JPH0622757A - Recombinant marek's disease virus and its production - Google Patents

Abstract

PURPOSE:To provide the excellent virus capable of inducing the immunity to Marek's disease and the immunoreaction against the proteins manifested by incorporated exogenous genes through inoculating into chickens, and to provide polyvalent live vaccine for chickens from the above virus. CONSTITUTION:The Us region of Marek's disease virus or a gene derived from the reiterated inverted sequence adjacent thereto is incorporated into a plasmid, which is also incorporated with an exogenous gene-manifestative cassette in order to manifesting an exogenous gene in said gene, thus preparing a recombinant plasmid. This plasmid is then introduced into Marek's disease virus genome to obtain the recombinant Marek's disease virus for excellent polyvalent live vaccine for chickens.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel viral vector capable of expressing a foreign gene product in a chicken cell or chicken body. Further, the present invention relates to the production of a multivalent live vaccine for chicken using this vector and the construction of a recombinant Marek's disease virus 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. One possible solution to this problem is to simply mix several existing vaccines. However, in the case of live vaccine, there is a problem of mutual interference of viruses, and in the case of inactivated vaccine mixture, there is a limit in capacity. In addition to these problems, mixing live vaccines with inactivated vaccines has the problem of lowering the titer due to adsorption of live vaccine antigens on gels (adjuvants), and conventional techniques have practically left problems. Has been done.

In view of these points, it has been attempted recently to use a viral vector as a second breakthrough measure. That is, it is a method of producing a multivalent live vaccine by incorporating genes of a plurality of vaccine antigens into one virus. By this method, it is considered possible to produce a multivalent vaccine which does not cause the above-mentioned problems of mutual interference of viruses or an increase in the inoculated amount when an inactivated vaccine is mixed.

To date, research aimed at using viruses as vectors 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, since some of these viruses belong to tumorigenic viruses, administration to humans or animals is not practical because of many problems in terms of safety. Further, even if there is no problem in the safety of the virus itself, when viewed from the viewpoint of the viral vector for birds that is the object of the present invention, since birds are not the original host, they are tentatively used as viral vectors for birds. Even if you do, you can't expect much from the effect.

From these points, it is considered to use avian poxvirus (eg, chicken fowlpox virus) as a vector, and it has already been investigated as a viral vector, and 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.

[0006] On the other hand, Marek's disease is a malignant tumor whose onset is protected by a vaccine, including humans and animals. The mechanism of defense is Marek's disease in birds such as chickens due to persistent infection of the vaccine virus throughout life. It is considered that humoral and cell-mediated immunity to Marek's disease virus is elicited and maintained for life, and as a result, tumorigenesis by virulent virus is controlled. In addition, since this virus vaccine is administered in the form of live cells infected with virus and proliferates in the form of cell-to-cell infection in vivo, unlike other vaccine viruses, it can be administered to primary chicks with almost no effect of migratory antibodies. Is a major feature.

[0007] 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 described above, in order to prepare a multivalent live vaccine using a Marek's disease virus vector having characteristics far superior to those of other virus vectors, it is necessary to first insert a foreign gene into the Marek's disease virus DNA. It is essential to find possible parts.

[0008] The TK gene and the gA antigen gene have been discussed as potential 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 there is no report of a recombinant virus inserted with a foreign gene into the TK gene. 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 (1991), No. 11).
1st Japanese Society of Veterinary Medicine].

[0009] Further, gA is a major glycoprotein produced by the virus together with gB, and although no neutralizing antibody such as gB has been produced so far when inoculated into an animal body, 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 such a situation, the present inventors have previously conducted research on the production of a recombinant Marek's disease virus that is effective even for a gene whose analysis has not yet progressed, and have conducted research on BamHI of the Marek's disease type I viral gene. We have found that a recombinant Marek's disease virus can be prepared by inserting a foreign gene into the -H fragment (Japanese Patent Application No. 63-22696).
No. 0).

[0011]

[Objective of the Invention] Since then, as a result of intensive studies on the development of a more effective viral vector, the present inventors have found that a site capable of integrating a foreign gene into the Marek's disease virus gene genome very efficiently and stably. I found it. Furthermore, it was confirmed that the recombinant Marek's disease virus constructed in this manner exhibits excellent growth stability not only in vitro but also in vivo without losing the characteristics of the original Marek's disease virus. It was confirmed to be extremely excellent as a viral vector used as a monovalent vaccine. That is, the present invention includes a novel recombinant Marek's disease virus which can be used as a vaccine for birds, a method for producing the same, and a multivalent live vaccine for birds and hormones using the recombinant Marek's disease virus. The present invention provides a vector for administering a physiologically active substance as described above into a chicken body.

[0012]

At present, as a vaccine against Marek's disease, attenuated Marek's disease type I virus (MDV) is used.
-Type I), turkey hepes virus (HVT), or a mixture of Marek's disease type II virus and turkey herpes virus. It is known that Marek's disease itself is caused by infection with type I virus, and therefore, it is basically necessary to use an attenuated strain of Marek's disease type I virus, which belongs to the same serological group, as a vaccine to prevent its onset. Conceivable. That is, in the present invention, when the recombinant Marek's disease virus of the present invention is used as a multivalent vaccine including Marek's disease vaccine, it is preferable to use Marek's disease type I virus.

The foreign gene insertion region in the preparation of the recombinant Marek's disease virus found by the present inventors is the Us region of the Marek's disease virus genome, and the repetitive inverted sequence. . Us area is MD
A gene region having a length of about 12 kb, which is located at the 3'end of V-DNA and is flanked at both ends by inverted repeat sequences. By incorporating a foreign gene into this region, it is possible to stably express the foreign gene of interest and to prepare a recombinant Marek's disease virus without losing or reducing the properties of the Marek's disease virus required as a Marek's disease vaccine. It will be possible. The Marek's disease virus Us region-derived gene fragment used in the present invention refers to a foreign gene introduction site capable of incorporating a foreign gene, for example, a Us region-derived gene fragment of at least about 1 kbp having an appropriate restriction enzyme recognition site. As a preferred example of such a Us region-derived gene fragment, the Marek's disease type I virus gene is a restriction enzyme EcoR.
A gene fragment of about 2.8 kbp obtained by treating with I and having a BalI site (A4 fragment) can be mentioned. In addition, the repetitive sequence spontaneously present in the repetitive inverted sequence disclosed in the present invention can be removed without affecting the growth of the virus, and like the above fragment,
This is a valuable region where a foreign gene can be inserted without affecting the virus growth.

