JPH099979A - Recombinant, polyvalent recombinant and its preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a polyvalent recombinant useful as a live vaccine by composing the recombinant of an acceptor plasmid containing a specific restriction cleavage site transduced thereinto and an inserted DNA fragment and simply transducing many genes into one site on the plasmid in a desired direction. SOLUTION: This recombinant is obtained by inserting an inserting DNA fragment having one end which is a section cleaved with a restriction enzyme SfiI and the other end that is a section cleaved with a restriction enzyme X (a restriction enzyme Bgll, etc.) other than the restriction enzyme SfiI in a site recognized with the restriction enzyme Sfil of an acceptor plasmid having one site recognized with the restriction enzyme SfiI. The resultant recombinant has only one site recognized with the restriction enzyme SfiI. One or more DNA fragments having a section cleaved with the restriction enzyme SfiI and a section cleaved with the restriction enzyme X other than the restriction enzyme SfiI at both terminals are further inserted into the recombinant to provide a polyvalent recombinant.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant, a method for producing the recombinant, and a polyvalent recombinant (plasmid, transformant, virus, etc.) obtained by using the plasmid.

[0002]

2. Description of the Related Art In recent years, recombinants such as transformed bacteria and recombinant viruses play an important role as drugs, drug raw materials, drug synthetic materials and the like. For example, an antigen protein can be produced using a transformed bacterium into which a pathogen-derived gene has been introduced, or a recombinant virus incorporating a pathogen-derived gene can be administered as a live vaccine. In particular, recently, it has been considered to introduce or integrate genes derived from a plurality of pathogens to construct polyvalent recombinants and obtain a highly safe live vaccine. This is because in vaccines using recombinants, if the recombinants express only one pathogen-derived gene, it is necessary to inoculate or administer multiple recombinants to immunize the organism against multiple pathogens. On the other hand, when a multivalent recombinant expressing genes derived from multiple pathogens is used, it is possible to immunize multiple pathogens with one recombinant and reduce the burden on the living body. It is thought that this is due to the advantage that it can be done.

By the way, conventionally, for the production of recombinants,
So-called vectors such as plasmids and phages are used. For example, in the case of introducing a gene into bacteria, a plasmid or phage in which the gene to be introduced is incorporated is constructed, and then transformation or transduction in which this is introduced into bacteria is carried out. In addition, when introducing a gene into a virus, a recombinant plasmid having a gene into which a gene to be introduced is inserted between non-essential regions for viral growth is constructed, and homologous recombination with the virus is caused by using this. A method for introducing a gene into a virus is generally known.

In the above-mentioned method, the constructed recombinant plasmid is used as an important tool for the production of recombinant bacteria or viruses. A plasmid having a pathogen-derived gene is generally constructed by cutting a part of the plasmid with a restriction enzyme and inserting the pathogen-derived gene therein. The restriction enzymes used here are
It recognizes the sequence of a specific gene and cleaves nucleotides at a certain position in or from the sequence, and many restriction enzymes are restriction enzymes that recognize 6 to 8 bases.
Among them, a 6-base recognition type restriction enzyme is widely used for constructing recombinants because of its large variety.

However, the probability that a site (cleavage site) to be cleaved by a restriction enzyme exists on a gene is 4 if the bases are completely randomly linked.
Since it is 4 to the 8th power (the number of bases to be recognized) of (the number of types of bases), the existence probability of a cleavage site by a restriction enzyme that recognizes 4 bases is highest, which is less suitable for constructing a polyvalent recombinant. Not not. Similarly, even with a restriction enzyme that recognizes 6 bases, the probability that there are multiple sites recognized by the same restriction enzyme increases as the size of the plasmid increases. When a pathogen-derived gene is inserted into a plasmid by using a restriction enzyme having a plurality of cleavage sites in one plasmid, a technique such as partial digestion is required, which makes the operation complicated. Further, when a restriction enzyme that recognizes 8 bases is used, the above-mentioned problem is solved because there are few recognition sites in the plasmid. However, when a gene derived from one pathogen is introduced and then the same restriction enzyme is used when introducing a gene derived from the second other pathogen, the cleavage points at both ends of the gene originally derived from the pathogen are also cleaved. Will be done. In this case as well, there are a plurality of the same restriction enzyme recognition sites, and a technique such as partial decomposition is required as in the case of using the 6-base recognition restriction enzyme. Furthermore, since the direction of the inserted gene cannot be controlled, it is necessary to obtain a recombinant plasmid and then clean it to obtain a plasmid having the desired orientation. Such a phenomenon naturally occurs regardless of the number of recognition bases, as long as a restriction enzyme having the same recognition site is used.

