JPH06292583A - Antigenic protein derived from marek disease virus, recombinant of gene thereof and vaccine using the same - Google Patents

Abstract

PURPOSE: To provide a vaccine for Marek's disease virus.

CONSTITUTION: The objective vaccine is prepared by using a recombinant prepared by the use of a gene coding for a tegument protein of Marek's disease virus.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antigenic protein derived from Marek's disease virus, a gene thereof, a recombinant having the gene, and an anti-Marek's disease vaccine containing the recombinant or the antigenic protein as an active ingredient.

[0002]

Marek's disease virus (MDV), a type of herpesvirus, causes an infectious lymphoproliferative disease in young and adult chickens, and infected chickens present with neuropathy and lymphoma. Marek's disease is a spontaneous malignancy initially controlled effectively by monovalent vaccination. However, in some areas the damage due to Marek's disease continues to increase worldwide, with conventional vaccines failing to provide sufficient protective efficacy. One of the major causes of vaccine breaks in commercial-reared chickens is that currently used vaccines do not provide sufficient protection against the emerging MDV virulent strain. Highly virulent MDV strains, such as the highly virulent vvMDVs strain,
Although isolated from the field, current vaccines have little protection against these virulent strains.
MDV mutants with increased virulence could ultimately render current vaccines ineffective. In addition to this, current vaccines are not ideal in that they are cell-dependent, i.e. they need to infect cells and freeze the infected cells in liquid nitrogen for storage and transport. Several glycoproteins of HSV of herpesviruses (eg gB, gC, gD, etc.) are known to induce an immune response (Blacklaws et al., Virolog).
y, 177, 727-736 (1990), Marti.
n et al. Gen. Virol. , 70, 1359-1
370 (1989)). The same applies to other herpesviruses, see Guo et al. Gen. v
irol. , 64, 2399-2406 (1990),
Britt et al. Virol. , 64, 1079-1
085 (1990) and Keller et al. Viro
l. , 52, 293-297 (1984) and the like. Several other structural or nonstructural proteins also have the potential to induce protective immunity against herpesviruses. The tegument region of the herpesvirus particle is an amorphous region, usually defined as the electron-dense part, located between the viral envelope and the capsid. Proteins belonging to this region have been demonstrated by confirming that they are not released by nonionic detergents and are not present in the viral capsid (Whittaker).
Et al., J. Virol. , 65, 2320-2326 (1
991)). The product of the tegument gene is called the herpesvirus alpha gene and controls the first gene expression in trans after infection. The first cloned herpesvirus tegument gene was α-trans-
It was a gene encoding the main component of α-TIF, which is called an inducing factor (α-TIF) gene.
Proteins encoded by other tegument genes such as UL47 and UL46 also play a role in regulating the expression of the alpha gene (McKnight).
Et al., J. Virol. , 61, 992-1001 (19
87)). However, this method could not be used for cloning the MDV tegument gene. This is because it was difficult to extract a large amount of MDV protein. It is therefore clear that producing monoclonal antibodies or polyclonal antisera against native MDV tegument protein is not an effective method. In fact, such a protein has not yet been identified. In addition to this, MDV and HSV are less likely to undergo immunological cross-reactivity, making it difficult to find the MDV tegument protein using polyclonal antisera against HSV and other herpesvirus tegument proteins. there will be. Buckma
sters et al. Gen. Virol. , 69, 2
In 033-2042 (1988), it was suggested that a gene homologous to the 12th gene of VZV is located close to gCh of MDV, but data regarding its sequence are not known yet. In addition, recently, the HSV-1 tegument protein VP22 encoded by the UL49 gene was used.
(Elliot and Meredith, J. et al.
Gen. Virol. , 73, 723-726 (199
2)).

[0003]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a result of earnest research to obtain a new anti-Marek's disease vaccine, the present inventors have found that the Marek's disease virus tegument protein corresponding to the tegument protein of herpes simplex virus We have found the encoding gene and completed the present invention.

[0004]

Thus, the present invention relates to an antigenic protein derived from Marek's disease virus, a gene thereof, a recombinant having the gene, and an anti-Marek's disease vaccine containing the antigenic protein or the recombinant as an active ingredient.

