JPH07255488A - Recombinant Marek's disease virus and its production method - Google Patents

Abstract

PURPOSE:To obtain the subject virus having a structure in which the gene region containing a repeating sequence in reiterated inverted sequence of Marek' s disease I type virus genom S region gene is substituted with a foreign gene manifestation cassette and useful and a polyvalent live vaccine for chicken, a vector, etc. CONSTITUTION:A foreign gene manifestation cassette is prepared by bending a foreign gene coding an antiinfectious antigen derived from a pathogen except Marek's disease I type virus to the downstream region of a promoter executing its function in an animal cell. The obtained foreign gene manifestation cassette is integrated into a fragment derived from the reiterated inverted sequence of S region of Marek's disease I type virus gene so as to be substituted for a section containing the repeating sequence existing in the fragment. This integration into Marek's I type virus genom is carried out by using the recombinant gene fragment obtained by the above-mentioned method, thus creating the objective recombinant Marek's disease virus useful as a virus vector and a polyvalent live vaccine for chicken capable of inducing immunity against Marek's disease and an immunological reaction against a protein manifested by the integrated foreign gene by inoculation into a chicken.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel recombinant virus capable of expressing a foreign gene product in a chicken cell or a chicken body and a method for producing the same. Further, the present invention relates to a multivalent live vaccine for chickens using the present recombinant virus, and a recombinant viral vector for in vivo administration of physiologically active substances such as hormones.

[0002]

2. Description of the Related Art In the current poultry farming field, breeding chickens, laying chickens,
Vaccination is an important pillar of hygiene, regardless of whether it is chicken or chicken. However, the actual situation of vaccination is so frequent that it can be said that it is overcrowded.
Therefore, the labor cost required for this is a great financial burden for the poultry farmer. Use of a viral vector is being attempted as a solution to this problem. That is, it is a method of producing a multivalent live vaccine by incorporating genes of a plurality of vaccine antigens into one virus.

Up to now, research aimed at using a virus as a vector has already been carried out on vaccinia virus,
It has been carried out with adenovirus, herpes simplex virus, retrovirus, etc., and all of them succeeded in expression of hepatitis B virus surface antigen (HBs antigen) or glycoprotein antigens such as rabies virus and herpes zoster virus in an in vitro system. ing. However, some of these viruses belong to tumorigenic viruses, and therefore administration to humans or animals has many problems in terms of safety and is not realistic. In addition, even if there is no problem with the safety of the virus itself in the administration of animals,
When these viruses are viewed from the viewpoint of viral vectors for birds, which is the object of the present invention, since birds are not the original host for these viruses, they are effective against these viruses as avian viral vectors. In terms of, you can't expect much.

From these points, it is considered to use avian poxviruses (for example, chicken fowlpox virus) as a vector, and it has already been studied as a viral vector. It is possible to insert a foreign gene into the viral DNA. It has been shown that However, for example, in the current poultry farming field, immunity against fowlpox is
Due to the short duration of immunity of fowlpox virus, it is usually necessary to inoculate several times with the vaccine virus (attenuated fowlpox virus or pigeonpox virus) during the breeding period of chickens. Therefore, when poxvirus is used as a viral vector, it is considered that frequent vaccination is required even if a viral vector incorporating a plurality of antigens is produced and used as a vaccine. In the case of poxvirus vector,
It has been clarified that the growth of poxvirus itself is largely inhibited by the transfer antibody against poxvirus, and as a result, a sufficient immune response to the inserted antigen cannot be obtained.

On the other hand, Marek's disease is a malignant tumor whose onset is protected by a vaccine including humans and animals. As a defense mechanism, by persistent infection with a vaccine virus throughout life, eliciting humoral and cellular immunity against Marek's disease virus in birds such as chickens,
It is thought to be maintained for the rest of its life, and as a result, it suppresses tumorigenesis by virulent viruses. In addition, this virus vaccine is administered in the form of live cells infected with virus, and since it proliferates in a cell-associated form in vivo, unlike other vaccine viruses, it is not neutralized by the transfer antibody, and therefore the primary chicks It is a major feature that it can be administered to.

Focusing on such characteristics of Marek's disease virus, in recent years, studies using Marek's disease virus as a vector have been conducted for the development of multivalent vaccines. As mentioned above, in order to prepare a multivalent live vaccine using a Marek's disease virus vector having characteristics far superior to other virus vectors, first, insert a foreign gene on the Marek's disease virus DNA. It is essential to find a possible site.

[0007] The TK gene and the gA antigen gene have been discussed as possible sites for insertion of foreign genes on Marek's disease virus DNA. However, T
It has been reported that Marek's disease virus, which has lost thymidine kinase activity due to mutation of the K gene, has reduced proliferative properties [P. Bandyopadyay et al (1987), 12th.
INTER-NATIONAL HERPESVIRUS WORKSHOP] and a recombinant virus in which a foreign gene is inserted into the TK gene has not been reported. On the other hand, it has been reported that the recombinant virus in which the LacZ gene is inserted into the gA antigen gene is unstable and cannot be purified, and neither is practical [Kato Atsushi et al. (1991), 111th Japanese Veterinary Society].

[0008] In addition, gA is a major glycoprotein produced by the virus together with gB, and no neutralizing antibody such as gB has been produced so far when inoculated into an animal body, but gA It is highly presumed that it induces cell-mediated immunity. Therefore, when considering that Marek's disease virus should have both functions as a vector and a vaccine, it is necessary to insert a foreign gene into this gA gene.
It was expected that mutating Lactobacillus may reduce the efficacy of the vaccine. In addition, the US2 gene has been reported as a site into which a foreign gene can be inserted.
In vivo proliferative properties are completely unknown. Furthermore, U
In the case of the recombinant in S2, only plaque level cloning was performed, and as a phenotype, even if the recombinant was found, non-recombinant parent strains were mixed in the plaque. There is no denying the possibility that they supplemented the function of.
That is, there is no proof that US2 is non-essential and practical as an insertion site for foreign genes.