[0006] Further, a method is known in which, when a gene is inserted, the original restriction enzyme cleavage site is eliminated on the resulting plasmid by terminal modification with a Klenow fragment of DNA polymerase or the like. However, since the original restriction enzyme cleavage site disappears, it becomes impossible to insert the gene into the same site again. In addition, since the ends are blunt, there is no difference in that the gene can be inserted in two directions. The fact that there are two insertion directions indicates the possibility that a plasmid in which the reading direction of the gene to be integrated is ligated in the forward direction and a plasmid in which the gene is integrated in the reverse direction can be obtained, which is not desirable in operation. . Of course, if different restriction enzymes are used, it is possible to insert multiple pathogen-derived genes at different locations, but it is necessary to consider the restriction enzyme cleavage site originally possessed by the plasmid, and the gene insertion interval It is not suitable for constructing polyvalent recombinants that require strict design, such as the use of promoters and promoters. In this way, a multivalent plasmid (a plasmid in which multiple genes are consecutively introduced into one plasmid) for obtaining a multivalent recombinant needs to be designed such as the insertion site and the direction of the inserted gene. , Its construction operation is extremely complicated,
And there was a complicated problem.

[0007]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have made diligent studies to solve the above-mentioned operational problems that are generally difficult in the prior art, and as a result, have found that a unique restriction enzyme cleavage site Two introduced plasmids (plasmid pair)
It was found that the use of E. coli can easily introduce a large number of genes into one site on a plasmid, and the direction of the gene to be introduced can be arbitrarily determined, thus completing the present invention.

[0008]

Thus, according to the present invention, firstly, the SfiI recognition site of a plasmid having one restriction enzyme SfiI recognition site (hereinafter sometimes referred to as an acceptor plasmid) has one end Is the restriction enzyme Sfi
A DNA fragment (hereinafter, referred to as the cut surface by I, and the other end by the restriction enzyme X other than the restriction enzyme SfiI).
(Sometimes referred to as an inserted DNA fragment), a recombinant having a single restriction enzyme SfiI recognition site obtained by insertion is provided, and the restriction enzyme X other than SfiI is BglI, AlwNI, DraIII, PflM.
I, BslI, MwoI and their isoschi
It is a preferred embodiment to be any one of the Zomer. Secondly, the recombinant of the present invention further comprises a restriction enzyme SfiI cleavage surface and a restriction enzyme X other than the restriction enzyme SfiI.
Provided is a multivalent recombinant obtained by inserting one or more DNA fragments having a cleavage plane at both ends. Third, 1
S of plasmid containing two restriction enzyme SfiI recognition sites
D having a restriction enzyme SfiI cutting surface and a restriction enzyme X cutting surface other than the restriction enzyme SfiI at both ends at the fiI recognition site
There is provided a method for producing a recombinant characterized by inserting an NA fragment, wherein the recombinant of the present invention obtained here can serve as an acceptor plasmid, so that the inserted DN is re-inserted.
The A fragment is incorporated into this plasmid to give a multivalent recombinant. Of course, the operation of incorporating the inserted DNA fragment into the acceptor plasmid again can be repeated,
Two or more foreign DNAs can be incorporated as insert DNA fragments. Therefore, the method for producing a recombinant provided in the present invention is useful as a method for producing a multivalent recombinant. Fourthly, there is provided a transformant transformed with the polyvalent recombinant of the present invention and a recombinant virus produced using the polyvalent recombinant of the present invention.

(Acceptor plasmid) In the present invention, the acceptor plasmid is a plasmid having one restriction enzyme SfiI recognition site. The restriction enzyme SfiI recognition sites _{here, 5'-GGCCN 1 N 2 N} 3 N 4
N ₅ GGCC-3 ′ (N _{1 to} N ₅ are each independently A,
C, G, or T). The restriction enzyme SfiI recognizes this sequence, and the three bases (N ₂ N ₃ N ₄ ) at the center of the sequence are projected to the 3'terminal side. DN
Cut A. Of course, restriction enzymes other than the restriction enzyme SfiI such as HindIII, EcoRI, HincI
The acceptor plasmid referred to in the present invention may have a site that is recognized by a restriction enzyme that is abundant in the multicloning sites of general plasmids such as I and BamHI. Such a plasmid can be produced arbitrarily.

