesvirus type 1 belonging to the subfamily rpesvirinae. (3) At least EcoRV-SmaI of the gene encoding the thymidine kinase of herpesvirus type 1
The recombinant feline herpesvirus type 1 according to (2) above, wherein the site is defective and a foreign gene is inserted into the SmaI site.

(4) The gene encoding the thymidine kinase has a DNA sequence that can hybridize at least with the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and also has an EcoRV cleavage site and a SmaI cleavage site. The recombinant feline herpesvirus type 1 according to (1) or (2) above, characterized in that (5) The gene encoding the thymidine kinase is
Having a DNA sequence capable of hybridizing at least with the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and SEQ ID NO: 1
Recombinant feline herpesvirus type 1 according to (4) above, which has an EcoRV cleavage site at the 618th position and an SmaI cleavage site at the 1074th position of SEQ ID NO: 1. (6) The feline herpesvirus type 1 is C7301
The recombinant feline herpesvirus type 1 according to (1) or (2) above, which is a strain. (7) The above-mentioned (2), wherein the foreign gene is at least one gene encoding a foreign immunogen.
Alternatively, the recombinant feline herpesvirus type 1 according to (3).

(8) The gene encoding the foreign immunogen comprises at least one pathogen selected from feline calicivirus, feline leukemia virus, feline parvovirus, feline immunodeficiency virus, feline coronavirus, necorotavirus, and feline chlamydia. The recombinant feline herpesvirus type 1 according to (7) above, which is at least one gene encoding a protein serving as an immunogen against Escherichia coli. (9) The recombinant feline herpesvirus type 1 according to (8) above, wherein the gene encoding the foreign immunogen is a gene encoding the capsid protein of feline calicivirus. (10) The above-mentioned (1) or (2), wherein a foreign gene capable of expressing an immunogen against feline calicivirus is inserted into the genome of the feline herpesvirus type 1 so that it can be expressed. Recombinant feline herpesvirus type 1. (11) The recombinant feline herpesvirus type 1 according to (2) or (3) above, wherein the foreign gene is at least one gene encoding a pharmaceutical or diagnostic polypeptide. (12) The recombinant feline herpesvirus type 1 according to (11) above, wherein the medicinal or diagnostic polypeptide is a lymphokine, interleukin, interferon, or cytokine. (13) The foreign gene is feline herpesvirus 1
The recombinant feline herpesvirus type 1 according to (2) or (3) above, which is expressed by a promoter derived from the type.

(14) The recombinant feline herpesvirus type 1 DNA according to any one of (1) to (13) above
Cultured cells transfected with. (15) Culture in which the transfer vector necessary for producing the recombinant feline herpesvirus type 1 according to any one of (1) to (13) above and the feline herpesvirus type 1 genomic DNA are cotransfected. cell. (16) A cell culture infected with the recombinant feline herpesvirus type 1 according to any one of (1) to (13) above. (17) A vaccine comprising at least the recombinant feline herpesvirus type 1 according to any one of (1) to (13) above. (18) A method for immunizing a cat against infectious diseases, which comprises administering the vaccine according to (17) above via the eye, nose and mouth of a cat, or by intramuscular, intravenous or subcutaneous injection.

In the present invention, at least EcoRV-Sm in the gene encoding thymidine kinase in the FHV-1 genome is used by genetic engineering (recombinant technology).
By deleting the aI site or inserting a foreign gene into the SmaI site, the in vitro virus growth ability can be maintained without mutating the gene controlling other growth characteristics and the gene encoding the immunogen. And, the cat was attenuated steadily and sufficiently. These recombinant viruses had a sufficient ability to proliferate in the body of cats and had excellent immunogenicity in the body of cats. Furthermore, by causing the above-mentioned recombination in the TK gene, it became possible to clearly contain biological and genetic markers as a vaccine strain and to distinguish it from a field strain having a TK ⁺ phenotype. . As mentioned above, TK
A relationship between loss of activity and attenuation in other α-herpesviruses has been suggested, but in vivo TK
The growth characteristics and attenuation of recombinant FHV-1 lacking the gene have not been reported in detail. In the present invention, the recombinant FHV-1 lacking the EcoRV-SmaI site in the TK gene was clearly demonstrated to be useful as a vaccine against cats.

The TK gene of the FHV-1 genome has a unique SmaI site, which is the foreign gene DN.
It is an effective site into which the A fragment can be inserted. Therefore, recombinant FHV-1 in which a foreign gene such as a gene encoding a foreign immunogen has been inserted into the SmaI site has sufficient attenuation, growth characteristics, and immunogenicity of FHV-1.
In addition to having biological and genetic markers, it is a safe and extremely useful recombinant multivalent live virus vaccine that can stably and sufficiently express foreign genes in cultured cells or host organisms. . In the vaccine of the present invention, the inserted foreign gene is stably expressed in cultured cells, and the recombinant virus as the vaccine is administered in vivo to the product of the foreign gene in the living body. The induction of neutralizing antibodies was clearly shown.

The present invention will be described in detail below. As FHV-1 that can be used in the present invention, any known strain can be used. Specifically, the thymidine kinase described in SEQ ID NO: 1 in the sequence listing (hereinafter, TK
FHV-1 having a gene sequence of TK capable of hybridizing with the gene sequence (also referred to as TK) (gene encoding TK; 269th to 1300th of SEQ ID NO: 1) and having a cleavage site by EcoRV and / or SmaI. Is preferred. Such strains include, for example, F
2 strains [Bittle, JL, and Rubic, WJ 1975. Immunoge
nic and protective effects of the F-2 strainof fel
ine viral rhinotracheitis virus. Am. J. Vet. Res.
36: 89-91.], A standard strain C27 strain [Crandell, R.
A. and Maurer, FD 1958. Isolation of a associated
with intranuclear inclusion bodies. Proc. Soc. Ex
p. Biol. Med. 97: 487-490.], C7301 and C7805 which are the isolates in Japan [Mochizuki, M., Kon.
ishi, S. and Ogata, M. 1977. Studies on cytopathog
enic viruses from cats with respiratory infection
s. III. Isolation and certain properties of feline
herpesviruses. Jpn. J. Vet. Sci. 39: 27-37.],
UC-D strain etc. can be mentioned. In this, C73
01 strain and UC-D strain are preferable, and more preferably C7.
301 strains can be used.

As shown in FIG. 1A, the FHV-1 genome has a length of about 126 kbp, a region of about 99 kbp represented by UL, and a region of about 27 kb represented by Us.
Comprised of p regions, the TK gene is reported to be located approximately in the center of the UL region, as described in Figure 1B. In addition, the present inventors have found that the TK gene of FHV-1 is located upstream of a gene homologous to gH [Med.
a, K., Kawaguchi, Y., Kamiya, N., Ono, M., Tohya,
Y., Kai, C. and Mikami, T. 1993. Identification an
d nucleotide sequenceof a gene in feline herpesvir
us type 1 homologous to the herpes simplexvirus ge
ne encoding the glycoprotein H. Arch. Virol. 132:
183-191.].

The recombinant feline herpesvirus of the present invention is
In one embodiment, at least EcoR in the TK gene
It is FHV-1 in which the V-SmaI site has been deleted, and other portions may have mutations such as deletion, substitution and insertion as long as the characteristics of the recombinant virus of the present invention are satisfied. Preferably, EcoRV- of the TK gene shown in SEQ ID NO: 1
It is preferable that 450 bp of SmaI site is deleted.

The recombinant feline herpesvirus of the present invention is
In another embodiment, it is FHV-1 in which a foreign gene is inserted into at least the SmaI site in the TK gene, and if the characteristics of the recombinant virus of the present invention are satisfied, other portions may be deleted, replaced, inserted, etc. It may have a mutation. Preferably, EcoRV- in the TK gene shown in SEQ ID NO: 1
The 450 bp fragment at the SmaI site was deleted, and
A foreign gene is inserted into the maI site. This results in better attenuation of FHV-1 and, when expressing a foreign gene, its expression is retained.

