JPH05244940A - Recombinant avipox virus and vaccine composed thereof - Google Patents

Abstract

PURPOSE:To provide a vaccine for infectious bursal disease virus produced by using a recombinant avipox virus integrated with cDNA originated from infectious bursal disease virus. CONSTITUTION:cDNA of an RNA large segment originated from infectious bursal disease virus is extracted and integrated into an avipox virus to construct a recombinant virus. The recombinant virus is suspended in a proper solvent to obtain the objective live vaccine for infectious bursal disease virus.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant avipox virus (hereinafter referred to as APV) and a vaccine comprising the same. More specifically, it is not essential for the proliferation of APV.
Infectious bursal disease virus in the NA region (hereinafter,
IBDV) and recombinant APV capable of expressing a polypeptide capable of inducing antibodies against IBDV) and a vaccine comprising thereof.

[0002]

2. Description of the Related Art Recently, vaccinia virus (hereinafter referred to as VV
A method for constructing a recombinant VV incorporating an exogenous DNA has been devised, and a method for using a recombinant VV using an infectious disease antigen-encoding DNA as a live vaccine has been proposed ( For example, JP-A-58-129971 and JP-B-61-501957). According to this method, various foreign DNAs can be incorporated depending on the purpose, and it is considered as a promising method for producing a new live vaccine.

However, due to the limited host region of VV, recombinant V is used in the production of live vaccines against chicken diseases such as infectious bursal disease.
It is almost impossible to apply the V method, and AP is used instead of VV for the production of live chicken vaccine.
It is suggested that the method of incorporating exogenous DNA into V is effective (Avian Diseases, Vol. 30, N.
o. 1, pp. 24-27, 1985). Actually, a recombinant live vaccine in which the HN gene or F gene of Newcastle disease virus is incorporated into APV has also been reported (JP-A-3-27284, etc.).

Regarding IBDV, the sequence of a cDNA encoding a large segment (hereinafter referred to as SegA) has been reported (J. gen. Virol., 71 , 1303).
~ 1312, 1990), the 40 kd protein encoded by SegA (hereinafter referred to as VP2) or 32
It has also been reported that an IBDV neutralizing epitope exists in the kd protein (hereinafter referred to as VP3) (J. gen.
Virol, 69 , pp. 631-640, 1988,
J. gen. Virol, 66 , 2693-2702.
P., 1985 etc.).

SegA of IBDV is a structural protein VP
2. Non-constitutive protein VP4 and structural protein VP3 are expressed as a series of polypeptides and processed to produce VP
2, VP4 and VP3.

When a chicken was inoculated with VP2 expressed in yeast, a neutralizing antibody against IBDV was observed to be induced, and it was reported that the obtained antiserum can be given a passive immunization against the chicken again to confer protection against IBDV infection. (Vaccin
e, Vol. 8, p. 549, 1990). Therefore,
CDNA or VP encoding SBDA of IBDV
It will be possible to develop a vaccine that can prevent the infection of IBDV by incorporating the cDNA encoding 2 into APV and expressing it. D. Baylias et al. Reported that if a recombinant APV in which a DNA encoding VP2 was fused with the lacZ gene of E. coli was inserted into the terminal region of the APV genome together with a promoter and inoculated into chickens, IBDV infection could be protected to some extent.
Pox and Irido virus in May 0
Reporting at Meeting.

However, in these experimental examples, the amount of recombinant APV inoculated was very large, the number of times of inoculation was required twice, and further, even in the chickens that survived the challenge with IBDV in the infection protection test. The infection with IBDV was found in the bursa of Fabricius, which was an insufficient result from the viewpoint of practical vaccine development. Furthermore, C.I. D. The recombinant constructed by Baylias et al.
VP2-1 is located at two terminal regions on the left and right of the PV genome.
Since the acZ gene has been inserted, there is a risk that it will become an unstable recombinant virus in APV that is susceptible to intramolecular homologous recombination, and from this aspect too, the difficulty as a practical vaccine cannot be ruled out.

[0008]

Under the above-mentioned conventional techniques, the inventors of the present invention aim to construct a proliferative recombinant APV in which a cDNA encoding a polypeptide that induces an anti-IBDV antibody is incorporated into genomic DNA. As a result of earnest research, a region coding for SegA was selected from the cDNA of IBDV, or a region having the same amino acid sequence as all or part of the amino acid sequence of SegA was included, and was substantially the same as SBDA derived from IBDV. CDNA encoding an antigenic polypeptide is transferred to APV
Incorporation into a specific genomic region that is non-essential for the growth of
The inventors have found that a recombinant APV capable of propagation can be obtained, and completed the present invention.

[0009]

Thus, according to the present invention, there is provided a recombinant APV capable of expressing a polypeptide capable of inducing an antibody against SBDA derived from IBDV, and a vaccine comprising the recombinant APV.