(Insert DNA Fragment) In the present invention, the DNA fragment to be inserted into the acceptor plasmid has a cleavage plane (that is, N _{2 of the} above SfiI recognition sequence) provided at both ends by the restriction enzyme SfiI of the acceptor plasmid.
Cross section where N ₃ N ₄ protrudes to the 3'side and N _2c N _3c N _4c is 3 '
One end is N ₂ N ₃ so that it can be connected to the
N ₄ is a cross section protruding to the 3 ′ side, and the other end is N _2c N
_3c N _4c has a cross section protruding to the 3'side. Further, only one end of this cross section is the same as the cut surface of the restriction enzyme SfiI, and the other end is not the cut surface of the restriction enzyme SfiI but the cut surface of the restriction enzyme X other than the restriction enzyme SfiI. In the present invention, the restriction enzyme X may be any restriction enzyme that gives a cutting surface with three bases protruding in the same direction as SfiI, since its cross section needs to be ligated to the SfiI cutting surface of the acceptor plasmid. Considering the convenience of the design of recombinant construction and the specificity of the recognition site, the restriction enzymes BglI, AlwNI, DraIII,
PflMI, BslI, MwoI, these isosc
Hizomer and the like are preferable as the restriction enzyme X, and among them, the restriction enzyme BglI capable of cleaving the restriction enzyme SfiI cleavage site is most preferable. In these restriction enzymes, the central base can be set arbitrarily.

Of course, the size of the inserted DNA fragment, the reading direction, etc. can be arbitrarily selected. Also,
The inserted DNA fragment contains a gene of interest (hereinafter, sometimes referred to as a gene of interest) to be expressed, such as an antigen gene or a marker gene.
Hybrid DNA in which a regulator gene such as a signal sequence is also ligated is used in a general recombination technique. Vectors such as plasmids may be used to produce such hybrid DNAs. In the present invention, such a vector may be called a donor. As a vector serving as a donor, a plasmid having a multiple cloning site is usually used.

The structure of the inserted DNA fragment is basically [restriction enzyme X cleavage site]-[target gene etc.]-[Restriction enzyme S].
fiI cleavage site], and the structure for obtaining an inserted DNA fragment using a donor is [restriction enzyme X cleavage site]-[cloning site (insertion of target gene etc.)]- [Restriction enzyme SfiI cleavage site]
Are connected in this order. The target gene is generally inserted into a donor-derived cloning site. The origins of various genes inserted here are mammals,
It may be a vertebrate or an invertebrate such as a bird, a fish or an insect, as well as a plant, a protist or a virus.

(Method for Producing Recombinant) In the present invention, the acceptor plasmid and the inserted DNA described above are used.
A fragment (recombinant plasmid) can be constructed using the fragment. As mentioned above, since the recombinant of the present invention also functions as an acceptor plasmid, a multivalent recombinant (multivalent recombinant plasmid) can be obtained by repeating the same operation.

As a concrete method for producing a recombinant,
The following methods are available. After constructing a plasmid serving as a donor by a conventional method, it is cleaved with restriction enzymes X and SfiI to obtain an inserted DNA fragment (gene cartridge), which is inserted into the SfiI cleavage site of the acceptor plasmid. Since N ₂ N ₃ N ₄ of the SfiI recognition sequence is not a palindromic sequence, the direction of the gene to be inserted is uniquely determined. When the restriction enzyme X cleavage site is present inside any gene to be introduced, the donor may be completely digested with SfiI and then partially digested with the restriction enzyme X. When BglI is used in restriction enzyme X, the SfiI site is automatically changed to B
Since it is also cut with glI, the gene cartridge can be prepared by simply cutting with BglI. In this way, any gene can be easily introduced into the SfiI site of the acceptor plasmid in a single direction. Since SfiI recognizes 8 bases, it can be said that there is almost no probability that an SfiI site having the exact same base sequence will occur. Therefore, the plasmid prepared here has only one SfiI cleavage site unless an SfiI cleavage site is present in any inserted gene.
It will have an I-cleavage site.

Furthermore, the plasmid thus obtained can be used again as an acceptor plasmid. That is, the obtained recombinant was digested with a restriction enzyme SfiI, a new inserted DNA fragment (gene cartridge) was excised from a separately prepared donor by the same method, and the operation of re-ligating was repeated, whereby a plurality of genes (identical Can be introduced continuously and simply in any direction.

(Transformation and Recombinant Virus) The (polyvalent) recombinant of the present invention obtained by the above method includes prokaryotic microorganisms such as Escherichia coli, Bacillus subtilis, Serratia, Salmonella, Actinomycetes, etc. A transformant can be obtained by transferring into a host cell such as a filamentous fungus (fungus), a eukaryotic microorganism such as yeast, a chicken embryo fibroblast, a higher eukaryotic cell such as a human bone marrow cell.