(DNA molecule) The DNA molecule of the present invention comprises three tegument proteins UL46h and UL4 having the amino acid sequences shown in SEQ ID NOs: 5, 4, 3, and 2.
It encodes a gene encoding 7h, UL48h, UL49h, or an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more homology with the gene.

The homology referred to here is calculated by the DNASIS method. All three strains of MDV serotypes are suitable for cloning tegument genes. Of these, viruses belonging to serotype 1 are preferred,
Examples of such viruses include GA strain (Ediso).
n et al, Avian Dis. , 12, 467
-476 (1968)), RB1B strain (Schate)
al, Avian Pathol. , 11, 593
-605 (1982)), CU2 strain (Smith et al.
al, Avian Dis. , 17, 727-736
(1973)), JM strain (Sevoian et a.
1, Vet. Med. , 57, 500-501 (196
2)), Md5 strain (Witter et al, Avi)
an Dis. , 24, 210-232 (1985)).
And so on.

The tegument protein of the present invention is homologous to HSV-1 UL46, UL47 and UL48, respectively, and is also homologous to VZV gene 12, gene 11, gene 10 and gene 9. H
It is known that when the SV-1 genome and the VZV genome are compared, they overlap in many gene regions, but they are not all located at the same position (Darrymple et.
al, Nucleic Acids Res. 1
3, 7865-7879 (1985); McGeoch.
et al, J .; Gen. Virol. , 69, 15
34-1574 (1988); Davidson, J .;
Gen. Virol. , 67, 1759-1816 (1
986).

Genes encoding tegument proteins (hereinafter referred to as tegument genes) are HSV-1 and V
It is located in the long unique region (UL region) near the glycoprotein C of ZV or its homologue. The MDV-derived gene was derived from the viral genome of the GA strain, BamHI-, in determining whether a homologue of the major tegument gene is present at similar positions on the HSV-1, VZV and other herpesvirus genomes. A restriction enzyme cleavage point map of the B fragment was created (Fig1).

From the above BamHI-B fragment, a 5.1 kb SmaI fragment (referred to as SmaJ) was added to pUC18 Sma.
It was cloned into the I site. Same 5.1 kb Sma
Two clones (pUCSmaI5.1 + and pUCSmaI5.1-) in which the J fragment was inserted in the opposite direction.
Was selected to generate a series of deletion mutants by ExoIII nuclease and Mung Bean nuclease treatment.

A series of deletion mutants of the above clones were subjected to the dideoxy chain termination method (Sanger e).
al, PNAS USA, 74, 5463-546.
7 (1977), and United States B
The whole nucleotide sequence of the cloned 5.1 kb SmaI fragment was determined by analysis with a sequence analysis kit manufactured by iochemical. Analyzed in both directions at least once over the entire region of the 5.1 kb SmaI fragment,
The sequence was determined. As a result, it was found that there were four complete open reading frames (ORFs), which were HSV-1 UL49, UL48, and UL, respectively.
47, and UL46, and VZV Gene 9, Gene 10, Gene 11, and Gene 12.

In order to obtain the complete ORF of the regions (UL46h and UL49h) corresponding to UL46 and UL49 of HSV-1, a 8.4 Kb Bam is obtained by the same operation as described above.
The HI-EcoRI fragment was obtained and similarly sequenced. The DNA sequence and the deduced amino acid sequence are shown in SEQ ID NO: 1.

(Recombinant DNA Molecule) The recombinant DNA molecule of the present invention comprises the above-mentioned at least one MDV tegument gene sequence and at least one additional DNA sequence linked thereto by genetic engineering. The term "additional DNA sequence" as used herein means a sequence that is not naturally linked to the MDV tegument gene.

(Recombinant virus) virus The virus used in the present invention is not particularly limited as long as it can express the integrated tegument gene. Specific examples of such viruses include poxviruses such as fowlpox virus and quailpox virus, baculovirus, and turkey herpesvirus. Among them, those belonging to the foulpox virus are preferable, and specific examples thereof include A
TCCVR-251, ATCC VR-250, ATC
C VR-229, ATCC VR-249, ATCC
VR-288, Nishigahara strain, Sishui strain, CEVA strain, CEV
Of the viruses derived from the A vaccine strain, FPV in the narrow sense such as those that form large plaques when infected with CEF (chicken embryo fibroblast) cells, and NP strains (such as fetal pox venom pox Nakano strain) in a narrow sense The virus that is closely related to FPV of E. coli and is used as a live fowlpox vaccine strain is exemplified. These are easily available such as being commercially available.