There has been no previous report that antibodies in the chickens inoculated with the recombinant Marek's disease virus persisted to the product of the gene inserted in the viral genome for a long period of time, and cells such as β-galactosidase were not present. The development of an excellent immunization method in which an antibody against a protein expressed therein is maintained for 4 months or more with a single inoculation at the time of initiation as shown in the present invention has never been reported. Therefore, when a protein expressed on an infected cell such as a membrane protein such as Newcastle disease virus (NDV) or infectious tracheitis virus (IBV) is expressed in the chicken body using the immunization method according to the present invention. Are expected to induce stronger immunity. In fact, the recombinant virus Marek's disease virus in which the NDV-F protein gene was inserted as disclosed in the present invention had a sufficient effect of protecting against Newcastle disease for about 4 months after inoculation. In addition, for a protein that does not originally have a structure to be expressed on the cell membrane and outside the membrane, a signal peptide can be added to its N-terminal [Nucleic Adids Research, 1
4, p4683-4690 (1986)], and the construction of a gene that can be secreted extracellularly and that is to be expressed on the cell membrane by adding an anchor region rich in hydrophobic amino acids to the C-terminus. It is possible to induce stronger immunity to the inserted gene expression product by expressing in such a form.

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 good in vitro growth property,
Pathogenicity, which is a manifestation of proliferative activity or proliferative activity, is clearly reduced [Bernard Meignier
Et al., The Herpes viruses 4, p265 (1985)]. Therefore,
The produced recombinant virus or recombinant vaccine is i
In order to determine whether it can be applied in vivo, 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 a chicken inoculation test of the produced recombinant virus and confirmed its effect. As a result, the recombinant Marek's disease virus prepared according to the present invention was confirmed to be persistently infected for 16 weeks or longer in the chicken body, and it was confirmed that the original proliferation of Marek's disease virus was not lost. In addition, its good immunogenicity was confirmed from the persistence of antibodies against Marek's disease virus and the long-term persistence of antibodies against β-galactosidase, which is the inserted gene product, and the excellent Marek's disease preventive effect and Newcastle disease preventive effect of recombinant viruses. Was done.

As described above, the recombinant virus produced according to the technique disclosed in the present invention proliferates and persistently infects in the chicken body and has not only a protective effect against the virulent Marek's disease virus but also a foreign gene product. It is also possible to raise antibodies. Such a good in vivo
The technique for producing a recombinant virus exhibiting the proliferative and immunogenic properties of Escherichia coli is most remarkable in the present invention, and
It is the first technology that has shown application, that is, the potential of a recombinant multivalent live vaccine.

That is, according to the present invention, when a foreign gene encoding a vaccine antigen against other diseases is incorporated into Marek's disease virus and inoculated into birds, the recombinant virus is produced by a mechanism similar to that of the original Marek's disease virus. It is expected that an antigen derived from a foreign gene will be expressed in the inoculated individual over a long period of time or life and the humoral or cellular immunity to the antigen will be induced and maintained. According to the present invention, it is possible to produce a multivalent live vaccine capable of immunizing not only Marek's disease but also a large number of pathogens with a single administration at the time of birth to birds such as chickens. Further, as shown by the fact that this recombinant virus expressed β-galactosidase in the body for a long time after inoculation, the present technology is not limited to a mere antigen administration system,
It can also be used as a valuable drug administration system for administering a physiologically active substance such as a hormone into a living body.

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 Us region derived from Marek's disease virus or a gene having a repetitive inversion sequence, and the objective is located downstream of a promoter derived from an animal cell or an animal virus. Has a foreign structural gene and a structure in which a transcription termination factor is further connected downstream thereof. still,
A promoter and a foreign structural gene, and if necessary, a gene fragment in which 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, and is therefore used in the present invention. The insertion plasmid is U derived from Marek's disease virus.
It can also be expressed as a plasmid having an s region or a repetitive inverted sequence gene and having a foreign gene expression cassette incorporated in the gene region. By using such a plasmid, the virus-derived gene fragment on the insertion plasmid can be isolated from the viral DN at the homologous portion.
As a result, the foreign gene fragment sandwiched between the viral gene fragments is integrated into the viral genome. In addition, the promoter derived from an animal cell or an animal virus as used herein is not limited to a particular one, including various promoters for expression of animal cells known so far.

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, (2) is a structural gene in which each viral fragment cloned in a plasmid is cut at a single site with an arbitrary enzyme or partially deleted to express a promoter that functions in an animal cell. 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, examples of the foreign gene incorporated into the genome of Marek's disease virus include various genes encoding proteins that can be vaccine antigens for various chicken diseases such as viral diseases, bacterial diseases and parasitic diseases. .
For example, in the preparation of a multivalent vaccine for chickens,
As a foreign gene to be incorporated therein, a gene encoding a Newcastle disease virus (NDV) antigen, for example, NDV
-Gene encoding F protein or HN protein, gene encoding glycoprotein of chicken infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBD)
V) a gene encoding a viral structural protein, eg V
A gene encoding P2, infectious bronchitis virus (I
The gene encoding the spike protein of BV) and the gene encoding the HA protein of Hemophilus paragallinarum which is the causative bacterium of infectious coryza.

In the present invention, as a concrete production example of a recombinant live vaccine, a construction method of a recombinant Marek's disease virus that protects against Newcastle disease and its effect will be mentioned. As a gene of the Newcastle disease virus to be inserted, A cDNA derived from the super attenuated strain D-26 is used.
The pathogenicity of Newcastle disease virus is determined by whether or not the F protein is a virulent form, or whether or not the HN protein is a virulent form. Therefore, when a recombinant virus is produced using the highly virulent F or HN gene, there is a risk of acquiring new pathogenicity which the vector virus originally did not have. In order to eliminate such a possibility, in the present invention, any gene derived from the attenuated ultra-attenuated strain D-26 is used. That is, according to the present invention, an extremely excellent live vaccine having safety in addition to a long-term effect not provided by conventional Newcastle disease vaccines can be produced.