Similarly, a recombinant virus can be constructed using the (polyvalent) recombinant of the present invention according to a conventional method. Of course, the virus to be used can be arbitrarily selected according to the purpose of use. For example, an orthopox virus typified by vaccinia virus; a pox virus such as an avipox virus typified by foulpox virus; a herpes simplex virus, a shoe Examples thereof include herpesviruses such as drevis virus and turkey herpesvirus; adenoviruses such as human adenovirus type 2 and human adenovirus type 5.

[0018]

INDUSTRIAL APPLICABILITY The multivalent recombinant of the present invention is useful as a multivalent vaccine, and according to the method of the present invention, a recombinant, a recombinant virus, which is a raw material of this multivalent vaccine,
A transformant can be easily obtained.

[0019]

The present invention will be described below with reference to examples.
The present invention is not limited to this.

(Example 1) Donor plasmid pGTP
Construction of s and pGTP7.5 (see FIG. 1) Synthetic DN at the HindIII-PstI site of pUC18
A (5'-AGCTGCCCCCCCGGCAAGCT
TGCA-3 '(SEQ ID NO: 1)), and then the synthetic DNA at the SalI-KpnI site of the resulting plasmid.
(5'-TCGACATTTTTATTGTAC-3 '
(SEQ ID NO: 2)), and two synthetic DNs at the SacI-EcoRI sites of the plasmid obtained here.
A (5'-AATTCGGCCGGGGGGGGCCAG
CT-3 '(SEQ ID NO: 3)) and (5'-GGCCC
CCCCGGGCG-3 '(SEQ ID NO: 4)) annealed was inserted, and finally Hind of this plasmid was inserted.
Plasmid pNZ1729R at the III-SalI site
(Yanagida et al., J. Viro
l. , 66, 1402-1408, 1992) by Hin.
A DNA fragment of about 140 bp obtained by digesting with dIII and SalI was inserted to construct a plasmid pGTPs.

Separately from this, HindII of pUC18
Synthetic DNA (5'-AGCTGCC) at the I-PstI site
CCCCCGGCAAGCTTGCA-3 ′ (SEQ ID NO: 1)), and then S of the plasmid obtained here.
Synthetic DNA (5'-TCGAC) at the alI-KpnI site
ATTTTTATGTAC-3 '(SEQ ID NO: 2)) was inserted, and the SacI-E of the plasmid obtained here was further inserted.
Two synthetic DNAs (5'-AATTCG at the coRI site
GCCGGGGGGGGCCAGCT-3 ′ (SEQ ID NO: 3) and 5′-GGCCCCCCCCGGCCG-3 ′
(SEQ ID NO: 4)) was annealed, and finally, the plasmid pNZ1037 (Ogawa et al., In the HindIII-SalI site of this plasmid was inserted.
Vaccine, 8, 486-490, 1990)
About 270b obtained by digestion with indIII and SalI
p DNA fragment was inserted into the plasmid pGTP7.5.
Was built.

This plasmid pGTPs [restriction enzyme B
glI recognition site]-[poxvirus synthetic promoter Ps]-[multiple cloning site for heterologous gene introduction]-[poxvirus initial transcription termination signal]-[restriction enzyme SfiI recognition site] The plasmid pGTP7.5 contains [restriction enzyme BglI recognition site]-[vaccinia virus 7.5 promoter]-[multiple cloning site for introduction of heterologous gene]-[poxvirus initial transcription termination signal]-[restriction enzyme SfiI]. The recognition site] is connected in this order. An inserted DNA fragment (gene cartridge) can be easily prepared by inserting the antigen gene to be inserted into the multiple cloning sites of these plasmids and cutting with BglI.

Example 2 Acceptor plasmid p
Construction of NZ1829R (see Figure 2) Plasmid pNZ1729R (Yanagida et al.
al. J. Virol. , 66, 1402-140
8, 1992) was digested with EcoRI and SacI, and two synthetic Ds described in SEQ ID NO: 3 and SEQ ID NO: 4 were added to this site.
NA (5'-AATTCGGCCGGGGGGGGCCA
GCT-3 '(SEQ ID NO: 3) and 5'-GGCCCC
Plasmid pNZ1829R was constructed by inserting a synthetic adapter containing an SfiI site that was annealed with CCCGGCCG-3 ′ (SEQ ID NO: 4)).