In the present invention, as shown in SEQ ID NO: 1, EcoR originally contained in the TK gene of FHV-1.
It may be a recombinant virus utilizing a cleavage site by V and / or SmaI, but may be artificially provided with such a cleavage site and a cleavage site by various other restriction enzymes by genetic engineering. However, recombinant viruses that utilize the original cleavage site for EcoRV and / or SmaI are preferred for operation.

When the TK gene is deleted as described above, the present invention includes the case where a slight DNA sequence is inserted into the deletion site due to the operation of gene recombination technology.

Here, the foreign gene is a DNA sequence different from the gene encoding the thymidine kinase, and its insertion prevents the proper expression of thymidine kinase. Such a DNA sequence may be derived from a virus, a prokaryotic cell, a eukaryotic cell, or a synthetic one. Examples of the foreign gene include a gene encoding a foreign immunogen, a gene encoding a pharmaceutical or diagnostic polypeptide or protein, and the like. Examples of a gene encoding a foreign immunogen include, for example, an immunogen against at least one pathogen of feline calicivirus, feline leukemia virus, feline parvovirus, feline immunodeficiency virus, feline coronavirus, feline chlamydia, and necrotavirus. Examples of the gene encoding the protein to be used include two or more of them. The gene encoding the foreign immunogen is preferably a gene encoding the capsid protein of feline calicivirus, a gene encoding the envelope and / or gag protein of feline immunodeficiency virus, and more preferably encoding the capsid protein of feline calicivirus. It is a gene that does.

Examples of the medicinal or diagnostic polypeptide or protein include lymphokines, interleukins such as feline IL-6, interferons, cytokines and the like.

As another aspect of the present invention, the above recombinant TK
There is a recombinant feline herpesvirus type 1 in which a foreign gene capable of expressing an immunogen against feline calicivirus is operably inserted into the gene-deficient feline herpesvirus type 1 genome. The foreign gene capable of expressing an immunogen against feline calicivirus is preferably a gene encoding a capsid protein. FH in which this gene can be inserted
The V-1 gene region may be any part as long as it is a site that can be expressed in a non-essential region for virus growth. Specific examples of the region include gene regions encoding gI and gE of FHV-1.

A foreign promoter may be inserted to express the foreign gene. Any known promoter may be used as such a promoter, and examples thereof include a retrovirus LTR promoter, an SV40 promoter, and a cytomegalovirus-derived promoter. In the present invention, an expression promoter derived from FHV-1 can be used by inserting it into the SmaI site of the TK gene shown in SEQ ID NO: 1, and it is not necessary to intentionally insert an exogenous promoter, which makes gene manipulation easier. Furthermore, FH
Since an expression promoter derived from V-1 can be used,
The expression of the foreign gene becomes more stable. When a gene is expressed by inserting an exogenous promoter during gene manipulation, the gene may not be stably expressed. Furthermore, when a recombinant multivalent vaccine using an exogenous promoter is inoculated into a host, the exogenous promoter is inserted into the host gene, and the gene is spread to the host of other species, which causes unexpected and serious adverse effects. May affect. This concern disappears if an FHV-1-derived expression promoter can be used in the present invention.

As described above, inoculation of the recombinant virus into which the gene encoding the foreign immunogen has been introduced into the cat's living body allows the gene encoding the foreign immunogen to be stably expressed in the living body. it can. Therefore, not only can an infectious immune defense response against FHV-1 be induced in a cat body, but an infectious immune defense response can also be induced against a specific pathogen having the foreign immunogen. As a result, even if these pathogens become infected after the virus inoculation,
You will not be affected and you will be able to protect completely and reliably. That is, the recombinant virus of the present invention is extremely effective as a multivalent vaccine.

As a method for producing the recombinant feline herpesvirus type 1 of the present invention, any known gene recombination technique can be used as long as it has the above constitution. As such a method, a transfer vector is prepared, co-transfected with viral DNA into cultured cells,
Co-transfection that causes homologous recombination (Co
-Transfection) method or a method of transfecting only a transfer vector into a cultured cell and then infecting it with a parent virus. Preferred is the co-transfection method.

As the cotransfection method, specifically, first, a genomic DNA fragment containing the TK gene of FHV-1 to be recombined is introduced into a vector such as a plasmid, cosmid, or phage and cloned. The vector is cleaved with various restriction enzymes such as SmaI and EcoRV, the TK gene is deleted using a known gene recombination technique, or the foreign gene is incorporated by a binding enzyme such as ligase to obtain the target transfer vector. Create. Here, as the vector that can be used, a known plasmid, cosmid, phage or the like can be used. Those having a multi-cloning site are preferable.

The above transfer vector and FHV-1
And the recombinant DNA grown by homologous recombination and culturing in the presence of thymidine arabinoside (hereinafter also referred to as araT). Grow in cells. In the culture containing araT, only the recombinant virus having the TK ⁻ phenotype can selectively grow. In the case of the present invention, the virus of the present invention can be easily selected by using araT without intentionally inserting a marker gene showing recombination such as β-galactosidase or analyzing the gene of the recombinant.

The method for producing a recombinant virus having a foreign gene can also be performed in the same manner as above. That is, as described above, a transfer vector in which a genomic DNA fragment of a cloning plasmid containing at least the TK gene of FHV-1 is cleaved with a restriction enzyme such as SmaI according to the insertion site, and one or more foreign genes are introduced into the site. Recombinant FHV-1 containing a foreign gene can be prepared in the same manner as described above. In addition, SmaI-
A recombinant FHV-1 containing a foreign gene can be prepared in the same manner as above by preparing a transfer vector in which one or more kinds of foreign genes have been introduced into the SmaI site of the above transfer vector lacking the EcoRV fragment.
In the present invention, S having a unique site for introducing a foreign gene
The ideal characteristic of the maI site is that the cut surface has blunt ends. A foreign gene can be introduced more easily than when it has a sticky end. The ends cleaved by many restriction enzymes are specific, and it is necessary to manipulate the terminal sequence of the inserted gene so that it can bind to the ends, which is extremely complicated. It can be said that setting the SmaI site as the insertion site is extremely ideal. The recombinant virus obtained above is analyzed for the nucleotide sequence of a gene, analysis by creating a restriction enzyme map, or hybridization and PC.
By performing analysis using the analysis technique based on the R method, it is possible to confirm accurate deletion or insertion.

In the present invention, a method for producing a vaccine containing the above recombinant virus is as follows. The above recombinant virus is grown in its cultured cells, its cell culture or cultured cells are separated, and the The recombinant virus can be recovered and used as a vaccine by adding an additive to the suspension or freeze-dried product, if necessary. Examples of the additives include carbohydrates (sorbitol, mannitol, starch, sucrose, glucose, dextran, etc.), stabilizers, proteins such as albumin and casein,
Bovine serum, skim milk, buffer such as phosphate buffer, aluminum hydroxide, aluminum phosphate, aluminum oxide,
Examples include oil emulsions, saponins, arbitrary adjuvants such as vitamins, etc., or two or more thereof.

In the present invention, the dose of recombinant virus is appropriately set depending on the age, weight, administration method of the cat and the type of the inserted foreign gene. A specific guideline is 10 ³ to 10 ⁷ PFU / one cat. When the vaccine of the present invention is administered to cats for immunization,
It can be carried out via the eye, nose and mouth of the cat or by intramuscular, intravenous, subcutaneous or intraperitoneal injection. When the recombinant vaccine of the present invention is involved in respiratory tract infection such as rhinotracheitis, the eye,
It is preferably administered via the nose and mouth. As a result, immunity is locally imparted to the mucosal epithelium of the eyes, nose and mouth, which is a natural route of infection, at an early stage, and the onset of a field virulent pathogen can be immediately protected.