Under these circumstances, the present inventors have previously conducted research on the production of a recombinant Marek's disease virus that is effective even for a gene that has not yet been analyzed so far, and BamHI of Marek's disease type I virus gene has been developed. It has been found that a recombinant Marek's disease virus can be prepared by inserting a foreign gene into the -H fragment (JP-A-2-76578).
issue).

[0010] After that, as a result of intensive studies on the development of a more effective viral vector, the present inventors found a site capable of integrating a foreign gene into the Marek's disease virus gene genome very efficiently and stably. (Japanese Patent Application No. 4-205933). At that time, we examined the insertion of foreign genes at 22 sites on the viral genome, but reported that only a few sites were able to stably insert foreign genes (No. 19). Times World Poltl
y congress Proceedings 1, p150 -p155).

[0011]

It has been confirmed that some of the recombinant Marek's disease viruses constructed according to the above-mentioned technique show good in vivo growth properties, and thus they are extremely useful as viral vectors as avian multivalent vaccines. It has been confirmed to be excellent. However, the effect tended to be reduced in the chicks carrying the transfer antibody against Marek's disease virus. In spite of some reports of recombinant virus construction in papers and academic societies, it is presumed that the recombinant virus growth inhibition by the transfer antibody is one of the reasons why it has not been applied in the field. In fact, even in Marek's disease virus, which has been administered in the form of virus-infected cells and thus has been the least susceptible to migratory antibodies, the recombinant virus has been shown to suppress in vivo growth in maternal antibody-bearing chicks. It was As described above, solving the problem of the transfer antibody possessed by the chicks has been a problem in practical use.

[0012]

[Means for Solving the Problems] As a new recombinant Marek's disease virus showing good growth and immunogenicity equivalent to that of the parent strain even in the presence of such a transfer antibody, the present inventors The present inventors have found that a recombinant Marek's disease virus can be prepared by incorporating a foreign gene into a specific region to produce a recombinant virus that solves the above problems, and have completed the present invention. That is,
A recombinant gene fragment was prepared in which the foreign gene expression cassette of interest was integrated into a fragment derived from the S region repeat inversion sequence of the Marek's disease type I virus gene so as to replace the part containing the repeat sequence present in the fragment. It was found that a recombinant Marek's disease virus capable of solving the above problems can be obtained by incorporating a foreign gene expression cassette into the Marek's disease type I virus genome using this gene fragment.

At present, as a vaccine against Marek's disease, attenuated Marek's disease type I virus (MDV-I type), turkey hepes virus (HVT), or Marek's disease II is used.
There is a mixture of type I virus and turkey herpes virus. It is known that Marek's disease itself is caused by infection with the type I virus, and therefore the protection against the onset of Marek's disease is related to the Marek's disease I belonging to the same serological type.
It is considered that the use of attenuated strains of type I virus as a vaccine is the basis. Also in the present invention, Marek's disease type I virus is used as a parent virus for recombination.

The foreign gene insertion region in the preparation of the recombinant Marek's disease virus found by the present inventors is the Us region present in the Marek's disease virus genome S region (the Us region is the 3'end of MDV-DNA. , Which is a gene region having a length of about 12 kb and flanked by repetitive inversion sequences at both ends.
ed repeat sequence: IRs or TRs), and the repeat sequence portion present therein serves as the insertion (substitution) region. Further, in the present invention, when a foreign gene expression cassette is incorporated, it is necessary to incorporate (replace) the foreign gene expression cassette so that the repetitive sequence contained in the repetitive inverted sequence is removed. Preferably, the IRs gene is used as the repetitive inverted sequence, and the repetitive sequence region contained in the IRs gene and its downstream (3 ′ side)
The foreign gene expression cassette is incorporated so that the corresponding gene region including the border between IRs and Us is removed. By preparing the recombinant virus in this manner, the target recombinant virus can be prepared without affecting the growth property of the Marek's disease virus itself on the side where the foreign gene is integrated.

By the way, usually, a virus in which a part of its constituent genes has been inactivated by insertion of a foreign gene or the like has a proliferative property in vitro or a proliferative property in vivo. One manifestation, pathogenicity, is clearly reduced [Bernard Meignier
Et al., The Herpes viruses 4, p265 (1985)]. Therefore, the produced recombinant virus or recombinant vaccine is
In order to determine whether it can be applied ivo, it is necessary to actually inoculate the chicken with the virus and confirm its proliferative property and immunogenicity. From this point of view, we conducted an inoculation test of the recombinant virus thus produced into field chicks. As a result, it has been found that in order to make the recombinant virus in vivo proliferative in view of its practicality, it should be performed not in SPF animals but in field chickens carrying various transfer antibodies. Using multiple recombinant viruses constructed so far (Japanese Patent Application No. 4-205933, etc.) and the recombinant virus of the present invention, in vivo proliferative tests in field chicks were performed. Recombinant Marek's disease virus
It was confirmed that, unlike conventional recombinant viruses, chicks carrying the transfer antibody also showed good in vivo growth.