The Marek's disease virus vector is useful not only as a protective antigen for these infections but also as a vector for administering 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, it is possible to incorporate these genes into a Marek's disease virus vector as a drug delivery system (DDS).

Regarding the method of selecting the recombinant virus in (4), a measurement system according to the foreign gene incorporated into the virus for expression is used. For example, when a recombinant Marek's disease virus expressing Newcastle disease virus F antigen is prepared for the purpose of multivalent vaccine with Newcastle disease vaccine, the desired Newcastle disease vaccine can be obtained by detecting NDV-F antigenicity. It is possible to select a recombinant Marek's disease virus that can become When an enzyme gene is incorporated, the recombinant Marek's disease virus can be screened using the enzyme activity as an index. As a specific example, as shown in the present Example, when a gene (LacZ gene) encoding β-galactosidase (β-gal) is incorporated into Marek's disease virus, and a recombinant virus expressing β-galactosidase is selected For example, by adding a substrate for β-galactosidase [eg, X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside)] to the agar solution overlaying the cell sheet, It is possible to color-code viral plaques with β-galactosidase activity.

Further, the cell f disclosed in the present invention is
Ree virus-producing LacZ (+) Marek's disease virus is used as a parent strain, and the LacZ gene is replaced with the target gene by homologous recombination (hereinafter referred to as the reverse method).
By selecting and cloning Z (-) plaques, it becomes possible to easily prepare a recombinant Marek's disease virus in which a foreign gene of interest is incorporated. This reverse method is an excellent method for preparing a recombinant Marek's disease virus in which a foreign gene of interest is incorporated. In conventional Marek's disease type I virus, cell f
Since almost no ree virus was produced, it was impossible to apply such a reverse method, but the problem is solved by using the cell free virus-producing Marek's disease virus of the present invention.

The present invention will be described in more detail below with reference to Preparation Examples and Examples, but the present invention is not limited thereto.

[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 2X103 were inoculated into the abdominal cavity of 1-day-old chicks, no onset or death occurred 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. ., 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
After 2) was added and ice-cooled for 30 minutes, pipetting was performed.
This solution was centrifuged at 2500 rpm for 10 minutes, and the supernatant was
0% (w / w) sucrose solution (0.02M-Tris-HCl, pH7.4,0.15M-
NaCl), centrifuged at 175KG for 2 hours, and then washed with 40% sucrose solution and 6%
The layer of capsid derived from Marek's disease virus formed in the middle of 0% 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% -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 the polylinker of pUC119 and then transduced into competent cells such as JM109. After culturing the obtained transformants overnight in LB medium,
The 30 # was infected with M13 phage (109 / ml or more) at 60 # and further shake-cultured overnight. 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, cat. No. 6030), a plasmid shortened stepwise was constructed, and ssDNA was similarly prepared.

Production of Recombinant Virus Primary CEF cultured overnight at 37 ° C. was harvested with EDTA-trypsin solution, washed, and then 5% bovine serum (BS) -added igloo 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 cells to the cuvette of Bio-Rad Gene Pulser (cat.No.165-2075), and insert. The insertion plasmid was added and pulsed according to the attached protocol to introduce the insertion plasmid into the virus-infected cells. Then, the cells were suspended in 15 ml of 5% BS-added E-EME (Japanese water), transferred to a petri dish (No. 3003 manufactured by Falcon) having a diameter of 10 cm, and cultured at 37 ° C. On the next day, dead cells that had not engrafted were removed together with the medium, and the newly harvested primary CEF was harvested (2nd CEF) at 500,000 cells /
15 ml of 5% -BS-added E-MEM suspended in ml was added. 4 at 37 ° C
After culturing for 7 to 7 days, chlorophenol red β-D-
A 1% -agarose / E-MEM solution (without phenol red) containing galactopyranoside (Seikagaku Corporation) at a concentration of 100 μg / 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 labeli
ng Kit (cat.No.150350) and TROPIX Southern-light
(cat. No. SL100) was used to prepare a probe and hybridize it according to the attached protocol. Briefly, linearized DNA was denatured by heating, and probe DNA was prepared using dNTPs containing digoxigenin-labeled dUTP by Klenow fragment with random primers as a substrate.
Was synthesized.

This probe was transferred to a high bond N + (Amersham Japan, cat.No.RPN.303) to which the target DNA was transferred.
Hybridize according to the protocol in (B),
Alkaline phosphatase labeled anti-digoxigenin sheep
Detection was performed using IgG. AMPPD was used as a substrate for alkaline phosphatase, and the generated specific luminescence was detected using an X-ray film (FUJIXEROX, X-OMAT).

Recovery of Recombinant Viral DNA Petri dish inoculated with virus (Falcon, cat. No. 1
When 50350) showed CPE on the entire surface, the supernatant was aspirated and proteinase K solution (proteinase K 1 mg / ml, 0.1M-Tr
isHCl.pH 9.0, 0.1M-NaCl, 0.001M-EDTA, 1% SDS) 2m
After being poured and treated at 37 ° C for 1 hour, the mixture was transferred to a conical tube (Falcon, cat. No. 2099) and treated at 37 ° C overnight.
Then, the phenol-treated supernatant was ethanol-precipitated,
After drying, it was dissolved in 100 μl of TE (10 mM-Tris.HCl.pH8.0, 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, cat.No.2099) and centrifuged at 1500 rpm for 30 minutes (K
UBOTA, KN-30F). Intermediate layer consisting of lymphocytes and monocytes (Buff
y coat) was collected, suspended again in PBS-containing 0.01% EDTA, centrifuged at 1000 rpm for 5 minutes and collected, and inoculated into CEF (2ndary) that had been cultured for 4 hours. I observed the day. MDV
For those in which CPE was not observed, the subculture was repeated up to the third generation for repeated judgment.

Fluorescent antibody method (FA) 61-554 strain 104PFU with CEF107cell 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.
Plasmid pASLacZ to which gene is connected (Japanese Patent Application No. 63-2269)
No. 60) was introduced into BMT-10 cells by electroporation, cultured for 2 days, and similarly fixed with acetone to -80
Stored at ° C.