The present invention also includes a recombinant multivalent vaccine, a pharmaceutical preparation, or a diagnostic preparation, which is formed by expressing a product of a foreign gene by the above recombinant virus.
In order to express the product of the foreign gene, it is necessary to use cultured cells transfected with the above-mentioned recombinant feline herpesvirus type 1 DNA of the present invention or to produce the above-mentioned recombinant feline herpesvirus type 1 of the present invention. Cultured cells co-transfected with a transfer vector and feline herpesvirus type 1 genomic DNA can be used. In addition, cell cultures infected with the above-mentioned recombinant feline herpesvirus type 1 of the present invention can also be used in the above various formulations. Here, the cell culture includes cultured cells, culture supernatant thereof, and the like. Further, by using the above-mentioned recombinant virus or the above foreign protein, an antiserum induced by a predetermined method, a polyclonal antibody or a monoclonal antibody is used to treat or diagnose a cat infected with the pathogen, or an anti-idiotypic antibody. Can be manufactured.

As an animal for which the recombinant virus and vaccine of the present invention can be used, any animal can be used so long as the virus can be propagated.
Preferably used for feline species. More preferably, it is used for so-called domestic animals or pet cats.

[0045]

The present invention will be described in more detail with reference to the following Examples, which, however, do not limit the present invention.

Example 1 Virus, Cell, and Culture Solution FHV-1 strain C7301 isolated from a cat with respiratory disease in Japan in 1973 [Mochizuki,
M., Konishi, S. and Ogata, M. 1977. Studies on cyto
pathogenic viruses from cats with respiratory infe
ctions. III. Isolation and certain properties of f
eline herpesviruses. Jpn. J. Vet. Sci. 39: 27-37.
] Was used as the parent strain for the construction of recombinant FHV-1. The parent virus and recombinant virus were grown in CRFK cells (Crandell feline kidney cell).
The culture medium of the cells was DMEM (Dulb supplemented with 8% inactivated fetal calf serum (FCS) and antibiotics).
ecco's modified Eagle's medium) (manufactured by Sigma Chemical Co., Missouri, USA) was used. After infection, the cells are FC
It was maintained in the same culture medium as described above except that S was removed (maintenance culture medium).

Analysis of the sequence of the TK gene of the C7301 strain of FHV-1 The present inventors have previously reported that the 6.6 kilobase pair (kb
p) EcoRI fragment was cloned, mapped and the 3746 bp SacI fragment within the fragment was sequenced [Maeda, K., Kawaguchi, Y., Kamiya,
N., Ono, M., Tohya, Y., Kai, C. and Mikami, T. 199
3. Identification and nucleotide sequence of a gen
e in feline herpesvirus type 1 homologous to the h
erpes simplex virus gene encoding the glycoprotein
H. Arch. Virol. 132: 183-191.]. In this document, it is reported that the TK gene of the C7301 strain of FHV-1 is located upstream of a gene homologous to gH. In order to sequence the TK gene of the strain, 1.8 kbp of BamHI- was added to each of the multiple cloning sites of plasmid Bluescript SK +.
HindIII fragment, 0.5 kbp BamHI-Hi
ndIII fragment and 0.8 kbp HindIII-Ec
The oRI fragment was subcloned. DNA sequencing was performed by ABI (Applied Biosystems, Foster City Ca
(Nada) Dye primer cycle sequencing method was used, and analysis was performed using an ABI 370A automatic sequencer.

The entire sequence of the TK gene of FHV-1 has been determined in the American FHV-1 strain UC-D [Nunberg, JK, Wright, DK, Cole GE, Petrovsk.
is, EA, Post, LE, Compton, T. and Gilbert, J.
H. 1989. Identification of the thymidine kinase ge
ne of feline herpesvirus: Use of degenerate oligo
nucleotides in the polymerase chain reaction to is
olate herpesvirus genehomologs. J. Virol. 63: 324
0-3249.]. Here, the sequence of the TK gene of the C7301 strain, which is an isolated standard strain in Japan, was determined as described above. There was no difference in the nucleotide sequence of the TK gene between the UC-D strain and the C7301 strain, which showed remarkable homology of the TK gene of FHV-1.

Recombinant FHV-1 lacking the TK gene
(Construction of transfer vector) Plasmid pUC1
In Fig. 9, a 19 kbp SalI fragment of the FHV-1C7301 strain (A fragment in Fig. 1A) [Rota, PR, Maes, RK
and Ruyechan, WT 1986. Physical characterizatio
n of the genome of feline herpesvirus-1. Virolog
y. 154: 168-179.] containing the TK gene
4. cloning the BamHI-SalI fragment of p.
The 5 kbp fragment (Fig. 2B) was mapped. The plasmid was named pfTK-SB and was used for the construction of transfer vector. pfTK-SB is E
3.3kbp EcoR digested with coRV and SalI
The V-SalI fragment was separated by agarose gel electrophoresis. The 3.3 kbp EcoRV-SalI fragment is an Eco plasmid of plasmid Bluescript (pBluescript) SK-.
Subcloned into RV-SalI site, pfTK
(Ev-Sa). Furthermore, pfTK-SB is Bam
1.7 kbp Ba separated by cutting with HI and SmaI
The mHI-SmaI fragment was transformed into Ba of pfTK (Ev-Sa).
Subcloned into the mHI-SmaI site. The plasmid was designated pfTK-SBΔSE (FIG. 1C),
It was used as a transfer vector.

(Creation and selection of recombinant FHV-1) C73
Viral DNA and transfer vector pfTK-SBΔSE extracted from CRFK-infected cells of strain 01
Is a calcium phosphate precipitation method [Graham, FL and van de
r Eb, AJ 1973. A new technique for the assay of
infectivity of human adenovirus 5 DNA. Virology. 5
2: 456-467.] Into CRFK cells. Recombinant viruses of interest were selected from among the propagated viruses in the presence of 100 μg / ml thymidine arabinoside (araT). The selected viruses were plaque purified 3 times as described below.

In order to produce a recombinant virus lacking the TK gene, the C7301 viral DNA and the transfer vector pfTK-SBΔS were introduced into CRFK cells.
E and E were co-transfected to obtain infected cells.
The virus lacking the TK gene was selected from the infected cells by adding araT, which inhibits the replication of the virus exhibiting the TK ⁺ phenotype, to the culture medium [Schinazi, RF, Willia
ms, CC, Fritz, ME and Nahmias, AJ 1981. The
effect of pyrimidine and purine nucleosides on the
feline (herpes) rhinotracheitis virus in feline to
ngue cells.pp681-682. In: The human herpesviruse
s (Nahmias, AJ, Dowdle, WR and Schinazi, RF e
ds), Elsevier / North-Holland Publishing Co., New Yo
rk.]. One of these araT resistant viruses was used to plaque purify three times and is referred to as C7301dlTK. To test whether C7301dlTK has a planned deletion in the TK gene, DNA of recombinant (C7301dlTK) and parent strain (C7301)
Was extracted with T by Southern blotting and PCR.
The K gene was analyzed.

(Extraction of viral DNA from infected cells)
C7301 at a multiplicity of infection (MOI) of 3 plaque forming units (PFU) per cell in monolayer cultures of CRFK cells.
The strain or recombinant virus (C7301dlTK) was infected. After the cytopathic effect (CPEs) has progressed sufficiently, the infected cells are washed once with phosphate buffer (PBS),
DNA extraction buffer [1% SDS, 0.1M Tris H
Cl (pH 9.0), 0.1 M NaCl and 1 mM EDTA] and then treated with 1 mg / ml pronase E overnight at 37 ° C. The viral DNA is [Hira
i, K., Nakajima, K., Ikuta, K., Kirisawa, R., Kawa
kami, Y., Mikami, T. and Kato, S. 1986. Similariti
es and dissimilarities in the structure and expres
sion of viral genomes of various virus strainsimmu
nologically related to marek's desease virus. Arc
h. Virol. 89: 113-130.], phenol extraction and ethanol precipitation were performed, and the mixture was dialyzed against distilled water three times.