In the present invention, the virus used for incorporating the cDNA encoding the polypeptide capable of inducing an antibody against SBDA derived from IBDV may be any strain as long as it is classified as APV.
Those capable of growing in poultry cells such as turkeys and ducks are preferable, and specific examples thereof include ATCC VR-25.
1, ATCC VR-250, ATCC VR-22
9, ATCC VR-249, ATCC VR-28
8. Foulpox virus (hereinafter referred to as FPV) such as Nishigahara strain, and Susui strain, and viruses closely related to FPV, such as NP strain (fetal poultry pox venom Nakano strain) Examples include viruses used as vaccine strains. All of these strains are commercially available and can be easily obtained.

In the present invention, c incorporated into APV
The DNA contains a portion having the same amino acid sequence as all or part of the amino acid sequence of SBDA derived from IBDV, and encodes a polypeptide having substantially the same antigenicity as SegA derived from IBDV. It is cDNA. Having substantially the same antigenicity as IBDV-derived SegA means that an antibody against IBDV-derived SegA can be induced. The IBDV-derived cDNA encoding SegA can be prepared using, for example, the IBDV strain GPF-1E. For example, c encoding SBDA derived from IBDV prepared from GPF-1E strain
DNA is approximately 3.3 kbp (Fig. 1 and SEQ ID NOs: 2, 3,
4) and is incorporated into a plasmid pIBDV of about 6.0 kbp which can be extracted from Escherichia coli NZ-9101 (accession number, Microtechnology Research Institute Co., Ltd. No. 12422) by a conventional method. It can also be synthesized from a known sequence by a known method. Seg substantially derived from IBDV
As long as it has the same antigenicity as A, the cDNA may be modified, that is, the nucleotide sequence may be replaced, inserted or deleted.

In carrying out the present invention, first, AP
A first recombinant vector incorporating a non-essential DNA region for V propagation is produced. Those that include a promoter that functions in APV in the same region are preferable.

As the non-essential DNA region for proliferation, the region described in JP-A-1-168279 can be used. Specific examples thereof include Eco of APV / NP strain DNA showing a restriction map as shown in FIG.
RI fragment (7.3 kbp), EcoRI-HindII
I fragment (about 5.0 kbp), BamHI fragment (about 4.0)
Kbp), HindIII fragment (about 5.2 kbp), and the like, which give rise to regions that undergo homologous recombination.

D having a promoter function referred to in the present invention
NA may have any nucleotide sequence as long as it can effectively function as a promoter in a transcription system possessed by APV, whether synthetic or natural, and an APV-specific promoter such as the promoter of the APV gene encoding thymidine kinase is Of course, DNA derived from viruses other than APV or DNA derived from eukaryotes or prokaryotes can naturally be used in the present invention as long as the above conditions are satisfied.

Specific examples of these promoters include, for example, Journal of Virology, 51 ,
VV promoters as exemplified on pages 662-669 (1984), specifically the VV gene promoter encoding the 7.5K polypeptide, the VV gene promoter encoding the 19K polypeptide, 42K.
A promoter of the VV gene encoding a polypeptide,
Examples include a VV gene promoter encoding thymidine kinase, a VV gene promoter encoding a 28K polypeptide, and the like. Also, Moss et al. (J. Mol. Biol., 210 , 749-76).
Page, 771 to 784, 1989), and a synthetic promoter synthesized with reference to it can also be used.

The production of the first recombinant vector may be carried out by a conventional method, for example, DN which is non-essential for APV growth.
After incorporating the A fragment into an appropriate vector, a promoter having a restriction enzyme cleavage sequence may be incorporated downstream of the fragment by utilizing the restriction enzyme cleavage point existing in the fragment.

Specific examples of the vector used include, for example, pBR322, pBR325, pBR327 and pB.
R328, pUC7, pUC8, pUC9, pUC19
Plasmids such as λ phage, phages such as M13 phage, pHC79 (Gene, 11 , p.291, 1
980) and the like.

In the present invention, the IBDV SegA is then placed downstream of the promoter of the first recombinant vector.
The second recombinant vector is prepared by inserting the region encoding the. The insertion method may also be carried out according to a conventional method, for example, by using a restriction enzyme cleavage point previously provided downstream of the promoter of the first recombinant vector, the IBDV-derived cD
A DNA encoding an easily detectable enzyme may be inserted under the control of the NA fragment and the promoter.

In constructing these first and second recombinant vectors, an Escherichia coli system, which can be easily genetically manipulated, may be used, and the plasmid vector used is not particularly limited as long as it is suitable for the purpose.

In the present invention, next, the second recombinant vector is introduced into a host cell previously infected with APV,
Recombinant APV is constructed by causing homologous recombination between vector DNA and viral DNA. The host cells used here are only required to be capable of proliferating APV, and specific examples thereof include chicken-derived cultured cells such as fetal chicken cells. Even if the cells are not made into cell lines, if they can be cultured for a necessary time, they are naturally included in the category of host cells. The term "culture" as used herein means to survive cells, and is not limited to culture using an artificial medium or the like, and may be one that survives as a tissue such as chicken egg chorioallantoic membrane. I do not care.