[0039] 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., Cat. No. 031506) was diluted 20-fold with PBS-, reacted at 37 ° C for 1 hour, washed, and observed under a fluorescence microscope.

ELISA β-gal (Toyobo code No.GAH-201) 20 ml was dispensed into 96 well flat bottom plate (Nunc cat.No.473768) by 100 μl per well and reacted overnight at 4 ° C., 0.15M After washing with -PBS (pH 7.3), blocking was performed with 1% BSA / 0.15M-PBS at 37 ° C for 2 hours.

The test serum was diluted 800-fold with PBS- and then diluted 100 times.
Add μl and incubate at 37 ℃ for 1 hour.
Double-diluted anti-chicken IgG POD-labeled rabbit antibody (Nordic) and TMBZ (Dojindo, cat.No.346-04031) were reacted, and the reaction was stopped with 1N-H2SO4, then OD450nm / 63
The absorbance at 0 nm was measured, and the value obtained by subtracting the absorbance at 630 nm from 450 nm was taken as the ELISA value.

[0042]

Examples Example 1: Cloning of 61-554 strain DNA 1 μg of 61-554 strain DNA was cleaved with HindIII and subjected to 0.7% -agarose gel electrophoresis to give a 17 kb fragment (hereinafter, HindIIIB fragment).
Was excised and cloned into pBR322. This HindI
After cutting the II-B fragment with EcoRI, it was subcloned into the plasmid pUC119 (hereinafter referred to as Δpuc119) in which the BamHI site to the HindIII site were deleted at the multicloning site (MCS) (pKHB). The EcoRI cleavage pattern of pKHB is shown in Fig. 1.

EcoRI fragment of 61-554 strain, KA3 (5.2 Kb), KA4 (2.
The positions of 8Kb) and KA5 (2.1Kb,) KA6 (2.4Kb) are shown in FIG.
The plasmids containing each fragment were designated pKA3, pKA4, pKA5, pKA, respectively.
I named it 6. Of these, the A5 and A6 fragments were shown to differ in length between viruses (Table 1).

[0044]

[Table 1]

Example 2: Production of K-A4BL At the BalI site of pKA4, pCH110 (Pharmacia cat. No. 27) was prepared.
-4508-01) was excised with BamHI and TthlllI and a blunt-ended 4.2 Kb fragment (SV40-LacZ) was inserted (pKA4B
L, Fig. 3).

After cutting pKA4BL with KpnI and linearizing it,
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-A4BL, the DNA extracted from KEF-infected CEF was cleaved with EcoRI and then subjected to Southern hybridization. LacZ excised with HindIII and BamHI from KA4 fragment and pCH110
The results of Southern hybridization using the gene (3.7K) as a probe are shown in FIG. KA4 fragment (lane 2), L
Only the expected size band was detected using either probe of the acZ gene (lane 1), confirming that the LacZ gene was inserted by homologous recombination and that the recombinant virus was purified. It was

Table 2 shows the virus recovery test and the anti-MDVFA antibody test results in SPF 1-day-old chicks inoculated with K-A4BL 7000 PFU. Virus recovery at week 1 and week 6 after inoculation was 4/5 and 5/5 (+), respectively, and anti-MDV FA antibody at week 6 was
It was 7/7 (+). On the other hand, anti-β-gal was observed in the 7th week serum.
The antibody titer was measured by ELISA. 7 as shown in FIG.
All cases showed higher values than the control group (HVT + SB-1 inoculation group),
All of these were also positive in FA using β-gal as an antigen, which has a lower sensitivity as an antibody detection system, and confirmed that anti-β-gal antibodies were produced in K-A4BL-inoculated chickens. Further, anti-β-gal antibody was positive in all cases even at 16th week, and virus recovery was also good at 2/7 (+) at 16th week. At this time, HVT or CVI988cl
The virus recovery rate from chickens inoculated with 20,000PFU of oneC was 2/12 (+) in all cases [Witter et al., Avian Diseases
31, p829-840 (1987)]. That is, the results of this time prove that this A4BL has a persistent infectivity equal to or higher than that of the original Marek's disease vaccine virus strain, and retains extremely preferable properties as a vector. Surprisingly, the antibody against β-gal was already positive at 4 weeks after the inoculation, and after all cases had been positively converted, the antibody titer (FA value) was 160 times or more over 4 months. The system proved to be a very good vaccination system.

Furthermore, all the plaques of the recombinant virus recovered from the peripheral blood at 6 weeks retain 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.

[0050]

[Table 2]

On the other hand, the virus recovery and the titer from the virus non-inoculated chickens raised in the same isolator as the recombinant virus inoculation group were negative, and it was revealed that this recombinant virus lacks the cohabitation infectivity. became. That is,
It has been shown to be very practical in that the recombinant virus remains in the inoculated individual.

Next, for the purpose of confirming the Marek's disease vaccine effect of K-A4BL, 2000 P of K-A4BL was intraperitoneally injected into the abdominal cavity of one-day-old chicks.
FU or 6000 PFU was inoculated, and one week later, the rats were challenged intraperitoneally with 5000 PFU of the virulent Marek's disease virus alabama strain. The animals were bred and observed for 10 weeks, and the effect was determined using the death or leg paralysis caused by Marek's disease during the period and the presence or absence of tumor lesions at necropsy after 10 weeks as an index. As a result, as shown in Table 3, all 10 animals died or developed in the non-immunized control group, while 20 in the K-A4BL inoculation group.
Only one case was observed in the 00 PFU inoculation group, and it was confirmed that this recombinant virus has sufficient immunogenicity as a Marek's disease vaccine.

[0053]

[Table 3]

Example 3: Inserter for producing recombinant virus
Construction of the transfection vector pKA4B pUC119 was digested with MflI and then electrophoresed on a 1% -agarose gel to select from the multiple cloning sites HindIII to XbaI sites (hereinafter referred to as cloning sites).
A 1.0 kb fragment containing pSV2-dhfr (AT
CC No. 371464) was inserted into the BglII site (pSV2-dl
n). From this plasmid, by using HindIII and BamHI with a cloning site upstream of the polyA addition signal.
A 1.0 kb fragment was cut out and inserted into the downstream of the SV40 early gene promoter of pCH110 in the form of replacing the LacZ gene (pSVEA). After cutting pSVEA with PvuII and BamHI, SV
40 early gene promoter cloning site and poly
A 1.3 kb fragment having an A addition signal was blunt-ended and then inserted into the BalI site of pKA4 to construct pKA4B (FIG. 6).