Example 3 Acceptor plasmid p
Construction of NZ6929Sfi (see FIG. 3) Plasmid pNZ98 (a plasmid having the F antigen gene derived from Newcastle disease virus described in JP-A-3-27284) is partially digested with SacI, and then Ba
Partial digestion with mHI was performed to recover a DNA fragment of about 1.9 kbp. On the other hand, plasmid pNZ1829R was added to BamH
After digested with I, it was further partially digested with SacI, and the above-mentioned BamHI-SacI DNA fragment of about 1.9 kbp was inserted to construct the desired plasmid pNZ5929. AvaI after cutting plasmid pNZ87 (a plasmid having the HN antigen gene derived from Newcastle disease virus described in JP-A-63-239239) with HindIII
The largest fragment obtained by partial digestion with I and plasmid p
A 95 bp fragment obtained by digesting NZ1829R with HindIII and BamHI was added to two kinds of synthetic DNAs (5′-GATCCAGCAT) shown in SEQ ID NO: 5 and SEQ ID NO: 6.
G-3 '(SEQ ID NO: 5) and 5'-GTCCATGC
The plasmid pNZ2929R was constructed by adding annealed TG-3 ′ (SEQ ID NO: 6)). After cutting this plasmid pNZ2929R with HindIII, Bam
Partial digestion with HI gives a fragment of approximately 1.9 kb. This fragment was made blunt-ended with Klenow fragment of DNA polymerase and then digested with SmaI to obtain plasmid p.
Inserted into NZ5929 to acceptor plasmid pNZ
6929Sfi was obtained.

(Example 4) Marek's disease virus (MD
V) Cloning of gE and gI genes Chicken fibroblasts (CEF; chic) infected with MDV GA strain
ken embrionic fibroblast) cells are collected from the petri dish by trypsin treatment, washed twice with PBS, and then
Proteinase K buffer (10 mM Tris-HCl (p
H7.8), suspended with 5 mM EDTA, 0.5% SDS), and proteinase K (Boehringe
r Mannheim) was added to give a concentration of 50 μg / ml. After leaving at 55 ° C for 2 hours, remove the protein twice with phenol / chloroform and
A double amount of ethanol was added, and the mixture was allowed to stand at -20 ° C for 20 minutes and then centrifuged to obtain genomic DNA of MDV GA strain.

To clone both the gI and gE genes from the genomic DNA of the MDV GA strain, Velicer
Based on the nucleotide sequence of the unique short region of the MDV GA strain (US Pat. No. 5,252,716), which was published by et al.
Seed DNA primer (5'-CGGGAGATCTG
CGATGTATGTACTACAATTA-3 ′ (SEQ ID NO: 7) and 5′-GGATCCCGCATCGA
CAATAAATT-3 '(SEQ ID NO: 8)) was used. MDV GA previously obtained using these primers
Polymerase chain using strain genomic DNA as a template
A DNA fragment having the entire length of the gI gene and a part of the gE gene was amplified by reaction (hereinafter referred to as PCR). The reaction composition was 8 μl of 10-fold concentrated PCR reaction buffer (200 mM Tris-HCl (pH 8.50) in 64.5 μl of an aqueous solution containing 20 pg of MDV genomic DNA.
4), 500 mM KCl, 25 mM MgCl ₂ ),
4 μl of 2.5 mM dNTPs, 0.5 μl of 5 U /
20 μM of each primer was added with 1 μl of Taq polymerase. The reaction conditions are denaturation 95 ° C. for 1 minute,
Annealing was performed at 55 ° C. for 2 minutes and polymerase reaction at 72 ° C. for 2 minutes, and 30 cycles were performed using a thermal cycler 480 manufactured by Perkin Elmer Cetus. As a result, a DNA fragment of about 1.5 kbp containing the entire length of the gI gene and a part of the base sequence on the 5'side of the gE gene was obtained.

After cleaving the MDV GA strain with various restriction enzymes, it was electrophoresed on a 0.8% agarose gel, the DNA fragment in the gel was transferred to a nylon membrane, and Southern High was used with the PCR product as a probe. Hybridization was performed. As a result, an approximately 1.1 kbp BamHI-KpnI DNA fragment containing the gI gene and g
About 6.0 kbp BglII-Hind containing E gene
It was confirmed that the III DNA fragment was hybridized. These DNA fragments were obtained from BamHI of pUC18.
-KpnI site and BamHI-HindIII site, and cloned into pUC-gI-1 and pUC-g, respectively.
E-1 was obtained. A BglII-HindIII DNA fragment of about 6.0 kbp cloned in pUC-gE-1 was cleaved with various restriction enzymes to prepare a restriction enzyme map.
Utilizing this, K of about 2.2 kbp containing gE gene
The pnI-HpaI DNA fragment was ligated with KpnI-of pUC18.
Subcloned into HincII site, pUC-gE
-2 was obtained. Sanger's dideoxy chain termination method (Sanger et al., Proc.
Natl. Acad. Soc. USA, 74, 5463
-5467, 1977) confirmed the insertion of the gE gene into this plasmid by examining the sequence of a series of deletion mutants of pUC-gE-2.