The recombinant virus produced according to the present invention showed a protective effect against the virulent Marek's disease virus even in field chicks having a transfer antibody, as compared with conventional recombinant viruses. Furthermore, it was confirmed that the recombinant virus of the present invention simultaneously elicits an antibody against a product expressed from a foreign gene integrated in the genome when inoculated into field chicks. In the present invention, it is most noteworthy in the present invention that the results showing good in vivo proliferability and immunogenicity were obtained even in the primary chicks having the transfer antibody.

The method for producing the recombinant Marek's disease virus of the present invention will be further described below. Generally, the following procedure is required to produce the recombinant virus of the present invention. (1) A part of viral DNA is cloned into a plasmid vector. (2) Insert a gene fragment containing a foreign gene constructed for expression into a plasmid into which a viral DNA fragment has been cloned to construct an insertion plasmid. (3) The insertion plasmid is transduced into virus-infected cells. (4) Select a recombinant virus carrying the foreign gene by an appropriate method.

The basic structure of an insertion plasmid used for incorporating a foreign gene into a virus has a gene having a repetitive inversion sequence adjacent to the Us region derived from Marek's disease virus, and is further aimed downstream of a promoter that functions in animal cells. Has a foreign structural gene and a structure in which a transcription termination factor is further connected downstream thereof. A gene fragment in which a promoter, a foreign structural gene, and, if necessary, each gene of a transcription termination factor is designed and recombined so that the foreign gene is transcribed and translated is referred to as a foreign gene expression cassette. The insertion plasmid used for has a repetitive inverted sequence gene derived from Marek's disease virus, wherein the foreign gene expression cassette is integrated so that the repetitive sequence part contained therein is replaced. Can also be expressed as a plasmid. By using such a plasmid, the virus-derived gene fragment on the insertion plasmid is replaced with the viral DNA at the homologous portion, and as a result, the foreign gene fragment sandwiched between the viral gene fragments is integrated into the viral genome.

In the present invention, first, as (1), the virus DN
A is cleaved with a restriction enzyme, and each fragment is separated by agarose gel electrophoresis and then recovered from the gel, and each fragment is cloned into a plasmid.

Next, as (2), a structural gene to be expressed in a promoter that functions in an animal cell is obtained by cutting each viral fragment cloned in a plasmid with one enzyme to cut or partially deleted it. The foreign gene is inserted in the form that is upstream of.

The method (3) is homologous recombination of a viral fragment containing a foreign gene into a viral DNA, which is usually carried out by simultaneously transducing cells with an infectious viral DNA and an insertion plasmid. In this method, a method of infecting a cultured cell with a virus and then introducing an insertion plasmid is used. Therefore, this method is a very simple recombination method, and it is possible to obtain a recombinant virus with extremely high efficiency by using the electroporation method as a transduction method.

At this time, as the foreign gene incorporated into the genome of Marek's disease virus, various genes encoding proteins that can be vaccine antigens of various chicken diseases such as viral diseases, bacterial diseases, parasitic diseases, that is, Marek's disease Examples include genes encoding protective antigens derived from pathogens other than disease virus. For example, in the preparation of a multivalent vaccine for chickens, a gene encoding the Newcastle disease virus (NDV) antigen, for example, a gene encoding the NDV-F protein or HN protein, a chicken infectious laryngotrachea, as a foreign gene to be incorporated Flame virus (ILT
V) a gene encoding a glycoprotein, a gene encoding a viral structural protein of infectious bursal disease virus (IBDV), for example, a gene encoding VP2, a gene encoding a spike protein of infectious bronchitis virus (IBV) And Hemophilus paragallinaru, which is the causative agent of infectious coryza
Examples include genes encoding the HA protein of m).

The recombinant Marek's disease virus of the present invention is useful not only as a protective antigen for infection as described above, but also as a viral vector for administration of chicken growth hormone or immunostimulatory substances. Furthermore, it is also useful for immunization for immunizing breeders in order to impart various useful antibodies to eggs. That is, the recombinant Marek's disease virus of the present invention is also used as a viral vector for a drug delivery system (DDS). Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples, but the present invention is not limited thereto.

[0024]

[Preparation Example] Virus strain I-type virus 61-554 strain isolated from the field was used. This strain is a virus isolated in 1986 from a 50-day-old broiler that had not been vaccinated with Marek's disease vaccine. SPF
In a test in which 2 × 10 ³ larvae of 1-day-old chick were inoculated into the abdominal cavity, there was no onset or death during the 10-week observation period, and no abnormalities such as tumor lesions were observed at autopsy.

Purification of Viral DNA After inoculating chicken embryo fibroblasts (CEF) with virus, when the cytopathic effect (CPE) is strongly exhibited, the virus-infected cells are harvested and the method of Hirai et al. [J.gen.Virol ., 45, p119 (197
9)], the viral DNA was purified. That is, first, virus-infected cells that strongly exhibited CPE had a double dose of 1% -NP40.
Solution (0.01M-Tris-HCl, pH7.4, 0.01M-NaCl, 0.0015M-MgCl
₂ ) was added and ice-cooled for 30 minutes, and then pipetting was performed.
This solution was centrifuged at 2500 rpm for 10 minutes, and the supernatant was
60% (w / w) sucrose solution (0.02M-Tris-HCl, pH7.4,0.15M
-NaCl), and after centrifuging at 175KG for 2 hours, add 40% sucrose solution
The layer of capsid derived from Marek's disease virus formed in the middle of a 60% sucrose solution was fractionated. Furthermore, this intermediate layer was again suspended in a 0.02M-Tris-HCl, pH7.4, 0.15M-NaCl solution, and centrifuged at 160 KG for 1 hour to be pelleted. These pellets were mixed with proteinase K (Boehringer Mannheim Yamanouchi) in 0.1% 1% -SDS solution (0.1% -Tris-HCl,
pH 7.4, 0.01M-EDTA, 1% -Sarcocinate; Hanai Chemical Co., Ltd.) and left overnight at 37 ° C. Then phenol treatment,
DNA was recovered by ethanol precipitation, and TE buffer (10 mM-Tr
It was dissolved in is-HCl, pH 8.0, 1 mM-EDTA), layered with a 10% to 30% glycerol gradient solution, and then centrifuged at 175 KG for 4 hours. Next, the solution was fractionated from the bottom of the tube, a fraction containing viral DNA was taken, and 10% -trichloroacetic acid was added to precipitate and collect the DNA.