After cloning the gene to be expressed at the cloning site of the present pKA4B, the recombinant Marek's disease virus having the gene can be easily produced by producing recombinant virus and performing homologous recombination according to Example 2. Can be done. By infecting cultured cells or chickens with the recombinant virus, the protein encoded by the gene can be efficiently expressed using the SV40 early gene promoter and polyA addition signal.

Example 4 Production of K-A4BF The fusion protein gene (F gene) derived from the ultra-attenuated NDV, D-26 strain was inserted into the SmaI site of pSVL (Pharmacia, Code No. 27-4509-01) ( pSVLF, Figure 7). This plasmid is EcoR
After digestion with I and SalI, the 4.3 kb fragment was recovered by electroelution and blunt-ended, and then the pKA4 BalI fragment was isolated.
The insertion plasmid was inserted into the site to construct the insertion plasmid pKA4BF (Fig. 7).

After linearizing the plasmid with PvuI, recombination was performed according to the procedure for producing a recombinant virus. However, the recombinant virus was cloned using monoclonal antibodies # 83 and # 313 [Y. Umino et a
l., J.gen.virol., 71, p1199 (1990)].

The cloning method of the recombinant virus by immunostaining is shown below. A few days after the recombination, the dish in which plaques appeared was washed with E-MEM medium, and then an isotonic solution containing monoclonal antibodies # 83 and # 313 was added to the dish at room temperature for 1 hour.
0 minutes ♪ reacted for 60 minutes. After washing, 100 times with isotonic solution
Add peroxidase-labeled anti-mouse antibody (Bio-Rad, code No.172-1011) diluted 200 times, and further add 1 at room temperature.
0 minutes ♪ reacted for 60 minutes. After washing, 3,3-Diam per 10ml
inobenzidine Tetrahydrochloride (DAB, Wako, code N
o.343-00901) 5 mg and hydrogen peroxide solution (Mitsubishi Gas Chemical Co., Ltd., H2O2
0.1M-Tris buffer (pH 7.5) containing 1.6 # (containing 31%) was added and reacted at room temperature for 5 minutes and 60 minutes to stain the plaque of the recombinant virus. Surround the plaque stained in brown with a ring-shaped object and recover the recombinant virus-infected cells by digesting only the inside of the ring with a trypsin solution containing EDTA at 0.1% and co-culturing with new CEF. Thus, the recombinant virus was purified. The recombinant virus was purified by repeating this operation and cell free treatment by ultrasonic treatment several times. The purified recombinant virus showed good in vitro growth comparable to that of the parent strain.

An immunological test was carried out to examine the protective effect of K-A4BF against Newcastle disease (ND). After inoculating 10-day-old K-A4BF into the abdominal cavity of one-day-old chick, it was attacked intramuscularly in the lower leg with a lethal dose of 10 4 virulent NDV Sato strain at 3, 9 and 16 weeks. As a result, in the non-immune control group, all developed or died during the 2-week observation period, whereas in the K-A4BF-inoculated group, no single bird developed or died, and this recombinant virus was sufficient for Newcastle disease. It was shown to have a vaccine effect (Table 4).

[0060]

[Table 4]

Example 5: Production of K-A4BHN The HN protein gene derived from strain D-26 was inserted into the SmaI site of pSVL. After partial digestion of this plasmid with EcoRI and SalI, a 4.5 kb fragment was recovered by the electroelution method, blunt-ended, and inserted into the BalI site of pKA4 to construct an insertion plasmid pKA4BF (FIG. 8). .

After linearizing the plasmid with PvuI, recombination was carried out according to the procedure for producing a recombinant virus. However, the cloning of the recombinant virus was performed using monoclonal antibodies # 193, # 142 and # 265 which recognize the HN protein.
[Y. Umino et al., J.gen.virol., 71, p1189 (1990)] was used according to the method for producing K-A4BF. The purified recombinant virus showed good in vitro growth comparable to that of the parent strain.

Example 6: Production of K-A3VL SV40-LacZ was inserted into the EcoRV site of pKA3 (FIG. 9). This was cut with KpnI and linearized, and then K-A3VL was produced according to the production of the recombinant virus.

To confirm the insertion position of the LacZ gene, K-
The DNA extracted from A3VL-infected CEF was cleaved with EcoRI and then subjected to Southern hybridization (FIG. 10). As a result of using the KA3 fragment and the LacZ gene as a probe, only the band of the expected size was detected in both cases, and it was confirmed that the LacZ gene was inserted by the homologous recombination and the recombinant virus was purified. It was Moreover, the plaques of K-A3VL were smaller than the plaques of the original virus before recombination, and their growth was obviously inhibited.

Example 7: Repeated distribution in IRs and TRs
Nucleotide sequence determination After cutting pKA5 with StuI, the excised fragment of about 220b was cloned into the SmaI site of pUC119 to determine the nucleotide sequence. When the result was compared with the nucleotide sequence of the GA strain (GenBank Accession Number M80595, M. Sakaguchi), it was revealed that the region corresponding to nucleotide numbers 614 to 822 was repeated twice. On the other hand, pKA6
As a result of examining the nucleotide sequence of, the region was repeated 5 times.

Further, the nucleotide sequence of the fragment corresponding to A6 of BC-1 strain was determined. As a result, in TRs almost the same as KA6,
It was confirmed that the nucleotide sequence of 278b showing a homology of 88% with the avian leukemia virus (ALV, RAV2) DNA was integrated and repeated 3.5 times. The position of the repeating sequence is shown in FIG. Sequence Listing: SEQ ID NO: 1 shows the nucleotide sequence of A6 fragment of BC-1 strain. Sequence Listing: The Us region gene is located at the 5'side (smaller base number direction) from the 853th nucleic acid in SEQ ID NO: 1, and the TRs region gene is located at the 3'side from the 854th nucleic acid (direction of increasing base number). Equivalent to. From the above results, it was revealed that there is a site into which the gene can be inserted in the portion of IRs and TRs which is in contact with the US region without affecting the virus growth.