(Southern Blot Analysis) The DNA extracted from the virus-infected cells as described above was digested with SalI or EcoRI, and then subjected to 0.5% agarose gel electrophoresis to separate the cut fragments into nylon membrane ( Biodyne, Pall Biosupport, New York, U
SA) according to the product description. 5.5 kbp BamHI-containing the region encoding TK (FIG. 1B)
The SalI fragment was subjected to [ ³² P] α-C using a nick translation kit (Berlinker, Germany).
It was labeled with TP and used as a probe. Hybridization is performed using the hybridization solution (5
0% formamide, 0.6 M NaCl, 0.2 M Tris HCl (pH 8.0), 0.02 M EDTA,
0.5% SDS, and 100 μg / ml denatured salmon sperm DNA) at 42 ° C. overnight. The membrane was SSC containing 0.3% SDS (0.3 M NaC).
1, 0.03 M sodium citrate dihydrate) × 0.
Washed 3 times at 42 ° C. for 30 minutes. The film was then exposed to X-ray film at -70 ° C.

The film developed after exposure is shown in FIG.
As shown in FIG. 2A, the parental C73 containing the TK gene.
The size of the 19 kbp SalI fragment observed in 01 DNA (lane 1) was slightly smaller for C7301dlTK (lane 2). On the other hand, as shown in FIG. 2B, for the large size fragment by the EcoRI fragment, the parent strain C7301 had a 6.6 kbp fragment (lane 1), while the C7301dlTK had a 5.7 kbp fragment (lane 2). ). The size difference seen between them is due to the 438 bp deletion that occurred at the EcoRV and SmaI sites and the EcoRI cut in 12 nucleotides from the multiple cloning site of pBluescript KS- inserted between the EcoRV and SmaI cuts. A new 424b caused by the addition of parts
p (C in FIG. 1). Although the 424 bp fragment was too small to be detected in this analysis, the 3.6 kbp fragment of both strains located immediately upstream of the TK gene was detected as expected (FIG. 2B).

The decrease in the sequence of C7301dlTK is
It was also confirmed by the R method. The result is shown in FIG. Sequences for the primers are based on our work and FHV-
1 known sequences of the TK gene [Nunberg, JK, Wright,
DK, Cole GE, Petrovskis, EA, Post, LE, Co
mpton, T. and Gilbert, JH 1989. Identification o
f the thymidine kinase gene of feline herpesvirus
: Use of degenerate oligonucleotides in the polym
erase chain reaction to isolate herpesvirus gene h
omologs. J. Virol. 63: 3240-3249.].
The sequences of the primers used were 11 'to 30'5'-GAACCATCCCCGTTCAGAAT-3'(upstream of TK) and 932 to 913 in the TK gene of FHV-1 capable of amplifying the target DNA fragment of 922 bp. 5'-CATTTCATCAGGGGTTCCTTC-3 '(downstream of TK).

PCR [Saiki, RK, Gelfand, DH, St
offel, S., Scharf, SJ, Higuchi, R., Horn, GT,
Mullis, KB and Erlich, HA 1988. Primer-directe
d intentionally amplification of DNA with a thermostab
le DNA polymerase. Science. 239: 487-491.] was carried out in a 0.5 ml microcentrifuge tube with a total volume of 50 μl. 1
μg of each viral DNA (C7301 strain or C73
01 dlTK strain), 1 μM of each primer, 200 μM of each dATP, dCTP, dGTP and dTT.
P, 10 mM Tris-HCl (pH 8.3), 50 m
M KCl, 1.5 mM MgCl ₂ , 0.001% gelatin, and 1 U of AmpliTaq DNA polymerase (Perkin Elmer, Connecticut, USA) were added to the reaction mixture. All reaction samples were coated with mineral oil (Sigma Chemical Co.). Then 25 cycles of denaturation (94 ° C; 1
Min), annealing (60 ° C; 1 min), extension (72 ° C;
2 minutes) was done. After amplification, the PCR products were analyzed by electrophoresis on 2% agarose.

The results of agarose gel electrophoresis analysis of the above PCR products are shown in FIG. Lane 1 is C7301
Lane 2, which is a strain, is an electrophoretic image of DNA extracted from infected cells of the C7301dlTK strain. Looking at the size of these bands, they are the same as above, 438bp
It can be seen that there is a deficiency. When the cotransfection method was performed using the transfer vector pfTK-SBΔSE, 450 bp in the TK gene of C7301 was obtained.
Was deleted, and a 12 bp nucleotide was inserted at the deleted site. Here, the inserted 12 bp nucleotide is a sequence derived from the multi-cloning site of -GAATTCCTGCAG-, pBluescript KS-, from the upstream side of the TK gene. These data indicate that the planned deletion (recombination) was introduced into the TK gene of the parental virus, and a loss of TK activity could be expected.

(Immunoblot Analysis and Indirect Fluorescent Antibody Method (IFA)) Furthermore, in order to confirm that no mutation was observed in the proteins of other immune antigens in C7301dlTK, immunoblot was performed using cat sera infected with C7301 strain. Analysis, and gp143 / 108, gp113,
Monoclonal antibody 22F4, which recognizes gp60,
Indirect fluorescent antibody analysis was performed using 17C11 and 41G4 [Horimoto, T., Limcumpao, JA, Tohya, Y, Tak.
ahashi, E. and Mikami, T. 1990. Feline herpesvirus
type 1 glycoproteins eliciting virus neutralizing
and hemagglutination-inhibiting antibodies. Arch.
Virol. 111: 127-132.]. Immunoblot analysis and indirect fluorescent antibody analysis (IFA) are carried out in known literature, for example, [Horimoto, T., Limcumpao, JA, Xuan, X., Ono,
M., Maeda, K., Kawaguchi, Y., Kai, C., Takahashi,
E., and Mikami, T. 1992. Heterogeneity of feline h
erpesvirus type 1 strains. Arch. Virol. 126: 283-2
92., Limcumpao, JA, Horimoto, T., Xuan, X., Taka
hashi, E. and Mikami, T. 1990. Immunological relat
ionship between feline herpesvirus type 1 (FHV-1) a
nd canine herpesvirus (CHV) as revealed by polyvalen
t and monoclonal antibodies. Arch. Virol. 111: 165-
176.] was used. These monoclonal antibodies bind antigenic determinants for virus neutralization in FHV-1. C7301 and C7301dlT
No difference was found in their reactivity with K.

(C7301dl which is a recombinant FHV-1
Proliferation property of TK in vitro) Next, C7301dl
The growth properties of TK and parent strain C7301 in CRFK cells were compared. CRFK cells were inoculated with each virus,
The amount of intracellular and extracellular virus produced at 0, 6, 12, 24, 48 and 72 hours post infection was determined. As a specific method, monolayer cultures of CRFK cells were prepared on the wells of 6-well plates and inoculated with a virus sample at an MOI of 3 PFU / cell. After 90 minutes of adsorption, the monolayer culture was washed 3 times with DMEM and incubated with 2 ml of maintenance medium per well at 37 ° C. After infection, infected cells were scraped together with the culture medium at various intervals by a rubber policeman and centrifuged at low speed to precipitate the infected cells. The culture supernatant at that time was used as an extracellular sample. Infected cells, which are the precipitate, were
The cells were washed once, suspended in 2 ml of the maintenance culture solution, subjected to freeze-thaw treatment three times, and further centrifuged at 4000 rpm for 10 minutes to remove cell debris. The resulting supernatant was used as an intracellular sample. Intracellular and extracellular samples were measured infectious titer of the virus as _{TCID 50 (log TCID 50 per50μl)} .

FIGS. 4A and 4B show the time-dependent changes in the growth of intracellular virus and extracellular virus, respectively. In FIG.
○ and □ represent C7301 strains, ● and ■ represent C7301
Represents the dlTK strain. A represents the virus titer of the intracellular virus, and B represents the virus titer of the extracellular virus. The intracellular or extracellular virus titers recovered from CRFK cells infected with parental C7301 or C7301dlTK increased rapidly, 24 hours after infection and 4 hours, respectively.
The maximum was reached in 8 hours. The growth patterns of both viruses were almost the same inside and outside the cells. When a TK gene-deficient recombinant virus was prepared in the F2 strain of FHV-1 in the same manner as above, the same favorable results as above were obtained.