Cloning of viral DNA 1 μg of purified Marek's disease virus DNA was cleaved with a restriction enzyme, and each fragment was separated by 0.7% -agarose gel electrophoresis.
It was eluted from the gel by the electroelution method, and recovered by phenol treatment and ethanol precipitation. The fragment thus obtained was inserted into pUC or PBR using T4 DNA ligase and transduced into an appropriate competent cell (for example, JM109) to obtain transformed Escherichia coli. Next, 100 μg / ml
After culturing in LB medium containing ampicillin, the intracellular plasmid was recovered by the alkaline method.

Determination of nucleotide sequence The gene fragment was cloned into a polylinker of pUC119 and then transduced into competent cells such as JM109. After culturing the obtained transformants overnight in LB medium,
30 μl of each was infected with 60 μl of M13 phage (10 ⁹ / ml or more), and further cultured overnight by shaking. After removing the bacterial cells by centrifugation, phages were recovered from the supernatant, and single-stranded DNA (ssDNA) containing the nucleotide sequence of the target gene fragment was prepared by a conventional method. The nucleotide sequence of the obtained ssDNA was determined using SEQUENASE V2.0 (TOYOBO) according to the attached protocol. If necessary, copy the gene fragment on the plasmid to Del for Kilo-Sequence.
Using the etion Kit (Takara, Catalog No. 6030), a plasmid shortened stepwise was constructed, and ssDNA was similarly prepared.

Recombinant virus production Primary CEF cultured overnight at 37 ° C was harvested with EDTA-trypsin solution, washed, and then 5% bovine serum (BS) -added Eagle-MEM (E-
Suspended at a cell concentration of 200,000 cells / ml in MEM;
40 ml was placed in a Falcon tissue culture flask (NO.3028), and about 800,000 Marek's disease virus-infected CEFs were inoculated into the tissue culture flask and cultured at 37 ° C. for 4 hours. Then harvest the cells again with EDTA-trypsin solution, wash the cells twice with PBS-, transfer 5 million of these cells to a biorad Gene Pulser (catalog number 165-2075) cuvette, and insert the insertion plasmid. And a pulse was added according to the attached protocol to introduce the insertion plasmid into the virus-infected cells. Next, 5% BS added E-EME
(Sun water) Floating in 15 ml, transferred to a petri dish with a diameter of 10 cm (Falcon No. 3003) and cultured at 37 ° C. The next day, dead cells that did not engraft were removed together with the medium, and the newly harvested primary CEF harvested (2nd CEF) was suspended at 500,000 cells / ml and E-MEM containing 5% BS was added. 15 ml was added. After culturing at 37 ℃ for 4 to 7 days, 100 μg of chlorophenol red β-D-galactopyranoside (Seikagaku Corporation)
A 1% -agarose / E-MEM solution (containing no phenol red) contained at a concentration of / ml was overlaid.

Red plaques showing β-gal activity that appeared within 5 to 60 minutes were subcultured to the next generation by plaque cloning. After repeating this operation several times, the virus-infected cells were gently disrupted using an ultrasonic disruptor to make the virus cell-free, and then cultured for 4 hours in CEF.
Culture for several days after inoculation of
The recombinant virus was purified ~ 3 times.

Southern hybridization Boehringer Mannheim Yamanouchi DIG-DNA labeling kit (catalog number 150350) and TROPIX Southern light (catalog number SL100) were used according to the attached protocol to prepare and Hybridization was performed. Briefly, linearized DNA was denatured by heating, and probe DNA was synthesized by using Klenow fragment with random primers to dNTPs containing digoxigenin-labeled dUTP as a substrate.

This probe was transferred to a high bond N + (Amersham Japan, Catalog No.
N.303B) according to the protocol, and detection was carried out using alkaline phosphatase-labeled anti-digoxigenin sheep IgG. AMPPD was used as a substrate for alkaline phosphatase, and the generated specific luminescence was detected using an X-ray film (Fuji Xerox, X-OMAT).

Recovery of Recombinant Viral DNA When the petri dish (Falcon, Catalog No. 150350) inoculated with the virus exhibited CPE on the entire surface, the supernatant was aspirated,
Proteinase K solution (Proteinase K 1 mg / ml, 0.1
(M-Tris / HCl, pH 9.0, 0.1M-NaCl, 0.001M-EDTA, 1% SDS)
2 ml was poured and treated at 37 ° C. for 1 hour, then transferred to a conical tube (Falcon, Catalog No. 2099) and treated at 37 ° C. overnight. Then, the phenol-treated supernatant was ethanol-precipitated, dried and then 100 μl of TE (10 mM-Tris.HCl.pH 8.0, 1 mM-
EDTA). For Southern hybridization, 2 μl of this was cut with a restriction enzyme and used.