Example 8: Creation of K-A5SL Since it was confirmed that the TRs and IRs flanking the US region have a site into which a foreign gene can be inserted, an insertion plasmid was constructed using pKA5. . First,
The pKA5 was cleaved with StuI to release a repeat sequence of about 220 bp, the 5.1 kb fragment was recovered by agarose gel electrophoresis, and then dephosphorylated with alkaline phosphatase. Next, pCH110 (Pharmacia cat.No.27-4508-01)
The fragment was excised with BamHI and TthlllI and the 4.2 Kb blunted fragment (SV40-LacZ) was inserted to construct the insertion plasmid pKA5SL (Fig. 12).

This plasmid was digested with KpnI and linearized, and then recombined according to the procedure for producing a recombinant virus. The purified recombinant virus is as good as the parent strain.
It showed in vitro proliferation.

Example 9: Determination of nucleotide sequence of KA4 pKA4 was digested with BalI and SmaI to give 1.1 kb and 4.9 kb.
And then each was recovered. The 1.1 kb fragment was cloned again into the SmaI site of Δpuc119, and the 4.9 kb fragment was subjected to self-ligation, and then ssDNA was similarly prepared according to the determination of the base sequence to determine the base sequence. The results are shown in the sequence listing: SEQ ID NO: 2 and FIG. An ORF consisting of 639 bases was found in this base sequence (US639).

Example 10: Cell free virus producing type K
-Creation of A4BL After inoculating 100,000 PFU of K-A4BL-infected cells with a primary CEF of 30 million into a petri dish having a diameter of 10 cm, CPE was exhibited on the entire surface 4
The supernatant was collected on the day. The supernatant was centrifuged at 1500 rpm for 10 minutes (KUBOTA KN-30F), and after adding 30% of the original CEF and 5% of BS, pH was adjusted with 0.7% sodium bicarbonate aqueous solution.
Was adjusted to around 7.4, and then inoculated into a petri dish having a diameter of 10 cm and cultured. After repeating this operation 5 times, the supernatant was further diluted 10 times with 5% BS-added E-MEM, and the second CE was performed.
After adding 500,000 F, 100 μl of each was inoculated into a 96-well plate and further cultured. After 5 days, cells were collected from the wells in which single plaques appeared, and inoculated into a petri dish having a diameter of 5 cm together with 10 million primary CEFs. The supernatant was inoculated into a 96-well plate, and the supernatant of the well in which a single plaque appeared was treated three times in the same manner to obtain a cell free virus high-producing clone. 200 to 500 PFU / ml of LacZ (+) virus was produced in the CEF supernatant cultured for 5 days after inoculation of this virus with 10,000 PFU. In contrast,
Only the virus produced under the same condition was produced.

The recombination efficiency by normal homologous recombination is 0.1
It is said to be around%. Therefore, 200 to 500 PFU / ml
By using K-A4BL, which produces the cell free virus of E. coli, as the parent strain, it is possible to efficiently produce the virus by the reverse method.

[0072]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3001 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Marek's disease virus sequence GAATTCCTTC CTTTAAAATG AAAGATGTCC AGGTAGACGA TGCGGGATTG TATGTGGTTG 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 AAAAGAATGG GTTCGGGTTA TACCGCTTGG 540 CTAAAAAATG ATATNCCGAA ATTNGNAACC CCTAGATTTA ATCCCACTGA TATGNACANA 600 TTTAAACTTA ATGGGATATA GTATATGGAC GTCTATATGA CGAGAGTAAA TAAACTGACA 660 CTGCAAATGA AGCTGATC TA 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 GACGCAAGGA CATATGGG CG 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 GCAAGGACAT ATGGGCGTAG ACG AAGCTAT 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 CGGAGAGTNC CGGTGGACCC GNGGTTGTGC TC GGANCGACTGACGTCGAGGANCCCT GGGNGNGCTCT

[0073]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 900 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Marek's disease virus sequence TGTTGCCTTT TTGTTGTATA TGAAGATATT TAATGTGGCG TTGAGCCTAA TGAGAGGAGA 60 ACGTGTTTGA ACACTGGA CGAGCGCCGT GTAAGATTAA AACATATTGG AGAGGTATGG 120 CCATGTGGTC TCTACGGCGC AAATCTAGCA GGAGTGTGCA ACTCCGGGTA GATTCTCCAA 180 AAGAACAGAG TTATGATATA CTTTCTGCCG GCGGGGAACA TGTTGCGCTA TTGCCTAAAT 240 CTGTACGCAG TCTAGCCAGG ACCATATTAA CCGCCGCTAC GATCTCCCAG GCTGCTATGA 300 AAGCTGGAAA ACCACCATCG TCTCGTTTGT GGGGTGAGAT ATTCGACAGA ATGACTGTCA 360 CGCTTAACGA ATATGATATT TCTGCTTCGC CATTCCACCC GACAGACCCG ACGAGAAAAA 420 TTGTAGGCCG GGCTTTACGG TGTATTGAAC GTGCTCCTCT TACACACGAA GAAATGGACA 480 CTCGGTTTAC TATCATGATG TATTGGTGTT GTCTTGGACA TGCTGGATAC TGTACTGTTT 540 CGCGCTTATA TGAGAAGAAT GTCCGTCTTA TGGACATAGT AGGTTCGGCA ACGGGCTGTG 600 GAATAAGTCC ACTCCCCGAA ATAGAGTCTT ATTGGAAACC TTTATGTCGT GCCGTCGCTA 660 CTAAGGGGAA TGCAGCAATC GGTGATGATG CTGAATTGGC ACATTATCTG ACAAATCTTC 720 GGGAATCGCC AACAGGAGAC GGGGAATCCT ACTTATAACT AATCGCACAA TTATTAATAG 780 GATTTTAGGA AAAACTGCTA CTAACGTTGT TTAAATAATA AAATTTTATT TTCAATAAGG 840 CATTACAGTTG CATGAATGATT

[Brief description of drawings]

FIG. 1 shows a schematic diagram of an EcoRI cleavage pattern of pKHB.