The construction of the recombinant APV may be carried out according to a conventional method, for example, EMBO J. , 1 , 841-
845, 1982, Proc. Nat. Acad.
Sci. USA, 81 , 7161-7165, 19
According to the description in '84, the second recombinant vector is introduced into the host cell infected with APV by electroporation, and the resulting virus population containing the recombinant virus is collected in a tissue culture medium, for example, Eagle's MEM.
The plaques grown by infecting the host cells cultured above may be used as recombinant virus candidate strains. Regarding the method for selecting a virus in which a DNA fragment encoding SBD of IBDV is integrated from among these candidate strains,
It may be performed according to a conventional method, for example, DN encoding an enzyme.
When the β-galactosidase gene is used as A, a halogenated indolyl-β- is formed after plaques are observed in the plaque forming medium of the recombinant APV.
Overlay with agarose medium containing D-galactoside,
The plaques that turn blue by incubation at 5 ° C and 5% CO ₂ may be selected.

The recombinant APV of the present invention functions as a live anti-IBDV vaccine by inoculating birds. The use as a vaccine may be the same as the use of a normal live vaccine, for example, about 10 ⁴ to 10 ⁸ plaque forming units are inoculated per chicken. In this case, the recombinant APV of the present invention is usually suspended in an isotonic solvent such as about 0.1 ml of physiological saline and then injected.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 Construction of recombinant vector-pNZ9929 containing IBDV SegA gene DNA

(1) Construction of the first recombinant vector (see FIGS. 3, 4 and 5) A region shown in FIG. 1 which is not essential for APV growth (about 7.3 k).
plasmid pNZ133 in which the EcoRI fragment of bp) was incorporated into the EcoRI site of pUC18 (JP-A 1-16).
No. 8279) APV DNA fragment of about 2.7 Kbp was further inserted into the EcoRI-HindIII site of pUC18 by blunt-end ligation to construct a plasmid pNZ133S of about 5.4 kbp (Fig. 3).

With pNZ133S, competent E. coli was transformed by a conventional method, and the transformed E. coli was cultured and stored. When it was used, it was extracted from the transformed Escherichia coli by a conventional method, freshly used, and used (hereinafter, when a new plasmid was constructed, the same treatment was performed).

Next, the HindIII-EcoRI multiple cloning site of pUC18 was inserted into the EcoRV site of pNZ133S to construct a plasmid pNZ133SL of about 5.4 kbp (see FIG. 4).

Further, shown in SEQ ID NO: 1 (however, the first
Synthetic promoter P-only the fourth base is single-stranded)
HindIII-Hin of the _N 7.5 _pNZ133SL
The first recombinant vector, approximately 5.4 kbp of plasmid pNZ133SL-P, was constructed by inserting into the cII site (see FIG. 5).

(2) Preparation of hybrid plasmid containing IBDV SegA gene DNA (see FIG. 5) A plasmid pIBDV incorporating the IBDV SegA gene DNA was obtained from Professor Katsuya Hirai, Faculty of Agriculture, Gifu University. The nucleotide sequence of this plasmid was analyzed by the dioxy method using M13 phage, and the known SegA gene sequence (J. gen. Virol., 71 , No. 1303 to
1312, 1990), it was confirmed that the fragment of the IBDV-derived fragment containing the inserted SegA gene encodes VP2, VP3, and VP4. The restriction map of this fragment is shown in FIG. The base sequences of the A fragment, B fragment, and C fragment of this fragment shown in FIG. 1 were as shown in SEQ ID NOs: 2, 3, and 4, respectively.

From the first amino acid sequence shown in SEQ ID NO: 2 to the 338th amino acid sequence shown in SEQ ID NO: 3 is VP2, and from the 339th amino acid sequence shown in SEQ ID NO: 3.
The 608th position is VP4, and the 609th position of the amino acid sequence shown in SEQ ID NO: 3 to the 11th position which is the last of the amino acid sequence shown in SEQ ID NO: 4 is VP3. In addition, the first to fourth base sequences of the base sequence shown in SEQ ID NO: 2 are pIBD.
Derived from the plasmid used to construct V, the 5th to 16th p
Derived from the additional fragment used for the construction of IBDV, from the 58th position to the 130th position of the base sequence shown in SEQ ID NO: 4 is IBD
V is derived, the 131st to 144th are derived from the additional fragment used for the construction of pIBDV, and the 145th to 161st are derived from linkers.

The plasmid pIBDV was used for E. coli NZ.
It is a plasmid of about 6.0 kbp that can be extracted by a conventional method from -9101 (accession number, Microbiology Research Institute No. 12422).