Virus recovery from chicken body After collecting 1 ml of blood with a syringe sucking heparin, P
BS- was added to make 4 ml, and this was gently layered on 3 ml of Ficoll-Pque (Pharmacia) placed in a conical tube (Falcon, Catalog No. 2099) and centrifuged at 1500 rpm for 30 minutes (Kubota, KN-30F). . Intermediate layer consisting of lymphocytes and monocytes (Bu
ffy coat) was collected, suspended again in PBS-containing 0.01% EDTA, centrifuged at 1000 rpm for 5 minutes, collected, and inoculated into CEF (2ndary) that had been cultured for 4 hours. I observed the day. MDV
For those for which no CPE was observed, the passage was repeated until the third generation.

Fluorescent antibody method (FA) 61-554 strain 10 ⁴ PFU together with CEF10 ⁷ cell cover glass (MATUN
(AMI.No.1,18x18mm) Inoculate into a petri dish with a diameter of 5 cm containing 3 pieces, incubate for 2 days, take out the cover glass, and leave at room temperature for 2
After fixing with acetone for 0 minutes, it was stored at -80 ° C. For detection of anti-β-gal antibody, LacZ was placed downstream of β-actin promoter.
A plasmid pASLacZ to which a gene is connected (JP-A-2-76578)
No.) was introduced into BMT-10 cells by electroporation, cultured for 2 days, and similarly fixed with acetone at -80 ° C.
Saved in.

Chicken serum was diluted 10-fold with PBS-and reacted overnight at 4 ° C., and then FITC-labeled anti-chicken IgG goat antibody (KIRKEGAA) was used.
RD & PERRY Lab., Catalog No. 031506) was diluted 20-fold with PBS-, reacted at 37 ° C for 1 hour, washed, and then observed under a fluorescence microscope.

[0036]

Examples Example 1: Cloning of 61-554 strain DNA 1 μg of 61-554 strain DNA was cleaved with HindIII and then subjected to 0.7% -agarose gel electrophoresis to cut a 17 kb fragment (HindIII-B fragment). It was cloned into pBR322 (pKHB).
The EcoRI cleavage pattern of pKHB is shown in Fig. 1. Of these, the A5 and A6 fragments were shown to differ in length between viruses (Table 1).

[0037]

[Table 1] nt: not tested

Example 2: Repeated distribution in IRs and TRs
Nucleotide sequence determination When pKA5 was mapped with a restriction enzyme present in the GA strain A5 fragment, about 220 bp excised by StuI was amplified in the IRs portion as shown in FIG. When the base sequence of 220 bp was determined, the base sequence of the GA strain (GenBank Ac
cession Number M80595, M. Sakaguchi) base numbers 614 to 822. On the other hand, as a result of similar mapping of pKA6, it was estimated that there were 5 repeats each having a unit of about 220 bp (FIG. 3).
Sequence Listing: The nucleotide sequence of the GA strain A5 fragment is shown in SEQ ID NO: 1. In the repeat inversion sequence of the 61-554 strain, the base number 61 in the sequence is 61.
The parts corresponding to 4 to 822 were repeated.

Further, the nucleotide sequence of the fragment corresponding to A6 of BC-1 strain was determined. As a result, it was confirmed that, in TRs similar to KA6, a 278 bp nucleotide sequence showing 88% homology with LTR of avian leukemia virus (ALV, RAV2) was incorporated and repeated 3.5 times. The position of the repeating sequence is shown in FIG. Sequence Listing: SEQ ID NO: 2 shows the nucleotide sequence of A6 fragment of BC-1 strain. Sequence Listing: Us region gene on the 5'side (smaller base number direction) from the 853th nucleic acid in SEQ ID NO: 1 and on the 3'side (incrementing base number) from the 854th nucleic acid
Corresponds to the gene in the TRs region. From the above results, I
It was revealed that there is a site into which the gene can be inserted without affecting the viral growth property in the portion of Rs and TRs in contact with the Us region.

Example 3: Generation of K-A5L pKHB was digested with HindIII and EcoRI to contain a KA5 fragment.
The 5.5 kb fragment was subcloned into pSP72. This plasmid was digested with NcoI and PstI to completely remove the repetitive sequence, and then excised with BamHI and TthlllI from pCH110 (Pharmacia Catalog No. 27-4508-01) at the same site. The 4.2 kb fragment (SV40-LacZ) obtained by excising the inserted SV40-LacZ again with BglII and BamHI was inserted (pKA5L, FIG. 5).

Next, pKA5L was cut with KpnI and linearized, and then K-A4BL was produced according to the production of recombinant virus. This virus showed good in vitro proliferation ability comparable to that of parent strain 61-554. In order to confirm the insertion position of the LacZ gene in K-A5L, DNA extracted from K-A5L-infected CEF was cleaved with EcoRI and then subjected to Southern hybridization. The results of Southern hybridization using US420 (FIG. 6) and pCH110 with LadZ gene (3.7K) excised with HindIII and BamHI as a probe are shown in FIG. No matter which probe was used, US420 (lane 2) or LacZ gene (lane 1), only the band of the expected size was detected, and the LacZ gene was inserted by homologous recombination, and the recombinant virus was purified. Was confirmed.

Table 2 shows the virus recovery test and anti-MDVFA antibody test results in SPF 1-day-old chicks inoculated with K-A5L 6000PFU. The virus recovery at 8 weeks after the inoculation was 4/5 (+), indicating good persistent infection. The anti-MDV FA antibody at the 4th week was 7/7 (+), and the antibody titer level tended to be higher than that when other recombinant viruses were inoculated (Fig. 7). On the other hand, the anti-β-galactosidase antibody titer of the serum at 4 weeks was measured by the fluorescent antibody method. As a result, all the antibodies against β-galactosidase showed high antibody titer (FA titer) of 160 times or more after 4 weeks after the inoculation, and all of them were positively converted. Was demonstrated to be an administration system that can sufficiently induce antibodies.