Non-essential region The non-essential region used in the present invention is not particularly limited. For example, about 7.3 Kbp EcoRI fragment of NP strain DNA, about 5.2 Kbp HindIII fragment, about 5.0 K.
bp EcoRI-HindIII fragment, approximately 4.0 Kb
Examples thereof include a BamHI fragment of p, a TK region of qualpox virus, a TK region of turkey herpesvirus, a baculovirus inclusion body region and the like (JP-A-1-168279).

Vector Containing Virus Non-Essential Region The vector containing a virus non-essential region used for constructing the vector for virus recombination used in the present invention is not particularly limited. For example, pBR322, pBR
325, pUC7, pUC8, pUC18 and other plasmids, λ phage, M13 and other phages, p
A vector such as cosmid such as HC79 may be treated with an appropriate restriction enzyme to incorporate the above-mentioned nonessential region of the virus.

Antigen Gene The antigen gene to be incorporated into the virus in the present invention is a gene encoding a Marek's disease tegument protein having the nucleotide sequence of SEQ ID NO: 2, 3, 4 or 5.
The gene or a fragment thereof encodes a fragment thereof, or a part of the amino acid sequence of the tegument protein which is modified to such an extent that the ability to induce a protective immune response is not impaired. An antigenic protein encoded by such a fragment or a partially modified sequence has an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 2, 3, 4, or 5 as a whole. , Thereby conferring a substantially equivalent immune response on the host. The fragment or a part of the sequence modified is 80 relative to the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5.
% Or more, preferably 90% or more, more preferably 9%
It has a homology of 5% or more. These genes may be incorporated into a recombinant one by one or multiple genes may be incorporated. In addition, genes encoding gBh, gCh, gDh, gHh, gIh and the like of Marek's disease are also included. It can also be incorporated.

Vector for recombination The vector for virus recombination used in the present invention contains at least a tegument gene and a nonessential viral region into which a promoter controlling it is inserted. It is sufficient to insert the above-mentioned antigen gene and the promoter that controls it into the virus-non-essential region of the vector containing the above-mentioned virus-non-essential region, and also insert the above-mentioned vector-derived antigen gene and the promoter that controls it. The fragment containing the thus-obtained non-essential region of the virus may be inserted into another vector. Further, a marker gene such as Escherichia coli lacZ gene and a promoter controlling it may be incorporated for purification of recombinant virus.

Promoter The promoter used here is not particularly limited as long as it functions as a promoter in a recombinant virus-infected host. For example, promoter of vaccinia virus gene encoding 7.5K polypeptide, 11K
Vaccinia virus gene promoter that encodes a polypeptide, vaccinia virus promoter that encodes thymidine kinase, polyhedrin promoter of baculovirus, and SV4 of turkey herpesvirus.
0 promoter and the like are exemplified, and as long as it functions as a promoter, it may be modified such as partially deleted, or may be synthetic.

Method for Producing Recombinant Virus The method for producing the recombinant virus is not particularly limited and may be performed by a conventional method. That is, by introducing a recombinant vector into a virus-infected cell in advance by, for example, the calcium phosphate coprecipitation method, homologous recombination occurs between the vector and the viral genomic DNA in the infected cell, resulting in recombinant virus. Be built. The obtained recombinant virus is infected with a host cell cultured in a medium such as Eagle MEM, and growing plaques are selected as candidates for the target recombinant virus. The candidate strain is purified by a hybridization method using the antigen gene incorporating this candidate strain as a probe, or a method of selecting an expression of the marker gene incorporated with the antigen gene, and the antigen encoded by the incorporated antigen gene is purified. The antibody may be confirmed to be the target recombinant virus using an immunoassay. For example,
In the case of a recombinant virus in which the lacZ gene is incorporated as a marker gene, β-galactosidase is expressed, and blue plaques are formed one after another in the presence of one of its substrates, Blu-o-gal. The host cell is not particularly limited as long as it can be infected with the virus to be used and propagates. For example, when FPV is used, chicken embryo fibroblasts (CEF cells), developing chicken chorioallantoic chondrocytes, etc. When baculovirus is used, examples include Spodoptera frugiperda cells, and when turkey herpes virus is used, duck fibroblasts (DEF cells) and the like.