After digesting pIBDV with PvuI, the DN
The cohesive end of the A fragment was made blunt by DNA polymerase. Further, this DNA fragment was digested with KpnI,
This was subjected to 1.5% agarose electrophoresis. About 3.2
The KpnI-PvuI fragment of the kbp SegA gene DNA was extracted from the agarose gel, and the KpnI-PvuI fragment of the SegA gene DNA was recovered by ethanol precipitation.

The pNZ133SL-P prepared in (1) was digested with HincII and KpnI, extracted with phenol / chloroform, and precipitated with ethanol for about 5.
The 4 kbp cleaved pNZ133SL-P was recovered.

Cleaved pNZ133SL-P DNA
And KpnI-Pv of SegA DNA of about 3.3 kbp
The ul fragment was ligated and the hybrid plasmid pNZ1 of about 8.7 kbp containing SegA gene DNA was ligated.
33SL-P · SegA was constructed.

(3) Introduction of Marker Gene (see FIGS. 6 and 7) pMA001 [Shirakawa et al., Gene, 2]
8 , p. 127, 1984] with BamHI.
About 3.3 kbp of β-galactosidase gene was recovered. On the other hand, pUC19 was digested with BamHI, extracted with phenol / chloroform, and recovered by ethanol precipitation.
The β-galactosidase gene prepared above was ligated to construct a hybrid plasmid pNZ66 of about 6.0 kbp.

The hybrid plasmid pNZ66 was transformed into Hi.
After complete digestion with ndIII, partial digestion with BamHI, extraction with phenol / chloroform treatment, and ethanol precipitation to recover about 6.0 kbp of cleaved pNZ66. It is shown in SEQ ID NO: 5 containing a promoter (P ₁₇ ) using ₁₇ bp of the promoter sequence of the gene encoding the 28-kilodalton polypeptide (the 5 ′ end is the HindIII sticky end and the 3 ′ end is the BamHI sticky end. ) Synthetic promoter and cleaved p
It was ligated with NZ66 to construct a hybrid plasmid pNZ66-P of about 6.0 kbp.

Next, the hybrid plasmid pNZ66-
PNZ digested P with KpnI, extracted with phenol / chloroform, and cleaved by ethanol precipitation
66-P was recovered. Cleaved pNZ66-P and a synthetic terminator shown in SEQ ID NO: 6 (a double-stranded DNA consisting of single-stranded DNA of the same sequence, wherein the 15th to 18th four bases are single-stranded) TT), and hybrid plasmid pN of about 6.0 kbp
Z66-PT was constructed.

The hybrid plasmid pNZ133SL-P.SegA constructed in (2) was digested with SacI, extracted with phenol / chloroform, and then ethanol-precipitated to give a cleaved pNZ13 of about 3.3 kbp.
3SL-P was recovered.

Further, pNZ66-PT was partially digested with SacI and then subjected to 1.5% agarose electrophoresis.
A DNA fragment of about 3.3 kbp was extracted from an agarose gel, and the P17-lacZ-TT fragment was recovered by ethanol precipitation.

The cleaved pNZ133SL-P and the above P17-lacZ-TT fragment were ligated to each other, and about 1
A 2 kbp hybrid plasmid pNZ9929 was constructed.

PNZ9929 corresponds to the second recombinant vector in the present invention.

Example 2 Recombinant APV containing SBD gene DNA of IBDV
Production of

Fetal chicken cells cultured in a 75 cm ² culture flask were cultivated with the APV / NP strain at 0.05 p. f. u. / Cell. Two hours after virus inoculation, 0.
400 μl of 1% trypsin was added to detach the infected fetal chicken cells from the culture flask. The infected chicken fetal cells that had been detached were treated with Saline G (0.14 M NaCl, 5 mM).
_{KCl, 1.1mM Na 2 HPO 4 ·} 6H 2 O, 1.
5 mM KH ₂ PO ₄ , 0.5 mM MgCl ₂ · 6H
After suspending in 5 ml of ₂ O and 0.011% glucose), centrifugation was performed at 1000 rpm for 5 minutes to recover the precipitate. Resuspend the precipitate in 5 ml of Saline G and
Centrifuge at 000 rpm for 5 minutes to collect the sediment, 700μ
1 of Suline G was suspended.

Next, 10 μg of the second recombinant vector pNZ9929 obtained in Example 1 was added to 100 μl of Salin.
Infected chicken embryo cell suspension 70 prepared in advance by dissolving in eG
After mixing with 0 μl, the mixture was transferred to a Gene Pulser cuvette and subjected to electroporation.