Forced infection experiment of cat using parent strain and recombinant C7301dlTK strain As a test animal, a cat (8) of about 3 months old was used. The cats of these experimental animals have not been vaccinated, do not show respiratory symptoms, have negative neutralizing antibody titers such as FHV-1, FCV, or ELIZA antibody titer, and further have no virus isolated. Also, breed for 1 week or more with each breeding gauge, wait for the body temperature etc. to stabilize, and then perform the experiment.

The viruses used in the infection experiment are the parent strain C7301 strain and the above-mentioned C7301dlTK strain. These viruses were forcibly infected into the orbits, nasal cavities, and oral cavity of 4 cats in a total amount of 10 ⁶ PFU, and observed for about 3 weeks. Cats infected with C7301 strains (C-1)-(C
-4), and cats infected with the C7301dlTK strain were designated (T-1) to (T-4).

Observation items and results of observation [Body weight and body temperature (rectal)]: The body weight and body temperature (rectal) of each individual were measured and observed daily. The results are shown in FIG. The results of the body temperature are shown in A of FIG. 5. The body temperature of the cat inoculated with the C7301 strain is significantly increased 3 days after the inoculation (0 day), whereas the cat inoculated with the C7301dlTK strain is shown. Showed almost no significant change in body temperature. As shown in FIG. 5B, the cats inoculated with the C7301dlTK strain showed an almost constant increase in body weight, whereas the cats inoculated with the C7301 strain had a weight gain of approximately 9 days after the inoculation. It did not increase, but tended to decrease.

[Neutralizing antibody titer]: The serum of each cat was collected every 3 days, and the neutralizing antibody titer in the serum was measured.
The transition of the antibody was observed. FIG. 6 shows the result. FIG.
As shown in, C730
It can be seen that the neutralizing antibody titer against FHV-1 is also sufficiently increased in the 1dlTK strain.

[Separation of virus]: In order to observe the excretion of virus, a wiping solution was collected from the orbit, nasal cavity and oral cavity of each cat over time, and based on the plaque forming unit (PFU) in CRFK cells. The amount of isolated virus was measured.
From 7 days before virus inoculation, it was sampled and measured every day or at least every 3 days. The result is shown in FIG. As shown in FIG. 7, the amount of virus isolated from the eyes, nose, and mouth of the C7301dlTK-inoculated cat was almost equal to that of the C7301 strain-inoculated cat. Also, in the above,
A DNA fragment was amplified from the TK gene in the genome of the virus isolated from each of the cats T-1 to T-4 and C-1 to C-4 by the PCR method using the above primer pair, The same was analyzed. FIG. 8 shows the result. From the results of FIG. 8, the virus isolated from the cats T-1 to T-4 inoculated with C7301dlTK had the TK gene deficiency as in the results of FIG. This suggests that the inoculated C7301dlTK strain retains the same deficiency as the inoculated virus even after sufficiently proliferating in the cat body, and using this method, it may be easily distinguished from the field strain C7301 having the TK gene. Clearly suggests.

[Clinical symptoms]: Clinical symptoms of cats inoculated with each virus were observed after inoculation. Clinical symptoms include dehydration, appetite, energy, runny nose, sneezing, salivation,
Conjunctivitis and respiratory mode were observed daily and scored based on the evaluation criteria shown in Table 1 below. The results are shown in Tables 2 to 5 below.

[0067]

[Table 1]

[0068]

[Table 2]

[0069]

[Table 3]

[0070]

[Table 4]

[0071]

[Table 5]

From the results shown in Tables 2 to 5, cats C-1 to C-4 inoculated with the C7301 strain have a significantly high clinical evaluation number (total points from 1 to 21 days; 65 to 65 days).
131), the cats T-1 to T-4 inoculated with C7301dlTK had a considerably small clinical evaluation number (total points from 1 to 21 days; 2-1).
8) It can be seen that there are almost no clinical symptoms. From the above results, it can be seen that the C7301dlTK strain is sufficiently attenuated, since there is almost no change in body weight and body temperature even after inoculation with C7301dlTK and almost no clinical symptoms. Furthermore, since the neutralizing antibody titer in the cat body was sufficiently increased in the C7301dlTK strain, and the virus isolation amount was almost the same as that in the C7301 strain in all of the eyes, nose, and mouth, the C7301dlTK strain was confirmed. It can be seen that the strain has sufficient immunogenicity and retains the ability to grow in the cat body.
Also, the virus T isolated from each inoculated cat
By analyzing the K gene, the C7301dlTK strain and the field strain can be clearly distinguished. Therefore, the C7301dlTK strain according to the present invention satisfied all of its attenuated virulence, immunogenicity, proliferative ability in cats, and retention of genetic markers.

Example 2 Using the same cat as in Example 1 above, a forced infection experiment was conducted according to the schedule shown in FIG. That is, group A has 3
Attenuated C7301dlTK strain was intramuscularly administered twice to rats in an amount of 10 ⁵ PFU / 1 ml at an interval of 3 weeks twice, and after 4 weeks, 10 ^{6 of the} strongly virulent C7301 strain was administered.
An amount of PFU / 1 ml was administered from the nose, mouth and eye to inoculate. Group B was the same as Group A above except that the culture supernatant of CRFK cells (not virally infected) was administered intramuscularly to the three cats instead of the C7301dlTK strain in Group A above. I inoculated.
In group C, two cats were inoculated twice with the C7301dlTK strain as in the case of group A above, and then the C
The 7301 strain was not inoculated, and instead, the culture supernatant of CRFK cells (not infected with virus) was administered through the nose, mouth and eye.

In the above three groups, body temperature (rectal), body weight, neutralizing antibody titer, and clinical symptoms were evaluated in the same manner as in Example 1 above. [Body temperature (rectal)] The results of body temperature are shown in FIG. C7 in advance
Group B, which is not vaccinated with the 301dlTK strain, had a body temperature of 40 when challenged with the C7301 strain.
The temperature rose above ℃, and the body temperature continued to rise for some time after that. In contrast, in Group A, which was previously inoculated with the C7301dlTK strain, the body temperature temporarily increased slightly when the C7301 strain was inoculated, but the degree of increase was lower than that in Group B above, and immediately thereafter, the body temperature in the steady state was increased. Returned to. On the other hand, in group C, C730
Even if the 1dlTK strain was inoculated twice, no increase in body temperature was observed.

[Weight] FIG. 11 shows changes in body weight. In both groups A and C, C7301dlTK
No abnormal changes in body weight due to inoculation of the strain were found. Furthermore, when the C7301 strain was inoculated, the weight was significantly reduced in Group B. On the other hand, in advance C
Group A vaccinated with the 7301dlTK strain showed little change in body weight.

[Neutralizing antibody titer] C73 of FHV-1 for attack
Before inoculating 01 strain, the serum of each cat was collected once a week and at least every 3 days after inoculating the strain, and the neutralizing antibody titer against FHV-1 in the serum was measured. The transition was observed. FIG. 12 shows the result. The antibody titer after inoculation with the C7301 strain was significantly increased after about 3 days in the group A inoculated with the C7301dlTK strain in advance. In contrast, Grape B, which had not been previously inoculated with the C7301dlTK strain, had an immune response slower than that of Group A, and C73
About 9 days after inoculation with 01 strain, the antibody titer increased relatively slowly. Therefore, Group A shows that immunization was sufficiently performed by inoculating the C7301dlTK strain twice intramuscularly, showing that the C7301dlTK strain is effective as an immunogen against FHV-1 in vivo. It was

[Clinical symptoms]: In the groups A and B, the clinical symptoms of the cats after challenge with the C7301 strain were observed for 4 weeks after the inoculation of the strains. Clinical symptoms include dehydration, appetite, energy, runny nose, sneezing, salivation,
Conjunctivitis, lacrimation, breathing mode, observe them daily,
It was scored based on the evaluation criteria shown in Table 1 above. The results are shown in Tables 6 to 8 below. Here, the cats of group A are (TC-1) to (TC-3), and the cats of group B are (CC-1) to (CC-3).