(Transformant) Antigen gene Same as recombinant virus

Vector, Transformed Cell, Transformed Microorganism The vector for constructing the recombinant vector having the above-mentioned gene is not particularly limited and may be, for example, pU.
C8, pUC9, pUC10, pUC11, pUC1
8, pUC19, pBR322, pBR325, pBR
327, pDR540, pDR720 and other plasmids, λgt11, λgt10, λEMBL3, λEMBL
4, phages such as Charon 4A, etc. are exemplified. A method for inserting the above gene into these vectors to generate a recombinant vector may be well known to those skilled in the art. For example, after the vector is cleaved with a restriction enzyme, the above-mentioned method is performed under the control of a promoter that functions in the host. The gene may be directly linked. Examples of the promoter used include lac promoter / operator, trp promoter, tac promoter, lpp promoter, PL promoter, amyE promoter, Ga17 promoter, PGK promoter, ADH promoter and the like.

In producing a recombinant vector for expressing these MDV-derived tegument proteins, the above-mentioned gene is once incorporated into an appropriate vector to produce a recombinant vector and subcloning is a method well known to those skilled in the art. These subcloned genes can be excised with an appropriate restriction enzyme and ligated to the above promoter to prepare a recombinant vector capable of producing a protein.

The vector that can be used for the above subcloning is not particularly limited, and it is pUC.
8, pUC9, pUC10, pUC11, pUC18,
pUC19, pBR322, pBR325, pBR32
7, pDR540, pDR720, pUB110, pI
J702, YEp13, YEp24, YCp19, YC
Examples of plasmids include p50, pAC373, pACTM1 and the like. Then, using the obtained recombinant vector, various suitable hosts are transformed to obtain a microorganism having an ability to produce an antigenic MDV-derived tegument protein or a fusion protein containing its amino acid sequence. be able to. The host used here can be selected in consideration of suitability for expression vector, stability of product, etc., and may be a prokaryotic cell or a eukaryotic cell. Specific examples include the genus Escherichia (eg, Escherichia coli), the genus Salmonella (eg, Salmonella typhimurium), actinomycetes, yeast, insect cells, avian cells, human cells and the like. A host transformed by introducing an appropriate expression vector can be cultured and grown under culture conditions well known to those skilled in the art. In addition, in producing a protein, conditions for inducing the action of a promoter can be selected. As a specific example thereof, taking the lac promoter operator as an example,
This is achieved by adding an appropriate amount of isopropylthio-β-D-galactopyranoside to the culture medium.

[0024] An anti-Marek's disease vaccine can be produced using the thus obtained transformed host according to a conventional method. Culturing of this host may be carried out under the conditions usually used for culturing microorganisms. For example, when Escherichia coli is used, it is cultured under aerobic conditions in LB medium at 37 ° C.

(Tegment Protein) The tegument protein of the present invention has an amino acid sequence represented by SEQ ID NOs: 2 to 4 obtained by the above-mentioned gene recombination technique or 80% or more, preferably 9% homologous to the amino acid sequence.
It is 0% or more, more preferably 95% or more. In addition, the tegument protein of the present invention may be an analog of those having these sequences. The term "analog" as used herein means a homologue to a natural tegument protein which induces a protective immune response in a host when administered, and the amino terminus of the amino acid of the above sequence. It may be a mutant in which any one of the carboxyl terminus and the central portion is deleted, or a mutant in which these deletions are combined. Further, the analog may be a mutant in which one or more amino acids are substituted with other amino acids, or may be one in which one or more amino acids are deleted / inserted, and a combination of these It may be Such a tegument protein can be produced by culturing the above-mentioned transformant. Alternatively, the recombinant virus described above can be produced by culturing it in an appropriate host cell. Produced tegument protein is Method in Enzymo
182, volume ("Guide to Prote"
in Purification "Murry P. D
It may be isolated and purified in accordance with the description in "Eutscher", published by Academic Press.

The thus obtained tegument protein can be used as a component vaccine by mixing it with a diluting / expanding material and the like by a conventional method. When used as a component vaccine, 0.1 μg per individual
The above may be administered, and the upper limit is not particularly limited as long as acute toxicity is not shown. The administration method may be subcutaneous, intravenous, intramuscular injection, or the like, and it is also possible to immunize the respiratory tract by spraying. Furthermore, this tegument protein can also be used as a diagnostic agent for Marek's disease virus infection.