Further, all the plaques of the recombinant virus recovered from the peripheral blood at 6 weeks retain the β-galactosidase activity, and the present vector is not limited to a mere transient antigen administration system, and the enzyme and hormone It has been proved to be an extremely excellent system as a DDS that continuously produces such substances in the body.

Next, for the purpose of confirming the Marek's disease vaccine effect of K-A5L, 6000 PFU of K-A5L was intraperitoneally administered to one-day-old chicks.
After inoculation, one week later, the cells were challenged intraperitoneally with a spleen cell suspension infected with the virulent Marek's disease virus alabama strain. 10 weeks breeding
The effect was determined by observing and declining the occurrence of death or leg paralysis due to Marek's disease during the period, and the presence or absence of a tumor lesion at necropsy after 10 weeks. As a result, as shown in Table 2, there was no significant difference in the anti-MD effect between the vaccines in the test using SPF chicks, but in the test in the field chicks carrying the transfer antibody, there was a large difference between the vaccines. Admitted. That is, 5 cases out of 10 in the KA3VL immunization group and 3 cases in the KA4BL group. On the other hand, only one case was observed in the K-A5L inoculation group, and this recombinant virus was superior to other recombinant viruses in the immunoassay in field chicks carrying the transfer antibody. Showed the effect. From the above, it was confirmed that the region disclosed in the present invention is an insertion site that is superior to other foreign gene insertion sites when considering application in the field.

[0045]

[Table 2]

[0046]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1839 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Marek's disease virus sequence GAATTCCTTC AGCCCAAAGC ACAACCGCGG GGTCCACCGG GACTCTCCGA CATAGCTCGA 60 GCCAAAAGGG AATTGTAGCC CAGGCAAGTG CAATGTTTTA ATTTGTTCTT CTCCCTCCCC 120 CACATAAAAA AACCACTGGA TCGTACAAGT ATACGAGTAT ATGGGTGGGG TGCCGTTTTA 180 TATAAACACA GCTTAGTTTG TTTGCCACGT CAAGGAAGGG CGGTGCATAT CTGTAAGTAA 240 ACAAAACTCG GGGTTCTGTA CGATTGGCCG GGGTCTTACA TGCTCGCCGA ATTGGATTTG 300 AGAATCAATC TTCCGACGGG TTTCCTGACT TGAACAGGGG AAAGGGGGAG GGGGAGTGTG 360 TTATCTTGTC GCGAACCAAT AAAATAGATT TGTGGCCTAA CGAGTTCTCT TTTTTTTTAT 420 ATCGCAAGTG TTAACGAAGC TATGGGACTA GTCTTTTCGT ACAAGTCTCA GACAAACCGC 480 GACCAAAAAA GCGCGGCCCT GTCGAAGAGG AAATACTGAA TCGACGAGCT AACCACAGCG 540 TGTCTCCTGG GTTAACCTCT CTATAGGCGG GGGGAAGTCC CTCTTATATT GGCGAGTGGC 600 CTAGGATTGA ATATTAGACC TCAGTTCCCT ACGGCGCTTT GAGGTAGAGG GAAGTTCTCA 660 GAGCTTGCAT ATGCAAAC GA GATGTTGTAG GAAAAAAAAA GAGGAACCGT GCCTTTCTCT 720 ACGCAGATGG GTCCCCCCCC CCCCCAAAAA AAAAAGGAAC CGTGCCTTCC TCCGCGCAAA 780 TAGGTGCCCC ACGAGGCCTC GGGGTCCGCG GGGCGGAGAG GGGAAAAAGA GTACGGTTCA 840 GGGGATATGA GAACAGCTGC GTATTTTCCC CGTGCATCTC ATACCGCCCA TTTTTGGGTA 900 GAGGTATATT TTTATTAGCC AAATCGTATT CCTGGAAGTT TGACAAAACC TGTCAAACTG 960 ACACGGTCCA AGCGAAACTC GAAAAAAAAA GGGGGGGGGG GAGAATATTC TGTAGGACCG 1020 GCAGAACTTC TCAAGGCAGA GGAAAGATAC ACATTATTTT TTGTTAGATT TAGGCAAGTT 1080 TTGCAGAACC TGCAGGGAAT GTATACACCA TCAAATCTAC TCGACTTATT GCTTGAGTCC 1140 AATTTAACAG AAATTAAAAT ATATTGATGT TGCGACATAT GCATCCTCGC ATATGGGGGT 1200 GGGACACAGG ACGATTATAT CCCCAGACAT GAACCTCAAA CTGCCATTTT GATCCCATCA 1260 TTGGAGAGAC AAATTCGCAT ACATCCTACT TATCGCACAC ATTGGATGTC GGTCTTTATT 1320 CAGGCCATAT CAGCTTTCAC GGGGGCAAAT TCGTATTCAT AGATCCGTCA TCGATGCAGC 1380 GCCAAACCGG ACATATGGAA GACAAAAAGA GAACCGGTTT GGAATCGCAG GGGACCGAGA 1440 ATGCTTTTTC AGATGGCAGA GATGGCAAAG ATGGATTGTT ACATGAAGGA ATTAATGAGC 1500 CCATTTTGAT TCCGTCTACC ATCGCAGA TC TCGAGGGGAT TCGTGAATTG GTCCGAAAAT 1560 TCCGTGGTCG TCTACTGCCC TTTGAAAAGT GTCCCGATTT TTGTCTGAGA ATTGGGGGTT 1620 TGGAGGCCAG CTTTCATAAA GGGCAGGAGG AGCTGTTAGA GTATTGTGAA GCACTTTATT 1680 TACCACAACC TGTTAAGATG GAAATAGTAG GCATTGTAGA CGATGTGCCA TGTCTGGCAA 1740 CGGGGATGCA ATTACTCATT CTTGTTGCCG AAGGGGGGAG AGGTATATGC CTATGAAGAA 1800 GATACTCTGC ATAAGTTAGC CACGAGTTTT TCCGAATTC 1839