[0078]

[Table 6]

[0079]

[Table 7]

[0080]

[Table 8]

From the results of Tables 6 to 8, C7301d is previously set.
The cats inoculated with the 1TK strain had a significantly low clinical evaluation number (total points from 1 to 28 days; 7-1.
7) It can be seen that there are almost no clinical symptoms. In contrast, cats that have not been previously inoculated with C7301dlTK
The number of clinical evaluation is quite high (total point from 1 to 28 days; 63 to 86), and it is understood that clinical symptoms are remarkable. The cats in Group C were all 0.

Example 3 Construction of Transfer Vector Containing Gene Encoding Capsid Protein of Feline Calicivirus (FCV) Document Virus Research, 30 (1993)
From pMCVII containing the open reading frame (2007 bp) of the gene encoding the capsid protein of FCV (capsid gene) described on pages 17 to 26, E
A fragment of the coRI site was cut out. The DNA sequence of the fragment is shown in SEQ ID NO: 2. The fragment is blunted (blunted) and treated with alkaline phosphatase (CIAP treatment)
Then, it was integrated into the SmaI site of pfTK-SBΔSE prepared above, and transferred to pTK-Ca.
p. It was created. This pTK-Cap. A schematic diagram of the constitution of is shown in C of FIG.

C7301dlTK containing the capsid gene
-Preparation of Cap strain Transfer vector pTK was prepared in the same manner as in Example 1.
-Cap. And CRFK-infected cell extract DNA of the parent strain C7301 were co-transfected into CRFK cells to prepare a recombinant virus in the same manner as in Example 1. The obtained recombinant virus was screened by a selective culture medium containing araT, and recombinant C7 containing the capsid gene was screened.
The 301dlTK-Cap strain was selected.

C7301dlTK-Ca obtained above
One of the p strains was selected and subjected to Southern blot and immunoblot analysis as described above to see if it contained the target capsid gene. [Southern Blot] C is operated in the same manner as in Example 1 above.
7301 strain, C7301dl TK strain and C7301dl
The TK-Cap strains were each SalI (lanes in FIG. 14-
) And EcoRI (from lane in FIG. 14) were electrophoresed and transferred to a nylon membrane. As a probe, by Nick Translation,
Using [ ³² P] α-CTP-labeled pfTK-SB (FIG. 14A) and pMCVII (FIG. 14B),
Hybridization was performed on the membrane. The film was then exposed to X-ray film at -70 ° C. The result is shown in FIG.

As shown in FIG. 14A, C7301dl
In the TK-Cap strain (with lane), there is a band with a shorter migration distance than other lanes. This is 2034
This is due to the insertion of an EcoRI fragment containing the bp capsid gene. In addition, as shown in B of FIG.
In the 01dlTK-Cap strain (with lane), pMC
A band hybridizing with the capsid gene in VII was observed at the same position as the above band. Therefore, C730
It can be seen that the 2007 bp capsid gene was correctly inserted into the 1dlTK-Cap strain.

[Immunoblot] Immunoblot analysis was carried out in the same manner as in Example 1 above. The result is shown in FIG. CRFK cells only (lane 1), CRFK cells inoculated with C7301dlTK strain (lane 2), C
7301dlTK-Cap strain inoculated (lane 3), the capsid gene in pMCVII expressed in COS-7 cells (lane 4), FCV at 45 ° C.
Infected RFK cells (lane 5) and FCV
Infected with CRFK cells at 37 ° C (lane 6)
Proteins were extracted from the cells and subjected to polyacrylamide gel electrophoresis. Transfer them to a nitrocellulose membrane,
A monoclonal antibody that recognizes gC of FHV-1 (Fig. 1
5A) 17c11 and a monoclonal antibody (B in FIG. 15) 4B1 that recognizes the F4 protein of FCV,
An immunoblot was performed.

As shown in FIG. 15A, g of FHV-1
C7301d with a monoclonal antibody that recognizes C
1TK strain and C7301dlTK-Cap strain are FHV-1
It can be seen that the antigen is expressed. As shown in FIG. 15B, FCV F4 protein (capsid protein)
The FC4 F4 protein is expressed by a monoclonal antibody that recognizes C7301dlTK-
Only the Cap strain. In addition, the size of the band reacted there was 8.8 k as compared with lanes 4, 5 and 6.
It corresponded to the Da TK-capsid fusion protein (showing a clear band) and the 7.4-kDa capsid (precursor) protein (unclear but clearly having a band). This is C7301dlTK-Cap
It corresponds to the expressed protein predicted from the nucleotide sequence of the gene that has undergone recombination in the strain. C7301dlTK-
The Cap strain also expressed a 7.4 kDa capsid (precursor) protein.

Therefore, according to the results of Southern blot and immunoblot analysis, the C7301dlTK-Cap strain contained two capsid genes in the TK gene and could react with two monoclonal proteins against the FCV capsid protein in cultured cells. Was revealed. [Indirect Fluorescent Antibody Analysis] Similar to the above immunoblot analysis, only CRFK cells and C7301dl were added to CRFK cells.
Proteins were purified from those inoculated with the TK strain and those inoculated with the C7301dlTK-Cap strain, and several monoclonal antibodies recognizing antigenic determinants for virus neutralization on the capsid protein of FCV shown in Table 9 below were used. Then, indirect fluorescent antibody analysis was performed. The results are shown in Table 9 below.
Shown in Here, −: no reaction at all, +: clearly reacted.

[0089]

[Table 9]

From the results in Table 9, C7301dlTK-C
Only those inoculated with the ap strain reacted with all of the respective monoclonal antibodies, and it can be seen that the FCV neutralizing epitope is sufficiently preserved. Therefore, C7301dlTK-Ca
It was revealed that the p strain is a recombinant virus capable of expressing the FCV immunogen.

Forced infection experiment on cats with C7301dlTK-Cap strain Forced infection experiments were carried out using SPF cats. That is, one SPF cat has a C7301dlTK strain, and two SPF cats have a C7301dlTK-Cap strain (No1 and No2)
Was administered twice a day at intervals of 12 days in the amount of 10 ⁶ PFU / 1 ml by intranasal and oral administration. For 24 days after inoculation,
The body temperature, body weight, clinical symptoms and neutralizing antibody titer were observed in the same manner as in Example 1. As a result, body temperature, body weight and clinical symptoms were not abnormally changed in all the inoculated cats. Further, as in Example 1, serum was collected every 3 days and the neutralizing antibody titer was measured. The results are shown in FIG. As shown in A of FIG. 16, the neutralizing antibody titer of FCV is C7301dlTK-.
It increased only in No1 and No2 inoculated with the Cap strain. In addition, the neutralizing antibody titer against FHV was 15 to 24 days after the inoculation of cats with C7301dlTK strain and N.
Increased only for cats inoculated with the O1 C7301dlTK-Cap strain. Also, the No. 2 C7301dlT
Cats inoculated with the K-Cap strain showed no increase on day 24, but it has been confirmed that the neutralizing antibody titer gradually increased thereafter. Therefore, from these results C730
It can be seen that the 1dlTK-Cap strain is capable of inducing a neutralizing antibody titer against both FHV-1 and FCV, has sufficient weak toxicity, and is useful as a recombinant bivalent vaccine.

[0092]

EFFECT OF THE INVENTION Attenuation is stably and sufficiently achieved by gene manipulation, and the ability to proliferate in cultured cells in vitro and the ability to proliferate cats in vivo are both sufficiently retained, and the immunogenicity is excellent. Furthermore, it is possible to provide a recombinant feline herpesvirus type 1 that clearly has biological and genetic markers that can be distinguished from field strains, and a vaccine containing the same. Furthermore, the gene manipulation enables stable and sufficient attenuation, and the proliferative ability in cultured cells in vitro and the proliferative ability in the cat in vivo are both sufficiently retained, and the immunogenicity of FHV-1 is excellent. Furthermore, it clearly has biological and genetic markers that can be distinguished from field strains, and the protein that is the product of a foreign gene such as a heterologous immunogen is stably and appropriately expressed, and the foreign protein is required for its expression. A recombinant feline herpesvirus type 1 that does not need to intentionally insert a heterologous promoter, and a vaccine containing the same can be provided.