FIG. 2 shows the position of each fragment of the 61-554 strains A3, A4, A5 and A6 on the genome.

FIG. 3 shows the construction of the insertion plasmid pKA4BL used for producing K-A4BL.

[Figure 4] DNA extracted from K-A4BL-infected CEF was cleaved with EcoRI, and then LacZ gene (lane 1) and KA4 fragment (lane)
The schematic diagram of the result of Southern hybridization performed using 2) as a probe is shown.

FIG. 5: 7-day-old chickens inoculated with K-A4BL 7000PFU
The anti-beta-gal ELISA antibody titer in serum at the age of week is shown.
(Circles of chickens vaccinated with the usual Marek bivalent vaccine
(Indicates the value of serum measured at 7 weeks of age)

FIG. 6 Insertion vector pKA4 used in the present invention
Shows the construction of B.

FIG. 7: Insertion plasmid pK used in the present invention
The structure of A4BF is shown.

FIG. 8: Insertion plasmid pK used in the present invention
The structure of A4BHN is shown.

FIG. 9 shows the construction of the insertion plasmid pKA3VL used for the production of K-A3VL.

FIG. 10: LacZ gene (lane 1) and KA3 fragment (lane) after cutting DNA extracted from K-A3VL-infected CEF with EcoRI
The schematic diagram of the result of Southern hybridization performed using 2) as a probe is shown.

FIG. 11 shows the positions of repetitive sequences in the K-554 strain and A5 fragment, and the positions of repetitive sequences in the A6 fragment of K-554 strain and BC-1 strain.

FIG. 12: Insertion plasmid used in the present invention
The structure of pKA5SL is shown.

FIG. 13 shows the nucleotide sequence of the ORI containing the BalI site of the 61-554 strain, A4 fragment, and US639.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 8, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction content]

Determination of nucleotide sequence The gene fragment was cloned into the polylinker of pUC119 and then transduced into competent cells such as JM109. The obtained transformant was cultured overnight in LB medium, and 30 μl thereof was cultured with M13 phage ( 10 ⁹ /
60 μl ) and further cultured overnight by shaking. After removing the bacterial cells by centrifugation, the phage was 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 obtained ssDNA
Using SEQUENASE V2.0 (TOYOBO), the nucleotide sequence was determined according to the attached protocol. If necessary, the gene fragment on the plasmid may be Ki
Lo-Sequence Deletion Kit
(Takara, cat. No. 6030) was used to construct a stepwise shortened plasmid.
Was prepared.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction content]

The fluorescent antibody technique (FA) 61-554 shares 10 ⁴ cover glass with the CFF ¹⁰ 7 cell the PFU (MATUNAMI.No.1,18
(x18 mm) 3 dishes containing 5 cm in diameter were inoculated and cultured for 2 days, then the cover glass was taken out, fixed with acetone for 20 minutes at room temperature, and stored at -80 ° C. In addition, a plasmid pAS in which the LacZ gene is connected downstream of the β-actin promoter for detecting anti-β-gal antibody
LacZ (Japanese Patent Application No. 63-226960) BMT-1
The cells were introduced into 0 cells by electroporation, cultured for 2 days, similarly fixed with acetone and stored at -80 ° C.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

After diluting the test serum 800-fold with PBS-,
100 μl of the solution was added and reacted at 37 ° C. for 1 hour, and then 5000-fold diluted anti-chicken IgG PO according to a conventional method.
D labeled rabbit antibody (Nordic Co.) and TMBZ (Dojin Kagaku, Cat.Nanba346-04031) is reacted, and after the reaction was stopped with _1N- H 2 SO _4, OD45
The absorbance at 0 nm / 630 nm was measured, and the value obtained by subtracting the absorbance at 630 nm from 450 nm was taken as the ELISA value.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction content]

[0050]

[Table 2]

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0056

[Correction method] Change

[Correction content]

Example 4: Preparation of K-A4BF pSVL (Pharmacia, Code No. 27-45)
09-01) has a super-attenuated NDV, D-2 at the SmaI site.
Fusion protein gene (F gene : H.
Sato et al. , Virus Research
h 7, 241-255 (1987) ) was inserted (p.
SVL F) . This plasmid was designated as EcoRI and SalI.
4.3k after electrolysis after digestion with
The fragment of b was recovered, blunt-ended, and then Bal of pKA4
Insertion plasmid pKA4 at the I site
The BF was constructed (Fig. 7).

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0057

[Correction method] Change

[Correction content]

After linearizing the plasmid with PvuI,
Recombination was performed according to the procedure for producing recombinant virus. However, the recombinant virus was cloned by monoclonal antibodies # 83 and # 313 [Y. U
mino et al. J. gen. virol. ，
71, p1189 (1990)].

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0058

[Correction method] Change

[Correction content]

The cloning method of the recombinant virus by immunostaining is shown below. A few days after the recombination, the petri dish in which plaque appeared was washed with E-MEM medium, an isotonic solution containing monoclonal antibodies # 83 and # 313 was added, and the mixture was reacted at room temperature for 10 minutes to 60 minutes. After washing, 100 with isotonic solution
Peroxidase-labeled anti-mouse antibody (Bio-Rad, code No. 172-10) diluted 2-fold to 200-fold
11) was added, and the mixture was further reacted at room temperature for 10 minutes to 60 minutes. After washing 3,3-diaminob per 10 ml
enzine Tetrahydrochlori
de (DAB, Wako, code No. 343-009)
01) 5 mg and hydrogen peroxide solution (Mitsubishi Gas Chemical Co., H ₂ O ₂
0.1M-Tris buffer (pH 7.5) containing 1.6 μl of 31%) was added and reacted at room temperature for 5 minutes to 60 minutes,
Recombinant virus plaques were stained. The brown-stained plaque was surrounded by a ring-shaped object, and only the inside of the ring was digested with a trypsin solution containing EDTA at 0.1% to recover the recombinant virus-infected cells.
Purification of recombinant virus was performed by co-culturing with EF. Cell fr by this operation and ultrasonic treatment
The ee procedure was repeated several times to purify the recombinant virus. The purified recombinant virus has a good in v
It showed proliferative properties in vitro.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0059

[Correction method] Change

[Correction content]

Newcastle disease (ND) of K-A4BF
In order to see the protective effect against, an immunological test was carried out.
After inoculating K-A4BF 10 ³ into the abdominal cavity of one-day-old chicks 3,
At the 9th and 16th weeks, the virulent NDV Sato strain 104 was lethal to the lower leg muscles. As a result, during the 2-week observation period,
All non-immune control groups developed or died whereas KA
The 4BF-inoculated group did not have one onset or death, indicating that this recombinant virus has a sufficient vaccine effect against Newcastle disease (Table 4).