SEQ ID NO: 2 Sequence length: 3001 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin organism name: Marek's disease virus sequence GAATTCCTTC CTTTAAAATG AAAGATGTCC AGGTAGACGA TGCGGGATTG TATGTGGTTGTG 60 TGGCTTTATA TAATGGACGT CCAAGTGCAT GGACTTACAT TTATTTGTCA ACCGGTGAAA 120 CACTATCNTT AAATGTGGAA TGTATATGAA AACTACCACA AGCCGGGATT TGGGTATAAA 180 TCATTTCTAC AGAACAGTAG TATCATCGAC GAAAATGAGG CTAGCGATTG GTCCAGCTCG 240 TCCATTAAAC GGAGAAATAA TGGTACTATC CTTTATGATA TTTTACTCAC ATCGCTATCA 300 ATTGGGGCGA TTATTATCGT CATAGTAGGG GGTGTTTGTA TTGCCATATT AATTAGGCGT 360 AGGAGACGAC GTCGCACGCG GGGGTTATTC GATGAATATC CCAAATATAT GACGCTACCA 420 GGAAACGATC TGGGGGGCAT GAATGTTCCG TATGATAATG CATGCTCTGG TAACCAAGTT 480 GAATATTATC AAGAAAAGTC GGATAAAATG AAAAGAATGA GTTCGGGTTA TACCGCTTGG 540 CTAAAAAATG ATATNCCGAA ATTNGNAACC CCTAGATTTA ATCCCACTGA TATGNACANA 600 TTTAAACTTA ATGGGATATA GTATATGGAC GTCTATATGA CGAGAGTAAA TAAACTGACA TGCAAATGA AGCTGATCTA TATTGTGCTT TATATTGGGA CAAACCACTC GCACAAGCTC 720 ATTCAACACA TCCACTCTTG GACAGCTTCA TGTTAAAATA AACTGTAAAT CATTCAATGA 780 TAATGGGAGA AGAATGTGAG CAAGGATCCA TGGTGTCTGC TTTTTATAGT ATCTACCGCA 840 ATGCTACATA TAAAATAAAA ATATACCTCT ACCCAAAAAT GGGCGGTATG AGATGCACGG 900 GGAAAATACG CAGCTGTTCT CATATCCCCT GAACCGTACT CTTTTTCCCC TCTCCGCCCC 960 GCGGACCCCG AGGCCTCGTG GGGCACCTAT TTGCGCGGAG GAAGGCACGG TTCCTTTTTT 1020 TTTTGGGGGG GGGGGACCCA TCTGCGTAGA NAAAGGCACG GTTCCTCTTT TTTTTTTCCT 1080 ACAACATCTC GTTTGCATAT GCAAGCTCTG AGAACTTCCC TCTACCTCAA AGCGCCGTAG 1140 GGAACTGAGG TCTAATATTC AATCCTAGGC CACTCGCCAA TATAAGAGGG ACTTCCCCCC 1200 GCCTATAGAG AGAGGCAGCC CGAAAATGGA GCAGTGTAAA GCAGTACATG GGTGGTGGTA 1260 TGAAACTTGC GAATCGGGCT GTAACGGGGC AAGGCTTGAC TGAGGGGACC ATAGTATGTA 1320 TAGGCNAAAG GCGGGGCTTC GGTTGTANGC GGTTAGGAGT CCCCTCAGGA TACAGTAGTT 1380 GCGCTTTTGC ATAGGGAGGG GGAAATGTAG TCAAATAGAG CCAGAGGCAA CTTGAATAGC 1440 CTAAAGACCA AATAAGGAAA AAGCAAGACA TTCCATATGC TCATTGGTGG CGACTAGATA 1500 AGGAAGGAAT G ACGCAAGGA CATATGGGCG TAGACGAAGC TATGTACGAT TATATAAGCT 1560 GTTGCCACCA TCAAAATAAA ACGCCATTTT ACCATTCACC ACATTGGTGT GCACCTGGGT 1620 AGATGGACAG ACCGTTGAGT CCCTAACGAT TGCGAACACC TGAATGAAGC AGAAGGCTTC 1680 ATTAATGTAG TCAAATAGAG CCAGAGGCAA CTTGAATAGC CTAAAGACCA AATAAGGAAA 1740 AAGCAAGACA TTCCATATGC TCATTGGTGG CGACTAGATA AGGAAGGAAT GACGCAAGGA 1800 CATATGGGCG TAGACGAAGC TATGTACGAT TATATAAGCT GTTCCACCAT CAAATAAACG 1860 CCATTTTACC ATTCACCACA TTGGTGTGCA CCTGGGTAGA TGGACAGACC GTTGAGTCCC 1920 TAACGATTGC GAACACCTGA ATGAAGGAGA AGGCCTCATT AATGTAGTCA AATAGAGCCA 1980 GAGGCAACTT GAATAGCCTA AAGACCAAAT AAGGGAAAAG CAAGACATTC CATATGCTCA 2040 TTGGTGGCGA CTAGATAAGG AAGGAATGAC GCAAGGACAT ATGGGCGTAG ACGAAGCTAT 2100 GTACGATTAT ATAAGCTGTT GCCACCATCA AATAAACGCC ATTTTACCAT TCACCACATT 2160 GGTGTGCACC TGGGTAGATG GACAGACCGN TGAGTCCCTN ACGATTGCGN ACACCTNAAT 2220 GAAGNNGAAG GCCTCATTAA TGNAGTCAAA TAGAGCCAGA GGCTAACTTG AATAGCCTAA 2280 AGGACCAAAT AAGGAAAAAG CAAGACATTC CATATGCTCA TTGGTGGCGA CTAGATAAGG 2340 AAGGAATGAC GCAAGGA CAT ATGGGCGTAG ACGAAGCTAT GTACGATTAT ATAAGCTAAA 2400 CCCAGGAGAC ACGCTGTGGT TAGCTCGTCG ATTCAGTATC CCCCCCNAAN GGCCCCCCCT 2460 TTTTNGGCCC CNGGTTTNCC NNAANCNTTG NCCAAAAANC CTAGCCCAAA AGCNNCGTAA 2520 NNCTTGGGAT NNTAAAAAAA ANGGAGAACN CGTAAGGCCA AAAAANCTAT TTTAATGGGT 2580 CCCCGACAAA NATAAACACA CTCCCCCCTC CCCCTTNCCC CTGTTCAAGT CAGNAAACCC 2640 GTCGNAAGAT TAATTCTCAA AATCCCAATN CGGCGAGCAT GTAAGACCCC GGCCAATCGT 2700 ACAGAACCCC GAGTTTTGTT TACTTGCAGA TATGCACCGC CCTTCCTTGA CGTGNCAAAC 2760 AAACTAAGCT GTGTTTATAT AAAACGGCAC CCNACCCATA TACTCGTATA CTTGTACGAA 2820 CCAGTGGTTT TTTTATGTGG GGGAGGGAGA AGGACAAATT AAAACATTGN ACTTGCCTGG 2880 GCTACAATTC CCTTTTGGCT CGAGCTATGT CGGAGATCNC NGGTCTGAGANTNC CGCTCG G