[0093]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1619 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: genomic DNA Origin: Feline herpesvirus type 1 Sequence features Characteristic symbol: virion feature how to determine the: E sequence AATTGTATAG TACATACACA ATCAGGTCGG CGACGACCCA AGTTAACCTA ACATGCTAGG 60 TACACGCCCT TAGCCTTTTT AAGAGACTCT GCGGATACAG AGCCGCCCAA TAAACACTCG 120 AGTCGGTCGG TATATACTCC ACTCGCAGAG GTCGAGGATA TATCGCGCTT GAGGACAGCA 180 TAAAAGCGAT TGTGGCATCG AATTCCAGCC CGGAGCCTCA ATCCGACACT GCGTCGTTGT 240 TCACGTTTCA TCATACACAG ATCAGACG ATG GCG AGT GGA ACC ATC CCC GTT 292 CAG AAT GAA GAG ATT ATT AAA TCA CAG GTG AAT ACT GTC CGC ATT TAC 340 ATA GAT GGT GCC TAT GGA ATA GGT AAG AGT TTA ACG GCG AAG TAC CTG 388 GTC AGA GCG GAT GAA AAT CGA CCG GGA TAT ACT TAC TAC TTC CCA GAA 436 CCA AA 436 CCA CTA TAC TGG CGT AGT CTC TTT GAA ACT GAT GTT GTC GGT GGT 484 ATC TAT GCC GTC CAG GAC CGG AAA CGA CGT GGT GAA TTA TCA GCT GAA 532 GAT GCT GCC TAT ATC ACC GCC CAC TAT CAA GCA AGA TTT GCC GCA CCA 580 TAC CTT CTT TTA CAT TCC AGA CTA TCC ACA ATA ACA GGA TAT CAG AAA 628 GTT GTA TGT GAG GAA CAC CCC GAC GTG ACC CTA ATC ATA GAT CAC 676 CCT CTC GCC TCT CTG GTC TGT TTC CCA CTC GCA AGA TAT TTT GTG GGT 724 GAT ATG ACT CTT GGG TCT GTA CTT AGT CTA ATG GCA ACA CTT CCA CGA 772 GAA CCT CCT GGT GGA AAT CTA GTT GTA ACA ACC TTG AAT GAG GAA 820 CAT TTG AAG CGT CTC AGG GGA CGC TCA AGA ACC GGA GAA CAG ATA GAC 868 ATG AAG CTA ATT CAC GCA CTA CGC AAT GTA TAT ATG ATG TTG GTA CAT 916 ACT AAG AAA TTT TTA ACA AAA AAT ACT AGT TGG CGT GAT GGG TGG GGG 964 AAG CTT AAA ATT TTC TCC CAC TAT GAA CGG AAT AGG CTC GTG GAA ACT 1012 ACA ATA GTT TCC GAT TCG ACG GAG TCA GAT TTA TGT GAC ACA TTA TTC 1060 AGT GTT TTC AAA GCC CGG GAG CTC TCC GAC CAA AA GGA GAT CTA CTT 1108 GAC ATG CAT GCA TGG GTC CTC GAT GGA CTT ATG GAA ACC CTC CAA AAT 1156 TTA CAG ATC TTT ACT TTA AAT CTG GAA GGA ACC CCT GAT GAA TGT GCC 1204 GCC GCC TTG GGA GCA CTG AGA CAA GAT GAT ATG ACA TTT ATA GCC 1252 GCA TGT GAT ATG CAC CGT ATA AGT GAA GCC TTG ACG ATA TAC CAT TAA 1300 ACATTAGTGG TGTTCCCTAT TACCCCCCTG TGGTGAATGT GTGGAGGTCA GGGGATAATT 1360 GTATAATGAC CATCGTTTCA TGAATAAAAT AACCGTGTGT GATGTGGATG TATTCATTAA 1420 TTGAATTTCT CTTCCGGTTT TAGATCTTTA TAAGCGTAAA ACTGGTGTTT TAAATCCAAG 1480 AGCCGGGTTC TTTGGAGGTT GGTCACATCA TCGCCACAGC CCGTGGATTC AAGCAATCTT 1540 ATGATGTGTT TGATAATATA CCTATCGATA TTCCTGATCA TTGTATCGAG GATGTTGACT 1600 GGTTTACCGA TGATGGATA 1619

SEQ ID NO: 2 Sequence length: 2034 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: genomic DNA Origin: Feline calicivirus Sequence features Characteristic symbol: virion Method of characterization: S-sequence GAATTCCGAA GTTTGAGC ATG TGC TCA ACC TGC GCT AAC GTG CTT AAA TAC 51 TAT GAT TGG GAT CCC CAC TTT AGG TTG ATC ATC AAT CCT AAC AAA TTT 99 CTC CCT ATT GGC TTC TGT GAC AAC CCC CTT ATG TGT TGC TAT CCA GAC 147 TTG CTT CCT GAA TTT GGA ACA GTG TGG GAC TGT GAC CAG TCA CCA CTG 195 CAA ATT TAT CTG GAA TCT ATC CTT GGA GAT GAT GAG TGG GCT TCA ACT 243 CAC GAG GCC ATC GAC CCC CCA GTACTCCT ATG CAC TGG GAC AGT GCT 291 GGC AAG ATC TTT CAG CCA CAC CCC GGT GTT TTG ATG CAT CAT CTC ATT 339 GGA GAG GTT GCA AAG GCT TGG GAC CCA AAC CTA CCG CTC TTC CGA TTG 387 GAG GCC GAT GAT GGA ACA ATC ATC CCC GAA CAG GGA ACT GCG GTT 435 GGT GGG GTT ATC GCT GAG CCT AGT GCC CAG ATG TCA ACT GCT GCT GAC 483 ATG GCC TCG GGG AAA AGC GTT GAC TCT GAG TGG GAG GCG TTT TTC TCT 531 TTC CAC ACT AGC GTC AAC TGG AGT ACA TCG GAA ACC CAA GGT AAG ATT 579 CTC TTC AAA CAA TCC CTA GGA CCT CTC CTT AAC CCT TAT CTC GAG CAC 627 TTG TCA AAG CTT TAC GTC GCA TGG TCT GGC TCT ATT GAA GTT AGG TTC 675 TCT ATT TCT GGC TCT GGT GTG TTC GGG GGT AAG CTT GCT GCC ATT GTT 723 GTG CCA CCA GGG GTT GAC CCT GTT CAG AGC CCG TCA ATG CAA TAC 771 CCC CAT GTT CTG TTT GAC GCT CGT CAG GTG GAA CCT GTC ATC TTC ACT 819 ATC CCC GAT CTA AGG AGC ACA CTG TAT CAC GTT ATG TCT GAC ACT GAT 867 ACC ACG TCT TTG GTT ATT ATG GTG TAT AAC GAT CTA AAC CCA TAT 915 GCT AAT GAC TCA AAC TCT TCT GGG TGT ATT GTC ACT GTA GAA ACC AAA 963 CCT GGA CCC GAT TTC AAA TTC CAC TTG TTG AAA CCC CCT GGT TCT GTG 1011 TTA ACT CAT GGC TCG ATT CCT TCG GAC CTG ATC CCC AAA TCA TCA TCC 1059 CTT TGG ATT GGT AAT CGC TAC TGG ACT GAC ATA ACC GAT TTT GTA ATT 1107 CGA CCC TTT GTG TTC CAA GCA AAC CGT CAT TTC GAC TTT AAC CAA GAA 1155 ACA GCT GGC TGG AGC ACA CCA AGA TTC AGG CCC ATC ACC ATT ACA ATT 1203 AGT GAA AAG AAT GGT TCA AAG TTA GGA ATT GGC GTT GCA ACC GAT TAC 1251 ATT ATC CCA GGG ATT CCT GAT GGC TGG CCA GAT ACT ACA ATT GCT GAT 1299 AAA TTG ATT CCC GCG GCA GAC TAT ATT ACC ACG GGG GAG GGG AAT 1347 GAC ATC AAA ACG GCT CAG GCC TAT GAC ACT GCA GCT GTG GTA AAG AAC 1395 ACC ACA AAT TTC CGA GGG ATG TAT ATC TGT GGT TCA TTG CAA CGG GCT 1443 TGG GGC GAT AAG AAG ATT TCA AAG ATT TCA A ACT GCC TTC ATA ACC ACC GCC ATC 1491 AGG GAC GGC AAC GAA ATC AAA CCA TCT AAC ACA ATT GAC ATG ACA AAG 1539 CTC GCC GTG TAC CAA GAT ACT CAT GTA GAG CAG GAA GTC CAA ACA TCT 1587 GAT GAC ACG CTT GCC CTC CTT GCC CTC GGT TAC ACT GGG ATT GGC GAG GAG GCA 1635 ATT GGC TCA AAT AGG GAC AGG GTA GTG CGC ATT AGC GTG CTA CCA GAA 1683 GCT GGG GCC CGT GGT GGC AAT CAC CCC ATC TTT TAC AAA AAC TCA ATT 1731 AAA TTG GGC TAT GTAATT AGA TCT ATC GAT GTG TTC AAT TCT CAA ATC 1779 TTG CAC ACA TCC AGA CAA CTG TCA CTT AAC CAC TAT CTG TTA CCT CCT 1827 GAC TCC TTT GCT GTT TAT AGA ATA ATT GAC TCA AAT GGC TCT TGG TTT 1875 GAC ATT GGT ATT GAT AGT GAA GGG TTC TCT TTT GTT GGT GTT TCT GAT 1923 ATT GGT AAA TTA GAA TTT CCT CTT TCA GCC TCC TAC ATG GGA ATA CAA 1971 TTG GCA AAA ATT CGC CTT GCC TCA AAC ATT AGG AGC AGA ATG ACT AAA 2019 TTA TGA ATTGAATTC 2034