(Example 5) Donor plasmid pGTPs
MDgB, pGTPsMDgE, and pGTP7.5
Construction of MDgI (see FIGS. 4 and 5) Plasmid pNZ29RMDgB (Yanagida)
et al. J. Virol. , 66, 1402-1
408, 1992) was digested with SalI and then partially digested with BamHI to obtain a fragment of about 2.9 kb which was used as a plasmid pG.
PGTP by inserting TPs into SalI and BamHI sites
sMDgB was obtained. PCR was carried out using pUC-gE-1 obtained in Example 4 as a template and the primers shown in SEQ ID NOs: 9 and 10. The reaction composition and conditions were the same as in Example 4. The resulting PCR product was BglII and SalI
After digestion with, a DNA fragment of about 1.5 kbp was recovered, and a synthetic DNA (5'-) for making a BglII site between the HindIII and PstI sites of the plasmid pUC18 was recovered.
AGCTAGATCTTGCA-3 ′ (SEQ ID NO: 1
Plasmid pUC18-X prepared by incorporating 1))
It was inserted into the BglII-SalI site of G and the plasmid p
UC-gE-PCR was constructed. The nucleotide sequence of the inserted DNA fragment was confirmed, but no mutation was found during PCR. This plasmid pUC18-gE-PCR
Digested with BglII and SalI, D1.5 of about 1.5 kbp
The NA fragment was recovered and BamHI-SalI of pGTPs was recovered.
It was inserted into the site to construct the plasmid pGTPs-gE.

(Example 6) Plasmid pNZ29RNDFHN-MDgB, p for producing multivalent recombinant fowlpox virus
NZ29RNDFHN-MDgBgE, and pNZ2
Construction of 9RNDFHN-MDgBgEgI (see FIG. 6) A fragment of about 3.0 kb obtained by digesting donor plasmid pGTPsMDgB with BglI was inserted into the SfiI site of acceptor plasmid pNZ6929Sfi to obtain plasmid pNZ29RNDFHN-MDgB. By using a sufficient amount of insert fragment, high integration efficiency was achieved without treatment with terminal phosphatase. Specifically, randomly examine the plasmids of 24 transformants,
Twelve had no insert, six had one insert, and the remaining six had two MDgBs inserted in tandem.

The plasmid pNZ29RN obtained here is
DFHN-MDgB was used again as an acceptor plasmid to try to insert the next gene. The donor plasmid pGTPsMDgE was digested with BglI and the resulting approximately 1.7 kb fragment was pNZ29RNDFHN-MDgB.
At the SfiI site of pNZ29RNDFHN-M
DgBgE was obtained. Donor plasmid p
About 1.4k after digesting GTP7.5MDgI with BglI
A bp DNA fragment was recovered. This DNA fragment was inserted into the SfiI site of the previously obtained pNZ29RNDFHN-MDgBgE to obtain the plasmid pNZ29RNDFHN-MD.
gBgEgI was constructed.

(Example 7) Preparation and purification of recombinant fowlpox virus A virus having a large plaque formation as a phenotype isolated from a fowlpox virus vaccine in monolayer CEF (Nazerian et al., Avian).
Dis. , 33, 458-465, 1989) 0.1.
Was infected with a multiplicity of infection. After 3 hours, these cells were detached by trypsin treatment to obtain a cell suspension. 2 × 10 ⁷ cells and 10 μg of the recombinant fowlpox virus producing plasmid pNZ29RNDFHN-MD from this suspension.
gB (Example 6) was mixed. The mixture of this cell and the plasmid for recombination was treated with Saline G (0.14M N
aCl, 0.5 mM KCl, 1.1 mM Na ₂ HP
_{O 4, 0.5mM MgCl 2 · 6H} 2 O, 0.011%
Glucose), and at room temperature, Gene Pulser (Bio-Rad) 3.0 KV / cm, 0.4 ms
Electroporated under ec conditions. The plasmid-introduced cells were then cultured for 72 hours at 37 ° C.
Cells were lysed by freezing and thawing once. The released recombinant virus was selected as follows.