For electroporation, Gene Pulser (manufactured by Bio-Rad) was used, and 6.2 kVc was used.
The measurement was performed under the conditions of m ⁻¹ , 0.1 msec, and room temperature. After electroporation, the mixture was allowed to stand for 10 minutes, and Eagle's MEM 5 was added and adjusted so that the fetal bovine serum concentration was 5%, L-glutamine concentration was 0.03%, and tryptose phosphate broth concentration was 10%. 0.0 ml
Was added. Then, the cells were cultured at 37 ° C. in a 5% CO ₂ incubator for 3 days, and the cultured cells were freeze-thawed 3 times.
5ml of recombinant APV solution containing SegA gene of BDV
Got

Example 3 Selection of Recombinant with Halogenated Indolyl-β-D-galactoside Selection of Recombinant by Color Selection

Fetal chicken cells cultured in a 10 cm petri dish were inoculated with the virus solution obtained in Example 2 and 2 hours later, the concentration of Bact Agar (manufactured by Difco) was 0.8% and the concentration of fetal calf serum was 5%. , L-glutamine concentration of 0.03%, tryptoose phosphate broth concentration of 10% added and adjusted, respectively, phenol red-free Eagle '
10 ml of s MEM was laminated and cultured at 37 ° C. in a 5% CO ₂ incubator for 6 days. To this, a bacto agar concentration of 0.8%, an L-glutamine concentration of 0.03%, a tryptose phosphate broth concentration of 10%, and a halogenated indolyl-β-D-galactoside concentration of 0.
10 ml of Eagle's MEM without phenol red, each of which was added and adjusted so as to have a concentration of 05%, was overlaid and cultured at 37 ° C. in a 5% CO ₂ incubator for 12 hours. Recombinant virus expresses β-galactosidase and turns halogenated indolyl-β-D-galactoside into blue. Therefore, both agar and cells around recombinant plaque develop blue color, which is easily distinguished from non-recombinant. It was possible. Recombinants were isolated from this blue plaque with a sterile Pasteur pipette. The obtained recombinant was named fNZ9929.

Example 4 Recombinant APV containing SBD gene DNA of IBDV
Purification of

The plaque isolated in Example 3 was treated with 1 ml of E
Suspend in agle's MEM and add 200 μl to 10 cm
Fetal chicken cells cultured in Petri dishes were inoculated. 2 hours later, Bact agar concentration 0.8%, fetal calf serum concentration 5
%, L-glutamine concentration 0.03%, tryptose phosphate broth concentration 10%, respectively added / adjusted and phenol red-free Eagle's M
10 ml of EM was laminated and cultured at 37 ° C. in a 5% CO ₂ incubator for 6 days. In addition, the concentration of bacto-agar 0.8%, the concentration of L-glutamine 0.03%, the concentration of tryptose phosphate broth 10%, the concentration of halogenated indolyl-β-D-galactoside 0.05%
10 ml of Eagle's MEM without phenol red added and adjusted so that
Culturing was carried out for 12 hours in a 5% CO ₂ incubator at ℃.
Since the recombinant virus produced blue plaques as in Example 3, the plaques were purified by isolating the recombinant virus plaques.

A second plaque was purified by repeating the same procedure as above except that the plaque isolated in Example 3 was replaced with the above isolated plaque.

As a result, in fNZ9929, about 500 plaques were produced per sheet in Example 3.
Recombinant plaques (blue plaques) appeared per 10 cm petri dishes, and about 200 plaques appeared in the first plaque purification. 90 recombinant plaques (15%) appeared in 3 petri dishes. In the second plaque purification, almost all plaques were recombinant.

Example 5 Expression of recombinants in cells (fluorescent antibody method)

Fetal chicken cells cultured on a tissue culture chamber slide were treated with m.p. o. i. = FN of 1.0 or 10
Inoculated with Z9929 (obtained in Example 3),
After culturing for 2 hours in a 5% CO ₂ incubator, Eagle's MEM supplemented with L-glutamine concentration of 0.03% and tryptose phosphate broth concentration of 10% was added. Then, after culturing at 37 ° C. in a 5% CO ₂ incubator, the medium was removed, and Dulbecco's PBS for tissue culture was used.
After washing with (-) (prepared from Nissui Pharmaceutical Co., Ltd. phosphate buffered saline adjustment powder) and air-drying, -20
The cells were fixed by treatment with acetone at 20 ° C. for 20 minutes. Check with a fluorescent antibody that uses anti-IBDV / chicken antibody as the primary antibody, and anti-chicken IgG antibody conjugated with isothiocyanate fluorescein as the secondary antibody.
It was confirmed that V has the ability to produce SegA protein.

Example 6 Measurement of Neutralizing Antibody Titer and Infection Protection Rate with Recombinant Avipoxvirus (fNZ9929) (1) Measurement of Neutralizing Antibody Titer 13 days old SPF chickens obtained in Example 3 on the wing membrane. fNZ
9929 at 4 × 10 ⁴ p. f. u inoculated. After inoculation,
Blood was collected at 3, 4, and 5 weeks to measure the neutralizing antibody titer.

(2) Measurement of infection protection rate After measurement of the neutralizing antibody titer at the 5th week, 0.5 ml of the IBDV virulent strain DV86-infected chicken Fabricius sac 20% emulsion per (1) SPF chicken was used. Was orally administered. After the administration, each chicken was observed for 5 days. The results of (1) and (2) are shown in Table 1.