(Live Vaccine) The live vaccine of the present invention is a non-cell-dependent vaccine comprising one or more recombinant viruses of the present invention. That is, separate recombinant viruses having the three types of genes disclosed in the present invention may be used alone, or two or three types of recombinant viruses may be used in combination. In addition to the recombinant virus, a carrier that does not have a pharmacological problem, such as physiological saline, may be contained. The method for preparing the vaccine of the present invention is not particularly limited. For example, cells capable of growing the virus used in the present invention are infected with the recombinant virus of the present invention and cultured until the recombinant virus grows.
Then, the cells are collected and disrupted. The cell lysate is separated by a centrifuge in a centrifuge tube into a precipitate and a non-cell-dependent high titer supernatant containing recombinant virus. This centrifuge supernatant, essentially free of host cells, but containing cell culture medium and recombinant virus, can be used as a vaccine according to the invention. Also, a physiologically acceptable physiological saline or the like may be reconstituted and used. It can also be used as a freeze-dried vaccine obtained by freeze-drying the centrifuged supernatant.

The vaccine of the present invention may be administered to poultry by any method as long as the recombinant virus in the vaccine can infect poultry to induce protective immunity. For example, it is possible to vaccinate the skin by scratching the skin for vaccination, or to vaccinate the poultry subcutaneously with an injection needle or other device. It is also possible to suspend the vaccine in the drinking water of poultry or mix it with the solid matter of the feed for oral inoculation. Furthermore, a method of inhaling a vaccine by aerosol or spray, an intravenous inoculation method, an intramuscular inoculation method, an intraperitoneal inoculation method and the like can also be used. The amount of inoculation is usually 1 for chickens, for example.
0 is a ⁴ to 10 ⁶ PFU (plaque forming units). For injection, this amount may be diluted with a physiologically acceptable liquid such as physiological saline to about 0.1 ml. The timing of inoculation is not limited, but for the purpose of immunization, it is preferable to inoculate at the age of one day, as in existing vaccines. The vaccine of the present invention is non-cell dependent and can be stored and used under normal conditions.
This can reduce costs and preserve in liquid nitrogen, which was necessary for existing cell-dependent vaccines,
It is possible to reduce troublesome procedures such as handling and inoculation. For example, by freeze-drying the recombinant virus of the present invention, the vaccine of the present invention can be stored, handled, and transported at room temperature (about 20 to 22 ° C) for a long period of time. Since it can be stored even in a frozen state, and the non-cell-dependent recombinant virus in this vaccine can be retained in water, for example, a suspension of the recombinant virus can be stored at −20 ° C.
It can be stored at 70 ° C by freezing.

[0029]

【Example】

(Example 1) Cloning of SmaI 5.1 Kb (Sma-J) fragment derived from BamHI-B fragment of MDV GA strain BamHI contained in plasmid pACYC184
-B fragment (from MDV GA strain, Fukuchi et al.,
J. Virol. , 51, 102-109 (198
4)) was treated with the restriction enzyme SmaI, and the 5.1 Kb fragment was purified by agarose gel electrophoresis. Plasmid pUC
After treating 18 with the restriction enzyme SmaI, it was treated with alkaline phosphatase derived from calf intestine to remove the 5'phosphate moiety. Then, the 5.1 Kb fragment obtained above was inserted to obtain two plasmids, pUCSma5.1 + and pUCSma5.1-. These plasmids have a 5.1 Kb fragment inserted in the opposite direction.

Example 2 Sequence Analysis of SmaI 5.1 Kb Fragment To prepare a series of defective strains pUCSmaI + and pUCSmaI +
UCSmaI-restriction enzyme BamHI and restriction enzyme Pst
After treatment with I, the treatment time was changed and treatment with endonuclease III was performed. Samples of the reaction mixture taken at different intervals were collected and treated with Mung Bean nuclease. After phenol / chloroform extraction, DNA was obtained by ethanol precipitation. The recovered DNA was self-ligated and introduced into competent E. coli. Approximately 100 clones were deleted from each of the two plasmids, and the size of the insert was examined.
Clones were selected for sequence analysis. The sequence of each clone was analyzed using ³⁵ S-dATP (manufactured by DuPont) and Sequenase (manufactured by United States Biochemical). As a result, the selection of clones was not appropriate in some regions, and sequence analysis could not be performed, so subcloning was performed using restriction enzymes to supplement the sequences.