Although progeny virus was released from the lysed cells, CEF was infected with a 10-fold serial dilution of the lysate, and 10 ml of an agar solution containing growth medium was overlaid. After solidifying the agar at room temperature, it was incubated at 37 ° C. until the appearance of typical fowlpox virus plaques. Bl
Another agar containing 600 μg / ml of uo-gal 10 m
1 was overlaid on each culture plate and further cultured at 37 ° C. for 24 hours. Blue plaques appeared at a rate of approximately 1% for all progeny viruses. These blue plaques were isolated, the contained virus was recovered, and the recombinant virus was further purified by the same method until all the formed plaques were stained blue with Blue-gal. This process usually ends in 3-4 times. This purified virus was named fNZ29RNDFHN-MDgB. This fNZ29RNDFHN-MDgB was analyzed by Southern hybridization, and it was confirmed that each gene was in the expected position. Plasmid p for producing the recombinant fowlpox virus shown in Example 6
NZ6929Sfi, pNZ29RNDFHN-MDg
BgE and pNZ29RNDFHN-MDgBgEgI were also introduced into fowlpox virus-infected cells by the same method as described above, and the resulting recombinant viruses were respectively transformed into fNZ.
6929Sfi, fNZ29RNDFHN-MDgBg
E, named NZ29RNDFHN-MDgBgEgI.

(Example 8) Anti-NDV antibody titer and anti-MDV antibody titer of chickens inoculated with recombinant virus Recombinant fowlpox virus 10 ⁴ PFU shown in Table 1 was applied to the right wing membrane of a 7-day-old SPF chicken with a puncture needle. Was inoculated. Serum was collected 3 weeks after the inoculation, and the anti-NDV antibody titer and anti-MDV antibody titer used for hemagglutination inhibition (hereinafter referred to as HI) of Newcastle disease were measured. The HI reaction in Newcastle disease, which is the basis of anti-NDV antibody titer, is 2
ND containing 4 hemagglutination units in 25 μl of double serial dilution
V antigen was added in an equal amount and mixed, and after standing and sensitizing for 10 minutes, 0.5
% Chicken red blood cell solution (50 μl) was added, and the mixture was left at room temperature for 45 minutes, and hemagglutination was observed. The HI value was expressed by the highest dilution ratio of serum in which hemagglutination inhibition was observed (HI value in the table).

The anti-MDV antibody titer was measured by the following method. MDV gB gene restriction enzymes EcoRV and Bam
The fragment obtained by digesting with HI was used as Sma of pATH-11.
Part of the MDV gB gene antigen protein polypeptide inserted into the I-BamHI fragment (353 amino acids)
Was expressed. 75% of this polypeptide has 10% SD
Add 750 μl of S and 6.675 ml of water and boil for 10 minutes. 0.1M bicarbonate buffer (pH 9.6)
292.5 ml, and add 100 μl / well Nun
It was dispensed on a 96-well U-bottom plate Maxi-sorb manufactured by C company and left overnight at 4 ° C. to prepare an MDV gB antigen plate. Block Ace diluted 2-fold from this plate
(Dainippon Pharmaceutical Co., Ltd.) overnight at 4 ° C. to block, and each well was serially diluted 2-fold with 10-fold diluted Block Ace to 37 ° C.
For 1 hour. The plate was washed 3 times with PBS containing 0.05% Tween 20 (hereinafter referred to as PBS-T), and 2000-fold diluted with HRP-conjugated rabbit anti-chicken IgG (H + L) from Bethyl was reacted at 37 ° C. for 1 hour. P
After being washed 3 times with BS-T, it was washed once with PBS, and the color was developed with a color developing solution (ABTS) manufactured by Sumitomo Bakelite Co., Ltd.
After reacting in a dark place for 30 minutes, Mo of Bio-Rad
4 with the absorbance at 490 nm as a control at del3550
The absorbance at 15 nm was measured, and the value was used as the MDV antibody titer.

As a result, as shown in Table 1, the recombinant fowlpox virus fNZ29R having the Marc disease gB gene in addition to the F and HN genes of Newcastle disease virus
Recombinant fowlpox virus fNZ6929Sfi (expressing F and HN genes of Newcastle disease virus) or fNZ29RMDgB-S (geck gene of Marek's disease) was expressed in chicken serum inoculated with NDFHN-MDgB; , Which is the recombinant virus produced in the examples of JP-A-520753.), An immune response equivalent to that obtained by immunizing the recombinant virus was obtained.