[Brief description of drawings]

[Fig. 1] Position of S region on MDV1 genome, and further HindI
The positions of the II-B fragment and its EcoRI fragment are shown. The positions of the repeating sequences in the 61-554 strain and BC-1 strain are shown.

FIG. 2 shows the position of the repeat sequence in the A5 fragment.

FIG. 3 shows the positions of repetitive sequences in the A6 fragment and BC-1 strain A6 fragment.

FIG. 4 shows the construction of the insertion plasmid pKA5L used for producing K-A5L.

FIG. 5 shows a theoretical diagram of lacZ gene insertion in K-A5L.

FIG. 6 shows a schematic diagram of the results of Southern hybridization carried out using the LacZ gene (lane 1) and US420 (lane 2) as probes after cutting the DNA extracted from K-A5L-infected CEF with EcoRI.

FIG. 7: Antibody titer in chickens inoculated with K-A5L. The comparison of the antibody titer with the conventional recombinant Marek's disease virus is shown. The upper row is S
The production of anti-MDV antibody in PF chickens, and the lower row shows the production of antibody in field chicks.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12N 15/09 ZNA C12R 1:92) C12R 1:92)

Claims (11)

[Claims] 1. A recombinant Marek's disease virus, wherein a gene portion containing a repetitive sequence contained in a repetitive inverted sequence of a Marek's disease type I virus genome S region gene is replaced with a foreign gene expression cassette. 2. The repetitive inverted sequence is an I sequence in an S region gene.
The recombinant Marek's disease virus of claim 1 which is Rs. 3. The recombinant malec according to claim 1, wherein the foreign gene expression cassette is a gene fragment in which a gene encoding a protective antigen derived from a pathogen other than Marek's disease type I virus is connected downstream of a promoter that functions in animal cells. Disease virus. 4. A foreign gene expression cassette in which a foreign gene is connected to the downstream of a promoter that functions in animal cells, is a fragment derived from the S region repeat inversion sequence of the Marek's disease type I virus gene, and the repeat sequence present in the fragment. And a method for producing a recombinant Marek's disease virus, characterized by incorporating the foreign gene expression cassette into the Marek's disease type I viral genome using the recombinant gene fragment thus obtained. . 5. The repetitive inverted sequence is I in the S region gene.
The method according to claim 4, which is Rs. 6. The S region IRs-derived fragment of the Marek's disease type I virus gene is an S region-derived A5 fragment (a gene fragment obtained by EcoRI treatment, which contains a boundary between IRs and Us). The method according to claim 4. 7. The method according to claim 4, wherein the portion containing the repetitive sequence is a gene region containing the repetitive sequence portion on the 3 ′ side in the IRs region and the boundary portion between IRs and Us. 8. The method according to claim 4, wherein the foreign gene expression cassette is a gene fragment in which a foreign gene encoding a pathogen-derived protective antigen other than Marek's disease type I virus is connected downstream of a promoter that functions in animal cells. . 9. An avian vaccine comprising the recombinant Marek's disease virus of claim 1. 10. The vaccine according to claim 9, wherein the bird is a chicken. 11. The avian bioactive substance administration vector comprising the recombinant Marek's disease virus according to claim 1, wherein the foreign gene incorporated in the foreign gene expression cassette is a gene fragment encoding a bioactive substance.