[0056]

[Table 1]

From these results, it was found that the flock inoculated with the recombinant avipox virus fNZ9929 induces neutralizing antibody. In addition, the recombinant avipoxvirus fNZ992 obtained from the survival number / total number × 100
The survival rate of the flock inoculated with 9 was 55%, and it was found that the recombinant avipox virus fNZ9929 protected the attack by the virulent strain of IBDV. From this, it was confirmed that the SBDA protein of IBDV expressed by fNZ9929 functions as a protective antigen for infection.

Recombinant avipoxvirus fNZ99
It is expected that a higher effect can be obtained by increasing the amount of 29 inoculated.

[0059]

INDUSTRIAL APPLICABILITY According to the present invention, thus, the IBDV-derived antigen code cD is present in a genomic region that is not essential for APV growth.
A stable, highly expressed antigen-recombinant APV that can be used as a live poultry vaccine, in which NA is expressibly incorporated together with DNA having a promoter function is obtained.

[0060]

[Brief description of drawings]

FIG. 1 is an explanatory view showing a restriction enzyme cleavage point map of SegA.

FIG. 2 is an explanatory view showing a restriction enzyme cleavage point map of a DNA region which is not essential for APV growth.

FIG. 3 is an explanatory diagram showing a method for constructing a hybrid plasmid.

FIG. 4 is an explanatory diagram showing a method for constructing a hybrid plasmid.

FIG. 5 is an explanatory view showing a method for constructing a hybrid plasmid.

FIG. 6 is an explanatory view showing a method for constructing a hybrid plasmid.

FIG. 7 is an explanatory diagram showing a method for constructing a hybrid plasmid.

[0061]

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 60 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic Promoter Sequence AGCTTTTTTT TTTTTTTTTT TTTGGCATAT AAATAATAAA TACAATAATT AATTACGCG

[0062]

[Sequence listing] SEQ ID NO: 2 Sequence length: 76 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin organism name: Poultry infectious bursal disease virus Strain Name: GPF-1E sequence GATCCGGGTA CCATGGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTGGA 60 TACGATCGGT CTGACCCCGG GGGAGTCTC CGGGGACAGG CCGTGCAG CA TGCA GCA ATG ATG ACG ACG ATC AGT AGC ATG ACG ACG ACG ACG ATC ACG ATC ATC

[0063]