[Procedure Amendment 9]

[Document name to be amended] Statement

[Name of item to be corrected] 0070

[Correction method] Change

[Correction content]

Example 10: cell free virus
Production of production type K-A4BL 100,000 PFU of K-A4BL infected cells were used as the primary CEF300.
After inoculating a petri dish with a diameter of 10 cm together with 0,000 pieces, CPE
The supernatant was collected on the 4th day when the whole surface was exposed. The supernatant was centrifuged at 1500 rpm for 10 minutes (KUBOTA KN
-30F), 30% of the original CEF and 5% of BS were added, and then the pH was adjusted to 7. with a 0.7% sodium bicarbonate aqueous solution.
After being corrected to around 4, a petri dish with a diameter of 10 cm was inoculated and cultured. After repeating this operation 5 times, the supernatant was further diluted 10 times with 5% BS-added E-MEM, and the second C
After adding 500,000 EFs, 100 μl of each was inoculated into a 96-well plate and further cultured. After 5 days, cells were collected from the wells where single plaques appeared, and the primary CEF1 was recovered.
A petri dish having a diameter of 5 cm was inoculated together with 10 million pieces. The supernatant was inoculated into a 96-well plate, and the supernatant of the well in which the single plaque appeared was treated three times in the same manner to obtain the cell.
A high-producing clone of the free virus was obtained. 200 to 500 PFU / ml of LacZ (+) virus was produced in the CEF supernatant cultured for 5 days after inoculation of this virus with 10,000 PFU. On the other hand, the supernatant of CVI988-infected cells, which is a vaccine strain cultured under similar conditions
Only 2-10 PFU / ml of virus was produced in some cases.

Claims (16)

[Claims] 1. A foreign gene expression cassette having a gene fragment derived from the Us region of a Marek's disease virus gene or a repetitive inverted sequence region adjacent thereto and having a foreign gene connected downstream of a promoter derived from an animal cell or an animal virus. Recombinant Marek's disease virus mutated by a plasmid incorporated in the gene fragment. 2. The recombinant Marek's disease virus according to claim 1, wherein the Marek's disease virus is Marek's disease type I virus. 3. A Marek's disease virus type I gene Hind is a gene fragment derived from the Us region of the Marek's disease virus gene.
The recombinant Marek's disease virus according to claim 2, which is a gene fragment of about 2.8 Kbp obtained by further treating the IIIB fragment with EcoRI. 4. A Marek's disease virus type I gene is a gene fragment derived from a repetitive inverted sequence of the Marek's disease virus gene.
The recombinant Marek's disease virus according to claim 2, which is a gene fragment containing a boundary between IRs and Us or TRs and Us, which is obtained by further treating the HindIII B fragment with EcoRI. 5. A foreign gene expression cassette in which a foreign gene is connected to the downstream of a promoter derived from an animal cell or an animal virus in a gene fragment derived from the Us region of the Marek's disease type I virus gene or the repetitive inversion sequence region adjacent thereto. A method for preparing a recombinant Marek's disease virus, which comprises integrating and incorporating a foreign gene into the Marek's disease type I virus genome using the recombinant gene fragment thus obtained. 6. A Marek's disease virus type I gene Hind is a gene fragment derived from the Us region of the Marek's disease virus gene.
The method for preparing the recombinant Marek's disease virus according to claim 5, which is a gene fragment of about 2.8 Kbp obtained by further treating the IIIB fragment with EcoRI. 7. A Marek's disease I using a foreign gene expression cassette.
The method for preparing a recombinant Marek's disease virus according to claim 5, wherein the site to be incorporated into the gene fragment derived from the Us region of the type I virus gene is a site cleaved by the restriction enzyme BalI. 8. A Marek's disease virus type I gene is a gene fragment derived from a repetitive inverted sequence of the Marek's disease virus gene.
The method for preparing the recombinant Marek's disease virus according to claim 5, which is a gene fragment containing a boundary between IRs and Us or TRs and Us, which is obtained by further treating the HindIII B fragment with EcoRI. 9. A foreign body having a gene fragment derived from the Us region of a Marek's disease virus gene or a repetitive inverted sequence region adjacent thereto and encoding a vaccine antigen other than Marek's disease downstream of a promoter derived from an animal cell or animal virus. A multivalent avian vaccine comprising a recombinant Marek's disease virus mutated with a plasmid in which a gene-connected foreign gene expression cassette is incorporated into the gene fragment. 10. The multivalent live vaccine according to claim 9, wherein the bird is a chicken. 11. The multivalent live vaccine according to claim 10, wherein the Marek's disease virus is Marek's disease type I virus. 12. A Marek's disease virus type I gene Hi is a gene fragment derived from the Us region of the Marek's disease virus gene.
About 2.8 Kbp of ndIII B fragment further treated with EcoRI
The multivalent live vaccine according to claim 11, which is a gene fragment of 13. A gene containing a boundary between IRs and Us or TRs and Us, which is a gene fragment derived from a repetitive inverted sequence of a Marek's disease virus gene, which is obtained by further treating a Marek's disease virus type I gene HindIII B fragment with EcoRI. The multivalent vaccine according to claim 11, which is a fragment. 14. The multivalent live vaccine according to claim 9, wherein the foreign gene encoding a vaccine antigen is the Newcastle disease virus antigen gene. 15. The multivalent live vaccine according to claim 14, wherein the Newcastle disease virus antigen gene is a gene derived from an attenuated Newcastle disease virus. 16. The vector for administering a physiologically active substance to birds, which comprises the recombinant Marek's disease virus according to any one of claims 1 to 4, wherein the foreign gene is a gene encoding a physiologically active substance.