(Example 3) 8.4K of MDV GA strain
Cloning and Sequencing of the bBamHI-EcoRI Fragment 8.4 KbBamHI-Ec which is a subfragment of the BamHI-B fragment of MDV GA strain in pUC18.
The oRI fragment was inserted and the resulting plasmid was designated as pUC B.
It was named am-Eco8.4. Multiple restriction enzymes were used to obtain several subclones. Sequence analysis of these subclones was performed by the Sanger dideoxy chain termination method. For sequence analysis from both directions, Oligos Etc. Inc. C
Several synthetic oligonucleotides from T USA were used. Major OR analyzed by computer analysis
The positional relationship of Fs is shown in FIG. The first ORF, UL49h, extends from the start codon ATG at base 718 to the stop codon TGA at base 1475, and has a peptide molecular weight of 27,654 daltons. There is a TATA sequence (bases 703 to 706) 15 bp upstream of this ORF, and it is presumed to be a promoter region. UL49
There was no AATAAA sequence that appeared to be a polyadenylation signal found between the h and UL48h gene regions. UL48h, the second ORF, is 1
From the start codon ATG at base 575 to the stop codon TAA at base 2856, the peptide molecular weight is 48,
It is 655 daltons. There is a TATATA sequence about 30 bp upstream of this ORF, which is presumed to be a promoter region. In addition, about 80 bp downstream of this ORF, AA
There is a TAAA sequence (bases 2940 to 2946), which is considered to be a polyadenylation signal.

The third ORF, UL47h, has 31
From the start codon ATG of base 03 to the stop codon TGA of base 5521, the peptide molecular weight is 91.8
It is 80 Daltons. T about 50bp upstream of this ORF
It has an ATAA sequence (bases 3043 to 3047) and is presumed to be a promoter region. UL47h
No AATAAA sequence was found between and UL46h. This has been reported by McKnight et al. (J. Virol., 61, 992-1001 (198) that there is no signal sequence between UL47 and UL46 of HSV-1.
7)). UL46, the fourth ORF,
From the start codon ATG of base 5665 to the stop codon TGA of base 7369, the peptide molecular weight is 6
It is 3,920 daltons. There is a TATA sequence (bases 5,620 to 5,623) at about 40 bp upstream of this ORF, and it is presumed to be a promoter region. In addition, there is an AAYAAAA sequence (bases 7,465 to 7,470) at 100 bp downstream of this ORF, which is presumed to be a polyadenylation signal. Furthermore, ORFs consisting of two 100 amino acids or more are 7,679-
It was found at bases 8,302 and 8,139-7,507 (which has a reverse reading). Estimated peptide molecular weights are 22,969 daltons and 23,50, respectively.
It is 6 Daltons. However, these putative peptides did not show significant identity with any of the putative peptides of many herpesviruses.