[Sequence listing] SEQ ID NO: 3 Sequence length: 2029 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin organism name: Chicken infectious bursal disease virus Strain name: GPF-1E sequence GGT GGC GTT TAT GCA CTA AAC GGC ACC ATA AAC GCC GTG ACC TTC CAA 48 Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr Phe Gln 1 5 10 15 GGA AGC CTG AGT GAA CTG ACA GAT GTT AGC TAC AAT GGG TTG ATG TCT 96 Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu Met Ser 20 25 30 GCA ACA GCC AAC ATC AAC GAC AAA ATT GGG AAT GTC CTA GTA GGG GAG 144 Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly Glu 35 40 45 GGG GTC ACC GTC CTC AGC TTA CCC ACA TCA TAT GAT CTT GGG TAT GTG 192 Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val 50 55 60 AGG CTC GGT GAC CCC ATT CCC GCT ATA GGG CTT GAC CCA AAA ATG GTA 240 Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys Met Val 65 70 75 80 GCC ACA TGT GAC AGC A GT GAC AGG CCC AGA GTC TAC ACC ATA ACT GCA 288 Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr Ala 85 90 95 GCC GAT GAT TAC CAA TTC TCA TCA CAG TAC CAA CCA GGT GGG GTA ACA 336 Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly Val Thr 100 105 110 ATC ACA CTG TTC TCT GCC AAC ATT GAT GCT ATC ACA AGC CTC AGC GTT 384 Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu Ser Val 115 120 125 GGG GGA GAG CTC GTA TTT CAT ACA AGC GTC CAA AGC CTT GTA CTG GGC 432 Gly Gly Glu Leu Val Phe His Thr Ser Val Gln Ser Leu Val Leu Gly 130 135 140 GCC ACC ATC TAC CTT ATA GGC TTT GAT GGG TCT ACG GTA ATC ACC AGA 480 Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Ser Thr Val Ile Thr Arg 145 150 155 160 GCT GTG GCC GCA GAC AAT GGG CTG ACG GNC GGC ACC GAC AAT CTT ATG 528 Ala Val Ala Ala Asp Asn Gly Leu Thr Xaa Gly Thr Asp Asn Leu Met 165 170 175 CCA TTC AAT CTT GTG ATT CCA ACC AAC GAG ATA ACC CAG CCA ATC ACA 576 Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro Ile Thr 180 185 190 TCC ATC AAA CTG GAG ATA GTG ACC TCC AAA AGT GGT GGT CAG GCA GGG 624 Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln Ala Gly 195 200 205 GAT CAG ATG TCA TGG TCG GCA AGT GGG AGC CTA GCA GTG ACG ATC CAT 672 Asp Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr Ile His 210 215 220 GGT GGC AAC TAT CCA GGG GSC CTC CGT CCC GTC ACA CTA GTA GCC TAC 720 Gly Gly Asn Tyr Pro Gly Xaa Leu Arg Pro Val Thr Leu Val Ala Tyr 225 230 235 240 GAA AGA GTG GCA ACA GGA TCT GTC GTT ACG GTC GCT GGG GTG AGC AAC 768 Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val Ser Asn 245 250 255 TTC GAG CTG ATC CCA AAT CCT GAA CTA GCA AAG AAC CTG GTT ACA GAG 816 Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val Thr Glu 260 265 270 TAC GGC CGA TTT GAC CCA GGA CCC ATG AAC TAC ACA AAA TTG ATA CTG 864 Tyr Gly Arg Phe Asp Pro Gly Pro Met Asn Tyr Thr Lys Leu Ile Leu 275 280 285 AGT GAG AGG GAN NNK CTT GGC ATC AAG ACC GTC TGG CCA ACA AGG GAG 912 Ser Glu Arg Xaa Xaa Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Glu 290 295 30 0 TAC ACT GAC TTT CGT GAG TAC TTC ATG GAA GTG GCC GAC CTC AAC TCT 960 Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser 305 310 315 320 CCC CTG AAG ATT GCA GGA GCT TTT GGC TTC AAA GAC ATA ATC CGG GCC 1008 Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile Arg Ala 325 330 335 ATA AGG AGG ATA GCT GTG CCG GTG GTC TCT ACA TTG TTC CCA CCT GCC 1056 Ile Arg Arg Ile Ala Val Pro Val Val Ser Thr Leu Phe Pro Pro Ala 340 345 350 GCT CCC CTA GCC CAT GCA ATT GGG GAA GGT GTA GAC TAC CTG CTG GGC 1104 Ala Pro Leu Ala His Ala Ile Gly Glu Gly Val Asp Tyr Leu Leu Gly 355 360 365 GAT GAG GCA CAG GCT GCT TCA GGA ACT GCT CGA GCC GCG TCA GGA AAA 1152 Asp Glu Ala Gln Ala Ala Ser Gly Thr Ala Arg Ala Ala Ser Gly Lys 370 375 380 GCA AGA GCT GCC TCA GGC CGC ATA AGG CAG CTG ACT CTC GCC GCC GAC 1200 Ala Arg Ala Ala Ser Gly Arg Ile Arg Gln Leu Thr Leu Ala Ala Asp 385 390 395 400 AAG GGG TAC GAG GTA GTC GCG AAT CTA TTC CAG GTG CCC CAG AAT CCC 1248 Lys Gly Tyr Glu Val Val Ala Asn Leu Phe Gln Val Pro Gln Asn Pro 405 410 415 GTA GTC GAC GGG AAT CTT GCT TCA CCT GGG GTA CTC CGC GGT GNA CAC 1296 Val Val Asp Gly Asn Leu Ala Ser Pro Gly Val Leu Arg Gly Xaa His 420 425 430 AAC CTC GAC TGC GTG TTA AGA GAG GGT GCC ACG CTG TTC CCT GTG GTC 1344 Asn Leu Asp Cys Val Leu Arg Glu Gly Ala Thr Leu Phe Pro Val Val 435 440 445 ATT ACG ACA GTG GAG GAC GCC ATG ACA CCN AAA GCA TTG AAC AGC AAA 1392 Ile Thr Thr Val Glu Asp Ala Met Thr Pro Lys Ala Leu Asn Ser Lys 450 455 460 ATG TTT GCT GTC ATT GAA GGC GTG CGA GAA GAC CTC CAA CCT CCA TCT 1440 Met Phe Ala Val Ile Glu Gly Val Arg Glu Asp Leu Gln Pro Pro Ser 465 470 475 480 CAA AGA GGA TCC TTC ATA CGA ACT CTC TCC GGA CAC AGA GTC TAT GGA 1488 Gln Arg Gly Ser Phe Ile Arg Thr Leu Ser Gly His Arg Val Tyr Gly 485 490 495 TAT GCT CCA GAT GGT GTA CTT CCA CTG GAG ACT GNG AGA GAC TAC ACN 1536 Tyr Ala Pro Asp Gly Val Leu Pro Leu Glu Thr Xaa Arg Asp Tyr Thr 500 505 510 GTT GTC CCA ATA GAT GAT GTC TGG GNN ACC AGC ATT ATG CTG TCC AAA 1584 Val Val Pro Ile Asp Asp Val Trp Xaa Thr Ser Ile Met Leu Ser Lys 515 520 525 GAT CCC ATA CCT CCT ATT GTG GGA AAC AGT GGA AAC CTA GCC ATA GCT 1632 Asp Pro Ile Pro Pro Ile Val Gly Asn Ser Gly Asn Leu Ala Ile Ala 530 535 540 TAC ATG GAT GTG TTT CGA CCC AAA GTC CCC ATC CAT GTA GCC ATG ACA 1680 Tyr Met Asp Val Phe Arg Pro Lys Val Pro Ile His Val Ala Met Thr 545 550 555 560 GGA GCC CTC AAT GCT TGT GGT GGG ATT GAG AAA GTA AGC TTT AGA AGC 1728 Gly Ala Leu Asn Ala Cys Gly Gly Ile Glu Lys Val Ser Phe Arg Ser 565 570 575 ACC ACC AAG CTC GCC ACT GCA CAC CGA CTT GGC CTC AAG TTG GCT GGT CCC 1776 Thr Lys Leu Ala Thr Ala His Arg Leu Gly Leu Lys Leu Ala Gly Pro 580 585 590 GGA GCA TTC GAT GTA AAC ACC GGG CCC AAC TGG GCA ACG TTT ATC AAA 1824 Gly Ala Phe Asp Val Asn Thr Gly Pro Asn Trp Ala Thr Phe Ile Lys 595 600 605 CGT TTC CCC CAC AAT CCA CGC SMC TGG SAC AGG CTC CCC TAC CTC AAC 1872 Arg Phe Pro His Asn Pro Arg Xaa Trp Xaa Arg Leu Pro Tyr Leu Asn 610 615 620 CTT CCA TAC CTT CCA CCC AAT GCA GCG CGC CAG TAC CAC CTT GCC ATG 1920 Leu Pro Tyr Leu Pro Pro Asn Ala Ala Arg Gln Tyr His Leu Ala Met 625 630 635 640 GCT GCA TCA GAG TTC AAA GAG ACC CCT GAA CTC GAG AGT GCC GTC AGG 1968 Ala Ala Ser Glu Phe Lys Glu Thr Pro Glu Leu Glu Ser Ala Val Arg 645 650 655 GCC ATG GAA GCA GCA GCC AAC GTG GAC CCA CTA TTC CAA TCT GCA CTC 2016 Ala Met Glu Ala Ala Ala Asn Val Asp Pro Leu Phe Gln Ser Ala Leu 660 665 670 AGT GTG TTC ATG T 2029 Ser Val Phe Met 675