[0036]

[Table 1]

(Example 9) Protective Effect against Infectious NDV in Chickens Inoculated with Recombinant Virus Recombinant fowlpox virus was treated with 10 ⁴ PFU of 7 with a puncture needle.
The right wing membrane was inoculated on day-old SPF chickens. 1 every 4 weeks after inoculation
0 ⁴ PFU of the highly virulent NDV Sato strain was inoculated into peach muscle. Thereafter, deaths due to Newcastle disease were recorded for 2 weeks. The results are shown in Table 2. All the chickens in the unvaccinated and non-recombinant fowlpox virus-inoculated groups died of Newcastle disease in response to the challenge test for highly virulent NDV, but fNZ2 containing both F and HN genes of Newcastle disease
9RNDFHN-MDgB and fNZ6929Sfi
Both showed complete protection. Thus, a multivalent recombinant virus vaccine can be easily prepared by incorporating a plurality of antigen genes by the method of the present invention.

[0038]

[Table 2]

[0039]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: AGCTGCCCCC CCGGCAAGCT TGCA 24

[0040]

[Sequence list]

 SEQ ID NO: 2 Sequence Length: 17 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: TCGACATTTT TATGTAC 17

[0041]

[Sequence list]

 SEQ ID NO: 3 Sequence Length: 22 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: AATTCGGCCG GGGGGGCCAG CT 22

[0042]

[Sequence list]

 SEQ ID NO: 4 Sequence Length: 14 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: GGCCCCCCCG GCCG 14

[0043]

[Sequence list]

 SEQ ID NO: 5 Sequence length: 11 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GATCCAGCAT G 11

[0044]

[Sequence list]

 SEQ ID NO: 6 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GTCCATGCTG 10

[0045]

[Sequence list]

 SEQ ID NO: 7 Sequence Length: 31 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: CGGGAGATCT GCGATGTATG TACTACAATT A 31

[0046]

[Sequence list]

 SEQ ID NO: 8 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: GGATCCCGCA TCGACAATAA ATT 23

[0047]

[Sequence list]

 SEQ ID NO: 9 Sequence Length: 32 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: GGGGAGATCT CATAATGTGT GTTTTCCAAA TC 32

[0048]

[Sequence list]

 SEQ ID NO: 10 Sequence Length: 28 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: GGGGGTCGAC GTCCATATAC TATATCCC 28

[0049]

[Sequence list]

 SEQ ID NO: 11 Sequence Length: 14 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: AGCTAGATCT TGCA 14

[0050]

[Brief description of drawings]

FIG. 1: Donor plasmids pGTPs and pGTP
It is drawing explaining the construction method of 7.5.

FIG. 2 is a drawing explaining a method for constructing an acceptor plasmid pNZ1829R.

FIG. 3 Acceptor plasmid pNZ6929Sfi
3 is a diagram illustrating a method of constructing the above.

FIG. 4 is a drawing explaining a construction method of a donor plasmid pGTPsMDgB.

FIG. 5: Donor plasmids pGTPsMDgE and p
It is drawing explaining the construction method of GTP7.5MDgI.

[Fig. 6] Plasmid p for producing multivalent recombinant fowlpox virus
NZ29RNDFHN-MDgB, pNZ29RNDF
HN-MDgBgE, and pNZ29RNDFHN-
It is drawing explaining the construction method of MDgBgEgI.

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

Claims (6)

[Claims] 1. A SfiI recognition site of a plasmid having one restriction enzyme SfiI recognition site, one end of which is a restriction enzyme S
It is the cut surface by fiI, and the other end is the restriction enzyme Sfi
A DNA fragment that is a cleavage surface with a restriction enzyme X other than I
Only one restriction enzyme Sf obtained by insertion
A recombinant having an iI recognition site. 2. A restriction enzyme X other than SfiI is BglI,
AlwNI, DraIII, PflMI, BsII, M
The recombinant according to claim 1, which is woI or any one of these isoschizomers. 3. The recombinant according to claim 1 or 2,
Further, a multivalent recombinant obtained by inserting one or more DNA fragments having a restriction enzyme SfiI cutting surface and a restriction enzyme X cutting surface other than the restriction enzyme SfiI at both ends. 4. The SfiI recognition site of a plasmid having one restriction enzyme SfiI recognition site, one end of which is the restriction enzyme S
A method for producing a recombinant, which comprises inserting a DNA fragment having a fiI cleavage surface and a restriction enzyme X cleavage surface other than the restriction enzyme SfiI at the other end. 5. A transformant transformed with the polyvalent recombinant plasmid according to claim 3. 6. A recombinant virus produced using the polyvalent recombinant plasmid according to claim 3.