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the constitution of a TK gene and a transfer vector.

FIG. 2 is an electrophoretic diagram showing the results of Southern blot analysis.

FIG. 3 is an electrophoretic diagram showing the results of PCR amplification of the TK gene of FHV-1.

FIG. 4 is a diagram showing the time course of virus growth in CRFK cells.

FIG. 5 is a diagram showing changes in body temperature and body weight with time when a cat was inoculated with the C7301 strain or the C7301dlTK strain.

FIG. 6 is a view showing a time change of neutralizing antibody titer when a C7301 strain or a C7301dlTK strain was inoculated into a cat.

FIG. 7 is a diagram showing the time-dependent changes in the amount of virus that can be isolated from a cat inoculated with the C7301 strain or the C7301dlTK strain.

FIG. 8: PCR of TK gene of virus isolated from cats inoculated with C7301 strain or C7301dlTK strain
It is a figure of electrophoresis showing the result of amplification.

FIG. 9 is a diagram showing a schedule for inoculating a cat with a virus.

FIG. 10 is a view showing a time change of body temperature when a C7301 strain and a C7301dlTK strain were sequentially inoculated into a cat.

FIG. 11 is a view showing a time change in body weight when a C7301 strain and a C7301dlTK strain were sequentially inoculated into a cat.

FIG. 12 is a view showing a time-dependent change in neutralizing antibody titer when a C7301 strain and a C7301dlTK strain were sequentially inoculated into a cat.

FIG. 13 is a schematic diagram showing the structure of a transfer vector having a capsid gene inserted therein.

FIG. 14 is an electrophoretic diagram showing the results of Southern blot analysis to confirm the insertion of the capsid gene.

FIG. 15 is an electrophoretic diagram showing the results of immunoblot analysis to confirm insertion of the capsid gene.

FIG. 16: C7301dlTK strain or C7301 in the cat
It is a figure which shows the time change of the neutralizing antibody titer with respect to FHV and FCV at the time of inoculating the dlTK-Cap strain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display C12R 1:92)

Claims (18)

[Claims] 1. The Herpesviridae Alpha
Recombinant feline herpesvirus 1 characterized in that at least the EcoRV-SmaI site of the gene encoding thymidine kinase in the genome of feline herpesvirus 1 belonging to the subfamily aherpesvirinae is deleted.
Type. 2. The Herpesviridae Alpha.
Recombinant feline herpesvirus type 1 characterized in that an exogenous gene is inserted at the SmaI site in the gene encoding thymidine kinase in the genome of feline herpesvirus type 1 belonging to the subfamily aherpesvirinae. 3. At least EcoRV-S of the gene encoding thymidine kinase of herpesvirus type 1.
The recombinant feline herpesvirus type 1 according to claim 2, wherein the maI site is deleted and a foreign gene is inserted into the SmaI site. 4. The gene encoding thymidine kinase has a DNA sequence capable of hybridizing at least with the DNA sequence set forth in SEQ ID NO: 1 in Sequence Listing, and Ec
The recombinant feline herpesvirus type 1 according to claim 1 or 2, which has an oRV cleavage site and an SmaI cleavage site. 5. The gene encoding thymidine kinase has a DNA sequence capable of hybridizing at least with the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and an EcoRV cleavage site and sequence at position 618 of SEQ ID NO: 1. The recombinant feline herpesvirus type 1 according to claim 4, which has a SmaI cleavage site at the 1074th position of No. 1. 6. The feline herpesvirus type 1 is C7
The recombinant feline herpesvirus type 1 according to claim 1 or 2, which is strain 301. 7. The recombinant feline herpesvirus type 1 according to claim 2, wherein the foreign gene is at least one gene encoding a foreign immunogen. 8. The gene encoding the foreign immunogen comprises:
At least one gene encoding a protein that is an immunogen against at least one pathogen of feline calicivirus, feline leukemia virus, feline parvovirus, feline immunodeficiency virus, feline coronavirus, feline corotavirus, and feline chlamydia. The recombinant feline herpesvirus type 1 according to claim 7, which is characterized in that 9. The gene encoding the foreign immunogen comprises:
The recombinant feline herpesvirus type 1 according to claim 8, which is a gene encoding a capsid protein of feline calicivirus. 10. The set according to claim 1, wherein a foreign gene capable of expressing an immunogen against feline calicivirus is operably inserted in the feline herpesvirus type 1 genome. Altered feline herpesvirus type 1. 11. The recombinant feline herpesvirus type 1 according to claim 2 or 3, wherein the foreign gene is at least one gene encoding a pharmaceutical or diagnostic polypeptide. 12. A pharmaceutical or diagnostic polypeptide comprising:
The recombinant feline herpesvirus type 1 according to claim 11, which is a lymphokine, interleukin, interferon, or cytokine. 13. The recombinant feline herpesvirus type 1 according to claim 2 or 3, wherein the foreign gene is expressed by a promoter derived from feline herpesvirus type 1. 14. A cultured cell transfected with the recombinant feline herpesvirus type 1 DNA according to any one of claims 1 to 13. 15. A cultured cell cotransfected with the transfer vector required for producing the recombinant feline herpesvirus type 1 according to any one of claims 1 to 13 and feline herpesvirus type 1 genomic DNA. . 16. A cell culture infected with the recombinant feline herpesvirus type 1 according to any one of claims 1 to 13. A vaccine comprising at least the recombinant feline herpesvirus type 1 according to any one of claims 1 to 13. 18. A method for immunizing a cat against an infectious disease, which comprises administering the vaccine according to claim 17 by eye, nose and mouth or intramuscular, intravenous or subcutaneous injection in a cat.