[0064]

[Sequence listing] SEQ ID NO: 4 Sequence length: 161 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin organism name: Poultry infectious bursal disease virus Strain name: GPF-1E sequence G CTG GAT CAG ACT GTC TCT GAT GAG GAC CTT GAG TGAGGC TCCTGGGAGT 50 Leu Asp Gln Thr Val Ser Asp Glu Asp Leu Glu 1 5 10 CTCCCGACAC CACCCCGCGC AGGTGTGGAC ACCAATTCGG CCCTACAACA TCTCAATTG 110TCG

[0065]

[Sequence listing] SEQ ID NO: 5 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic promoter Sequence AGCTTGAGCT CGGATCGTTG AAAAAATAAT ATA 33

[0066]

[Sequence listing] SEQ ID NO: 6 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic terminator Sequence ATTTTTATAA AAATGTAC
18

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 15/86 // C12P 21/02 8214-4B (C12P 21/02 C12R 1:92) (72 ) Inventor Shigemi Aoyama 370-13 Kikugawa, Mizuguchi-cho, Koga-gun, Shiga Prefecture (72) Inventor Takeshi Yamaguchi 1-2501 -49 Kibogaoka-honcho, Konan-cho, Koga-gun, Shiga Prefecture Inventor Yoshikazu Iriya Fukakusa, Fushimi-ku, Kyoto-shi, Kyoto Prefecture 151, Omagaya Manjoshiki-cho (72) Inventor Yukino Hayashi 5-15-5 Nomura-cho, Kusatsu-shi, Shiga Prefecture (72) Inventor Ikuhiro Yoneda 873, Taio-cho, Kohoku-ku, Yokohama-shi, Kanagawa (72) Setsuko Okawa Kanagawa 17-13 Shinohara Nishi-cho, Kohoku-ku, Yokohama-shi, Japan (72) Inventor Kamogawa Sachi-shi 2153-42, Eritani-cho, Kanazawa-ku, Yokohama-shi, Kanagawa

Claims (4)

[Claims] 1. A cDNA encoding a polypeptide having substantially the same antigenicity as an RNA large segment derived from infectious bursal disease virus in a genomic region that is non-essential for the growth of avipox virus. Recombinant avipox virus incorporated. 2. The recombinant avipoxvirus according to claim 1, wherein the cDNA is under the control of a promoter. 3. A cDNA having the whole or a part of the nucleotide sequences of SEQ ID NOs: 2, 3 and 4 incorporated thereinto.
Or the recombinant avipox virus according to 2. 4. An anti-IBDV vaccine comprising the recombinant avipoxvirus according to claim 1, 2, or 3.