(Example 4) Expression of UL48h gene in Escherichia coli PCR method of Saiki et al. (Science 230, 1)
350-1354 (1985)), the UL48h gene was amplified. CCCGGAT shown in SEQ ID NO: 6
CCA AAAATGGAGG CAAATATGAG
TTT and CCCGTCGACT A shown in SEQ ID NO: 7
PUCSma5.1 + obtained in Example 1 obtained by digesting TTATAAAGT ACTGATGGTA GT with the restriction enzyme EcoRI was used as a template. BamHI-SalI of 1.3Kb is P
Obtained by CR and cloned into pUC18. The inserted fragment was confirmed, but no mutation was found during PCR. 1.3 Kb BamHI-Sal
The I fragment was labeled with the tac promoter (de Boer et al., P
NAS USA, 78, 21 (1983)) and rrnB.
Ribosomal RNA transcription terminator (Brosius et al.
Mol. Biol. , 148, 107 (1981)) to prepare the expression vector pTacUL48h. E. coli TG1 (Amersham, UK),
pKK223-3 (manufactured by Gibco BRL, MD U
SA-derived plasmid pT1 or pTacUL4
8h was introduced. Plasmid pT1 and pTacUL48
Escherichia coli TG1 having h and Escherichia coli TG1 having no plasmid were cultured in an LB medium at 37 ° C. overnight. However, when culturing TG1 containing the plasmid, 50 μg / ml
Ampicillin (Gibco BRL, MD US
A) was used (Maniatis et al., M
oleclar Cloning (1982)). Next, 0.1 ml of each cultivated E. coli TG1 was newly prepared 1
It was added to 0 ml of LB medium and cultured for 2 hours. However, when TG1 having a plasmid was cultured, 50 μg / ml of ampicillin (manufactured by Gibco BRL, MD US
The medium to which A) was added was used. In addition, IPTG (Gibco BRL, MDU, so that the final concentration is 1 mM
SA) was added to each medium and cultured for 4 hours. Next, the culture solution was centrifuged (10 minutes × 10,000 G) to obtain a cell pellet. This pellet is used as a sample buffer (Laem
mli, Nature, 227, 680-685 (19
70)), and boiled. After centrifuging this, the supernatant was analyzed by SDS-PAGE (Laemml.
i, Nature, 227, 680-685 (197).
0)). As a result, as shown in lane 1 of FIG.
50,000 dalton protein is pTacUL48h
Was detected in a sample of E. coli with. From this molecular weight, the obtained protein is UL derived from MDV GA strain.
It turned out to be 48h.

[Sequence List] SEQ ID NO: 1 Sequence length: 8433 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Circular type Sequence type: DNA sequence

[Chemical 1]

[Chemical 2]

[Chemical 3]

[Chemical 4]

[Chemical 5]

[Chemical 6]

[Chemical 7]

[Chemical 8]

[Chemical 9]

[Chemical 10]

[Chemical 11]

[Chemical 12]

SEQ ID NO: 2 Sequence length: 249 amino acids Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence:

[Chemical 13]

[Chemical 14]

SEQ ID NO: 3 Sequence length: 407 amino acids Topology: Linear Sequence type: Amino acid Sequence type: Protein sequence

[Chemical 15]

[Chemical 16]

[Chemical 17]

SEQ ID NO: 4 Sequence length: 806 amino acids Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

[Chemical 18]

[Chemical 19]

[Chemical 20]

SEQ ID NO: 5 Sequence length: 568 amino acids Sequence type: Amino acid Topology: Linear array:

[Chemical 21]

[Chemical formula 22]

[Chemical formula 23]

SEQ ID NO: 6 Sequence length: 33 base pairs Sequence type: Nucleic acid Number of strands: Single strand Sequence type: DNA sequence

[Chemical formula 24]

SEQ ID NO: 7 Sequence length: 32 base pairs Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence:

[Chemical 25]

[Brief description of drawings]

FIG. 1 shows the restriction map of the BamHI-B fragment of the MDV genome and the location of the ORF of the gc homolog.

FIG. 2 E. It is a flowchart which shows the procedure of preparing the expression vector of UL48h in E. coli .

FIG. 3 E. It is a pattern diagram of the expressed protein on SDS-PAGE which shows the expression of UL48h in E. coli .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area C12N 7/01 // C12N 1/21 7236-4B C12P 21/02 C 8214-4B (C12N 1 / 21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Lucy F. Lee United States 48823 Whittier Drive, East Lansing, Michigan 954 (72) Inventor Noboru Yanagida United States 48823 East Lansing, MI, A-1, South Shore Drive 1640

Claims (8)

[Claims] 1. A DNA molecule encoding a polypeptide having an amino acid sequence having a homology of 80% or more with an amino acid sequence selected from SEQ ID NO: 2, 3 or 4. 2. A recombinant DNA molecule comprising the DNA molecule according to claim 1 and at least one additional DNA sequence. 3. A recombinant vector containing the DNA sequence of the DNA molecule of claim 1. 4. A cell, microorganism, or virus having the DNA molecule of claim 1 or 2. 5. A recombinant virus comprising the DNA molecule of claim 1. 6. An isolated or purified polypeptide having an amino acid sequence having a homology of 80% or more with an amino acid sequence selected from SEQ ID NO: 2, 3 or 4. 7. A live vaccine for anti-Marek's disease, which comprises the recombinant virus of claim 5 as an active ingredient. 8. An anti-Marek's disease vaccine comprising the polypeptide of claim 6 or a pharmacologically acceptable salt thereof as an active ingredient.